Animals

Two hundred and sixteen male Sprague–Dawley rats, weighing between 200 g and 300 g, were obtained from the Animal Experimental Center at Sichuan University. They were maintained in the animal facility and kept in an air-conditioned room at 21 °C with a 12 h light-dark cycle. Standard rat chow and water were provided ad libitum. Animal experiments were performed in accordance with protocols that were approved by the ethical committee of the State Key Laboratory of Oral Diseases, Sichuan University (protocol No.WCCSIRB-D-2014-084).

The rats were randomly assigned to 6 groups: force group (group A; n = 36), pseudo-force group (group B; n = 36), force + TRPV1 antagonists (SB366791, positive control) group (group C; n = 36), force + normal saline group (group D; n = 36), force + TRPV1 shRNA lentivirus group (group E; n = 36), and force + blank lentivirus group (group F; n = 36). Rats were anaesthetised with an intraperitoneal injection of 7% chloral hydrate in normal saline (NS) solution at the dose of 0.06 mL·g−1 body weight, and then fixed Ni-Ti alloy closed-coil springs were ligated to the rats with ligation wires (0.2 mm in diameter) between the left maxillary first molar and the upper incisor of the rats to simulate orthodontic forces. In all force groups, the springs delivered a 40 g force measured, as by a force meter (Tiantian, Changsha, China), while a 0 g force was used for the pseudo-force group. Rats were euthanized by decapitation after being anaesthetised with pentobarbital sodium (50 mg·kg−1·bw) on days 0, 1, 3, 5, 7, and 14 (n = 6 for each group per day). The rats in all groups that were euthanized on day 14 were used in the orofacial pain assessments on days 0, 1, 3, 5, 7, and 14. Moreover, rats that were euthanized on day 0 did not receive any interventions and were chosen as the baseline control for each group. The details of animal use in different groups are depicted in Supplementary Fig. 1. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research43.

Lentivirus vector preparation

A lentivirus vector encoding an enhanced red fluorescence protein was recombined with the rat TRPV1 RNA interference sequence (sense: CAGATAACACAGTTGACAA). The recombined sequence was amplified with a polymerase chain reaction (PCR) assay. Viral vectors were packaged and harvested by transfection into 293 T cells, and this was followed by visualisation under a fluorescent microscope. The viral titre was determined using TaqMan PCR and expressed as transducing unit (TU) per mL. The viral vector titre was 1.0 × 109 TU·mL−1. In addition, blank lentivirus vectors that did not contain the RNAi sequence were simultaneously prepared as a control.

TG inoculation

A 15 μL aliquot of the TRPV1 antagonist solution (SB366791, Sigma-Aldrich, USA), 15 μL of a normal saline solution, 10 μL of a TRPV1 shRNA lentivirus solution (virus 3.3 × 105 TU·μL−1, polybrene 6 × 10−3 TU·μL−1, Qiagen, USA), and 10 μL of a blank lentivirus vector (virus 3.3 × 105 TU·μL−1, polybrene 6 × 10−3 TU·μL−1) were inoculated into the TG of animals in groups C, D, E, and F, respectively. The TRPV1 antagonist was inoculated into the TG of group C rats 30 min before spring mounting, which was followed by TG inoculations on days 1, 3, 5, 7, and 14. The group D rats were treated similarly but received normal saline TG inoculations. On each inoculation day, all of the animals in these two groups were inoculated, and six rats were sacrificed four hours after the inoculations. The TG were rapidly harvested and placed in liquid nitrogen for PCR and western blot analyses. The animals in groups E and F were inoculated with either the TRPV1 shRNA lentivirus or the blank lentivirus seven days before the orthodontic springs were applied. Six rats in each group were euthanized on days 0, 1, 3, 5, 7, and 14, and the TG were harvested.

Immunofluorescence staining

Twelve rats that received the same inoculations as the group E rats but did not have springs inserted were euthanized, and the TG were harvested and subjected to immunofluorescence staining analyses on days 1, 3, 5, and 7 after the lentiviral inoculation. The TG specimens were embedded in a cutting compound (OCT) at an optimal temperature and cut at a 14 μm thickness along the TG macroaxis in a freezing microtome. The prepared sections were deparaffinized, rinsed with phosphate-buffered saline (PBS) three times, blocked with a 3% bovine serum albumin (BSA) solution, and incubated for 30 min after fixation in acetone for 15 min. Thereafter, the sections were stained with a primary rabbit anti-TRPV1 monoclonal antibody (1:500, Abcam) and incubated for another 45 minutes at 37 °C followed by rinsing with PBS three times for 5 min. The sections were further stained with an immunofluorescent tetramethylrhodamine (TRITC)-labelled secondary antibody (1:100), incubated for 1 h at 37 °C, rinsed with PBS and finally mounted onto slides for observation under a fluorescence microscope (DM4000B, Leica, Germany).

Verification of TG transduction in vivo

Twelve rats were euthanized to verify the transduction of the viral vectors into the TG using an in vivo fluorescence imaging system (In-Vivo Xtreme, Bruker, Germany) on days 1, 3, 5, and 7 after TRPV1 shRNA lentivirus inoculation and prior to the application of the orthodontic force springs. All rats were routinely imaged on days 1, 3, 5, and 7 after lentivirus inoculation, and three rats were imaged on each day. Cherry was selected as the fluorescence reporter gene, and it has a fluorescence detector with excitation/emission wavelengths of 587/610 nm.

