缺血后适应对大鼠全脑缺血再灌注损伤的影响
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摘要
研究背景大量研究试图寻找可减轻或预防脑缺血再灌注损伤的方法或药物,并且已经在动物模型上获得一定成功,但是这些方法和药物很少能成功用于临床。缺血预处理对心脏和神经系统缺血再灌注损伤具有明显的保护作用,但是在临床实践中预处理只适用于缺血再灌注可预期的情况下。尽快恢复灌注是减轻缺血导致的损伤和行为障碍的最有效方法,然而再灌注也可能加重损伤。许多研究发现,在再灌注早期,大量活性氧自由基产生和钙超载导致了缺血再灌注损伤。
近期研究提示,在冠状动脉再灌注开始时,反复的短暂再灌注和再阻断具有内在的保护作用,这一现象称为“缺血后适应”。由于突然发生的缺血很难预测,而缺血后适应发生在缺血后,因此受到广泛关注。临床有很多情况可以导致全脑缺血,如淹溺、心搏骤停及大血管和心脏手术中的严重低血压等,而脑比其他器官对缺血更敏感。最近,一些实验结果显示,大鼠缺血后适应可减小局灶性脑缺血再灌注后梗死面积,减轻全脑缺血再灌注损伤和脊髓缺血后损伤,然而其分子机制尚未完全清楚。
本实验首先改良Pulsinelli四血管阻塞大鼠全脑缺血模型,利用此改良模型评估缺血后适应对大鼠全脑缺血再灌注损伤的保护作用,并探讨其可能机制。
第一部分大鼠四血管阻塞全脑缺血模型的改良
目的:改良四血管阻塞法大鼠全脑缺血模型,以提高模型的成功率。
方法:100只雄性SD大鼠随机分为2组:组Ⅰ:Pulsinelli法四血管阻塞模型;组Ⅱ:改良四血管阻塞模型。组Ⅰ大鼠椎动脉永久性电凝阻断,双侧颈总动脉游离备用,第二日,直视下用动脉夹夹闭颈总动脉10min造成短暂脑缺血。组Ⅱ大鼠模型进行了改进:双侧颈总动脉游离后,分别用15cm长的10号外科丝线一端经椎旁间隙穿出到颈后,另一端绕过同侧颈总动脉经胸锁乳突肌和颈外静脉间隙穿出至颈后,固定后备用。第二日在颈部收紧丝线阻断颈总动脉10min造成短暂脑缺血。模型成功的标准采用临床指标:翻正反射完全、持续消失,对非伤害性刺激无反应。为了鉴别模型失败的原因,对不成功的大鼠进行全脑血管DSA。
结果:①与组Ⅰ相比,组Ⅱ符合模型成功标准的大鼠显著增加,两组因呼吸衰竭、肺水肿和惊厥而剔除的大鼠没有统计学差异;②两组椎动脉烧灼失败率没有统计学差异。
结论:改良四血管阻塞大鼠全脑缺血模型可提高模型的成功率而不增加并发症。
第二部分:缺血后适应对大鼠全脑缺血再灌注损伤的保护作用
目的:研究缺血后适应对大鼠全脑缺血再灌注的保护作用。
方法:43只雄性SD大鼠随机分为3组:sham组(12只,假手术组)、I/R组(16只,缺血10min再灌注组)和Postcond组(15只,缺血10min后,在再灌注早期行后适应处理,即灌注30s再阻断10s,如此3个周期)。再灌注24h后,每组5只大鼠被断头取脑,左脑用干湿重法计算脑水含量。右侧海马检测MDA和SOD活性。每组7只大鼠再灌注72h后进行Nissl染色观察海马CA1区神经元破坏程度。
结果:①I/R组有4只(其中1只因呼吸衰竭,3只因惊厥)剔除出实验,Postcond组有3只(其中1只因呼吸衰竭,2只因惊厥)剔除出实验,I/R组与Postcond组相比差异无显著性(P＞0.05);②脑组织水含量的测定结果表明,全脑缺血再灌注后脑组织的含水量都明显升高(P＜0.05),但是Postcond组较I/R组脑水含量少(P＜0.05);③与sham组相比,全脑缺血再灌注后海马中的SOD活性均有不同程度的降低(P＜0.05),I/R组降低更为显著(P＜0.05),与sham组比较,全脑缺血再灌注后海马中MDA含量明显增高(P＜0.05),而I/R组增高更为显著(P＜0.05);④Sham组海马细胞核膜完整,核仁清楚,偶可见退变锥体细胞。I/R组再灌注3日后,在CA1区可观察到大量退变神经元;与I/R组相比,Postcond组海马CA1区退变神经元明显减少(P＜0.05)。
结论:缺血后适应对大鼠全脑缺血再灌注损伤具有保护作用,可减少海马神经元的破坏。其保护作用可能与抑制脂质过氧化有关。
第三部分:缺血后适应对大鼠全脑缺血再灌注损伤后神经行为学的影响
目的:通过神经损害评分(NSS)和Morris水迷宫研究缺血后适应对大鼠全脑缺血再灌注损伤后近期及远期神经行为学的影响。
方法:96只雄性SD大鼠随机分为3组(同第二部分)。于再灌注后6h、12h、24h、48h及96h进行NSS评分。分别于再灌注后5日及28日开始,每组各取16只分别进行水迷宫实验评价大鼠的空间学习和记忆能力。
结果:①与sham组相比,I/R组和Postcond组NSS评分增加,但是Postcond组增加较少(P＜0.05);②水迷宫实验结果方差分析显示,I/R组和Postcond组有认知障碍,I/R组更明显(P＜0.05),在同一实验日期,Postcond组平均潜伏期较I/R组短(P＜0.05)。
结论:缺血后适应对大鼠全脑缺血再灌注损伤引起的近期和远期认知障碍有保护作用。
第四部分:缺血后适应对大鼠全脑缺血再灌注损伤后神经元凋亡的影响
目的:研究缺血后适应对大鼠全脑缺血再灌注损伤后神经元凋亡的影响及caspase-3和bcl-2在其中的作用机制。
方法:45只雄性SD大鼠随机分为3组(同第二部分)。再灌注后72h每组5只大鼠生理盐水、多聚甲醛灌注后端头取脑,用TUNEL染色法计算各组海马CA1区凋亡的神经元个数。再灌注后24h时每组5只大鼠生理盐水、多聚甲醛灌注后断头取脑行免疫组织化学检测海马CA1区caspase-3和bcl-2蛋白的表达,另每组各取5只大鼠深麻醉下断头取脑,分离海马,行Western-Blot检测caspase-3和bcl-2蛋白的表达。
结果:①再灌注后72h时,I/R组与Postcond组神经元凋亡增多,I/R组更严重(P＜0.05);②I/R组和Postcond组于缺血再灌注后24h可见caspase-3和bcl-2表达增高,与I/R组相比,Postcond组caspase-3表达更低,而bcl-2表达更高(P＜0.05)。
结论:缺血后适应可减轻神经细胞凋亡,caspase-3与bcl-2可能参与了其机制。
第五部分:缺血后适应对大鼠全脑缺血再灌注损伤后海马区炎症反应的影响
目的:研究缺血后适应对大鼠全脑缺血再灌注损伤后炎症反应的影响及可能机制。
方法:75只雄性SD大鼠随机分为3组(同第二部分)。再灌注24h,每组5只大鼠深麻醉下断头取脑,分离海马,用MPO试剂盒检测海马MPO活性,凝胶电泳迁移率实验(EMSA)检测海马NF-KB的DNA结合活性。分别于再灌注后0h、4h、12h、24h及72h每组各取5只大鼠深麻醉下断头取脑,分离海马,行Real-Time PCR检测海马TLR4基因mRNA的表达。
结果:①缺血后适应对全脑缺血再灌注后海马MPO活性、NF-KB的DNA结合活性的升高有抑制作用;②Real-Time PCR结果显示:缺血再灌注后各时间点TLR-4基因mRNA表达上调,24h达高峰,缺血后适应对TLR-4基因mRNA表达上调具有明显的抑制作用。
结论:缺血后适应可抑制大鼠全脑缺血再灌注损伤后MPO活性和NF-KB的DNA结合活性,并抑制TLR4基因mRNA的上调,其对脑缺血再灌注损伤的保护作用可能与减轻炎症反应有关。
Background Extensive research has been aimed at finding effective strategies and drugs to ameliorate or prevent brain ischemia and reperfusion(I/R) injury.However,few have been successfully applied in clinical practice,although several strategies and drugs have been shown to decrease ischemic damage in the brain in animal models.Ischemic preconditioning has been shown to provide powerful protection against I/R injury in both the heart and nervous system.However,its clinical application is only possible for cases in which the occurrence of I/R is predictable.Rapid initiation of reperfusion is the most effective treatment to reduce injury and the behavioral deficits caused by ischemia. However,reperfusion also has the potential to introduce additional injury;many studies have shown that overproduction of reactive oxygen species and overloading of calcium occur in the early reperfusion period and lead to reperfusion injury.
