外周代谢型谷氨酸受体5在颞下颌关节疼痛大鼠三叉神经节表达

目的探讨外周代谢型谷氨酸受体5(m Glu R5)在颞下颌关节炎性疼痛中的作用。方法 SD大鼠36只,随机均分为6组,正常对照组(不做任何处理),阴性对照组(右侧颞下颌关节内注射50μl生理盐水),完全弗氏佐剂组(CFA组,右侧颞下颌关节内50μl CFA注射),MPEP-1组(1mm 2-甲基-6苯基乙炔嘧啶+50μl CFA),MPEP-2组(2.5mm MPEP+CFA),MPEP-3组(5mm MPEP+CFA)。将CFA注入右侧颞下颌关节腔内,诱导大鼠颞下颌关节疼痛。24h后检测各组大鼠的机械疼痛阈值,用Western-blot法和RT-PCR方法检测各组大鼠三叉神经节内m Glu R5和ERK1/2的蛋白和m RNA表达变化。结果 CFA组大鼠摆头阈值较正常组和阴性对照组明显下降(0.01),MPEP-3组明显高于CFA组(0.05)。三叉神经节内m Gu Rl 5和ERK1/2 m RNA及蛋白表达水平CFA组明显高于正常对照组和阴性对照组(0.05),MPEP-3组明显低于CFA组(0.05)。结论 m Glu R5参与颞下颌关节炎性疼痛,可能与ERK1/2信号通路相关。     

三叉神经节内降钙素基因相关肽的分布及其在伤害性刺激条件下的变化

目的研究三叉神经节内降钙素基因相关肽(calcitoningenerelatedpeptide,CGRP)的分布及其在伤害性刺激条件下的变化.方法将10%福尔马林150～200μl注入8只Wistar大鼠面口部皮下,1/2、2、12h取材,用ABC法行免疫组化染色,图像分析仪测量阳性细胞的积分光密度.结果(1)CGRP阳性细胞以神经节的外周为著,多为中等细胞(89%).(2)染色深浅与细胞的大小无关.(3)面口部伤害性刺激条件下,节细胞内CGRP阳性产物的积分光密度在F1/2h组较对照组下降(P＜0.05);F2h组较对照组升高(P＜0.001);F12h组较对照组无明显差异(P＞0.05).结论(1)福尔马林刺激后,三叉神经节细胞中CGRP的积分光密度发生时间相关性变化.(2)在三叉神经系统,CGRP参与伤害性刺激的传递.     

大鼠PAG内Nogo-A和CGRP参与痛觉调制的研究

目的:探讨Nogo-A在痛觉调制过程中的作用以及它在大鼠中脑导水管周围灰质(periaqueductal gray,PAG)内与降钙素基因相关肽(CGRP)和内源性阿片系统在痛觉调制过程中的相互关系。方法:选用雄性SD大鼠32只,随机等分为4组:其中空白对照组8只,分别向PAG内注射0.9%生理盐水1μl;痛敏模型对照组、吗啡组和纳洛酮组各8只,首先单侧后爪掌心皮下注射1%的福尔马林0.1 ml复制痛敏大鼠模型,3 d后分别向PAG内注射等量0.9%生理盐水、吗啡和纳洛酮各1μl。60 min后取大鼠大脑PAG区,采用免疫组化法观测各组大鼠PAG内CGRP和Nogo-A免疫反应阳性神经元的表达差异。结果:与空白对照组比较,模型对照组大鼠PAG内CGRP的表达量显著增高(P<0.01),Nogo-A显著降低(P<0.01);与模型对照组比较,吗啡组大鼠PAG内CGRP的表达量显著降低(P<0.05),Nogo-A显著增高(P<0.01),而纳洛酮组PAG中CGRP的表达量显著增高(P<0.01),Nogo-A显著降低(P<0.05)。结论:Nogo-A和CGRP参与了痛觉调制过程,但Nogo-A与CGRP之间有反向调节作用;Nogo-A与内源性阿片系统之间可能也存在一定的功能联系。     

