阿尔茨海默病5XFAD小鼠模型早期多模态7.0T磁共振成像研究

发布时间：2018-06-12 04:34

本文选题：阿尔茨海默病 + 5XFAD小鼠　；参考：《东南大学》2017年博士论文

【摘要】：研究背景阿尔茨海默病(Alzheimer's disease,AD),又称老年性痴呆,是最常见的老年期痴呆。目前AD病因不清,发病机制不明,起病隐匿,缺乏明确的诊断标志物,尚无有效根治措施。早期诊断与及时干预是延缓AD疾病进展的关键措施。AD的诊断标准开始于1984年,近年来不断修订,2011年至今新修订的AD诊断标准采用“情节记忆功能受损”结合“AD生物标志物异常”的新诊断模式,将AD定义为一个包含临床前期(preclinicalAD)、轻度认知障碍期(mild cognitive impairment due to AD)与阿尔茨海默病痴呆期(dementia due to AD)的延续疾病谱。然而,目前现有的生物标志物检测在AD早期阶段的临床可行性较低。因此,寻找AD早期阶段新的生物标志物,为AD治疗打开关键的时间窗是AD研究的热点。磁共振成像(magnetic resonance imaging,MRI)具有无创伤、可重复性、时间/空间分辨率高、成像快速、成本相对较低等优点,已成为目前探讨AD生物标志物的核心工具之一。研究发现的可辅助AD诊断的MRI生物标记物主要分为结构和功能两大类,如以海马为主的内侧颞叶结构萎缩和后扣带功能代谢改变。然而,以AD患者为对象的研究通常始于疾病的中晚期,对于AD发病早期不能进行深入地研究。因此转基因AD实验动物为AD疾病早期的研究提供了理想的模型。此外,近年来随着高场强动物磁共振(micro-MRI)的出现,进一步提高了早期AD诊断的敏感性,并可进一步探讨AD行为学改变、MRI特征与病理改变之间的关系。因此,本研究选择携带5个家族性基因突变的APP/PS1转基因模型小鼠(transgenic mice with five familial AD,5XFAD)和正常野生型 C57BL/6 小鼠为研究对象,采用多维度动物行为学测试评估其情感和认知功能,继而,结合7.0T高场强动物多模态MRI (T2加权结构成像、T2map成像和锰增强磁共振成像)进行纵向随访研究,主要探讨(1) 5XFAD小鼠疾病早期是否存在脑组织微结构完整性改变?脑组织微结构改变是否随着疾病进展加重?脑组织微结构改变是否与情感和/或认知障碍相关?(2)5XFAD小鼠疾病早期是否存在神经元活性改变?脑功能改变是否随着疾病进展加重?脑功能改变是否与情感和/或认知障碍相关?最终,希望通过以上两部分研究加深我们对AD早期情感和/或认知功能损害的神经网络机制的了解,为寻找AD早期阶段MRI生物标记物提供动物层面的依据。第一部分5XFAD小鼠早期脑组织微结构完整性及其对空间认知功能影响的研究目的:探讨5XFAD小鼠早期脑组织微结构完整性损害模式及其与空间认知功能的关系。方法:(1)本研究采用1、2、3和5月龄四个时间点的5XFAD模型小鼠和野生型C57BL/6小鼠,采用蔗糖水偏爱实验、旷场实验、高架十字迷宫实验和Morris水迷宫实验对动物进行行为学评估。(2)用7.0Tmicro-MRI完成每个时间点两组小鼠的多模态脑成像(T2加权和T2map)。分别采用基于体素的分析方法(voxel-based analysis,VBA)和感兴趣区分析方法(region of interest,ROI)对比分析各时间点5XFAD小鼠和野生型小鼠灰质脑区的T2弛豫时间值(T2 relaxation time,T2)差异。此外,利用ROI进一步分析海马各亚区:背侧海马角/安蒙角1区(cornu Ammonisl,CA1)、背侧海马角3区(CA3)、背侧齿状回(dentate gyrus, DG)、腹侧CA1、腹侧CA3和腹侧DG)的T2值和体积。(3)通过相关分析评价海马各亚区的T2值与认知评分之间的相关性。结果:(1)行为学结果:1、2、3和5月龄的5XFAD小鼠和野生型小鼠在蔗糖水偏爱实验、旷场实验和高架十字迷宫实验中的行为学表现无明显差异。Morris水迷宫结果:与同月龄野生型小鼠相比,1月龄5XFAD小鼠前5天学习训练期的潜伏期时间无明显差异,第6天记忆检测过程中目标象限停留时间明显减少(p = 0.0365) ; 2和3月龄5XFAD小鼠学习训练期第3天至站台的潜伏期时间延长(p= 0.0024, 0.0300),记忆检测过程中目标象限停留时间明显减少(P 0.001, = 0.011) : 5月龄5XFAD小鼠学习训练期第3、4和5天至站台的潜伏期时间延长(p = 0.0154, 0.0154, 0.0011),记忆检测过程中目标象限停留时间明显减少(= 0.034)。(2) MRI结果:与同月龄野生型小鼠相比,1月龄5XFAD小鼠各脑区的T2值无明显差异;VBA和ROI两种方法都显示2、3和5月龄的5XFAD小鼠海马区(hippocampus,Hip) T2值明显降低(p=0.0420,0.0182, 0.0001),此外,VBA还显示5月龄的5XFAD小鼠的前扣带皮层(the cingulate cortex,cg)和杏仁核(amygdala,Amy)的 T2值降低(p 0.05)。海马各亚区ROI分析显示:2和3月龄的5XFAD小鼠的背侧CA1、CA3、DG和腹侧CA1的 T2 值明显降低(2 月龄:p = 0.0214, 0.0030, 0.0369,0.0369; 3 月龄:p = 0.0119,0.0031,0.0119, 0.0119),5月龄的 5XFAD小鼠的背腹侧 CA1、CA3、和 DG的 T2值均明显降低(p = 0.0045, 0.001,0.0036,0.0007,0.0001,0.0045)。1、2、3和 5月龄的5XFAD小鼠海马各亚区体积与野生型小鼠相比无明显差异。(3)相关分析结果:3和5月龄的5XFAD小鼠背侧CA1及5月龄5XFAD小鼠背侧DG区的T2值与水迷宫实验的第5天潜伏期负相关(r = -0.6188, -0.5190,-0.5240),2、3和5月龄的5XFAD小鼠背侧CA1的T2值与水迷宫实验的目标象限停留时间正相关(r = 0.6144,0.6645, 0.5629);腹侧海马各亚区的T2值均没有发现与这两者相关。结论:5XFAD小鼠早期存在海马区组织微结构完整性受损,并随疾病进展加重。背侧海马区组织微结构完整性损害与其空间认知功能状态相关。本研究从动物模型在体影像的层面证实了海马在AD早期的重要作用,且背侧海马的T2值可能成为辅助AD早期诊断及监测AD进展的生物标记物之一。第二部分5XFAD小鼠早期脑神经元活性及其对空间认知功能影响的研究目的:探讨5XFAD小鼠早期脑神经元活性改变模式及其与空间认知功能的关系。方法:(1)本研究采用1、2、3和5月龄四个时间点的5XFAD模型小鼠和野生型C57BL/6小鼠,采用蔗糖水偏爱实验、旷场实验、高架十字迷宫实验和Morris水迷宫实验对动物进行(基线期)行为学评估。(2)利用7.0Tmicro-MRI完成每个时间点两组小鼠的基线期T1加权成像(T1 weighted imaging,T1WI);继而腹腔注射Mnc12溶液,间隔24小时,共注射4次(每次13.95 mg/kg),最后一次注射后24小时进行锰强磁共振成像(manganese-enhanced magnetic resonance imaging,MEMRI)。(3) MEMRI 成像后每天观察两组小鼠日常行为状态,并于首次注射MJncl2溶液后12天再次完成旷场实验和Morris水迷宫实验。