摘要
缺血性脑损伤(中风)是临床上高发病之一,尽管脑缺血的损伤与保护机制一直是人们研究的重点,但是临床上对于脑缺血性中风,仍缺乏有效防治措施,目前越来越多的证据表明体内阿片系统在中风的机制中有重要作用。
以往传统的阿片研究主要集中在阿片和内源性阿片肽对于痛感觉、情绪、自主功能、免疫调节具有广泛的生理和行为效应,最近,研究人员在缺血缺氧性神经元保护机理中发现DOR(delta-opioid receptors,DOR)的重要作用,DOR系统可能参与缺血缺氧神经元的保护和神经发生,例如:1.乌龟脑内独特的DOR高密度分布及其神经元的耐缺血缺氧性可能与神经元的缺血缺氧有关。2.DOR激动剂可以延长缺氧小鼠的存活时间,并且这种保护作用可以被DOR而非MOR,KOR的选择性受体拮抗剂所阻断。3.DOR激动剂可以减轻谷氨酸对培养的大脑皮层神经元兴奋性毒性损伤,活化MOR(mu-opioid receptor,MOR)和KOR(kappa-opioidreceptor,KOR)对谷氨酸所致神经元损伤无保护性作用。这些研究都支持,DOR在体内脑缺血缺氧神经元损伤及保护有重要作用。
脑源性神经营养因子(BDNF)是神经营养因子超家族的一员,BDNF在神经保护中有重要作用。在局灶性脑缺血,cAMP-反应元件结合蛋白(CREB)活化产生p-CREB可以提高BDNF的表达从而促进海马齿状回的神经保护。文献报道DOR激动剂DPDPE(D-Pen2,D-Pen5-enkephalin)能够快速激活CREB形成p-CREB,呈剂量依赖性,而且这种作用可被DOR特异性拮抗剂Naltrindole所拮抗。侧脑室给予非肽类的DOR激动剂(+)BW373U86可以提高BDNF mRNA在前脑皮层表达,并且这种效应可以被DOR拮抗剂Naltrindole所拮抗,而非MOR拮抗剂naltrexone和KOR拮抗剂nor-binaltorphimine。因此,在明确DOR参与神经元的缺血缺氧保护的基础上,研究DOR功能的改变对p-CREB/CREB及BDNF的影响,从而探讨DOR参与神经元的缺血缺氧保护的可能机制,是本课题研究的主要内容之一。
针刺治疗中风在我国已有数千年的临床实践,早在《黄帝内经》、《甲乙经》中即有记载。长期以来针刺治疗中风取得了一定的效果,针刺作为中国传统医学的一个重要组成部分,针刺治疗中风,因其经济、有效、便利在全世界范围已得到专家患者的认可。
大量研究表明,EA可以调节脑内阿片系统,而且近来也有文献报道:脑缺血前给予EA有缺血预保护作用,可以减低MCAO缺血再灌大鼠脑梗塞体积,而且这种保护作用,可以被腹腔注射DOR拮抗剂naltridole所拮抗,针刺可以提高缺血皮层的周边区BDNF表达,从而参与了脑缺血损伤的保护过程;我室以前的工作也表明针刺可以提高缺血和再灌时细胞外BDNF的水平,EA可以拮抗抑郁症模型中p-CREB表达的下降,并且提高BDNF的水平。因而,探讨针刺抗神经元的缺血缺氧损伤的神经保护作用是否与DOR有关,并且在此过程中是否有p-CREB/CREB及BDNF的参与,也是本课题研究的又一重要内容。
大量研究表明,哺乳动物脑内终生存在神经发生,并且被脑缺血所加强。促进损伤区的神经再生替代死亡神经元已经成为缺血损伤治疗的潜在的有效的方法。文献报道,近来有研究表明,DOR可能与神经发生(增殖、迁移和分化)有关,在SVZ区,其神经元、神经胶质和少突胶质细胞亚群分别表达MOR、DOR、KOR。长期使用选择性DOR拮抗剂Naltrindole诱导大脑皮层前腹带区星型胶质细胞增生,DOR激动剂SNC80抑制星型胶质细胞增生。这些研究都支持DOR可能在神经发生中起重要作用。而EA可以加强大鼠局灶性脑缺血后纹状体的神经发生,以及提高BDNF的表达,因此,探讨DOR与EA在脑缺血非急性期神经保护与神经元发生中的作用及p-CREB/CREB和BDNF在其中的变化,是本课题研究的第三方面内容。
综上所述,本课题拟在MCAO/再灌模型上,对DOR在脑缺血及针刺脑缺血的作用,作如下的系列探讨:1.明确DOR参与神经元的缺血缺氧保护的基础上,研究DOR功能的改变对p-CREB/CREB及BDNF的影响,从而探讨DOR参与神经元的缺血缺氧保护的可能机制。2.阐明EA抗神经元的缺血缺氧损伤的作用是否与DOR有关,并且探讨在此过程中是否有p-CREB/CREB及BDNF的参与。3.探讨脑缺血时DOR与EA的神经保护作用与神经发生的关系。
实验结果如下:
1.DOR在脑缺血/再灌注损伤的作用及与p-CREBICREB、BDNF的关系
1.1 DOR激动剂TAN-67、拮抗剂Naltrindole对MCAO/再灌大鼠的影响
大鼠随机分为8组:sham组,MCAO+aCSF组,MCAO+TAN-67 3 mM组,MCAO+TAN-67 6 mM组,MCAO+TAN-67 20 mM组,MCAO+Naltrindole 2 mM组,MCAO+Naltrindole 5 mM,MCAO+Naltrindole 10 mM组,缺血1hr再灌24hr。
1.1.1 TAN-67、Naltrindole对大鼠局部脑血流的影响
在MCAO术前侧脑室注射人工脑脊液,不同剂量的DOR激动剂(TAN-67,3mM,6 mM,20 mM)和拮抗剂(Naltrindole,2 mM,5mM,10 mM),通过侧脑室注射DiI检测侧脑室注射部位正确与否,用激光多普勒血流仪记录缺血侧皮层脑血流值。缺血前可以记录到稳定的血流基础值,因此,血流值可以用百分比来表示。所有动物均在缺血过程中和再灌30min时记录脑血流值,脑缺血发生后,顶叶皮层局部脑血流值骤然下降,仅为缺血前血流值的30%以下。在缺血持续的1h内,在该处血流值基本稳定在此低水平,甚至再灌时脑血流值也维持在低水平。侧脑室注射TAN-67(3 mM,6 mM,20 mM)、Naltrindole(2 mM,5 mM,10 mM)后,脑血流值与侧脑室注射aCSF缺血大鼠变化相似,维持在缺血前基础血流值的20-30%,提示药物并不影响缺血区皮层局部脑血流。
1.1.2 TAN-67、Naltrindole对大鼠脑梗死体积的影响
雄性SD大鼠,缺血1hr再灌24hr。应用Leica Q500图像处理系统,测定CV染色脑片的脑梗塞区域及损伤侧、对照侧半个脑片的面积脑梗死体积。结果显示,与缺血组相比,MCAO+TAN-67 6 mM明显降低梗死体积,然而MCAO+NAI 10mM则显著增加梗死体积。
1.1.3 TAN-67、Naltrindole对大鼠神经功能缺损评分的影响
缺血1hr再灌24hr,进行神经功能缺损评分,评分采用0-5级评分标准,结果显示,在所有缺血大鼠中MCAO+TAN-67 6 mM组评分最高,大鼠神经功能缺损程度最低,而MCAO+Naltrindole 10 mM组评分最低,大鼠神经功能缺损程度最高。
1.1.4 TAN-67、Naltrindole对DOR蛋白表达的影响
在TAN-67、Naltrindole对大鼠脑梗死体积影响的基础上,动物分为6组,MCAO组,MCAO+TAN67 6 mM组,TAN67 6mM组(不行MCAO手术),MCAO+Naltrindole 10 mM组,Naltrindole 10 mM group(不行MCAO手术)。
雄性SD大鼠,缺血1hr再灌24hr,共聚焦显微镜显示DOR免疫阳性标记细胞形态为神经元样,脑内分布广泛,在非缺血侧皮层及纹状体高表达,在缺血侧皮层及纹状体表达下降。免疫荧光双标显示,DOR与MAP-2共存。
Western blot分析显示,缺血1hr再灌24hr,DOR蛋白含量(36KD,60KD)在缺血侧纹状体表达下降。MCAO+NAI 10 mM组与缺血组相比DOR蛋白含量显著下降,MCAO+TAN-67 6 mM可以部分抑制MCAO所诱导的DOR蛋白表达下降;DOR蛋白(48KD)在缺血侧纹状体的表达没有变化。正常大鼠侧脑室注射Naltrindle 10 mM和TAN-67 6 mM后,与假手术组相比,DOR蛋白(36KD,48KD和60KD)表达没有显著性变化。
1.2 DOR激动剂TAN-67、拮抗剂Naltrindole对MCAO大鼠p-CREB/CREB蛋白表达的影响
雄性SD大鼠,缺血1hr再灌24hr,用共聚焦显微镜检查p-CREB(红色)/CREB(绿色),形态多为圆形或椭圆形,在脑内分布广泛,非缺血侧皮层及纹状体高表达,缺血侧皮层及纹状体表达下降。
Western blot分析显示,缺血1h再灌24hr,p-CREB/CREB蛋白(43KD)含量在缺血侧纹状体表达下降。MCAO+NAI 10 mM组与缺血组相比,CREB蛋白表达显著下降,MCAO+TAN-67 6 mM可以部分抑制MCAO所诱导的CREB蛋白表达下降;正常大鼠侧脑室注射Naltrindle 10 mM和TAN-67 6 mM,与假手术组相比对CREB蛋白表达没有显著性差异,MCAO+TAN67 6 mM组,MCAO+NAI10 mM组与假手术组相比,p-CREB蛋白表达下降。
