三重脑震荡鼠认知行为障碍与脑多巴胺能神经元变化研究

发布时间：2018-08-21 07:48

【摘要】： 目的：建立三重脑震荡(multiple cerebral concussion，MCC)大鼠模型，观察MCC大鼠脑多巴胺能神经元、神经纤维变化的规律，探讨与MCC大鼠认知行为障碍相关脑区的多巴胺能神经元变化，以了解三重脑震荡鼠认知行为障碍的神经生物学基础。方法：应用金属单摆打击装置复制大鼠MCC模型48只，大鼠随机分为1天、2天、4天、8天、16天和24天组共6个三重脑震荡组(n=8)，另设一正常对照组(n=8)。Morris水迷宫(MWM)分别于伤后1～7天、8～14天、15～21天连续7天检测大鼠的学习记忆能力。用抗TH和抗DBH单克隆抗体行免疫组化S-P法，联合标记中脑黑质致密区、腹侧被盖区、前额叶皮质、前扣带皮质、尾壳核、伏隔核、嗅结节、额顶叶皮质、后扣带皮质和内嗅皮质多巴胺能神经。用光镜对大鼠中脑黑质致密区、腹侧被盖区的TH阳性细胞进行观察和计数，并对大脑前额叶皮质、前扣带皮质、尾壳核、伏隔核、嗅结节、额顶叶皮质、后扣带皮质和内嗅皮质的TH阳性纤维密度和强弱进行观察和半定量检测；用图像分析系统分别对上述脑区的TH阳性细胞和神经纤维进行定量分析。结果：(1)在Morris水迷宫测试中，，MCC后16d组的第9、10、13、14天与对照组相比差异具有显著性(P＜0.05)：8d、24d组找到平台的时间与对照组相比有所延长，但差异无显著性(P＞0.05)。在无平台探测实验中，与对照组相比，MCC后8d、16d组大鼠在原有平台第三象限停留时间明显减少，差异具有显著性(P＜0.05)；MCC后24d组在原有平台第三象限停留时间稍有减少，差异无显著性(P＞0.05)。(2)MCC后脑黑质致密区和腹侧被盖区TH阳性细胞计数分析结果：在黑质致密区TH阳性细胞数以伤后1天开始升高，8天达到高峰，与对照组相比4天、8天组有显著性差异(p＜0.05)；与对照组相比，腹侧被盖区2天、4天、8天、16天组有显著性差异(p＜0.05)，以4天组TH阳性细胞数达到高峰，24天组基本恢复。(3)MCC后脑黑质致密区和腹侧被盖区TH阳性细胞图像分析结果：TH免疫反应阳性表达在黑质致密区以伤后1天开始升高，4天达到高峰，与对照组相比有显著性差异(p＜0.01)，8天开始减弱，与对照组相比仍有显著性差异(p＜0.05)，24天组基本恢复。与对照组相比，腹侧被盖区2天、4天、8天、16天组有显著性差异(p＜0.05)，以2天组阳性反应最强(p＜0.01)，24天组基本恢复。(4)大鼠脑前额叶皮质、前扣带皮质、尾壳核、伏隔核、嗅结节、额顶叶皮质、后扣带皮质和内嗅皮质TH阳性纤维密度和强弱半定量分析结果：与对照组相比，伤后1天、4天、16天组伏隔核TH阳性纤维密度和强弱明显增强(p＜0.008)。嗅结节于伤后1天、8天组，TH免疫反应阳性纤维的密度和强弱高于对照组，差异具有显著性(p≤0.008)。内嗅皮质于伤后8天组，TH免疫反应阳性纤维的密度和强弱明显高于对照组(p＜0.008)。前额叶皮质、前扣带皮质、尾壳核、额顶叶皮质、后扣带皮质TH免疫反应阳性纤维的密度和强弱有不同程度的增强，但与对照组相比无显著差异(p＞0.008)。(5)MCC后不同脑区TH阳性纤维图像分析结果：伤后4天、8天组，前额叶皮质多巴胺能神经纤维免疫反应阳性显著高于对照组(p＜0.05)，4天组反应达高峰，24天组仍高于对照组。与对照组相比，各损伤组前扣带皮质显著高于对照组(p≤0.01)，16天组反应达高峰，24天组仍未恢复。伤后1天、4天、16天组，尾壳核TH阳性纤维显著高于对照组(p＜0.05)，1天组反应达高峰，24天组仍高于对照组。与对照组相比，伏隔核TH阳性纤维于伤后4天、8天、16天、24天组显著增高(p＜0.01)，8天组反应达高峰，24天组仍未恢复。嗅结节TH阳性纤维1天、8天组与对照组相比有显著差异(p＜0.05)，8天组反应达高峰，24天组基本恢复。与对照组相比，各损伤组额顶叶皮质TH阳性纤维明显增高(p＜0.05)，1天组反应达高峰，24天组仍未恢复。后扣带皮质TH阳性纤维于伤后1天、2天组与对照组相比有显著差异(p＜0.05)，2天组反应达高峰，24天组基本恢复。内嗅皮质于伤后1天、2天、8天组明显高于对照组(p＜0.05)，2天组反应达高峰，24天组仍未恢复。以上各部位免疫组化DBH反应均为阴性。结论：1．金属单摆式闭合性机械打击装置能成功建立三重脑震荡大鼠模型；2．三重脑震荡后大鼠出现中期学习记忆功能障碍；3．三重脑震荡后，大鼠大脑皮质、尾壳核、伏隔核、嗅结节、黑质致密区、腹侧被盖区TH阳性表达出现不同程度的增高。4．三重脑震荡后大鼠行为学测试结果在不同时间点的变化与TH阳性表达产物在不同时间点的变化趋势基本一致。5．三重脑震荡后大鼠认知行为障碍可能与伤后脑内多巴胺能神经元变化有一定关系。
[Abstract]:AIM: To establish a rat model of multiple cerebral concussion (MCC), observe the changes of dopaminergic neurons and nerve fibers in the brain of MCC rats, and explore the changes of dopaminergic neurons in the brain regions associated with cognitive and behavioral disorders in MCC rats, so as to understand the neurobiological basis of cognitive and behavioral disorders in rats with MCC. METHODS: Forty-eight MCC rats were randomly divided into 6 triple concussion groups (n=8) and a normal control group (n=8). Morris water maze (MWM) was used to test the learning and memory abilities of rats 1-7 days, 8-14 days, 15-21 days after injury. Immunohistochemical S-P method was used to label the TH positive cells in substantia nigra compact area, ventral tegmental area, prefrontal cortex, anterior cingulate cortex, caudate putamen nucleus, nucleus accumbens, olfactory nodule, frontal parietal cortex, posterior cingulate cortex and endoolfactory cortex. The density and intensity of TH-positive fibers in prefrontal cortex, anterior cingulate cortex, caudate putamen nucleus, nucleus accumbens, olfactory tubercle, frontal-parietal cortex, posterior cingulate cortex and inner olfactory cortex were observed and counted, and the TH-positive cells and nerve fibers in these areas were quantitatively analyzed by image analysis system. Results: (1) In the Morris water maze test, there was significant difference between the control group and the MCC group on the 9th, 10th, 13th and 14th day after MCC (P The residence time in the third quadrant of the original platform was slightly decreased in the 24 days after MCC, but there was no significant difference (P > 0.05). (2) The number of TH positive cells in the substantia nigra compact area and the