(4)利用ROI对比分析各时间点5XFAD小鼠和野生型小鼠灰质脑区的信号强度值(signal intensity,SI)差异,其中同一只小鼠注射Mnc12前后的两次T1WI的平均信号强度差值[SI=100% × (SI注射锰后- SI基线期)/SI基线期] 作为反映MR2+聚集浓度的指标。(5)通过相关分析评价ROI的信号强度值与认知评分之间的相关性。结果:(1)行为学结果:1、2、3和5月龄的5XFAD小鼠和野生型小鼠在蔗糖水偏爱实验、旷场实验和高架十字迷宫实验的行为学表现无明显差异。Morris水迷宫结果:与野生型小鼠相比,1月龄5XFAD小鼠前5天的学习训练期的潜伏期时间无明显差异,第6天记忆检测过程中目标象限停留时间明显减少(p=0.0365) ; 2和3月龄5XFAD小鼠学习训练期第3天至站台的潜伏期时间延长(p=0.0024 , 0.0300),记忆检测过程中目标象限停留时间明显减少(p 0.001,= 0.011) ; 5月龄5XFAD小鼠学习训练期第3、4和5天至站台的潜伏期时间延长(p = 0.0154,0.0154,0.0011),记忆检测过程中目标象限停留时间明显减少(p = 0.034)。(2) MRI结果:与同月龄野生型小鼠相比,1月龄5XFAD小鼠各脑区的SI值无明显差异;2月龄5XFAD小鼠海马区(Hip)SI值明显增加(p = 0.0167); 3月龄5XFAD小鼠海马区和内嗅皮层区(entorhinal cortex,ERC) SI 值明显增加(= 0.0107,0.0684); 5 月龄 5XFAD 小鼠尾壳核(caudate-putamen,CPu)、压后皮层区(theretrosplenial cortex, RSC)、HIP 和 ERC 的 SI 值明显增加(= 0.0228,0.0795,0.0167, 0.0684)。(3)相关分析结果:5 月龄 5XFAD 小鼠Hip的SI值与水迷宫实验的第5天潜伏期正相关(r = 0.8668)。3和5月龄的5XFAD小鼠Hip的SI值与水迷宫实验的目标象限停留时间负相关(r = -0.8269,-0.8364) ; 2、3和5月龄的5XFAD小鼠ERC的SI值与目标象限停留时间负相关(r = -0.9204, -0.8576, -0.8386)。各月龄的野生型小鼠Hip和ERC的SI值均没有发现与这两者相关。结论:5XFAD小鼠早期存在认知相关脑区(特别是海马和内嗅皮层)的神经元活性增加,并随疾病进展明显加重。海马和内嗅皮层的神经元活性与其空间认知功能状态相关。本研究从动物模型在体影像的层面证实了海马和内嗅皮层在AD早期阶段的重要作用,且早期神经元活性可能成为辅助AD早期诊断的生物标记物之一。
[Abstract]:Alzheimer's disease (AD), also known as Alzheimer's disease, is the most common senile dementia. At present, the etiology of AD is unclear, the pathogenesis is unknown, the onset of the disease is hidden, there is no clear diagnostic marker, and there is no effective radical cure. Early diagnosis and time intervention are the key measures to delay the progress of AD disease, the diagnostic mark of.AD. Starting in 1984, it has been revised in recent years. From 2011 to date, the newly revised AD diagnostic standard uses the new diagnostic model of "impaired episodic memory function" combined with "AD biomarker anomaly", and defines AD as a period containing the preclinical (preclinicalAD), the mild cognitive impairment (mild cognitive impairment due to AD) and the Artz sea The persistent disease spectrum of dementia due to AD. However, the clinical feasibility of existing biomarker detection at the early stage of AD is low. Therefore, finding new biomarkers at the early stage of AD and opening the key time window for AD treatment are the hot spots of AD research. Magnetic resonance imaging (magnetic resonance imaging, MRI) has a low clinical feasibility. No trauma, repeatability, high time / space resolution, rapid imaging and low cost, it has become one of the core tools to explore AD biomarkers. The MRI biomarkers that can be diagnosed by AD are divided into two major categories, structure and function, such as the atrophy of the medial temporal lobe and the posterior cingulate of the hippocampus. Functional metabolic changes. However, the study of AD patients usually begins in the middle and late stages of the disease and can not be deeply studied in the early stages of the onset of AD. Therefore, transgenic AD experimental animals provide an ideal model for the early study of AD disease. In addition, in recent years, with the emergence of high field dynamic magnetic resonance (micro-MRI), it has been further improved. The sensitivity of early AD diagnosis can be further explored in the relationship between AD behavioral changes, MRI characteristics and pathological changes. Therefore, this study selected 5 familial gene mutations