Another endogenous protective strategy,termed "ischemic postconditioning," has been recently reported,in which several repeated cycles of brief reperfusion and reocclusion of the coronary artery were applied at the onset of full reperfusion.Because ischemia cannot be predicted and happens suddenly,ischemic postconditioning,which can be applied after ischemia,is attracting considerable attention.In clinical practice,many accidents lead to global ischemia,such as drowning,cardiac arrest,and marked hypotension during cardiac surgery,and the brain is intrinsically more vulnerable to ischemia than other organs.Recent studies demonstrated ischemic postconditioning can reduce infarct size after focal brain I/R injury and ameliorate neuronal injury after global cerebral I/R injury and spinal cord ischemia in rat.However,the molecular mechanisms underlying this phenomenon are not completely understood.
In the current study,we estimated the protective effect of the ischemic postconditioning against neuronal loss and behavioral deficits after global cerebral I/R using an animal model modified from Pulsinelli's four-vessel occlusion model in rats. Furthermore,to explore the mechanisms,we also determined whether the change of the expression of Caspase-3,Bcl-2,NF-ΚB,TLR-4 are involved.
Section One:Modifying the Four-vessel Occlusion Method to Establish the Global Brain Ischemic Model in Rats
Objective:To modify the four-vessel occlusion rat model to improve its reliability.
Methods:One hundred male Sprague-Dawly rats were randomly divided into 2groups:groupⅠ(Pulsinelli 4-VO model) and groupⅡ(modified 4-VO model).In group Ⅰ,the vertebral arteries were permanently electrocauterized and then the bilateral common carotid arteries were freed from surrounding tissues.On the following day,both common arteries were occluded for 10min with aneurysm clips to induce transient cerebral ischemia.In groupⅡ,after the bilateral common carotid arteries were freed,the four-vessel occlusion rat model was modified by the placement of 15-cm length of 10-gauge surgical silk through the cervical region of the rat such that one end of the suture traversed the paravertebral gap to the nape of the rat's neck and the other end traversed the gap between external jugular vein and nutator to the nape.The two ends are secured through a 8-gauge urethral catheter to the nape with adhesive tape.When occlusion of the common carotid arteries(the vertebral arteries having been permanently occluded 24 hours earlier),the cervical suture is tied and slowly tightened to encircle and occlude the common carotid arteries and the collateral blood vessels in the neck muscles.The clinical criteria for successful four-vessel occlusion is complete and lasting loss of the righting reflex even after innocuous stimulation.To detect the cause of unsuccessful four-vessel occlusion,the failures were examined with digital subtraction angiography(DSA).Under the guidance of fluoroscopy,a microduct was placed at aortic arch via left femoral artery and radiopaque contrast medium was injected.The success rate of vertebral arteries occlusion was observed.
Results:①Compared with that in groupⅠ,the percentage of rats that meet the criteria for successful 4-VO in groupⅡsignificantly increased.There were no significant differences between groupⅠand groupⅡin the percentage of rats removd because of respiratory failure and seizure.②Significant differences were not noted between groupⅠand groupⅡin the percentage of rats in which one of the vertebral arteries failed to be permanently electrocauterized.
Conclusions:The modified 4-VO to estabish the rat global cerebral ischmia model can improve the achievement ratio of the model.
Section 2:Protective Effect of Ischemic Postconditioning on Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objective:To study the effects of ischemic postconditioning on global cerebral ischemia in rats.
Method:Forty-three male Sprague-Dawley rats were randomly divided into 3 groups: group Sham(sham operation),group Control(ten-minute ischemia and persistent reperfusion) and group Postcond(ten-minute ischemia and postconditioning at early reperfusion consisting of 3 cycles of 30-second/10-second reperfusion/reocclusion followed by persistent reperfusion).After reperfusion 24h later,five rats per group were decapitated and brain were removed.The left brain water content were measured by wet-dry weight method and the MDA content and SOD activity in the right hippocampus were measured.After reperfusion 72h later,Nissl staning were used to campare the neuronal damage in the CA1 layer of the hippocampus。
Results:①The rats in I/R group,in comparison with in sham and postcond,had a higher brain water content.Significant differences were noted in the brain water content between sham and Postcond group.②Significant differences were noted in the level of SOD and MDA among sham,I/R group,and Postcond group.③Compared with that in sham group,the live neurons in hippocampal CA1 in I/R group and Postcond group significantly decreased after ischemia-reperfusion.The change was relieved in group Postcond.
Conclusions:Ischemic postconditioning have a neuroprotective effect against global cerebral ischemia-reperfuion injury in rats.It can decrease the damage of hippocampal.Its neuroprotective effects may be related to the inhibited lipid peroxidation.
Section 3:Influence of Ischemic Postconditioning on Neurological and Behavioral Outcomes after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objective:To study the immediate and delayed influence of ischemic postconditioning on neuroethology after global cerebral ischemia and reperfusion injury in rats.
Methods:Ninety-six male Sprague-Dawlay rats were randomly allocated to 3 groups: sham group,I/R group and Postcond group.Neurological severity scores(NSS) were tested at 1hr,6hr,12hr,24hr,48hr,96hr after reperfusion.Five and 28 days after reperfusion,Morris water maze test was emplyed to assess spatial learning and memory per group respectively and to analyze the effects of postconditioning.
Results:①Compared to the sham group,the NSSs increased in I/R group and Postcond group,and the change was relieved in Postcond group.②Cognition impairment was found in the I/R and Postcond groups,and the statistic analysis showed that there were significant differences between I/R and Postcond groups in ANOVA.Shortened mean escape latency was detected in the Postcond group compared with the the I/R group during the same trial times.
Conclusions:Ischemic postconditioning exerted an immediate and delayed protective effect on cognitive impairment caused by global cerebral ischemia-reperfusion injury in rats。
Section 4:Influence of Ischemic Postconditioning on Neural Apoptosis after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Obejective:To study the influence of ischemic postconditioning on neural apoptosis and to explore the role of caspase-3,bcl-2 in the mechanism of neural apoptosis after global cerebral ischemia and reperfusion(I/R) injury in rats.
Methods:The levels of neural apoptosis were measured by transferase dUTP-bition nick end labeling(TUNEL) and the expression of caspase-3 and bcl-2 were measured by immunohistochemical method and Western-Blot after reperfusion 24hr later.
Results:①Compared to the sham group,the levels of neural apoptosis increased in I/R group and Postcond group,and the change was relieved in Postcond group at reperfusion 72hr.②The expression of caspase-3 and bcl-2 increased in I/R group and Postcond group at reperfusion 24h,and compared to that in I/R group,the expression of caspase-3 and bcl-2 decreased and the expression of bcl-2 increased in Postcond group.
Conclusions:Ischemic postconditioning can relieve neural apoptosis and the mechanism may be related to the change of the expression of caspase-3 and bcl-2.
Section 5:Effects of Ischemic Postconditioning on Inflammatory Reaction and NF-κB,MPO,and TLR-4 after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objectives:To investigate the effect of ischemic postconditioning on inflammatory reaction after ischemia-reperfusion injury in rats and possible mechanism.
Methods:MPO activity in hippocamp was detected by MPO kit after reperfusion 24h later.The activity of NF-κB were measured by EMSA.The expression of TLR4 mRNA were detected by Real-Time PCR after reperfusion 4h,12h,24h,and 72h.
Results:Significant increase of activity of MPO and NF-κB were shown after reperfusion in I/R group and Postcond group.The expression level of TLR4 mRNA increased after reperfusion,came to the highest point after 24h,then decreased in the following time.Postconditioning could inhibit activity of MPO and NF-κB,and down-regulate the expression of TLR4 mRNA.
Conclusions:Postconditioning can inhibit the activity of MPO and NF-κB and down-regulate the expression of TLR4 mRNA and attenuated inflammatory injury after ischemia- reperfusion injury in rats.The mechanism of its neuroprotective effect against global cerebral ischemia-reperfusion injury may be by inhibiting inflammatory reaction.
