【作者】 黄勇华；

【导师】 张微微；

【作者基本信息】 第三军医大学 ， 神经病学， 2009， 博士

【摘要】 研究的背景和目的:脑白质疏松(leukoaraiosis, LA)由Hachinski于1986年首先提出,系指脑室周围或皮质下区(半卵圆中心) CT上的弥漫性低密度带或MRI T2加权像上的弥漫性高信号。白质疏松主要发生于反复低灌注区域的脑组织,一般为弥散性改变,也称为白质改变(white matter change, WMC)、白质损害(white matter lesions, WMLs)或白质高信号(white matter hyperintensity, WMH)等。组织学上表现为节段性髓鞘脱失、反应性星形细胞增生,部分部位可发现轴突断裂和神经胶质细胞残骸,伴有巨噬细胞吞噬碎片现象。白质疏松是认知功能损害的特征性改变之一。近期的一项研究表明,WMH与认知、步态、精神和排尿障碍相关。伴LA的痴呆患者痴呆程度更严重,且痴呆程度与LA程度明显相关,说明LA可能与智能有关。另外,老年人LA与神经心理障碍相关,发生脑血管意外的危险性明显增加,伴有LA的患者发生急性脑梗死进行溶栓治疗时出血转化的几率比无LA者明显增加。脑白质疏松可见于65岁以上四分之一的人群,但其确切病因及发病机制尚未阐明。缺血性脱髓鞘和小血管病是脑白质疏松发病机制的两个重要学说。有研究发现,高血压动脉硬化与LA密切相关。Wiszniewska等认为,长期高血压、糖尿病可能会造成脑内小动脉硬化和狭窄,脑血流下降时白质区易发生缺血性改变。血管周围引流通路是将组织间液(interstitial fluid, ISF)和溶质从脑中清除的有效淋巴引流途径。理论模型表明组织间液和溶质经血管周围引流的动力来自于血管搏动。由于年龄的增长,血管逐渐硬化,动脉博动幅度减小,从而影响脑白质组织间液从血管周围引流通路的回流。组织间液的引流受阻也可能参与引起脑白质的液体增多(白质疏松),从而减少脑白质的灌注储备。近年越来越多的研究表明,炎症反应是脑缺血后继发性损害的机制之一。脑缺血后的炎症反应是一个级联放大过程,脑缺血后缺血灶内及其周围存在明显的炎症细胞浸润、黏附分子、细胞因子和化学增活素表达增加,同时外周白细胞、C-反应蛋白和各种细胞因子,如肿瘤坏死因子-a、白介素-1和白介素-6等水平增高。因此,干预炎症过程的某些环节,降低炎症反应的损害,有可能成为治疗缺血性损伤的有效方法之一。白质损伤与氧化性应激、凋亡和炎症损伤关系密切。有研究发现ICAM增高的水平与头颅MRI白质高信号的进展相关。另一项前瞻性研究提示血循环中的内皮细胞激活标志物(ICAM-1、VCAM-1)与LA进展相关,此相关性可能存在因果关系,并提示内皮细胞激活可能在LA的发病机制中起重要作用。脑小血管内皮细胞表达的细胞黏附分子在生理上参与脑组织正常结构和功能的维持,保证内皮细胞层的连续性、通透血脑屏障等功能,在病理过程中具有介导炎性细胞向正常或炎症组织移动或定位等功能,其中由脑微血管内皮细胞表达的免疫球蛋白超家族和选择素家族两类黏附分子与缺血性脑损伤的发生、发展密切相关。ICAM-1和VCAM-1是脑小血管内皮细胞激活的标志。ICAM-1和VCAM-1主要由活化内皮细胞表达。脑血管内皮损伤后可出现炎症反应,而炎症的产生、发展与VCAM-1的异常表达密切相关。白细胞从血管内渗出是整个炎症过程的重要组成部分,白细胞的移行是通过其在血管壁上附壁滚动、激活、紧密黏附和移行四个步骤完成的,其中激活较为关键。参与激活的黏附分子ICAM-1外,还有VCAM-1,而激活后的过程也由VCAM-1和ICAM-1等介导。Lipoic acid是一种具有神经保护作用的代谢性抗氧化剂,分子生物学和基因分析揭示lipoic acid具有调节内皮细胞的行为学和基因表达的功能,能抑制培养的人体主动脉内皮细胞ICVM-1和VCAM-1mRNA水平的表达。研究表明其能抑制VCAM-1和ICAM-1在中枢神经系统内皮细胞的表达而具有免疫调制功能。目前大鼠双侧颈总动脉结扎导致脑部低灌注状态是研究缺血性白质损害的理想模型。实验证明双侧颈总动脉结扎后,脑部的局部脑血流和脑葡萄糖利用率都显著下降,是理想的低灌注模型。研究表明,在低灌注状态下,白质区域发生髓鞘脱失和轴突丧失情况,并且伴有胶质激活情况。在低灌注区域的白质内也发现了少突胶质细胞DNA碎裂情况,提示存在凋亡情况。本课题拟:(1)动物实验部分:以双侧颈总动脉结扎(LBCCA)致低灌注制成缺血性白质损害大鼠为研究对象,模拟人类白质疏松的循环病理基础,对缺血性白质损害的脑微小血管的结构(特别是内皮细胞)及其功能改变进行系统性观察,探讨缺血性白质损害的发病机制,以期为针对白质损害的治疗研究提供理论基础。(2)人尸解标本:利用常规组织化学和免疫组化技术对脑白质疏松患者的脑小动脉结构改变进行深入研究,探讨脑小动脉结构改变与功能的异常以及与脑白质疏松发病机制之间的关系。方法:动物实验部分:(1)建立双侧颈总动脉结扎(LBCCA)致前脑慢性低灌注大鼠缺血性白质损害模型,以假手术组为对照。采用的雄性Wistar大鼠(重190-220g) 144只,随机分为空白对照组、缺血1d、缺血3d、缺血1w、缺血2w、缺血4w、干预对照、干预1d、干预3d、干预1w、干预2w、干预4W共12组,每组12只大鼠。对照组仅暴露双侧颈总动脉而不结扎,缺血组行双侧颈总动脉结扎(术后皮下注射与干预组等量的生理盐水),干预组双侧颈总动脉结扎术后每天皮下注射100mg/kg的lipoic acid。lipoic acid用生理盐水溶解,缺血组皮下注射等量生理盐水。术后同样条件饲养,按计划不同时间点处死,断头取脑。(2)采用免疫荧光双标技术观察对照组、缺血组及干预组不同时间点脑白质小血管内皮细胞von Willebrand factor (vWF)、ICAM-1和VCAM-1表达变化,同时使用Luxol Fast Blue观察白质损害程度及CD11b/c、GFAP分别标记小胶质细胞/巨噬细胞激活变化、反应性星形细胞增生程度,并采用病理图文分析系统对空白对照组、缺血组及干预组的组化染色及免疫荧光结果进行量化,用统计学方法进行比较。(3)大鼠脑组织ICAM-1 mRNA、VCAM-1mRNA RT-PCR定量分析:每组取6只大鼠,麻醉后迅速断头取脑,取前囟后2mm-4mm脑组织置入液氮迅速冷冻后转入-70℃冰箱保存。待全部动物脑组织收集完毕后将抽提RNA,合成cDNA后行PCR分析定量分析,引物如下:VCAM-1 :上游引物: 5′-GGAAGCTGGAACGAAGTATC-3′;下游引物: 5′-TCCAGAGTCTTCCATCCTCA-3′。ICAM-1:上游引物5′-AGGTGT GAT ATC CGG TAG AA3′;下游引物5′CCT TCTAAG TGG TTG GAA CA 3′。计量数据的统计处理方法采用方差分析(one-way ANOVA),多重比较采用LSD法和SNK法,计数数据的统计处理方法采用卡方检验方法,P<0.05为差异有显著统计学意义。