Elevated TRPV1 expression in the central nucleus of the amygdala participates in initial pain and subsequent anxiety-related behaviors in mice undergoing orthodontic tooth movement

Abstract

Objectives

Pain is a common issue for individuals undergoing orthodontic treatment, with some even discontinuing treatment due to the pain experienced. Furthermore, orthodontic pain is often accompanied by negative emotional responses such as anxiety and unease. This study aims to investigate the role of transient receptor potential vanilloid type 1 (TRPV1) in the mouse’s central nucleus of the amygdala (CeA), focusing on its involvement in nociception and anxiety during experimental orthodontic tooth movement (OTM).

Methods

Twenty-five 8-week-old male C57BL/6J mice were randomly divided into five groups: blank, 0 centinewton (cN), 10 cN, 30 cN, and 50 cN, based on the force applied for OTM(n = 5). Behavioral responses were assessed at different time points. Face-rubbing behavior and Von Frey test were used as a measure of nociception during experimental OTM. Both Open Field (OF) and Elevated Plus Maze (EPM) tests were conducted to evaluate anxiety-related behaviors. Immunohistochemistry was used to study the localization of TRPV1 in the CeA(n = 5), while Western blotting was employed to measure its expression levels(n = 3).

Results

During the experimental OTM, all groups of mice exhibited nociception, with the 50 cN group showing the highest level of face rubbing and the minimum force value of von frey test on the first day (1 d). Mice subjected to different force values also showed different degrees of anxiety-related behavior. The anxiety-related indexes in the 30 cN group and 50 cN group were significantly higher than in the 0 cN group on the 28 d (P < 0.05). Immunofluorescence staining revealed that the TRPV1 expression in the 30 cN-1 d group was higher than in the 0 cN-1 d group (P < 0.001). Western blotting analysis showed that the TRPV1 expression in the CeA of the 30 cN group was significantly higher compared to the 0 cN-1 d group during experimental OTM, reaching its peak on the 1 d and 28 d time point (P < 0.001).

Conclusions

The present study demonstrates that orthodontic procedures can induce pain and emotional changes, which may be associated with increased TRPV1 expression in the central amygdala.

Clinical significance

The study provides preliminary evidence that TRPV1 in CeA is involved in both nociception and anxiety behavior during orthodontic tooth movement.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-026-08652-5.

Background

Pain is described as an unpleasant sensory or emotional experience accompanied by real or potential tissue damage, and most orthodontic patients encounter pain during their treatment [1]. Furthermore, individuals suffering from orthodontic pain often experience negative emotions such as anxiety and discomfort [2–5]. Research indicates that up to 30% of individuals undergoing orthodontic treatment may experience anxiety [2]. A prior study has suggested that pain is the primary cause responsible for initiating these emotional responses [6]. Orthodontic treatment-related pain may arise at various stages, including the application of bands, the initial engagement of wires, the insertion of separators, the use of elastics, the implementation of rapid maxillary expansion, and the removal of braces [7]. In animal studies, orthodontic tooth movement (OTM) as a way of tooth movement, has been proved to be associated with pain and emotions such as anxiety [7].

In clinical practice, nonsteroidal anti-inflammatory drugs (NSAIDs) are occasionally employed to manage orthodontic pain. Numerous studies have highlighted that NSAIDs can reduce the rate of tooth movement and extend the duration of orthodontic treatment [8]. The efficacy of alternative treatment methods, such as low-level laser and mechanical therapies, is still controversial [9]. Therefore, it is imperative to identify a more effective treatment method with minimal adverse reactions. In this context, molecular drug targeting therapy is the focus of many scientists, with the amygdala being a focal point of research since its activation has been observed in both humans and rodents experiencing pain [10].

The central nucleus of the amygdala (CeA), a main component of the amygdala, receives nociceptive inputs via the spino-parabrachial-amygdala pathway and sends this information to the hypothalamus, substantia innominata dorsalis, and brainstem nucleus. These areas regulate defensive behavior responses. Activation of the amygdala was observed in various inflammatory and neuropathic pain models, including formalin test [11], colitis [12], and nerve injury [13] .Our previous study has explored the involvement of CeA in orthodontic pain experience during tooth movement [14]. Numerous pain-associated agents and receptors contribute to the manifestation of orthodontic pain and the overall transmission of pain information. For example, TRPV1, a polymodal nociceptor, is widely expressed across brain regions, including the cortex, hippocampus, central amygdala, dentate gyrus, hypothalamus, striatum, thalamus, substantia nigra (SN) pars compacta, trigeminal nerve nucleus, and the inferior olive [15]. Both exogenous and endogenous physical and chemical stimuli can activate TRPV1, which led to its identification as a potential target for managing inflammatory pain [16].