医用臭氧可缓解颞下颌关节骨关节炎的疼痛

背景:颞下颌关节骨关节炎引起的疼痛严重影响了患者的生活质量及心理健康。研究发现医用臭氧能有效缓解颞下颌关节骨关节炎疼痛,但对其镇痛作用及机制尚不清楚。目的:探讨医用臭氧对颞下颌关节骨关节炎疼痛的作用及可能机制。方法:采用随机数字表法将24只SD大鼠分成4组,每组6只,除对照组外,模型组、空气组、医用臭氧组通过关节内注射碘乙酸钠的方法建立颞下颌关节骨关节炎模型,造模1周后,空气组、医用臭氧组大鼠颞下颌关节内分别注射洁净空气、医用臭氧,1次/周,共3次。造模前及造模后28 d内,采用Von Frey机械疼痛测量技术衡量大鼠颞下颌关节机械痛阈值;造模后28 d取材,应用ELISA法检测血清和颞下颌关节液中白细胞介素1β水平,苏木精-伊红染色观察颞下颌关节软骨组织病理学变化,免疫组化染色分析颞下颌关节内缺氧诱导因子1α、环氧合酶2表达水平。结果与结论:①与对照组相比,模型组大鼠造模后1,3,7,14,21,28 d的颞下颌关节机械痛阈值降低(P<0.01);与模型组、空气组相比,医用臭氧组大鼠造模后28 d的颞下颌关节机械痛阈值升高(P<0.01);②与对照组相比,模型组大鼠血清与关节液中的白细胞介素1β质量浓度升高(P<0.01);与模型组、空气组相比,医用臭氧组大鼠血清与关节液中的白细胞介素1β质量浓度降低(P<0.01);③苏木精-伊红染色显示模型组和空气组软骨结构紊乱,呈退行性改变;医用臭氧组软骨结构紊乱程度轻于模型组、空气组;④免疫组化染色显示,与对照组相比,模型组大鼠颞下颌关节内缺氧诱导因子1α、环氧合酶2表达升高(P<0.01);与模型组、空气组相比,医用臭氧组大鼠颞下颌关节内缺氧诱导因子1α、环氧合酶2表达降低(P<0.01,P<0.05)。结果表明,医用臭氧可通过减少关节内缺氧诱导因子1α、白细胞介素1β和环氧合酶2表达来缓解SD大鼠颞下颌关节骨关节炎引起的疼痛。     

偏侧咀嚼大鼠三叉神经节内PPTAmRNA、CGRPmRNA及其相应蛋白的表达变化

目的　观察偏侧咀嚼大鼠模型TG中SP ,CGRP的蛋白含量及PPTAmRNA和CGRPmRNA表达的变化。方法　 2 4只Wister大鼠随机均分为 4组 ,拔除实验组动物右侧上、下颌磨牙 ,人为造成偏侧咀嚼 ,1个月和三个月后 ,取三叉神经节 ,分别进行免疫组化和原位杂交反应。结果　免疫组化结果显示 ,两个实验组TG内SP免疫阳性神经元百分比与对照组相比显著降低 (P <0 .0 5 ) ,每一组内左右侧比较无差异。CGRP含量 1M组双侧均比对照组显著减少 (P<0 .0 1) ,且咀嚼侧比非咀嚼侧增多 (P<0 .0 5 ) ;3M组咀嚼侧与对照组比无差异 ,非咀嚼侧低于对照组 (P <0 .0 1) ,咀嚼侧高于非咀嚼侧 (P <0 .0 1)。原位杂交结果显示 ,实验组非咀嚼侧TG中PPTAmRNA阳性神经元的数量均较对照组明显增高 (P<0 .0 1) ,咀嚼侧较对照组有增多趋势 ,但无统计学意义 ,实验组非咀嚼侧阳性神经元数量明显高于咀嚼侧 (P <0 .0 5 ) ;实验组非咀嚼侧TG中CGRPmRNA阳性神经元的数量均较对照组明显增多 (P <0 .0 1或P<0 .0 5 ) ,IM组咀嚼侧均较对照组增多 (P<0 .0 5 ) ,实验组非咀嚼侧阳性神经元数量明显高于咀嚼侧 (P<0 .0 5 )。结论　SP ,CGRP可能参与颞下颌紊乱综合征发病机制     

外源性bFGF对坐骨神经钳夹损伤后脊髓DRG感觉神经元CGRP变化的影响

目的:观察外源性碱性成纤维细胞生长因子(bFGF)对坐骨神经损伤后大鼠背根神经节(DRG)和脊髓后角内降钙素基因相关肽(CGRP)变化的影响。方法:成年雄性Wistar大鼠随机分成正常组、阳性对照组和bFGF组。阳性对照组动物右侧坐骨神经钳夹损伤,bFGF组动物右侧坐骨神经损伤后给予bFGF,在不同时间点运用免疫荧光技术结合图像分析检测相应背根节和脊髓后角CGRP的变化。结果:bFGF处理组术侧DRG内中小型神经元和脊髓后角内的CGRP表达明显高于阳性对照组,积分光密度值相比(P<0.05);但DRG内大型神经元内CGRP表达没有明显变化。结论:结果提示外源性bFGF能明显促进损伤后同侧DRG中小型神经元和脊髓后角CGRP的合成,对DRG内大型神经元中CGRP的表达没有明显影响。     