人体标本研究部分:(1)利用本科现有的人体尸解标本,脑白质疏松患者20例,研究脑白质疏松患者皮质、白质内径<50um的脑小动脉内径、外径、血管壁厚度和血管弹性指数( [SI= 1-(内径/外径)])进行定量分析,并与无脑白质疏松患者进行比较。(2)采用免疫组化方法对脑白质疏松组和对照组内径<50μm的脑小动脉进行胶原I、III、IV、V、VI和Laminin染色,并采用病理图文分析系统定量分析,探讨脑白质疏松的发病机制。结果:一、动物实验部分(1)双侧颈总动脉结扎模型组大鼠白质可见髓鞘脱失、反应性星形细胞增生,小胶质细胞/巨噬细胞激活,假手术组大鼠脑组织无以上病理改变。(2)RT-PCR显示与对照组比较,缺血组脑小血管内皮细胞ICAM-1、VCAM-1的mRNA表达在1d开始增加,3天达到高峰,以后逐渐下降。干预组脑小血管内皮细胞ICAM-1、VCAM-1的表达显著减少,两组比较有显著差异性(P<0.01);(3)免疫荧光双标技术显示缺血组脑小血管内皮细胞ICAM-1、VCAM-1表达与RT-PCR显示的脑小血管内皮细胞ICAM-1、VCAM-1mRNA的表达结果基本一致,1d增加,3天达到高峰,以后逐渐下降。干预组脑小血管内皮细胞ICAM-1、VCAM-1的表达显著减少,与缺血组比较有显著差异性(P<0.01);(4)彩色病理图文分析系统显示缺血组大鼠脑小胶质细胞/巨噬细胞活化3d开始增加,7d达到高峰,以后保持平稳水平,干预组脑小胶质细胞/巨噬细胞活化明显减少,定量分析两组有显著性差异(P<0.01);(5)彩色病理图文分析系统显示缺血组大鼠脑白质反应性星形细胞增生7d开始明显,14天达到高峰,干预组白质反应性星形细胞增生明显减轻,定量分析两组有显著性差异(P<0.01);(6)Luxol fast blue染色显示假手术组白质无明显损害;缺血组的胼胝体、内囊和外囊白质损害7d开始明显,14d、28d可见显著损害,干预组白质损害显著减轻,Nakaji分级评分两组比较有显著差异性(P<0.01)。二、人体尸解标本部分:(1)脑白质疏松组的胶原I、III、V和胶原VI免疫组化染色阳性横切面积百分比与对照组有显著差异性(P<0.001);胶原IV和laminin在基底膜明显增加,两组之间有显著差异性(P<0.001)。(2)脑白质疏松患者脑小动脉的内径、外径、血管壁厚度和血管弹性指数与对照组有明显差异性,有统计学意义。脑白质疏松组的小动脉血管壁厚度明显厚于对照组(P<0.01),管腔明显小于对照组(P<0.01)。脑白质疏松组的小动脉硬化指数明显高于相邻皮质小动脉和对照组白质的硬化指数(P<0.01)。结论:(1)双侧颈总动脉结扎导致脑部低灌注状态是研究缺血性白质损害的理想模型。(2)脑小血管内皮细胞功能障碍,ICAM-1、VCAM-1表达增加,星形胶质细胞反应性增生、小胶质细胞/巨噬细胞激活可能在双侧颈总动脉结扎致低灌注大鼠缺血性白质损害的病理机制中起重要作用;lipoic acid对慢性缺血性白质损害有潜在的治疗价值。(3)白质损害的早期病理机制中涉及脑小血管内皮细胞激活,稳定脑小血管内皮细胞功能对白质损害的进展有保护作用;(4)脑白质疏松患者的脑小动脉管腔明显减小,血管硬化指数明显高于对照组,小动脉硬化的血管壁增厚和纤维化与异常细胞外间质的堆积相关,这种病理改变不仅影响到脑白质疏松患者白质血供的减少,同时可能影响到脑白质组织间隙液的通过血管周的引流,从而加速脑白质疏松的发展。更多还原

【Abstract】 Background and Purpose: The term leukoaraiosis (from the Greek leuko, white, and araiosis, rarefaction) was introduced in 1986 by Hachinski, Potter and Merskey to designate bilateral and symmetrical areas in the periventricular and centrum semiovale white matter that appeared hypodense on CT scans and hyperintense on T2-weighted MRI. Leukoaraiosis (LA) is used interchangeably with the term white matter lesions (WMLs) on MRI and seen to some degree in more than half of the routine scans in older persons. Leukoaraiosis can reflect a broad public health problem, which is caused by a cognitive impairment ranging from mild slowness of thinking to full-blown subcortical dementia. As many as 80% of patients with leukoaraiosis have some degree of gait disorder. This association is independent of age, gender, previous stroke and hypertension. Furthermore, deterioration of gait is associated with progression of leukoaraiosis. Other features include bladder instability and mood disturbance. The presence of leukoaraiosis has been identified as a marker of less favorable prognosis also in the acute stroke settings. In particular, white matter changes have been associated with an increased risk of hemorrhagic transformation of the brain infarct in patients subjected to thrombolysis. One-quarter of subjects aged 65 years or over are affected by some degree of white matter changes. The well-known vascular risk factors, however, do not fully explain