【作者】 关艳中；

【导师】 高天明；

【作者基本信息】 南方医科大学 ， 神经生物学， 2007， 博士

【摘要】 中风是最常见的危及生命的神经疾病，中风是继心脏病和癌症之后的第三位致死疾病，并且在老年人当中，它是主要的致残性疾病。在寻找药物治疗靶点的同时，预防和康复仍是主要的治疗策略，尽管这种策略减少致残和致死率效果还相对较差。大多数脑缺血源于血管问题而发生，并且由于神经元死亡而造成功能障为临床特点，因此几十年来，脑缺血的研究主要集中在脑血流、脑血管和神经元上。近些年才认识到星形胶质细胞、少突胶质细胞和小胶质细胞在缺血脑组织死亡、保护脑功能、促进神经再生等方面起着重要作用。在脑缺血中，根据抗炎化合物或一氧化氮合酶、环氧化酶2抑制剂能够减轻缺血脑损伤，并且能够改善脑缺血动物的预后，现在认为“炎症”是一种起着重要作用的机制。过去的二十几年的研究表明脑组织有内在的免疫系统，主要由小胶质细胞介导的慢性炎症，被认为是在许多神经疾病如阿耳茨海默(氏)病、帕金森(氏)病、及脑中风进行性神经元死亡中起重要作用。脑缺血导致的脑损伤一般在缺血后数小时至数天发生，尤其在全脑缺血模型，观察到海马CA1锥体神经元损伤发生在缺血后3天左右，提示有一种机制在脑缺血后对神经细胞延迟性死亡在起作用。近来的研究表明，在脑缺血后，有炎性细胞浸润到缺血脑区，并且在缺血脑区有几种致炎因子如：诱导型一氧化氮合酶、环氧化酶2及一些细胞因子大量表达。最近有人证明小胶质细胞上表达N型乙酰胆碱受体，并且此受体的激活可以抑制小胶质细胞释放几种炎性因子。根据以上研究结果提示，我们采用大鼠全脑缺血模型，用胆碱能受体激动剂通过抑制中枢小胶质细胞的炎性增殖来降低缺血再灌注引起的中枢炎症反应，从而保护海马神经元迟发性死亡。本实验做全脑缺血再灌注模型均使用200±20g雄性wistar大鼠，参照改良的Pulsinelli四血管闭塞法制作全脑缺血模型。结果发现缺血前腹腔注射烟碱组有显著的神经保护作用，使存活神经元由缺血对照组的11.9％增加到74.3％，但是这种预防作用的临床意义不理想，因为要在中风发作之前给予药物保护似乎不现实，目前临床很难准确预测中风发作的精确时机。所以，我们要探讨，在缺血后不同时间给予该药物，观察其对全脑缺血再灌注大鼠海马CA1区神经元迟发性死亡的治疗作用。当缺血后2小时给予0.5mg／kg／次烟碱腹腔注射，3次／天，给药7天观察到7只大鼠，海马CA1区神经元存活数目为114.2±17.6个／mm~2，为原有神经元数目的54.6％，差异显著。当缺血后6小时给予0.5mg／kg／次烟碱腹腔注射，3次／天，给药7天观察到6只大鼠，海马CA1区神经元存活数目为58.7±13个／mm~2，为原有神经元数目的28.1％，差异显著。当缺血后12小时给予0.5mg／kg／次烟碱腹腔注射，3次／天，给药7天观察到5只大鼠，海马CA1区神经元存活数目为42±10个／mm~2，为原有神经元数目的20.1％，差异显著。可见，全脑缺血再灌注后给予烟碱腹腔注射，给药时间越早，治疗效果越好，缺血后2小时给药可以保护半数以上的神经元免于死亡，至缺血后12小时给药仍有保护作用。为了观察烟碱是否能抑制缺血再灌注引起的中枢小胶质细胞炎反应，我们按上述时间给药，7天后观察海马CA1区小胶质细胞数目的反应，假手术组双侧海马CA1区小胶质细胞(OX-42阳性细胞)数目平均为26.5±4.5个／mm~2，缺血生理盐水组8只大鼠双侧海马CA1区小胶质细胞数目为283.4±24个／mm~2，即在全脑缺血再灌注7天时，海马CA1区活化的小胶质细胞数目为原有数目的10.7倍，当缺血后2小时给予0.5mg／kg／次烟碱腹腔注射，3次／天，给药7天观察到7只大鼠，海马CA1区小胶质细胞数目为62.9±11.5个／mm~2，为原有小胶质细胞数目的2.4倍，差异显著。当缺血后6小时给予烟碱0.5mg／kg／次，海马CA1区小胶质细胞数目为82.3±17.6个／mm~2，为原有小胶质细胞数目的3.1倍，差异显著。当缺血后12小时给予0.5mg／kg／次烟碱腹腔注射，海马CA1区小胶质细胞数目为151.3±24.6个／mm~2，为原有小胶质细胞数目的5.6倍，差异显著。可见，在全脑缺血再灌注模型中，海马CA1区小胶质细胞的数目是显著增多的，给药时间越早，对海马CA1区小胶质细胞数目的抑制作用越明显，至缺血后12小时给药，仍有较为明显的抑制作用。由此实验结果可知，烟碱对缺血再灌注引起的海马CA1区小胶质细胞数目反应性增多有显著的抑制作用，此效果具有剂量依赖性。为了进一步探讨烟碱是否能直接影响小胶质细胞的增殖过程，我们做了体外培养小胶质细胞实验。无菌条件下，取新生8只Wistar大鼠皮层做细胞混合培养，培养14天，收获前1日换液1次，轻摇培养瓶以去除贴壁未牢的细胞成分，吸出后加入新鲜培养液，此时所得细胞成分即绝大部分为小胶质细胞，经小胶质细胞特异性标记OX-42鉴定纯度为＞98％。培养一天待小胶质细胞适应条件后，即纯化后第二天，观察生长良好的细胞开始加药观察药物效果。结果显示，烟碱对原代培养小胶质细胞小胶质细胞的数目有抑制作用，此作用在加药48小时效果较好，且浓度为10uM时效果更为显著，在小胶质细胞系同样得到了类似的结果。进一步观察烟碱是否能抑制(粒细胞巨噬细胞集落刺激因子)GM-CSF引起的原代培养小胶质细胞的数目增多，结果显示，烟碱对GM-CSF引起的原代培养小胶质细胞的数目增多有抑制作用，此作用在加药48小时效果较好，且浓度为10uM时效果更为显著。我们在小胶质细胞系(BV-2)重复了上述实验，同样得到了类似的结果，接着，我们又观察了另一种胆碱能受体激动剂卡巴胆碱，对小胶质细胞系在自然情况下及GM-CSF引起的小胶质细胞的存活数目的影响，也得到了类似的效果。我们又探讨了胆碱能受体激动剂烟碱抑制小胶质细胞的数量是否是通过抑制其增殖过程实现的，用BrdU(溴化脱氧尿嘧啶核苷)掺入法观察了烟碱的上述效应，结果发现，对照组小胶质细胞的BrdU阳性率为30.7±7.9％，GM-CSF有效诱导小胶质细胞增殖，BrdU阳性率为45.8±10.5％，10nM浓度的烟碱即可明显减少GM-CSF诱导的小胶质细胞BrdU阳性细胞数，BrdU阳性率降为31.6±4.0％，随着烟碱浓度的增大，其抑制GM-CSF诱导的小胶质细胞BrdU阳性率的作用越显著，浓度增加至10uM时，对小胶质细胞的BrdU阳性率抑制最为显著，BrdU阳性率降为14.1±5.2％，烟碱浓度增加至100uM时，其抑制作用略有回升，BrdU阳性率变为15.9±4.6％。可见，烟碱能有效抑制炎性细胞因子GM-CSF诱导的小胶质细胞炎性增殖。综上所述，胆碱能受体激动剂可以减少全脑缺血模型海马神经元迟发性死亡的程度，效果显著，并且明显减少海马CA1区小胶质细胞增殖。胆碱能受体激动剂使体外培养小胶质细胞的数目减少，并且能减少GM-CSF引起小胶质细胞增殖过程，我们可以得出结论，胆碱能受体激动剂可能通过抑制小胶质细胞炎性增殖，并且保护脑缺血大鼠海马神经元迟发性死亡。这为脑中风的治疗提供了一条新的途径。更多还原

