【作者】 桂莉；

【导师】 郑健；

【作者基本信息】 第三军医大学 ， 神经病学， 2009， 博士

【摘要】 缺血性卒中是一类常见于中老年人的严重疾病,具有发病率高、死亡率高的特点。即使幸存,也大多遗留永久性残疾。它不仅严重威胁人类健康,还给社会和家庭造成巨大的负担。目前治疗急性脑缺血的措施主要包括早期血供的恢复与缺血神经元的保护治疗。但恢复血液再灌注促进神经功能恢复的治疗措施具有严格的时间窗限制(<3h),病人往往因为各种原因错过了最佳的治疗时机。而超过治疗时间窗再恢复血流,不但不能减轻因缺血造成的神经元损伤,反而可能因为再灌注引起的系列病理效应造成更为严重的伤害。而在缺血性脑保护方面,至今尚无确切有效的方法被临床采纳。临床研究未能证实自由基清除剂、钙离子拮抗剂和谷氨酸受体抑制剂等在动物实验证实有效的脑保护药物对脑梗死患者有效。因此探索新的治疗缺血性卒中的方法非常必要。腺苷(Adenosine,ADO)广泛存在于各种组织器官中,调节各种重要的生理功能。在中枢神经系统(central nervous system,CNS)腺苷是一种重要的神经调质,通过与其特异的受体结合参与了多种神经系统疾病的病理生理过程,如Parkinson病、Alzheimer病和睡眠障碍等。目前已明确的腺苷受体有4种亚型,即A1R、A2AR、A2BR和A3R。其中,A1R、A2AR与腺苷的亲和力最强,且在脑内的分布及数量也远远超过其他两种受体。动物实验研究发现在局灶性脑缺血发生后,脑内腺苷水平迅速升高。因此推测腺苷及其受体在缺血性脑损伤的发生中扮演着重要角色。研究表明当腺苷A1R被激活时对脑缺血具有神经保护作用,但激活A1R后严重的外周效应,如镇静、心动过缓、低血压等却限制了它在脑缺血治疗中的应用。近期研究发现,阻断腺苷A2A受体对急性缺血性脑损伤具有显著的神经保护作用,减小脑梗死体积,促进神经功能恢复。但其具体机制仍未明确。对A2A受体的研究发现,无论在生理或缺血状态下腺苷或A2AR模拟剂作用于神经元突触前膜上的A2A受体能增加谷氨酸释放进入突触间隙。此外,腺苷或A2AR模拟剂作用于小胶质细胞上的A2A受体可以促进其增生和活化,促使炎症因子释放增多,从而增强局部炎症反应。因而推测阻断A2A受体对缺血性脑损伤的保护作用机制可能包括:(1)抑制谷氨酸的释放;(2)减轻局部炎症反应而减轻缺血触发的炎性损害。遗憾的是,至今罕有直接证据证明阻断腺苷A2A受体后产生的神经保护作用与抑制了谷氨酸的过度释放有关,更不明确对谷氨酸释放的影响发生在缺血期还是再灌注期。也不明确阻断腺苷A2A受体对局部炎性过程的影响是否减轻缺血性脑损伤。本研究观察A2AR基因敲除对脑缺血及再灌注诱发的脑内氨基酸递质释放的影响,以及对脑缺血/再灌注后脑内炎性反应的调控及其与脑损伤的关系,以探讨阻断腺苷A2A受体对缺血性脑损伤的保护作用机制。方法:引进腺苷A2AR基因敲除(A2AR gene knockout,A2AR/KO)杂合子小鼠交配繁殖,PCR法鉴定A2AR/KO小鼠和野生型(wild-type littermates, A2AR /WT)小鼠。参照Huang等方法制备大脑中动脉局灶缺血模型(middle cerebral artery occlusion, MCAo)。分别测定A2AR/KO小鼠与A2AR/WT小鼠在缺血期与再灌注期神经功能缺损程度;甲酚紫染色(Cresyl violet stain,CV stain)结合图像分析方法测定脑梗死体积,观察A2AR缺失对缺血期及再灌注期脑梗死体积的影响。运用活体微透析技术动态收集两组小鼠在缺血前、缺血中及再灌注后纹状体区细胞外液,并结合改良的高效液相色谱荧光法(high performance liquid chromatography - fluorescence ,HPLC-FL)检测微透析液中谷氨酸、天冬氨酸、gamma-aminobutyric acid (GABA)与甘氨酸浓度。免疫组化与免疫荧光染色技术观察腺苷A2AR缺失对缺血/再灌注后梗塞周边区小胶质细胞与星形胶质细胞数量与形态变化的影响。结果:1.稳定地建立了小鼠MCAo模型,建模成功率为90%,建模后神经功能评分在1-3分的小鼠所占比例为91.1%。经2,3,5-三苯基氯化四氮唑(2,3,5-Triphenyltetrazolium Chloride,TTC)染色或CV染色证实MCAo后评分在1-3分的小鼠在缺血侧MCA供血区脑梗死部位与范围较固定。2.建立了一种稳定的能同时测定脑组织细胞外液中多种氨基酸的HPLC-FL方法。该方法能在18min内完成样品检测,快速、有效地将样品中谷氨酸、天冬氨酸、GABA、甘氨酸等多种氨基酸类物质分离,出峰好、重复性强、精密度高。3.脑缺血及再灌注后缺血中心区细胞外液谷氨酸、天冬氨酸、GABA和甘氨酸的水平两度升高,形成“双峰”样过度释放。4.腺苷A2AR缺失抑制脑缺血诱导的氨基酸的释放。A2AR缺失减轻缺血造成的神经功能缺损(与A2AR/WT组比较,P<0.01),减小缺血2h脑梗死体积(与A2AR/WT组比较,P<0.01)。5.腺苷A2AR缺失抑制再灌注诱导的氨基酸的再次过度释放。A2AR缺失减轻再灌注后神经功能缺损程度(与A2AR/WT组比较,P<0.05),减小再灌注22h脑梗死体积(与A2AR/WT组比较,P<0.01)。6.腺苷A2AR缺失抑制脑缺血/再灌注后局部胶质细胞反应,抑制小胶质细胞与星形胶质细胞的增生(与A2AR/WT组比较,P<0.01),抑制胶质细胞形态的活化样改变。结论:1.本实验建立了一种一次性检测脑组织细胞外液中多种氨基酸类神经递质的HPLC方法。该方法使样品中多种氨基酸类神经递质的检测更加便捷、灵敏、重复性好,不仅缩短了检测耗时,还降低了成本。该检测方法值得在科研及临床中推广。2.腺苷A2AR缺失对缺血性神经元损伤的保护作用是通过抑制缺血及再灌注诱导的谷氨酸等氨基酸类神经递质的过度释放实现的。该保护作用不仅发生在缺血急性期,还发生在再灌注期,能分别减轻急性缺血期及再灌注后神经功能缺损程度与脑梗死体积。3.腺苷A2AR缺失对缺血性神经元损伤的保护作用还归因于抑制了缺血/再灌注后局部的炎症反应。抑制了缺血后局部胶质细胞反应,使小胶质细胞与星形胶质细胞的增生程度降低。对局部炎症细胞的抑制作用是A2AR缺失减轻缺血/再灌注导致的脑损伤的另一重要原因。4.本研究为开拓缺血性脑损伤的神经保护新途径与作用靶点奠定理论基础;为腺苷A2AR抑制药物进行临床研究选择给药时机奠定基础。更多还原