1-3 DOR激动剂TAN-67、拮抗剂Naltrindole对MCAO大鼠BDNF蛋白表达的影响
雄性SD大鼠,缺血1hr再灌24hr,用共聚焦显微镜检查BDNF免疫阳性标记细胞在脑内分布广泛,非缺血侧皮层及纹状体高表达,缺血侧皮层及纹状体表达下降。免疫荧光三标显示DOR、CREB和BDNF在神经元中共存。
Western blot分析显示,缺血1hr再灌24hr,BDNF蛋白(21KD)含量在缺血侧纹状体表达下降。MCAO+NAI 10 mM与缺血组相比显著下降,MCAO+TAN-67 6mM可以部分抑制MCAO所诱导的BDNF蛋白表达下降;正常大鼠侧脑室注射Naltrindle 10 mM和TAN-67 6 mM,与假手术组相比对BDNF蛋白表达没有显著性差异。
小结:1.DOR拮抗剂Naltrindole加重脑缺血损伤,激动剂TAN-67具有明显的神经保护效应,提示DOR可能具有内源性抗脑缺血损伤的神经保护作用。2.在大鼠缺血侧纹状体,胞浆内及细胞膜上DOR表达均有下降,DOR拮抗剂不改变胞浆内48KD DOR蛋白的表达,但进一步抑制了细胞膜上36KD和60KD DOR蛋白的表达,提示DOR蛋白在细胞内不同部位的表达与其神经保护效应的发挥可能有一定关系。3.脑缺血后纹状体内p-CREB、CREB及BDNF的表达均有下降,DOR拮抗剂可进一步下调CREB及BDNF的表达,DOR激动剂上调CREB及BDNF的表达,提示CREB及BDNF可能参与了DOR的抗脑缺血损伤效应。
2.DOR在EA抗脑缺血中的作用及其与p-CREB/CREB和BDNF的关系
大鼠分为5组,分别为:sham组,MCAO组,MCAO+EA组,MCAO+sham EA组和MCAO+2 mM Naltrindole+EA组。MCAO+EA所选择穴位是“水沟(GV 26)和内关(PC 6)”。
2.1 EA,sham EA,MCAO,MCAO+2 mM Naltrindole+EA对大鼠脑梗死体积的影响
雄性SD大鼠,缺血1hr再灌24hr,MCAO术前侧脑室注射2mM Naltrindole,灌注开始时给予EA治疗,持续30min,应用Leica Q500图像处理系统,测定CV染色脑片的脑梗塞区域及损伤侧、对照侧半个脑片的面积,并计算脑梗死体积。结果显示,与缺血组相比,EA降低梗死体积,NAI 2 mM翻转EA的抗脑缺血作用,增加梗死体积。sham EA没有抗脑缺血效应。与MCAO组相比,EA增加神经功能缺损评分,而MCAO+2mM Naltrindole神经功能缺损评分低。
2.2 EA,MCAO,MCAO+2 mM Naltrindole+EA对大鼠DOR蛋白表达的影响
Western blot分析显示:缺血1hr再灌24hr,DOR蛋白含量(36KD,60KD)在缺血侧纹状体表达下降,与sham组相比有明显差异,EA使DOR蛋白表达升高,NAI2 mM翻转EA升高DOR蛋白含量的作用(36KD,60KD)。不同组别间DOR蛋白含量(48KD)在缺血侧纹状体没有变化。
2.3 EA,MCAO,MCAO+2 mM Naltrindole+EA对大鼠p-CREB/CREB蛋白表达的影响
Western blot分析显示,缺血1hr再灌24hr,p-CREB/CREB蛋白(43KD)含量在缺血侧纹状体表达下降。EA可以使CREB蛋白表达上升;NAI 2 mM翻转EA升高缺血侧CREB蛋白含量的作用。与sham组相比,EA组、MCAO组、MCAO+Naltrindole 2 mM+EA组p-CREB表达均下降。
2.4 EA,MCAO,MCAO+2 mM Naltrindole+EA对大鼠BDNF蛋白表达的影响
Western blot分析显示,缺血1hr再灌24hr,BDNF蛋白(21KD)含量在缺血侧纹状体表达下降。EA可以使BDNF蛋白表达上升;NAI 2 mM翻转EA升高缺血侧BDNF蛋白含量的作用。与sham组相比EA组、MCAO组、MCAO+Naltrindole 2mM+EA组BDNF表达均下降。
小结:1.EA“水沟、内关”具有明显的神经保护效应,DOR拮抗剂Naltrindole2mM能够翻转EA对MCAO缺血/再灌大鼠的神经保护作用,提示DOR可能是针刺抗脑缺血损伤中的一个重要环节。2.脑缺血后胞浆内及细胞膜上DOR表达下降,EA可明显上调细胞膜上DOR蛋白的表达,DOR拮抗剂则翻转EA对DOR表达的改变,提示EA的抗脑缺血损伤效应可能与其对DOR表达的改变有关。3.脑缺血后CREB、p-CREB及BDNF的表达均下降,EA可明显上调CREB和BDNF的表达,DOR拮抗剂翻转EA的作用,提示EA对CREB与BDNF的调节过程可能有DOR的参与。
3.DOR和EA在神经发生中的作用
大鼠随机分为7组:sham组,MCAO组,MCAO+EA组,MCAO+sham-EA组,MCAO+2 mM Naltrindole+EA组,MCAO+10 mM Naltrindole组,MCAO+6 mMTAN-67组。雄性SD大鼠,缺血1hr再灌7天,EA穴位为“水沟(GV 26)-内关(PC6)”。
3.1 EA,sham-EA,MCAO,MCAO+Naltrindole 2 mM+EA,MCAO+Naltrindole 10 mM,MCAO+TAN-67 6 mM对大鼠脑梗死体积的影响
雄性SD大鼠,缺血1hr再灌7天,应用Leica Q500图像处理系统,测定CV染色脑片的脑梗塞区域及损伤侧、对照侧半个脑片的面积,并计算脑梗死体积。结果显示:与缺血组相比,EA和6 mM TAN-67均可降低梗死体积,NAI 10 mM增加了脑梗死体积,而NAI 2 mM翻转EA的抗脑缺血作用,增加梗死体积。
3.2 EA,sham-EA,MCAO,MCAO+Naltrindole 2 mM+EA,MCAO+Naltrindole 10 mM,MCAO+TAN-67 6 mM对BrdU免疫阳性细胞的影响
MCAO+10 mM Naltrindole促进BrdU阳性细胞在缺血侧纹状体的表达,而EA和MCAO+TAN-67 6 mM降低BrdU阳性细胞的表达,MCAO+Sham EA、MCAO+Naltrindole 2 mM+EA和MCAO+aCSF组相比,BrdU免疫阳性细胞数目没有显著性差异。
小结:1.多次EA“水沟、内关”具有明显的神经保护效应,多次给予DOR拮抗剂Naltrindole 2mM能够翻转多次EA对MCAO缺血/再灌大鼠的保护作用,提示在EA抗脑缺血损伤的保护作用中,DOR可能是一个重要的中间环节。2.脑缺血后BrdU阳性细胞数明显增多,EA及DOR激动剂TAN-67处理后,BrdU阳性细胞数有所减少,DOR拮抗剂则进一步增加了BrdU阳性细胞数。提示,脑缺血损伤后的神经发生很可能与脑损伤程度有关。
综上所述,本工作获得以下结论:
1.DOR拮抗剂Naltrindole加重脑缺血损伤,激动剂TAN-67具有明显的神经保护效应,提示DOR可能是抗脑缺血损伤的一个重要环节,同时CREB及BDNF表达的改变可能是DOR发挥神经保护作用的重要途径。
2.EA的抗脑缺血损伤效应能被DOR拮抗剂Naltrindole翻转,提示DOR可能参与了EA的神经保护作用,CREB及BDNF表达的改变可能是DOR参与EA神经保护作用的重要环节。
3.DOR激动剂TAN-67及EA均可减少脑缺血后BrdU阳性细胞数目,而DOR拮抗剂增加BrdU阳性细胞数目,提示大鼠脑缺血/再灌注损伤后的神经发生很可能与脑损伤程度有关。
Ischemic stroke is a high-incidence disease in clinic.Studying the mechanism of ischemic brain injury and neuronal protection in stroke has been a major focus on clinical research,however,clinically effective and safe methods for reducing ischemic brain injury have not yet been well established.Evidence has accumulated to indicate that opioids in the body are involved in the pathogenesis of stroke.
