β-淀粉样蛋白增加Wistar大鼠癫痫发作易感性的实验研究
发布时间：2018-11-09 21:09
【摘要】：目的研究β-淀粉样蛋白(Beta amyloid protein,Aβ)对癫痫(Epilepsy,EP)病理机制发生发展的影响。从疾病发病机制的角度,探讨EP发作易感性增加与阿尔茨海默氏病(Alzheimer’s disease,AD)病理改变之间的相关性。方法选用6-8周成年Wistar大鼠40只,随机分为β-淀粉样蛋白1-42(Aβ1-42)+匹罗卡品(Pilocarpine,PILO)组、Aβ1-42组、磷酸盐缓冲液(Phosphate buffer solution,PBS)+PILO组、PBS组,每组各10只。采用脑立体定位仪将Aβ1-42注入Wistar大鼠海马CA3区,2周后观察大鼠Morris水迷宫实验,记录逃避潜伏期、目标象限内游泳时间比例和穿越平台次数,应用统计学分析软件,检测大鼠AD模型是否成功。建模成功后通过腹腔(Intraperitoneal,IP)注射氯化锂-匹罗卡品(Lithium chloride Pilocarpine,LI-PILO)制备大鼠慢性EP发作模型,依据Racine分级标准,记录EP发作潜伏期(达到IV级及以上发作所需时间)、发作程度和死亡率;采用改良的Timm’s染色方法观察各组大鼠海马齿状回(Dentate gyrus,DG)苔藓纤维芽生(Mossy fiber sprouting,MFS)水平。结果制备大鼠AD模型:注射Aβ1-42后,AD模型组和PBS对照组大鼠各死亡1只,解剖未发现明显人为死亡原因,推测可能的致死原因为大鼠之间存在个体差异;Morris水迷宫实验数据分析结果,AD模型组与PBS对照组大鼠相比逃避潜伏期显著延长(P0.01),目标象限内游泳时间比例缩短(P0.05),穿越平台的次数也明显减少(P0.05),差异均有统计学意义,表明AD模型制备成功。制备大鼠EP模型:依据Racine分级标准,Aβ1-42+PILO组与PBS+PILO组相比,EP发作潜伏期显著缩短(P0.01),发作程度更重(P0.05);Aβ1-42+PILO组与PBS+PILO组、PBS组相比死亡率均显著增加,分别为P=0.0294、P=0.0108。大鼠脑组织病理学实验:改良的Timm’s染色结果显示,Aβ1-42+PILO组与PBS+PILO组比较海马DG MFS染色强度显著增强(P0.05);在Aβ1-42组也观察到海马DG MFS染色强度增强,但和PBS+PILO组相比无统计学差异(P0.05);PBS+PILO组和PBS组比较MFS染色强度显著增强(P0.01);Aβ1-42组和PBS组比较MFS染色强度也显著增强(P0.01)。结论通过海马CA3区注射Aβ1-42,可以导致大鼠出现与海马有关的学习和记忆功能受损,采用Morris水迷宫实验从行为学上进一步验证上述功能障碍,证明Aβ可以诱导大鼠出现痴呆样行为改变。在Aβ存在的基础上诱导大鼠出现EP发作易感性是增加的,说明Aβ对于EP发生有一定的促进作用,从疾病病理学角度阐明AD与EP发作密切相关。组织化学染色观察到Aβ1-42+PILO组海马DG MFS存在高强度染色,进一步从病理生理学机制上验证Aβ诱导大鼠EP发作易感性增加的结构基础可能与MFS水平显著增加有关。尽管在Aβ1-42组没有EP发作,该组大鼠海马DG也出现MFS染色增强,这表明MFS不仅是EP发作的结果,而是代表在这些大鼠中固有的异常网络连接。在EP发作的“二次打击”学说中,Aβ可能的作用机制是导致大脑出现第一次脑损伤。EP发作易感性增加与AD发病机制之间具有显著的相关性。
[Abstract]:Objective to study the effect of 尾 -amyloid protein (Beta amyloid protein,A 尾 on the pathogenesis of epilepsy (Epilepsy,EP). To explore the relationship between the increased susceptibility of EP and the pathological changes of Alzheimer's disease (Alzheimer's disease,AD) from the point of view of the pathogenesis of the disease. Methods Forty adult Wistar rats aged 6-8 weeks were randomly divided into 尾 -amyloid 1-42 (A 尾 1-42) pilocarpine (Pilocarpine,PILO) group, A 尾 1-42 group, phosphate buffer (Phosphate buffer solution,PBS PILO group and PBS group (10 rats in each group). The A 尾 1-42 was injected into the CA3 area of the hippocampus of Wistar rats with a brain stereotactic locator. The Morris water maze test was observed two weeks later. The escape latency, the proportion of swimming time in the target quadrant and the times of crossing the platform were recorded, and the statistical analysis software was used. To detect the success of AD model in rats. The chronic EP seizure model of rats was established by intraperitoneal injection of lithium-pilocarpine (Lithium chloride Pilocarpine,LI-PILO) into abdominal cavity (Intraperitoneal,IP). According to the Racine grading standard, the incubation period of EP attack was recorded (the time required to reach IV grade and above). Severity and mortality; The (Mossy fiber sprouting,MFS levels of mossy fiber sprouts in dentate gyrus (Dentate gyrus,DG) of rats in each group were observed by modified Timm's staining. Results the AD model of rats was established: after injection of A 尾 1-42, one rat died in the AD model group and one in the PBS control group. No obvious cause of human death was found in the dissection, and the possible cause of death was inferred to be the individual difference between the rats. The results of Morris water maze test showed that the escape latency of AD model group was significantly longer than that of PBS control group (P0.01), and the proportion of swimming time in target quadrant was shortened (P0.05). The frequency of crossing the platform was also