This experiment further studied the changes of nociceptive sensation, anxiety-related behavior and the expression of TRPV1 in CeA during tooth movement. For this purpose, a combination of behavioral and molecular biology methods has been employed, building upon the findings of previous studies. The objective of this study was to highlight the significant involvement of TRPV1 in tooth movement-induced pain and anxiety within the CeA. It also contributes valuable insights into the complex network of pain regulation and provides a theoretical foundation for developing efficient strategies to manage orthodontic pain. In this study, the null hypothesis was defined as the absence of statistically significant differences between groups (P > 0.05), specifically: (1) there is no difference in pain or anxiety between mice subjected to orthodontic tooth movement and control mice, i.e., orthodontic tooth movement does not induce pain or anxiety in mice; and (2) there is no significant difference in the expression of TRPV1 in the central amygdala between orthodontic tooth movement model mice and control mice, i.e., orthodontic tooth movement does not significantly increase TRPV1 expression in the central amygdala.

Material and methods

Subjects and experimental design

In this study, male C57BL/6J mice (20–25 g) were used to explore the involvement of TRPV1 in tooth movement-induced nociception. The Experimental Animal Center of Xi’an Jiaotong University Health Science Center (Xi’an, China) supplied all the mice used in this study. The mice were housed in plastic cages with access to food and water, following a 12-hour light and 12-hour dark schedule, in a temperature-controlled room (18–20 °C). The animals were allowed a minimum of 5 days to acclimate to the housing conditions prior to the start of the experiments. The sample size for each experiment was determined based on our previous experience with similar investigations [14].

This study involved a total of 71 male mice, among which 25 were subjected to behavioral testing with each group comprising 5 mice according to the resource equation approach [17]. Immunofluorescence was performed on 10 mice, also in groups of five. Western blot (WB) analysis was conducted on 36 mice, with each group consisting of 3 mice. Initially, twenty-five mice were randomly categorized into five identical groups according to the force applied to induce tooth movement: the blank group (control), 0 Centinewton (cN, 10 cN, 30 cN, and 50 cN groups( n = 5). A one-month loading cycle was employed for the mice to replicate the clinical orthodontic force application duration. Each group was evaluated at intervals of 0.5 h, 2 h, 4 h, 1 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d post-application of their respective force. During these intervals, face rubbing experiments, Von Frey tests were conducted. open field (OF) experiments and elevated plus maze (EPM) tests were conducted on 1 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d (Fig. 1). The choice of time is based on the previous research group and other study [18]. After analyzing the outcome data and reviewing literature references [19], a force of 30 cN was selected for further experiments.

Fig. 1.

Flow chart of the study

In the experiment, observer blinding was applied, whereby the assessors were unaware of the animal grouping. Behavioral tests were fully recorded on video, and the assessors subsequently scored the video files after the order had been randomized and the group labels had been concealed. Before the formal experiment, a pilot study was conducted until the kappa coefficient reached above 0.8, at which point data collection for the formal experiment commenced.

To investigate the distribution of TRPV1 in the CeA, immunofluorescence staining was performed on CeA samples from the 30 cN-1d and 0cN-1d groups. TRPV1 expression was detected using Western blotting after applying a 30 cN force at various time intervals, compared to the 0 cN-1d group. All necessary measures were taken to reduce animal distress in compliance with established ethical standards for animal research. All animal testing procedures were conducted following the ARRIVE guidelines. All mice were euthanized at the end of the experiment by intraperitoneal injection of pentobarbital sodium (50 mg/kg) combined with manual cervical dislocation [20].

Experimental orthodontic tooth movement (OTM)

General anesthesia was administered for all procedures, using pentobarbital sodium (30 mg/kg) via intraperitoneal injection. A stainless steel spring appliance (Changsha Tiantian, China) was utilized to achieve mesial movement of the left maxillary first molar. Next, the upper incisor was connected to the opposite end of the coil spring [21]. To prevent early loss or displacement of the device, a thin layer of fluid composite(West Lake Biotech, China). was applied to the upper incisors. In the blank group, no surgical or orthodontic manipulation was performed [22]. However, in the 0 cN group, the first molars and anterior incisors were ligated, but the spring was not stretched. During the experiment, all mice were provided with powdered food and their body weights were monitored to maintain a range of 20–25 g. Data from mice that did not meet the weight criteria were excluded from the results.