雌激素对去势大鼠髁突软骨雌激素受体基因表达的影响

目的探讨不同浓度的雌二醇(E2)对去势大鼠髁突软骨雌激素受体(ER)基因ERα和ERβ表达的影响。方法将SD大鼠分为对照组(control)、假手术组(sham)、去势组(OVX)、及时和延迟替代治疗组(OVX/17β-E2),每天静脉注射不同浓度雌二醇(5×10、5×10~2和5×10~3μg/kg),阴道上皮脱落细胞涂片监测雌激素水平,用Western blot和real-time PCR法检测大鼠颞下颌关节髁突软骨ERα和ERβ的蛋白及mRNA表达。结果去势大鼠阴道上皮细胞MI指数180/12/8,表明雌激素水平低下(P0.05)。及时补给生理浓度雌二醇(5×10~2μg/kg)能维持髁突软骨ER于正常范围,延迟补给生理浓度雌二醇不能恢复或改善ER表达;及时和延迟补给高浓度(5×10~3μg/kg)及低浓度(50μg/kg)E2均抑制ER蛋白和mRNA合成(P0.05),且高浓度效应显著。结论及时补充合理浓度的雌激素对去势大鼠颞下颌关节的正常生理功能具有重要意义。     

关节腔注射医用臭氧对早期颞下颌骨关节炎模型大鼠髁突组织学的影响

背景：在临床以及动物实验中均已证实关节腔内注射医用臭氧可有效改善颞下颌关节炎的症状，减缓疾病的发展，但注射医用臭氧的最理想浓度及其范围仍有争议。目的：观察颞下颌关节腔内注射不同质量浓度医用臭氧对颞下颌骨关节炎大鼠髁突软骨的影响。方法：从42只SD大鼠中随机抽取6只作为健康对照组，剩余大鼠采用关节腔内一次性注射碘乙酸钠法建立颞下颌骨关节炎模型，在建模成功的36只大鼠中选择6只作为模型参照组，其余的30只为20,30,40 mg/L医用臭氧组，每组10只。建模成功后医用臭氧组大鼠关节腔内分别注射质量浓度为20,30,40 mg/L医用臭氧，每周1次，连续注射3周，模型参照组和健康对照组分别注射等体积生理盐水，末次注射后1周，取单侧颞下颌关节组织，ELISA法检测髁突软骨中血管内皮生长因子水平，并取另一侧颞下颌关节组织进行苏木精-伊红染色和番红O-固绿染色，应用改良Mankin’s评分法评价颞下颌关节组织病理变化程度。结果与结论：(1)与健康对照组相比，模型参照组、医用臭氧20,30,40 mg/L组大鼠髁突软骨中血管内皮生长因水平显著升高(P <0.05)；与模型参照组、医用臭氧40...     

鞘内注射P2X7受体拮抗剂BBG对大鼠行为学和脊髓PLA2表达的影响

目的:探讨鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)在福尔马林模型大鼠中的镇痛作用及对脊髓磷脂酶A2(PLA2)表达的影响。方法:雄性Wistar大鼠鞘内置管成功后鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)后所有的大鼠左侧后肢爪掌背侧皮下注射福尔马林(5%,50μl),观察大鼠在第一相(0~10 min)和第二相(10~60 min)的疼痛行为学比,变化(自发缩足次数)。免疫组织化学方法检测脊髓背角PLA2蛋白的表达与变化。结果:各组间大鼠第一相相比自发缩足次数没有明显的差异;第二相与对照组相比鞘内1μg、5μg及腹腔10μg BBG注射组相比,鞘内10μg BBG注射组大鼠的疼痛行为学明显减少(自发缩足次数,P0.05)。与此同时,鞘内10μg BBG注射组大鼠脊髓背角浅层内PLA_2免疫阳性产物的表达与其它组比较明显减少(P0.05)。结论:鞘内注射10μg BBG可减轻福尔马林诱导的炎性痛觉过敏,可能是通过抑制脊髓背角浅层PLA2的表达而发挥作用。     