significantly reduced (P0.05), the differences were statistically significant, indicating that the AD model was successfully prepared. EP model of rats: according to the Racine grading standard, the latency of EP attack in A 尾 1-42 PILO group was significantly shorter than that in PBS PILO group (P0.01), and the severity of EP attack was more serious (P0.05). The mortality of A 尾 1-42 PILO group was significantly higher than that of PBS PILO group and PBS group, respectively. Histopathological experiment of rat brain: the results of modified Timm's staining showed that the intensity of DG MFS staining in hippocampus of A 尾 1-42 PILO group was significantly higher than that of PBS PILO group (P0.05). The intensity of DG MFS staining in hippocampus was also increased in A 尾 1-42 group, but there was no significant difference compared with PBS PILO group (P0.05); PBS PILO group and PBS group significantly increased MFS staining intensity (P0.01); The staining intensity of MFS in A 尾 1-42 group was significantly higher than that in PBS group (P 0.01). Conclusion injection of A 尾 1-42 into the CA3 region of the hippocampus can result in impaired learning and memory related to the hippocampus in rats. The Morris water maze test is used to further verify the above dysfunction. It was proved that A 尾 could induce dementia-like behavior changes in rats. On the basis of the existence of A 尾, the susceptibility to EP attack in rats was increased, which indicated that A 尾 could promote the occurrence of EP. From the point of view of disease pathology, it was shown that AD was closely related to EP attack. Histopathological staining showed that there was high intensity staining of hippocampal DG MFS in A 尾 1-42 PILO group. It was further demonstrated from the pathophysiological mechanism that the structural basis of the increased susceptibility to EP attack in rats induced by A 尾 1-42 PILO might be related to the significant increase in MFS level. Although there was no EP attack in group A 尾 1-42, MFS staining was also observed in hippocampus DG of this group, indicating that MFS is not only the result of EP attack, but also represents the inherent abnormal network connection in these rats. In the "second strike" theory of EP attack, the possible mechanism of A 尾 is to cause the first brain injury. There is a significant correlation between the increased susceptibility to EP attack and the pathogenesis of AD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1;R749.16
本文编号：2321566
[Abstract]:Objective to study the effect of 尾 -amyloid protein (Beta amyloid protein,A 尾 on the pathogenesis of epilepsy (Epilepsy,EP). To explore the relationship between the increased susceptibility of EP and the pathological changes of Alzheimer's disease (Alzheimer's disease,AD) from the point of view of the pathogenesis of the disease. Methods Forty adult Wistar rats aged 6-8 weeks were randomly divided into 尾 -amyloid 1-42 (A 尾 1-42) pilocarpine (Pilocarpine,PILO) group, A 尾 1-42 group, phosphate buffer (Phosphate buffer solution,PBS PILO group and PBS group (10 rats in each group). The A 尾 1-42 was injected into the CA3 area of the hippocampus of Wistar rats with a brain stereotactic locator. The Morris water maze test was observed two weeks later. The escape latency, the proportion of swimming time in the target quadrant and the times of crossing the platform were recorded, and the statistical analysis software was used. To detect the success of AD model in rats. The chronic EP seizure model of rats was established by intraperitoneal injection of lithium-pilocarpine (Lithium chloride