【作者】 邢岩；

【导师】 崔丽英；

【作者基本信息】 中国协和医科大学 ， 神经病学， 2007， 博士

【摘要】 背景和目的:1990年Kitagawa等发现全脑1次短暂缺血即缺血预处理后并未引起明显的脑神经损伤,但对再次致死性缺血损伤产生了明显的保护作用,这种保护作用即为脑缺血耐受(Ischemic Tolerance,IT),第一次亚致死性短暂缺血即为缺血预处理(Ischemic preconditioning,IP)。脑缺血耐受现象的发现引起了人们很大的兴趣,因为理解其机制有助于开拓预防缺血性神经元损伤的新措施。近年来发现许多预处理可以诱导脑缺血耐受包括非致死性亚低温、非致死性缺血、亚致死性缺氧或高压氧和扩散性抑制,但这些预处理方法大部分会对机体造成潜在的损伤。寻找无创安全能有效用于临床的诱导脑缺血耐受方法至关重要。经颅磁刺激(TMS)临床上已成为研究中枢和周围神经运动传导通路的重要电生理手段。近几年开展的经颅重复磁刺激(rTMS)是将一定频率和强度的磁脉冲以成串刺激的方式以一定间隔连续发放。由于其独特的生物学效应及无创安全的优点,目前正逐渐用于治疗方面的研究。除了对重症抑郁和运动障碍等神经精神疾病有确切疗效外,近几年多项研究还表明不同频率和强度的rTMS能促进脑梗死后患肢运动、语言、时空间忽略、学习记忆等功能的康复及抑郁情绪的改善。本研究室既往动物实验研究也初步表明rTMS对缺血后再灌注脑损伤有明显的保护作用。因此,设想rTMS有可能成为研究探讨脑缺血耐受现象安全方便、行之有效的优良工具。基于这样的设想,目前有日本学者动物实验研究探讨了rTMS预处理诱导脑缺血耐受的作用,发现rTMS预处理可增加脑缺血后海马神经元缺血耐受能力,减轻后继的海马神经元迟发性缺血性损伤,但诱导脑缺血耐受的机制未做进一步探讨。而且该研究是应用沙土鼠(Gerbil)制作全脑缺血模型,因沙土鼠缺少脑底动脉环(Willis环),解剖特点与人类相差较大。相比之下,SD大鼠有完整的Willis环,与人类脑血管构成相似,因此本实验应用SD大鼠制作一侧大脑中动脉局灶脑缺血模型,模拟人类缺血脑卒中的过程,通过观察rTMS预处理对大鼠大脑中动脉梗塞90min再灌流损伤后早期脑梗死体积、神经功能及脑血流的影响,以探讨rTMS预处理应用于各种原因造成的急性脑缺血损伤的预防治疗的可能性。方法:成年健康雄性SD大鼠15只,其中实验组随机取10只各分5只入rTMS预处理5次组和1次组,另5只入对照组。预处理与右侧大脑中动脉栓塞(middlecerebral artery occlusion,MCAO)90min再灌流模型造模之间间隔为48h。rTMS1次组制作缺血再灌流模型前48h给予1次rTMS预处理,5次组予造模前2～7天每天1次,连续5天共5次rTMS刺激预处理,对照组造模前48h给予rTMS1次假刺激。各组于再灌流24h和72h给予神经功能评分,最后1次评分后处死取脑TTC染色测算脑梗死体积。比较再灌流损伤不同时间的神经功能评分及再灌流72h后的脑梗死体积的变化,分析rTMS预处理对这些观察指标的影响。另外,本实验还取15只SD大鼠应用激光多普勒血流系统(laser doppler flowtry,LDF)观察了rTMS刺激对正常健康大鼠脑血流(cerebral blood flow,CBF)的影响,以观察rTMS对大鼠神经功能的影响是否与干扰脑血流变化有关。结果:本研究发现MCAO90min再灌流损伤使SD大鼠大脑出现局灶性梗死灶以及不同程度的神经功能障碍。而rTMS预处理可减轻大鼠MCAO90min再灌流损伤的神经功能障碍程度,缩小梗死体积。rTMS预处理5次组优于1次处理组。rTMS刺激后刺激侧和对侧脑血流均有轻微短暂升高,但刺激前后CBF变化无显著统计学差异,未见引起脑血流下降现象。结论:本实验结果提示rTMS预处理有助于减轻急性期脑缺血再灌流损伤,对急性期缺血脑组织有保护作用。rTMS可能通过诱导脑缺血耐受减轻缺血后脑梗死体积和神经功能障碍程度。rTMS诱导脑缺血耐受的作用可能有累积效应。rTMS未引起脑血流下降,故其诱导脑缺血耐受的机制不同于经典的亚致死性脑缺血预处理。有必要进一步探索其诱导脑缺血耐受的分子及病理生理机制。创新点:首次观察了连续rTMS预处理对大鼠局灶性大脑中动脉梗塞再灌流模型的神经功能和脑梗死体积及脑血流的影响。背景和目的:明确缺血耐受的机制有助于寻求预防缺血性脑损伤的新方法,但预处理诱导脑缺血耐受的确切机制还不完全清楚。研究表明短暂局灶性脑缺血预处理并不能使再次缺血时脑组织能量代谢及血流增加,蛋白合成抑制剂CHX能明显抑制缺血预处理诱导的脑保护作用,认为脑缺血预处理引发或改变了基因的转录过程,导致脑组织基因表达的变化,引发一系列基因的转录和翻译,经24h诱导才能合成具有保护作用的蛋白质。许多重要的分子和信号通路可能参与了预处理诱导的脑缺血保护。既往研究证实即早基因cFos对缺血等外界刺激可迅速作出反应进行表达,是反映神经细胞功能状态的敏感指标,在神经细胞分化和神经系统可塑性方面扮演着重要的角色。cFos的表达与脑缺血具有密切关系,而且cFos的高表达早于细胞凋亡的形态学和生化学变化之前。cFos表达可能为细胞凋亡的前兆。热休克蛋白是一类广泛存在于原核及真核细胞内,在受热、缺血、缺氧、感染等各种应激因素作用下发生热休克反应而产生的一类新的蛋白质,又称应激蛋白。HSP在蛋白质转位、折叠等生理过程中充当了分子伴侣的角色,并且通过对变性蛋白的再折叠参与了细胞修复过程。其中研究最多的HSP70是HSP70-KDa家族中结构保守且应激后生成最多的一类。局灶缺血研究已经证实半暗带中的HSP70诱导表达,可在代谢压力状态逆转后促进蛋白复性从而起内源性保护作用。血管内皮生长因子(vascular endothelial growthfactor,VEGF)由Ferrara等在牛垂体滤泡星状细胞体外培养液中首先纯化出来又称血管通透因子(vascular permeability factor,VPF),是一种内皮细胞特异的有丝分裂原,通过与其受体结合而发挥促进内皮细胞增殖,加速新生血管形成,提高血管通透性等生物学特性。我们第一部分实验的结果提示高频高强rTMS预处理可改善局灶脑缺血再灌流损伤后的神经功能状态,减小脑梗死体积,但其机制尚不明确。故此部分实验继续利用SD大鼠大脑中动脉梗塞再灌流模型为观察对象,探讨了rTMS预处理对cFos蛋白、HSP70蛋白及VEGF的影响,以期从分子水平进一步了解rTMS预处理诱导脑缺血耐受的作用机制。方法:采用免疫组化染色定性和蛋白印迹法半定量技术,由分子水平探讨不同rTMS参数预处理对大鼠脑缺血再灌流损伤后cFos基因、热休克蛋白(HSP70)和血管内皮生长因子(VEGF)阳性神经细胞及蛋白的表达影响。将大鼠按照不同的不同的预处理时间和次数分组,制作MCAO再灌流模型前48h和2～7天分别给予不同参数组合的rTMS刺激,分别于再灌流后3h和24h处死取脑,测定上述指标的变化,并观察不同预处理时间和次数及不同的rTMS刺激参数对这些指标的影响。结果:本实验发现MCAO大鼠缺血再灌流损伤后3h缺血侧脑组织大量cFos蛋白表达,再灌流24h时缺血侧脑组织多量HSP70和少量VEGF表达,均显著高于假手术组。高频高强rTMS预处理能显著下调cFos蛋白表达,使缺血区cFos表达量减少并上调HSP70和VEGF表达。高频低强预处理对上调HSP70和VEGF的表达也有较弱的作用。高频高强5次预处理作用优于1次处理。低频低强处理对上述蛋白的调节未见明显作用。结论:一定频率的rTMS刺激预处理可对后发的局灶缺血脑组织提供神经元保护作用,作用途径可能与调节缺血继发的cFos、HSP70及VEGF蛋白表达有关。高频作用显著,而当刺激频率一定时,高强度刺激较低强度刺激产生的上述效应更明显。rTMS刺激对蛋白表达的调节可能有比较弱的生物学累积效应,需要进一步观察寻求最佳诱导刺激时间总量。此结果为进一步探讨rTMS预处理诱导的对脑缺血损伤的神经保护作用提供了一些基本依据。创新点:首次观察了rTMS刺激预处理对大鼠大脑中动脉梗塞再灌流损伤后蛋白表达的影响,探讨了其基本作用机制。发现一定频率的rTMS刺激预处理对缺血早期神经元可提供保护作用,可能与调节缺血继发的相关蛋白表达有关。更多还原

