MPTP损伤对食蟹猴嗅球神经递质表达的影响
发布时间：2018-08-15 15:17
【摘要】：研究目的 不同于传统的限于运动系统认知，帕金森病更可以视为是以嗅觉障碍为主要症状而以运动障碍为次要症状的疾病。作为嗅觉通路中重要枢纽的嗅球在该疾病中的病理改变亦被广泛研究。MPTP非人灵长类帕金森病模型是迄今最理想的研究帕金森病发病机制的动物模型。 本研究旨在通过免疫组化的方法观察食蟹猴嗅球神经解剖和神经化学的特征,包括:1.食蟹猴嗅球组织结构学特点；2.食蟹猴嗅球各主要神经递质的表达情况,胶质细胞的分布；3.MPTP损伤对食蟹猴嗅球神经递质表达，胶质细胞和细胞凋亡的影响，MPTP损伤对食蟹猴嗅球中各主要神经递质表达、凋亡信号、胶质细胞的影响，这些知识可以给研究帕金森病嗅觉障碍积累一定的背景知识。 第一部分MPTP食蟹猴帕金森病模型的建立 方法 成年健康食蟹猴6只，在动物中心严格按照AAALAC (Association forAssessment and Accreditation of Laboratory Animal Care，国际实验动物评估和认可管理委员会）规定和要求对动物进行日常的饲养、关怀和管理。采用小剂量连续给药方法，，即0.2mg/kg MPTP-HCl溶液,于下肢静脉缓慢注入，每天早晨1次（9AM）。采用个体化原则，以出现典型的临床症状为注射终点。 结果 注射MPTP后10～14（12.0±1.67）天，临床评分达到10(10.5±0.55)分，此时所有实验猴均逐渐出现典型的运动损害症状，包括运动迟缓、随意运动活动减少、屈曲体态、姿势步态不稳，肌强直、间断性僵直发作及姿势性或静止性震颤。在其后约1周的时间（开始注射MPTP后18.8±2.9天）临床症状持续进展并进入相对稳定期，此时临床评分为（19.5±4.8）分。 第二部分MPTP损伤对食蟹猴嗅球神经递质表达的影响 方法 摘取模型组和对照组食蟹猴嗅球后制成石蜡切片。我们首先选择的抗体针对的抗原物质为单胺类神经递质DA、5-HT。两种抗体分别染模型组和对照组各25张石蜡切片，再从25张食蟹猴嗅球石蜡切片中随机选取5张，选取视野，显微镜摄片，分析免疫组化图片，测平均光密度，计阳性细胞数。 我们也选择了氨基酸类神经递质GABA和ChAT，研究方法同上。多巴胺系统与帕金森病颇具关联，这里我们除了研究DA，还研究多巴胺通路上的信号分子受体和转运体蛋白，分别为DARPP32D1R D2R DAT。 结果 与对照组比较，MPTP组的单胺类神经递质DA和5-HT的阳性细胞数和阳性纤维量均减少。 与对照组比较，MPTP组氨基酸类神经递质GABA的阳性细胞数和阳性细胞量均减少。 与对照组比较，MPTP组ChAT阳性细胞数与阳性纤维量均减少。 与对照组比较，MPTP组DARPP32D1R D2R DAT阳性细胞数与阳性纤维量均减少，其中以DAT减少程度尤为显著。 第三部分MPTP损伤对食蟹猴嗅球胶质细胞的影响 方法 以抗Iba1抗体、抗GFAP抗体、抗TNF-α抗体用SABC法染色，每组从25张食蟹猴嗅球石蜡切片中随机选取5张，显微镜摄片，分析免疫组化图片，测平均光密度，计阳性细胞数。从而观察MPTP损伤对食蟹猴嗅球胶质细胞的影响。 结果 与对照组比较，MPTP组Iba1阳性物质增多，提示小胶质细胞有增殖。 与对照组比较，MPTP组GFAP阳性物质增多，提示星形胶质细胞有增殖。 与对照组比较，MPTP组TNF-α阳性物质增多，提示有炎性反应。 第四部分MPTP损伤对食蟹猴嗅球细胞凋亡的影响 方法 用抗Bcl2和抗caspase3抗体以SABC法染色，每组从25张食蟹猴嗅球石蜡切片中随机选取5张，选取视野后显微镜摄片，分析免疫组化图片，测平均光密度，计阳性细胞数。观察MPTP损伤对食蟹猴嗅球凋亡信号的影响。 结果 与对照组比较，MPTP组Bcl2阳性物质减少，caspase3阳性物质增多，提示细胞凋亡异常，呈上升。 结论 食蟹猴嗅球的组织结构是环状的多层结构。 MPTP损伤造成了食蟹猴嗅球神经递质表达的减少。 MPTP损伤造成了食蟹猴嗅球胶质细胞的增生和炎症反应，免疫反应。
[Abstract]:research objective
Parkinson's disease is more likely to be seen as a disease characterized by olfactory impairment than motor impairment, unlike traditional motor system cognition. The pathological changes of olfactory bulb, an important hub in olfactory pathway, are also extensively studied in this disease. MPTP non-human primate Parkinson's disease model is the most ideal study to date. Animal models of the pathogenesis of Parkinson's disease.
