【作者】 刘文；

【导师】 龙星；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2021， 博士

【摘要】 第一部分大鼠TG中与TMJOA慢性疼痛有关的TMJ传入神经元特征目的:慢性疼痛是颞下颌关节骨关节炎(TMJOA)的主要症状之一,而TMJ的感觉神经主要来自三叉神经节(TG)。TG神经元根据直径可将其分为小型、中型和大型三种类型。中、小型TG神经元一般为C型和Aδ型神经元,可分为无髓的降钙素基因相关肽阳性(CGRP~+)肽能神经元和植物凝集素B4阳性(IB4~+)非肽能神经元,介导热痛、机械痛等伤害性刺激;相反,大型TG神经元一般为Aβ型神经元,是有髓神经丝蛋白200阳性(NF200~+)神经元,检测和传递触觉等无害性刺激。因此,本研究的目的是探讨何种类型的TG神经元支配TMJ并参与MIA诱导的TMJOA慢性疼痛发生,这对进一步了解TMJOA慢性疼痛的机制非常重要。研究方法:大鼠TMJ关节腔内注射碘乙酸钠(MIA;50μl;1mg/50μl)构建TMJOA慢性疼痛大鼠模型,注射生理盐水(50μl)作为对照组。MIA注射后第9天将荧光示踪剂(Dil,10μl,2.5mg/ml)注入关节腔逆行标记支配TMJ的TG神经元(即Dil~+TG神经元),MIA注射后第14天获取大鼠TG组织;荧光显微镜下测量原代培养的Dil~+TG神经元直径;组织免疫荧光检测Dil~+TG神经元中CGRP、IB4和NF200的表达。结果:逆行追踪实验结果显示实验组大鼠TG中Dil~+神经元的数量较对照组明显增加。原代培养TG神经元直径测量结果组内分析显示,在对照组和实验组大鼠Dil~+TG神经元中,中、小型神经元占比显多于大型神经元;免疫荧光结果组内分析显示,在对照组大鼠Dil~+TG神经元中,IB4~+神经元占比多于CGRP~+和NF200~+神经元,而在实验组大鼠Dil~+TG神经元中,CGRP~+和IB4~+神经元占比明显多于NF200~+神经元。此外,原代培养TG神经元直径测量结果组间比较显示,与对照组大鼠相比较,TMJOA慢性疼痛大鼠TG中的中、小型Dil~+神经元的数量明显增加,而大型Dil~+神经元的数量未见明显变化;免疫荧光结果组间比较显示,与对照组大鼠相比较,TMJOA慢性疼痛大鼠TG中CGRP~+Dil~+和IB4~+Dil~+神经元数量明显增加,而NF200~+Dil~+神经元的数量未见明显变化。结论:MIA诱导的TMJOA慢性疼痛主要动员了大鼠Dil~+TG神经元中的小型和中型神经元以及CGRP~+和IB4~+神经元,这些神经元可能参与TMJOA慢性疼痛发生。第二部分褪黑素在TMJOA慢性疼痛中的作用目的:慢性疼痛是TMJOA患者就诊的主要原因之一,给临床治疗带来了巨大挑战。目前,主要用于缓解TMJOA疼痛的药物如非甾体类抗炎药疗效不理想且存在诸多副作用。近年来,有关褪黑素(MT)与慢性疼痛的研究明显增多,同时发现MT在慢性头痛、纤维肌痛、肠易激综合征等慢性疼痛中有良好的镇痛作用,但很少有研究涉及MT对TMJOA慢性疼痛的作用。本研究的目的是探讨MT在MIA诱导的大鼠TMJOA慢性疼痛中的作用以及两种MT膜受体(MT1R和MT2R)在大鼠外周TG神经元中的表达。研究方法:收集TMJOA和髁突肥大(CH,对照组)患者的关节滑液,应用酶联免疫吸附试验(ELISA)检测关节滑液中MT的含量。大鼠TMJ关节腔内注射MIA或生理盐水后第14天收集大鼠TMJ关节滑液,应用ELISA检测关节滑液中MT的含量。应用Von Frey电子疼痛测量仪和大鼠痛苦表情量表(RGS)分别评估MIA诱导的大鼠TMJOA模型接受MT(20μl;0.5、1.0、2.0mg;TMJ关节腔注射)治疗前后大鼠的机械痛和自发痛。关节腔注射Dil逆行标记支配TMJ的TG神经元。应用免疫荧光检测大鼠TG组织中MT1R和MT2R的表达。结果:TMJOA患者关节滑液中的MT含量显著高于对照组,MIA诱导的大鼠TMJ关节滑液中的MT含量也明显高于对照组,结果有统计学意义。MIA诱导的大鼠TMJ关节腔内注射MT呈剂量依赖性地改善了MIA诱导大鼠的缩头阈值(HWT)和RGS评分。与对照组大鼠相比较,MT2R在MIA诱导大鼠的Dil+TG神经元中表达上调,而MT1R未见明显改变。结论:MT能有效地缓解MIA诱导的大鼠TMJOA慢性疼痛,而外周TG神经元上的MT2R可能参与这一过程。第三部分褪黑素调控TMJOA慢性疼痛大鼠TG神经元敏化和钙信号目的:由于急性疼痛向慢性疼痛转变的机制非常复杂,慢性疼痛的具体机制尚未阐明,因此目前TMJOA慢性疼痛的治疗效果并不理想。尽管第二部分研究已经证明MT能有效缓解MIA诱导的大鼠TMJOA慢性疼痛,但是MT缓解TMJOA慢性疼痛的具体机制仍未阐明。因此,本研究的目的是在前两部分研究的基础上进一步探讨MT在TMJOA慢性疼痛中的镇痛机制,即MT是否能通过MT2R下调TMJOA慢性疼痛大鼠外周TG神经元的敏化和细胞内钙离子浓度。研究方法:大鼠TMJ关节腔内注射Dil逆行标记支配TMJ的TG神经元。在MIA诱导的大鼠TMJOA模型中,在经4P-PDOT预处理或不经4P-PDOT预处理(20μl;10μg;MT给药前30min)的TMJ关节腔内注射MT(20μl;2.0mg),应用Von Frey电子疼痛测量仪和RGS检测大鼠的机械痛和自发痛;应用免疫荧光检测大鼠TG中MT2R与CGRP/IB4/NF200的共表达。用MT孵育(100μM;30min)经4P-PDOT预处理或不经4P-PDOT预处理(10μM,孵育30min,MT给药前30min)的原代培养TG神经元,应用Fluo-3 AM钙成像技术检测大鼠TG神经元的细胞内钙离子浓度。结果:免疫荧光结果显示,在MIA诱导的TMJOA慢性疼痛大鼠TG中,MT2R主要与CGRP和IB4共表达,很少和NF200共表达;关节腔注射MT抑制了MIA诱导的TMJOA慢性疼痛大鼠TG中CGRP+和IB4+神经元细胞数上调,而4P-PDOT关节腔预处理逆转了MT的作用;此外,MT关节腔注射抑制了MIA诱导的TMJOA慢性疼痛大鼠TG中CGRP+MT2R+和IB4+MT2R+神经元细胞数上调,而4P-PDOT关节腔预处理逆转了MT这一作用。行为学研究结果显示,在MIA诱导的TMJOA慢性疼痛大鼠中,4P-PDOT关节腔注射呈剂量依赖性地逆转了MT所改善的HWT值和RGS评分。原代培养TG神经元的钙成像结果显示,在MIA诱导的TMJOA慢性疼痛大鼠中,Dil+TG神经元的荧光强度明显高于Dil-TG神经元,但在对照组大鼠中,Dil+TG神经元与Dil-TG神经元的荧光强度无明显差异;此外,MT孵育Dil+TG神经元抑制了MIA诱导的细胞内钙离子浓度升高,但4P-PDOT预处理逆转了MT的作用。结论:MT通过MT2R下调TMJOA慢性疼痛大鼠TG中CGRP+和IB4+神经元的敏化以及Dil+神经元的细胞内钙离子浓度,从而缓解MIA诱导的大鼠TMJOA慢性疼痛。更多还原