近期研究提示,在冠状动脉再灌注开始时,反复的短暂再灌注和再阻断具有内在的保护作用,这一现象称为“缺血后适应”。由于突然发生的缺血很难预测,而缺血后适应发生在缺血后,因此受到广泛关注。临床有很多情况可以导致全脑缺血,如淹溺、心搏骤停及大血管和心脏手术中的严重低血压等,而脑比其他器官对缺血更敏感。最近,一些实验结果显示,大鼠缺血后适应可减小局灶性脑缺血再灌注后梗死面积,减轻全脑缺血再灌注损伤和脊髓缺血后损伤,然而其分子机制尚未完全清楚。
本实验首先改良Pulsinelli四血管阻塞大鼠全脑缺血模型,利用此改良模型评估缺血后适应对大鼠全脑缺血再灌注损伤的保护作用,并探讨其可能机制。
第一部分大鼠四血管阻塞全脑缺血模型的改良
目的:改良四血管阻塞法大鼠全脑缺血模型,以提高模型的成功率。
方法:100只雄性SD大鼠随机分为2组:组Ⅰ:Pulsinelli法四血管阻塞模型;组Ⅱ:改良四血管阻塞模型。组Ⅰ大鼠椎动脉永久性电凝阻断,双侧颈总动脉游离备用,第二日,直视下用动脉夹夹闭颈总动脉10min造成短暂脑缺血。组Ⅱ大鼠模型进行了改进:双侧颈总动脉游离后,分别用15cm长的10号外科丝线一端经椎旁间隙穿出到颈后,另一端绕过同侧颈总动脉经胸锁乳突肌和颈外静脉间隙穿出至颈后,固定后备用。第二日在颈部收紧丝线阻断颈总动脉10min造成短暂脑缺血。模型成功的标准采用临床指标:翻正反射完全、持续消失,对非伤害性刺激无反应。为了鉴别模型失败的原因,对不成功的大鼠进行全脑血管DSA。
结果:①与组Ⅰ相比,组Ⅱ符合模型成功标准的大鼠显著增加,两组因呼吸衰竭、肺水肿和惊厥而剔除的大鼠没有统计学差异;②两组椎动脉烧灼失败率没有统计学差异。
结论:改良四血管阻塞大鼠全脑缺血模型可提高模型的成功率而不增加并发症。
第二部分:缺血后适应对大鼠全脑缺血再灌注损伤的保护作用
目的:研究缺血后适应对大鼠全脑缺血再灌注的保护作用。
方法:43只雄性SD大鼠随机分为3组:sham组(12只,假手术组)、I/R组(16只,缺血10min再灌注组)和Postcond组(15只,缺血10min后,在再灌注早期行后适应处理,即灌注30s再阻断10s,如此3个周期)。再灌注24h后,每组5只大鼠被断头取脑,左脑用干湿重法计算脑水含量。右侧海马检测MDA和SOD活性。每组7只大鼠再灌注72h后进行Nissl染色观察海马CA1区神经元破坏程度。
结果:①I/R组有4只(其中1只因呼吸衰竭,3只因惊厥)剔除出实验,Postcond组有3只(其中1只因呼吸衰竭,2只因惊厥)剔除出实验,I/R组与Postcond组相比差异无显著性(P＞0.05);②脑组织水含量的测定结果表明,全脑缺血再灌注后脑组织的含水量都明显升高(P＜0.05),但是Postcond组较I/R组脑水含量少(P＜0.05);③与sham组相比,全脑缺血再灌注后海马中的SOD活性均有不同程度的降低(P＜0.05),I/R组降低更为显著(P＜0.05),与sham组比较,全脑缺血再灌注后海马中MDA含量明显增高(P＜0.05),而I/R组增高更为显著(P＜0.05);④Sham组海马细胞核膜完整,核仁清楚,偶可见退变锥体细胞。I/R组再灌注3日后,在CA1区可观察到大量退变神经元;与I/R组相比,Postcond组海马CA1区退变神经元明显减少(P＜0.05)。
结论:缺血后适应对大鼠全脑缺血再灌注损伤具有保护作用,可减少海马神经元的破坏。其保护作用可能与抑制脂质过氧化有关。
第三部分:缺血后适应对大鼠全脑缺血再灌注损伤后神经行为学的影响
目的:通过神经损害评分(NSS)和Morris水迷宫研究缺血后适应对大鼠全脑缺血再灌注损伤后近期及远期神经行为学的影响。
方法:96只雄性SD大鼠随机分为3组(同第二部分)。于再灌注后6h、12h、24h、48h及96h进行NSS评分。分别于再灌注后5日及28日开始,每组各取16只分别进行水迷宫实验评价大鼠的空间学习和记忆能力。
结果:①与sham组相比,I/R组和Postcond组NSS评分增加,但是Postcond组增加较少(P＜0.05);②水迷宫实验结果方差分析显示,I/R组和Postcond组有认知障碍,I/R组更明显(P＜0.05),在同一实验日期,Postcond组平均潜伏期较I/R组短(P＜0.05)。
结论:缺血后适应对大鼠全脑缺血再灌注损伤引起的近期和远期认知障碍有保护作用。
第四部分:缺血后适应对大鼠全脑缺血再灌注损伤后神经元凋亡的影响
目的:研究缺血后适应对大鼠全脑缺血再灌注损伤后神经元凋亡的影响及caspase-3和bcl-2在其中的作用机制。
方法:45只雄性SD大鼠随机分为3组(同第二部分)。再灌注后72h每组5只大鼠生理盐水、多聚甲醛灌注后端头取脑,用TUNEL染色法计算各组海马CA1区凋亡的神经元个数。再灌注后24h时每组5只大鼠生理盐水、多聚甲醛灌注后断头取脑行免疫组织化学检测海马CA1区caspase-3和bcl-2蛋白的表达,另每组各取5只大鼠深麻醉下断头取脑,分离海马,行Western-Blot检测caspase-3和bcl-2蛋白的表达。
结果:①再灌注后72h时,I/R组与Postcond组神经元凋亡增多,I/R组更严重(P＜0.05);②I/R组和Postcond组于缺血再灌注后24h可见caspase-3和bcl-2表达增高,与I/R组相比,Postcond组caspase-3表达更低,而bcl-2表达更高(P＜0.05)。
结论:缺血后适应可减轻神经细胞凋亡,caspase-3与bcl-2可能参与了其机制。
第五部分:缺血后适应对大鼠全脑缺血再灌注损伤后海马区炎症反应的影响
目的:研究缺血后适应对大鼠全脑缺血再灌注损伤后炎症反应的影响及可能机制。
方法:75只雄性SD大鼠随机分为3组(同第二部分)。再灌注24h,每组5只大鼠深麻醉下断头取脑,分离海马,用MPO试剂盒检测海马MPO活性,凝胶电泳迁移率实验(EMSA)检测海马NF-KB的DNA结合活性。分别于再灌注后0h、4h、12h、24h及72h每组各取5只大鼠深麻醉下断头取脑,分离海马,行Real-Time PCR检测海马TLR4基因mRNA的表达。
结果:①缺血后适应对全脑缺血再灌注后海马MPO活性、NF-KB的DNA结合活性的升高有抑制作用;②Real-Time PCR结果显示:缺血再灌注后各时间点TLR-4基因mRNA表达上调,24h达高峰,缺血后适应对TLR-4基因mRNA表达上调具有明显的抑制作用。
结论:缺血后适应可抑制大鼠全脑缺血再灌注损伤后MPO活性和NF-KB的DNA结合活性,并抑制TLR4基因mRNA的上调,其对脑缺血再灌注损伤的保护作用可能与减轻炎症反应有关。
Background Extensive research has been aimed at finding effective strategies and drugs to ameliorate or prevent brain ischemia and reperfusion(I/R) injury.However,few have been successfully applied in clinical practice,although several strategies and drugs have been shown to decrease ischemic damage in the brain in animal models.Ischemic preconditioning has been shown to provide powerful protection against I/R injury in both the heart and nervous system.However,its clinical application is only possible for cases in which the occurrence of I/R is predictable.Rapid initiation of reperfusion is the most effective treatment to reduce injury and the behavioral deficits caused by ischemia. However,reperfusion also has the potential to introduce additional injury;many studies have shown that overproduction of reactive oxygen species and overloading of calcium occur in the early reperfusion period and lead to reperfusion injury.
Another endogenous protective strategy,termed "ischemic postconditioning," has been recently reported,in which several repeated cycles of brief reperfusion and reocclusion of the coronary artery were applied at the onset of full reperfusion.Because ischemia cannot be predicted and happens suddenly,ischemic postconditioning,which can be applied after ischemia,is attracting considerable attention.In clinical practice,many accidents lead to global ischemia,such as drowning,cardiac arrest,and marked hypotension during cardiac surgery,and the brain is intrinsically more vulnerable to ischemia than other organs.Recent studies demonstrated ischemic postconditioning can reduce infarct size after focal brain I/R injury and ameliorate neuronal injury after global cerebral I/R injury and spinal cord ischemia in rat.However,the molecular mechanisms underlying this phenomenon are not completely understood.
In the current study,we estimated the protective effect of the ischemic postconditioning against neuronal loss and behavioral deficits after global cerebral I/R using an animal model modified from Pulsinelli's four-vessel occlusion model in rats. Furthermore,to explore the mechanisms,we also determined whether the change of the expression of Caspase-3,Bcl-2,NF-ΚB,TLR-4 are involved.
Section One:Modifying the Four-vessel Occlusion Method to Establish the Global Brain Ischemic Model in Rats
Objective:To modify the four-vessel occlusion rat model to improve its reliability.
