【作者】 周爱民；

【导师】 李文斌；

【作者基本信息】 河北医科大学 ， 神经病学， 2002， 博士

【摘要】 腺苷机制是解释脑预缺血(cerebral ischemic preconditioning，CIP)神经保护作用的一个重要学说，即CIP引起脑细胞合成和释放腺苷(adenosine，ADO)增加，后者与其特异性受体结合发挥对神经元的保护作用。其证据是，ADO类物质可以替代CIP诱导产生脑缺血耐受(brain ischemic tolerance，BIT)，而用DPCPX等阻断腺苷受体(adenosine receptors，AR)使CIP的保护作用消失。但是，由于存在高效的失活机制，CIP引起ADO浓度的升高，往往不能持续到损伤性缺血发生时。因此，在BIT诱导过程中AR的数量增多和活性增强可能发挥更重要的作用。为证实这一设想，本项研究在诱导大鼠海马缺血耐受的同时，应用放射性配基法观察海马细胞膜AR的变化及其与BIT形成的关系，并在此基础上采用免疫组化方法检测CIP对腺苷A1受体(A1 adenosine receptors，A1AR)和腺苷A2b受体(A2b adenosine receptors，A2bAR)表达的影响，进一步明确是何种亚型AR的变化在CIP保护机制中发挥作用。 1 全脑缺血预处理诱导大鼠海马缺血耐受的实验研究 本实验通过四血管闭塞(4-vessel occlusion，4VO)致全脑缺血大鼠模型，观察CIP持续时间和其与后续损伤性缺血间隔时间对BIT形成的影响。健康SD大鼠54只，戊巴比妥钠腹腔麻醉下颈部背侧纵切口约1.5cm，暴露第一颈椎横突，将预热的电烙针插入翼状孔凝闭双侧椎动脉。术后1d在乙醚麻醉下游离双侧颈总动脉，待动物清醒后，挟闭双颈动脉阻断血流进行全脑CIP及损伤性缺血。分为4组进行实验：①假手术组(Sham组，n=6)：只暴露双侧颈总动脉，不阻断血流。②缺血3min组(n=6)：挟闭双颈总动脉3min。③缺血6min组(n=6)：挟闭双颈总动脉6min。④缺血预处理组：动物36只，进一步分为6组，每组n=6，分别为：3min-1h-6min(挟闭双颈总动脉3min作为缺血预处理，再灌注1h后再挟闭6min，下同)、3min-12h-6min、 中文摘要3min．id．6min、3min．3d．6min、lmin．id－6min、smin．id．6min组。 挟闭颈总动脉过程中，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，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，冠状切取视交叉后1至4mm脑组织，常规石蜡切片spin厚，硫莫染色。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①级：无神经元死亡；l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人计数 CA区每 lmm区段内未死亡的锥体细胞数目，取其平均数为字经元密度（neuronal density，ND）。 结果发现，Sham组和缺血3min组海马无明显损伤，组织学分级多为0司级，CAI区ND分别为223土26、218土17。缺血6min组海马 CA、CAZ区有明显损伤，组织学分级多为 2—3级，CA区 ND为63士23，与幼am、缺血3min组相比明显升高（P＜0刀5）或降低（P＜ o．01）。缺血预处理组中，3minl 和 3min习d6min组海马损伤不明显，CA、CAZ区组织学分级与缺血 6min组相比明显降低（P＜0刀5人 CA区ND分别为 189士30和 186土32，与缺血6min组相比明显升高 （P＜0＊1）。lmin．id．6min不 3min．12h6min组海马损伤与缺血 6min组类似，CAI区ND分别为76士28、68土19，均明显低于Sham和缺血 3min组（P＜0．of）。smin－id－6min和 3min－lh－6min组海马损伤更为严重，CA、CAZ区组织学分级多为 3级，CA3和 CA4区组织学分级也呈增高趋势；CAI区ND分别为42士17、18士9，均明显低于Sham和缺血3min组（P功．of人其中后者还明显低于缺血6min组？刃．05人 以上结果提示：＊）全脑缺血3min不足以引起大鼠海马神经元损伤，而缺血 6min可引起海马 CA、CAZ区明显的延迟性神经元死亡 uelnyed neuron death，DND人C）以 3min缺血作为预处理，可以诱导大鼠海马产生缺血耐受，对间隔1或3d后6min缺血引起的海马DND产生保护作用。③ lmin和 smin CIP不能对海马神经无产生保护，提示诱导海马缺血耐受需要恰当的预处理程度（缺血3min左右人N）3minCIP不能对间隔 lh和 12h后6min缺血引起的损伤产生保护，提示CIP与后续损伤性缺血间隔过短，不足以诱导产生BIT，其适宜间隔应为1至爿左右。 2 中文摘要 二脑预缺血引起大鼠海马细胞膜腺昔受体数量和活性升高及其对神经 元的保护作用 在CIP诱导海马缺血耐受形成过程中，采用放射性配基法检测海 马细胞膜AR数量和亲和力的变化，以探讨BIT形成与N变化之间 的关系。健康SD大鼠96只，按以上手术方法凝闭双侧椎动脉并游离 双颈总动脉，分4组进行实验。①假手术组uham组，n＝6）：只暴 露颈总动脉不阻断血流，术后饲养 3d处死取材。＠单纯预缺血组u 组人动物 30只，暴露颈总动脉，间隔 id后挟闭双颈总动脉 3min，分 别于再灌注 4h（更多还原