【Abstract】 Part 1.Characteristics of TMJ afferent neurons related to TMJOA chronic pain within the TG in ratsObjective:Chronic pain is one of the major symptoms of temporomandibular joint osteoarthritis(TMJOA),and the sensory nerve of TMJ mainly comes from the trigeminal ganglion(TG).TG neurons can be classified into three types based on the soma diameter:small,medium and large.Small-and medium-sized TG neurons are regarded as C-type and Aδ-type neurons,which can be categorized as unmyelinated Calcitonin Gene-Related Peptide positive(CGRP~+)peptidergic neurons and Isolectin B4 positive(IB4~+)nonpeptidergic neurons,which transmit nociceptive stimuli such as thermal pain and mechanical pain.In contrast,large myelinated TG neurons are generally regarded as Aβ-type neurons,marked with neurofilament 200(NF200~+),which detect and transmit innocuous stimuli such as tactile.Therefore,the aim of this study is to explore the type of TG neurons innervated TMJ and participated in the occurrence of TMJOA chronic pain induced by MIA,which is very important to better understanding the mechanism of TMJOA chronic pain.Methods:Intra-TMJ injection of monosodium iodoacetate(MIA;50μl;1mg/50μl)was conducted to establish a rat model of TMJOA chronic pain,with injection of saline(50μl)as a control group.At day 9 after MIA injection,a fluorescent tracer(Dil,10μl,2.5mg/ml)was injected into TMJ to retrograde label the TG neurons(i.e.Dil~+TG neurons)innervating TMJ.The TG tissue of rats was harvested at day 14 after MIA injection.The diameter of the primary cultured Dil~+TG neurons was measured under fluorescence microscope,and the expression of CGRP,IB4 and NF200 in Dil~+TG neurons was detected by immunofluorescence.Results:Retrograde tracing showed that the number of Dil~+TG neurons in TMJOA rats was significantly higher than that in the control group.The intra-group analysis of the diameter measurement of primary cultured TG neurons showed that in the Dil~+TG neurons of the control and experimental groups,the percentages of small-and medium-sized neurons was significantly higher than that of large-sized neurons.The intra-group analysis of immunofluorescence showed that the percentage of IB4~+neurons was higher than that of CGRP~+and NF200~+neurons in the Dil~+TG neurons of control rats,while the percentages of CGRP~+and IB4~+neurons was significantly higher than that of NF200~+neurons in the Dil~+TG neurons of experimental rats.Furthermore,comparison of diameter measurement of primary cultured TG neurons between groups showed that the number of small-and medium-sized Dil~+TG neurons in rats with TMJOA chronic pain was