【Abstract】 Background and purpose: In 1990 Kitagawa found that transient ischemia of whole brain not only didn’t lead to obvious injury on neurons but also conspicuously prevent from subsequent lethal ischemic damage.This phenomenon is called as ischemic tolerance(IT) and the first sublethal transient ischemia is named as ischemic preconditioning(IP) . The phenomenon of ischemic tolerance is of considerable interest because understanding its mechanism could lead to new treatments for preventing ischemic neuronal damage. Data from subsequent studies have revealed that other manipulations besides sublethal ischemia also induce ischemic tolerance, including hypo-oxygen or hyperbaric oxygen, hypothermia or hyperthermia, chemical drugs, cortical spreading depression (CSD) .Although several manipulations may induce tolerance, most are invasive or have the potential for causing direct neuron injury or initiating molecular processes that are also engaged following injury. In terms of potential clinical applications, it is considerably important to develop techniques for inducing ischemic tolerance without causing injury or dysfunction and ideally using noninvasive manipulations.At the basis of the principle of the electromagnetic induction, the transcranial magnetic stimulation (TMS) has been a kind of important electricalphysiological examine methods clinically. As a new noninvasive and good tolerance technique, the volunteers and the patients can accept TMS easily. Repetitive transcranial magnetic stimulation (rTMS) emerged in the last decade which is the development of single pulse TMS and can produce trains of magnetic stimulation with certain intensity and frequency,then affect the functional state of the local stimulus and remote sites. Owning to its specific biophysiology characteristic and the perfect merit of no pain, noninvasive, safe and good tolerance, the technique of rTMS has been applied gradually to therapeutic fields .Besides the ascertained improvement effects on major depression or other dyskinesia diseases, rTMS has been recently noticed to be worthy in improving ischemic vascular disorders including limb movement founctions , language capabilities, visual and spatial sensory, studying and memoring damage. The past studies of our laboratory have also showed that rTMS could affect the pathophysiological conditions of ischemic stroke and could protect the ischemic neurons.So we had consideration for applying the safe and useful rTMS technique to exploring the phenomenon of ischemic tolerance. Altough studies in Japanese have been noticed that rTMS induces ischemic against DND of hippocampal neurons following an otherwise lethal ischemic insult, the mechanism of rTMS wasn’t be explored.Morever that study used gerbils as experimental model, which haven’t Willis circles intracalvarium .In contrast, SD rats have intact Willis and similar structure with human’ cerebral blood vessel.So the present study demonstrate the impact of rTMS pretreatment on the infarct volume and neurofunction at the acute stage of middle cerebral artery occlusion and reperfusion injury using SD rats as focal cerebral ischemic model and mimicking the stroke course in humans.Methods: SD rats were used as research model. At 48h after rTMS were applied to them, the rats were