免疫荧光法观察正畸大鼠牙髓CGRP的变化

目的:观察正畸牙齿移动不同时期大鼠牙髓组织中降钙素基因相关肽(CGRP)阳性神经纤维的变化。方法:采用冷冻切片和间接免疫荧光方法观察正畸大鼠牙髓组织CGRP阳性神经纤维的表达变化情况。结果:CGRP阳性神经纤维在大鼠牙髓组织中分布较丰富,在根髓呈粗大的束状;在冠髓呈条索状、串珠样,并在髓顶造牙本质细胞下层,成网状分布。正畸加力后3d大鼠牙髓CGRP阳性神经纤维数量开始增加;加力7d达到最高;至撤力后28d下降到正常水平。结论:CGRP阳性神经纤维参与了正畸牙齿移动大鼠牙髓组织的反应过程。     

Expression of Nav1.9 channels in human dental pulp and trigeminal ganglion

There is a higher incidence of local anesthetic failure in endodontic patients experiencing pulpal hyperalgesia. Up-regulation of Nav1.9, a voltage-gated sodium channel isoform, might play a key role in local anesthetic failure because Nav1.9 channels increase neuronal excitability and have low sensitivity to blockade by local anesthetics. Immunocytochemistry was used to examine Nav1.9 channel expression in axons of symptomatic (painful) versus asymptomatic human dental pulp and to determine Nav1.9 expression levels in neuronal somata of the human trigeminal ganglion. Nav1.9 channel immunoreactivity on pulpal axons was significantly increased in painful teeth. Nav1.9 channels were expressed in membranes and cytoplasm of human trigeminal ganglion neurons, with the highest expression in small neuronal somata. Nav1.9 expression in the trigeminal ganglion coupled with increased expression in symptomatic pulp might contribute to hypersensitivity of inflamed pulps and local anesthetic failure. Furthermore, the present study suggests that Nav1.9 channels are potential targets for novel anesthetics.     

大鼠正畸牙移动过程中TRPV1和CGRP在三叉神经节中的表达变化

目的建立SD大鼠正畸牙移动模型,研究牙移动过程中,三叉神经节(trigeminal ganglion,TG)中瞬时受体电位香草酸亚型1（transient receptor potential vanilloid 1,TRPV1）及降钙素基因相关肽(calcitonin gene related peptide,CGRP)的表达位置及表达量的变化规律,进而探讨其在正畸疼痛中的作用机制。方法将66只SD大鼠随机分为空白对照组（6只）、假手术组（6只）和实验组（54只）。建立正畸牙移动模型,实验组大鼠施加50 g力后,分别于4、8 h、1 d（1 d组按力值大小分为1 d-30 g、1 d-50 g、1 d-80 g 3个亚组）、3、5、7、14 d,随机处死6只,切取三叉神经节,采用免疫荧光技术分别检测大鼠三叉神经节中TRPV1及CGRP的表达位置及表达量变化。采用SPSS16.0软件包对数据进行统计学分析。结果免疫荧光染色显示,TRPV1及CGRP主要表达于小型及中型神经元。随着加力时间的延长,三叉神经节中TRPV1免疫反应阳性（TRPV1-IR）神经元百分比及CGRP免疫反应阳性（CGRP-IR）神经元百分比增加,1~3 d相继达到高峰,之后逐渐降低,回落至初始水平,且两者随加力力值增大而呈现增高趋势。结论大鼠正畸牙移动过程中,三叉神经节内TRPV1及CGRP的表达随加力时间及加力力值改变呈现规律性变化,提示TRPV1及CGRP可能在正畸疼痛的发生机制中具有重要作用。     

实验性牙移动三叉神经节内降钙素基因相关肽改变

目的：观察实验性牙移动后,初级感觉神经元兴奋性递质降钙素基因相关肽（CGRP）的改变。分析正畸疼痛时,初级感觉神经元痛信号发生、传导及敏感化机制。方法：制备大鼠实验性牙移动动物模型,采用免疫荧光染色法和RT-PCR法分别观察不同时间点三叉神经节内CGRP免疫阳性结构的改变及CGRP mRNA表达的变化。结果：实验性牙移动后,实验侧三叉神经节（TG）内CGRP-ir节细胞以及CGRP mRNA的表达强于对侧。结论：实验性牙移动后,初级感觉神经元中痛感受兴奋性神经递质CGRP的合成、表达增加。表明实验性牙移动影响了初级感觉神经元痛信号的发生、传导,使之致敏。     

