血管性痴呆小鼠模型的建立及其空间学习记忆能力的评价
发布时间：2018-09-03 17:01
【摘要】：目的:血管性痴呆(Vascular Dementia, VD)是由各种脑血管因素,如脑缺血、脑出血等导致的脑组织损伤,从而引起以认知功能障碍为主要特征的痴呆综合征。VD是一种慢性进行性疾病,也是导致老年期痴呆的一个重要原因。在欧美等西方国家,VD是导致痴呆的第二位原因,而在一些亚洲国家VD则是导致痴呆的最主要原因。近年来,随着全球人口的老龄化,VD的发病率逐年增高,严重威胁老年人的身体健康和生活质量。 建立稳定、可复制的动物模型对于研究VD的发生发展机制、防治措施及相关药物的疗效评价至关重要。目前,国内外常用的VD模型主要以大鼠和小鼠为研究对象。大鼠模型的制备方法有血管阻断法VD模型、去脑皮层VD模型、高血压复合VD模型、VD自发模型、静脉注入四氧化三铁VD模型等。小鼠VD动物模型则主要为不完全性脑缺血再灌注模型,其中较经典的是双侧颈总动脉缺血—再灌注加尾部放血法。目前,国内外关于VD动物模型制备的研究和探讨虽然取得了很大的进步,但仍然存在着诸多问题,如:痴呆模型制作标准不统一;可重复性差;个别方法操作复杂,动物存活率低;动物模型的病理生理学改变与临床患者差异较大,不利于长期观察等。理想的VD动物模型,理论上应与临床患者的病理损害一致,并伴有动物的智能损害;同时,模型的制备还应具有容易操作、稳定可靠、重复性强、便于各种电生理检查及智能测试的特点。但是,目前尚无完全达到此种理想程度的动物模型。 八臂迷宫实验通过观察动物寻找食物过程中所走的路程长度及典型错误的次数评价动物的空间学习记忆能力,能较客观地反映动物的痴呆程度。此外,除了行为学指标,病理组织学结果也是判断VD模型成功与否的一项重要依据。海马作为参与学习与记忆功能的重要结构,对脑组织缺血缺氧非常敏感,反复、适时的脑缺血—再灌注会导致海马CA1区神经元受损。因此,观察海马神经元的损伤程度有助于判断VD模型的成功情况。 综上所述,本实验旨在既往实验基础上制备四种小鼠VD模型,通过八臂迷宫实验检测其空间学习记忆能力;同时辅以病理组织学观察海马CA1区锥体神经元损伤情况,以评价模型的效果,为VD的基础研究及疾病防治研究提供实验依据。 动物分组及方法:150只雄性成年昆明小鼠,体重34±2.5 g,实验室环境中饲养1周,随机分为以下5组(n=30): (1) Sham组:仅暴露双侧颈总动脉,不阻断血流; (2)双侧夹闭20分钟模型:用无损伤小动脉夹夹闭双侧颈总动脉20 min,松开血管夹再灌流10 min,反复3次,同时尾端放血0.3 ml; (3)双侧夹闭30分钟模型:用无损伤小动脉夹夹闭双侧颈总动脉30 min,松开血管夹再灌流10 min,反复3次,同时尾端放血0.3 ml; (4)右侧结扎+左侧夹闭模型:用无菌丝线永久结扎右侧颈总动脉,同时用无损伤小动脉夹夹闭左侧颈总动脉30 min,松开血管夹再灌流10 min,反复3次; (5)左侧结扎+右侧夹闭模型:用无菌丝线永久结扎左侧颈总动脉,同时用无损伤小动脉夹夹闭右侧颈总动脉30 min,松开血管夹再灌流10 min,反复3次。 以上各组动物根据再灌注后的时间进一步分为15天、30天及45天亚组,每个亚组10只动物:分别于术后第15天、30天、45天开始八臂迷宫实验,记录小鼠找到所有食物走过的路程长度(路程)以及小鼠进入无食物臂的典型错误次数(典型错误),连续观察10天以反映其空间学习记忆能力情况;后随机选取6只断头取脑,常规石蜡切片,硫堇染色,确定CA1区锥体神经元迟发性死亡(delayed neuronal death, DND)情况。参照Kitagawa和Kato分级方法,对海马CA1区组织学改变进行分级,标准如下:0级,无神经元死亡;Ⅰ级,散在的神经元死亡;Ⅱ级,大片的神经元死亡;Ⅲ级,几乎全部的神经元死亡。高倍镜下计数双侧海马CA1区1 mm区段内胞膜完整、胞核饱满、核仁清楚的锥体细胞数目,每侧海马CA1区各计数3个区段,取平均数为神经元密度(neuronal density),作为判定迟发性神经元损伤程度的依据。 结果: 1空间学习记忆能力的变化 1.1四种VD模型小鼠同一时间点空间学习记忆能力的差异 1.1.1四种VD模型小鼠15天空间学习记忆能力的差异 与sham组相比,双侧夹闭20分钟模型组、双侧夹闭30分钟模型组、左侧结扎+右侧夹闭模型组小鼠的路程及典型错误均无明显改变(P0.05);而右侧结扎+左侧夹闭模型组,路程明显延长、典型错误明显增多(P0.05),提示小鼠的学习记忆能力明显降低。 1.1.2四种VD模型小鼠30天空间学习记忆能力的差异 与sham组比较,双侧夹闭20分钟模型组小鼠的路程及典型错误无明显变化(P0.05);而双侧夹闭30分钟模型组、左侧结扎+右侧夹闭模型组及右侧结扎+左侧夹闭模型组的小鼠,路程明显增加、典型错误也明显增多(P0.05)。并且,上述各组中右侧结扎+左侧夹闭模型组的路程及典型错误变化最为明显,提示小鼠的学习记忆能力最差。 1.1.3四种VD模型小鼠45天空间学习记忆能力的差异 与sham组比较,上述4组VD模型组小鼠的路程及典型错误均明显增加(P0.05),其中右侧结扎+左侧夹闭模型组小鼠的结果最为显著。 1.2同一模型组小鼠不同时间点空间学习记忆能力的差异sham组三个时间点路程及典型错误比较,无显著性差别(P0.05)。因此,随机取其一作为4种VD模型的sham组。 1.2.1双侧夹闭20分钟模型 与sham组比较, 15天模型组及30天模型组小鼠的路程无明显延长、典型错误无明显增多(P0.05),而45天模型组小鼠的路程及典型错误均明显增加(P0.05)。 