Opioids have a wide range of physiologic and behavioral effects on pain perception,mood,motor control and autonomic function.Their effects are mediated by opioid receptors.In addition,previous studies have suggested that delta opioid receptors protect neurons against ischemia/hypoxic stress.For example:1.The high density and high affinity of delta-opioid receptors may be important in the tolerance to hypoxia in the turtle brain.2.Systemic administration of opioid receptor agonists prolonged the survival period during hypoxia in mice in whole animal experiments and this protection is selectively blocked by delta- but not by mu- or kappa-opioid receptor antagonists.3.Delta-,but not mu- and kappa- opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.These findings support the concept that the d-opioid receptor plays an important role in neuronal protection.
Brain-derived neurotrophic factor(BDNF) is a member of the neurotrophin family.BDNF is recognized to play an important role in the neuroprotection. Activation of cAMP-response-element-binding protein(CREB) after focal cerebral ischemia increase in CREB phosphorylation can increases BDNF expression and stimulates neuroprotection in the adult dentate gyrus.It is reported that delta-opioid receptor agonist[D-Pen(2,5)]enkephalin(DPDPE) produced a dose-dependent increase in CREB phosphorylation and the effect was reversed by Naltrindole,which is a highly selective deltaopioid receptor antagonist.Intracerebroventricular administration of a selective non-peptidic DOR agonist(+)BW373U86,increased BDNF mRNA expression in the frontal cortex through a DOR-mediated mechanism because these effects were blocked by the DOR antagonist Naltrindole,but not by the micro-opioid receptor(MOR) antagonist naltrexone(NTX) or the kappa-opioid receptor antagonist nor-binaltorphimine.Therefore,to ensure the role of DOR in MCAO/reperfusion injury and to investigate its mechanism from p-CREB/CREB and BDNF in the pathogenesis of stroke is the main target of the present study.
In traditional Chinese medicine,acupuncture for ischemic stroke is much better than other treatments with convenience availability,cheapness and convinced by both experts and patients of ischemic stroke all over the world.Compared with the increasingly application of acupuncture in the treatment of stroke,the basic studies of these protective mechanisms are still limited so far.
Numerous reports have indicated that Electroacupuncture(EA) can activate the endogenous opioid system during the treatments.It is reported that the EA preconditioning reduced brain infarct volume,and administration of Naltrindole attenuated the brain infarct volume reduction induced by EA preconditioning.It is reported that EA may increase expression of BDNF in the peripheral cortex of the infarction,and it would be protective effect against cerebral ischemia.In the previous work of our laboratory,we detected that EA could enhance extracellular BDNF level during ischemia and reperfusion.EA can reverse the decreased levels of p-CREB in rat model of depression and increase the BDNF expression.Then,another part of our studies was to explore the relationship between DOR and EA in MCAO/reperfusion injury,and to investigate its mechanism involving p-CREB/CREB and BDNF in the pathogenesis of stroke.
It is well documented that neurogenesis exists in the adult mammalian brain throughout the life,and it is strengthened by cerebral ischemia.Adult neurogenesis as a treatment to replace dying neurons is therefore a potentially significant therapeutic strategy for ischemic injury.It is reported recently that DOR is related with neurogenesis,the subpopulations of neurons,astrocytes,and oligodendrocytes in the SVZ and striatum differentially express mu-,delta-,and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner.The selective delta-opioid receptor antagonist Naltrindole,increase astrogliosis in the cingulate cortex,but SNC-80 decrease astrogliosis.MCAO mediates the phosphorylation of CREB which drives a prominent synthesis of BDNF and this BDNF synthesis induces neurogenesis.Electroacupunctrue enhances striatal neurogenesis in adult rat brains after a transient cerebral middle artery occlusion and up-regulates BDNF.Then,the third part of our studies was to explore the relationship berween DOR and EA in neurogenesis after MCAO/reperfusion injury,and to investigate its mechanism from p-CREB/CREB and BDNF in the neurogenesis after stroke.
By using the molecular biological techniques,including immunohistochemistry and Western-Blot,the present study was designed to explore:(1) the role of DOR and the mechanism in MCAO/reperfusion injury with p-CREB/CREB and BDNF;(2) the role of DOR in EA anti-ischemic efficacy and the mechanism with p-CREB/CREB and BDNF;(3) the role of DOR and EA in neurogenesis,and the relationship between p-CREB/CREB and BDNF.
The results are following:
1.Role of DOR and its mechanism in MCAO/reperfusion injury with p-CREB/CREB and BDNF
1.1 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on MCAO/reperfusion rats
Rats were divided into eight groups:sham group,middle cerebral occlusion group(MCAO),MCAO+TAN67 3 mM group,MCAO+TAN67 6mM group, MCAO+TAN67 10 mM group,MCAO+Naltrindole 2mM group,MCAO+Naltrindole 5mM group group,MCAO+Naltrindole 10mM group.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.
1.1.1 Effects of Naltrindole and TAN-67 on rCBF
Regional cortical focal cerebral blood flow(rCBF) of ischemic hemisphere was monitored by laser Doppler Flowmetry.Steady state baseline values were recorded before MCAO so that all regional CBF(rCBF) data were expressed as percentages of the respective basal values.The rCBF was monitored continuously during focal ischemia till the first 30 min of reperfusion in all experimental groups.When the middle cerebral artery is obstructed successfully,local blood flow decreased immediately to lower than 30%of pre-ischemia(baseline) and persisted lower level during the whole ischemia stage and even to stage of reperfusion.The results indicated that no significant differences were observed between the MCAO (aCSF+MCAO),[MCAO+Naltrindole(2 mM,5mM,10 Mm)]and[MCAO+TAN-67 (3mM,6 mM,20 mM)]groups.