in APP/PS1 transgenic mice (transgenic mice with five familial AD, 5XFAD) and normal wild type C57BL/6 mice. Animal behavioral tests assess their emotional and cognitive functions, and then combine 7.0T high field animals with multimodal MRI (T2 weighted structural imaging, T2map imaging and manganese enhanced magnetic resonance imaging) for longitudinal follow-up studies. (1) whether there is a change in brain tissue integrity in the early stage of the disease of 5XFAD mice and the change in the microstructure of the brain tissue Is the brain tissue changes associated with emotional and / or cognitive impairments? (2) is there a change in neuronal activity in the early stage of the 5XFAD mouse disease? Is brain function change aggravated with disease progression? Is brain function change associated with emotion and / or cognitive impairment? Finally, hope to deepen the study through the above two parts Our understanding of the neural network mechanism of AD early emotion and / or cognitive impairment provides the basis for finding the animal level for the early stage of AD MRI biomarkers. Part 1 the study of the early brain microstructure integrity and its effect on spatial cognitive function in the early part of the 5XFAD mice: To explore the early brain tissue microstructure in the 5XFAD mice. The relationship between the pattern of integrality damage and its spatial cognitive function. Methods: (1) this study uses 5XFAD model mice and wild type C57BL/6 mice with 1,2,3 and 5 month old time points, using sucrose water preference experiment, open field experiment, elevated cross maze experiment and Morris water maze test to evaluate the behavior of animals. (2) 7.0Tmicro-MRI finished with 7.0Tmicro-MRI The multimodal brain imaging (T2 weighted and T2map) of two groups of mice at each time point were compared with the voxel based analysis (voxel-based analysis, VBA) and the region of interest analysis (region of interest, ROI), respectively, to analyze the T2 relaxation time values of the gray matter brain regions of 5XFAD mice and wild type mice at each time point. In addition, ROI was used to further analyze the value and volume of the hippocampal subregions: dorsal hippocampal corner / amono angle 1 (cornu Ammonisl, CA1), dorsal hippocampal angle 3 (CA3), dorsal dentate gyrus (dentate gyrus, DG), ventral CA1, ventral CA3 and ventral DG). (3) correlation analysis was used to evaluate the correlation between the values of the hippocampus subregions and the cognitive score. Fruit: (1) behavioral results: there was no significant difference in behavioral performance between 1,2,3 and 5 month old 5XFAD mice and wild type mice in sucrose water preference experiment, open field experiment and elevated cross maze test. Compared with the same month old wild type mice, there was no significant difference in the latency time of the training period of the 1 month old 5XFAD mice in the first 5 days. In the process of sixth day memory detection, the residence time of the target quadrant decreased significantly (P = 0.0365); the incubation period of the 2 and 3 month old 5XFAD mice was prolonged from third days to the platform (p= 0.0024, 0.0300), and the target quadrant residence time in the memory detection process was significantly