1.2.2双侧夹闭30分钟模型 结果显示,与sham组相比,15天模型组小鼠的路程及典型错误均无明显增加(P0.05),而30天模型组、45天模型组小鼠的路程明显增加(P0.05),并且典型错误也明显增多(P0.05)。 1.2.3右侧结扎+左侧夹闭模型 与sham组相比,15天模型组、30天模型组及45天模型组小鼠的路程延长及典型错误增加(P0.05),并且30天模型组及45天模型组小鼠的空间学习记忆能力最差。 1.2.4左侧结扎+右侧夹闭模型 与sham组相比,15天模型组小鼠的路程、典型错误均无统计学差异(P0.05),而30天模型组、45天模型组小鼠的路程延长、典型错误增加,与sham组相比有统计学差异(P0.05)。 2海马CA1区组织病理学评价 2.1四种VD模型小鼠同一时间点海马CA1区组织病理学变化 2.1.1四种VD模型小鼠15天组海马CA1区组织病理学变化Sham组小鼠海马CA1区有3~5层锥体细胞,排列整齐致密、细胞形态完整、胞核饱满、核仁清晰,无细胞损伤。双侧夹闭20分钟模型组、双侧夹闭30分钟模型组及左侧结扎+右侧夹闭模型组海马CA1区无明显的神经元损伤。然而,右侧结扎+左侧夹闭模型组小鼠海马CA1区锥体神经元出现明显损伤,与sham组比较,HG明显升高(P0.05),ND值显著降低(P0.05)。 2.1.2四种VD模型小鼠30天组海马CA1区组织病理学变化双侧夹闭20分钟模型组小鼠海马CA1区无明显的DND,神经元密度略有降低、组织学分级略有升高,但与sham组比较无明显的统计学差异(P0.05)。双侧夹闭30分钟模型组、右侧结扎+左侧夹闭模型组、左侧结扎+右侧夹闭模型组小鼠海马CA1区均出现了明显的DND,与sham组相比,HG显著升高(P0.05),ND值显著降低(P0.05)。其中右侧结扎+左侧夹闭模型组海马CA1区损伤最为严重。 2.1.3四种VD模型小鼠45天组海马CA1区组织病理学变化 结果显示,与sham相比,4个模型组小鼠海马CA1区均出现明显的DND,HG显著升高(P0.05),ND值显著降低(P0.05)。上述变化以右侧结扎+左侧夹闭模型组最明显。 2.2同一模型组小鼠不同时间点海马CA1区组织病理学变化 sham组三个时间点海马CA1区锥体神经元的HG、ND值比较,无显著性差别(P0.05)。因此,随机取其一作为4种VD模型的sham组。 2.2.1双侧夹闭20分钟模型 15天模型组及30天模型组小鼠海马CA1区无明显的DND。与sham组相比,45天模型组海马CA1区出现明显的DND,其HG显著升高(P0.05),ND值显著降低(P0.05)。 2.2.2双侧夹闭30分钟模型 结果显示,15天模型组小鼠海马CA1区无明显的DND。而30天模型组、45天模型组海马CA1区锥体神经元损伤严重,与sham相比HG显著升高(P0.05),ND值显著降低(P0.05)。 2.2.3右侧结扎+左侧夹闭模型 本VD模型小鼠在15天、30天和45天组海马CA1区均出现了明显的DND,与sham组相比,HG显著升高(P0.05),ND值显著降低(P0.05),其中30天、45天模型组改变最为明显。 2.2.4左侧结扎+右侧夹闭模型 硫堇染色显示,15天模型组小鼠海马CA1区无明显的DND,与sham组比较,HG和ND无统计学意义(P0.05)。30天模型组、45天模型组小鼠海马CA1区锥体神经元损伤严重,与sham组比较HG显著升高(P0.05)、ND值显著降低(P0.05)。 结论: 所建立的4种VD模型中,小鼠分别在不同时段表现出空间学习记忆障碍及海马CA1区锥体神经元的延迟性死亡,其中右侧结扎+左侧夹闭模型组小鼠最为显著。
[Abstract]:Objective: Vascular dementia (VD) is a kind of dementia syndrome characterized by cognitive impairment caused by various cerebrovascular factors, such as cerebral ischemia and cerebral hemorrhage. VD is a chronic progressive disease and an important cause of senile dementia. VD is the second leading cause of dementia, and in some Asian countries VD is the main cause of dementia. In recent years, with the aging of the global population, the incidence of VD increases year by year, seriously threatening the health and quality of life of the elderly.