重度磨耗对大鼠三叉神经节中CGRP表达的影响

目的：探讨重度磨耗对大鼠三又神经节中降钙素基因相关肽（calcitonin gene—related petide,CGRP）表达的影响。方法：8周龄雄性SD大鼠75只,随机平均分为操作对照组、空白对照组和实验组,每组各25只,实验组用人工逐次磨低大鼠牙冠高度的方法建立重度磨耗动物模型。各组分别于干预后3、7、14、21、28天处死5只动物,取出双侧三叉神经节,通过免疫荧光染色的方法观察三叉神经节中CGRP的表达变化。结果：与空白对照组和操作对照组相比,实验组3天组和7天组的CGRP阳性神经元数量明显增加（P〈0．05）,14天组和21天组CGRP阳性神经元数量有所减少,但仍明显多于对照组（P〈0．05）,28天组基本恢复正常（P〉0．05）。结论：重度磨耗可导致大鼠三叉神经节内CGRP表达的可逆性变化。     

降钙素基因相关肽对人牙髓细胞的矿化影响

目的：分析降钙素基因相关肽（calcitonin gene-related peptide,CGRP)对人牙髓细胞促矿化功能的影响，探讨CGRP在牙髓炎症状态下表达量增多的生物学意义。方法：人牙髓细胞分实验组和对照组培养，实验组加入适量的CGRP。分别测定细胞的碱性磷酸酶活性、骨钙素含量、I型胶原mRNA表达量变化。结果：培养3、6、9、12、15d,5个时间点实验组比对照组细胞碱性磷酸酶活性增强、骨钙素含量增加，且有显著性差异（P＜0．01）。培养6d的实验组的细胞I型胶原mRNA表达比对照组明显高。结论：CGRP刺激牙髓细胞分化，促进矿化形成，CGRP表达增高将有助于修复性牙本质形成。     

The effect of orthodontic forces on calcitonin gene-related peptide expression in human dental pulp

Introduction

The purpose of this study was to quantify the effect of moderate and severe orthodontic forces on calcitonin gene-related peptide (CGRP) expression in healthy human dental pulp.

Methods

Thirty human dental pulp samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, teeth were divided into 3 groups of 10 premolars each: (1) the control group: healthy premolars without application of orthodontic forces; (2) the moderate force group: a 56-g force was applied to the premolars for 24 hours; and (3) the severe force group: a 224-g force was applied to the premolars for 24 hours. All dental pulp samples were processed, and CGRP was measured by radioimmunoassay.

Results

Greater CGRP expression was found in the severe force group followed by the moderate force group. The lower CGRP values were for the control group. The Kruskal-Wallis test showed statistically significant differences between groups (P < .0001). Least significant difference (LSD) post hoc tests showed statistically significant differences in CGRP expression between the control group and the severe force group (P < .0001) but not with the moderate force group (P = .06). Differences between the moderate and severe force groups were statistically significant (P < .0001).

Conclusions

CGRP expression in human dental pulp increases when teeth are submitted to severe orthodontic forces.     

咬合创伤大鼠三叉神经节中PPTA mRNA表达的变化

目的:观察咬合创伤后,三叉神经节中前速激肽原A (PPTA) mRNA表达的变化。方法:制备大鼠咬合创伤模型,采用分子杂交方法,观察咬合创伤后7、15、30天时,三叉神经节中PPTA mRNA表达的变化。结果:持续性咬合创伤后PPTA mRNA表达高于对照侧。咬合创伤7天,PPTA mRNA表达增高趋势明显高于15天和30天组。15天和30天两组间PPTA mRNA的表达增高没有明显差异。结论:咬合创伤后,三叉神经节PPTA mRNA表达增加,初级感觉神经元合成SP前体增加。     

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current in submandibular ganglion

Objective

The control of saliva secretion is mainly under parasympathetic control. The submandibular ganglion (SMG) is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. The aim of this study was to investigate if adrenomedullin (ADM) and/or calcitonin gene-related peptide (CGRP) modulate voltage-dependent calcium channel (VDCCs) current (I_Ca) in SMG.

Design

The profile of CGRP and ADM actions in SMG was studied using the whole-cell configuration of the patch-clamp technique.

Results

Both ADM and CGRP facilitated I_Ca. These facilitations were attenuated by intracellular dialysis of the anti-Gα_s-protein and pretreatment of SQ22536 (an adenylate cyclase inhibitor).