introduced to middle cerebral artery occlusion for 90min and then reperfusion; The control group was set with sham rTMS treatment; other experimental conditions were just the same as the rTMS group. At 24 h, 72 h after the reperfusion, the extent of modified neurological severity scores(NSS) were evaluated. At 72h after reperfusion,the rats were decapitated rapidly and infarct volumes were measured. Changes in these parameters and effects of rTMS preconditioning on these parameters were analyzed. Cerebral blood flow(CBF) effected by rTMS was also measured using Laser Dopplar Flowery(LDF) in present study.Results: The results showed that ischemic reperfusion injury produced distinct focal infarction and neurological deficits, which presented with the growing of neurofunction scores, and deteriorated along with the prolongation of reperfusion time. And rTMS preconditioning could reduce neurological scores as well as infarct volume, and improve neurofunction. Especially at 72 h after reperfusion, the neurological scores of rats receiving rTMS were statistically better than the control group. The change of CBF effected by rTMS was little and not obvious.Conclusions: These results suggested that rTMS preconditioning may provide important neuroprotective role in acute stage of cerebral ischemic reperfusion injury, and should be explored further for its corresponding mechanisms in order to find new assistant therapeutic strategy for ischemic stroke. On the other hand, as the animal sample in this study is limited, and some methods should be perfected further, the conclusion should be validated further.Innovation: It is the first time that we investigate the effects of rTMS preconditioning on the rats motor cortical excitability and neurofunction and the infarction in acute stage ofthe middle cerebral artery occlusion reperfusion injury. Background and purpose: Up to date,much ischemic tolerance phenomenon has been found, however the certain mechanism hasn’t been fully understood.Some studies showed that transient ischemic preconditioning couldn’t increase the energy metabolism and blood flow during the subsequent severe ischemia.Protein synthesis inhibitor CHX could surely inhibit the protection iuduced by ischemic pretreatment, which suggested that preconditioning may leading to serial gene transcription and translation by inducing or changing the course of transcription , and finally synthesize protective protein after 24h.Many important molecular and signal paths may involveled in the brain protective fouction induced by preconditioning.It has been noticed that early gene cFos is an sensitive index reflecting nerve cell fountional status which could present instant response to outside stimulation and play an important role in nerve cell defferentiation and nervous system plasticity. The expression of cFos has close relation with brain ischemia.Moreover the high expression of cFos emerges before the change of morph and biochemics of cell apoptosis, and the expression of cFos may be the premonition of apoptosis.Hot shock protein(HSP) is a kind of new proteins which generally reside in prokaryotic and eucaryotic cells and may be induced by stress such as hot, ischemia and hypoxygen, so named as stress protein.HSP may act as molecular chaperones during protein translocation and folding which participate in cytothesis through redoubling of degenerative