In total, 365 male Sprague–Dawley rats (200-250 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 (WCCSIRB-D-2015-006). Following general anesthesia with intraperitoneal injection of sodium pentobarbital (30 mg·kg−1), rats were placed in supine positions, and intraoral NiTi alloy closed-coil springs were ligated between left upper first molars and incisors to mimic orthodontic tooth movement. Four initial force levels were used in this study, i.e., 0 g, 20 g, 40 g and 80 g. Force magnitudes were determined through a force meter (Tiantian, Changsha, China). Rats were euthanized by decapitation following anesthesia with pentobarbital sodium (50 mg·kg−1) six hours following drug administrations. Rats that did not receive any intervention were regarded as the baseline control for each group.
2.2 Drug administration into TG
The administration of drugs and lentivirus vectors into TG was conducted according to our previous study with modifications that shaving was not performed to avoid its impact on behavioral testing (23). Following general anesthesia with intraperitoneal injection of sodium pentobarbital (30 mg·kg−1), the injected region was disinfected with 75% ethanol. The injected position is between tympanic bulla and the posterior edge of mandibular ramus. The injected direction is middle upper, perpendicular to the long axis of rat body and 15 degree to coronary plane. When the needle reached trigeminal fossa, the drug was slowly injected at a constant speed. The injection process should last 1 min, and remained in situ for 1 min before withdrawn. Specifically, 15 μl nociceptin (10 nM), 15 μl UFP-101 (10 nM) or 15 μl normal saline was administered right after spring placement (0h) and on 1st day, 3rd day, 5th day and 7th day to evaluate the effects of N/OFQ on pain and CGRP expression. UFP-101 ([Nphe1, Arg14, Lys15] N/OFQ-NH2) is a specific peptide antagonist of N/OFQ which has been reported to have high efficacy and durability in vivo (24). N/OFQ-overexpressing lentivirus vector suspension (10 μl), blank vector suspension (10 μl) or normal saline (10 μl) were administered into TG one week after spring placement (40-g initial force) to assess the effects of N/OFQ-based gene therapy on pain.
2.3 Orofacial pain assessment (rat grimace scale, RGS)
The assessment of orofacial pain was performed through rat grimace scale (RGS) six hours following drug administrations, strictly according to our previous study (25). The RGS scoring was performed based on videotaping by two experienced investigators independently and in duplicate. These two investigators were blind to the group information. In brief, rats were placed in transparent cubicles and videotaped for 30 min for each testing session. For each session, 10 images of facial expressions for each rat were extracted for scoring. The RGS scoring was performed based on the facial expression changes in orbit, nose, ear and whisker (Figure 1). The surrogate pain levels were obtained by subtracting the baseline RGS scores from the ones for testing sessions.
2.4 Tissue processing and analysis
For immunostaining, V1/V2 of TG were placed in liquid nitrogen and cryosectioned at a thickness of 10 μm by using microtome (Thermo Fisher Scientific, USA). The tissue samples were immunostained with primary antibodies against CGRP (1:200, Ab36001, Abcam, Cambridge, UK) and FITC-labelled rabbit anti-goat secondary polyclonal antibody IgG (1:100, Ab150077, Abcam, Cambridge, UK). Image observation and acquisition were performed by using a fluorescence microscope (Leica, Germany).
For western blot, V1/V2 of TG tissue samples were homogenized through RIPA lysis buffer with PMSF (Beyotime Biotechnology, China). Following electrophoretic separation, proteins were transferred onto PVDF membranes and blocked with 5% skim milk in TBST solution. Afterwards, sealed PVDF membranes were incubated in the primary polyclonal antibody against FLAG (F1804, Sigma, USA), N/OFQ (Ab216413, Abcam, Cambridge, UK) or β-actin (SC-69879, Santa-cruz, USA), then washed with TBST and incubated with secondary polyclonal antibody goat anti-mouse IgG (SC-2005, Santa-cruz, USA). The protein blot densities were analyzed using ImageJ Software (NIH, Bethesda).
Simple Western (WES) analysis was performed on a WES system (ProteinSimple, WS-2471). The V1/V2 of TG tissues were homogenized with RIPA lysis buffer plus PMSF (Beyotime Biotechnology, China). After dilution and degeneration, the extracted proteins, along with the primary polyclonal antibody against CGRP (Ab36001, Abcam, Cambridge, UK), N/OFQ (Ab216413, Abcam, Cambridge, UK) or β-actin (SC-69879, Santa-cruz, USA), horseradish peroxidase (HRP)-conjugated secondary polyclonal antibody IgG (PS-MK15, Protein Simple, USA) and chemiluminescent substrate, were put into the microplate as instructed. The target band intensity of Western Blot results was analyzed using Compass Software. The specificity of antibodies was pre-validated by the antibody manufacturer Abcam (https://www.abcam.com/nociceptin-antibody-ab216413.html). Moreover, in our study, the specificity of primary antibodies was validated according to their corresponding proteins’ specific molecular sizes (20 kD for N/OFQ and 14 kD for CGRP).
Real-time PCR was conducted to determine the mRNA levels of N/OFQ (Pnoc gene) and CGRP (Calca gene) with GAPDH being the reference. PCR was performed using specific primers for rat CGRP (forward primer: GAAGAAGAAGCTCGCCTACTGG, reverse primer: CTGTCCAAGCTAGAGCCCTCA, expected size: 110 bp) and Pnoc (forward primer: GCTCACGTCCGCTGCTCTTTA, reverse primer: TCCACCTCATCGGCCTCATCT, expected size: 147bp). Total RNA was extracted from TG via Trizol RNA Extraction Kit (Pufei, Shanghai, China) and cDNA reversely transcribed through the M-MLV test kit (M1705, Promega). The expressions were quantified in a LightCycler480 (Roche, Switzerland) RT-PCR platform with the SYBR Premix Ex Taq (Takara, Dalian, China). The thermal profile was set at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and then at 60 °C for 30 s.
2.5 Lentivirus vector construction
The lentivirus vector was constructed as previously described (23). As shown in Figure 5A, a lentivirus vector GV320 (Shanghai Genechem, China) containing red fluorescence (Cherry) and vector marker (3FLAG) was recombined with rat Pnoc gene. Pnoc was expressed under ubiquitin promoter, and m-cherry under SV40 promoter. Thus, the two genes were under two independent promoters. The specific sequences retrieved from GenBank (NM_013007) are displayed in Additional file 1. The recombined sequence was amplified with PCR and DNA sequencing performed for sequence verification. Viral vectors were packaged and harvested by transfecting 293T cells, followed by visualization through fluorescence microscope and verification of Pnoc expression through WB. The viral titer was determined to be 2×108 TU/ml. All the procedures involving use of lentivirus vectors were performed in the laboratory with biosafety level 2 (BSL2) containment according to General Guidelines for Biosafety in Microbiology and Biomedical Laboratories (WS233-2002).
2.6 Statistical analysis
Results are depicted as mean ± standard error of the mean (SEM). Two-way ANOVA with repeated measures were used to analyze the effects of force magnitudes (0 g, 20 g, 40 g and 80 g), time (0 d, 1 d, 3 d, 5d, 7 d and 14 d) and their interactions on RGS scores and the expression levels of N/OFQ. One-way ANOVA with Bonferroni post hoc test was employed to analyze the differences in PNOC expression, CGRP expression and orofacial pain among different time points in each group. All the statistical analyses were performed in SPSS 19.0 and Graphpad Prism 6.0, with a p value less than 0.05 being considered as statistical significance.