大鼠实验性牙齿移动中三叉神经节P2X3受体表达变化的研究

目的 :研究探索正畸牙移动中加力后ATP受体P2X3在三叉神经节的表达变化的规律。方法 :本实验以雄性SD大鼠为实验对象 ,模拟临床矫治的牙移动过程 ,应用免疫组织化学、蛋白质印迹法及原位杂交技术研究。结果 :1.大鼠牙齿加力后 ,利用免疫组化方法观察到P2X3受体免疫反应阳性增高 ,并呈现一定的时间规律 :实验后 1天开始出现显著性变化 ,实验后 3天达高峰 ,7天后下降至与对照组基本一致。 2 .western免疫印迹法发现三叉神经节中出现P2X3受体的分子量为 4 5kd的蛋白质条带 ,大鼠牙齿加力移动后三叉神经节中P2X3受体蛋白量变化呈现一定时间规律 ,与免疫组化结果基本一致。 3.大鼠牙齿加力移动后激活了三叉神经节中P2X3mRNA表达。这一变化有一定的时间性 ,与免疫组化的结果基本一致。结论 :1.大鼠牙齿加力后 ,观察到P2X3受体免疫反应阳性及mRNA阳性增高 ,并呈现一定的时间规律 ,与正畸牙移动疼痛时间暗合。暗示P2X3受体的变化在正畸牙移动疼痛中可能的作用。 2 .P2X3受体作为一个已知疼痛传递的离子通道 ,牙移动过程中在蛋白及基因水平均有表达变化 ,呈现出一种上调及短时一过性变化的趋势 ,表明牙移动中的伤害存在 ,同时也反映了P2X3与牙移动伤害表达密切相关 .其作用机制还有待进一步探索。     

大鼠实验性牙移动中P2X3受体在牙周膜的表达变化研究

目的 探讨大鼠实验性牙移动中P2X3受体在牙周膜的表达变化,初步了解P2X3受体与正畸牙移动疼痛信息传递的关系.方法 54只雄性SD大鼠（体重200～250 g）随机分为空白组（5只）、对照组（14只）和实验组（35只）.选择左侧上颌第一磨牙作为观察对象,制备大鼠正畸牙移动不同时间点牙周膜组织切片.结果 正畸牙移动加力后,牙周膜压力侧和张力侧P2X3受体免疫阳性表达增加,呈短时上调规律,两侧相同时间点进行两两比较,结果无统计学意义.结论 正畸牙移动疼痛信息传递过程中P2X3受体在牙周膜压力侧与张力侧呈一过性上调规律,推测P2X3受体与牙移动伤害表达密切相关,但其作用机制还有待进一步研究.     

正畸力作用下大鼠牙周组织中CCR1及其相关配体的表达

目的 研究正畸力加力后CC类趋化因子受体1（CCR1）及其相关配体（CCL3、CCL5、CCL7）的mRNA在大鼠牙周组织中的表达变化规律,以探讨CCR1在正畸牙移动的作用.方法 将35只8周龄体重（220±25）g雄性SD大鼠随机分为7组,每组5只,空白对照组大鼠不安装实验装置,其余大鼠安装实验装置.安装实验装置大鼠随机选择一侧上颌第一磨牙为实验牙,对侧第一磨牙作为对照牙,采用镍钛拉簧建立大鼠正畸牙移动模型,力值50 g.实验动物分别于加力后0h、12h、1d、3d、7d、14 d处死,取大鼠上颌第一磨牙周围牙周组织应用实时荧光定量PCR法进行研究.结果 大鼠牙齿加力后,观察到牙周组织内CCR1及其配体的mRNA表达量呈现一定的时间规律：加力后12 h,CCR1、CCL3、CCL5、CCL7的mRNA表达均开始上升,CCR1 mRNA表达在3d达高峰（F=1.745,P=0.021）,CCL3、CCL5mRNA表达在1d达高峰（F=19.976,PCCL3 =0.019; F=17.114,PCCL5=0.008）,CCL7表达差异无统计学意义.CCR1及其配体的mRNA转录水平在加力时7d均下降至与对照组基本一致.结论 正畸力作用下,大鼠牙周组织中CCR1及其相关配体mRNA表达出现一过性变化,呈现短时上调规律,推测CCR1及其相关配体表达与正畸牙移动过程及牙周骨改建可能相关.     