Conclusions

ADM and CGRP facilitates VDCCs mediated by Gα_s-protein and adenylate cyclase in SMG.     

实验性牙移动过程中三叉神经节内神经生长因子受体TrkA的改变

目的观察实验性牙移动时,神经生长因子受体TrkA在三叉神经节内的改变。方法采用免疫荧光方法观察大鼠实验性牙移动后,三叉神经节内TrkA免疫阳性细胞的改变。结果实验性牙移动加力24 h组3、d组、1周组,实验侧三叉神经节内TrkA免疫阳性神经元较对侧增多;2周组实验侧三叉神经节TrkA免疫阳性神经元与对照组相比差异无显著性。结论实验性牙移动时,实验侧三叉神经节内TrkA免疫阳性神经元增多。     

Vanilloid receptor 1 expression in human tooth pulp in relation to caries and pain

AIMS: To investigate the presence of vanilloid receptor 1 (TRPV1) in human dental pulp and to correlate any expression with caries and pain. METHODS: Permanent mandibular first molars were collected and categorized as intact or grossly carious. Grossly carious teeth were further categorized as carious asymptomatic or carious painful samples. Coronal pulps were removed and processed for indirect immunofluorescence using antibodies raised against TRPV1 and a neuronal marker, either protein gene product 9.5 or alpha-smooth muscle actin, in conjunction with Ulex europaeus agglutinin 1 lectin to fully label the pulp vasculature. RESULTS: Analysis revealed that TRPV1 labeling was not confined to pulpal nerve fibers. TRPV1 was also consistently expressed within pulp microvasculature. Expression of neuronal TRPV1 was significantly increased throughout the pulp in grossly carious samples (P < .05). No significant differences were found between carious asymptomatic and carious painful samples. A significant increase in vascular TRPV1 expression was observed in arterioles present in the midcoronal pulp in carious painful compared with carious asymptomatic samples (mean area +/- SEM [%] of TRPV1 to vascular labeling; 6.48% +/- 4.5% for carious asymptomatic teeth, n = 9; 31.21% +/- 9.6% for carious painful teeth, n = 9; P = .02). CONCLUSION: Expression of TRPV1 in pulpal nerve fibers undergoes marked changes with caries. This may be of relevance in the development of pulpal inflammation, but its relationship to dental pain is still unclear. However, vascular TRPV1 expression does appear to be positively correlated with dental pain, thus providing new insights into symptomatic pulpitis.     

正畸牙齿移动过程中大鼠三叉神经节CGRP mRNA表达变化

目的：观察正畸牙齿移动不同时期大鼠三叉神经节CGRP mRNA表达变化。方法：采用反转录。聚合酶链反应（RT-PCR）技术检测正畸加力和撤力后不同时期大鼠三叉神经节CGRP mRNA水平的变化。结果：加力后2h CGRP mRNA的表达量没有明显增加,至加力后8h开始增加,加力1d-1W达到最高峰,至撤力后2～4WCGRP mRNA的表达量开始下降,但CGRP mRNA水平仍然明显高于对照组（P〈0．05）。结论：CGRP可能不仅参与早期正畸牙周组织的炎症损伤过程,而且可能参与了后期的组织修复重建过程。     

Calcitonin gene-related peptide receptor expression in healthy and inflamed human pulp tissue

AIM: To use radioreceptor analysis for comparing calcitonin gene-related peptide (CGRP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation. METHODOLOGY: Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another eight pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In four of these premolars, inflammation was induced prior to pulp collection. All the samples were processed and labelled with 125I-CGRP. Binding sites were identified by 125I-CGRP and standard CGRP competition assays. RESULTS: CGRP receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. The Kruskal-Wallis and Mann-Whitney (post-hoc) tests showed statistically significant differences between the groups (P < 0.05). CONCLUSION: CGRP receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.     

Increase of galanin in trigeminal ganglion during tooth movement

It is known that nerve fibers containing neuropeptides such as galanin increase in the periodontal ligament during experimental tooth movement. However, the origin of galanin-containing nerve fibers in the periodontal ligament remains unclear. This study was conducted to examine our hypothesis that the increased galanin nerve fibers have a sensory neuronal origin, and that the peptide is associated with pain transmission and/or periodontal ligament remodeling during experimental tooth movement. In control rats, galanin-immunoreactive trigeminal ganglion cells were very rare and were observed predominantly in small ganglion cells. After 3 days of experimental tooth movement, galanin-immunoreactive trigeminal ganglion cells significantly increased, and the most marked increase was observed at 5 days after experimental tooth movement. Furthermore, their cell size spectrum also significantly changed after 3 and 5 days of movement: Medium-sized and large trigeminal ganglion cells began expressing, and continued to express, galanin until 14 days after experimental tooth movement. These findings suggest that the increase of galanin in the periodontal ligament during experimental tooth movement at least partially originates from trigeminal ganglion neurons and may play a role in pain transmission and/or periodontal remodeling.     