significantly increased compared with the control group,while the number of large-sized Dil~+TG neurons remained unchanged.Comparison of immunofluorescence between groups showed that the number of CGRP~+Dil~+and IB4~+Dil~+TG neurons in rats with TMJOA chronic pain was significantly increased compared with the control group,but the number of NF200~+Dil~+TG neurons remained unchanged.Conclusion:MIA-induced TMJOA chronic pain mainly mobilized small-and medium-sized and CGRP-and IB4-positive Dil~+TG neurons in rats,which may be involved in the occurrence of TMJOA chronic pain.Part 2.The role of melatonin in TMJOA chronic painObjective: Chronic pain is the main cause for TMJOA patients to seek medical care,and it is also a major challenge for clinicians.At present,the drugs to relieve TMJOA pain,such as non-steroidal anti-inflammatory drugs,have unsatisfactory effects and many side effects.While researches linking MT to chronic pain have increased significantly over recent years,and MT has been found to have a good analgesic effect in fibromyalgia,irritable bowel syndrome,and chronic headaches.However,few studies have investigated the contribution of MT to TMJOA chronic pain.The aim of this study was to investigate the role of MT in rats suffering from TMJOA chronic pain induced by MIA and the expression of two kinds of MT membrane receptors(MT1R and MT2R)in peripheral TG neurons of rats.Methods: The TMJ synovial fluid of patients with TMJOA and condylar hyperplasia(CH,control group)was collected,and the content of MT in TMJ synovial fluid was determined by enzyme-linked immunosorbent assay(ELISA).The TMJ synovial fluid of rats was collected at day 14 after intra-TMJ injection with MIA or saline,and the content of MT in TMJ synovial fluid was determined by ELISA.The Von Frey and Rat Grimace Scale(RGS)were used to assess mechanical pain and spontaneous pain in TMJOA rats induced by MIA before and after treatment with MT(20 μl;0.5,1.0,2.0 mg;intra-TMJ injection).Dil was injected into TMJ to label the TG neurons innervating TMJ.The expression of MT1 R and MT2 R in TG of rats was detected by immunofluorescence.Results: MT levels were higher in the synovial fluid of patients with TMJOA pain than in the control group,and MT levels in synovial fluid samples from rats induced by MIA were also significantly higher compared with the control group,these results were statistically significant.Intra-TMJ injection of MIA-induced rats with MT dose-dependently ameliorated the HWT and RGS score in MIA-induced rats.The expression of MT2 R in Dil+TG neurons was significantly higher in MIA-induced rats than that in control rats,while the expression of MT1 R remained unchanged.Conclusion: MT can effectively relieve TMJOA chronic pain of rats induced by MIA,and MT2 R in peripheral TG