reduced (P 0.001, = 0.011): 5 month old 5XFAD mice learning training period 3,4 The latency of the 5 days to the platform was prolonged (P = 0.0154, 0.0154, 0.0011), and the target quadrant time in the memory detection process was significantly reduced (= 0.034). (2) MRI results: compared with the same month old wild type mice, the T2 values in the brain regions of 1 month old 5XFAD mice were not significantly different; the VBA and ROI two methods showed 2,3 and 5 month old 5XFAD mice hippocampus. The T2 value of the region (hippocampus, Hip) decreased significantly (p=0.0420,0.0182, 0.0001). In addition, VBA also showed that the anterior cingulate cortex of 5 month old 5XFAD mice (the cingulate cortex, CG) and amygdala (amygdala, Amy) decreased (0.05). Significantly decreased (2 month old: P = 0.0214, 0.0030, 0.0369,0.0369; 3 month old: P = 0.0119,0.0031,0.0119, 0.0119), and 5 month old of 5XFAD mice on the dorsal ventral CA1, CA3, and DG significantly decreased (P = 0.0045, 0.001,0.0036,0.0007,0.0001,0.0045).1,2,3 and 5 month old of the hippocampal subregions of.1,2,3 and 5 month old were less obvious than those of wild type mice. (3) correlation analysis results: 3 and 5 month old of the dorsal CA1 and 5 month old 5XFAD mice in the dorsal side of the 5XFAD mice were negatively correlated with the fifth day incubation period of the water maze test (r = -0.6188, -0.5190, -0.5240). 2,3 and 5 month old 5XFAD mouse dorsal CA1 was positively correlated with the target quadrant time of the water maze experiment. 0.5629): the T2 values in the subregions of the ventral hippocampal subregions were not found to be related to the two. Conclusion: the microstructural integrity of the hippocampal tissue was impaired in the early hippocampal tissue in the 5XFAD mice and increased with the progression of the disease. The tissue integrity damage in the dorsal hippocampus was related to its spatial cognitive function. The important role of hippocampus in the early AD, and the T2 value of the dorsal hippocampus may be one of the biomarkers to assist the early diagnosis of AD and monitor the progress of AD. The study of the early brain neuron activity and its influence on spatial cognitive function of the second part of 5XFAD mice: To explore the pattern of early brain neuron activity change and its space in 5XFAD mice. Methods: (1) the study adopted 5XFAD model mice and wild type C57BL/6 mice with four time points of 1,2,3 and 5 month old. Using sucrose water preference experiment, open field experiment, elevated cross maze experiment and Morris water maze experiment, the animal (baseline) behavior assessment was carried out. (2) each time was completed by 7.0Tmicro-MRI. The baseline T1 weighted imaging (T1 weighted imaging, T1WI) of the two groups of mice was followed by intraperitoneal injection of Mnc12 solution, 24 hours interval, 4 times (13.95 mg/kg per time), and 24 hours after the last injection (manganese-enhanced magnetic resonance imaging, MEMRI). (3) two mice were observed every day after the imaging. After the first injection of MJncl2 solution, 12 days after the first injection, the open field experiment and the Morris water maze experiment were completed again. (4) the difference between the signal intensity (signal intensity, SI) of