【Abstract】 Ischemic apoplexy, a serious disorder, is of high morbility and fatality. According to epidemiological analysis, most of the survivors have remain permanent disability. Nowadays, recovery of the blood fluid at the ealier phage and protection of the ischemic neuron are the main strategies in in the treatment of cerebral infarction (CI ) . However, as the reperfusion of the blood fluid is limited by the time window (<3 h ), the patients always lossed the optimal opportumity for therapy, which could aggravate the injury induced by the reperfusion beyond the time window. Up to now, limited efficiecy measures have been applied in clinic to protect the ischemic neuron from injury. The brain protecting drugs such as calcium ion antagonist and glutamate receptor antagonist were failed in clinic trails. Therefore, it is urgent to explore a new strategy for curing ischemic apoplexy.Adenosine is a ubiquitous homeostatic substance released from most cells, including neurones and glia. Once in the extracellular space, adenosine modifies cell functioning by operating the receptors. The adenosine is also serves as a neuromodulator under physiological conditions in central nervous system (CNS ), such as Parkinson disease, Alzheimer disease, Dyssomnia, epilepsy, cognitive disorder, depression, anxiety and so on. Extracellular adenosine acts through multiple Gprotein coupled receptors (adenosine receptor subtypes A1,A2A, A2B and A3) to exert a variety of physiological effects. Adenosine levels rise markedly in response to ischemia, which has long been known to act predominantly as a neuroprotectant under this condition.Recent studies have demonstrated that the inactivation of adenosine A2A receptor (A2AR) protects against brain injury caused by cerebral ischemia in various animal models. In these previous studies, pathohistological lesions were assessed after reperfusion or after 24 h persistent focal cerebral ischemia. Therefore, it is still unclear whether A2AR blockade protects against ischemic neuronal injury during acute ischemia, reperfusion or both stages.The underlying neurochemical mechanism remains to be determined. A2AR activation may promote glutamate release in the brain under both normoxic and ischemic conditions, facilitate the inflammatory activities of inflammatory cells. Therefore, we infer that the beneficial effects of A2AR inactivation on ischemic brain injury attributed to reduced glutamate outflow and inhibitory inflammatory activities after ischemia and reperfusion.However, few direct evidence has been obtained to determine the effect of the A2AR inactivation on glutamate outflow and glia reaction in cerebral ischemia. In this study, we critically evaluated the effect of A2AR inactivation on ischemic brain injury in both the ischemic and reperfusion stages using an A2AR knock-out mouse (A2AR-KO) model. In addition, we observed the glia reaction at the delay injury stage after reperfusion, in order to reveal the underly mechanisim of the the neuroprotective effect of the A2A adenosine receptor deficiency.Method: The PCR protocol used to genotype the mice, A2AR gene knockout mice(A2AR-KO) and wild-type littermates(A2AR /WT). Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCA) with an 8-0 nylon monofilament with a silicon-coated tip to establish the MCAo model according to Huang, et al. Evaluating the neurological deficits at ischemic phase and reperfusion phase respectively. Measuring the infarction volumes of the A2AR/KO mice and A2AR/WT mice by Cresyl violet(CV)staining and image analysis system, in order to investigate the impact on the infarction volumes at ischemic stage and reperfusion stage by the A2AR/KO. Collecting the extracellular fluid of the two groups by microdialysis before the ischemia and during the ischemia/reperfusion, then determining the glutmate, aspartate, gamma-aminobutyric acid (GABA) and glycine content by high performance liquid chromatography -fluorescene detection (HPLC-FL). Immunohistochemical staining was used to observe the glia cells quantity and morphologic changes influenced by the inactivation of the A2AR.Result: 1. The mice MCAo model was established stably. The successful rate of execution was 90% and the neurological deficit scoring of 1-3 grades was 91.1% of all these MCAo-mice. After the 2,3,5-Triphenyltetrazolium Chloride(TTC)and CV staining, it was proved that the infarction degrees and areas after the MCAo were stability with neurological deficit scoring of 1-3 grades.2. Establishing a reliable HPLC-FL detecting technique, which could separat and determin multi-amino acids including glutmate, aspartate, GABA, glycine and so on within 18 min. The modified HPLC-FL was a more quick , reliable and sensitive method to measure the amino acid neurotransmitters in brain.3. The extracellular concentration of glutmate, aspartate, GABA and glycine increased in ischemia phase and reperfusion phase respectively, which became a“double-peaks”excessive release.4. Adenosine A2AR deficiency reduced ischemia-induced amino acid neurotransmitters excessive release in striata. A2AR deficiency ameliorates neurological deficit (P<0.01 compared with the A2A/WT), and attenuates cerebral infarct volume (P<0.01 compared with the A2A/WT) after transient MCAo.5. Adenosine A2AR deficiency reduced the excessive release of these amino acid neurotransmitters once more induced by reperfusion. A2AR deficiency ameliorates neurological deficit (P<0.05 compared with the A2A/WT), and attenuates cerebral infarct volume (P<0.01 compared with the A2A/WT) after 22h reperfusion.6. Adenosine A2AR deficiency inhibited the glial cells responding after ischemia/reperfusion. inhibiting the hyperplasy of the microglial cells and astrocytes, the number of positive CD11b and GFAP significantly reduced in A2AR/KO group (P<0.01 compared with the A2A/WT), (P<0.01 compared with the A2A/WT), and inhibiting the morphological change responding to activation.Conclusion:1. The modified HPLC technique is a ideal measuring method could detect multi-amino acid neurotransmitters in the samples in one time, which improve the sensitivenes and reproducibility, save the time, cut down the cost. 2. Adenosine A2AR deficiency inhibits ischemia- and reperfusion-induced excessive release of glutamate and other amino acid neurotransmitters in striata, protects against ischemic cerebral injury in both ischemia and reperfusion phases, ameliorating the neurological deficit and attenuating the cerebral infarct volumes at last.3. Blockading the adenosine A2AR could inhibit the glial cells responding induced by ischemia/reperfusion, depressing the hyperplasy degree of the the microglial cells and astrocytes. Inhibition of the inflammatory cells could be an other important reason to attenuating ischemic cerebral injury by the A2AR inactivation.更多还原