铁螯合剂对血管性痴呆小鼠模型的保护作用及机制研究

发布时间：2018-07-06 17:40

  本文选题：铁 + 螯合剂　； 参考：《第二军医大学》2012年博士论文

【摘要】：背景 血管性痴呆(vascular dementia,VaD)是各种脑血管病引起的获得性智能损害和认知障碍的综合征,为一种慢性进行性疾病。在导致痴呆的众多病因中,VaD是仅次于Alzheimer病(Alzheimer's disease, AD)的第二大病因。随着全球人口的老龄化和脑血管病发病率的增高,VaD的发病率也逐年增高,严重危害着人类健康,并给社会和家庭造成沉重的经济压力。就临床实际意义而言,VaD是目前唯一可以预防的痴呆类型,可能较AD更有预防和治疗价值,早期合理防治VD可以减轻社会和家庭的沉重负担。正因如此,近年来VaD逐渐成为人们关注的热点。 VaD的发病机制可能与以下几个因素有关：中枢胆碱能系统功能障碍、氧自由基生成增加、中枢RNA和蛋白质合成减少、局部炎症反应及机体免疫异常等。由于发病机制的多样化,现有治疗手段或药物(如胆碱酯酶抑制剂、N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受体拮抗药等)均不能完全控制血管性痴呆的所有症状或者延缓甚至逆转其进程。 最近的研究表明,金属离子尤其是铁离子与神经退行性疾病的发生关系密切[4]。已有动物实验表明,脑组织中铁离子过负荷,与阿尔茨海默病(AD)、帕金森病(PD)等的发病直接相关。用铁离子螯合剂来治疗神经退行性疾病的动物模型,取得了比较乐观的效果。 越来越多的证据表明AD与VaD经常相互伴随发病,两者在病因学、危险因素、发病机制、病理学、症状学和疾病转归等方面都有显著的重叠。然而铁离子在血管性痴呆中的作用却并无深入探究。 目的 本研究旨在运用两血管法制备血管性痴呆小鼠模型,观察不同时间点小鼠行为学及脑损伤的关系。并利用铁螯合剂——甲磺酸去铁胺对动物模型进行干预,观察其是否对血管性痴呆模型具有保护或治疗效应并初步探讨其可能的作用机制。 第一部分血管性痴呆小鼠模型的建立和评价 方法：采用二血管法即双侧颈总动脉完全阻断1小时后再灌注的方法造模,并于造模后第3天开始Morris水迷宫训练,第3、7、14、21天测悬尾实验静止不动时间,第7、14、21天测定位航行实验上台潜伏期和空间探索实验跨越平台次数,第21天测Open filed实验休息时间。第21天观察小鼠脑组织病理改变(HE染色、尼氏染色)。 结果：小鼠缺血再灌注组与假手术组相比,悬尾实验静止不动时间、水迷宫定位航行实验上台潜伏期明显延长,空间探索实验跨越平台次数明显减少,差异均有统计学意义(P0.05)；但旷场实验休息时间,两组间差异无明显统计学意义(P0.05)模型组不同时间点之间比较,随着再灌注时间的延长,悬尾静止不动时间逐渐延长,定位航行上台潜伏期逐渐缩短,具有统计学意义(P0.05)；但空间探索跨越平台次数的增多未见统计学意义(P0.05)。病理HE染色示模型组较假手术组海马区细胞层次不清、胞体变小。Nissl染色示模型组较假手术组海马神经元形态不规则,着色不均匀,尼氏小体含量明显减少。 结论：二血管法制备的血管性痴呆小鼠模型,成模率高,稳定性好。 