VD小鼠模型的建立及其被动逃避反应和空间学习记忆的改变

发布时间：2018-03-07 11:22

  本文选题：血管性痴呆　切入点：小鼠　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:血管性痴呆(vascular dementia,VD)主要是因脑组织血液循环障碍所致的一种获得性、进行性认知功能障碍综合征,是最常见的老年期痴呆原因之一。随着社会人口的老龄化和脑血管疾病发病率的增高,VD的发病率也随之增高,严重地威胁着老年人的身体健康和生存质量。 但是,VD确切的发病机制尚不清楚,临床也缺乏有效的预防和治疗药物。建立理想的VD动物模型是探讨其发病机制,研制有效的防治药物并进行评价的关键。目前常用的VD动物模型有:血管阻断VD模型、血管内栓塞VD模型、高脂血症VD模型、VD自发模型和光化学诱导VD模型等,均以模拟人类VD发病特点为基础而建立,种类较多,但效果各异,尚缺乏稳定可靠的VD动物模型。 用于VD研究的动物首先应有与人类相似的脑血管解剖特点,其次应该便于进行学习记忆的行为学测试。考虑到小鼠在具备上述要求的同时,价格低廉,繁殖快,生存率高,实验操作简便。因此,本实验选用昆明小鼠,建立五种VD小鼠模型,并通过避暗箱实验和Morris水迷宫对上述五种模型中小鼠学习记忆的改变进行检测,同时应用硫堇染色观察小鼠海马神经元损伤情况,再结合小鼠大脑的大体改变对上述各VD模型的效果进行比较,以筛选出较为稳定可靠的VD模型,为后续VD发病机制及有效防治措施的研究奠定基础。 方法:将105只28-32克健康雄性昆明小鼠随机分为以下7组(n=15): (1)正常组:正常小鼠,不做任何处理。 (2)Sham组:只暴露双侧颈总动脉,但不阻断血流。 (3)10分钟模型组:分别暴露双侧颈总动脉,反复夹闭双侧颈总动脉3次,每次夹闭10分钟,每次间隔10分钟,于第一次夹闭颈总动脉的同时尾动脉放血0.3 ml。 (4)20分钟模型组:分别暴露双侧颈总动脉,反复夹闭双侧颈总动脉3次,每次夹闭20分钟,每次间隔10分钟,于第一次夹闭颈总动脉的同时尾动脉放血0.3 ml。 (5)30分钟模型组:分别暴露双侧颈总动脉,反复夹闭双侧颈总动脉3次,每次夹闭30分钟,每次间隔10分钟,于第一次夹闭颈总动脉的同时尾动脉放血0.3 ml。 (6)左侧结扎模型组:分别暴露双侧颈总动脉,左侧颈总动脉永久性结扎,右侧颈总动脉反复夹闭3次,每次夹闭30分钟,每次间隔10分钟。 (7)右侧结扎模型组:分别暴露双侧颈总动脉,右侧颈总动脉永久性结扎,左侧颈总动脉反复夹闭3次,每次夹闭30分钟,每次间隔10分钟。 各组动物在相同环境下饲养4周。其中正常组动物直接于饲养4周后,其余各组动物于术后4周时,首先通过避暗箱实验和Morris水迷宫检测其学习记忆能力的改变;待行为学实验结束后取材,采集小鼠大脑大体标本图像,观察其大体改变,同时应用硫堇染色,观察海马神经元损伤情况。 结果: 1小鼠学习记忆能力的改变 1.1被动逃避反应的改变——避暗箱实验 饲养或术后第29天,开始对所有动物进行避暗箱测试,采用被电次数(次/3 min)和避暗潜伏期(s)作为学习记忆的检测指标。 结果显示:正常组小鼠的被电次数是1.7±1.0。与正常组相比,Sham组小鼠的被电次数(1.8±0.8)无显著变化。与正常组和Sham组相比,各模型组的被电次数均有增加的趋势,但无统计学意义。 正常组小鼠的避暗潜伏期为267.0±52.9。与正常组相比,Sham组小鼠的避暗潜伏期(247.9±101.5)无显著性变化,而左侧结扎模型组小鼠的避暗潜伏期(198.9±103.6)和右侧结扎模型组小鼠的避暗潜伏期(186.4±111.5)均显著缩短(P0.05)。其余10分钟模型组、20分钟模型组和30分钟模型组,与正常组和Sham组相比,均有一定的缩短趋势,但无统计学意义。 以上结果表明,左侧结扎模型组小鼠和右侧结扎模型组小鼠在被动逃避反应中显示出明显的学习记忆能力的下降。 1.2空间学习记忆能力的改变——Morris水迷宫实验 避暗箱实验结束后,对所有实验动物进行Morris水迷宫测试,以定向航行阶段的寻台潜伏期(s)和空间搜索阶段的平台所在象限滞留时间(s)、寻台次数(次)作为学习记忆的检测指标。 结果显示:在定向航行实验阶段,正常组小鼠定向航行5 d的寻台潜伏期逐渐缩短。与正常组小鼠相比,Sham组小鼠的寻台潜伏期无显著性变化。与正常组、Sham组、10分钟模型组和20分钟模型组相比,30分钟模型组和右侧结扎模型组小鼠的寻台潜伏期均显著延长(P0.05);并且,右侧结扎模型组小鼠的寻台潜伏期还显著长于左侧结扎模型组小鼠的寻台潜伏期(P0.05);10分钟模型组、20分钟模型组和左侧结扎模型组小鼠的寻台潜伏期均无显著性变化。 