大鼠实验性牙移动中配基门控阳离子通道P2X3受体在牙髓中的表达变化

目的 探讨大鼠实验性牙移动过程中配基门控阳离子通道P2X3受体在牙髓中表达变化,初步了解P2X3受体在正畸牙移动过程中的可能作用.方法 将54只雄性SD大鼠(体重200～250 g)随机分为空白组(5只)、对照组(14只)和实验组(35只).选择左侧上颌第一磨牙作为观察对象,获取不同时间点牙髓组织切片,进行免疫组织化学...     

成骨特异转录因子 Runx2 在去势大鼠正畸牙移动牙周组织中的表达

目的：研究成骨特异转录因子Runx2在去势大鼠正畸牙移动过程中牙周组织内的表达规律。方法：选用3月龄雌性未孕SD大鼠50只,随机分为去势组（OVX）与假手术组（Sham）各25只。全麻下行大鼠去势术或假手术,术后饲养3个月。构建大鼠上颌第一磨牙正畸牙移动模型,并分别于牙移动0d、1d、3d、7d与15d处死去势组大鼠和假手术组大鼠各5只,取上颌第一磨牙周围牙周组织,行H-E染色和免疫组织化学染色后观察正畸牙移动牙周组织的改建。结果：去势大鼠与假手术大鼠正畸牙移动过程中牙周组织的改建基本相似,即张力区以成骨为主,压力区以破骨为主。去势组大鼠牙移动15d张力区成骨细胞及压力区破骨细胞均显著多于假手术组大鼠（尸〈0．05）。大鼠正畸牙移动过程中牙周组织Runx2表达先升后降。牙移动1d、3d、7d张力区与压力区Runx2表达均显著高于0d（P〈0．05）,15d与0d则无显著差异。去势组大鼠牙移动牙周组织Runx2表达变化与假手术组大鼠基本一致,但是去势组1d压力区和7d张力区Runx2表达均显著高于假手术组大鼠（P〈0．05）。结论：去势大鼠正畸牙移动过程中的牙周组织改建符合正畸牙移动的基本规律,但其牙周组织的改建更为活跃。去势大鼠正畸牙移动牙周组织内Runx2的表达增强,这可能与活跃的牙周组织改建相关,其机制需进一步研究。     

大鼠牙周炎牙正畸移动后整合素β_3mRNA变化的实验研究

目的研究大鼠牙周炎牙及其正常牙在正畸移动中整合素β3mRNA的表达变化.方法选用10周龄雄性成年SD大鼠96只,随机分为正常牙组、牙周炎牙组,每组48只.分别在加力后0 d、12 h、1 d、3 d、5 d、7 d每一时间点处死8只动物,制备标本.采用原位杂交检测牙周炎牙与正常牙正畸移动后β3mRNA转录水平的变化.结果正常牙组和牙周炎牙组加力后12 h和3 d时的牙周膜内破骨细胞整合素β3mRNA阳性强度均较加力前有明显增加(P＜0.001);其余时间点未见整合素β3mRNA阳性表达.在加力各时间点,两组整合素β3mRNA的表达强度无显著性差异(P＞0.05).结论在正常牙及牙周炎牙移动早期,牙周膜和牙槽骨骨髓腔内有整合素β3mRNA表达,提示整合素β3可能参与破骨细胞前体向破骨细胞转化及破骨细胞迁移和粘附.     