【Abstract】 Stroke is the most common life-threatening neurological disease, and is the thirdleading cause of death after heart disease and cancer, and in the elderly it is a majorsource of disability leading to institutionalization. While pharmacological therapy toreduce ischemic damage is being pursued, prevention and rehabilitation are still theonly strategies, albeit relatively inefficient; to reduce the disability and lethality of thedisease. Most forms of cerebral ischemia initiate from some sort of vascular problem,and are clinically defined by a loss of function due to the death of neurons. Fordecades, therefore, cerebral ischemia research was mainly focused on blood flow,cerebral vasculature, or neuronal cells. It is a rather recent concept that astrocytes,oligodendrocytes, as well as microglial cells, play significant roles in the demise ofbrain tissue after cerebral ischemia, in addition to protecting brain function andenhancing survival and regeneration under these conditions. Accumulating evidenceover the past two decades has indicated that the brain has an active endogenousimmune system. Chronic inflammation, predominantly mediated by microglial cells,is thought to play an important role in the progressive neuronal death seen in manyneurological diseased states, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and stroke. The activation of microglia involves a change in morphology, andexpression and release of molecules such as inducible nitric oxide synthase (iNOS)and nitric oxide (NO), cyclooxygenase (COX)-2 and prostaglandins (PG) as well asproinflammatory cytokines and reactive oxygen species (ROS).The brain damage produced by cerebral ischemia maturates over a period ofseveral hours or days. Especially in global ischemia, a delayed hippocampal damageis observed 3 days or so after the insult in CA1 pyramidal neurons, suggesting thatmechanisms that develop slowly after ischemia have a role in ischemic cell death.Recent studies have shown that inflammatory cells infiltrate the ischemic brain area(24), and several proinflammatory genes or mediators, such as iNOS,cyclooxygenase-2, and cytokines are strongly expressed in the ischemic brain.Inflammation is now recognized as a significant contributing mechanism in cerebralischemia because antiinflammatory compounds or inhibitors of iNOS andcyclooxygenase-2 reduce ischemic damage and improve the outcome of animals afterischemic insult. It has been observed that N-cholinergic receptors were expressed onmicroglia and regulated some inflammatory factors released from these cells. Beingsuggested from the above cultures, we studied whether nicotine has neuroprotectiveeffects on delayed neuronal death in cerebral ischemia rat through anti-inflammationparthway.Transient cerebral ischemia was performed by