Face rubbing

Mice were placed in a transparent plastic enclosure measuring 40 cm × 40 cm × 40 cm, for the observation of intentional face rubbing behavior. This enclosure was situated in a space with a background noise level of 45 dB. Before the video recordings, the mice were allowed a minimum of 15 min for acclimatization to the new environment. The mice’s behavior was recorded by two cameras for 15 min at each designated time interval. The equipment was thoroughly cleaned with a 75% ethanol solution during intervals between experiments. Two independent observers, who were blinded to the treatment groups, subsequently analyzed the recorded test session. For each animal, the observational value was determined by the average of the two separate assessments, as previously described [23].

Von Frey [24, 25]

In order to measure the mechanical pain threshold of the perioral area, the lower half of the mouse’s body was placed in a cylindrical restraint device (inner diameter 25 mm), and the mice were controlled by the tail while allowing free movement of the head and forelimbs. Von Frey monofilaments (North Coast Medical, Gilroy, CA) ranging from 0.008 g to 2 g were applied. After 10 min adaptation period in the restraint device, when the mice were calm, its perioral area was stimulated with von Frey monofilaments, and the test intensity was increased one by one. Each stimulation intensity was stimulated for 5 times with an interval of 30 s. When the perioral area was stimulated by von Frey wire, the mice were judged to be positive when they appeared the following symptoms: mice turned their head to von Frey wire and sniffed von Frey wire; when stimulation was applied, the mice moved their head slowly or retreated quickly; mice grasped and occluded in order to avoid contact with vonFrey wire. The mice scratched the stimulated perioral area and showed a series of uninterrupted facial washing movements. After repeated measurement for 5 times in each mouse, the minimum number of vonFrey filaments that induced at least 3 positive reactions was the threshold.

Open field (OF)

The mice were placed in a transparent acrylic enclosure with a floor area measuring 40 × 40 cm and a wall height of 40 cm. The floor, square in shape, was divided into sixteen smaller squares measuring 10 cm × 10 cm, with one square in the center designated as the central square. To record the spontaneous activity of each mouse, which was initially positioned in the middle of the floor, two cameras were positioned 180 degrees apart on opposite sides of the enclosure. Each mouse was observed for 15 min. The equipment was thoroughly cleaned with a 75% ethanol solution during intervals between experiments. The first 15-minute segment of the recordings was then assessed by two observers who were blinded to the treatment groups. The mean of two assessments was adopted as the observational measure for each mouse. Scoring was based on the three criteria: (1) number of crossing (at least 3 feet across each square); (2) duration of the central grids; (3) number of central grid crossing [26].

Elevated plus maze (EPM)

A white acrylic EPM with a height of 55 cm was used, comprising two open arms (30 × 5 cm) and two closed arms (30 × 5 × 15 cm), with a central neutral zone (NZ) located at the intersection. Mice were initially placed at the beginning of one arm in the NZ, facing away from the experimenter’s position. They were then allowed to freely explore the maze for 10 min, during which their movements were recorded for subsequent analysis. The time each mouse spent in either the open or closed arms was counted only when all four paws were placed on the arm. The time spent in the NZ was counted every time a mouse placed at least one paw in that area, as previously described [26]. These data were used to additionally calculate % open entries (i.e., open entries/total entries x 100) and % time spent in open and closed arms (e.g., open time/300 × 100) [27]. For each mouse, the observational value was calculated as the average of the assessments made by the two observers. Both the OF and EPM tests are well-established methods for assessing anxiety in rodents [26].

Micro-CT

A micro-computed tomography (micro-CT) system with a resolution of 1.5 μm (PerkinElmer, Germany) was used to capture tooth movement distance in mice. The CT images were analyzed using VG Studio 2.1 software. The mouse head was positioned within a cylindrical scan volume of 4 cm × 4 cm × 4 cm, symmetrically aligned with the three spatial axes. The distance of the most prominent point from the distal side of the first molar to the mesial side of the second molar of the maxillary was measured and analyzed [28].

Immunohistochemistry

Immunohistochemical analysis was conducted to detect the presence of TRPV1 in CeA of mice from the 30 cN-1d and 0 cN-1d groups during experimental OTM. The mice were euthanized using an overdose of pentobarbital sodium (100 mg/kg) and then subjected to transcardial perfusion with 50 mL of saline solution (0.9% NaCl) followed by 100 mL of 4% paraformaldehyde (PFA). Post-perfusion, their brains were extracted and fixed in a 4% PFA solution for 12–24 h. Subsequently, the brains were immersed in a 30% sucrose solution for an additional 48 h for cryoprotection. Brain sections of 25 μm thickness were obtained by cutting the specimens using a cryostat microtome(CryoStar, britain) in -20 ℃. These sections were stored at 4 °C until they were ready for immunohistochemistry [29].