the gray matter brain regions of the 5XFAD mice and the wild type mice at each time point was contrasted with ROI, and the average signal intensity of two times before and after the injection of Mnc12 in the same mouse was observed. The degree difference [SI=100% x (SI after SI baseline SI baseline period) /SI baseline] as an indicator to reflect the concentration of MR2+ aggregation. (5) correlation analysis was used to evaluate the correlation between the signal intensity values of ROI and the cognitive score. Results: (1) behavioral results: 1,2,3 and 5 month old 5XFAD mice and wild mice in sucrose water preference experiment, open field experiment and There was no significant difference in the behavioral performance of the elevated cross maze experiment with.Morris water maze. Compared with the wild type mice, there was no significant difference in the latency time of the 1 month old 5XFAD mice at the first 5 days of the learning and training period, and the target quadrant residence time in the sixth day memory detection was significantly reduced (p=0.0365); 2 and 3 month old 5XFAD mice were in the training period. The latency time of the 3 days to the platform was prolonged (p=0.0024, 0.0300). The retention time of target quadrants in the memory detection process was significantly reduced (P 0.001, = 0.011), and the latency time of the 3,4 and 5 days to the platform was prolonged (P = 0.0154,0.0154,0.0011) in the learning and training period of 5 month old 5XFAD mice, and the retention time of the target quadrant in the memory detection process was significantly reduced (P = 0.034) (2) (2) MRI results: compared with the same month old wild type mice, there was no significant difference in the SI value of the brain regions of 1 month old 5XFAD mice; the SI value of the hippocampus (Hip) of the 2 month old 5XFAD mice increased significantly (P = 0.0167); the 3 month old 5XFAD mice hippocampus and the inner olfactory cortex region (entorhinal cortex, ERC) significantly increased (= 5 month old); 5 month old mice Caudate-putamen (CPu), theretrosplenial cortex, RSC, SI value of HIP and ERC increased significantly (= 0.0228,0.0795,0.0167, 0.0684). (3) correlation analysis results: 5 month old 5XFAD mice Hip SI values were positively correlated with the fifth day incubation period of water maze experiment (0.8668) and 5 month old The target quadrant time of the labyrinth experiment was negative correlation (r = -0.8269, -0.8364); the SI value of ERC in 2,3 and 5 month old of 5XFAD mice was negatively correlated with the stay time of the target quadrant (r = -0.9204, -0.8576, -0.8386). Neuronal activity in the brain region (especially in the hippocampus and the olfactory cortex) increased significantly with the progression of the disease. The activity of neurons in the hippocampus and the inner olfactory cortex was related to the state of spatial cognitive function. This study demonstrated the important role of the hippocampus and the olfactory cortex at the early stage of AD from the animal model at the body image level, and the early neuronal activity. Sex may be one of the biomarkers for early diagnosis of AD.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16;R-332

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