occluding 4 major arteriessuppllying the brain ,i.e.,the bolateral vertibral and common carotid arteries,modifying the original method by Pulsinelli on adult mate wistar rats(body weight200+20g). We found that pretreatment of nicotine 30 minutes before ischemia areneuroprotective against ischemic stroke. However this prevention is not suitable onclinical therapy, it is not humanistic to apply nicotine on healthy people before theyare subject to stroke. Therefore we observed nicotine’s neuroprotective effects at different time after ischemia against reperfusion.In sham operated rats, the number ofneurons in the CA1 pyramidal cell layer was 209.5±35个/mm2(n=8), whereas 7 daysafter 15-minute global ischemia,the number was decreased to 11.9% (25±7个/mm2).Nicotine-posttreated rats at 2, 6, 12 hours after ischemia had 54.6%(114.2±17.6个/mm2), 28.1%(58.7±13个/mm2), 20.1%(42±10个/mm2) of the neuron profilesleft in the CA1 pyramidal cell layer respectively. The neuroprotection wasstatistically significant in every animal group(P＜0.05), and the effect of nicotine isdose-dependent. Treatment of the same dose of saline did not provide any protection.Therefore, the earlier posttreatment of nicotine after ischemia, the betterneuroprotection. The neuroprotective effects of nicotine are still statisticallysignificant even applied 12h after ischemia.In order to determine the effect of nicotine on microglia inflammatory reactioninduced by reperfusion after ischemia, we observed the number of microglia in theCA1 pyramidal cell layer 7 days after 15-minute global ischemia. In sham operatedrats, the number of microglia in the CA1 pyramidal cell layer was 26.5±4.5个/mm2(n=8), whereas 7 days after 15-minute global ischemia,the number was increased to10.7 times (283.4±24个/mm2). Nicotine-posttreated rats at 2,6,12 hours afterischemia had 2.4 times(62.9±11.5个/mm2), 3.1 times(82.3±17.6个/mm2), 5.6times(151.3±24.6个/mm2) of the microglia profiles left in the CA1 pyramidal celllayer respectively. The anti-inflammation of nicotine was statistically significant inevery animal group(P＜0.05), and the effect of nicotine is dose-dependent. Treatmentof the same dose of saline did not provide any anti-inflammation effects. Therefore,the earlier posttreatment of nicotine after ischemia, the better anti-inflammationeffects. The anti-inflammation effects of nicotine are still statistically significant evenapplied 12h after ischemia.In order to study whether nicotine has a direct antiinflammtory effects on microglia, we performed the following experiments on cultured microglia in vitro.Microglial cultures were prepared from 10-14 day mixed primary glial culturesobtained from the cerebral cortex of 1-day-old rats, as previously described.Microglial cells, harvested from the mixed primary glial cultures by mild shaking,were resuspended in DMEM supplemented with 10 % fetal calf serum, and plated