1.1.2 Effects of Naltrindole and TAN-67 on Infarct Volume
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Rats were received intracerebroventricular injection with DOR antagonist(Naltrindole,2 mM,5mM,10 mM) and agonist(TAN-67,3mM,6 mM,20 mM) or vehicle,starting at 30 min before MCAO and continuing until the time of death.ICV injection was confirmed by the use of fluorescent dye DiI.Infarct volume was quantified by cresyl violet-stained brain sections at 24 h of reperfusion with Leica Q500 IW Image Processing System. The results showed TAN-67 6 mM treatment decreased infarct volume,whereas NAI 10 mM treatment increased infarct volume,compared with the MCAO groups.
1.1.3 Effects of Naltrindole and TAN-67 on Neurological scores
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Neurological deficits were evaluated at 24 h of reperfusion with 0～5 grade method.MCAO+TAN-67 6 mM group's neurological scores were the highest,and MCAO+Naltrindole 10 mM group's neurological scores were the lowest.
1.1.4 Effects of Naltrindole and TAN-67 on DOR protein expression
Based on the effects of Naltrindole and TAN-67 on infarct volume,the experiment animals were divided into six groups:sham group,middle cerebral occlusion group(MCAO),MCAO+TAN67 6 mM group,TAN67 6mM group (without MCAO),MCAO+TAN67 10 mM group,Naltrindole 10 mM group(without MCAO).
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.DOR protein levels at 24 h of reperfusion were measured by laser scanning confocal microscope(LSCM). DOR-positive cells,exhibiting neuronal-like morphology,were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.
Western blot analysis indicated that DOR protein level(36KD and 60KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum. MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups;MCAO+TAN-67 6 mM partly inhibited the MCAO-induced decrement experession of DOR protein(36KD and 60KD) in the ipsilateral striatum.DOR protein level(48KD) was not changed in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects to DOR protein level(36KD,48KD and 60KD) compared with the sham groups.
1.2 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on p-CREB/CREB protein expression in MCAO rats
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion,p-CREB/CREB protein levels at 24 h of reperfusion were measured by laser scanning confocal microscope(LSCM).p-CREB (green)/CREB(red)-positive cells were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.
Western blot analysis indicated that p-CREB/CREB protein level(43KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum. MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups;MCAO+TAN-67 6 mM partly inhibited MCAO-induced decrement of p-CREB/CREB protein expression in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects on CREB protein level,compared with the sham(C) groups,p-CREB was low in MCAO+TAN67 6 mM,MCAO+NAI 10 mM groups.
1.3 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on BDNF protein expression in MCAO rats
The animals were divided as the 1.2 groups.
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.BDNF protein levels at 24h of reperfusion were measured by laser scanning confocal microscope(LSCM). BDNF-positive cells were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.Triple fluorescent signals also showed DOR,CREB,and BDNF co-localized in neurons.
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that BDNF protein level(21KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups; MCAO+TAN-67 6 mM partly inhibited MCAO-induced BDNF protein expression decrease in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects to BDNF protein level.
Summary:1.Activation of DOR with DOR agonist TAN-67(6 mM i.c.v.) reduced MCAO-induced brain ischemia injury.DOR antagonist Naltrindole(10 mM i.c.v.),aggravated MCAO-induced brain ischemia injury.This study demonstrates that activation of DOR in the brain protect neurons from MCAO-induced brain ischemia injury.DOR might be one of the endogenous protective factors in the anti-ischemic efficacy.2.In the MCAO rats,DOR expression in cytoplasm and membrance were all decreased,the Naltrindole did not change the protein in 48KD, but inhibited the 60KD protein in the membrance.It shows that the DOR expression in different area of cell may have different function and the neuroprotective function of DOR may be related with its distribution.3.The content of p-CREB/CREB and BDNF in the striatum decreased after MCAO/reperfusion injury,DOR antagonist can downregulation the degree of CREB and BDNF further,DOR agonist can increase DOR expression.It shows that DOR might be one of the key protective factors in the anti-ischemic efficacy
2.Role of DOR in EA anti-ischemic efficacy and the mechanism with p-CREB/CREB and BDNF
Based on the 1.1.2 exprements,2 mM Naltrindole could be used in EA experiment.Rats were divided into five groups:sham group,MCAO group,EA group, MCAO+2 mM Naltrindole +EA group.EA was delivered to "ShuiGou(GV 26) and NeiGuan(PC 6)".
2.1 Effects of EA,MCAO,MCAO+2 mM Naltrindole +EA on Infarct Volume and neurological scores
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Rats(J) were received intracerebroventricular injection with Naltrindole(2mM),starting at 30 min before MCAO.At the beginning of reperfusion EA treatment was given.Infarct volume was quantified by cresyl violet-stained brain sections at 24 h of reperfusion with Leica Q500 IW Image Processing System.The results showed EA treatment decreased infarct volume,and NAI 2 mM trEAtmeaent reversed EA anti-ischemic effects and increased infarct volume compared with the MCAO groups.Sham EA had no anti-ischemic effects.Furthermore,the neurological deficits analyzed at 24-hour reperfusion were medianed in the MCAO groups,with a mild neurological score of (2.76±0.28).The EA group's neurological scores were the highest and MCAO+2mM Naltrindole group's neurological scores were the lowest.
2.2 Effects of EA,MCAO,MCAO+ Naltrindole 2 mM+EA on DOR protein expression
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that DOR protein level(36KD and 60KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO groups resulted in a decrease in the ipsilateral striatum compared with the sham groups;EA increased DOR protein expression.NAI 2 mM reversed EA induced DOR protein(36KD and 60KD) expression increase in the ipsilateral striatum.DOR protein level(48KD) was not changed in the ipsilateral striatum.
2.3 Effects of EA,MCAO,MCAO+Naltrindole 2 mM+EA on p-CREB/CREB protein expression
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that p-CREB/CREB protein level(43KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO groups resulted in a decrease in the ipsilateral striatum compared with the sham groups;EA increased CREB protein expression.NAI 2 mM reversed EA induced CREB protein expression increase in the ipsilateral striatum,compared with the sham,EA,MCAO,MCAO+Naltrindole 2 mM+EA groups p-CREB was low.
2.4 Effects of EA,MCAO,MCAO+Naltrindole 2 mM+EA on BDNF protein expression
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that BDNF protein level(21KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.EA increased BDNF protein expression.NAI 2 mM reversed EA induced BDNF protein expression increase in the ipsilateral striatum. compared with the sham,EA,MCAO,MCAO+Naltrindole 2 mM+EA groups BDNF was low.
Summary:1.EA on "ShuiGou(GV 26)-NeiGuan(PC 6)" acupoints had anti-ischmic efficacy in the early stage of stroke,that shows EA had neuroprotective efficacy.MCAO+2mM NAI reversed EA anti-ischmic efficacy.These results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA in the early stage of stroke.2.In the MCAO rats,DOR expression in cytoplasm and membrance were all decreased,EA can increase DOR protein expression and MCAO+2mM NAI reversed EA induced DOR increase.The neuroprotective function of EA may be related with the changes of the DOR.3.The content of p-CREB/CREB and BDNF in the striatum decreased after MCAO/reperfusion injury,DOR antagonist can downregulation the degree of CREB and BDNF further,DOR agonist can increase DOR expression and the effects could be reversed by 2mM NAI.It shows that DOR might be communicated with CREB and BDNF in EA neuroprotectiv efficency.
3.Effect of DOR and EA in neurogenesis
Rats were divided into seven groups:sham group,MCAO group,EA group, sham-EA group,MCAO+ 2 mM Naltrindole +EA group,MCAO+ 10 mM Naltrindole group,MCAO+ 6 mM TAN-67 group.EA was applied to"ShuiGou(GV 26)-NeiGuan (PC 6)" acupoints.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 7 days reperfusion.
3.1 Effects of EA,sham-EA,MCAO,MCAO+ Naltrindole 2 mM+EA,MCAO+ Naltrindole 10 mM,MCAO+ TAN-67 6 mM on Infarct Volume
Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 7 days reperfusion.Infarct volume was quantified by cresyl violet-stained brain sections at 7 days of reperfusion with Leica Q500 IW Image Processing System.The results showed EA and 6 mM TAN-67 treatment decreased infarct volume.NAI 10 mM treatment increased infarct volume compared with the MCAO groups.
