选择性毁损脚桥被盖核（PPTg）类胆碱能神经元对帕金森（PD）大鼠步态的影响

发布时间：2018-06-03 21:58

本文选题：帕金森病 + 脑桥被盖核　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究目的:当帕金森病进展到晚期阶段,有大约一半的患者会出现轴性症状,比如:严重的步态和姿势障碍(更具体的说,冻僵步态和跌倒),并且不被左旋多巴与STN-DBS治疗改善。近年来,有越来越多的证据表明,电刺激脑桥核(PPN-DBS)在缓解轴性症状中发生起着至关重要的作用。通过研究PD中的PPN和它的功能将有助于理解轴性症状的病理基础和PPN-DBS治疗的机制。因此,我们可以合理推测难治性轴性症状的发生与胆碱能神经元变性更相关,而不是多巴胺能神经元变性。在本研究中,我们进一步探讨PPN胆碱能神经元在大鼠发生运动障碍中的作用。特别是轴性运动障碍。研究方法本实验选用42只健康成年雄性SD大鼠随机分四组:(1)生理盐水注射假手术组(2)6-羟基多巴胺(6-OHDA)毁损单侧黑质致密部(SNc)组(3)anti-ChAT-SAP毁损单侧脚桥被盖核(PPTg)组(4)6-羟基多巴胺(6-OHDA)毁损单侧黑质致密部(SNc)合并anti-ChAT-SAP毁损同侧脚桥被盖核(PPTg)组(联合毁损组)。anti-ChAT-SAP是一种带皂素(SAP)(皂类植物种子中提取的可使核糖体失活蛋白)的特异性抑制胆碱乙酰转移酶(ChAT)的兔源性抗体毒药。右侧定位内侧前脑束(MFB)注射4ul6-OHDA。靶向抗胆碱乙酰转移酶IgG免疫毒素(anti-ChAT-SAP)注入右侧PPTg毁损胆碱能神经元,通过注射相同剂量的生理盐水到右侧MFB和PPTg造成假毁损。我们使用Catwalk XT系统(Noldus信息技术,瓦赫宁恩,荷兰)分析自由活动的大鼠的步态。在毁损术后重新进行行为学评估的过程中,除了假手术组,其他大鼠在测试中均表现出轻到中度的冻僵步态(起步延迟)。由于冻结步态(起步延迟)而不能完成整个阶段的(包括少于三次通过通道的)计入冻结步态的数量,计算每一组中冻僵步态数量占测试阶段的全部数量的百分比。最后完成行为学测试后,SNc通过酪氨酸羟化酶(TH)免疫组织化学染色法如前所述。此程序适用于PPTg的ChAT免疫组织化学染色方法,染色后,使用Leica显微摄像程序组获取图片。根据光学分馏器原理对SNc中TH+的神经元或者PPTg中ChAT+的神经元数量进行定量分析评估。以每6张覆盖了完整SNc或者PPTg的切片取得的均值为计数值。数据以完整一侧相应区域的百分比记录。研究结果在SNc毁损组DA神经元的平均损失90.19%,单纯SNc毁损组与联合毁损组之间没有组间差异。在PPTg毁损组的胆碱能神经细胞的损失达到了 77.62%,在联合毁损组达到81.75%,两者与假手术组相比明显降低。单纯PPTg毁损组与联合毁损组胆碱能细胞的损失没有显着差异。Catwalk行为学分析系统捕捉动态和静态的步态参数。损伤后,与假手术组和PPTg毁损组相比SNc毁损组和联合毁损组的跑动时长、站立时间和步替循环明显增加步幅长度和摆动速度明显下降。SNc毁损组与联合毁损组无显着差异。最大接触面积和平均强度的相互作用只出现在左侧爪印(对侧脑损伤)。与假手术组和PPTg毁损组相比,左前爪和左后爪在SNc毁损组和联合毁损组中这两个步态参数显著下降。而SNc毁损组和联合毁损组之间无差异。与假手术组和PPTg毁损组比较,SNc毁损组和联合毁损组的爪(前或后)间宽距参数明显增加,而在前肢只有微弱增加。在SNc毁损组和联合毁损组中所有足印的终末双足着地时间都有显著增加。并且联合毁损组高于SNc毁损组。在四组中SNc毁损组和联合毁损组出现了最严重的冻结步态症状(57.58%,p0.05)。从某种程度上说,与假手术组相比,单纯毁损SNc也会诱发大鼠出现FOG(26.66%,p0.05,)。假手术和PPTg毁损组大鼠无显著差异(0%vs.13.88%)。在PPTg毁损组中没有发现胆碱能神经元与步态参数变化存在相关性。在联合毁损组,只有左侧肢体的终末双足着地时间与胆碱能神经元存在相关(偏相关系数:左前肢:-0.77,P = 0.04;左后肢:-0.79,P = 0.02),其他参数无显著相关性。研究结论结果表明,单纯毁损PPTg大鼠不会引起步态异常。但是6-OHDA毁损多巴胺神经元合并PPTg胆碱能神经元毁损可加重PD大鼠的冻僵步态症状。我们的数据表明,PPN胆碱能神经元在PD患者FOG的发生过程中起着至关重要的作用。后续研究可以更进一步探究PPN胆碱能神经元与参与PD相关的结构或神经元在PPN-DBS治疗机制的关系。
[Abstract]:Research purposes: when Parkinson's disease progresses to a late stage, about half of the patients have axial symptoms, such as severe gait and postural disorders (more specifically, frozen gait and fall), and not treated by levodopa and STN-DBS. In recent years, there is more evidence that the electrical stimulation of the pontine nucleus (PPN-DBS) is relieved. The study of PPN and its function in PD will help to understand the pathological basis of axial symptoms and the mechanism of PPN-DBS therapy. Therefore, we can reasonably speculate that the occurrence of intractable axial symptoms is more related to the degeneration of cholinergic neurons, rather than the degeneration of dopaminergic neurons. In the study, we further explored the role of PPN cholinergic neurons in dyskinesia in rats, especially axonal dyskinesia. The study method selected 42 healthy adult male SD rats randomly divided into four groups: (1) physiological saline injection sham group (2) 6- hydroxyl dopamine (6-OHDA) damaged unilateral substantia nigra dense part (3) anti-C HAT-SAP destruction of unilateral foot bridge tegmentum (PPTg) group (4) 6- hydroxyl dopamine (6-OHDA) damage unilateral substantia nigra dense part (SNc) combined with anti-ChAT-SAP damage in the same lateral foot bridge tegmental nucleus (PPTg) group (joint damage group).Anti-ChAT-SAP is a kind of specific inhibitory choline B with saponin (SAP) (the ribosome inactivation protein extracted from the seed of soap plant) The right localization medial forebrain bundle (MFB) injected 4ul6-OHDA. target to the cholinergic acetyltransferase IgG immuno toxin (anti-ChAT-SAP) injecting the right PPTg damage to the cholinergic neurons by injecting the right side of the medial forebrain bundle (ChAT) into the right side. We used the Catwalk XT system (Nold) by injecting the same dose of the saline to the right MFB and PPTg. Us information technology, wakningen, Holland) analysis of the gait of free active rats. In the process of reassessment of behavior after