For immunostaining, the tissue sections underwent three washes with phosphate-buffered saline (PBS) and were then treated with Quickblock™ Blocking Buffer for Immunol Staining (Beyotime Biotechnology, China) at room temperature for 15 min. The sections were incubated with rabbit anti-TRPV1 Ig (1:200; ab203103,Abcam, UK) at 4 °C for 48 h. Afterward, the sections were exposed to the secondary antibody (1:500, 488-linked AffiniPure donkey anti-rabbit IgG (H + L)) for 24 h at 4 °C. The excess of secondary antibody solution was washed off before observation. Finally, the relative expression levels of each group were analyzed using Image J software (National Institutes of Health).

Western blotting

TRPV1 expression in the CeA tissue was detected using Western blotting following the application of 30 cN-forces at various time intervals, including the 0 cN-1 d group in the experiment OTM and the blank group. To extract protein from the CeA tissue, RIPA lysis buffer and a mixture of protease inhibitors (Roche, Germany) was used. The protein concentration of the samples was determined using the BCA protein concentration determination kit (Boster, China). Next, the membrane was incubated with the rabbit polyclonal anti-TRPV1 antibody (1:800, ab203103, Abcam, UK) along with either horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Sigma-Aldrich, USA) as primary and secondary antibodies, respectively. Following the rinsing process, the membrane was incubated with the luminescent agent, and then exposed for analysis. This procedure was repeated three times. According to the total protein determined by Bio-Rad without staining, the gray value of each group was analyzed using Image lab software (Bio-Rad, USA).

Statistical analysis

Statistical analysis was performed using SPSS 17.0 (IBM, Armonk, NY, USA). All data are presented as the mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using the t-test, while comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test. Statistical significance was set at P < 0.05. In the figures, significance levels are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001; # P < 0.05, ## P < 0.01, ### P < 0.001.

Results

Micro-CT and Rate of Tooth Movement

Tooth movement is presented in Fig. 2. After established the OTM model, the tooth movement rate of 30 g group was the most ideal group. We speculated that the force of 10 g was small, resulting in limited effects. In contrast, the force of 50 g was too large to move the tooth properly.

Fig. 2.

Micro-CT performance of OTM. A OTM model diagram. B OTM image of 30 g 28d. C OTM distance in each group of 28d (mm). n = 5, ** P < 0.01, *** P < 0.001

Behavioral tests in mice

Directed face rubbing behavior and von frey tests during experimental orthodontic tooth movement (OTM)

The results of face rubbing tests (Fig. 3A) indicated a gradual increase in the duration of face rubbing time in the 30 cN group, starting at 0.5 h, reaching its peak on the first day (1 d), and subsequently decreasing. Notably, the duration of face rubbing was significantly higher in the 30 cN group compared to the 0 cN group at 4 h, 1 d, and 3 d( P < 0.05, P < 0.01, P < 0.05). In the 10cN group, an increase in face rubbing was observed beginning at 4 h, reaching its peak at 1 d, and then decreasing. Similarly, the 50 cN group showed a peak in face rubbing on the first day, followed by a decrease. The duration of face rubbing was significantly higher in the 50 cN group compared to the 0 cN group at 1 d ( P < 0.001). The results of von frey tests (Fig. 3B) indicated a gradual decrease of the thresholds for mechanical pain of the perioral area, starting at 0.5 h, reaching its peak on the first day (1 d), and subsequently increasing. Additionally, 10 cN, 30 cN, 50 cN group thresholds were lower on the 0.5 h, 2 h, 4 h, 1 d, and 3 d days compared to the 0 cN group( P < 0.001, P < 0.001, P < 0.001).followed by an increase. The threshold decreased in all three experimental groups, but the threshold decreased more in 30cN group and 50cN group.

Fig. 3.

Effect of OTM on the facial rubbing behavior and Von Frey test in mice. A facial rubbing behavior; B Von Frey test; To compensate for the risk of device detachment and death, the initial setting was for each group to consist of 8 mice. However, it was ensured that at the final time point of each group, 5 mice were available for measurement. The symbols “*”, “**”, and “***” indicate statistically significant comparisons with the blank group at the same time point, where P < 0.05, P < 0.01, and P < 0.001, respectively. The symbols “#”, “##”, and “###” indicate statistical comparisons with the 0 cN group at the same time point, corresponding to P < 0.05, P < 0.01, and P < 0.001, respectively

Anxiety-related behavior during experimental OTM

The results of the open field test (Fig. 4) showed that in terms of the number of grid crossings, the 30 cN group exhibited significantly fewer crossings than the 0 cN group at 14 d and 28 d (P < 0.05, P P < 0.01), while the 50 cN group showed significantly fewer crossings than the 0 cN group at 7 d (P < 0.05). Regarding the time spent in the central grid, the 30 cN group spent significantly less time than the 0 cN group at 7 d, 21 d, and 28 d (P < 0.01, P < 0.001, P < 0.001), whereas the 50 cN group spent significantly less time than the 0 cN group at 7 d, 14 d, and 28 d (P < 0.001, P < 0.05, P < 0.05). In terms of the number of entries into the central grid, the 30 cN group had significantly fewer entries than the 0 cN group at 7 d, 21 d, and 28 d (P < 0.01, P < 0.001, P < 0.001), while the 50 cN group had significantly fewer entries than the 0 cN group at 7 d and 28 d (P < 0.01, P < 0.05).