Orofacial pain assessment

For the animals in groups C, D, E, and F that were sacrificed on day 14, orofacial pain levels were assessed using the RGS between 7:00 pm and 9:00 pm on days 0, 1, 3, 5, and 7 in a room with a level of background noise less than 45 dB following the protocols described previously44. Briefly, the tested rats were placed individually into transparent cubicles with a 20.0 cm × 10.5 cm × 9.0 cm volume. After acclimation to the environment for 15 min, the rats were continuously videotaped for 30 min. For each rat on each assessment day, ten facial expression images were extracted every 3 min to be used for the RGS scoring. The RGS was scored independently by two analysts by examining the facial expression changes in the orbits, nose, ears, and whiskers of the rats. The two analysts’ scores of the pain levels were averaged. For each rat, the RGS scores that were determined before the interventions were applied were used as the baseline RGS. Differences in the RGS scores between the baseline and experimental tooth-movement groups were regarded as the surrogate pain levels for each rat at each assessment time point.

The real-time RT-PCR assay

Total RNA was extracted from the TG using the Takara MiniBEST Universal RNA Extraction Kit (Takara, Shiga, Japan) according to the manufacturer’s protocols. Then, cDNA was reverse transcribed using the M-MLV test kit (M1705, Promega) following the manufacturer’s recommendations.

Rat TRPV1 (NM_031983) mRNA expression was quantified in the TG samples using triplex RT-PCR performed in a LightCycler480 (Roche, Switzerland) RT-PCR platform with the SYBR Premix Ex Taq (Perfect Real-time, TAKARA, Dalian, China) according to the manufacturer’s protocol. To quantify the amount of specific mRNA expression in the samples, a standard curve was generated for normal rat TG samples. GAPDH served as an internal standard. PCR was performed using specific primers for rat GAPDH (forward primer: TTCAACGGCACAGTCAAGG, reverse primer: CTCAGCACCAGCATCACC, expected size: 114 bp) and TRPV1 (forward primer: AAGGATGGAACAACGGGCTAG, reverse primer: TCCTGGTAGTGAAGATGTGGG, expected size: 127 bp). The final reaction volume of 12 µL consisted of 6 µL of the SYBR Premix Ex Taq, 0.3 μL of the 5 µmol·L−1 primer mix, 0.6 μL of the reverse transcription nucleic acid template, and 5.1 μL of RNase-free H 2 O. The thermal profile was set at 95 °C for 2 min and 94 °C for 10 s, followed by 40 cycles at 60 °C for 30 s and 40 cycles at 60 °C for 10 s. Relative mRNA transcript level calculations were performed using the comparative CT method (ΔΔCT). The average CT values of the three complex holes for each sample were calculated and marked as CT TRPV1 and CT GAPDH , ΔΔCT = N(CT TRPV1 − CT GAPDH ) − (CT TRPV1 − CT GAPDH ).

Western blot analysis

The TG tissues were cut into pieces and homogenised with the RIPA lysis buffer plus phenylmethanesulfonyl fluoride (PMSF) at a ratio of 20 mg to 150–250 μL, resulting in a final concentration of 1 mm. The homogenate was incubated on ice for 30 min followed by centrifugation at 14 000 × g for 5 min. The supernatant was stored at −80 °C before analysis. The lysates were run on SDS-PAGE and separated by electrophoresis (Tanon, Shanghai, China). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk in a Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature. The sealed PVDF membranes were incubated in the primary TRPV1 goat polyclonal antibody (1:200, Santa Cruz, USA) for 2 h at room temperature, washed with TBST four times and incubated with the second HRP-conjugated goat anti-rabbit antibody (Beyotime Biotechnology, China) for 1 h. The protein blot densities were analysed using ImageJ Software (National Institutes of Health, Bethesda), which marked the target intensity band as A TRPV1 and the internal reference intensity band (GAPDH) as A GAPDH ; protein expression at the corresponding time point = A TRPV1 /A GAPDH .

Statistical analyses

Statistical analyses were performed using SPSS 16.0 (SPSS, Chicago, Illinois, USA) and GraphPad Prism 6.0 software (GraphPad Software, San Diego, USA). The results are depicted as the means ± the standard deviation (SD). A one-way ANOVA (Bonferroni post hoc test) was employed to analyse the differences between TRPV1 expression and orofacial pain among different time intervals in each group. A two-way ANOVA with repeated measures was used to examine the effects of time (0, 1, 3, 5, 7, and 14 d), groups (the force group vs. the pseudo-force group, the force + TRPV1 antagonist group vs. the force + normal saline group, the force + TRPV1 shRNA lentivirus group vs. the force + blank lentivirus group), and the interactions with both TRPV1 expression and orofacial pain. For the two-group comparisons at each time interval, the Bonferroni post hoc test was used if the pretest for normality was not rejected at the 0.05 significance level. P values less than 0.05 were considered statistically significant.