重度磨耗对大鼠外周及初级神经中枢影响的初步研究

发布时间：2018-06-18 17:29

本文选题：大鼠 + 重度磨耗　；参考：《第四军医大学》2014年硕士论文

【摘要】：研究背景：重度磨耗（severe occlusal attrition，SOA）作为口腔临床上常见的慢性疾病，是导致颞下颌关节紊乱病（temporomandibular joint disorder，TMD）的一个重要原因，常伴随着疼痛、下颌运动异常、颞下颌关节（temporomandibular joint，TMJ）区弹响和杂音等症状，其中咀嚼肌和颞下颌关节区疼痛常为患者就诊的首要原因。前期关于重度磨耗的基础研究主要集中于口腔局部组织的反应，研究证实重度磨耗会引起咀嚼肌和颞下颌关节发生适应性改建，但是针对重度磨耗引起的口颌面部疼痛以及神经系统相关反应的研究较少。在其他口颌面部神经病理性痛和炎性痛的模型中发现，伤害性刺激可引起外周感觉神经中枢三叉神经节（trigeminal ganglion，TG）内降钙素基因相关肽（calcitonin gene-related peptide，CGRP）表达增多，脑干及延髓三叉神经脊束核（spinal nucleus of trigeminal nerve，SpⅤ）区域内的细胞丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPK）通路激活，磷酸化p38（phospho-p38，p-p38）蛋白的表达水平上调。因此，本研究拟以疼痛作为研究重点，分析重度磨耗后口颌面部疼痛的阈值变化及神经系统的相关反应。研究目的：建立并评价重度磨耗的动物模型，观察重度磨耗后大鼠咬肌机械压痛敏感程度的变化，并利用相关实验技术手段观察三叉神经节、脑干及延髓内的相关神经递质的变化，以分析重度磨耗后大鼠外周及初级中枢神经系统的反应。研究方法： 1.采用外科显微镜下逐渐均匀降低大鼠上颌后牙牙冠的方法建立重度磨耗动物模型，观察大鼠的表现型及体重变化，利用行为学方法检测大鼠咬肌机械压痛的敏感程度。 2.利用免疫荧光染色方法对重度磨耗后大鼠三叉神经节内CGRP表达水平的变化进行分析。 3.利用免疫荧光染色和蛋白免疫印迹方法对重度磨耗后大鼠脑干及延髓中p38MAPK表达水平的变化进行分析。研究结果： 1.采用外科显微镜下逐渐均匀降低大鼠上颌后牙牙冠的方法成功建立了重度磨耗的动物模型，建模后大鼠表现型无明显异常。3d时咬肌机械压痛阈值显著降低，持续到21d，第28d时基本恢复到基线水平。 2.重度磨耗可引起大鼠三叉神经节上颌牙槽神经神经元相关区域内的CGRP表达水平的升高。建模后的3d CGRP阳性神经元的数量显著增加，达到峰值，随后3wCGRP阳性神经元的数量有所减少但仍明显高于对照组，第28d时基本恢复到正常水平。 3.重度磨耗后大鼠脑干及延髓三叉神经脊束核相关区域内p38MAPK通路被激活，建模后3d p-p38蛋白表达显著上调，达到峰值后开始逐渐减少，激活状态持续3w，第28d时p-p38蛋白的表达基本恢复到正常水平。结论： 1.本实验成功建立了重度磨耗的动物模型，行为学方法检测发现大鼠重度磨耗后可以出现一过性的咬肌机械压痛敏感性增高。 2.重度磨耗后大鼠三叉神经节上颌牙槽神经神经元相关区域内的CGRP表达水平一过性升高，，说明咬肌疼痛敏感性与三叉神经节内CGRP表达水平相关，进一步证实CGRP在此模型疼痛的发展过程中发挥了作用。 3.重度磨耗后大鼠脑干及延髓三叉神经脊束核相关区域内p-p38蛋白表达一过性增加，提示MAPK通路在重度磨耗建模后被激活，p38MAPK通路在此模型疼痛的调控中发挥着重要作用。
[Abstract]:Research background:
Severe occlusal attrition (SOA), as a common chronic disease in the oral cavity, is an important cause of the temporomandibular joint disorder (temporomandibular joint disorder, TMD), often accompanied by pain, abnormal mandibular movement, ringing and murmurs in the temporomandibular joint (temporomandibular joint, TMJ) area, including Tsui. The primary cause of pain in the masticatory and temporomandibular joint areas is often the primary cause of the patient's treatment. The basic research on severe abrasion is mainly focused on the response of the local tissue in the early stage. The study confirms that the severe attrition will cause the adaptation to the masticatory muscles and the temporomandibular joint, but the pain and the nerve system caused by severe wear and maxillofacial pain and the nervous system There are few studies on the related reactions. In the models of other oral and maxillofacial neuropathic pain and inflammatory pain, it is found that nociceptive stimulation can cause the increase in the expression of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and the trigeminal trigeminal spinal tract of the brain and medulla. The cell mitogen activated protein kinase (mitogen-activated protein kinases, MAPK) pathway in the nucleus (spinal nucleus of trigeminal nerve, Sp V) is activated and the expression level of phosphorylation p38 (phospho-p38) protein is up-regulated. Therefore, this study intends to focus on pain as a key point to analyze the threshold of pain in the oral and maxillofacial region after severe attrition. Changes in response to the nervous system.
The purpose of the study is:
To establish and evaluate the animal model of severe abrasion, observe the changes in the sensitivity of the masseter mechanical tenderness in the rats after severe abrasion, and observe the changes of the related neurotransmitters in the trigeminal ganglia, the brainstem and the medulla with relevant experimental techniques, in order to analyze the response of the peripheral and primary central nervous system in the severe attrition rats.
Research methods:
1. using the surgical microscope to gradually reduce the crown of the maxillary posterior teeth, a severe wear animal model was established to observe the changes of the phenotype and weight of the rats, and the sensitivity of the mechanical tenderness of the masseter muscle in rats was detected by the behavior method.
2. the expression of CGRP in trigeminal ganglion of severe wear rats was analyzed by immunofluorescence staining.
3. the expression of p38MAPK in brainstem and medulla oblongata was analyzed by immunofluorescence staining and Western blotting.
The results of the study:
1. the animal model of severe attrition was successfully established by the method of gradual uniform reduction of the maxillary posterior teeth crown under the surgical microscope. The threshold of the mechanical tenderness of the masseter muscle was significantly reduced when the model of the model had no obvious abnormal.3d. It continued to 21d, and the baseline was basically restored to the baseline at 28d.
2. severe attrition could increase the level of CGRP expression in the maxillary alveolar nerve neurons in the rat trigeminal ganglia. The number of 3D CGRP positive neurons after modeling increased significantly and reached the peak. Then the number of 3wCGRP positive neurons decreased but was still significantly higher than that of the control group. At the time of 28d, the number of positive neurons recovered to the normal level.
3. the p38MAPK pathway in the brain stem and the nucleus of the spinal trigeminal nucleus in the rats after severe abrasion was activated. After modeling, the expression of 3D p-p38 protein increased significantly. After the peak, the activation state gradually decreased and the activation state continued to be 3W. The expression of p-p38 protein at 28d was basically restored to normal level at the time of 28d.
Conclusion:
1. the animal models of severe attrition were successfully established in this experiment. The behavior method test found that the sensitivity of the mechanical tenderness of the masseter muscle increased after the severe attrition of the rats.
2. the level of CGRP expression in the maxillary alveolar nerve neurons in the trigeminal ganglion neurons of the rats after severe attrition increased, which indicates that the sensitivity of the masseter muscle pain is related to the level of CGRP expression in the trigeminal ganglia, and further confirms the role of CGRP in the development of this model.
3. the expression of p-p38 protein in the brain stem and the nucleus of the spinal trigeminal nucleus in the rats after severe attrition increased, suggesting that the MAPK pathway was activated after the modeling of severe attrition, and the p38MAPK pathway played an important role in the regulation of the model pain.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781

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