the incidence of LA. The neuropathological appearances corresponding to leukoaraiosis are neuronal loss, ischemic demyelination, and gliosis. Ischemic demyelination and small-vessel disease have been assumed as underlying pathological processes in the evolution of this entity. The pathogenesis of cerebral small vessel disease (SVD) is poorly understood, but endothelial activation and dysfunction may play a causal role. The exact etiological factors and the pathomechanism of LA remain to be identified.Cerebral arteries not only deliver blood to the brain, but their walls also act as drainage pathways for interstitial fluid (ISF) and solutes from the brain. More specifically, ISF and solutes drain from the brain along the basement membranes between the smooth muscle cells in the tunica media. Amyloid-β(Aβ) is deposited in the basement membranes of arteries in cerebral amyloid angiopathy (CAA) in elderly humans. This strongly suggests that ISF and solutes drain from the human brain along artery walls and that changes occurring in the walls of arteries with age impair such drainage. Several factors may be responsible for the impaired drainage of ISF and solutes from the white matter in LA in elderly brains. Mathematical models suggest that the motive force for the drainage of ISF along artery walls is the contrary wave that follows each pulse wave and that the motive force would decrease when the amplitude of the pulse wave is reduced in stiffened arteriosclerotic arteries. Both arteriosclerosis and arteriolosclerosis in the elderly brain are characterised by loss of smooth muscle cells and deposition of collagen in artery walls. Such a change in architecture would interfere with the drainage pathways that depend upon intact smooth muscle cell basement membranes in the walls of the arteries.Brain microvascular endothelial cells, the inner liner of blood-brain barrier, are involved in many physiological and pathophysiological processes in the brain such as transport of nutrients from blood to brain, export of critical toxins from brain, transmigration of circulating leukocytes and formation of new blood vessels. Endothelial dysfunction, which can lead to breakdown of the blood-brain barrier, impaired cerebral autoregulation and prothrombotic changes, is believed to be important in mediating disease. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important adhesion molecules that are upregulated during endothelial activation. Both belong to the immunoglobulin superfamily and have adhesion molecules on leukocytes as ligands, namely the integrins CD11/CD18 for ICAM-1 and very late antigen-4 (VLA-4) for VCAM-1. Cytokines formed immediately after ischemia stimulates the expression of adhesion molecules on endothelial cells and leukocytes, leading to leukocyte adherence and extravasation into brain parenchyma. White matter lesions can be experimentally induced in rat brains under chronic cerebral hypoperfusion by