ventral tegmental area increased from 1 day after MCC. Compared with the control group, there were significant differences in the ventral tegmental area of 2 days, 4 days, 8 days and 16 days (p < 0.05), and the number of TH positive cells in the 4 days group reached the peak, and the number of TH positive cells in the 24 days group basically recovered. (3) The image scores of TH positive cells in the substantia nigra compact area and ventral tegmental area after MCC. Results: The expression of TH immunoreactivity in substantia nigra dense area increased from 1 day to 4 days after injury, and reached the peak at 4 days. There was significant difference between the control group and the control group (p < 0.01). There was a significant difference (p < 0.05). The TH positive fibers in the prefrontal cortex, anterior cingulate cortex, caudate putamen nucleus, nucleus accumbens, olfactory nodule, frontal parietal cortex, posterior cingulate cortex and inner olfactory cortex were the strongest in the 2-day group (p < 0.01) and recovered basically in the 24-day group. The density and intensity of TH-positive fibers in the nucleus accumbens were significantly increased (p < 0.008). The density and intensity of TH-immunoreactive fibers in the olfactory nodules were significantly higher than those in the control group on the 1st and 8th day after injury (p < 0.008). The density and intensity of TH-immunoreactive fibers in the inner olfactory cortex were significantly higher than those in the control group on the 8th day after injury (p < 0.008). The density and intensity of TH immunoreactive fibers in the cortex, anterior cingulate cortex, caudate putamen nucleus, frontal and parietal cortex, and posterior cingulate cortex were increased in varying degrees, but there was no significant difference between the two groups (p > 0.008). (5) The results of image analysis of TH immunoreactive fibers in different brain regions after MCC: the prefrontal cortex at 4 and 8 days after injury, the prefrontal cortex dopaminergic nerve fibers. Compared with the control group, the anterior cingulate cortex of each injury group was significantly higher than that of the control group (p < 0.01). The reaction peaked in the 16 days group and did not recover in the 24 days group. The TH positive fibers in the caudate putamen nucleus were significantly higher in the 1, 4 and 16 days group than that in the control group (p < 0.05). Compared with the control group, the TH-positive fibers in the nucleus accumbens increased significantly on the 4th, 8th, 16th and 24th day after injury (P < 0.01), the reaction peaked in the 8th day group and remained unchanged in the 24th day group. Compared with the control group, the TH-positive fibers in the frontal and parietal cortex of each injury group were significantly higher (p < 0.05), the reaction peaked in the first day group, but did not recover in the 24 day group. Immunohistochemical DBH reaction was negative in all the above parts. Conclusion: 1. Metal pendulum closed mechanical percussion device can successfully establish the rat model of triple cerebral concussion; 2. Middle-term learning after triple cerebral concussion in rats. Memory impairment; 3. After triple concussion, the TH positive expression in the cerebral cortex, caudate putamen, nucleus accumbens, olfactory nodule, substantia nigra compact area, ventral tegmental area increased in varying degrees. 4. Behavioral changes of rats after triple concussion at different time points and the trend of TH positive expression products at different time points. 5. Cognitive and behavioral disorders in rats after triple concussion may be related to the changes of dopaminergic neurons in the brain.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

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