第二部分铁螯合剂对血管性痴呆小鼠模型的作用 方法：将在我校实验动物中心购得的雄性ICR小鼠160只随机分为4组：假手术组、血管性痴呆模型+生理盐水对照组、血管性痴呆模型+甲磺酸去铁胺(50mg/kg)干预组及血管性痴呆模型+甲磺酸去铁胺(100mg/kg)干预组,每组40只。各组再次随机分成4亚组,每组10只,分别标记术后3d、7d、14d、21d。同实验第一部分制作动物模型,再灌注时给予甲磺酸去铁胺或等量生理盐水干预,之后2天相同时间点再次给予去铁胺或等量生理盐水干预。术后3、7、14、21天四个时间点每组中的各亚组10只小鼠分别进行行为学检测、取血及脑组织匀浆行铁含量检测。 结果：去铁胺干预组较生理盐水空白对照组术后7、14、21d的Morris水迷宫定位航行实验上台潜伏期明显缩短,悬尾不动时间也明显缩短(P0.05)；而去铁胺高剂量组与低剂量组相比,上述指标差异无统计学意义(P0.05)。水迷宫空间探索实验在再灌注21d时去铁敏不同剂量干预组较生理盐水空白对照组跨越站台次数明显增多(P0.05)；而7、14d时,各组间无明显统计学差异(P0.05)。再灌注14天时旷场实验小鼠休息时间,各模型组间无明显差异(P0.05) 血清铁及脑组织匀浆铁检测表明：生理盐水模型组较假手术组,四个时间点血清及脑组织匀浆铁含量均明显增高,有统计学意义(P0.05)；去铁胺干预组较生理盐水空白对照组,术后3、7、14三个时间点,血清及脑组织匀浆铁含量均明显减少,有统计学意义(P0.05) 结论： 1、铁螯合剂可改善血管性痴呆小鼠与认知有关的行为学指标； 2、血管性痴呆小鼠的血清和脑组织铁含量明显升高； 3、铁螯合剂干预血管性痴呆小鼠后,血清和脑组织铁含量明显降低,说明铁螯合剂可能是通过螯合血及脑组织中铁来发挥神经保护作用的。 第三部分铁螯合剂对血管性痴呆小鼠模型的神经保护作用机制探讨 方法：实验动物分组和干预以及血和脑组织标本收集同第二部分。小鼠大脑左半球投入4%多聚甲醛后固定48小时后投入20%蔗糖/PB溶液4℃浸泡至组织沉底。行冰冻切片,选取额叶皮层区域分别行免疫组化GST-pi染色和MBP染色,记数前额叶GST-pi阳性的少突胶质细胞的数目以及测定MBP Density/Area。小鼠大脑右半球脑组织匀浆,分别检测脑组织总超氧化物歧化酶活力,抑制羟自由基能力、丙二醛含量。血清测定乙酰胆碱含量、胆碱酯酶活力。 结果：去铁胺干预组较生理盐水空白对照组术后3、7、14、21d脑组织抑制羟自由基能力增强、丙二醛含量减少、T-SOD活力增强,差异均有统计学意义(p0.05)血清乙酰胆碱含量、胆碱酯酶活力在各组间无明显统计学差异(P0.05)。病理结果提示：单纯模型组与假手术组相比,术后各时间点额叶皮层下少突胶质细胞数目明显减少,前额叶皮层髓鞘碱性蛋白(MBP) Density/Area明显降低,差异有统计学意义(P0.05)；而去铁敏干预组与生理盐水空白对照组相比,术后各时间点额叶皮层下少突胶质细胞数目增多、前额叶皮层Density/Area升高,有统计学意义(p0.05) 结论： 1、铁螯合剂可阻断氧化应激反应,减少缺血缺氧诱导的血管性痴呆小鼠模型羟自由基和丙二醛生成,增强SOD活力。这种作用能持续至缺血再灌注21天。 2、铁螯合剂可以促进血管性痴呆小鼠的髓鞘修复,促进少突胶质细胞的增生具有胶质细胞保护作用。这种作用在缺血再灌注3周内均比较明显。
[Abstract]:Background

