【作者】 叶建宁；

【导师】 李露斯；

【作者基本信息】 第三军医大学 ， 神经病学， 2007， 博士

【摘要】 研究背景:脑卒中,一直是威胁人类生命与健康的重要疾病,其中缺血性卒中,约占70-80%。据统计,全世界每年约有1500万人发生脑卒中,仅美国就有近500万,在我国该病发病率约为120～180/10万,死亡率约为60～120/10万,幸存者75%丧失劳动力,40%重度致残,每年直接或间接经济损失高达数百亿元。尤其是,当今世界及我国人口老化日趋加速的情况下,脑卒中的发病率更有日渐增高趋势,其危害性更加突出。然而,这类疾病一旦发生,可选择的治疗方法往往有限,早前阶段的预防与治疗将是主要的原则。并从目前大量的研究报告显示,慢性脑低灌注,极有可能是脑卒中、老年期痴呆等疾病早前阶段,且在中老年人群中,约2/3处于慢性低灌注状态。因此,人们也形象地称之为“威胁中老年人生命与健康的隐形杀手”!慢性脑低灌注,是一种常见的脑缺血损伤,临床上也称慢性脑血流灌注不足(Chronic cerebral hypoperfusin,CCH),是指由不同原因导致的大脑慢性、供血减少,从而引发的脑缺血、缺氧,进而出现的一系列脑功能障碍的慢性脑损伤。早期由于症状体征并不明显,脑的结构和功能可能并没有严重的损伤,因此往往并不引起病患者的重视,但实际上,大脑某些结构,如某些细胞或亚细胞成分,功能分子等可能已经产生变化,如果长期得不到控制或纠正,最终可能进展为不可逆性脑损伤。由于慢性低灌注损伤,早期往往是可逆的。由此,给该疾病的治疗及相关疾病预防带来了希望,也给相关研究带来了更多价值。但长期以来,关于脑损伤与修复,及其神经生物学研究中,神经元学说始终是主轴,胶质细胞功能及其研究受到忽略,相关研究并未得到同步进展,胶质细胞研究至少落后数十年。事实上,在大脑神经网络中胶质细胞的作用远比以往所认识的要活跃得多,重要得多。在脑缺血性损伤的研究中,以往认为神经元和灰质可能具有高度敏感性而脑白质,少突胶质细胞对缺血可能具有较高的耐受性。但目前人们逐渐对其认识有重大转变,根据分子病理学研究显示,慢性低灌注损伤极有可能是导致白质损害主要原因,特别是老年期脑白质改变的重要途径,但少突胶质细胞(Oligodendrocyte,OL)对慢性低灌注反应性、损害特点及机制研究虽逐渐受到重视,但相关研究仍然有限。因此,本研究首先通过采用双侧颈总动脉部分狭窄方法建立老年大鼠慢性低灌注损伤模型基础上,运用组织病理学、超微结构等方法对损伤后轴索、髓鞘以及少突胶质结构功能损害进行观察与评价,同时并运用免疫组织化学和免疫萤光、免疫印迹技术以及神经电生理和行为学技术,并分别观察了与大脑信息处理和传导相关的重要结构:轴突-髓鞘连结装置Ranvier’s结区的分子Caspr2表达和分布变化;动物认知与中枢传导功能变化;以及髓鞘标志蛋白MPB、PLP的变化;OPCs活化改变;并对各指标变化特点及可能机制进行了初步分析,其结果可能对临床卒中、VD等相关疾病发生、发展转归判断及其防治有重要指导作用。本研究共分三部分:一、可控性双侧颈总动脉部分狭窄致慢性脑低灌注模型建立及脑组织病理学与超微结构评价1.方法利用针线法,采用SD老年大鼠,无菌技术条件下,行颈正中分离并游离出双侧颈总动脉,在颈总动脉近心侧距颈内动脉和颈外动脉分叉处约1.5 cm,置相应型号(0.5号或不同型号)注射针头,用丝线将颈总动脉与针头紧扎,然后取出针心,致双侧颈总动脉部分狭窄,血流灌注减少,从而导致慢性低灌注,程度可控;在低灌注后2周和1、3月,行脑组织病理形态学和电镜观察。2.结果(1)模型动物,除麻醉意外,无死亡率,且稳定性和可重复性好,低灌注程度效果可靠、肯定;(2)病理组织学检查,脑组织无局限性或弥散性脑梗死,但脑组织白质纤维成分变得疏松,随时相延长,3月时纤维结构相对紊乱;细胞成分,皮层、海马神经元有缺失,CA1区明显,并与对照组比较有显著性差异(P<0.05);(3)电镜检测,慢性低灌注损伤后轴突、髓鞘、少突胶质细胞具有明显改变。①轴突内有囊泡和致密颗粒,结构模糊,有脊崩解、破坏、线粒体空化现象;髓鞘可见板层结构排列紊乱,有的出现融合,亦有髓鞘局部变形,髓鞘空泡化,髓鞘与轴突间隙增宽,部分出现髓鞘脱失,髓鞘变薄、轴索脱失。②少突胶质细胞电子密度增高,胞浆内及细胞间隙可见残余小体;③部分海马CA1神经元有损害,出现核仁消失或变小,胞浆电子密度增高,粗面内质网、高尔基体扩张,线粒体有嵴紊乱和空化,部分皱折甚至分叶,核仁增大和靠边,胞浆游离核糖体增多,有的线粒体畸形,出现包含物。二、慢性脑低灌注对老年大鼠脑组织Caspr2表达及认知与电生理功能的影响1.方法将老年SD大鼠,随机分为,老年慢性低灌注损伤组(n=15)和老年非慢性低灌注损伤对照组(即仅有手术过程,而未行颈动脉狭窄脑血流灌注处正常水平,n=12),在建模完成后于第2周、1、3月,分别对低灌注损伤组和对照组采用神经行为学,电生理学以及免疫萤光组织化学和免疫印迹方法,行认知行为(Morris水迷宫)测定、以及中枢传导功能,内嗅皮层—海马(即穿通通路,Perforant pathway)传导潜伏期测定、以及脑白质轴突-髓鞘连结装置Ranvier’s结近结侧区Caspr2表达进行定性和定量分析。2.结果(1)慢性低灌注损伤组,定位航行试验(place navigtion)逃避潜伏时较对照组明显延长(p<0.05):空间探索试验(spalial probe),穿越平台次数明显减少,路径、及第一次穿越平台潜伏期与对照组比较均有明显统计差异(p<0.05);(2)中枢传导功能,穿通通路传导潜伏期明显较对照组延长(p<0.05),并随时相延长传导速度减退愈明显。(3)白质区域,特别是胼胝体等部位密集成束神经纤维内Caspr2表达,低灌注损伤组呈明显下调(p<0.05),各时相组比较也有明显差异(p<0.05)。(4)其中,Caspr2表达定量分析并与逃避潜伏时、穿越平台潜伏期,中枢传导潜伏期有明显相关性(各相关系数检验,p<0.05)。三、慢性低灌注时老年大鼠脑组织髓鞘蛋白MBP、PLP变化及其少突胶质前体细胞的活化改变。1.方法动物组别,既设立老年慢性低灌注损伤组和老年非慢性低灌注损伤对照组,为更进一步比较青年与老年在慢性低灌注损害中的差异,同时增加设立了青年慢性低灌注损伤组和相应对照组。指标选择,同样采用免疫组织化学和免疫印迹技术方法,并进一步观察了,慢性低灌注损伤时脑白质髓鞘标志蛋白MBP、PLP的变化,以及OPCs的特异标志蛋白NG2、O4的表达和分布的变化。2.结果(1)NG2、O4阳性细胞,在青老年大鼠皮层、海马以及皮质下白质各脑区均有分布,并与O4表达分布趋于一致;但慢性低灌注损伤后,NG2阳性细胞数,青老年均呈明显减少,有统计学差异(P<0.05);青年与老年组之间比较也有差异(P<0.05),青年组NG2增殖更明显;(2)慢性低灌注损伤后,白质MBP与PLP表达均呈显著下降,并随时相延长呈渐进性表达下降。PLP免疫印迹分析,老年低灌注损伤组与老年对照组比较有显著差异(P<0.05),各时相组比较表达逐渐减弱(P<0.05)。本研究主要结论如下:1.本研究,采用双侧颈总动脉部分狭窄方法建立了老年大鼠慢性低灌注模型。该模型从形态学观察显示,具有与临床低灌注脑白质损伤影象学相一致的特性,没有明显梗死灶,却具有明确的髓鞘、轴索以及少突胶质细胞等成分的超微结构改变;且具有稳定性和可重复性好,贴近临床,作为模拟慢性低灌注损害是一种较可靠的成功模型。2.在此基础上,采用形态学和超微结构方法,重点对少突胶质细胞结构易损特点进行了进一步观察和分析,进一步证实了少突胶质细胞对慢性缺血同样具有易损性。并提示临床相关治疗既要注意神经元也要注意少突胶质的保护治疗才是最完整的治疗选择。3.本研究发现,低灌注损伤后脑白质Ranvier’s结区分子Caspr2表达明显下调,并具有与认知与中枢传导功能下降明显相关性,提示Caspr2改变可能是慢性低灌注损伤脑内传导和白质功能损害重要环节和可能的分子基础。4.本实验证实,慢性低灌注损伤时动物具有明显认知和中枢传导功能损害,随时相呈渐进性减退趋势,但病理形态学显示并无明显梗死灶。因而提示,在临床上超早期对中老年病患者进行神经心理、认知功能、诱发电位、甚至功能影象学检测可能对慢性低灌注损害程度、转归判断以及是否进展为卒中或VD可能具有预警信号作用。5.本实验证实,髓鞘蛋白MBP、PLP表达下降,提示慢性低灌注时少突胶质细胞具有明显损害;同时,也进一步提示作为髓鞘蛋白MBP、PLP可为本病评价是较敏感指标。6.本实验发现,少突胶质前体细胞(OPCs)具有明显增殖活化,且老年大鼠较青年大鼠明显减弱,推测可能为慢性低灌注损伤后的一种代偿适应或修复机制,OPCs活化并可能受老年因素影响,其具体机制值得并仍有待进一步探讨。更多还原