Establishing a stable and reproducible animal model is very important to study the mechanism of VD, the prevention and treatment measures and the efficacy evaluation of related drugs.At present, the commonly used models of VD at home and abroad mainly focus on rats and mice.The preparation methods of rat models include VD model by blocking blood vessels, VD model by removing cerebral cortex and VD model by combining hypertension. The animal model of VD in mice is mainly incomplete cerebral ischemia-reperfusion model, among which the classical method is bilateral common carotid artery ischemia-reperfusion plus tail bleeding. However, there are many problems, such as: the standard of making dementia model is not uniform; the repeatability is poor; individual methods are complex and the survival rate of animals is low; the pathophysiological changes of animal models are quite different from clinical patients, which is not conducive to long-term observation. The ideal animal model of VD should be consistent with the pathological damage of clinical patients in theory, and accompanied by movement. At the same time, the preparation of the model should be easy to operate, stable and reliable, repeatable, easy to conduct various electrophysiological examinations and intelligent testing. However, there is no animal model to achieve this ideal level.
The eight-arm maze experiment can objectively reflect the dementia degree of animals by observing the distance and the number of typical errors in the process of looking for food. In order to participate in the important structure of learning and memory function, it is very sensitive to cerebral ischemia and hypoxia. Repeated cerebral ischemia-reperfusion can lead to damage of neurons in hippocampal CA1 area. Therefore, it is helpful to judge the success of VD model to observe the degree of damage of hippocampal neurons.
To sum up, this experiment aims to prepare four kinds of mice VD models on the basis of previous experiments, and test their spatial learning and memory ability by eight-arm maze experiment, and observe the damage of pyramidal neurons in hippocampal CA1 area with pathological histology, so as to evaluate the effect of the model, and provide experimental basis for the basic research of VD and disease prevention and treatment.
Animal grouping and methods: 150 adult male Kunming mice weighing 34 + 2.5 g were randomly divided into the following five groups (n = 30):
(1) group Sham: only bilateral carotid arteries were exposed without blocking blood flow.