【Abstract】 The adenosine (ADO) mechanism is an important explaining for the protective effects of cerebral ischemic preconditioning (CIP) on neurons. The protective effects are usually referred to as brain ischemic tolerance (BIT). CIP lead to increases in synthesis and release of adenosine. The released adenosine combines with its specific receptors to play protective effects on neurons. The hypothesis was supported by evidences that the protective effects of CIP might be resembled by ADO or ADO analogues and blocked by adenosine receptor (AR) antagonists such as DPCPX. Because there are efficient inactivating mechanisms for adenosine, the enhanced level of ADO induced by CIP might not last to the occurrence of the following severe ischemic insult. So increases in number and activity of AR might play a more important role in the induction of BIT. To testify the assumption, changes of AR in rat hippocampal cellular membranes in the induction of BIT were observed using the radioligand binding method. In order to clarify which kind of subtype of AR contributes to the protective effect of CIP, changes in expression of hippocampal Al adenosine receptor (Al AR) and A2b adenosine receptor (A2bAR) in the induction of BIT were further observed using immunohistochemistry.1 Experimental study on the induction of ischemic tolerance of hippocampal neurons by CIP in ratsThe effects of duration of CIP and intervals between the CIP and the following ischemic insult on BIT were investigated using rat four-vessel occlusion global cerebral ischemic model. Fifty-four healthy SD rats were used. They were anesthetized with pentobarbital by abdominal administration. An incision of skin, 1.5cm in length, directly overlying thefirst cervical vertebra was made on the dorsal side of the neck behind the occipital bone. The paraspinal muscles were separated from the middle, and the right and left alar foramina of the first cervical vertebra were exposed. An electrocautery needle burned in advance was inserted though each alar foramen and both vertebral arteries were electrocauterized and permanently occluded. Silver ball electrodes were mounted on the parietal bone to record the electroencephalogram (EEG). The both common carotid arteries of the rats were exposed at Id after the operation under light ether anesthesia. Bilateral carotid arteries were occluded with aneurysmal clip to produce CIP and ischemic insult after the rat recoveryed from the ether anesthesia. The animals were divided into 4 groups: (1) the sham-operated group (n=6), exposing bilateral carotid arteries but without blocking blood flow; (2) 3-min ischemia group (n=6); (3) 6-min ischemia group; (4) ischemic preconditioning group (n=36), which were further divided into the following 6 groups (n=6 in each group): 3min-lh-6min (3min ischemic preconditioning-Ih reperfusion-6min ischemia, the same as the following) group; 3min-12h-6min group; 3min-ld-6min group; 3min-3d-6min group; lmin-ld-6min group; 5min-ld-6min group. During bilateral common carotid arteries occlusion, the pupils enlarged and EEG showed decreases in frequency and amplitude, even approach isoelectric level, indicated the production of global cerebral ischemia. Seven days after the operation or the last time of ischemia, the animals were decapitated and a brain slice l-4mm behind optical chiasm including hippocampus were excised coronally. After fixation and dehydration, the excised brain slices were imbedded in paraffin, sectioned in 8jim thickness, and stained with Lauth’s staining. Histological changes of the hippocampus were divided into the following 4 grades under light microscope: grade 0, no neuron death; grade 1, scattered single neuron death; grade 2, death of many neurons; grade 3, death of almost complete neurons. The neuronal density (ND) of hippocampal CA1 subfield was determined by measuring the number of the surviving pyramidal neurons per 1mm linear length of the CA1.It was found that the rats in the sham-operated and 3-min ischemia groups showed no significant neuronal damage更多还原