Methods:One hundred male Sprague-Dawly rats were randomly divided into 2groups:groupⅠ(Pulsinelli 4-VO model) and groupⅡ(modified 4-VO model).In group Ⅰ,the vertebral arteries were permanently electrocauterized and then the bilateral common carotid arteries were freed from surrounding tissues.On the following day,both common arteries were occluded for 10min with aneurysm clips to induce transient cerebral ischemia.In groupⅡ,after the bilateral common carotid arteries were freed,the four-vessel occlusion rat model was modified by the placement of 15-cm length of 10-gauge surgical silk through the cervical region of the rat such that one end of the suture traversed the paravertebral gap to the nape of the rat's neck and the other end traversed the gap between external jugular vein and nutator to the nape.The two ends are secured through a 8-gauge urethral catheter to the nape with adhesive tape.When occlusion of the common carotid arteries(the vertebral arteries having been permanently occluded 24 hours earlier),the cervical suture is tied and slowly tightened to encircle and occlude the common carotid arteries and the collateral blood vessels in the neck muscles.The clinical criteria for successful four-vessel occlusion is complete and lasting loss of the righting reflex even after innocuous stimulation.To detect the cause of unsuccessful four-vessel occlusion,the failures were examined with digital subtraction angiography(DSA).Under the guidance of fluoroscopy,a microduct was placed at aortic arch via left femoral artery and radiopaque contrast medium was injected.The success rate of vertebral arteries occlusion was observed.
Results:①Compared with that in groupⅠ,the percentage of rats that meet the criteria for successful 4-VO in groupⅡsignificantly increased.There were no significant differences between groupⅠand groupⅡin the percentage of rats removd because of respiratory failure and seizure.②Significant differences were not noted between groupⅠand groupⅡin the percentage of rats in which one of the vertebral arteries failed to be permanently electrocauterized.
Conclusions:The modified 4-VO to estabish the rat global cerebral ischmia model can improve the achievement ratio of the model.
Section 2:Protective Effect of Ischemic Postconditioning on Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objective:To study the effects of ischemic postconditioning on global cerebral ischemia in rats.
Method:Forty-three male Sprague-Dawley rats were randomly divided into 3 groups: group Sham(sham operation),group Control(ten-minute ischemia and persistent reperfusion) and group Postcond(ten-minute ischemia and postconditioning at early reperfusion consisting of 3 cycles of 30-second/10-second reperfusion/reocclusion followed by persistent reperfusion).After reperfusion 24h later,five rats per group were decapitated and brain were removed.The left brain water content were measured by wet-dry weight method and the MDA content and SOD activity in the right hippocampus were measured.After reperfusion 72h later,Nissl staning were used to campare the neuronal damage in the CA1 layer of the hippocampus。
Results:①The rats in I/R group,in comparison with in sham and postcond,had a higher brain water content.Significant differences were noted in the brain water content between sham and Postcond group.②Significant differences were noted in the level of SOD and MDA among sham,I/R group,and Postcond group.③Compared with that in sham group,the live neurons in hippocampal CA1 in I/R group and Postcond group significantly decreased after ischemia-reperfusion.The change was relieved in group Postcond.
Conclusions:Ischemic postconditioning have a neuroprotective effect against global cerebral ischemia-reperfuion injury in rats.It can decrease the damage of hippocampal.Its neuroprotective effects may be related to the inhibited lipid peroxidation.
Section 3:Influence of Ischemic Postconditioning on Neurological and Behavioral Outcomes after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objective:To study the immediate and delayed influence of ischemic postconditioning on neuroethology after global cerebral ischemia and reperfusion injury in rats.
Methods:Ninety-six male Sprague-Dawlay rats were randomly allocated to 3 groups: sham group,I/R group and Postcond group.Neurological severity scores(NSS) were tested at 1hr,6hr,12hr,24hr,48hr,96hr after reperfusion.Five and 28 days after reperfusion,Morris water maze test was emplyed to assess spatial learning and memory per group respectively and to analyze the effects of postconditioning.
Results:①Compared to the sham group,the NSSs increased in I/R group and Postcond group,and the change was relieved in Postcond group.②Cognition impairment was found in the I/R and Postcond groups,and the statistic analysis showed that there were significant differences between I/R and Postcond groups in ANOVA.Shortened mean escape latency was detected in the Postcond group compared with the the I/R group during the same trial times.
Conclusions:Ischemic postconditioning exerted an immediate and delayed protective effect on cognitive impairment caused by global cerebral ischemia-reperfusion injury in rats。
Section 4:Influence of Ischemic Postconditioning on Neural Apoptosis after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Obejective:To study the influence of ischemic postconditioning on neural apoptosis and to explore the role of caspase-3,bcl-2 in the mechanism of neural apoptosis after global cerebral ischemia and reperfusion(I/R) injury in rats.
Methods:The levels of neural apoptosis were measured by transferase dUTP-bition nick end labeling(TUNEL) and the expression of caspase-3 and bcl-2 were measured by immunohistochemical method and Western-Blot after reperfusion 24hr later.
Results:①Compared to the sham group,the levels of neural apoptosis increased in I/R group and Postcond group,and the change was relieved in Postcond group at reperfusion 72hr.②The expression of caspase-3 and bcl-2 increased in I/R group and Postcond group at reperfusion 24h,and compared to that in I/R group,the expression of caspase-3 and bcl-2 decreased and the expression of bcl-2 increased in Postcond group.
Conclusions:Ischemic postconditioning can relieve neural apoptosis and the mechanism may be related to the change of the expression of caspase-3 and bcl-2.
Section 5:Effects of Ischemic Postconditioning on Inflammatory Reaction and NF-κB,MPO,and TLR-4 after Global Cerebral Ischemia and Reperfusion(I/R) Injury in Rats
Objectives:To investigate the effect of ischemic postconditioning on inflammatory reaction after ischemia-reperfusion injury in rats and possible mechanism.
Methods:MPO activity in hippocamp was detected by MPO kit after reperfusion 24h later.The activity of NF-κB were measured by EMSA.The expression of TLR4 mRNA were detected by Real-Time PCR after reperfusion 4h,12h,24h,and 72h.
Results:Significant increase of activity of MPO and NF-κB were shown after reperfusion in I/R group and Postcond group.The expression level of TLR4 mRNA increased after reperfusion,came to the highest point after 24h,then decreased in the following time.Postconditioning could inhibit activity of MPO and NF-κB,and down-regulate the expression of TLR4 mRNA.
Conclusions:Postconditioning can inhibit the activity of MPO and NF-κB and down-regulate the expression of TLR4 mRNA and attenuated inflammatory injury after ischemia- reperfusion injury in rats.The mechanism of its neuroprotective effect against global cerebral ischemia-reperfusion injury may be by inhibiting inflammatory reaction.
引文
[1]WA Pulsinelli,JB Brierley.A new model of bilateral hemispheric ischemia in the unanesthetized rat.Stroke,1979;10;267-272.
[2]Smith ML,Bendek G,Dahlgren N,et al.Models for studying long-term recovery following global cerebral ischemia in the rat.2.A 2-vessel occlusion model.Acta Neurol Scand,1984,9(6):385-401.
[3]Kameyama M,Suzuki J,Shirane R,Ogawa A.A new model of bilateral hemispheric ischemia in the rat--three vessel occlusion model.Stroke,1985,16;489-493.
[4]Mitsuo Yamaguchi,John W.Calvert,et al.One-stage anterior approach for four-vessel occlusion in rat.Stroke,2005,36:2212-2214.
[5]李兵,章翔等.改良四血管阻塞法建立大鼠全脑缺血模型.中华神经外科疾病研究杂志,2005,4(2):110-113.
[6]WA Pulsinelli,JB Brierley.The Four-Vessel Occlusion Rat Model:Method for Complete Occlusion of Vertebral Arteries and Control of Collateral Circulation.Stroke,1988,19:913-914
[7]Alps BJ,Hass WK.The potential beneficial effect of nicardipine in a rat model of transient forebrain ischemiao Neurology.1987,37:809-814.
[8]Schmidt-Kastner R,Paschen W,Grosse Ophoff B,et al.A modified four-vessel occlusion model for inducing incomplete forebrain ischemia in rats.Stroke.1989,20:938-946.
[1]Vinten-Johansen J, Zhao ZQ, et al. Postconditioning-A new link in nature's armor against myocardial ischemia-reperfusion injury. Basic Res Cardiol. 2005; 100: 295-310.
[2]Murry CE, Jennings RB, et al. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124-1136.
[3]Perez-Pinzon MA. Neuroprotective effects of ischemic preconditioning in brain mitochondria following cerebral ischemia. J Bioenerg Biomembranes. 2004; 36: 323-327.
[4]Zhao ZQ, Corvera JS, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003; 285: 579-588.
[5]Elliott KAC. Measurement of experimentally induced brain swelling and shrinkage. Am J Physiol. 1949; 157: 122-9.
[6] 包新民,舒斯云,主编．大鼠脑立体定位图谱。北京,人民卫生出版社,第1版,1991；32-50．
[7]DuchenLW. General pathology of neurons and neuroglia. In: Adams JH, Duchen LW, eds. Greenfield Sneuropathology. 5th ed. London: Edward Arnold, 1992; 1-68.
[8]Levine S, Saltzman A, Kumar AR. A method for peripheral chromatolysis in neurons of trigeminal and dorsal root ganglia, produced in rats by lithium. J Neurosci Methods. 2004; 132: 1-7.
[9]Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia. Ann Neurol. 1982; 11:499-502.