(2) 20-minute bilateral clamping model: the bilateral common carotid artery was clamped with non-invasive small artery clamp for 20 minutes, the clamp was loosened and then perfused for 10 minutes, repeated 3 times, and the tail bleeding was 0.3 ml.
(3) 30-minute bilateral clamping model: the bilateral common carotid artery was clamped with non-invasive small artery clamp for 30 minutes, the clamp was loosened and then perfused for 10 minutes, repeated three times, and the tail bleeding was 0.3 ml.
(4) The right common carotid artery was permanently ligated with hyphal-free thread, and the left common carotid artery was clamped with non-invasive small artery for 30 minutes. The clamp was loosened for 10 minutes and then perfused for 3 times.
(5) The left common carotid artery was permanently ligated with hyphal-free thread, and the right common carotid artery was clamped with noninvasive small artery for 30 minutes. The clamp was loosened for 10 minutes and then perfused for 3 times.
Each group was further divided into 15 days, 30 days and 45 days subgroups according to the time after reperfusion. Each subgroup consisted of 10 animals. The eight-arm maze test was performed on the 15th, 30th and 45th days after reperfusion. The length of the journey (the journey) for which the mice found all the food and the number of typical errors (the typical error) for which the mice entered the food-free arm were recorded. After 10 days of observation, 6 severed heads were randomly selected to observe their spatial learning and memory abilities. The delayed neuronal death (DND) in CA1 region was determined by routine paraffin section and staining with thionine. Neuronal death; Grade I, scattered neuron death; Grade II, large neuron death; Grade III, almost all neuron death. At high power microscopy, the number of pyramidal cells in 1 mm segment of bilateral hippocampal CA1 region was counted intact, the nucleus was plump, and the nucleolus was clear. The average number of pyramidal cells in each hippocampal CA1 region was 3 segments, and the average number was neu. Ronal density) as a basis for judging the degree of delayed neuronal damage.
Result:
1 changes in spatial learning and memory abilities
1.1 the difference of spatial learning and memory ability between four VD mice at the same time.
Differences in learning and memory ability of 15 mouse models in four 1.1.1 VD models
Compared with sham group, there were no significant changes in the course and typical errors in the 20-minute clipping model group, 30-minute clipping model group, left ligation + right clipping model group (P 0.05), while the right ligation + left clipping model group, the course was significantly prolonged and the typical errors were significantly increased (P 0.05), suggesting that the learning and memory abilities of mice were significantly decreased. Low.
Differences in learning and memory ability of 30 mouse models in four 1.1.2 VD models
Compared with sham group, there were no significant changes in the distance and typical errors of mice in bilateral clamping 20 minutes model group (P 0.05), while the distance and typical errors of mice in bilateral clamping 30 minutes model group, left ligation + right clamping model group and right ligation + left clamping model group were significantly increased (P 0.05). The changes of distance and typical errors were the most obvious in the ligation + left clipping model group, suggesting that the learning and memory abilities of the mice were the worst.
Differences in learning and memory ability of 45 mouse models in four 1.1.3 VD models
Compared with sham group, the routing and typical errors of VD model mice in the above four groups were significantly increased (P 0.05), and the results of right ligation + left clipping model mice were the most significant.
1.2 There was no significant difference in the spatial learning and memory abilities between the same model group and the sham group at different time points (P 0.05). Therefore, one of the four VD models was randomly selected as the sham group.
1.2.1 bilateral occlusion 20 minute model
Compared with the sham group, the 15-day model group and 30-day model group showed no significant prolongation of the distance and no significant increase in typical errors (P 0.05), while the 45-day model group showed significant increase in the distance and typical errors (P 0.05).
1.2.2 bilateral occlusion 30 minute model
The results showed that compared with the sham group, the 15-day model group had no significant increase in the distance and typical errors (P 0.05), while the 30-day model group and the 45-day model group had significant increase in the distance (P 0.05) and the typical errors (P 0.05).
1.2.3 ligation + left clipping model
Compared with sham group, the 15-day model group, 30-day model group and 45-day model group had longer distance and more typical errors (P 0.05), and the 30-day model group and 45-day model group had the worst spatial learning and memory ability.