The aim of this study was to observe the neuroanatomical and neurochemical characteristics of the olfactory bulb in cynomolgus monkeys by immunohistochemical methods, including: 1. histological characteristics of the olfactory bulb in cynomolgus monkeys; 2. expression of major neurotransmitters and distribution of glial cells in the olfactory bulb of cynomolgus monkeys; 3. MPTP injury induced neurotransmitter expression in the olfactory bulb of cynomolgus, glial cells and cell apoptosis. The effects of MPTP injury on the expression of major neurotransmitters, apoptosis signals and glial cells in the olfactory bulb of cynomolgus monkeys can provide some background knowledge for the study of Parkinson's disease.
Part one establishment of MPTP cynomolgus Parkinson disease model
Method
Six healthy adult cynomolgus monkeys were raised, cared for and managed in animal centres in strict accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) regulations and requirements. Fluid, slowly injected into the lower extremity veins, once a day in the morning (9AM). Individualized principles were adopted, with typical clinical symptoms as the end point of injection.
Result
From 10 to 14 days after MPTP injection, the clinical score reached 10 (10.5 (+ 0.55) points. At this time, all the monkeys gradually developed typical symptoms of motor impairment, including slow movement, decreased voluntary movement, flexion posture, instability of postural gait, muscular rigidity, intermittent stiffness attacks and postural or static tremors. (18.8 (+ 2.9) days after MPTP injection) the clinical symptoms progressed continuously and entered a relatively stable phase, with a clinical score of (19.5 (+ 4.8).
The second part is the effect of MPTP damage on the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
Method
Paraffin sections were made from the olfactory bulbs of the model and control groups. Firstly, the antigens targeted by the antibodies against DA and 5-HT were stained with 25 paraffin sections of the model group and 25 paraffin sections of the control group, and then 5 paraffin sections were randomly selected from 25 paraffin sections of the olfactory bulbs of the cynomolgus monkeys. Immunohistochemical analysis was used to measure the mean optical density and count the number of positive cells.
We also chose amino acid neurotransmitters GABA and ChAT, the same methodology as above. The dopamine system is associated with Parkinson's disease. Here we study not only DA, but also the receptors and transporters of the dopamine pathway, DARPP32D1R D2R DAT.
Result
Compared with the control group, the number of DA and 5-HT positive cells and the amount of positive fibers in MPTP group were decreased.
Compared with the control group, the number of positive neurons and the number of positive cells of amino acid neurotransmitters GABA in group MPTP were decreased.
Compared with the control group, the number of ChAT positive cells and the number of positive fibers in group MPTP decreased.
Compared with the control group, the number of DARPP32D1R D2R DAT positive cells and the amount of DAR DAT positive fibers in MPTP group were decreased, especially the decrease of DAT.
The third part is the effect of MPTP damage on the glial cells in the olfactory bulb of cynomolgus monkeys.
Method
Anti-Iba1 antibody, anti-GFAP antibody and anti-TNF-alpha antibody were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from 25 paraffin sections in each group. Immunohistochemical images were analyzed. Mean optical density was measured and the number of positive cells was counted.
Result
Compared with control group, Iba1 positive substance increased in group MPTP, suggesting microglia proliferation.
Compared with the control group, GFAP positive substance increased in group MPTP, suggesting astrocyte proliferation.
Compared with the control group, there was an increase in TNF- alpha positive substance in group MPTP, suggesting an inflammatory response.
The fourth part is the effect of MPTP damage on the apoptosis of olfactory bulb cells in cynomolgus monkeys.
Method
Anti-Bcl-2 and anti-caspase-3 antibodies were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from each group. Immunohistochemical images were taken under microscope after visual field. The average optical density was measured and the number of positive cells was counted. The effect of MPTP injury on the apoptotic signal of the olfactory bulb of cynomolgus monkeys was observed.
Result
Compared with the control group, the positive substance of Bcl 2 decreased and caspase 3 increased in MPTP group, suggesting that apoptosis was abnormal and increased.
conclusion
The structure of the olfactory bulb of cynomolgus monkeys is a circular multilayer structure.