【Abstract】 Backgrounds:Brain stroke has been a critical disease threatening human lives and health. Of the patients with it, 70-80 % of them suffered from ischemic stroke. It is reported that the disease occurs in 15 million people in the world annually and 5 million of them are Americans. In China, its morbidity is about 120-180/100000 and the mortality 60-120/100000. A percentage of 75% of the surviving patients loss their labor and 40% of them are severely disabled. The annual direct and indirect economic loss is as high as several hundred billion yuan.In particular, the human aging of society in the world and China is increasing, thus the mobidities of stroke and senile dementia are also increasing. The prevention and treatment of stroke and senile dementia becomes a great challenge in the fields of geriatrics and neurology. Such disease is difficult to recover. Therefore, the early prevention is the major principle.Large numbers of researches show that chronic hypoperfusion injury is more likely to be the early stage of stroke and senile dementia. Meanwhile, about 2/3 of the middle-aged and aged people are in the state of it. Thus chronic hypoperfusion injury is called a potential killer for life and health of the middle-aged and aged.Chronic hypoperfusion injury, clinically called chronic cerebral hypoperfusion(CCH), refers to chronic and extensive ischemia of cerebral tissue due to various reasons resulting in cerebral ischemia and hypoxia to cause chronic cerebral injury presenting as a series of cerebral functional disorders. And it is usually reversible at the early stage, which gives hope to treatment of chronic hypoperfusion injury, and prevention of stroke and senile dementia, and brings more valuable points for studies.For a long time, researchers have paid close attention to neurobiological features, post-injury repair of nurons. Glial cells are considered to have the effects of supporting and noruishing. As such, the studies on glial cells have been ignored. In fact, the effects of glial cells in cerebral nervous network are more complicated and important than we have expected. In addition, few studies have been conducted to investigate the effects of glial cells after cerebral ischemic injury.Consequently, the model of chronic hypoperfusion injury was established in aged rats by partial narrowing of bilateral common carotid arteries. Then pathological and ultrastructural examinations were employed to observe and evaluate the damage in axon, myelin sheath and oligodendrocyte. The relevant critical strucutures of oligodendrocytes in white matter such as axon-myelin sheath connecting Ranvier’s reagion and its associated structures were observed and morphological, cognitive and behavorial as well as electrophysiological features determined to primarily study and evaluate the chronic hypoperfusion. The results of which might be of great guidance for judgment of chronic hypoperfusion injury degree and reversion and prevention and treatment of stroke and VD . This study was divided into the following 3 parts:Part I Establishment of model of chronic hypoperfusion injury by controllable partial stenosis of bilateral common carotid artery and pathological