1.2.4 ligation plus right clipping model
Compared with the sham group, there was no significant difference in the typical errors of the 15-day model group (P 0.05), while the 30-day model group and 45-day model group had longer and more typical errors, which was significantly different from the sham group (P 0.05).
2 histopathological evaluation of hippocampal CA1 area
2.1 histopathological changes in hippocampal CA1 area of four VD mice at the same time point.
2.1.1 Histopathological changes in CA1 area of hippocampus of four VD model mice in 15 days Sham group had 3-5 layers of pyramidal cells in CA1 area of hippocampus, arranged neatly and tightly, with complete cell morphology, full nucleus, clear nucleolus and no cell damage. However, the pyramidal neurons in the CA1 area of the hippocampus in the right ligation + left clipping model group were significantly damaged. Compared with the sham group, HG increased significantly (P 0.05), and ND decreased significantly (P 0.05).
2.1.2 Histopathological changes of CA1 area in hippocampus of mice with three kinds of VD after 30 days occlusion for 20 minutes showed no obvious DND, slightly decreased neuron density and slightly increased histological grade in CA1 area of hippocampus of mice with bilateral occlusion for 30 minutes. There was no significant difference between the two groups (P 0.05). DND was found in the CA1 area of the hippocampus in the left ligation + right clipping model group, and HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05) compared with the sham group.
Histopathological changes in hippocampal CA1 area of mice in 45 days of 2.1.3 four VD models
The results showed that compared with sham, DND, HG and ND were significantly increased (P 0.05) and decreased (P 0.05) in CA1 area of hippocampus in the four model groups.
2.2 histopathological changes in hippocampal CA1 area of mice in the same model group at different time points
There was no significant difference in HG and ND values of pyramidal neurons in hippocampal CA1 region at three time points in sham group (P 0.05).
2.2.1 bilateral occlusion 20 minute model
There was no significant DND in CA1 area of hippocampus in 15-day model group and 30-day model group. Compared with sham group, DND appeared in CA1 area of hippocampus in 45-day model group, and HG increased significantly (P 0.05), ND decreased significantly (P 0.05).
2.2.2 bilateral occlusion 30 minute model
The results showed that there was no obvious DND in CA1 area of hippocampus in 15-day model group, but in 30-day model group, pyramidal neurons in CA1 area of hippocampus in 45-day model group were severely damaged, HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05) compared with sham.
2.2.3 ligation + left clipping model
DND in hippocampal CA1 region of VD model mice appeared on 15 days, 30 days and 45 days. Compared with sham group, HG increased significantly (P 0.05), ND decreased significantly (P 0.05), and the change was most obvious in 30 days and 45 days model group.
2.2.4 ligation plus right clipping model
Thionine staining showed that there was no significant DND in CA1 area of hippocampus in the 15-day model group, and there was no significant difference in HG and ND between the sham group and the 15-day model group (P 0.05). Pyramidal neurons in CA1 area of hippocampus in the 45-day model group were severely damaged, and HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05).
Conclusion:
Among the four VD models, mice showed spatial learning and memory impairment and delayed neuronal death in hippocampal CA1 region at different time points, especially in the right ligation + left clipping group.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R749.16;R-332
本文编号：2220612
[Abstract]:Objective: Vascular dementia (VD) is a kind of dementia syndrome characterized by cognitive impairment caused by various cerebrovascular factors, such as cerebral ischemia and cerebral hemorrhage. VD is a chronic progressive disease and an important cause of senile dementia. VD is the second leading cause of dementia, and in some Asian countries VD is the main cause of dementia. In recent years, with the aging of the global population, the incidence of VD increases year by year, seriously threatening the health and quality of life of the elderly.
Establishing a stable and reproducible animal model is very important to study the mechanism of VD, the prevention and treatment measures and the efficacy evaluation of related drugs.At present, the commonly used models of VD at home and abroad mainly focus on rats and mice.The preparation methods of rat models include VD model by blocking blood vessels, VD model by removing cerebral cortex and VD model by combining hypertension. The animal model of VD in mice is mainly incomplete cerebral ischemia-reperfusion model, among which the classical method is bilateral common carotid artery ischemia-reperfusion plus tail bleeding. However, there are many problems, such as: the standard of making dementia model is not uniform; the repeatability is poor; individual methods are complex and the survival rate of animals is low; the pathophysiological changes of animal models are quite different from clinical patients, which is not conducive to long-term observation. The ideal animal model of VD should be consistent with the pathological damage of clinical patients in theory, and accompanied by movement. At the same time, the preparation of the model should be easy to operate, stable and reliable, repeatable, easy to conduct various electrophysiological examinations and intelligent testing. However, there is no animal model to achieve this ideal level.