在空间搜索实验阶段,60 s内正常组小鼠的平台所在象限滞留时间是21.3±8.1,寻台次数是4.1±2.1。与正常组相比,Sham组小鼠的平台象限滞留时间(18.1±8.8)、寻台次数(2.7±1.8),均无显著性差异;而10分钟模型组的平台象限滞留时间(15.1±7.9)、寻台次数(2.5±1.9),30分钟模型组的平台象限滞留时间(14.4±6.0)、寻台次数(2.1±1.9),左侧结扎模型组的平台象限滞留时间(15.1±7.3)、寻台次数(2.0±1.6),以及右侧结扎模型组的平台象限滞留时间(14.0±5.8)、寻台次数(2.2±1.4)均显著减少(P0.05)。与Sham组相比,仅右侧结扎模型组的平台象限滞留时间显著缩短(P0.05)。20分钟模型组的平台象限滞留时间(16.9±8.8)、寻台次数(3.1±2.1)与正常组和Sham组相比,均无显著性变化。 以上结果表明,10分钟模型组、30分钟模型组、左侧结扎模型组和右侧结扎模型组小鼠的空间学习记忆能力均有不同程度的下降,其中以右侧结扎模型组小鼠下降最为显著。 2小鼠大脑的形态学改变 各组小鼠于行为学实验后取材,首先在体视显微镜下观察小鼠大脑的大体外观改变。结果显示:正常组小鼠的大脑外观晶莹剔透,脑组织饱满、无缺损。Sham组小鼠的大脑外观与正常组小鼠相似,脑组织饱满、无明显缺损。各模型组中,除了10分钟模型组小鼠未见明显的脑组织缺损外,其余4个模型组中,均有部分小鼠的脑组织出现明显的梗死灶。其中,20分钟模型组中出现明显梗死灶的小鼠占20%;30分钟模型组中占13%;左侧结扎模型组中占13%;右侧结扎模型组中占33%。 以上结果表明,右侧结扎模型组小鼠脑组织大体损伤的成功率最高。 3小鼠海马的组织病理学改变 对整个海马的组织病理学评价结果显示:正常组和Sham组小鼠海马各区锥体神经元排列致密、整齐,胞核饱满,核仁清晰;组织学分级(Histological grade,HG)均为0～Ⅱ级,其中HG 0～Ⅰ级的分别占87%和93%。 相比之下,10分钟模型组和20分钟模型组小鼠海马各区锥体细胞排列较整齐,核固缩现象较少;其中,10分钟模型组小鼠HG 0～Ⅰ级的占60%,HGⅡ级的占33%,HGⅢ级的占7%;20分钟模型组小鼠HG 0～Ⅰ级的占66%,HGⅡ级的占27%,HGⅢ级的占7%;与正常组和Sham组相比,此两组的HG均有升高的趋势,但无统计学意义。 30分钟模型组部分小鼠海马可见片段性的锥体细胞缺失,HG 0～Ⅰ级的占67%,HGⅡ级的占20%,HGⅢ级的占13%;与正常组和Sham组相比,该组的HG也仅有升高的趋势,但无统计学意义。 左侧结扎模型组和右侧结扎模型组小鼠海马片段性的锥体细胞缺失显著增多;其中,左侧结扎模型组HG 0～Ⅰ级的占47%,HGⅡ级的占33%,HGⅢ级的占20%;右侧结扎模型组HG 0～Ⅰ级的占47%,HGⅡ级的占26%,HGⅢ级的占27%;与正常组和Sham组相比,此两组的HG均显著升高(P0.05),并且右侧结扎模型组小鼠的HG还显著高于30分钟模型组小鼠的HG(P0.05)。 以上结果表明,左侧结扎模型组和右侧结扎模型组造成的小鼠海马神经元损伤较为严重,并且,以右侧结扎模型组海马神经元损伤最为严重。 结论: 本实验建立的5种VD小鼠模型中,右侧结扎模型组造成的小鼠大脑大体损伤的成功率最高,引起的海马神经元的损伤最为严重;该组小鼠的被动逃避反应和空间学习记忆能力的降低最为显著,且表现较为稳定。
[Abstract]:Objective: vascular dementia (vascular dementia VD) is mainly due to an acquired brain blood circulation disorder caused by the progressive cognitive dysfunction syndrome, is one of the most common cause of dementia in the elderly. With the aging of the population and the incidence of cerebrovascular diseases increased, the incidence of VD is also increased, a serious threat to the health and quality of life of the elderly.