Fig. 4.

Open Field (OF) test. A number of crossing (B) duration spent in the central grid, and (C) the number of central grid crossing. To compensate for the risk of device detachment and death, the initial setting was for each group to consist of 8 mice. However, it was ensured that at the final time point of each group, 5 mice were available for measurement. The symbols “*”, “**”, and “***” indicate statistically significant comparisons with the blank group at the corresponding time point, where P < 0.05, P < 0.01, and P < 0.001, respectively. The symbols “#”, “##”, and “###” indicate statistical comparisons with the 0cN group at the corresponding time point, where P < 0.05, P < 0.01, and P < 0.001, respectively

The results of the elevated plus maze (EPM) test (Fig. 5) showed that in terms of the percentage of time spent in the open arms, the 30 cN group spent significantly less time than the 0 cN group at 28 d (P < 0.05), whereas the 50 cN group spent significantly less time than the 0 cN group at 5 d and 21 d (P < 0.05, P < 0.05). Regarding the percentage of time spent in the closed arms, the 30 cN group spent significantly less time than the 0 cN group at 28 d (P < 0.05), while the 50 cN group spent significantly less time than the 0 cN group at 5 d, 7 d, and 28 d (P < 0.05, P < 0.01, P < 0.05).

Fig. 5.

Elevated Plus Maze (EPM) test. A open entries% (B) Time in open arms%, and (C) Time in closed arms%. To compensate for the risk of device detachment and death, the initial setting was for each group to consist of 8 mice. However, it was ensured that at the final time point of each group, 5 mice were available for measurement. The symbols “*”, “**” indicate statistically significant comparisons with the blank group at the corresponding time point, where P < 0.05, P < 0.01, respectively. The symbols “#” indicate statistical comparisons with the 0 cN group at the corresponding time point, where P < 0.05

In the above experiments, the 30 cn group exhibited the greatest tooth movement distance, while both the 30 cn and 50 cn groups showed relatively pronounced responses in behavioral tests. In clinical orthodontic tooth movement, excessive force may slow tooth movement but simultaneously cause significant pain. For mice, 50 cn may represent an excessive force value. Therefore, to better approximate the orthodontic tooth movement force, 30 cn was ultimately selected as the force value for subsequent experiments in mice.

Distribution of TRPV1 in the CeA following experimental OTM

To investigate the distribution of TRPV1 in CeA during experimental OTM, the 30 cN-1 d and 0 cN-1 d groups were selected for immunofluorescence analysis. TRPV1 immunoreactivity was observed in CeA, indicated by the green fluorescence (Fig. 6A-H). The intensity of immunofluorescence staining in the 30 cN-1 d group was significantly higher when compared to the 0 cN-1 d group (P < 0.001, Fig. 6I).

Fig. 6.

Effect of OTM on TRPV1 expression in the CeA of mice. A-G Immunofluorescence micrographs of the CeA in both 30 cN-1 d and 0 cN-1 d groups during experimental OTM (n = 5). TRPV1 expression is identified by green fluorescence. G Statistical comparison of the relative brightness of immunofluorescence between the 30 cN-1 d and 0 cN-1 d groups. The symbol “***” indicates a significant difference when compared to the 0 cN-1 d group (P < 0.001). a: Olivary pretectal nucleus (OPT); b: Basolateral amygdaloid nucleus, anterior part (BLA); Scale bar = 100 μm

Changes in TRPV1 expression in the CeA following experimental OTM

The changes in TRPV1 expression in the CeA during experimental OTM were assessed by Western blotting analysis (Fig. 7). The results indicated that, in comparison to the 0 cN-1 d group, TRPV1 expression in the 30 cN-4 h group increased significantly (P < 0.001). This expression reached its peak on the first day (P < 0.001), remained significantly elevated on the third day (P < 0.001), and then decreased to levels that were not statistically different from the control group. However, a significant increase was again observed on the 28th day (P < 0.001). In addition, the data analysis revealed two distinct peaks in TRPV1 protein expression: one on the first day and another on the 28th day.

Fig. 7.