3.2 Effects of EA,sham-EA,MCAO,MCAO+Naltrindole 2 mM+EA,MCAO+ Naltrindole 10 mM,MCAO+ TAN-67 6 mM on BrdU immunostaining
10 mM MCAO+Naltrindole promoted BrdU+ cells in the stratum EA and MCAO+TAN-67 6 mM decreased BrdU+ cells.Sham EA and MCAO+aCSF had no effect on BrdU immunostaining.
Summary:1.Cumulative EA on "ShuiGou(GV 26)-NeiGuan(PC 6)" acupoints and TAN-67 had anti-ischmic efficacy in the late stage of stroke.NAI 2 mM reversed EA anti-ischmic efficacy,these results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA in the late stage of stroke.2.BrdU positive cells were increased after MCAO/reperfusion injury,EA and TAN-67 can reduced the BrdU positive cells,Naltrindole increased BrdU positive cells.It shows that the proliferation after ischemia will be related with the degree of injury.
Conclusion:
1.Activation of DOR protected neurons against MCAO induced brain ischemia injury,whereas antagonist of DOR further enhanced ischemic injury.DOR might be one of the key factors in the anti-ischemic efficacy.
2.Naltrindole reversed EA anti-ischmic efficacy.These results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA.The change of CREB and BDNF might be one of the key factors in the anti-ischemic efficacy of DOR and EA in the stroke.
3.BrdU positive cells were increased after MCAO/reperfusion injury,EA and TAN-67 can reduced the BrdU positive cells.That shows EA and DOR in neurogenesis after MCAO/reperfusion may be related with the degree of MCAO/reperfusion injury.
引文
[1]Martin,LJ.Neuronal cell death in nervous system development,disease,and injury(Review)[J].Int J Mol Med,2001.7(5):455-478.
[2]Xia Y,Haddad GG,et al..Major difference in the expression ofδ-and μ-opioid receptors between turtle and rat brain.J Comp Neurol.2001,436:202-210.
[3]Boutin H,Jauzac P,Dauphin F,et al.Maximal densities of μ,δ,and κ receptors are differentially altered by focal cerebral ischemia in the mouse.Brain Res.1998,(787):237-241,.
[4]Horiuchi T,Kawaguchi M,Furuya H,et al.The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats.Anesth Analg.2004,99(1):235-40.
[5]Borlongan CV,Oeltgen PR,Su T-P,Wang Y.Delta opioid peptide(DADLE)protects against ischemia-reperfusion damage in the striatum and cerebral cortex.Soc Neurosci Abstr 1999,24:979;.
[6]Ferrer I,Krup insk i J,Goutan E,et al.Brain2derived neuro trophic factor reduces co rtical cell death by ischem ia after m iddle cerebral artery occlusion in the rat(J)Acta Neuropathol(Berl),2001;101(3):2292-2381
[7]Saarelainen T,L ukkafinen JA,Koponen S,et al.Transgenic mice overexpressing truncated trkB neurotrophin receptors in neurons show increased susceptibility to cortical injury after focal cerebral ischem ia(J.Mol Cell Neuro sci,2000;16(2):872-961
[8]Kokaia Z,Zhao Q,Kokaia M,et al.Regulation of brain - derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage[J].Exp Neuro,1995,136:73-88.
[9]Arai S,Kinouchi H,Akabane A,et al.Induction of brain- derived neurotrophic factor(BDNF) and the receptor trkB mRNA filowing middle cerebral artery occlusion in rat[J].Neurosci Lett,1996,211(1):57- 60.
[10]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].Cereb Blood FlowMetab,1997,17:500- 506.
[11]Zhang H,Watson SJ and Ko MC,et al.Endogenous opioids upregulate brain-derived neurotrophic factor mRNA through delta- and micro-opioid receptors independent of antidepressant-like effects.Eur J Neurosci.2006,23(4):984-94
[12]Tabuchi A,Nakaoka R,Amano K,Yukimine M,Andoh T,Kuraishi Y,Tsuda M.??Differential activation of brain-derived neurotrophic factor gene promoters Ⅰ and Ⅲby Ca2+ signals evoked via L-type voltage-dependent and N-methyl-D-aspartate receptor Ca2+ channels.J Biol Chem 2000;275:17269-17275. [13]Tao X,Finkbeiner S,Arnold DB,Shaywitz AJ,Greenberg ME.Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism.Neuron 1998;20:709-726. [14]Tao X,West AE,Chen WG,Corfas G;Greenberg ME.A calcium-responsive transcription factor,CaRF,that regulates neuronal activity-dependent expression of BDNF.Neuron 2002;33:383-395. [15]Chen WG,West AE,Tao X,Corfas G,Szentirmay MN,Sawadogo M,Vinson C,Greenberg ME.Upstream stimulatory factors are mediators of Ca2+-responsive transcription in neurons.J Neurosci 2003;23:2572-2581. [16]Timmusk T,Palm K,Lendahl U,Metsis M.Brain-derived neurotrophic factor expression in vivo is under the control of neuron-restrictive silencer element.J Biol Chem 1999;274:1078-1084. [17]D.Y Zhu,J.S Wei,Y.M Lu,et al.Activation of cAMP-response-element-binding protein(CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus.PNAS.2004.101(25):9453-9457 [18]熊杰,石学敏。针刺治疗急性脑梗死的临床研究近况[J]。江西中药,2004,7:60-62
[19]应赛霞 程介士。电针对沙鼠脑缺血及再灌注时脑电活动的影响[J]。针刺研究,1994,19(1):29-32 [20]Wei GW(魏光伟),Huang YL,Wu GC(吴根诚),Cao XD(曹小定).Regulation of glial cell line-derived neurotrophic factor expression by EA after transient focal cerebral ischemia.[J].Acupunct.Electrother.Res.2000,25:81-90 [21]Guo JC(郭景春),Li R,Zhao P,Cheng JS(程介士)。Effect of taurine in combination with electroacupuncture on neuronal damage following transient focal cerebral ischemia in rats[J].Acupunct Electrother.Res,2002,27(2):129-136 [22]Xu ZF(许贞峰),Wu GC(吴根诚),Cao XD(曹小定)。Effect of EA on the expression of interlukin-1 β mRNA after transient focal cerebral ischeimia.[J].Acupunct.Electrother.Res.2002,27:29-35 [23]Zhao P(赵鹏),Huang YL,Cheng JS(程介士).Taurine antagonizes calcium overload induced by glutamate or chemical hypoxia in cultured rat hippocampus neurons[J].Neurosci Lett 1999,268(1):25-28
[24]Zhao P(赵鹏),Huang ZN,Chen G,Cheng JS(程介士).Electro-acupuncture attenuates nitric oxide release from rat striatum after transient middle cerebral artery occlusion[J].Acupunct Electrother Res,2000,25(2):101-107
[25]Guo JC(郭景春),Gao HM,Chen J,Zhao P,Cao XD,Li Y,Cheng JS.Modulation of the gene expression in the protective effects of electroacupuncture against cerebral ischemia:a cDNA microarray study.Acupunct Electrother Res.2004,29(3-4):173-86
[26]XIONG Li-ze,YANG Jing,LU Zhi-hong,et al.Involvement of δ-and μ-opioid receptors in the delayed cerebral ischemic tolerance induced by repeated electroacupuncture preconditioning in rats.Chinese Medical Journal 2007;120(5):394-399
[27]Lucia Pagani,Luigi Manni and Luigi Aloe.Effects of electroacupuncture on retinal nerve growth factor and brain-derived neurotrophic factor expression in a rat model of retinitis pigrnentosa Brain Research,2006,1092:198-206.
[28]Su-Jin Yun,Hye-Jung Lee,Eunjoo H.Lee.Effect of electroacupuncture on the stress-induced changes in brain-derived neurotrophic factor expression in rat hippocampus.Neuroscience Letters,2002,318:85-88.