大鼠正畸牙移动压力侧牙槽骨中cath K mRNA表达的研究

目的研究组织蛋白酶K（cathepsin K,cath K）mRNA在大鼠正畸牙移动压力侧牙槽骨中的表达变化及时间分布特点,探讨正畸牙移动中牙周改建的分子生物学机制。方法选用80只6周龄SD雄性大鼠建立正畸牙移动模型,分别在加力后2d、5d、7d、10d和14d处死动物各16只,HE染色观察牙周组织改建的变化,TRAP染色计数压力侧破骨细胞数量;实时定量PCR（real-time quantitative PCR）检测cath K mRNA表达变化及时间分布特点。结果cath K mRNA表达随加力时间的增加而增加,在加力后的第7天开始下降。这与牙周组织形态学的变化以及TRAP染色阳性破骨细胞数目的变化规律相一致。结论在生物机械力的诱导下,cathK参与了正畸牙移动骨改建过程中有机基质的降解,cath K mRNA随正畸加力时间的增加出现规律性变化。     

正畸力作用下兔牙周组织中雷帕霉素靶蛋白和核糖体蛋白S6激酶的表达

目的探讨正畸牙移动过程中雷帕霉素靶蛋白（mTOR）和核糖体蛋白S6激酶（p70 S6K）在牙周组织改建中的作用。方法选用24只日本大耳白兔建立正畸牙移动的动物模型，将实验动物上颌右侧戴矫治器，作为实验侧；左侧未戴矫治器，作为对照侧。分别在戴矫治器后3、5、7、14 d各处死6只实验动物。采用苏木精-伊红染色、逆转录聚合酶链反应（RT-PCR）及Western blot免疫印迹分析方法观察牙周组织形态学变化及mTOR和p70 S6K表达的变化。结果组织形态学观察可见，实验侧牙周组织较对照侧有明显改建，牙槽骨由致密变得疏松，细胞的排列由有序变得紊乱，牙槽骨的骨壁由平整变得凹凸不平，出现了破骨细胞及骨陷窝。RT-PCR结果显示，加力3 d后牙周组织中p70 S6K mRNA表达增强，7 d后牙周组织中p70 S6K mRNA明显增强，随后缓慢下降，与对照侧相比，其差异有统计学意义（P＜0.01）。Western blot免疫印迹分析与RT-PCR结果一致。结论在正畸牙移动过程中，兔牙周组织中mTOR和p70 S6K的表达明显增强，提示mTOR和p70 S6K参与牙周组织改建，并在牙周组织改建中起重要作用。     

大鼠正畸牙移动过程中牙周组织NGF的表达变化

目的了解大鼠正畸牙移动过程中牙周组织内神经生长因子(nerve growth factor,NGF)的变化,探讨NGF在正畸牙移动过程中的作用。方法将85只雄性SD大鼠随机分为17组,其中空白对照组(0d组)、实验加力和对照不加力组各6h、12h、24h、3d、5d、7d、14d、21d组。选择左上第一磨牙作为实验牙,制备大鼠正畸牙移动不同时间牙周组织切片,进行免疫组织化学研究。结果正畸牙移动过程中,牙周组织内NGF表达发生改变。NGF表达量在正畸模型加载后迅速上调,实验组和对照组分别于5d、1d时表达水平达到最高,所有观察区域牙周膜内NGF表达显著增加,且实验组明显高于对照组。结论NGF在正畸牙移动过程中牙周组织改建的多个阶段起作用,参与了牙周膜早期的炎症反应及修复和晚期的改建。     