Vascular dementia ( VaD ) is a syndrome of acquired intellectual impairment and cognitive disorder caused by various cerebrovascular diseases , and is a chronic progressive disease . In many causes of dementia , VaD is the only second major cause of Alzheimer ' s disease ( AD ) . With the aging of global population and the increasing incidence of cerebrovascular disease , VaD is the only type of dementia that can be prevented .

The pathogenesis of VaD may be related to several factors : the dysfunction of central cholinergic system , the increase of oxygen free radicals , the decrease of central RNA and protein synthesis , the local inflammatory response and the abnormal organism immunity , etc . Due to the diversification of the pathogenesis , the existing treatment methods or drugs ( such as cholinesterase inhibitor , N - methyl - D - aspartate ( N - methyl - D - aspartate , nmd A ) receptor antagonist , etc . ) can not completely control all the symptoms of vascular dementia or delay or even reverse its progression .

Recent studies have shown that metal ions , especially iron ions , are closely associated with the onset of neurodegenerative diseases . Animal experiments have shown that the overload of iron ions in brain tissue is directly related to the pathogenesis of Alzheimer ' s disease ( AD ) and Parkinson ' s disease ( PD ) .

There is an increasing number of evidence that AD and VaD are frequently associated with each other , both in etiology , risk factors , pathogenesis , pathology , symptoms , and disease outcomes . However , the role of iron ions in vascular dementia is not deeply explored .

Purpose

The purpose of this study was to use two - vessel method to prepare the model of vascular dementia mice , to observe the relationship between behavior and brain injury in mice at different time points .

Establishment and evaluation of mouse model of vascular dementia in the first part

Methods : A two - vessel method was used to block the total occlusion of bilateral common carotid artery for 1 hour , and the Morris water maze training was started on the 3rd day after the molding . The latency and space exploration experiments on the 3rd , 7th , 14th and 21st day were carried out . On the 7th , 14th and 21st day , the latency and the space exploration experiment were measured across the platform . On the 21st day , the pathological changes of the brain tissues were observed ( HE staining and Nye staining ) .

Results : Compared with the sham operation group , the experimental results showed that the latency period of the experiment was prolonged obviously , and the space exploration experiment significantly decreased the number of stages and the difference was statistically significant ( P0.05 ) .
However , there was no significant difference between the two groups ( P0.05 ) .
However , there was no statistical significance in the spatial exploration of the number of stages across the platform ( P0.05 ) . The pathological HE staining showed that the level of cells in the hippocampus of sham operation group was not clear , and the cell body became smaller . Nissl staining showed that the morphological irregularity , non - uniform coloring and the content of Nissl ' s small body were obviously reduced in the sham operation group .

Conclusion : The model of vascular dementia mice prepared by two - vessel method has high forming rate and good stability .

Effect of the second part of iron chelating agent on the model of vascular dementia mice

Methods : 160 male ICR mice were randomly divided into four groups : sham operation group , vascular dementia model + physiological saline control group , vascular dementia model + methanesulfonic acid deferrioxamine ( 50mg / kg ) intervention group and vascular dementia model + methanesulfonic acid deferrioxamine ( 100mg / kg ) intervention group .

Results : Compared with saline control group , the latency period was significantly shorter than that of saline control group ( 7 , 14 , 21 d ) , and the duration of suspension tail was shortened obviously ( P0.05 ) .
Compared with the low - dose group , there was no significant difference in the above - mentioned indexes ( P0.05 ) .
No significant difference was found between the groups at 7 and 14 days ( P0.05 ) . There was no significant difference between the model groups ( P0.05 ) .

The levels of iron and iron in serum and brain tissue of normal saline group were significantly higher than those in sham operation group and four time points ( P0.05 ) .
The levels of iron in serum and brain tissue were significantly decreased at 3 , 7 and 14 hours after deironing and compared with normal saline control group ( P0.05 ) .

Conclusion :

1 . The iron chelating agent can improve the behavioral indexes of vascular dementia mice and cognition ;


2 . The iron content in serum and brain tissue of vascular dementia mice was significantly increased .


3 . The iron content of serum and brain tissue decreased significantly after the iron chelating agent was used in the treatment of vascular dementia mice , indicating that the iron chelating agent could play a role of neuroprotection by chelating blood and iron in brain tissue .

Neuroprotective effect of the third part of iron chelating agent on the model of vascular dementia mice

Methods : The group and intervention of experimental animals and blood and brain tissue samples were collected in the same second part . After 48 hours , the brain left hemisphere of mice was fixed for 48 hours and then immersed in 20 % sucrose / PB solution at 4 鈩，

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