the damage, other rats showed mild to moderate frozen gait in the test, except for the sham operation group (starting delay). The whole stage was not completed due to the freezing step (starting delay). The number of frozen gait in the three passage was counted, and the percentage of the total number of frozen gait in each group was calculated. After the final behavioral test, the SNc immunohistochemical staining of tyrosine hydroxylase (TH) was described as before. This program is suitable for the ChAT immunohistochemical staining method of PPTg and dyeing. Then, the images are obtained using the Leica microcamera group. According to the optical fractionator principle, the number of neurons in the neurons of TH+ in the SNc or the number of ChAT+ in the PPTg is quantitatively evaluated. The mean value obtained with every 6 slices covered with complete SNc or PPTg is calculated. The data is recorded with the percentage of the corresponding region on the complete side. The average loss of DA neurons in the SNc damage group was 90.19%. There was no difference between the simple SNc damage group and the combined damage group. The loss of cholinergic neurons in the PPTg damage group reached 77.62%, and 81.75% in the combined damage group, which was significantly lower than that in the sham group. The cholinergic activity in the simple PPTg damage group and the combined damage group was fine. The loss of cell was not significantly different from the.Catwalk behavioral analysis system to capture dynamic and static gait parameters. After injury, the running time of the SNc damage group and the combined damage group was longer than the sham operation group and the PPTg damage group. The standing time and the step cycle significantly increased the length and the swinging speed of the.SNc damage group and the joint damage group. There was no difference. The interaction between the maximum contact area and the average strength only appeared in the left claw print (contralateral brain damage). Compared with the sham operation group and the PPTg damage group, the two gait parameters of the left front and left posterior claw in the SNc damage group and the combined damage group were significantly decreased. There was no difference between the SNc damage group and the combined group. Compared with the PPTg damage group, the width parameters of the claw (before and after) in the SNc damage group and the joint damage group increased significantly, but only a faint increase in the forelimb. All the end of foot prints in the SNc damage group and the combined damage group increased significantly. And the joint destruction group was higher than the SNc damage group. In the four groups, the SNc damage group and the joint destruction were destroyed. The most serious frozen gait symptoms (57.58%, P0.05) were found in the loss group. To some extent, compared with the sham group, the simple destruction of SNc could induce FOG (26.66%, P0.05,) in rats. There was no significant difference between the sham operation and the PPTg damage group (0%vs.13.88%). The changes of the cholinergic neurons and gait parameters were not found in the PPTg damage group. Correlation. In the combined damage group, only the left limb terminal bipedal time was associated with cholinergic neurons (partial correlation coefficient: left forelimb: -0.77, P = 0.04; left hind limbs: -0.79, P = 0.02), and no significant correlation was found in other parameters. The results of the study showed that the simple destruction of PPTg rats would not cause abnormal gait, but 6-OHDA was much damaged. Our data suggest that PPN cholinergic neurons play a vital role in the occurrence of FOG in PD patients. Further studies can further explore the PPN cholinergic neurons associated with PD related structures or neurons in the treatment of PD in the PPN-DBS treatment. The relationship of the therapeutic mechanism.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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