protein. Of HSP70-KDa family , HSP70 has conservative structure and is the maximum produced after stress.Studies for focal ischemia have showed HSP70 induced in penumbra could promote protein renaturation after motabolism stress status retroconversing and act as endogenous protective fouction. Vascular Endothelial Growth Factor (VEGF) was firstly purificated from the culture fluid of stellate cell in cattle pituitary gland follicle by Ferrara et al which is also named as vascular permeability factor (VPF) .VEGF is a kind of specific mitogens in endothelial cell, which could promote endothelial cell proliferation through integrating with its receptors.Thus it could accelerate the neovascularization and enhance thevasopermeability.Methods: The rats were randomly assigned to the two groups according to different reperfusion time.The rats in experimental groups were given rTMS stimulation with different stimulus parameters for one time or five time.At 48h After the last rTMS were applied to them, the rats were introduced to middle cerebral artery occlusion for 90min and then reperfusion; The control group was set with sham rTMS treatment; other experimental conditions were just the same as the rTMS group. At 3h or 24 h after the reperfusion, the rats were decapitated to determine the protein expression level of cFos or HSP and VEGF respectively using immunohistochemistry staining and western blotting in order to evaluate the effects of rTMS preconditioning on molecular level.Results: The results suggested that different amount of expression of protein cFos , HSP70 and VEGF in ischemic area after ischemic reperfusion injury and microamount in sham operation groups. rTMS preconditioning with different stimulus parameters could produce different effects on the expression of protein above-mentioned. Compared with the intensity, we found that the stimulus frequency could arise much apparent effects, and high frequency (20Hz) especially in contrast to the low frequency (5Hz). High frequency could decrease the expression of cFos positive neurons and opposite in terms of HSP70 and VEGF positive ones. When the frequency is fixed, the effects of high stimulus intensity (2MT) would be more distinct than the low intensity (1.2MT). These impacts would be more evident with rTMS stimulition time.Conclusions: Our experiments primarily suggested that the rTMS preconditioning with high frequency (20Hz) could provide neuroprotection in the subsequent acute stage of cerebral ischemia reperfusion injury; and when the rTMS frequency is limited, we found that the higher the stimulus intensity, the distinct its effects.Morever the effect of rTMS on protein expression probably has time cumulative effect. And the corresponding mechanisms of inducing ischemic torelance by rTMS may involve with protein expression modulating. These conclusions may provide some basic evidence on the influence of rTMS preconditioning on the subsequent acute stage of ischemic stroke.Innovation: It is the first time that we explore the effects of rTMS preconditioning on protein expression after the rats middle cerebral artery occlusion reperfusion injury, and investingate its corresponding mechanisms. We found that the rTMS pretreatment could provide neuroprotection at the early stage of the cerebral ischemia reperfusion injury; the underlying mechanisms may involve the protein expression modulating.更多还原