Effect of OTM on TRPV1 expression in the CeA. A The protein bands representing TRPV1 expression in the CeA for the blank group, 0 cN-1 d group, and 30 cN groups at various time points: 0.5 h, 2 h, 4 h, 1 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d (n = 3). B Relative TRPV1 protein expression. The symbol “***” indicates a significant difference when compared with the 0 cN-1 d group (P < 0.001)

Discussion

Orthodontic treatment often results in significant pain and emotional distress. It is estimated that approximately 95% of individuals experience discomfort, pain, and even anxiety during their treatment, potentially impacting their psychological well-being [2]. This pain can lead some patients to discontinue their ongoing orthodontic treatment [30]. Typically, adults dedicate approximately 24 months to orthodontic care, which requires a considerable level of adherence. Thus, alleviating pain associated with orthodontic tooth movement may positively influence patient compliance.

Current scientific understanding suggests that Orthodontic pain is a kind of inflammatory pain accompanied by tooth movement [31]. The essence of tooth movement is the inflammatory process of continuous reconstruction of tension side and pressure side. The blood vessels in the periodontal tissue of the compressed side were compressed, resulting in local ischemia. Ischemia can lead to hypoxia of related periodontal cells including fibroblasts, followed by increased anaerobic activity of cells leading to acidosis, which promotes the release of nitric oxide (NO) from periodontal endothelial cells and fibroblasts. NO increases the permeability of periodontal vessels, and then a large number of white blood cells, including neutrophils, macrophages, monocytes, mast cells and T cells, are recruited into periodontal tissue [32–35]. In response to local inflammation, cells release various chemokines, cytokines and inflammatory mediators to further amplify local inflammation, and their products act on sensory endings to cause pain, such as interleukin-1(IL-1), interleukin-6(IL-6), prostaglandins, interferon-gamma (IFN–γ) and vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha(TNF- α) and macrophage colony stimulating factor (M-CSF) [36–38].

On the other hand, some ion channels transduce locally generated acidic signals into pain signals. Acid-sensing ion channel 3 (ASIC3), a hydrogen ion channel receptor, has been well established to be expressed in periodontal sensory endings and trigeminal nerves. Transient receptor potential vanilloid type 1 (TRPV1), another acid sensor, is activated at pH values below 7.2 and 5.3, respectively [39, 40]. These ion channels mediate the transmission of pain sensation to the trigeminal ganglion, where they stimulate trigeminal neurons to release several neurogenic mediators, including calcitonin gene-related peptide (CGRP) and substance P (SP), to both the central nervous system, including the trigeminal nucleus, and the peripheral tissues such as the periodontium [41–43]. The released neurogenic mediators cause local vasodilation and increased inflammation, which further promotes the release of CGRP and other substances from trigeminal sensory endings. This series of reactions amplifies orthodontic pain in a positive feedback loop. The pain signal is projected to the second-order neuron located in the medulla oblongata, the trigeminal nucleus caudalis. Subsequently, fibers originating from the caudalis nucleus form the trigeminal lemniscus, which transmits the signal to the contralateral thalamus, specifically the posterior ventral nucleus. Afterward, the thalamus sends fibers to numerous brain regions, including the amygdala, before projecting to the sensory cortex. Finally, the information about orthodontic pain is integrated and converges [44].

Various evaluation methods have been developed to assess maxillofacial and oral nociception in rodents, including face-rubbing activity, RGS coding, and empty chewing [44]. In this study, face-rubbing activities were the primary evaluation method chosen. Rubbing behaviors, such as wiping the mouth, flailing the forelimbs, grasping the ears, stroking the face, shaking the head, licking the paws, and rubbing the chin, encompass a range of actions. For C57, a small black mouse, it is difficult and error to observe the subtle expression changes and small movements of RGS and empty chewing, and we chose face rubbing test and von frey test as pain indicators, which are reliable and consistent indicators, representing the most robust behavioral response to nociception induced by tooth movement in experimental settings [45, 46].

The study rejected the null hypothesis, indicating that orthodontic tooth movement in mice was associated with pain and anxiety, and that the expression of TRPV1 in the central amygdala was significantly increased in the orthodontic tooth movement model mice. After establishing the experimental orthodontic tooth movement (OTM) model in C57BL/6J mice, it was observed an increase in nociception levels, evidenced by face-rubbing behaviors, in the 30 cN group. These results suggest a trend of increasing nociception initially, which then decreases, with the highest duration observed on the first day. In von frey test, the threshold decreased in all three experimental groups on the 0.5 h, 2 h, 4 h, 1 d, and 3 d days, but the threshold decreased more in 30cN group and 50cN group.