[10]Zhao ZQ, Vinten-Johansen J. Postconditioning: reduction of reperfusion-induced injury. Cardiovasc Res. 2006; 70:200-211.
[11] Obal D, Dettwiler S, Favoccia C, et al. The influence of mitochondrial K_(ATP)~+-channels in the cardioprotection of preconditioning and postconditioning by sevoflurane in the rat in vivo. Anesth Analg. 2005; 101:1252-1260.
[12] Kin H, Zatta AJ, Lofye MT, etal. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res. 2005; 67: 124-133.
[13]Iliodromitis EK, Georgiadis M, Cohen MV, etal. Protection from postconditioning depends on the number of short ischemic insults in anesthetized pigs. Basic Res Cardiol. 2006; 101:502-507.
[14]Dosenko VE, Nagibin VS, Tumanovskaya LV, etal. Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytes. Fiziol Z. 2006; 52:15-24.
[15]Staat P, Rioufol G, Piot C, etal. Postconditioning the human heart. Circulation. 2005; 112: 2143-2148.
[16]Zhao H, Robert M Sapolsky, Gary K Steinberg. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. Journal of Cerebral Blood Flow & Metabolism. 2006; 26: 1114-1121.
[17]Jozef Burda, Viera Danielisov'a, Miroslava N'emethov'a, et al. Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain. Cellular and Molecular Neurobiology. 2006; 26: 1141-1151.
[18]Viera Danielisov'a, Miroslava N'emethov'a, Miroslav Gottlieb, et al. The Changes in Endogenous Antioxidant Enzyme Activity After Postconditioning. Cellular and Molecular Neurobiology. 2006; 26: 1181-1191.
[19]Ashish K. Rehni, Nirmal Singh. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked b(?)havioral deficits in mice. Pharmacological Reports. 2007; 59: 192-198.
[20]Ikeda Y, Long DM, The molecular basis of brain injury and brain edema: The role of oxygen free radicals. Neurosurgery. 1990; 27: 1-11.
[21]Chan PH, Kawase M, Murakami K, et al. Overexpression of SOD1 in transgenic rats protects vulnerable neurons against ischemic damage after global cerebral ischemia and reperfusion. J Neurosci. 1998; 18:8292-9.
[22]Kim GW, Lewen A, Copin J. Neuronal expression of the DNA repair protein Ku 70 after ischemic preconditioning corresponds to tolerance to global cerebral ischemia. Stroke. 2001; 32: 2388-93.
[23]Kawase M, Murakami K, Fujimura M, et al. Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency. Stroke. 1999; 30:1962-8.
1 WA Pulsineili,JB Brierley.A new model of bilateral hemispheric ischemia in the unanesthetized rat.Stroke.1979;10:267-272.
2 Chen JL,Li Y,Wang L,et al.Therapeutic benefit of intraveous administration of bone marrow stromal cells after cerebral ischemia in rats.Stroke.2001;32:1005-11.
3 Charles V Vorhees,Michael T Williams.Morris water maze:procedures for assessing spatial and related forms of learning and memory.Nature Protocols.2006;1:848-858.
4 钟乐,王霞,余小河,等.新生大鼠缺氧缺血性脑损伤后的远期行为学测试.中国当代儿科杂志.2005:7:247-250。
5 Chen J,Li Y,Chopp M.Intracerebral transplantation of bone marrow with BDNF after MCAO in rat.Neuropharmacology.2000;39:711-716.
6 Hamm RJ,Pike BR,O'Dell DM,et al.The rotarod test:an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury.J Neurotrauma.1994;11:187-196.
7 Ding Y,Zhou Y,Lai Q,et al.Long-term neuroprotective effect of inhibiting poly(ADP-ribose)poly merase in rats with middle cerebral artery occlusion using a behavioral assessment.Brain Res.2001;915:210-7.
8 王新陆,王中琳.缺血性中风后遗症模型的建立与评价.世界科学技术——中医药现代化基础研究,2004,6(3):14.
9 Zhao H,Robert M Sapolsky and Gary K Steinberg.Interrupting reperfusion as a stroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats.Journal of Cerebral Blood Flow & Metabolism.2006;26:1114-21.
10 Jozef Burda,Viera Danielisov'a and Miroslava N'emethov'a.Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.Cellular and Molecular Neurobiology.2006;26:1141-51.
11 Morris,R.G.M.Spatial localization does not require the presence of local cues.Learn.Motiv.1981;12:239-260.
12 Stewart,C.A.& Morris,R.G.M.The water maze in Behavioural Neuroscience,Volume Ⅰ,A Practical Approach(ed.Sahgal).107-122(IRL Press at Oxford University Press,Oxford,1993).
13 Morris,R.G.M.An attempt to dissociate 'spatial-mapping' and 'working-memory' theories of hippocampal function in Neurobiology of the Hippocampus(ed.Seifert) 405-432(Academic Press,New York,1993).
[1]Kerr J F,Wyllie A H,Currie AR.Apoptosis,Br J Cancer.1972;2:239-242.
[2]Otsuki Y,Li Z,Shibata MA.Apoptotic detection methods--from morphology to gene.Prog Histochem Cytochem.2003;38:275-339.
[3]Froelich-Ammon S J,Patch MW,Osheroff N,et alo Topoisomerase I binds to ellipticine in the absence or presence of DNA.Characterization of enzyme-drug interactions by fluorescence spectroscopy.J Biol Chem.1995;270:14998-15004.
[4]Dawson Y M,Dawson V L.Protection of brain from ischemia.Cerebrovascular Disease.1997;7:349-352.
[5]MacManus JP,Buchan AM,Hill IE,et al.Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.Nemosci Lett.1993;164:89-92.
[6]Marlangue CC,Represa MA,Popcvici T,et al.Apoptosis and necrosis after reversible focal ischemia:An in situ DNA fragmentaition analysis.J Cereb Blood Flow Metab.1996;6:186-94.
[7]MacManus JP,Hill IE,Preston E,et al.Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.J Cereb Blood Flow Metab.1995;15:728-37.
[8]Heng Zhao,Robert M Sapolsky and Gary K Steinberg.Interrupting reperfusion as a stroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats.J Cereb Blood Flow Metab.2006;26:1114-21.
[9]Jozef Burda,Viera Danielisov'a,Miroslava N'emethov'a,et al.Delayed post-conditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.Cellular and Molecular Neurobiology,2006,26,1141-51.
[10]Schulz JB,Weller M,Moskowitz MA.Caspase as treatment targets in stoke and neurodegenerative disease.Ann Neurol.1999;45:421-9.
[11]Zhou P,Chou J,Olea RS,et al.Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD:a structure basis for specific adaptor/caspase interaction.Proc Natl Acad Sci.1999;96:11265-70.
[12]Ashkenazi A,Dixit VM.Death receptors:signaling and modulation.Science.1998;281:1305-8.
[13]Rao RV,Hermel E,Castro-Obregon S,et al.Coupling endoplasmic reticulum stress to the cel death program:mechan ism ofcaspase activation.J Biol Chem.2001;276:33869-74.
[14]张文华,周璐,徐淑军等,Caspase-3激活和细胞色素c释放在脑缺血再灌注损伤中的作用.山东大学学报(医学版).2007;45:1193-2000。
[15]Chen J,Nagayama T,Jin K,et al.Induction of Caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia.J Neurosci.1998;18:4914-28.
[16]Ni B,Wu X,Su Y,et aio Transient global cerebral ischemiainduces a prolonged espression of the Caspase-3 mRNA in rat hippocampal CA1 pyramidal neurons.J Cereb Blood Flow Metab.1998;18:248-256.
[17]Graham S H,Chen J,Clark R S,et al.Bcl-2 family gene products in cerebral ischemia and traumatic brain injury.J Neurotrauma.2000;17:831-841.
[18]Hata R,Gillardon F,Nfichaelidis T M,et alo Targeted disruption of the bcl-2 gene in the mice exacerbates focal ischemic brain injury.Mebtab Brain Dis.1999;14:117-24.
[19]Chen J,Graham SH,Chan PH,et al.Bcl-2 is expressed in neurons that survive focal ischemia in the rat.Neuroroporto 1995;6:394-398.
[20]Linnik MD,Zahos P,Geschwind MD,et al.Expression of bcl-2 from defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia.Stroke.1995;26:1670-75.
[21]韩雪梅,熊文激,胡国中,等.金纳多对大鼠全脑缺血再灌注损伤后Fas和Bcl-2蛋白表达的影响.吉林大学学报(医学版),2004,5:381-383.
[22]Shimizu S,Nagayama T,Jin KL,et alo Bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain.Cereb blood Flow Metab.2001;21:233-243.
[23]Chen J,Simon P,Nagayama T,et alo Suppression of endogenous bcl-2 expression by antisense treatment exacerbates ischemic neuronal death.Cereb Blood Flow Metab.2000;20:1033-39.
[1]Zhang RL, Chopp M, Chen H, et al. Temporal profile of ischemic tissue damage, neutrophile response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neurol Sci. 1994; 125:3-10.