[29]陈英辉,黄显奋.电针对MCAO大鼠皮层神经营养因子表达的影响.中风与神经疾病杂志,2000,17:1392141.
[30]Jarskog,LF and Gilmore JH Developmental expression of Bcl-2 protein in human cortex[J].Brain Res Dev Brain Res,2000.119(2):225-230.
[31]Abe-Dohmae,S,Harada N,et al.Bcl-2 gene is highly expressed during neurogenesis in the central nervous system[J].Biochem Biophys Res Commun,1993.191(3):915-921.
[32]Ferrer,I,Tortosa A,et al.Increased expression of bcl-2 immunoreactivity in the developing cerebral cortex of the rat[J].Neurosci Lett,1994.179(1-2):13-I6.
[33]Martinou,JC,Dubois-Dauphin M,et al.Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia [J].Neuron,1994.13(4):1017-1030.
[34]Menezes,JR,Marins M,et al.Cell migration in the postnatal subventricular zone [J].Braz J Med Biol Res,2002.35(12):1411-1421.
[35]Carlen M,Cassidy R.M,Brismar H,et al.Functional integration of adult-born neurons,Curr Biol,2002;12:606-8.
[36]Seaberg RM,vander Kooy D.Adult rodent neurogenic regions:the ventricular subependyma contains neural stem cells,but the dentate gyrus contains restricted progenitors.J Neurosci,2002;22:1784-1793.
[37]Tropepe V,Morshead CM,van der Kooy D,et al.Transforming growth factor-alpha null and senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma.J Neurosci,1997;17:7850-7859.
[38]Stiene-Martin A,Knapp PE,Martin K,et al.Opioid system diversity in developing neurons,astroglia,and oligodendroglia in the subventricular zone and striatum:impact on gliogenesis in vivo.Glia.2001 Oct;36(1):78-88.
[39]Narita M.,Kuzumaki N and SuzukiT,et al.Age-related emotionality is associated with cortical δ-opioid receptor dysfunction-dependent astrogliosis.Neurosci,2006;137,1359-1367.
[40]Scott R,Whittemore,K.Magnuson,et al.Mitogen and Substrate Differentially Affect the Lineage Restriction of Adult Rat Subventficular Zone Neural Precursor Cell Populations Experimental Cell Research,1999;252:75-95
[41]Youzhen Wang,Jeffrey D,Macklis,et al.Cortical Interneurons Upregulate Neurotrophin sin Vivoin Response to Targeted Apoptotic Degeneration of Neighboring Pyramidal Neurons.Experimental Neurology,1998;154:389-402
[42]Kim E.H,Kim Y.J,Lee H.J,et al.Acupuncture increases cell proliferation in dentate gyrus after transient global ischemia in gerbils,Neurosci Lett,2001;297:21-4.
[43]Zeng-jin YANG,Di-Han SHENG,Feng-Yan SUN et al,et al.Electroacupunctrue enhances striatal neurogenesis in adult rat brains after a transient cerebral middle artery occlusion.Acupunct & Electro-therapeutics Res,2005;30:185-199.
[44]Xi-Bin Liang,Xiao-Min Wang,Ji-Sheng Han,et al.Long-term high-frequency electro-acupuncture stimulation prevents neuronal degeneration and up-regulates BDNF mRNA in the substantia nigra and ventral tegrnental area following medial forebrain bundle axotomy Molecular Brain Research,2002,108:51-59
[1]Longa EZ,Weinstein PR,Cummins R,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke 1989,20(1):84-91
[2]Ginsberg MD.Adventures in the pathophysiology of brain ischemia:penumbra,gene expression,neuroprotection:the 2002 Thomas Willis Lecture[J].Stroke,2003,34(1):214-23.
[3]Mohammad-Reza Zarrindast,Mousa Sahebgharani.Effect of Alpha-Adrenoceptor Agonists and Antagonists on Imipramine-Induced Antinociception in the Rat Formalin Test Pharmacology 2002,64:201-207
[4]Nakatomi,Kuriu T.Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors[J].Cell,2002,110(4):429-441.
[5]Williams JT,Christie M J,Manzoni O,et al.Cellular and synaptic adaptations mediating opioid dependence.Physiol Rev.2001,81(1):299-343.Review.
[6]Xia Y,Haddad GG,Zhang J,et al.Major difference in the expression ofδ-and μ-opioid receptors between turtle and rat brain.J Comp Neurol.2001,436:202-210.
[7]Shimada N,Graf R,Heiss WD.Ischemia-induced accumulation of extracellular amino acids in cerebral cortex,white matter,and cerebrospinal fluid.J Neurochem.1993,60(1):66-71.
[8]Zhang J,Haddad GG,Xia Y.Delta-,but not mu- and kappa-,opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.Brain Res 2000,885:143-53.
[9]Pez Pinz MA,Mumford PL,Rosenthal M.Anoxia preconditioning in hippocampal slices:role of adenosine.Neuroscience,1996,75(3):687-694
[10]Ma MC,Zhao P,Xia Y,et al.Oxygen-sensitive {delta}-opioid receptor-regulated survival and death signals:novel insights into neuronal preconditioning and protection.J Biol Chem.2005;280(16):16208-18.
[11]Zhang J,Zhao P,Xia Y.Neuroprotective role of delta-opioid receptors in cortical neurons.Am J Physiol Cell Physiol.2002,282(6):C 1225-34.
[12]Hosobuchi Y,Baskin DS,Woo SK.Reversal of induced ischemic neurologic deficit in gerbils by the opiate antagonist naloxone.Science,1982,215(1):69- 72
[13]Olinger OP,Adams HP,JR Marler.High-dose intravenous naloxone for the treatment of acute ischemia.Stroke,1990,21:721.[2]
[14]JO Skarphedinsson,P Thoren.Endorphin mechanisms are responsible for the beneficial effects of opioid antagonists on cerebral function during relative cerebral ischaemia in rats.Acta physiologica scandinavica 132:33,281-288.
[15]DM Bofetiado,KP Mayfield and LG D'Alecy.Alkaloid delta agonist BW373U86increases hypoxic tolerance.Anesthesia & Analgesia,1996(182):1237-1241,
[16]Furui T.Potential protection by a specific kappa-opiate agonist U-50488H agonist membrane failure in acute ischemic,brain.Neuro Med Clir(Tokyo) 1993,33:133-138.
[17]KP Mayfield,LG D'Alecy J.Delta-1 opioid agonist acutely increases hvpoxic tolerance[J].J Pharmacol Exp Ther,1994,268:74-77.
[18]Boutin H,Jattzac P,Dauphin F,et al.Maximal densities of μ,δ,and κ receptors are differentially altered by focal cerebral ischemia in the mouse.Brain Res.1998,(787):237-241.
[19]Horiuchi T,Kawaguchi M,Furuya H,et al.The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats.Anesth Analg.2004,99(1):235-40.
[20]Borlongan CV,Oeltgen PR,Wang Y.Delta opioid peptide(DADLE) protects against ischemia-reperfusion damage in the stfiatum and cerebral cortex.Soc Neurosci Abstr.1999 24:979.
[21]Nagase H,,Takezawa Y,Endoh T.Rational drug design and synthesis of a highly selective nonpeptide delta-opioid agonist,(4aS~*,12aR~*)-4a-(3-hydroxyphenyl)-2-methyl-1,2,3,4,4a,5,12,12a-octahydropyrido[3,4-b]acridine(TAN-67).Chem Pharm Bull(Tokyo).1998,46(11):1695-702.
[22]Ivone Gomes,Shail K,Sharma.Monoclonal antibody to the N opioid receptor acts as an agonist in dual regulation of adenylate cyclase in NG108-15 cells FEBS Letters 1999(456):126-130
[23]Ferrer I,Krup insk i J,Goutan E,et al.Brain derived neuro trophic factor reduces co rtical cell death by ischemia after middle cerebral artery occlusion in the rat(J).Acta Neuropathol(Berl),2001;101(3):2292-2381
[24]Saarelalnen T,L ukkarinen JA,Koponen S,et al.Transgenic mice overexpressing truncated trkB neurotrophin receptors in neurons show increased susceptibility to cortical injury after focal cerebral ischemia J.Mol Cell Neuro sci,2000;16(2):872-961
[25]Kokala Z,Zhao Q,Kokaia M,et al.Regulation of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage[J].Exp Neuro,1995,136:73-88.
