【作者】 刘惠卿；

【导师】 李文斌；

【作者基本信息】 河北医科大学 ， 神经病学， 2003， 博士

【摘要】 脑缺血预处理（cerebral ischemic preconditioning，CIP）诱导的脑缺血耐受（brain ischemic tolerance，BIT）的产生涉及神经介质、受体及基因的表达等一系列过程。在BIT的诱导过程中，细胞在信号启动与转导，基因复制与转录，蛋白合成与修饰等环节发生相应的变化。在信号的启动环节，腺苷机制受到众多学者的重视。我室新近研究表明，BIT诱导过程中，腺苷受体的数量及亲和力均增加，进一步丰富了BIT诱导的腺苷机制的研究。但腺苷受体激动剂作为预处理对神经元所产生的保护作用较CIP所诱导的保护作用弱，说明还有其它机制参与这一过程。近年来，一氧化氮（NO）在缺血缺氧耐受诱导中的作用引起了人们的兴趣。离体研究表明，NO在神经元缺血缺氧预处理保护机制中发挥重要作用。如在离体大鼠海马脑片的研究发现，缺氧预处理可显著减少其后严重缺氧所引起的诱发电位波幅的下降，而一氧化氮合酶（NOS）阻断剂7-硝基吲哚（7-NI）可阻断CIP的这种作用，提示原生型NOS（cNOS）产生的NO参与了缺氧预处理对神经元的保护作用。近来有人应用培养神经细胞氧-葡萄糖剥夺（OGD）耐受模型，系统地研究了NO在神经元OGD耐受诱导中的作用，发现给与NOS抑制剂可阻断OGD耐受的诱导，给NO供体可替代OGD诱导耐受，表明NO的产生既是神经元OGD耐受形成的必要条件，又是充分条件。这些研究结果为研究整体状态下NO在BIT诱导中的作用提供了线索。 在新生鼠缺氧预处理模型中，应用非特异性NOS抑制剂L-NNA完全阻断了缺氧预处理对神经元的保护作用，而对神经元型NOS（nNOS）及诱生型NOS（iNOS）阻断剂7-NI和氨基胍则无效，提示<WP=5>由内皮型NOS（eNOS）产生的NO介导了保护作用。有学者对单纯全脑缺血3 min和6 min 以及6 min缺血前1天给予3 min CIP三组大鼠海马CA1区锥体细胞NADPH-黄递酶活性的表达情况进行比较，发现三种情况下NADPH-黄递酶表达的变化基本一致，即在损伤和未损伤的大鼠海马组织中均可见NADPH-黄递酶活性表达的增高，并且增高的时相也一致，说明NO的存在不一定与损伤相关联，但NO的升高是否参与整体状态下BIT的诱导，尚需进一步确定。本研究利用大鼠全脑BIT模型，观察BIT诱导过程中，NOS活性、NO生成以及NOS抑制剂L-NAME对BIT诱导的影响，在整体水平探讨NO在BIT诱导中的作用，并对NO参与BIT诱导的信号转导机制做了初步探讨。1. 全脑缺血耐受模型中缺血时间及性别对CIP保护效应的影响本实验通过观察CIP持续时间、CIP与后续损伤性缺血间隔时间、损伤性缺血持续时间及性别对CIP保护效应的影响，选择能使CIP发挥最大保护效应的时间参数建立理想的BIT模型。采用四血管闭塞法（4-vessel occlusion，4VO），制作大鼠全脑缺血耐受模型。永久凝闭椎动脉的Wistar大鼠108只，按动物性别分为两组进行实验。 1. 雄性大鼠组，分为4组：①Sham组（n=6）；只暴露双侧颈总动脉，不阻断血流；②缺血3 min组（n=6）：挟闭双颈总动脉3 min；③损伤性缺血组（n=18）：根据阻断血流时间的不同，进一步分为6 min、10 min和15 min组（每组n=6），分别挟闭双颈总动脉6 min、10 min和15 min；④CIP＋损伤性缺血组（n=24）：根据CIP与损伤性缺血时间间隔以及损伤性缺血时间的不同，分为4个亚组，每组n=6，分别为3min-3d-6min（挟闭双颈总动脉3 min作为CIP，再灌注3 d后再挾闭6 min，下同）、3min-3d-10min、3min-3d-15min和3min-1d-10min组。<WP=6>2. 雌性大鼠组，分为5组：①Sham组（n=6）；只暴露双侧颈总动脉，不阻断血流；②缺血2.5 min组（n=6）：挟闭双颈总动脉2.5 min；③缺血3 min组（n=6）：挟闭双颈总动脉3 min；④（n=12）：根据阻断血流时间的不同进一步分为6 min、10 min组（每组n=6），分别挟闭双颈总动脉6 min、10 min；⑤CIP＋损伤性缺血组（n=24）：根据预缺血及损伤性缺血时间的不同，进一步分为4组（每组n=6），分别为2.5min-3d-6min（挟闭双颈总动脉2.5 min作为CIP，再灌注3 d后再挾闭6 min，下同）、3min-3d-6min、2.5min-3d-10min和3min-3d-10min组。椎动脉凝闭术后2天，在乙醚麻醉下分离双侧颈总动脉，待动物清醒后进行挟闭，阻断血流。挾闭双侧颈总动脉期间，可观察到大鼠瞳孔散大，脑电波频率变慢，波幅逐渐缩小甚至呈等电位线，翻正反射消失，证明产生脑缺血。所有大鼠在术后或末次缺血再灌后饲养7天，断头取脑，硫堇染色，在光学显微镜下观察海马组织形态并对其组织学改变进行分级（0级：无神经元死亡；1级：散在的神经元死亡；2级：成片的神经元死亡；3级：几乎全部的神经元死亡），取双侧平均等级作为统计值。高倍镜下计数海马CA1区每1mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目，每张切片双侧海马各计数7个区段取平均数为神经元密度（neuronal density, ND）。计算以下参数对CIP的保护效应作定量分析：①保护数（protection number，PN）：CIP+损伤性缺血组与损伤性缺血组的ND值之差；②保护指数（protection index，PI）：保护数与sham组ND值之比；③增长指数（growth index，GI）：保护数与损伤性缺血组ND值之比。结果显示，雄性大鼠中， Sham组?更多还原