[2]Zhang GH, Lu P, W ang YL. Effects of dipfluzine on expressions of E-selectin, P-selectin, and ICAM-1 in brain ischemia-reperfusion rats. Yao Xue Xue Bao. 2005; 40: 1091-95.
[3]Medzhitov R, Preston-Hurburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388: 394-397.
[4]Oyama J, Blais C Jr. Liu X, et al. Reduced myocardial ischemia reperfusion injury in toll-like receptor 4-deflcient mice. Circulation. 2004; 109: 784 -789.
[5]Stephenson D, Yin T, Smalstig EB, et al. Transcription factor nuclear factor kappa B is activated in neurons after focal cerebral ischemia. J Cereb Blood Flow Metab. 2000; 20: 592-603.
[6]Sun Z, Andersson R. NF-kappa Bactivation and inhibition: a review. Stroke. 2002; 18: 99-106.
[7]Podrez EA, PoliakovE, Shen Z, et al. A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions. J Biol Chem. 2002; 277: 38517-38523.
[8]Singh H, Sen R, Baltimore D, et al. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature. 1986; 319: 154-8.
[9]Shen W, Zhang C, Zhang G. Nuclear factor-KB activation is mediated by NMDA and non-NMDA receptor and L-type voltage-gated Ca2+ channel following severe global ischemia in rat hippocampus. Brain Res. 2002; 933: 23-30.
[10]Salminen M, Spitz F, Fiszman MY, et al. Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (Ml) for unknown proteins. J Mol Biol. 1995; 253: 17-31.
[1 l]Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab. 2000, 2:1011-32.
[12]Lj C, Ha T, Kelley J,et al. Modulating Toll-like receptor mediated signaling by (1,3)-β-D-glucan rapidly induces cardioprotection. Cardiovasc Res. 2004; 61: 538-547.
[13]Kielian T. Tol 1-like receptors in central nervous system glial inflammation and homeostasis. J Neurosci Res. 2006; 83: 711-730.
[14]Cao CX, Yang QW, Lv FL, et al. Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun. 2007; 353: 509-514.
[15]Caso JR, Pradillo JM, Hurtado O, etal. Toll-like receptor 4 is involed in brain damage and inflammation after experimental stroke. Circulation. 2007; 115: 1599-1608.
[1]Kato, H, Liu, et al. MK-801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus. Neurosci. Lett, 1992; 139: 118-121.
[2]Garnier, Ying P, and Swanson R. A. Ischemic preconditioning by caspase cleavage of poly(ADPribose) polymerase-1. J Neurosci. 2003; 23: 7967-7973 .
[3]Vlasov, Korzhevskii, and Polyakova. Ischemic preconditioning of the rat brain as a method of endothelial protection from ischemic/reperfusion injury. Neurosci Behav Physiol. 2005; 35: 567-572 .
[4]Becker. Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. Proc Natl Acad Sci. 1997;94: 10873-10878 .
[5]Frenkel et al. Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke. J Neurol Sci. 2005;233: 125-132.
[6]Chen Y, et al. Mucosal tolerance to E-selectin provides cell-mediated protection against ischemic brain injury. Proc Natl Acad Sci. 2003;100: 15107-15112 .
[7]Kariko, Weissman, and Welsh. Inhibition of toll-like receptor and cytokine signaling - a unifying theme in ischemic tolerance. J Cereb Blood Flow Metab. 2004;24: 1288-1304.
[8]Gonzalez-Zulueta, et al. Requirement for nitric oxide activation of p21ras/extracellular regulated kinase in neuronal ischemic preconditioning. Proc Natl Acad Sci. 2000;97: 436-441.
[9]Jones and Bergeron. Hypoxia-induced ischemic tolerance in neonatal rat brain involves enhanced ERK1/2 signaling. J Neurochem. 2004;89: 157-167.
[10]Yoshida, Nakakimura, Cui YJ, et al. Adenosine Al receptor antagonist and mitochondrial ATP-sensitive potassium channel blocker attenuate the tolerance to focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 2004;24: 771-779.
[11]Wick, et al. Neuroprotection by hypoxic preconditioning requires sequential activation of vascular endothelial growth factor receptor and Akt. J Neurosci. 2002;22: 6401-6407.
[12]Raval, Dave K R, Mochly-Rosen D, et al. PKC is required for the induction of tolerance by ischemic and NMDAmediated preconditioning in the organotypic hippocampal slice. J Neurosci. 2003;23: 384-391.
[13]Wingrove JA and O'Farrell PH. Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila. Cell. 1999;98: 105-114.
[14]Cho S, et al. Obligatory role of inducible nitric oxide synthase in ischemic preconditioning. J Cereb Blood Flow Metab. 2005;25: 493-501.
[15]Ravati A, Ahlemeyer B, Becker A, et al. Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-KB. J Neurochem. 2001;78: 909-919.
[16]Kapinya K., Penzel R, Sommer C, et al. Temporary changes of the AP-1 transcription factor binding activity in the gerbil hippocampus after transient global ischemia, and ischemic tolerance induction. Brain Res. 2000; 872: 282-293.
[17]Ma D, et al. Xenon preconditioning reduces brain damage from neonatal asphyxia in rats. J Cereb Blood Flow Metab. 2006; 26: 199-208.
[18]Blondeau N, Widmann C, Lazdunski M, et al. Activation of the nuclear factor-KB is a key event in brain tolerance. J Neurosci. 2001 ;21: 4668-4677.
[19]Ryu H, et al. Sp1 and Sp3 are oxidative stressinducible, antideath transcription factors in cortical neurons. J Neurosci. 2003; 23: 3597-3606.
[20]Bernaudin M, et al. Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxiainducible factor-1 and its target genes, erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood Flow Metab. 2002;22: 393-403.
[21]Ralph GS, et al. Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 . and HIF-2 . in a primary neuronal model of hypoxia. J Cereb Blood Flow Metab. 2004;24: 245-258.
[22]Bernaudin M, Tang Y, Reilly M, et al. Brain genomic response following hypoxia and re-oxygenation in the neonatal rat. Identification of genes that might contribute to hypoxia-induced ischemic tolerance. J Biol Chem. 2002; 277: 39728-38.
[24]Douen AG, et al. Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes. J Neurochem 2000;75:812-818.
[25]Paschen W, Mies G. Effect of induced tolerance on biochemical disturbances in hippocampal slices from the gerbil during and after oxygen/glucose deprivation. Neuroreport 1999;10: 1417-1421.
[26]Bordet R, et al. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J Cereb Blood Flow Metab. 2000;20: 1190-1196.
[27]Brambrink AM, et al. Tolerance-inducing dose of 3-nitropropionic acid modulates bcl-2 and bax balance in the rat brain: a potential mechanism of chemical preconditioning. J Cereb Blood Flow Metab. 2000;20: 1425-1436.
[28] Wu C, et al. Different expression patterns of Bcl-2, Bcl-xl, and Bax proteins after sublethal forebrain ischemia in C57Black/Crj6 mouse striatum. Stroke 2003;34: 1803-8.
[29]Storkebaum E, Lambrechts D, Carmeliet P. VEGF:once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 2004;26: 943-954.
[30]Chen J, Graham SH, Zhu RL, et al. Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab. 1996; 16: 566-577.
[31]Dawson DA, Furuya K, Gotoh J, et al. Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion. J Cereb Blood Flow Metab. 1999; 19: 616-623.
[32]Ding YH, et al. Exercise preconditioning ameliorates inflammatory injury in ischemic rats during reperfusion. Acta Neuropathol. 2005;109: 237-246.
[33]Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia. 2005;50: 307-320.
[34]Kato H, Kogure K, Araki T, et al. Astroglial and microglial reactions in the gerbil hippocampus with induced ischemic tolerance. Brain Res. 1994 ;664: 69-76.
[2]Smith ML,Bendek G,Dahlgren N,et al.Models for studying long-term recovery following global cerebral ischemia in the rat.2.A 2-vessel occlusion model.Acta Neurol Scand,1984,9(6):385-401.
[3]Kameyama M,Suzuki J,Shirane R,Ogawa A.A new model of bilateral hemispheric ischemia in the rat--three vessel occlusion model.Stroke,1985,16;489-493.
[4]Mitsuo Yamaguchi,John W.Calvert,et al.One-stage anterior approach for four-vessel occlusion in rat.Stroke,2005,36:2212-2214.
[5]李兵,章翔等.改良四血管阻塞法建立大鼠全脑缺血模型.中华神经外科疾病研究杂志,2005,4(2):110-113.
[6]WA Pulsinelli,JB Brierley.The Four-Vessel Occlusion Rat Model:Method for Complete Occlusion of Vertebral Arteries and Control of Collateral Circulation.Stroke,1988,19:913-914
[7]Alps BJ,Hass WK.The potential beneficial effect of nicardipine in a rat model of transient forebrain ischemiao Neurology.1987,37:809-814.
[8]Schmidt-Kastner R,Paschen W,Grosse Ophoff B,et al.A modified four-vessel occlusion model for inducing incomplete forebrain ischemia in rats.Stroke.1989,20:938-946.