[26]Arai S,Kinouchi H,Akabane A,et al.Induction of brain-derived neurotrophic factor(BDNF) and the receptor trkB mRNA filowing middle cerebral artery occlusion in rat[J].Neurosci Lett,1996,211(1):57- 60
[27]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].Cereb Blood FlowMetab,1997,(17):500- 506.
[28]Tabuchi A,Nakaoka R,Amano K,Yukimine M,Andoh T,Kuraishi Y,Tsuda M.Differential activation of brain-derived neurotrophic factor gene promoters Ⅰ and Ⅲ by Ca2+ signals evoked via L-type voltage-dependent and N-methyl-D-aspartate receptor Ca2+ channels.J Biol Chem 2000,(275):17269-17275.
[29]D.Y Zhu,J.S Wei,Y.M Lu,et al.Activation of cAMP-response-element-binding protein(CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus.PNAS 2004.101(25):9453-9457
[30]Bilecki W,Hollt V,Przewlocki.Acute delta-opioid receptor activation induces CREB phosphorylation in NG108-15 cells.Eur J Pharmacol.2000;390(1-2):1-6
[31]Masaaki,OKA Michiko,UKAI Yojiro.Reversal by NS-7,a neuroprotective compound,of the decrease in transcription factor CREB mRNA expression in rat brain after permanent middle cerebral artery occlusion.Neuroscience letters 2001,(305):193-196
[32]Nina G,Pabelloa,Keller Jr,et al.Regional expression of constitutive and inducible transcription factors following transient focal ischemia in the neonatal rat:influence of hypothermia Brain Res.2005 Mar 15;1038(1):11-21
[33]Tanaka K,Nagata E,Fukuuchi Y,et al.Immunohistochemical analysis of cyclic AMP response element binding protein phosphorylation in focal cerebral ischemia in rats.Brain Res.1999,818(2):520-6
[34]Cui J K,Chung YH,Liu PK,et al.Suppression of post-ischemic hippocampal nerve growth factor expression by a c2fos antisense oligodeoxynucleotide[J].J Neurosci,1999,19(4):1335-1344.
[1]曾杰红。针刺疗法治疗中风机理研究进展概况[J]。针灸临床杂志,2001,17(1):53-57
[2]Guo JC(郭景春),Li R,Zhao P,Cheng JS(程介士).Effect of taurine in combination with electroacupuncture on neuronal damage following transient focal cerebral ischemia in rats[J].Acupunct Electrother Res,2002,27(2):129-136
[3]Guo JC(郭景春),Gao HM,Chen J,Zhao P,Cao XD,Li Y,Cheng JS.Modulation of the gene expression in the protective effects of electroacupuncture against cerebral ischemia:a cDNA microarray study.Acupunct Electrother Res,.2004,29(3-4):173-86
[4]Zhang R.X.,Lao L.,Berman B.M.Involvement of opioid receptors in electroacupunctureproduced anti-hyperalgesia in rats with peripheral inflammation.Brain Res.2004,1020:12-17.
[5]Zhang Y.Q.,Ji G.C.,Zhao Z.Q.Kynurenic acid enhances electroacupuncture analgesia in normal and carrageenan-injected rats.Brain Res.2003,966:300-307.
[6]Zhang Y.Q.,Ji G.C.,Zhao Z.Q.Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats.Pain,2002,99:525-535.
[7]Restivo L.,Middei S.,Passino E.,Formisano R.Ammassari-Teule M.Potentiation of ischemia-related behavioral alterations by electro-acupuncture in gerbils.Funct.Neurol.,2004,19:19-23.
[8]Chen J.,Huang C.,Cheng J.S.Expression of interleukin-6 mRNA in ischemie rat brain after electroacupuncture stimulation.Acupunct.Electrother.Res.2003,28:157-166.
[9]Chiu J.H.,Chung M.S.,Ho L.T.Different central manifestations in response to electroacupuncture at analgesic and nonanalgesic acupoints in rats:a manganese-enhanced functional magnetic resonance imaging study.Can.J.Vet.Res.2003,67:94-101.
[10]Lin J.G.,Lo M.W.,Sun W.Z.The effect of high and low frequency electroacupuncture in pain after lower abdominal surgery.Pain,2002,99:509-514.
[11]熊杰,石学敏。针刺治疗急性脑梗死的临床研究近况[J]。江西中药,2004,7:60-62
[12]应赛霞 程介士。电针对沙鼠脑缺血及再灌注时脑电活动的影响[J]。针刺研究,1994,19(1):29-32
[13]Wei GW(魏光伟),Huang YL,Wu GC(吴根诚),Cao XD(曹小定)。Regulation of glial cell line-derived neurotrophic factor expression by EA after transient focal cerebral ischemia[J].Acupunct.Electrother.Res.2000,25:81-90
[14]Xu ZF(许贞峰),Wu GC(吴根诚),Cao XD(曹小定).Effect of EA on the expression of intedukin-1 13 mRNA after transient focal cerebral ischeimia[J].Acupunct.Electrother.Res.2002,27:29-35
[15]Zhao P(赵鹏),Huang YL,Cheng JS(程介士).Taurine antagonizes calcium overload induced by glutamate or chemical hypoxia in cultured rat hippocampus neurons[J].Neurosci Lett 1999,268(1):25-28
[16]Zhao P(赵鹏),Huang ZN,Chen G,Cheng JS(程介士)。Electro-acupuncture attenuates nitric oxide release from rat striatum after transient middle cerebral artery occlusion[J].Acupunct Electrother Res,2000,25(2):101-107
[17]XIONG Li-ze,YANG Jing,LU Zhi-hong,et al.Involvement of δ-and μ-opioid receptors in the delayed cerebral ischemic tolerance induced by repeated electroacupuncture preconditioning in rats.Chinese Medical Journal 2007;120(5):394-399
[18]Lucia Pagani,Luigi Manni and Luigi Aloe.Effects of electroacupuncture on retinal nerve growth factor and brain-derived neurotrophic factor expression in a rat model of retinitis pigrnentosa Brain Research,2006,1092:198-206.
[19]Su-Jin Yun,Hye-Jung Lee,Eunjoo H.Lee.Effect of electroacupuncture on the stress-induced changes in brain-derived neurotrophic factor expression in rat hippocampus.Neuroscience Letters,2002,318:85-88.
[20]陈英辉,黄显奋.电针对MCAO大鼠皮层神经营养因子表达的影响.中风与神经疾病杂志,2000,17:1392141.
[21]D.Y Zhu,J.S Wei,Y.M Lu,et al.Activation of cAMP-response-element-binding protein(CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus.PNAS 2004.101(25):9453-9457
[22]卢峻;时宇静;图娅;电针对抑郁症模型大鼠脑磷酸化cAMP反应元件结合蛋白表达的影响中国中医基础医学杂志2006,12(05):380-382
[23]Bilecki W,Hollt V,Przewlocki.Acute delta-opioid receptor activation induces CREB phosphorylation in NG108-15 cells.Eur J Pharmacol.2000;390(1-2):1-6.
[1]Mazurova Y,Rudolf E,Latr I,Osterreicher J.Proliferation and differentiation of adult endogenous neural stem cells in response to neurodegenerative process within the striatum.Neurodegener Dis.2006;3(1-2):12-8.Review.
[2]Abrous,DN,Koehl M,et al.Adult neurogenesis:from precursors to network and physiology[J].Physiol Rev,2005.85(2):523-569.
[3]Lois,C and Alvarez-Buylla A Long-distance neuronal migration in the adult mammalian brain[J].Science,1994.264(5162):1145-1148.
[4]Cameron,HA and McKay RD Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus[J].J Comp Neurol,2001.435(4):406-417.