The present study employed coil springs to induce orofacial nociception related to OTM. Numerous studies have indicated that coil springs may not provide consistent forces due to changes in the spring’s length as a result of tooth movement. Hence, the term ‘initial force’ is more appropriate, as previously indicated [46]. Overall, this animal model for OTM has been well-established and validated in numerous studies [47–49], including our research [50]. Interestingly, in the 0 cN group, an increase in behaviors consistent with nociception was observed, potentially linked to the nociception caused by the springs in the mouth [44]. Furthermore, the ligature thread used could exert a force by inserting itself between the teeth. As a result, we expected some temporary behavioral changes in the 0 cN group. Literature suggests that animals undergoing experimental OTM would have experienced emotional distress within the first 48 h, consistent with the present experimental findings [51]. Numerous clinical and fundamental investigations have shown a positive association between pain and anxiety [52].

In this study, the results of OF and EPM showed that the 30 cN and 50 cN groups exhibited different levels of anxiety-related behavior, whereas the 10 cN group did not exhibit any noticeable anxiety-related behaviors. OF and EPM were tested on the same batch of mice at different time points, and repeated tests may also make them more adapted to behavioral tests, which could affect the experimental results. This influence can be alleviated to some extent by comparing with the control group.

The nociception associated with orthodontic tooth movement, as evidenced by facial rubbing actions, was observed from 4 h to 28 d. Interestingly, anxiety-related behavior was mostly detected during the later stages, while nociception was more prominent during the earlier stages. This pattern suggests that the onset of anxiety-related behavior occurs after the onset of nociception. Notably, the majority of anxiety-related behaviors was not observed until two weeks post-nociception onset, consistent with our previous findings [53].

Roeska and colleagues, in experimental models of spinal nerve ligation, showed that rats with chronic compression injury exhibited anxiety-related behavior 21 days after the surgery, which was alleviated by pain relievers like morphine and gabapentin [54]. Similarly, Seminowicz reported that rats showed increased body temperature and touch sensitivity shortly after spinal nerve ligation; however, signs of anxiety-related behavior were only observed several weeks following the pain onset [54]. Also, Mingjie Zhang and colleagues demonstrated that rats with chronic migraine exhibited anxiety-related behavior three weeks following a similar procedure [55]. While Seminowicz and colleagues assessed their rats at multiple time intervals (5, 10, 15, 20, and 25 weeks post-nerve injury), Mingjie Zhang and Roeska conducted behavioral observations at 3 to 4 weeks post-surgery, hypothesizing that anxiety-related behavior would manifest after the pain onset. The findings presented in this study corroborate and expand upon these observations, indicating that the anxiety-related behavior in mice undergoing OTM occurs later than the initial nociceptive experience.

Some scholars use 10 cN as the force value of the study [56], while some scholars think that 30 cN is more valuable as the force value [57], such as N. Chen [28]. In this study, the 30 cN force resulted in the greatest extent of tooth movement and the most pronounced behavioral changes in mice; therefore, it was selected as the force value for subsequent investigations. Besides monitoring nociception and anxiety-related behaviors, the Western blotting analysis indicated that the expression of TRPV1 in CeA exhibited dual peaks. In both mice and humans, the amygdala, which serves as the primary center for emotional integration, plays a crucial role in generating emotions related to pain and the corresponding somatic responses, such as avoidance behavior [53]. It is widely recognized that the amygdala links pain and emotion. As an essential part of the limbic forebrain, the amygdala comprises approximately twelve nuclei, forming a complex network [53]. The CeA functions as the primary efferent core, extending from the amygdala to the brainstem and hypothalamus. It integrates inputs from other amygdala nuclei, which encompass nociceptive sensory information. TRPV1 is one of several receptors operating within the CeA [45]. Upregulation of TRPV1 expression can occur in response to various stimuli, including inflammation, tissue injury, and exposure to certain chemicals. For example, proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), can induce TRPV1 expression in DRG neurons and other tissues. Additionally, oxidative stress and reactive oxygen species (ROS) can also induce TRPV1 expression. Downregulation of TRPV1 expression can occur through several mechanisms, including desensitization, internalization, and degradation of the channel. Desensitization occurs when the channel is exposed to prolonged stimulation, leading to a reduction in channel sensitivity and a decrease in the probability of channel opening. Internalization occurs when the channel is taken into the cell, leading to a reduction in the number of channels available at the cell surface. Degradation of the channel can occur through proteasomal degradation or lysosomal degradation, leading to a reduction in channel expression [58–60]. The TRPV1 sensitization process is an intracellular mechanism that can promote channel gating or enhance the current evoked during subsequent activation of TRPV1. TRPV1 is a multimodal receptor, and its sensitization and endogenous regulatory pathways act through kinases such as protein kinase C and A (PKC and PKA) and Ca2+/calmodulin-dependent kinase II (CAMKII) at phosphorylation sites. After inflammatory tissue injury, endogenously released inflammatory mediators (ATP, serotonin, bradykinin NGF, prostaglandins, chemokines, histamine, or proteases) can regulate the activity of TRPV1 through intracellular pathways associated with their specific GPCRs [61]. For example, in transfected HEK293 cells and DRG neurons, ATP increases the activation of TRPV1 induced by capsaicin through the purinergic receptor P2Y1, reby enhancing the activity of TRPV1 in the PKC-dependent pathway [62]. Numerous studies have shown that TRPV1 is not only associated with pain but also with anxiety [63]. Based on the behavioral findings of this study and others, it can be inferred that the increased expression of TRPV1 observed after 24 h was primarily related to pain, while the increase at 28 d was more likely associated with emotional aspects such as anxiety. Typically, the pain associated with tooth movement returns to normal levels within approximately 7–14 days. Therefore, the augmented expression of TRPV1 observed before this period could be attributed to the nociception. This indicates that there is a certain association between the expression of TRPV1 and the changes in pain and anxiety during tooth movement. TRPV1 in brain cannot receive direct temperature or PH stimulation, but can affected by some factors such as endogenous vanilloids/cannabinoids, which activate the TRPV1 in neurons and thus modulate synaptic strength, and then continue to transmit information through other media [64]. Studies have indicated that following exposure to the agonist capsaicin, TRPV1 induces transient activation of both ERK1/2 and p38 MAPK, manifested as increased phosphorylation [65]. Other research has also observed mTOR phosphorylation and altered expression of caspase-3 [66], suggesting that these factors may serve as downstream signaling pathways of TRPV1.