onuncoated plastic wells at a density of 1.25×105 cells/cm2. Cells were allowed toadhere for 20 min and then washed to remove non-adhering cells, the percents ofOX-42 positive cells is more than 98%. After a 24 h of incubation, the medium wasreplaced with fresh medium containing the substance(s) under study. Microglial cellswere stimulated with different concentrations of nicotine for different periods, anddetermined the microglia viability through MTT method. We found that nicotinedecreased the number of primary cultured microglia in 24 h,48 h, 72 h groupsrespectively, and the most appearent effect concentration is 10μM treated for 48 h.The similar effects of nicotine was observed on microglia cell line(BV-2). We furtherdetermined whether nicotine could decrease the number of survival primary culturedmicroglia pretreated with GM-CSF, microglial cells were prestimulated for 30 minwith different concentrations of nicotine, and then stimulated for 24 h, 48 h, 72 h inthe presence of 5 ng/ml GM-CSF. The results show that nicotine could decrease thenumber of survival primary cultured microglia pretreated with GM-CSF at differenttime points, and the most significant effect concentration is 10μM treated for 48 h.The similar effects of nicotine was observed on microglia cell line(BV-2). Then weobserved that carbachol, another cholinergic agonist, decreased the number ofmicroglia(BV-2), and the number of microglia induced by GM-CSF.At last, we determined whether nicotine decreased the number of microgliathrough inhibiting the proliferation of microglia, indicated by thymidine analog5’-bromo-2’deoxyuridine-5’-monophosphate (BrdU) positive cell proportions. We found that nicotine inhibited the proliferation of micrglia dose-dependently. Wefurther found that there are 30.7±7.9% BrdU positive cells of the DAPI positive cellsin control groups, and 45.8±10.5% BrdU positive cells of the DAPI positive cells inGM-CSF treated groups. Then BrdU positive cells decreased to 31.6±4.0% with10nM nicotine, and to 14.1±5.2% with 10μM nicotine, the most significant dose. Thedata suggested that nicotine could inhibit the proliferation of microglia induced byGM-CSF dose-dependently, which is a inflammtory factor.To conclude, Nicotine could increase the number of neurons left in the CA1pyramidal cell layer after ischemia, and decrease the number of microglia in the CA 1pyramidal cell layer 7 days after 15-minute global ischemia dose-dependently.cholinergic agonists decreased the number of micrglia, and they inhibited thespontaneous proliferation of microglia, as well as the proliferation induced byGM-CSF. All the data above suggests that cholinergic agonists are neuroprotective ondelayed neuronal death in the CA 1 of hippocampus in global ischemia rats and inhibitmicroglia activation even applied 12 hours after ischemia. Our results supplied a newmethod to cure stroke.更多还原