【Abstract】 The mechanism of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP) involves cascades of events including release of neurotransmitter, activation of receptors and gene expression. During the induction of BIT, some changes happen in the processes of signal triggering and transduction, gene duplication and transcription and protein synthesis and modification in cells. In the link of signal triggering, the role of adenosine underling the mechanism of BIT have drawn close attention of scholars. Recent researches of our lab showed that both the amount and the affinity of adenosine receptors increased during the induction of BIT, which enriched the studies on adenosine mechanism of BIT. However, the neuroprotection provided by the agonist of adenosine receptors acting as a pharmacological preconditioning in place of CIP was weaker than that provided by CIP, which suggested that there might be other mechanisms implicating in the induction of BIT. In recent years, the role of nitric oxide (NO) in the induction of hypoxic/ischemic tolerance has aroused the interests of researchers. Some in vitro studies showed that NO played an important role in the induction of neuronal hypoxic/ischemic (HI) tolerance induced by HI preconditioning. For example, a hypoxic insult could decrease the amplitude of evoked potentials of the hippocampal slices, while an anoxic preconditioning could prevent the decrease. The effect of CIP could be blocked by the nitric oxide synthase (NOS) inhibitor 7-NI. The results suggested that NO produced by the constitutive NOS (cNOS) might be involved in the protective effect of<WP=16>hypoxic preconditioning. Recently, the effect of NO on the induction of oxygen-glucose deprivation (OGD) tolerance was systematically studied in cultured neurons. It was found that the OGD tolerance of the neurons was blocked by NOS inhibitor and was induced by NO donor as a preconditioning instead of OGD. It showed that the production of NO was both essential and sufficient condition for the induction of OGD tolerance. These results mentioned above provided a clue for studying the effect of NO on the induction of BIT in vivo. In the neonatal rat hypoxic preconditioning model, the administration of non-specific NOS inhibitor L-NNA could block completely the protective effect of the preconditioning on neurons, but the administration of neuronal NOS (nNOS)-specific inhibitor 7-NI and the inducible NOS (iNOS)-specific inhibitor aminoguanidine (AG) were not effective, which indicated that NO produced by the endothelial NOS isoform (eNOS) contributed to the protective effects. Comparing the expression of NADPH-diaphorase activity of CA1 pyramidal neurons of rats subjected to 3 min ischemia as well as 6 min ischemia with and without CIP before 3 d, it was found that the changes of the expression of NADPH-diaphorase activity in the 3 groups were basically similar, such as that an increase in the expression of NADPH-diaphorase activity was seen both in the damaged and non-damaged CA1 regions, and the time courses of increased expression of NADPH-diaphorase activity among the 3 groups were coincidence, which suggested that NO production might not be related certainly to neuronal damage. However, whether the increase of NO production contributes to the induction of BIT has remained to be clarified.The present study was undertaken to explore the role of NO in the induction of BIT in vivo and the possible signaling mechanism underling the involvement of NO in the induction of BIT. For the aim, the effects of NOS activity, NO production and NOS inhibitor L-NAME on the induction of<WP=17>BIT and changes of phosphorylated-ERK1/2 (phospho-ERK1/2) expression induced by CIP as well as the effect of L-NAME on the changes were investigated in rat global cerebral ischemic model. 1. The effect of duration of ischemia and sex on the protective effect of CIP in rat global cerebral ischemic model The effects of 3 parameters, including the duration of CIP, interval between the CIP and the更多还原