正畸力作用下兔牙周组织中PI3K的表达

目的：探讨正畸力作用下兔牙周组织中PI3K在正畸牙移动牙周组织改建过程中的作用。方法：选用24只日本大耳白兔建立正畸牙移动的动物模型,将实验动物上颌右侧戴矫治器,作为实验组;左侧未戴矫治器,作为对照组。分别在戴矫治器后3、5、7、14d各处死6只实验动物。用实时荧光定量PCR及Western blot免疫印迹分析方法对PI3K表达的变化进行检测。结果：实时荧光定量PCR结果显示,加力3d后牙周组织中PI3KmRNA表达增强,7d后牙周组织中PI3KmRNA表达明显增强,随后缓慢下降,与对照侧相比,其差异有统计学意义（P〈0.01）。Western blot免疫印迹分析与RQ-PCR结果一致。结论：在正畸牙移动过程中,兔牙周组织中PI3K的表达明显增强,提示PI3K参与牙周组织改建,并在牙周组织改建中起重要作用。     

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。     

正畸牙齿移动过程中大鼠三叉神经节PPTA 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PPTA 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PPTA mRNA水平的变化情况。结果：加力后2h大鼠三叉神经节PPTA mRNA的表达量开始增加，加力8h至加力3d达到最高，至撤力后2～4周PPTA mRNA的表达量开始下降，但PPTA mRNA水平仍然明显高于对照组。结论：正畸牙齿移动过程中大鼠三叉神经节PPTA mRNA表达发生了明显的变化。提示P物质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。     

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。     

Increased expression of TRPV1 in the trigeminal ganglion is involved in orofacial pain during experimental tooth movement in rats

To investigate whether transient receptor potential vanilloid type 1 (TRPV1) is involved in pain induced by experimental tooth movement, experiments were performed in male Sprague‐Dawley rats weighing 200–250 g. Directed face‐grooming behavior was used to evaluate nocifensive behavior in rats during experimental tooth movement. The distribution of TRPV1 in the trigeminal ganglion (TG) was evaluated by immunohistochemistry, and its expression was detected by western blotting at several time points following the application of various magnitudes of force during tooth movement. Immunohistochemical analysis revealed that TRPV1 was expressed in TG, and its expression was increased after experimental tooth movement. Western blot results also showed that experimental tooth movement led to a statistically significant increase in expression of TRPV1 protein in TG. Meanwhile, the time spent on directed face‐grooming peaked on day 1 and thereafter showed a gradual decrease. In addition, both the change in TRPV1 expression in the TG and directed face‐grooming behavior were modulated in a force‐dependent manner and in concert with initial orthodontic pain responses. Our results reveal that TRPV1 expression is modulated by experimental tooth movement and is involved in tooth‐movement pain.     

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。     

Behavioural responses and expression of P2X3 receptor in trigeminal ganglion after experimental tooth movement in rats

Objective

To explore the role of P2X₃ receptor in pain induced by experimental tooth movement.

Design

Male Sprague-Dawley rats weighing 200-300 g were used. P2X₃ receptor distribution in the caudal one-third portion of the trigeminal ganglion (TG) was studied by IHC. Next, the changes of P2X₃ expression were detected by Western blotting 4 h, 1 d, 2 d, 3 d, 5 d, 7 d, 14 d after tooth movement. We then developed a behaviour pain model associated with directed mouth wiping. Finally, the effect of TNP-ATP on nociceptive-like behaviour was evaluated.

Results

Our results showed that P2X₃ receptors were expressed mainly in small- and medium-sized cells and experimental tooth movement led to an increase in staining of mandibular P2X₃ receptors. In addition, following experimental tooth movement, the expression of P2X₃ receptor in TG was statistically significantly up-regulated from days 1 to 5, with a peak on day 3. It was also found that the time spent on directed mouth wiping was dramatically increased by experimental tooth movement from days 1 to 7. The rhythm change of P2X₃ receptor expression in TG and the mouth wiping behaviour were in concert with the initial orthodontic pain responses. The directed mouth wiping behaviour was modulated in a force-dependent manner and could be attenuated by peripheral and systemic morphine. Furthermore, peripherally administered TNP-ATP could exert an analgesic effect on this pain model.

Conclusion

These results suggest that directed mouth wiping behaviour can be a reliable measurement of pain following experimental tooth movement in rats. The P2X₃ receptor is important in the development and maintenance of tooth movement pain and thus may be peripheral targets for analgesics in orthodontic pain control.     