permanent occlusion of both common carotid arteries.WM lesions have a strong relationship with oxidative stress, apoptosis and inflammatory damage. ICAM levels are related to progression of WMH on MRI. A prospective study design indicates the likelihood that this association is causal and supports a role of endothelial cell activation in the pathogenesis of LA. Studies of circulating endothelial markers are beginning to hint at a specific pattern of endothelial function that is associated with leukoaraiosis.Lipoic acid, a neuroprotective metabolic antioxidant has been used to improve glycemic control, treat polyneuropathies associated with diabetes mellitus, and mitigate toxicities associated with heavy metal poisoning. Biological assays and genomic analysis revealed that lipoic acid can modulate endothelial cell behavior and gene expression. Lipoic acid can inhibit expression of ICAM-1 and VCAM-1 at the mRNA level in cultured human aortic endothelial cells Administration of lipoic acid to patients with the metabolic syndrome can improve endothelial function and reduce proinflammatory markers.In the present study, (1) we induced WMLs in a rat chronic cerebral hypoperfusion model by permanent occlusion of both common carotid arteries. We then tested the hypothesis that the ICAM-1 and VCAM-1 upregulation after chronic cerebral hypoperfusion correlates with endothelial dysfunction, which leads to inflammatory damage and subsequent WM lesions. (2) we test the hypothesis that disruption of the structure of artery walls by the deposition of collagen is significantly greater in patients with LA than in normal white matter in age-matched controls. We performed immunohistochemistry on the components of extracellular matrix of the walls of arterioles in LA patients and in controls. In addition to immunohistochemical studies, we present a quantitative analysis of the pathology of arterioles, including the external diameter of arterioles, diameter of arteriolar lumina, thickness of arteriolar walls and sclerotic indices of arterioles in the white matter and cerebral cortex.Methods Part I: One hundred and fourty-four adult male Wister rats weighing 190 to 220g were divided at random into the following 3 groups: (1)the Lipoic acid group (n=60) were provided daily 1.1ml s.c. injections of LA at 100 mg/kg after ligation of both common carotid arteries (LBCCA); (2) the saline ( vehicle ) group: rats of this group (n=60) underwent LBCCA but were provided daily 1.1 ml s.c. injections of saline( vehicle ); and (3) the control sham-operated vehicle- and lipoic acid-treated groups: these rats (n=6 for each subgroup) underwent the same aforementioned protocol except for LBCCA. RT-PCR and double immunofluorescence for ICAM-1, VCAM-1, endothelial cells (staining positive for von Willebrand factor, vWF), reactive astrocyte (staining positive for glial fibrillary acidic protein, GFAP) and activated microglia/macrophages (CD11b/c