[1]Vinten-Johansen J, Zhao ZQ, et al. Postconditioning-A new link in nature's armor against myocardial ischemia-reperfusion injury. Basic Res Cardiol. 2005; 100: 295-310.
[2]Murry CE, Jennings RB, et al. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124-1136.
[3]Perez-Pinzon MA. Neuroprotective effects of ischemic preconditioning in brain mitochondria following cerebral ischemia. J Bioenerg Biomembranes. 2004; 36: 323-327.
[4]Zhao ZQ, Corvera JS, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003; 285: 579-588.
[5]Elliott KAC. Measurement of experimentally induced brain swelling and shrinkage. Am J Physiol. 1949; 157: 122-9.
[6] 包新民,舒斯云,主编．大鼠脑立体定位图谱。北京,人民卫生出版社,第1版,1991；32-50．
[7]DuchenLW. General pathology of neurons and neuroglia. In: Adams JH, Duchen LW, eds. Greenfield Sneuropathology. 5th ed. London: Edward Arnold, 1992; 1-68.
[8]Levine S, Saltzman A, Kumar AR. A method for peripheral chromatolysis in neurons of trigeminal and dorsal root ganglia, produced in rats by lithium. J Neurosci Methods. 2004; 132: 1-7.
[9]Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia. Ann Neurol. 1982; 11:499-502.
[10]Zhao ZQ, Vinten-Johansen J. Postconditioning: reduction of reperfusion-induced injury. Cardiovasc Res. 2006; 70:200-211.
[11] Obal D, Dettwiler S, Favoccia C, et al. The influence of mitochondrial K_(ATP)~+-channels in the cardioprotection of preconditioning and postconditioning by sevoflurane in the rat in vivo. Anesth Analg. 2005; 101:1252-1260.
[12] Kin H, Zatta AJ, Lofye MT, etal. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res. 2005; 67: 124-133.
[13]Iliodromitis EK, Georgiadis M, Cohen MV, etal. Protection from postconditioning depends on the number of short ischemic insults in anesthetized pigs. Basic Res Cardiol. 2006; 101:502-507.
[14]Dosenko VE, Nagibin VS, Tumanovskaya LV, etal. Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytes. Fiziol Z. 2006; 52:15-24.
[15]Staat P, Rioufol G, Piot C, etal. Postconditioning the human heart. Circulation. 2005; 112: 2143-2148.
[16]Zhao H, Robert M Sapolsky, Gary K Steinberg. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. Journal of Cerebral Blood Flow & Metabolism. 2006; 26: 1114-1121.
[17]Jozef Burda, Viera Danielisov'a, Miroslava N'emethov'a, et al. Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain. Cellular and Molecular Neurobiology. 2006; 26: 1141-1151.
[18]Viera Danielisov'a, Miroslava N'emethov'a, Miroslav Gottlieb, et al. The Changes in Endogenous Antioxidant Enzyme Activity After Postconditioning. Cellular and Molecular Neurobiology. 2006; 26: 1181-1191.
[19]Ashish K. Rehni, Nirmal Singh. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked b(?)havioral deficits in mice. Pharmacological Reports. 2007; 59: 192-198.
[20]Ikeda Y, Long DM, The molecular basis of brain injury and brain edema: The role of oxygen free radicals. Neurosurgery. 1990; 27: 1-11.
[21]Chan PH, Kawase M, Murakami K, et al. Overexpression of SOD1 in transgenic rats protects vulnerable neurons against ischemic damage after global cerebral ischemia and reperfusion. J Neurosci. 1998; 18:8292-9.
[22]Kim GW, Lewen A, Copin J. Neuronal expression of the DNA repair protein Ku 70 after ischemic preconditioning corresponds to tolerance to global cerebral ischemia. Stroke. 2001; 32: 2388-93.
[23]Kawase M, Murakami K, Fujimura M, et al. Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency. Stroke. 1999; 30:1962-8.
1 WA Pulsineili,JB Brierley.A new model of bilateral hemispheric ischemia in the unanesthetized rat.Stroke.1979;10:267-272.
2 Chen JL,Li Y,Wang L,et al.Therapeutic benefit of intraveous administration of bone marrow stromal cells after cerebral ischemia in rats.Stroke.2001;32:1005-11.
3 Charles V Vorhees,Michael T Williams.Morris water maze:procedures for assessing spatial and related forms of learning and memory.Nature Protocols.2006;1:848-858.
4 钟乐,王霞,余小河,等.新生大鼠缺氧缺血性脑损伤后的远期行为学测试.中国当代儿科杂志.2005:7:247-250。
5 Chen J,Li Y,Chopp M.Intracerebral transplantation of bone marrow with BDNF after MCAO in rat.Neuropharmacology.2000;39:711-716.
6 Hamm RJ,Pike BR,O'Dell DM,et al.The rotarod test:an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury.J Neurotrauma.1994;11:187-196.
7 Ding Y,Zhou Y,Lai Q,et al.Long-term neuroprotective effect of inhibiting poly(ADP-ribose)poly merase in rats with middle cerebral artery occlusion using a behavioral assessment.Brain Res.2001;915:210-7.
8 王新陆,王中琳.缺血性中风后遗症模型的建立与评价.世界科学技术——中医药现代化基础研究,2004,6(3):14.
9 Zhao H,Robert M Sapolsky and Gary K Steinberg.Interrupting reperfusion as a stroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats.Journal of Cerebral Blood Flow & Metabolism.2006;26:1114-21.
10 Jozef Burda,Viera Danielisov'a and Miroslava N'emethov'a.Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.Cellular and Molecular Neurobiology.2006;26:1141-51.
11 Morris,R.G.M.Spatial localization does not require the presence of local cues.Learn.Motiv.1981;12:239-260.
12 Stewart,C.A.& Morris,R.G.M.The water maze in Behavioural Neuroscience,Volume Ⅰ,A Practical Approach(ed.Sahgal).107-122(IRL Press at Oxford University Press,Oxford,1993).
13 Morris,R.G.M.An attempt to dissociate 'spatial-mapping' and 'working-memory' theories of hippocampal function in Neurobiology of the Hippocampus(ed.Seifert) 405-432(Academic Press,New York,1993).
[1]Kerr J F,Wyllie A H,Currie AR.Apoptosis,Br J Cancer.1972;2:239-242.
[2]Otsuki Y,Li Z,Shibata MA.Apoptotic detection methods--from morphology to gene.Prog Histochem Cytochem.2003;38:275-339.
[3]Froelich-Ammon S J,Patch MW,Osheroff N,et alo Topoisomerase I binds to ellipticine in the absence or presence of DNA.Characterization of enzyme-drug interactions by fluorescence spectroscopy.J Biol Chem.1995;270:14998-15004.
[4]Dawson Y M,Dawson V L.Protection of brain from ischemia.Cerebrovascular Disease.1997;7:349-352.
[5]MacManus JP,Buchan AM,Hill IE,et al.Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.Nemosci Lett.1993;164:89-92.
[6]Marlangue CC,Represa MA,Popcvici T,et al.Apoptosis and necrosis after reversible focal ischemia:An in situ DNA fragmentaition analysis.J Cereb Blood Flow Metab.1996;6:186-94.
[7]MacManus JP,Hill IE,Preston E,et al.Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.J Cereb Blood Flow Metab.1995;15:728-37.
[8]Heng Zhao,Robert M Sapolsky and Gary K Steinberg.Interrupting reperfusion as a stroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats.J Cereb Blood Flow Metab.2006;26:1114-21.
[9]Jozef Burda,Viera Danielisov'a,Miroslava N'emethov'a,et al.Delayed post-conditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.Cellular and Molecular Neurobiology,2006,26,1141-51.
[10]Schulz JB,Weller M,Moskowitz MA.Caspase as treatment targets in stoke and neurodegenerative disease.Ann Neurol.1999;45:421-9.
[11]Zhou P,Chou J,Olea RS,et al.Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD:a structure basis for specific adaptor/caspase interaction.Proc Natl Acad Sci.1999;96:11265-70.
[12]Ashkenazi A,Dixit VM.Death receptors:signaling and modulation.Science.1998;281:1305-8.
[13]Rao RV,Hermel E,Castro-Obregon S,et al.Coupling endoplasmic reticulum stress to the cel death program:mechan ism ofcaspase activation.J Biol Chem.2001;276:33869-74.
[14]张文华,周璐,徐淑军等,Caspase-3激活和细胞色素c释放在脑缺血再灌注损伤中的作用.山东大学学报(医学版).2007;45:1193-2000。
[15]Chen J,Nagayama T,Jin K,et al.Induction of Caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia.J Neurosci.1998;18:4914-28.
[16]Ni B,Wu X,Su Y,et aio Transient global cerebral ischemiainduces a prolonged espression of the Caspase-3 mRNA in rat hippocampal CA1 pyramidal neurons.J Cereb Blood Flow Metab.1998;18:248-256.
[17]Graham S H,Chen J,Clark R S,et al.Bcl-2 family gene products in cerebral ischemia and traumatic brain injury.J Neurotrauma.2000;17:831-841.