and ultrastructural evaluation of cerebral tissueMethodsThe suturing method was used and aged rats employed. Under the asepsis condition, the bilateral common carotid arteries were separated. A syringe needle was bound with the common carotid arteries at a lower place about 1.5cm to the bifurcation of internal and external carotid arteries .The syringe needle was then with drawn to cause partial narrowing of bilateral common carotid arteries and reduction in blood infusion of cerebral tissue at a controllable degree. Two weeks and 1 and 3 months after the hypoperfusion, pathomorphological and electron microscopic examinations of the cerebral tissue were performed.Results1. There was no death of the rats except for anesthesia-induced one. The model had good stability and repeatability and the hypoperfusion was of reliable results.2. Pathological examination showed that there was no focal and diffusive cerebral infarction. However, the fibrotic components in the white matter became rare. In 3 months after the chronic hypoperfusion, the fibrous components were relatively mixed. There was loss of cell compoents, in cortical and hippocampus neurons , especially CA1, and they were significantly more obvious than in the control group (P<0.05).3. Electron microscopy revealed that there were significant changes in axons, myelin sheath and oligodendrocytes. a) Vesicles and dense granule intensive particles of opaque structure, axon decomposition, destruction and mitochondrial vocuolation were seen in the axons. The layer structure of the myelin sheath was disarranged in mass. There were fusion, local deformation and vacuolation of myelin sheath, The gap increased distance between the sheath and axons widened,and partial loss of the sheath was observed.. b) The electronic density of the oligodendrocytes increased and there were corpuscles in cytoplasm and cellular interstitia. c) Part of CA1 neurons in hippocampus were damaged, which is presented as nucleolus disappearance or reduction in size, increase in electronic density of the cytoplasm, dilatation of the reticulum and Golgi body, disorder and vacuolation of mitochondria. Meanwhile, some mitochondria were malformed to have contents.Part II Effects of chronic cerebral hypoperfusion on expression of Caspr2 in cerebral tissue and cognitive and electrophysiological functions in aged ratsMethods:The aged rats were divided into the CCH group (n=15) and control group (n=12). The model of chronic hypoperfusion was established in the experimental group while no partial narrowing of the bilateral common carotid arteries was performed in the control. In 2 weeks and 1 and 3 months after hypoperfusion, cognitive and behavorial , central conductive function, perforant-pathway transduction speed, expression of Caspr2 in Ranvier’s node were determined with behavioral method, electrophysiological method, immunofluorescent method and immune blotting, respectively.Results:1.The escape latency was significantly prolonged, times of crossing platform in spatial probing markedly decreased in the experimental group as compared with the control (P<0.05) ,The first time passing hidden platform also prolonged significantly(P<0.05).2. The perforant-pathway conductive time was remarkably longer in the expenimental group than in the control (P<0.05). Meanwhile, the transduction speed was more rapidly decreased with time prologation in the experimental group.3. The Caspr2 expression in the white