Pilocarpine,LI-PILO) into abdominal cavity (Intraperitoneal,IP). According to the Racine grading standard, the incubation period of EP attack was recorded (the time required to reach IV grade and above). Severity and mortality; The (Mossy fiber sprouting,MFS levels of mossy fiber sprouts in dentate gyrus (Dentate gyrus,DG) of rats in each group were observed by modified Timm's staining. Results the AD model of rats was established: after injection of A 尾 1-42, one rat died in the AD model group and one in the PBS control group. No obvious cause of human death was found in the dissection, and the possible cause of death was inferred to be the individual difference between the rats. The results of Morris water maze test showed that the escape latency of AD model group was significantly longer than that of PBS control group (P0.01), and the proportion of swimming time in target quadrant was shortened (P0.05). The frequency of crossing the platform was also significantly reduced (P0.05), the differences were statistically significant, indicating that the AD model was successfully prepared. EP model of rats: according to the Racine grading standard, the latency of EP attack in A 尾 1-42 PILO group was significantly shorter than that in PBS PILO group (P0.01), and the severity of EP attack was more serious (P0.05). The mortality of A 尾 1-42 PILO group was significantly higher than that of PBS PILO group and PBS group, respectively. Histopathological experiment of rat brain: the results of modified Timm's staining showed that the intensity of DG MFS staining in hippocampus of A 尾 1-42 PILO group was significantly higher than that of PBS PILO group (P0.05). The intensity of DG MFS staining in hippocampus was also increased in A 尾 1-42 group, but there was no significant difference compared with PBS PILO group (P0.05); PBS PILO group and PBS group significantly increased MFS staining intensity (P0.01); The staining intensity of MFS in A 尾 1-42 group was significantly higher than that in PBS group (P 0.01). Conclusion injection of A 尾 1-42 into the CA3 region of the hippocampus can result in impaired learning and memory related to the hippocampus in rats. The Morris water maze test is used to further verify the above dysfunction. It was proved that A 尾 could induce dementia-like behavior changes in rats. On the basis of the existence of A 尾, the susceptibility to EP attack in rats was increased, which indicated that A 尾 could promote the occurrence of EP. From the point of view of disease pathology, it was shown that AD was closely related to EP attack. Histopathological staining showed that there was high intensity staining of hippocampal DG MFS in A 尾 1-42 PILO group. It was further demonstrated from the pathophysiological mechanism that the structural basis of the increased susceptibility to EP attack in rats induced by A 尾 1-42 PILO might be related to the significant increase in MFS level. Although there was no EP attack in group A 尾 1-42, MFS staining was also observed in hippocampus DG of this group, indicating that MFS is not only the result of EP attack, but also represents the inherent abnormal network connection in these rats. In the "second strike" theory of EP attack, the possible mechanism of A 尾 is to cause the first brain injury. There is a significant correlation between the increased susceptibility to EP attack and the pathogenesis of AD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1;R749.16
【参考文献】
相关期刊论文 前2条
1 许韬;陈阳美;;阿尔茨海默病和癫痫:异常兴奋的神经网络和病理学表现[J];中华神经科杂志;2014年10期
2 刘阳;韩春雷;孟凡刚;;匹罗卡品癫痫模型研究进展[J];中华神经外科杂志;2014年01期
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