The eight-arm maze experiment can objectively reflect the dementia degree of animals by observing the distance and the number of typical errors in the process of looking for food. In order to participate in the important structure of learning and memory function, it is very sensitive to cerebral ischemia and hypoxia. Repeated cerebral ischemia-reperfusion can lead to damage of neurons in hippocampal CA1 area. Therefore, it is helpful to judge the success of VD model to observe the degree of damage of hippocampal neurons.
To sum up, this experiment aims to prepare four kinds of mice VD models on the basis of previous experiments, and test their spatial learning and memory ability by eight-arm maze experiment, and observe the damage of pyramidal neurons in hippocampal CA1 area with pathological histology, so as to evaluate the effect of the model, and provide experimental basis for the basic research of VD and disease prevention and treatment.
Animal grouping and methods: 150 adult male Kunming mice weighing 34 + 2.5 g were randomly divided into the following five groups (n = 30):
(1) group Sham: only bilateral carotid arteries were exposed without blocking blood flow.
(2) 20-minute bilateral clamping model: the bilateral common carotid artery was clamped with non-invasive small artery clamp for 20 minutes, the clamp was loosened and then perfused for 10 minutes, repeated 3 times, and the tail bleeding was 0.3 ml.
(3) 30-minute bilateral clamping model: the bilateral common carotid artery was clamped with non-invasive small artery clamp for 30 minutes, the clamp was loosened and then perfused for 10 minutes, repeated three times, and the tail bleeding was 0.3 ml.
(4) The right common carotid artery was permanently ligated with hyphal-free thread, and the left common carotid artery was clamped with non-invasive small artery for 30 minutes. The clamp was loosened for 10 minutes and then perfused for 3 times.
(5) The left common carotid artery was permanently ligated with hyphal-free thread, and the right common carotid artery was clamped with noninvasive small artery for 30 minutes. The clamp was loosened for 10 minutes and then perfused for 3 times.
Each group was further divided into 15 days, 30 days and 45 days subgroups according to the time after reperfusion. Each subgroup consisted of 10 animals. The eight-arm maze test was performed on the 15th, 30th and 45th days after reperfusion. The length of the journey (the journey) for which the mice found all the food and the number of typical errors (the typical error) for which the mice entered the food-free arm were recorded. After 10 days of observation, 6 severed heads were randomly selected to observe their spatial learning and memory abilities. The delayed neuronal death (DND) in CA1 region was determined by routine paraffin section and staining with thionine. Neuronal death; Grade I, scattered neuron death; Grade II, large neuron death; Grade III, almost all neuron death. At high power microscopy, the number of pyramidal cells in 1 mm segment of bilateral hippocampal CA1 region was counted intact, the nucleus was plump, and the nucleolus was clear. The average number of pyramidal cells in each hippocampal CA1 region was 3 segments, and the average number was neu. Ronal density) as a basis for judging the degree of delayed neuronal damage.
Result:
1 changes in spatial learning and memory abilities
1.1 the difference of spatial learning and memory ability between four VD mice at the same time.
Differences in learning and memory ability of 15 mouse models in four 1.1.1 VD models
Compared with sham group, there were no significant changes in the course and typical errors in the 20-minute clipping model group, 30-minute clipping model group, left ligation + right clipping model group (P 0.05), while the right ligation + left clipping model group, the course was significantly prolonged and the typical errors were significantly increased (P 0.05), suggesting that the learning and memory abilities of mice were significantly decreased. Low.
Differences in learning and memory ability of 30 mouse models in four 1.1.2 VD models
Compared with sham group, there were no significant changes in the distance and typical errors of mice in bilateral clamping 20 minutes model group (P 0.05), while the distance and typical errors of mice in bilateral clamping 30 minutes model group, left ligation + right clamping model group and right ligation + left clamping model group were significantly increased (P 0.05). The changes of distance and typical errors were the most obvious in the ligation + left clipping model group, suggesting that the learning and memory abilities of the mice were the worst.