牙齿损伤时神经生长因子和降钙素基因相关肽在三叉神经节中的表达变化

目的　探讨神经生长因子 (NGF)和降钙素基因相关肽 (CGRP)在大鼠磨牙损伤后不同时段三叉神经节中表达变化。方法　制备大鼠磨牙机械损伤的模型 ,对其三叉神经节中NGF和CGRP进行免疫组化染色。结果　在正常三叉神经节中NGF低水平表达 ,CGRP呈阳性反应 ,损伤后 5h组二者表达增强 ;3d组NGF染色强性性 ;损伤 5d后CGRP染色强阳性 ,NGF表达开始减弱 ;损伤 9d后两者表达恢复到正常水平。结论　牙齿损伤后三叉神经节内NGF和CGRP表达增强 ,提示NGF和CGRP可能参与并促进牙齿损伤后的痛过敏及神经再生修复     

Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund's adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V(h)=-60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 mum), voltage-dependent transient K(+) current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K(+) current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund's adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K(+) current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.     

电针对内脏痛模型大鼠结肠壁、背根节和脊髓内AchE和CGRP表达的影响

目的:观察电针治疗内脏痛对结肠壁、背根节和脊髓内胆碱酯酶(AchE)和降钙素基因相关肽(CGRP)免疫阳性物质表达的影响,以探讨AchE和CGRP免疫阳性物质在痛觉传递中的作用及电针镇痛的机制。方法:26只SD大鼠随机分为正常对照组(6只)、内脏痛组(VP组,10只)和电针+内脏痛组(EA+VP组,10只)。电针取双侧"足三里"穴刺激60 min,运用酶组织化学技术和免疫组织化学技术,观察电针刺激对福尔马林所诱发的大鼠盆腔内脏痛时,结肠壁、背根节和脊髓内AchE和CGRP免疫阳性物质表达的影响。结果:内脏痛组大鼠在注射福尔马林后,结肠壁、背根节和脊髓内AchE免疫阳性物质的表达显著高于正常组(P<0.01),CGRP免疫阳性物质的表达显著低于正常组(P<0.01);而电针+内脏痛组,结肠壁、背根节和脊髓内AchE免疫阳性物质的表达显著低于内脏痛组(P<0.01),CGRP免疫阳性物质的表达又显著高于内脏痛组(P<0.01)。结论:电针对大鼠急性盆腔内脏痛具有预防性治疗作用,Ach和CGRP可能参与了电针对内脏痛的调节作用。     

Calcitonin gene-related peptide receptors in rat trigeminal ganglion do not control spinal trigeminal activity

Calcitonin gene-related peptide (CGRP) is regarded as a key mediator in the generation of primary headaches. CGRP receptor antagonists reduce migraine pain in clinical trials and spinal trigeminal activity in animal experiments. The site of CGRP receptor inhibition causing these effects is debated. Activation and inhibition of CGRP receptors in the trigeminal ganglion may influence the activity of trigeminal afferents and hence of spinal trigeminal neurons. In anesthetized rats extracellular activity was recorded from neurons with meningeal afferent input in the spinal trigeminal nucleus caudalis. Mechanical stimuli were applied at regular intervals to receptive fields located in the exposed cranial dura mater. α-CGRP (10(-5) M), the CGRP receptor antagonist olcegepant (10(-3) M), or vehicle was injected through the infraorbital canal into the trigeminal ganglion. The injection of volumes caused transient discharges, but vehicle, CGRP, or olcegepant injection was not followed by significant changes in ongoing or mechanically evoked activity. In animals pretreated intravenously with the nitric oxide donor glyceryl trinitrate (GTN, 250 μg/kg) the mechanically evoked activity decreased after injection of CGRP and increased after injection of olcegepant. In conclusion, the activity of spinal trigeminal neurons with meningeal afferent input is normally not controlled by CGRP receptor activation or inhibition in the trigeminal ganglion. CGRP receptors in the trigeminal ganglion may influence neuronal activity evoked by mechanical stimulation of meningeal afferents only after pretreatment with GTN. Since it has previously been shown that olcegepant applied to the cranial dura mater is ineffective, trigeminal activity driven by meningeal afferent input is more likely to be controlled by CGRP receptors located centrally to the trigeminal ganglion.     