Limitations

This study still has several limitations. The sample size was determined primarily based on pilot experiment results and the resource equation method, resulting in a relatively small overall sample size. Future studies should expand the sample size for further validation. In addition, the statistical analysis was relatively simple. These limitations may have an impact on the interpretation of the present findings. The current experimental evidence in this study is primarily correlative, and future studies are required to provide more direct experimental evidence, such as inhibition or knockdown of TRPV1 activity in the central amygdala. Meanwhile, existing evidence indicates upregulation of TRPV1 within the central nervous system, with particular attention to the CeA; however, the functional connectivity between the CeA and peripheral pathways remains to be experimentally validated. Therefore, it remains unclear whether the observed central TRPV1 expression results from local synthesis or is peripherally mediated. Elucidating this precise mechanism represents an important goal for future research, and further evaluation of its downstream signaling pathways is needed, such as the TRPV1–ERK1/2–CREB and TRPV1–mTOR–S6K1 pathways, which are associated with pain and emotional responses. Although TRPV1 is a key pain mediator during inflammatory processes, our findings are based on a mouse model. Confirmation of its potential as a therapeutic target requires validation in long-term models and with more comprehensive experimental approaches. Therefore, subsequent studies will explore the role of the CeA in regulating anxiety associated with orthodontic treatment. Finally, as pain and emotional responses result from the interaction of various nuclei within the central nervous system, our future studies will also investigate the interactions between these nuclei and the CeA to identify neural pathways involved in the regulation of orthodontic pain.

Conclusion

The present study demonstrates that orthodontic procedures can induce pain and emotional changes, which may be associated with increased TRPV1 expression in the central amygdala. These findings lay a foundation for further investigation into the functional role of TRPV1 in pain and emotional regulation during orthodontic tooth movement.

Authors' contributions

YX H and HQ proposed the experiment and led the supervision of the entire experimental process. DF and RW completed mouse modeling and molecular-biology-related experiments. YN G, YF J, SY L, XY L and ZP H completed behavioral tests and video analysis. The manuscript was written by DF The final version of the manuscript was contributed to by all authors and has been approved by them.

Funding

The National Natural Science Foundation of China Youth Science Foundation Project (ProgramNo.82001087) provided support for this article.

Key Research and Development Program of Shaanxi ( Program No.2021SF-047 ).

Data availability

The datasets produced and/or examined in this ongoing investigation cannot be accessed by the public due to copyright restrictions. However, they can be obtained from the corresponding author (Hu Qiao) upon a reasonable inquiry.

Declarations

Ethics approval and consent to participate

The experiments were conducted following the Guidelines for the Care and Use of Laboratory Animals provided by the National Institutes of Health. The protocols for the animal experiment were approved by the Biomedical Ethics Committee of the Health Science Center at Xi’an Jiaotong University (Protocol number: 2020 − 386).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.