neurons may be involved in this process.Part 3.Melatonin modulates the sensitization and calcium signals of TG neurons from rats of TMJOA chronic painObjective: As the transition mechanism of pain from acute to chronic is very complicated and the specific mechanism of chronic pain has not yet been elucidated,the therapeutic effect of TMJOA chronic pain is not ideal at present.Although Part 2 has suggested that MT can effectively relieve TMJOA chronic pain induced by MIA in rats,the mechanism of MT in relieving TMJOA chronic pain is not clear.Therefore,the aim of this study is to further explore the analgesic mechanism of MT in TMJOA chronic pain based on the previous two parts,that is,whether MT can down-regulate the sensitization and the intracellular calcium concentration of peripheral TG neurons from rats of TMJOA chronic pain by MT2 R.Methods: Dil was injected into TMJ of rats to retrograde label the TG neurons innervating TMJ.In MIA-induced TMJOA rats,MT was injected into TMJ with or without 4P pretreatment,the Von Frey and RGS were used to assess mechanical pain and spontaneous pain in rats,and the co-expression of MT2 R and CGRP/IB4/NF200 in TG of rats was detected by immunofluorescence.Primary cultured TG neurons with or without 4P-PDOT pretreatment were incubated with MT,Fluo-3AM calcium imaging technique was used to detect the concentration of calcium ion in TG neurons of rats.Results: Immunofluorescence staining demonstrated that MT2 R was heavily colocalized with both CGRP and IB4 but comparatively little with NF200 in TG neurons in TMJOA chronic pain rats induced by MIA.Intra-TMJ injection of MT inhibited the up-regulation of CGRP+ and IB4+ TG neuron numbers in TMJOA chronic pain rats induced by MIA,while these effects were reversed by 4P-PDOT pretreatment.In addition,intra-TMJ injection of MT inhibited the up-regulation of CGRP+MT2R+ and IB4+MT2R+ TG neuron numbers in TMJOA chronic pain rats induced by MIA,and these effects were reversed by 4P-PDOT pretreatment.Behavioral studies showed that pretreatment with 4P-PDOT dose-dependently reversed the MT-ameliorated HWT and RGS score in MIA-induced TMJOA rats.Calcium imaging of primary cultured TG neurons showed that the value of fluorescent intensity was significantly higher in Dil+TG neurons than that in Dil-TG neurons extracted from TMJOA chronic pain rats induced by MIA,while there was no difference between Dil+ and Dil-neurons in control rats.Furthermore,MIA-increased intracellular calcium concentration was abolished in MT-incubated Dil+TG neurons,but this effect was abrogated by 4P-PDOT pretreatment.Conclusion: MT could down-regulate the sensitization of CGRP+ and IB4+ TG neurons and the intracellular calcium concentration of Dil+TG neurons via MT2 R,which relieves TMJOA chronic pain of rats.更多还原