细胞外信号调节激酶在偏头痛大鼠模型中的表达

目的探讨细胞外信号调节激酶(ERK)信号转导通路在偏头痛大鼠模型中的作用。方法 48只雄性SD大鼠分为正常对照组、生理盐水对照组和模型组,采用皮下注射硝酸甘油(GTN)法制做大鼠偏头痛模型。应用免疫组化技术及Western印迹法,分别观察各组大鼠硬脑膜、三叉神经节(TG)及三叉神经颈复合体细胞外信号调节激酶(ERK)、磷酸化ERK(p-ERK)的表达。结果 ERK、p-ERK在各组大鼠硬脑膜、三叉神经节及三叉神经颈复合体细胞中均呈阴性表达,模型组大鼠p-ERK表达水平明显高于生理盐水对照组及正常对照组(P<0.05)。结论偏头痛大鼠p-ERK过表达,ERK可能参与了偏头痛痛觉信号的传导,在偏头痛发病机制中发挥作用。     

神经性痛和炎性痛大鼠伏核内CGRP mRNA表达的变化

为了研究神经性痛和炎性痛大鼠伏核内CGRP mRNA表达的变化,本研究分别制备神经性痛及炎性痛大鼠动物模型,并运用实时定量逆转录聚合酶链式反应(PCR)技术,观察大鼠伏核内CGRP mRNA含量的改变。实验中以β-actin为内参,记录大鼠伏核内CGRP mRNA表达产物的CT值,并计算每一样品对应其内参的相对含量,分析随时间变化致痛因素对大鼠伏核内CGRP mRNA表达产物含量的影响。结果显示:神经损伤组5、10、20、30d及炎症组3d,CGRP mRNA的表达比正常对照组明显增高(P<0.05);而在炎症组7d时,CGRP mRNA的表达与对照组相比无显著性差异(P>0.05)。以上结果提示CGRP作为一种神经递质或调质参与痛觉的调制过程,其主要是在炎症早期发挥作用。     

免疫复合物致痛与甲醛致痛大鼠疼痛行为的比较

目的: 建立免疫复合物致痛模型并与甲醛致炎性痛模型比较,观察大鼠疼痛行为、局部炎症反应及p38 MAPK在脊髓表达的改变,探讨免疫复合物所致疼痛与普通炎性介质致痛的不同特点及发病机制。方法: 成年SD健康大鼠30只,随机分为正常对照组、甲醛组及免疫复合物组,每组10只。每组中各取5只大鼠分别鞘内注 射DMSO(对照组)或p38 MAPK抑制剂SB203580。各组分别在大鼠右后足底皮下注入20 μL PBS、甲醛及免疫复合物,于30 min、1 h、2 h、4 h、8 h、12 h测定疼痛行为,并于12 h后取大鼠脊髓采用Western blotting测定p38及活化p-p38蛋白表达。结果: (1) 致痛模型比较:甲醛致痛后大鼠后足立即出现红肿及自发痛,疼痛阈值30 min内迅速下降之后随时间推移缓解,脊髓活化的p-p38表达增加。免疫复合物组大鼠注射部位无红肿表现,疼痛阈值缓慢下降,至注射后8 h达到低谷,脊髓p38无明显活化。(2) p38 MAPK抑制剂SB203580可使甲醛组大鼠局部炎症反应及疼痛阈值下降,但对免疫复合物组及对照组大鼠无效。结论: p38 MAPK活化参与了甲醛所致炎症性疼痛的机制,但不参与免疫复合物致痛机制。本实验建立的抗体复合物致痛模型表现出与甲醛炎性痛不同的疼痛特点。     

The role of calcitonin gene-related peptide on the increase in transient receptor potential vanilloid-1 levels in trigeminal ganglion and trigeminal nucleus caudalis activation of rat

Calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid-1 (TRPV1) play an important role in the development of pain and migraine pathogenesis. Increase in plasma CGRP levels is associated with delayed migraine-like attacks in migraine patients. Although several lines of evidence have indicated a key role of CGRP in migraine pain, its mechanisms remain unclear. In this study, we aimed to investigate the functional role of CGRP on trigeminal nociceptive pathway by determining the alteration in TRPV1 levels in trigeminal ganglion (TG) and the activation of trigeminal nucleus caudalis (TNC) of rat. Post intravenous injection of CGRP (600 ng/kg) at 60 min significantly increased the levels of TRPV1, CGRP, phosphorylated protein kinase C and phosphorylated cyclic AMP responsive element-binding protein in TG of rats. The number of small and medium TRPV1 and CGRP positive immunostaining neurons accompanying with co-localization of TRPV1 with CGRP neurons were significantly increased in TG of CGRP-injected rats. The sustained increase in c-Fos expression in TNC neurons was also observed in CGRP-injected rats. These results indicate that CGRP may participate in trigeminal nociceptive system sensitization by induced increase in TRPV1 and CGRP levels in TG neurons and activation of the central neurons in TNC.