Differences in learning and memory ability of 45 mouse models in four 1.1.3 VD models
Compared with sham group, the routing and typical errors of VD model mice in the above four groups were significantly increased (P 0.05), and the results of right ligation + left clipping model mice were the most significant.
1.2 There was no significant difference in the spatial learning and memory abilities between the same model group and the sham group at different time points (P 0.05). Therefore, one of the four VD models was randomly selected as the sham group.
1.2.1 bilateral occlusion 20 minute model
Compared with the sham group, the 15-day model group and 30-day model group showed no significant prolongation of the distance and no significant increase in typical errors (P 0.05), while the 45-day model group showed significant increase in the distance and typical errors (P 0.05).
1.2.2 bilateral occlusion 30 minute model
The results showed that compared with the sham group, the 15-day model group had no significant increase in the distance and typical errors (P 0.05), while the 30-day model group and the 45-day model group had significant increase in the distance (P 0.05) and the typical errors (P 0.05).
1.2.3 ligation + left clipping model
Compared with sham group, the 15-day model group, 30-day model group and 45-day model group had longer distance and more typical errors (P 0.05), and the 30-day model group and 45-day model group had the worst spatial learning and memory ability.
1.2.4 ligation plus right clipping model
Compared with the sham group, there was no significant difference in the typical errors of the 15-day model group (P 0.05), while the 30-day model group and 45-day model group had longer and more typical errors, which was significantly different from the sham group (P 0.05).
2 histopathological evaluation of hippocampal CA1 area
2.1 histopathological changes in hippocampal CA1 area of four VD mice at the same time point.
2.1.1 Histopathological changes in CA1 area of hippocampus of four VD model mice in 15 days Sham group had 3-5 layers of pyramidal cells in CA1 area of hippocampus, arranged neatly and tightly, with complete cell morphology, full nucleus, clear nucleolus and no cell damage. However, the pyramidal neurons in the CA1 area of the hippocampus in the right ligation + left clipping model group were significantly damaged. Compared with the sham group, HG increased significantly (P 0.05), and ND decreased significantly (P 0.05).
2.1.2 Histopathological changes of CA1 area in hippocampus of mice with three kinds of VD after 30 days occlusion for 20 minutes showed no obvious DND, slightly decreased neuron density and slightly increased histological grade in CA1 area of hippocampus of mice with bilateral occlusion for 30 minutes. There was no significant difference between the two groups (P 0.05). DND was found in the CA1 area of the hippocampus in the left ligation + right clipping model group, and HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05) compared with the sham group.
Histopathological changes in hippocampal CA1 area of mice in 45 days of 2.1.3 four VD models
The results showed that compared with sham, DND, HG and ND were significantly increased (P 0.05) and decreased (P 0.05) in CA1 area of hippocampus in the four model groups.
2.2 histopathological changes in hippocampal CA1 area of mice in the same model group at different time points
There was no significant difference in HG and ND values of pyramidal neurons in hippocampal CA1 region at three time points in sham group (P 0.05).
2.2.1 bilateral occlusion 20 minute model
There was no significant DND in CA1 area of hippocampus in 15-day model group and 30-day model group. Compared with sham group, DND appeared in CA1 area of hippocampus in 45-day model group, and HG increased significantly (P 0.05), ND decreased significantly (P 0.05).
2.2.2 bilateral occlusion 30 minute model
The results showed that there was no obvious DND in CA1 area of hippocampus in 15-day model group, but in 30-day model group, pyramidal neurons in CA1 area of hippocampus in 45-day model group were severely damaged, HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05) compared with sham.
2.2.3 ligation + left clipping model
DND in hippocampal CA1 region of VD model mice appeared on 15 days, 30 days and 45 days. Compared with sham group, HG increased significantly (P 0.05), ND decreased significantly (P 0.05), and the change was most obvious in 30 days and 45 days model group.
2.2.4 ligation plus right clipping model
Thionine staining showed that there was no significant DND in CA1 area of hippocampus in the 15-day model group, and there was no significant difference in HG and ND between the sham group and the 15-day model group (P 0.05). Pyramidal neurons in CA1 area of hippocampus in the 45-day model group were severely damaged, and HG was significantly increased (P 0.05) and ND was significantly decreased (P 0.05).
Conclusion:
Among the four VD models, mice showed spatial learning and memory impairment and delayed neuronal death in hippocampal CA1 region at different time points, especially in the right ligation + left clipping group.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R749.16;R-332
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