However, the exact mechanism of VD is not clear, and lack of clinical prevention and treatment of drug effective. Establish the ideal animal model of VD is to explore its pathogenesis, key to the development of effective drugs and evaluation. At present VD animal models are commonly used: VD vascular occlusion model, endovascular embolization in VD model. Hyperlipidemia, VD model, VD model and VD model of photochemically induced spontaneous, both to simulate the characteristics of human VD pathogenesis based, more categories, but the effect is different, the lack of VD animal model is stable and reliable.
For the characteristics of cerebral vascular anatomy research of VD animal should first be similar to humans, secondly should facilitate the learning and memory behavior were tested. Considering the mice in with the above requirements at the same time, low price, fast breeding, high survival rate, the operation is simple. Therefore, the selection of Kunming mice, five VD mice were established model, and by avoiding dark box test and Morris water maze to change the above five models of learning and memory of mice were detected at the same time were observed in mice hippocampal neurons injury by thionine, combined with the change of the mouse brain in each VD model to compare the effects of selected VD model is more stable and reliable. The study laid the foundation for the follow-up of VD pathogenesis and effective prevention and control measures.
Methods: 105 28-32 gram healthy male Kunming mice were randomly divided into 7 groups (n=15).
(1) normal group: normal mice, do not do any treatment.
(2) group Sham: only bilateral common carotid artery was exposed, but the blood flow was not blocked.
(3) 10 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 10 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(4) 20 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 20 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(5) 30 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 30 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(6) the left ligation model group was exposed to bilateral common carotid arteries, the left common carotid artery was ligated permanently, and the right common carotid artery was clipped repeatedly for 3 times. Each clamp was closed for 30 minutes, and the interval between each time was 10 minutes.
(7) the right side ligation model group: the bilateral common carotid arteries were exposed respectively, the right common carotid artery was permanently ligated, and the left common carotid artery was clipped repeatedly for 3 times. Each clamp was closed for 30 minutes, and the interval between each time was 10 minutes.
Each animal feeding for 4 weeks in the same environment. The normal animal directly after 4 weeks feeding, the rest in each animal after 4 weeks, first by avoiding dark box test and Morris water maze test the ability of learning and memory behavior change; to the end of the experiment were collected, the mouse brain specimen images. To observe the general change, at the same time using thionin staining, observed the injury of hippocampal neurons.
Result:
1 changes in learning and memory ability of mice
1.1 change of passive avoidance response: dark box experiment
On the twenty-ninth day after raising or after operation, all animals were tested in the dark box test. The number of times of electricity (/3 min) and dark latency (s) were used as indicators for learning and memory.
The results showed that the number of times of electric charge in normal group was 1.7 + 1.0.. Compared with the normal group, the number of times of electric shock in group Sham (1.8 + 0.8) did not change significantly. Compared with the normal group and Sham group, the number of times of electric charge increased in all models, but there was no statistical significance.
In the normal group the dark incubation period was 267 + 52.9. compared with the normal group, Sham group of mice the avoiding latency was (247.9 + 101.5) no significant changes, but the left ligation model mice stepthrough latency (198.9 + 103.6) and right ligation model mice stepthrough latency (186.4 + 111.5) were significantly shortened (P0.05). The remaining 10 minutes group, 20 minutes and 30 minutes in model group compared with model group, normal group and Sham group had a tendency to reduce, but not statistically significant.
The above results showed that the mice in the left ligation model group and the right ligation model group showed a significant decline in the learning and memory ability in the passive avoidance response.
The change of learning and memory in 1.2 space -- Morris water maze experiment
After the end of the dark box experiment, the Morris water maze test was carried out on all the experimental animals. The incubation period (s) of the navigation phase and the quadrant residence time (s) of the platform in the spatial search stage were selected. The number of search times (Times) was used as a detection index for learning and memory.
The results showed that: in the navigation experiment stage, normal mice navigation 5 d eacape latency shortened gradually. Compared with the normal mice, Sham mice eacape latency had no significant change. With the normal group, Sham group, model group, 10 minutes and 20 minutes compared to the model group, the model group and 30 minutes the ligation model mice eacape latency were significantly prolonged (P0.05); and, on the right side of ligation model mice eacape latency significantly longer than the left ligation model mice eacape latency (P0.05); 10 minutes 20 minutes in model group, model group and ligation of left model group mice were eacape latency no significant change.
鍦ㄧ┖闂存悳绱㈠疄楠岄樁娈，

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