Tooth injury increases expression of the cold sensitive TRP channel TRPA1 in trigeminal neurons

Objective

Transient receptor potential (TRP) channels, a family of structurally related proteins have been implicated in the sensation of pain and hyperalgesia caused by exogenous and endogenous agonists, as well as touch, pH, and temperature. The objective of this study was to determine the effects of tooth injury on the expression of the cold sensitive channel TRPA1, in the trigeminal ganglion, the primary source of sensory and nociceptive innervation of teeth.

Design

We analyzed TRPA1 expression in a rodent model of tooth injury, by Western blot analyses of proteins extracted from trigeminal ganglia.

Results

We found that TRPA1 was selectively increased in trigeminal ganglia innervating injured teeth when compared to TRPA1 expression in trigeminal ganglia innervating healthy teeth.

Conclusions

Our results provide the first evidence of increased expression of a cold-sensitive TRP channel in trigeminal ganglia after pulp exposure, and are consistent with the possibility that increased expression and function of TRPA1 in trigeminal neurons contributes to hyperalgesia and allodynia following tooth injury.     

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.     

Effect of endomorphin‐2 on orofacial pain induced by orthodontic tooth movement in rats

Endomorphin‐2 demonstrates potent antinociceptive effects in various pain models. The objectives of the present study were to explore the role of endomorphin‐2 in the modulation of orofacial pain induced by orthodontic tooth movement in rats. An orthodontic pain model was established in male Sprague‐Dawley rats by ligating coiled springs to mimic orthodontic force (40 g). On days 0, 1, 3, 5, 7, and 14 following orthodontic tooth movement, bite force was recorded as a surrogate measure of orthodontic pain. Ipsilateral trigeminal ganglia, trigeminal nucleus caudalis, and periodontal tissues were harvested for immunostaining. Endomorphin‐2, endomorphin‐2 + naloxone (a non‐selective opioid receptor antagonist), naloxone, and saline were injected into trigeminal ganglia and periodontal tissues to explore the role of endomorphin‐2 on orthodontic pain. The results showed that following orthodontic tooth movement, endomorphin‐2 expression levels in trigeminal ganglia were elevated on days 1, 3, 5, and 7. Orthodontic pain levels were increased on days 1, 3, and 5. The administration of endomorphin‐2 into both trigeminal ganglia and periodontal tissues alleviated orthodontic pain. Moreover, the effects of endomorphin‐2 could be blocked by naloxone completely in trigeminal ganglia but only partially in periodontal tissues. Therefore, endomorphin‐2 plays an important role in the modulation of orthodontic pain both centrally and peripherally, probably through different pathways.     

Changes in TrkB-like immunoreactivity in rat trigeminal ganglion after tooth injury

The purpose of this study was to characterize the impact of tooth injury on the distribution of tyrosine receptor kinase B (TrkB) among trigeminal ganglion neurons and assess the time course for tooth injury-induced TrkB distribution changes. In addition, we sought to further characterize the subpopulation of the afferents expressing TrkB receptors. Fifteen adult male Sprague-Dawley rats were studied. Pulpal inflammation was induced and ganglia were subsequently harvested and processed at different time points. Standard immunohistochemical fluorescence techniques were used to visualize TrkB-like immunoreactivity and isolectin B4 binding. Results indicate that full-length TrkB receptors are present in 36.6% of trigeminal ganglion neurons. This percentage decreases for the first 48 h and then increases to 41% by 7 days after tooth injury. Finally, TrkB appears to be present in a large percentage (54%) of isolectin B4+ neurons, suggesting that it is present in nociceptive afferents. These data highlight the fact that even mild injury results in sustained changes in nociceptive circuitry and raise the possibility that the brain-derived neurotrophic factor/TrkB system may contribute to persistent pain after tooth repair.