[18]Hata R,Gillardon F,Nfichaelidis T M,et alo Targeted disruption of the bcl-2 gene in the mice exacerbates focal ischemic brain injury.Mebtab Brain Dis.1999;14:117-24.
[19]Chen J,Graham SH,Chan PH,et al.Bcl-2 is expressed in neurons that survive focal ischemia in the rat.Neuroroporto 1995;6:394-398.
[20]Linnik MD,Zahos P,Geschwind MD,et al.Expression of bcl-2 from defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia.Stroke.1995;26:1670-75.
[21]韩雪梅,熊文激,胡国中,等.金纳多对大鼠全脑缺血再灌注损伤后Fas和Bcl-2蛋白表达的影响.吉林大学学报(医学版),2004,5:381-383.
[22]Shimizu S,Nagayama T,Jin KL,et alo Bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain.Cereb blood Flow Metab.2001;21:233-243.
[23]Chen J,Simon P,Nagayama T,et alo Suppression of endogenous bcl-2 expression by antisense treatment exacerbates ischemic neuronal death.Cereb Blood Flow Metab.2000;20:1033-39.
[1]Zhang RL, Chopp M, Chen H, et al. Temporal profile of ischemic tissue damage, neutrophile response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neurol Sci. 1994; 125:3-10.
[2]Zhang GH, Lu P, W ang YL. Effects of dipfluzine on expressions of E-selectin, P-selectin, and ICAM-1 in brain ischemia-reperfusion rats. Yao Xue Xue Bao. 2005; 40: 1091-95.
[3]Medzhitov R, Preston-Hurburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388: 394-397.
[4]Oyama J, Blais C Jr. Liu X, et al. Reduced myocardial ischemia reperfusion injury in toll-like receptor 4-deflcient mice. Circulation. 2004; 109: 784 -789.
[5]Stephenson D, Yin T, Smalstig EB, et al. Transcription factor nuclear factor kappa B is activated in neurons after focal cerebral ischemia. J Cereb Blood Flow Metab. 2000; 20: 592-603.
[6]Sun Z, Andersson R. NF-kappa Bactivation and inhibition: a review. Stroke. 2002; 18: 99-106.
[7]Podrez EA, PoliakovE, Shen Z, et al. A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions. J Biol Chem. 2002; 277: 38517-38523.
[8]Singh H, Sen R, Baltimore D, et al. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature. 1986; 319: 154-8.
[9]Shen W, Zhang C, Zhang G. Nuclear factor-KB activation is mediated by NMDA and non-NMDA receptor and L-type voltage-gated Ca2+ channel following severe global ischemia in rat hippocampus. Brain Res. 2002; 933: 23-30.
[10]Salminen M, Spitz F, Fiszman MY, et al. Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (Ml) for unknown proteins. J Mol Biol. 1995; 253: 17-31.
[1 l]Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab. 2000, 2:1011-32.
[12]Lj C, Ha T, Kelley J,et al. Modulating Toll-like receptor mediated signaling by (1,3)-β-D-glucan rapidly induces cardioprotection. Cardiovasc Res. 2004; 61: 538-547.
[13]Kielian T. Tol 1-like receptors in central nervous system glial inflammation and homeostasis. J Neurosci Res. 2006; 83: 711-730.
[14]Cao CX, Yang QW, Lv FL, et al. Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun. 2007; 353: 509-514.
[15]Caso JR, Pradillo JM, Hurtado O, etal. Toll-like receptor 4 is involed in brain damage and inflammation after experimental stroke. Circulation. 2007; 115: 1599-1608.
[1]Kato, H, Liu, et al. MK-801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus. Neurosci. Lett, 1992; 139: 118-121.
[2]Garnier, Ying P, and Swanson R. A. Ischemic preconditioning by caspase cleavage of poly(ADPribose) polymerase-1. J Neurosci. 2003; 23: 7967-7973 .
[3]Vlasov, Korzhevskii, and Polyakova. Ischemic preconditioning of the rat brain as a method of endothelial protection from ischemic/reperfusion injury. Neurosci Behav Physiol. 2005; 35: 567-572 .
[4]Becker. Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. Proc Natl Acad Sci. 1997;94: 10873-10878 .
[5]Frenkel et al. Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke. J Neurol Sci. 2005;233: 125-132.
[6]Chen Y, et al. Mucosal tolerance to E-selectin provides cell-mediated protection against ischemic brain injury. Proc Natl Acad Sci. 2003;100: 15107-15112 .
[7]Kariko, Weissman, and Welsh. Inhibition of toll-like receptor and cytokine signaling - a unifying theme in ischemic tolerance. J Cereb Blood Flow Metab. 2004;24: 1288-1304.
[8]Gonzalez-Zulueta, et al. Requirement for nitric oxide activation of p21ras/extracellular regulated kinase in neuronal ischemic preconditioning. Proc Natl Acad Sci. 2000;97: 436-441.
[9]Jones and Bergeron. Hypoxia-induced ischemic tolerance in neonatal rat brain involves enhanced ERK1/2 signaling. J Neurochem. 2004;89: 157-167.
[10]Yoshida, Nakakimura, Cui YJ, et al. Adenosine Al receptor antagonist and mitochondrial ATP-sensitive potassium channel blocker attenuate the tolerance to focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 2004;24: 771-779.
[11]Wick, et al. Neuroprotection by hypoxic preconditioning requires sequential activation of vascular endothelial growth factor receptor and Akt. J Neurosci. 2002;22: 6401-6407.
[12]Raval, Dave K R, Mochly-Rosen D, et al. PKC is required for the induction of tolerance by ischemic and NMDAmediated preconditioning in the organotypic hippocampal slice. J Neurosci. 2003;23: 384-391.
[13]Wingrove JA and O'Farrell PH. Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila. Cell. 1999;98: 105-114.
[14]Cho S, et al. Obligatory role of inducible nitric oxide synthase in ischemic preconditioning. J Cereb Blood Flow Metab. 2005;25: 493-501.
[15]Ravati A, Ahlemeyer B, Becker A, et al. Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-KB. J Neurochem. 2001;78: 909-919.
[16]Kapinya K., Penzel R, Sommer C, et al. Temporary changes of the AP-1 transcription factor binding activity in the gerbil hippocampus after transient global ischemia, and ischemic tolerance induction. Brain Res. 2000; 872: 282-293.
[17]Ma D, et al. Xenon preconditioning reduces brain damage from neonatal asphyxia in rats. J Cereb Blood Flow Metab. 2006; 26: 199-208.
[18]Blondeau N, Widmann C, Lazdunski M, et al. Activation of the nuclear factor-KB is a key event in brain tolerance. J Neurosci. 2001 ;21: 4668-4677.
[19]Ryu H, et al. Sp1 and Sp3 are oxidative stressinducible, antideath transcription factors in cortical neurons. J Neurosci. 2003; 23: 3597-3606.
[20]Bernaudin M, et al. Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxiainducible factor-1 and its target genes, erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood Flow Metab. 2002;22: 393-403.
[21]Ralph GS, et al. Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 . and HIF-2 . in a primary neuronal model of hypoxia. J Cereb Blood Flow Metab. 2004;24: 245-258.
[22]Bernaudin M, Tang Y, Reilly M, et al. Brain genomic response following hypoxia and re-oxygenation in the neonatal rat. Identification of genes that might contribute to hypoxia-induced ischemic tolerance. J Biol Chem. 2002; 277: 39728-38.
[24]Douen AG, et al. Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes. J Neurochem 2000;75:812-818.
[25]Paschen W, Mies G. Effect of induced tolerance on biochemical disturbances in hippocampal slices from the gerbil during and after oxygen/glucose deprivation. Neuroreport 1999;10: 1417-1421.
[26]Bordet R, et al. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J Cereb Blood Flow Metab. 2000;20: 1190-1196.
[27]Brambrink AM, et al. Tolerance-inducing dose of 3-nitropropionic acid modulates bcl-2 and bax balance in the rat brain: a potential mechanism of chemical preconditioning. J Cereb Blood Flow Metab. 2000;20: 1425-1436.
[28] Wu C, et al. Different expression patterns of Bcl-2, Bcl-xl, and Bax proteins after sublethal forebrain ischemia in C57Black/Crj6 mouse striatum. Stroke 2003;34: 1803-8.
[29]Storkebaum E, Lambrechts D, Carmeliet P. VEGF:once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 2004;26: 943-954.
[30]Chen J, Graham SH, Zhu RL, et al. Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab. 1996; 16: 566-577.
[31]Dawson DA, Furuya K, Gotoh J, et al. Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion. J Cereb Blood Flow Metab. 1999; 19: 616-623.
[32]Ding YH, et al. Exercise preconditioning ameliorates inflammatory injury in ischemic rats during reperfusion. Acta Neuropathol. 2005;109: 237-246.
[33]Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia. 2005;50: 307-320.
[34]Kato H, Kogure K, Araki T, et al. Astroglial and microglial reactions in the gerbil hippocampus with induced ischemic tolerance. Brain Res. 1994 ;664: 69-76.