matter was significantly down-regulated in the experimental group as compared with the control (P<0.05). Furthermore, there was marked difference between the 2 groups at different phases.4 .The level of Caspr2 expression was significantly correlated to escape latency, crossing platform latency and central conductive latency (P<0.05).Part III Changes in expression of myelin sheath protein MBP and PLP and activation of oligodendricyte precursor cells during chronic cerebral hypoperfusion in aged ratsMethods:We established the aged CCH groups , aged CCH control groups, young experimental and young control groups. The changes in MBP and PLP levels and expression of NG2 and 04 protein were investigated.Results:1. In aged and young experimental groups, the NG2-positive cells distributed in cortex, hippocampus and subcontical white matter, which is consistent with distribution of 04 protein. However, the cells were significantly decreased in the 2 experimental groups after chronic hypoperfusion (P<0.05). There was also maked difference between the aged and young experimental group (P<0.05). The proliferation of NG2-positive cells was more obvious in the young experimental group.2. After the chronic hypoperfusion injury, the expression of both MBP and PLP in the white matter was markedly decreased and this decrease was more and more rapid along with time prolongation. PLP was significantly lower in the aged experimental group than in the aged control (P<0.05). There was significant difference between the 2 groups at different phases (P<0.05).The major conclusions in this study are as follows:1. The established model is the same as clinical cases of chronic cerebral hypoperfusion in imaging features. Meanwhile, it has good stability and repeatability and can be used to simulate chronic cerebral hypoperfusion2. The pathological and ultrastructural examinations show that the oligodendrocytes are highly sensitive and susceptible during chronic hypoperfusion injury, which suggests that both neurons and oligodendrocytes should be protected in clinical practice.3. The Caspr2 expression in Ranvier’s node is significantly decreased after chronic hypoperfusion injury and the decrease is significantly correlated to decrease in cognitive and central conductive functions, which suggests that change in Caspr2 might be the critical link to and possible molecular basis for cerebral functional damage after chronic hypoperfusion injury.4. The finding that cognitive and central transduction functions are markedly reduced after chronic hypoperfusion injury in animals suggests that very early determination of cognitive function and evoked potentials in middle-aged and aged patients are of the role of alert for judgment of severity of chronic hypoperfusion injury, and possible development to cerebral infarction and senile dementia.5. MBP and PLP expression decreased, which suggested that oligodendrocytes was marked injuried during chronic hypoperfusion. The result also showed that MBP and PLP may be used as sensitive parameter for judgeing white damage.6. There is obvious proliferation and activation of Oligodendrocyte precursor cells (OPCs), OPCs and the proliferation is remarkably reduced in aged rats as compared with the young ones, which might be a mechanism of compensation or repair after chronic hypoperfusion injury. The activation of the OPCs might also be affected by aging, the mechanism of which needs to be further clarified.更多还原