Expression of insulin-like growth factor-1 receptor in human pulp tissue

Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation and differentiation. The purpose of this study was to use a radioreceptor assay to evaluate whether IGF-1 receptors are present in human pulp and to determine whether differences in its expression are found in the pulp tissue of teeth having incomplete or complete root development. Twenty pulps were obtained from freshly extracted human third molars; they were then processed and labeled with I-IGF-1. The results showed IGF-1 receptor expression in all human pulp samples. t test revealed statistically significant higher expression in the pulps from teeth having incomplete root development (P <0.005). Given the functions of this growth factor system in other tissues, the present findings are consistent with the hypothesis that IGF-1 contributes toward forming and mineralizing dental tissues as well as in pulp-repairing processes.     

Effect of capsaicin treatment or inferior alveolar nerve resection on dentine formation and calcitonin gene-related peptide- and substance P-immunoreactive nerve fibres in rat molar pulp

The aim of this study was to investigate whether decreased sensory innervation induced by capsaicin treatment or axotomy of the inferior alveolar nerve has an effect upon dentine formation in the rat first molar. Dentine formation was visualized by intravital injection of Procion brilliant Red H8BS and denervation was verified immunohistochemically for the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. The observation times were 6 weeks for the capsaicin-treated group and 11 days for the axotomized group. Capsaicin injections caused a consistent reduction in numbers of CGRP- and substance P-immunoreactive fibres in the pulps and a somewhat smaller reduction in the periodontal tissues. Unilateral axotomy of the inferior alveolar nerve induced an almost complete loss of immunoreactive fibres in the pulp and in the mesial gingiva of the first molar. Dentine formation at the mesial pulp horn and at the central pulp floor was significantly reduced in both groups compared to controls. The results suggest that sensory neuropeptides such as CGRP and substance P may play a part in dentine formation.     

三叉神经痛患者血液降钙素基因相关肽的观察

目的 :观察三叉神经痛发作时是否有降钙素基因相关肽参与 ,加深对三叉神经痛发病机理的认识。方法 :用放射免疫法检测 7例三叉神经痛患者疼痛发作时患侧颈外静脉血、肘静脉血及术后患侧颈外静脉血中降钙素基因相关肽的含量。结果 :发现疼痛发作时患侧颈外静脉血中降钙素基因相关肽含量显著高于肘静脉血及术后患侧颈外静脉血中降钙素基因相关肽的含量。结论 :我们认为 :三叉神经痛的痛支神经过度合成和释放降钙素基因相关肽可能促进了P物质的释放 ,导致阵发性剧烈疼痛 ,并在局部形成神经源性炎症。     

Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp

AIM: To determine whether leucocyte infiltration during neurogenic inflammation in the pulp is regulated by neuropeptides via inducing the release of proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) from human dental pulp. METHODOLOGY: Cultured primary pulp cells and pulp tissue explants were stimulated with substance P (SP) and/or calcitonin gene-related peptide (CGRP). IL-8 or MCP-1, secreted from cultured cells or produced in pulp explants, was analysed by enzyme-linked immunosorbent assay. RESULTS: Substance P induced IL-8 secretion from cultured pulp cells (approximately threefold increase over control, P < 0.05) and from pulp tissue explants (two- to three fold). SP only minimally to moderately induced MCP-1 (approximately two fold) in cultured pulp cells. While MCP-1 induction in cultured pulp cells was detected after 24 h of SP stimulation, no induction was observed in pulp tissue. CGRP did not induce IL-8, but moderately increased MCP-1 production (approximately three fold) in cultured pulp cells. There was no synergistic induction of MCP-1 by SP plus CGRP stimulation of pulp cells. CONCLUSIONS: Substance P is a stronger inducer of IL-8 production in dental pulp than CGRP. IL-8 is more strongly induced than MCP-1 by SP, suggesting a more important role for IL-8 than MCP-1 in leucocyte infiltration during neurogenic inflammation in dental pulp.