MPTP damage reduced the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
MPTP damage caused the proliferation and inflammatory reaction of the cells in the olfactory bulb of cynomolgus monkeys.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
[Abstract]:research objective
Parkinson's disease is more likely to be seen as a disease characterized by olfactory impairment than motor impairment, unlike traditional motor system cognition. The pathological changes of olfactory bulb, an important hub in olfactory pathway, are also extensively studied in this disease. MPTP non-human primate Parkinson's disease model is the most ideal study to date. Animal models of the pathogenesis of Parkinson's disease.
The aim of this study was to observe the neuroanatomical and neurochemical characteristics of the olfactory bulb in cynomolgus monkeys by immunohistochemical methods, including: 1. histological characteristics of the olfactory bulb in cynomolgus monkeys; 2. expression of major neurotransmitters and distribution of glial cells in the olfactory bulb of cynomolgus monkeys; 3. MPTP injury induced neurotransmitter expression in the olfactory bulb of cynomolgus, glial cells and cell apoptosis. The effects of MPTP injury on the expression of major neurotransmitters, apoptosis signals and glial cells in the olfactory bulb of cynomolgus monkeys can provide some background knowledge for the study of Parkinson's disease.
Part one establishment of MPTP cynomolgus Parkinson disease model
Method
Six healthy adult cynomolgus monkeys were raised, cared for and managed in animal centres in strict accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) regulations and requirements. Fluid, slowly injected into the lower extremity veins, once a day in the morning (9AM). Individualized principles were adopted, with typical clinical symptoms as the end point of injection.
Result
From 10 to 14 days after MPTP injection, the clinical score reached 10 (10.5 (+ 0.55) points. At this time, all the monkeys gradually developed typical symptoms of motor impairment, including slow movement, decreased voluntary movement, flexion posture, instability of postural gait, muscular rigidity, intermittent stiffness attacks and postural or static tremors. (18.8 (+ 2.9) days after MPTP injection) the clinical symptoms progressed continuously and entered a relatively stable phase, with a clinical score of (19.5 (+ 4.8).
The second part is the effect of MPTP damage on the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
Method
Paraffin sections were made from the olfactory bulbs of the model and control groups. Firstly, the antigens targeted by the antibodies against DA and 5-HT were stained with 25 paraffin sections of the model group and 25 paraffin sections of the control group, and then 5 paraffin sections were randomly selected from 25 paraffin sections of the olfactory bulbs of the cynomolgus monkeys. Immunohistochemical analysis was used to measure the mean optical density and count the number of positive cells.
We also chose amino acid neurotransmitters GABA and ChAT, the same methodology as above. The dopamine system is associated with Parkinson's disease. Here we study not only DA, but also the receptors and transporters of the dopamine pathway, DARPP32D1R D2R DAT.
Result
Compared with the control group, the number of DA and 5-HT positive cells and the amount of positive fibers in MPTP group were decreased.
Compared with the control group, the number of positive neurons and the number of positive cells of amino acid neurotransmitters GABA in group MPTP were decreased.
Compared with the control group, the number of ChAT positive cells and the number of positive fibers in group MPTP decreased.
Compared with the control group, the number of DARPP32D1R D2R DAT positive cells and the amount of DAR DAT positive fibers in MPTP group were decreased, especially the decrease of DAT.
The third part is the effect of MPTP damage on the glial cells in the olfactory bulb of cynomolgus monkeys.
Method
Anti-Iba1 antibody, anti-GFAP antibody and anti-TNF-alpha antibody were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from 25 paraffin sections in each group. Immunohistochemical images were analyzed. Mean optical density was measured and the number of positive cells was counted.
Result
Compared with control group, Iba1 positive substance increased in group MPTP, suggesting microglia proliferation.
Compared with the control group, GFAP positive substance increased in group MPTP, suggesting astrocyte proliferation.
Compared with the control group, there was an increase in TNF- alpha positive substance in group MPTP, suggesting an inflammatory response.
The fourth part is the effect of MPTP damage on the apoptosis of olfactory bulb cells in cynomolgus monkeys.
Method
Anti-Bcl-2 and anti-caspase-3 antibodies were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from each group. Immunohistochemical images were taken under microscope after visual field. The average optical density was measured and the number of positive cells was counted. The effect of MPTP injury on the apoptotic signal of the olfactory bulb of cynomolgus monkeys was observed.
Result
Compared with the control group, the positive substance of Bcl 2 decreased and caspase 3 increased in MPTP group, suggesting that apoptosis was abnormal and increased.
conclusion
The structure of the olfactory bulb of cynomolgus monkeys is a circular multilayer structure.
MPTP damage reduced the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
MPTP damage caused the proliferation and inflammatory reaction of the cells in the olfactory bulb of cynomolgus monkeys.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
【共引文献】
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