β-淀粉樣蛋白增加Wistar大鼠癲癇發(fā)作易感性的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-11-09 21:09
【摘要】：目的研究β-淀粉樣蛋白(Beta amyloid protein,Aβ)對(duì)癲癇(Epilepsy,EP)病理機(jī)制發(fā)生發(fā)展的影響。從疾病發(fā)病機(jī)制的角度,探討EP發(fā)作易感性增加與阿爾茨海默氏病(Alzheimer’s disease,AD)病理改變之間的相關(guān)性。方法選用6-8周成年Wistar大鼠40只,隨機(jī)分為β-淀粉樣蛋白1-42(Aβ1-42)+匹羅卡品(Pilocarpine,PILO)組、Aβ1-42組、磷酸鹽緩沖液(Phosphate buffer solution,PBS)+PILO組、PBS組,每組各10只。采用腦立體定位儀將Aβ1-42注入Wistar大鼠海馬CA3區(qū),2周后觀察大鼠Morris水迷宮實(shí)驗(yàn),記錄逃避潛伏期、目標(biāo)象限內(nèi)游泳時(shí)間比例和穿越平臺(tái)次數(shù),應(yīng)用統(tǒng)計(jì)學(xué)分析軟件,檢測(cè)大鼠AD模型是否成功。建模成功后通過(guò)腹腔(Intraperitoneal,IP)注射氯化鋰-匹羅卡品(Lithium chloride Pilocarpine,LI-PILO)制備大鼠慢性EP發(fā)作模型,依據(jù)Racine分級(jí)標(biāo)準(zhǔn),記錄EP發(fā)作潛伏期(達(dá)到IV級(jí)及以上發(fā)作所需時(shí)間)、發(fā)作程度和死亡率;采用改良的Timm’s染色方法觀察各組大鼠海馬齒狀回(Dentate gyrus,DG)苔蘚纖維芽生(Mossy fiber sprouting,MFS)水平。結(jié)果制備大鼠AD模型:注射Aβ1-42后,AD模型組和PBS對(duì)照組大鼠各死亡1只,解剖未發(fā)現(xiàn)明顯人為死亡原因,推測(cè)可能的致死原因?yàn)榇笫笾g存在個(gè)體差異;Morris水迷宮實(shí)驗(yàn)數(shù)據(jù)分析結(jié)果,AD模型組與PBS對(duì)照組大鼠相比逃避潛伏期顯著延長(zhǎng)(P0.01),目標(biāo)象限內(nèi)游泳時(shí)間比例縮短(P0.05),穿越平臺(tái)的次數(shù)也明顯減少(P0.05),差異均有統(tǒng)計(jì)學(xué)意義,表明AD模型制備成功。制備大鼠EP模型:依據(jù)Racine分級(jí)標(biāo)準(zhǔn),Aβ1-42+PILO組與PBS+PILO組相比,EP發(fā)作潛伏期顯著縮短(P0.01),發(fā)作程度更重(P0.05);Aβ1-42+PILO組與PBS+PILO組、PBS組相比死亡率均顯著增加,分別為P=0.0294、P=0.0108。大鼠腦組織病理學(xué)實(shí)驗(yàn):改良的Timm’s染色結(jié)果顯示,Aβ1-42+PILO組與PBS+PILO組比較海馬DG MFS染色強(qiáng)度顯著增強(qiáng)(P0.05);在Aβ1-42組也觀察到海馬DG MFS染色強(qiáng)度增強(qiáng),但和PBS+PILO組相比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);PBS+PILO組和PBS組比較MFS染色強(qiáng)度顯著增強(qiáng)(P0.01);Aβ1-42組和PBS組比較MFS染色強(qiáng)度也顯著增強(qiáng)(P0.01)。結(jié)論通過(guò)海馬CA3區(qū)注射Aβ1-42,可以導(dǎo)致大鼠出現(xiàn)與海馬有關(guān)的學(xué)習(xí)和記憶功能受損,采用Morris水迷宮實(shí)驗(yàn)從行為學(xué)上進(jìn)一步驗(yàn)證上述功能障礙,證明Aβ可以誘導(dǎo)大鼠出現(xiàn)癡呆樣行為改變。在Aβ存在的基礎(chǔ)上誘導(dǎo)大鼠出現(xiàn)EP發(fā)作易感性是增加的,說(shuō)明Aβ對(duì)于EP發(fā)生有一定的促進(jìn)作用,從疾病病理學(xué)角度闡明AD與EP發(fā)作密切相關(guān)。組織化學(xué)染色觀察到Aβ1-42+PILO組海馬DG MFS存在高強(qiáng)度染色,進(jìn)一步從病理生理學(xué)機(jī)制上驗(yàn)證Aβ誘導(dǎo)大鼠EP發(fā)作易感性增加的結(jié)構(gòu)基礎(chǔ)可能與MFS水平顯著增加有關(guān)。盡管在Aβ1-42組沒(méi)有EP發(fā)作,該組大鼠海馬DG也出現(xiàn)MFS染色增強(qiáng),這表明MFS不僅是EP發(fā)作的結(jié)果,而是代表在這些大鼠中固有的異常網(wǎng)絡(luò)連接。在EP發(fā)作的“二次打擊”學(xué)說(shuō)中,Aβ可能的作用機(jī)制是導(dǎo)致大腦出現(xiàn)第一次腦損傷。EP發(fā)作易感性增加與AD發(fā)病機(jī)制之間具有顯著的相關(guān)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1;R749.16
本文編號(hào)：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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1;R749.16
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 許韜;陳陽(yáng)美;;阿爾茨海默病和癲癇:異常興奮的神經(jīng)網(wǎng)絡(luò)和病理學(xué)表現(xiàn)[J];中華神經(jīng)科雜志;2014年10期
2 劉陽(yáng);韓春雷;孟凡剛;;匹羅卡品癲癇模型研究進(jìn)展[J];中華神經(jīng)外科雜志;2014年01期
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