staining) were analyzed at baseline and at 1, 3, 7, 14 and 28 days after hypoperfusion. The severity of the WM lesions in the corpus callosum, internal capsule, and external capsule of both hemispheres was graded by Luxol fast blue staining. Part II: Twenty brains were obtained from elderly LA patients (12 females and 8 males, mean age 66.4±2.9 years, range 62 to 78 years); ten brains from elderly individuals (5 females and 5 males, mean age 64.3±7.1 years, range 60 to 73 years) without known cerebrovascular disease served as controls. (1)We performed a quantitative analysis of the pathology of arterioles, including the external diameter of arterioles, diameter of arteriolar lumina, thickness of arteriolar walls and sclerotic indices of arterioles in the white matter and cerebral cortex. (2)We used immunocytochemistry to quantify changes to the extracellular matrix in arterioles of cerebral white matter (WM) in 20 autopsy cases with leukoaraiosis and in 10 controls.Results: (1) RT-PCR and double immunofluorescence analysis of white matter from rats that had received lipoic acid (100 mg/kg/day) exhibited markedly reduced expression of ICAM-1 and VCAM-1 over endothelial cells compared with that of rats receiving saline( vehicle) (P<0.01). In the rats treated with lipoic acid, the WM lesions after chronic cerebral hypoperfusion were significantly less severe, and the number of reactive astrocyte (GFAP staining) and activated microglia/macrophages (CD11b/c staining) were also significantly lower as compared with the saline(vehicle)-treated rats (P<0.01). (2) The ratio of the area immunoreactive for collagen types I , III, V, and VI to the cross-sectional area of arterioles was significantly higher in the LA patients compared to controls (P<0.001). Collagen IV and laminin were increased in the basement membrane. The walls of WM arterioles were significantly thicker (P<0.01), and lumina were significantly smaller (P<0.01). Arterioles had a significantly higher more sclerotic index (1-(internal/external diameter)) in LA than in adjacent cortex or control white matter (P<0.01).Conclusions: (1) These findings indicate that endothelial dysfunction plays a critical role in overexpression of ICAM-1 and VCAM-1, glial cell activation and WM lesions after chronic cerebral hypoperfusion and suggest the potential value of lipoic acid as a therapeutic tool in cerebrovascular WM lesions. (2) Our results also provide support for endothelial activation being involved in early pathogenesis of WM lesions and suggest that therapies that stabilize the endothelium may have a role in preventing WM lesions progression. (3) Our results suggest that fibrosis and thickening of the walls of arterioles in cerebral white matter in LA are due to the accumulation of extracellular matrix components. Although these changes may result in decreased perfusion, they could also impede perivascular drainage of interstitial fluid from white matter in LA.更多还原