[5]Kato,T,Yokouchi K,et al.Continual replacement of newly-generated olfactory neurons in adult rats[J].Neurosci Lett,2001.307(1):17-20.
[6]Leker,RR and Shohami E Cerebral ischemia and trauma-different etiologies yet similar mechanisms:neuroprotective opportunities[J].Brain Res Brain Res Rev,2002.39(1):55-73
[7]Shacka,JJ and Roth KA Regulation of neuronal cell death and neurodegeneration by members of the Bcl-2 family:therapeutic implications[J].Curr Drug Targets CNS Neurol Disord,2005.4(1):25-39.
[8]Parent,JM Injury-induced neurogenesis in the adult mammalian brain[J].Neuroscientist,2003.9(4):261-272.
[9]Stiene-Martin A,Knapp PE,Martin K,et al.Opioid system diversity in developing neurons,astroglia,and oligodendroglia in the subventricular zone and striatum:impact on gliogenesis in vivo.Glia.2001 Oct;36(1):78-88.
[10]Narita M.,Kuzumaki N and SuzukiT,et al.Age-related emotionality is associated with cortical δ-opioid receptor dysfimcfion-dependent astrogliosis.Neurosci,2006;137,1359-1367.
[11]Scott R,Whittemore,K.Magnuson,et al.Mitogen and Substrate Differentially Affect the Lineage Restriction of Adult Rat Subventricular Zone Neural Precursor Cell Populations Experimental Cell Research,1999;252:75-95
[12]Youzhen Wang,Jeffrey D,Macklis,et al.Cortical Interneurons Upregulate Neurotrophin sin Vivoin Response to Targeted Apoptotic Degeneration of Neighboring Pyramidal Neurons.Experimental Neurology,1998;154:389-402
[13]Pierre R.Blanquet,Betty Fournier et al.Identification of a biphasic signaling pathway involved in ischemic resistance of the hippocampal dentate gyrus Experimental Neurology,2006,202(2),357-372
[14]Kim E.H,Kim Y.J,Lee H.J,et al.Acupuncture increases cell proliferation in dentate gyrus after transient global ischemia in gerbils,Neurosci Lett,2001;297:21-4.
[15]Zeng-jin YANG,Di-Han SHENG,Feng-Yan SUN et al,et al.Electroacupunctrue enhances striatal neurogenesis in adult rat brains after a transient cerebral middle artery occlusion.Acupunct & Electro-therapeutics Res,2005;30:185-199.
[16]Xi-Bin Liang,Xiao-Min Wang,Ji-Sheng Han,et al.Long-term high-frequency electro-acupuncture stimulation prevents neuronal degeneration and up-regulates BDNF rnRNA in the substantia nigra and ventral tegrnental area following medial forebrain bundle axotomy Molecular Brain Research,2002,108:51-59
[17]Zhang RL,Zhang ZG,Zhang L,Chopp M.Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia.Neuroscience.2001;105:33-41
[18]Arvidsson A,Kokaia Z,Lindvall O.N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke.Eur J Neurosci.2001 Jul;14(1):10-8
[19]Van Kampen,Theo Hagg,Harold A.Robertson.Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation.European Journal of Neuroscience.2004,(19) 9:2377-2387
[1]Williams JT,Christie MJ,Manzoni O.Cellular and synaptic adaptations mediating opioid dependence.Physiol Rev.2001 Jan;81(1):299-343.Review.
[2]Stéphane Allouche,Ahmed Hasbi,Jocelyne Polastron,et al.Pharmacological δ1-and δ2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE:no evidence for distinct molecular entities.Biochemical Pharmacology.2000,59(8):915- 925.
[3]Xia Y,Haddad GG,et al.Major difference in the expression of δ-and μ-opioid receptors between turtle and rat brain.J Comp Neurol.436:202-210,2001.
[4]Boutin H,Jauzac P,Dauphin F,et al.Maximal densities ofμ,δ,and κ receptors are differentially altered by focal cerebral ischemia in the mouse.Brain Res.787:237-241,1998.
[5]Horiuchi T,Kawaguchi M,Furuya H,et al.The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats.Anesth Analg.2004 Jul;99(1):235-40.
[6]Borlongan CV,Su T-P,Wang Y.Delta opioid peptide(DADLE) protects against ischemia-reperfusion damage in the striatum and cerebral cortex.Sac Neurosci Abstr 24:979;1999.
[7]Zhang J,Haddad GG,Xia Y.Delta-,but not mu- and kappa-,opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.Brain Res 2000;885:143-53.
[8]Ma MC,Zhao P,Xia Y,et al.Oxygen-sensitive {delta}-opioid receptor-regulated survival and death signals:novel insights into neuronal preconditioning and protection.J Biol Chem.2005;280(16):16208-18.
[9]Hauser KF,Houdi AA,Turbek CS,et al Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro:differential impact of mu and delta receptor activation on proliferation and neufite elongation.Eur J Neurosci.2000;12(4):1281-93.
[10]Persson AI,Thorlin T,Bull C,et al.Mu- and delta-opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors.Eur J Neurosci.2003 Mar;17(6):1159-72.
[ll]Stiene-Martin A,Knapp PE,Martin K,et al.Opioid system diversity in developing neurons,astroglia,and oligodendroglia in the subventricular zone and striatum:impact on gliogenesis in vivo.Glia.2001 Oct;36(1):78-88.
[12]Eisch A J,Michel Barrot,Schad,et al.Opiates inhibit neurogenesis in the adult rat hippocampus.Proc Natl Acad Sci,2000;97(13):7579-7584.
[13]Narita M.,Kuzumaki N and SuzukiT,et al.Age-related emotionality is associated with cortical δ-opioid receptor dysfunction-dependent astrogliosis.Neurosci,2006;137,1359-1367.
[14]Djoher N.A,Muriel K,Michel L.M.Adult Neurogenesis:From precursors to network and physiology Physiol.Rev,2005;85:523-569.
[15]Arvidsson A,Kokaia Z,Lindvall O,et al.Neuronal replacement from endogenous precursors in the adult brain after stroke.Nat Med,2002;8:963-970.
[16]Robin J,Lichtenwalner,Jack M Parent.Adult neurogenesis and the ischemic forebrain.J Cereb Blood Flow Metab,2006;26:1-20.
[17]Stiene-Martin A,Knapp PE,Martin K,et al.Opioid system diversity in developing neurons,astroglia,and oligodendroglia in the subventricular zone and striaturn:impact on gliogenesis in vivo.Gila,2001 Oct;36(1):78-88.
[18]Zhang H,Watson SJ and Ko MC,et al.Endogenous opioids upregulate brain-derived neurotrophic factor mRNA through delta- and micro-opioid receptors independent of antidepressant-like effects.Eur J eurosci.2006 Feb;23(4):984-94.
[19]Mattson M.P,Duan W,et al.Modification of brain aging and neurodegenerative disorders by genes,diet,and behavior.Physiol.Rev,2002;82,637-672.
[20]Yuen E.C.Mobley W.C.Early BDNF,NT-3,and NT-4 signaling events.Exp.Neurol,1999;159,297-308.
[21]Obermeier A,Schlessinger J.and Ullrich A,et al.Identification of Trk binding sites for SHC and phosphatidylinositol 3 c-kinase and formation of a multimedc signaling complex.J.Biol.Chem.1993;268,22 963-22 966.
[22]Finkbeiner S.,Harris K.M.and Greenberg M.E,et al.CREB:a major mediator of neuronal neurotrophin responses.Neuron,1997;(19),1031-1047.
[23]Rhee S.G.Bae Y.S.Regulation of phosphoinositide-specific phospholipase C isozymes.J.Biol.Chem,1997;272,15 045-15 048.
[24]Minichiello L,Davies A.M.and Klein R,et al.Point mutation in trkB causes loss of NT4-dependent neurons without major effects on diverse BDNF responses.Neuron 1998;21,335-345.
[25]Chen P,Xie H.and Wells A.Mitogenic signaling from the EGF receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism.Mol.Biol.Cell,1996;7,871-881.