Animals and Surgery
ICR male mice weighing 26-30 g were provided by the Experimental Animal Center at Nantong University. Tlr8-/- mice were developed by CAYN Company (Suzhou, China). Tlr7-/- mice were purchased from Jackson Laboratory (stock number 008380). All animals were housed in standard clear plastic cages under controlled ambient temperature (22-24 °C) with a reversed 12:12 hour dark/light cycle, and allowed ad libitum to access to water and food. The experimental and surgical procedures in this study were reviewed and approved by the Animal Care and Use Committee of Nantong University. Animal treatments were performed in accordance with the guidelines of International Association for the Study of Pain.
The pIONL surgery was made as described by Zhang et al. [20, 21]. In brief, mice were anesthetized with compound anesthetics, and then the oral cavity was opened to locate the left tendon of masseter muscle on the top wall of oral cavity in supine position. In front of the tendon, a 1 mm incision was made to expose the infraorbital nerve. The TNP was established by ligating one half of the infraorbital nerve (ION) with 6-0 silk suture. The mucous membrane of the incision was adhered with the Tissue Adhensive (3M Vetbond, USA). The sham operation was just made an incision on the mucous membrane without damaging the infraorbital nerve.
DNA extraction and genotyping
About 2 mm diameter of the mouse ear was cut from each Tl8-/- or Tlr7-/- mouse, and then was used to extract DNA with the phenol-chloroform method. The primers of genotyping for Tl8-/- mice are as following: forward primer: 5′-GCA GTT GAC GAT GGT TGC ATT-3′, and reverse primer: 5′-TGA CGT GCT TTT GTC TGC TG-3′. A 50 μL reaction volume of PCR amplification buffer was used, including 200 ng DNA, 25 μl 2×Taq PCR MasterMix (Tiangen Biotech), and 1.0 μM TLR8 genotyping primers. Reactions initially were denatured at 95 °C for 5 minutes, 35 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and then the final extension at 72 °C for 2 min. The genotyping protocol of Tlr7-/- mice was used as described by Jackson Laboratory. Primers included common forward primer olMR8628: 5′-AGG GTA TGC CGC CAA ATC TAA AG-3′, wild type reverse primer olMR8629: 5′-ACC TTT GTG TGC TCC TGG AC-3′, and mutant reverse primer olMR8630: 5′-TCA TTC TCA GTA TTG TTT TGC C-3′. For PCR amplification, 200 ng DNA was used in a 50 μL reaction volume containing 25 μl 2×Taq PCR MasterMix (Tiangen Biotech), 0.5 μM primers of olMR8628, olMR8629, and olMR8630. Reactions initially were denatured at 94 °C for 2 minutes, 10 cycles at 94 °C for 20 s , 65 °C for 15 s (-0.5 °C per cycle decrease), 68 °C for 10 s, and then 28 cycles at 94 °C for 15 s , 60°C for 15 s, 72 °C for 10 s, and then the final extension at 72 °C for 2 min. Amplicons were separated using 1.5% agarose gel, stained with DuRed (Biotium) and photographed with GelDoc-It Ts Imaging System (UVP, USA).
Drugs and administration
VTX-2337 was purchased from Active Biochem (Hongkong, China). Tlr8 siRNA and negative control siRNA (NC siRNA) were designed by RiboBio [16]. RVG-9R peptide was purchased from AnaSpec (USA). For peri-infraorbital nerve injection, the Tlr8 siRNA (2 µg) or NC siRNA was mixed with RVG-9R peptide (molar ratio=1:10) [16, 22]. Intra-TG injection was performed with a 29 G syringe (Becton, Dickinson and Company, USA). After deeply anesthetized with the isoflurane, the head of mouse was hold by one hand. The tip of needle was inserted subsequently through the infraorbital foramen, infraorbital canal and foramen rotundum, finally was positioned in the TG [23]. Different doses (10 ng. 50 ng, and 100 ng) of VTX-2337 (5 ml) were slowly injected.
Facial pain behavioral test
The behavioral test environment was kept at 22-24 °C and 40-60% humidity. Before facial behavioral test, the mice habituate the behavioral test cage for 30 min every day for three days in the behavioral test environment. The von Frey filaments (0.02 g and 0.16 g) were used to stimulate whisker pad innervated by the ION, and the response of the mice was recorded. Each filament was applied three times on the ipsilateral whisker pad. The mean score of three measurements was calculated according to the following criteria: score 0, no response; score 1, exploratory behavior - the mice detect the von Frey filament; score 2, slight withdrawal response - the mice slowly move their face backward to the stimulation; score 3, quick and intense withdrawal response with lifting paw; score 4, wiping face with forepaw less than three times toward the stimulated facial area; score 5, wiping face with forepaw more than three times toward the stimulated facial area [24, 25].
RNA collection and Real-time RT-PCR
According to the manufacture’s protocol of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), the total RNA in TG tissue was collected. The quality and quantity of RNA was checked on the NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The cDNA was reversed from RNA using the first Strand cDNA Synthesis Kit (Takara, Japan). Real-time PCR was performed in A&B Applied Biosystems using the SYBR Green Master Mix (Vazyme, China). The following primers were used in PCR reaction: TLR8 forward: 5′-ACC TGA GCC ACA ATG GCA TTT AC-3′, and TLR8 reverse: 5′-TTG CCA TCA TTT GCA TTC CAC-3′; TNF-a forward: 5′-GTT CTA TGG CCC AGA CCC TCA C-3′, TNF-a reverse: 5′-GGC ACC ACT AGT TGG TTG TCT TTG-3′; IL-1β forward: 5′-TCC AGG ATG AGG ACA TGA GCA C -3′, IL-1β reverse: 5′-GAA CGT CAC CCA GCA GGT TA-3′; IL-6 forward: 5′-CCA CTT CAC AAG TCG GAG GCT TA-3′, and IL-6 reverse: 5′-CCA GTT TGG TAG CAT CCA TCA TTT C-3′; GAPDH forward: 5′-AAA TGG TGA AGG TCG GTG TGA AC-3′, and GAPDH reverse: 5′-CAA CAA TCT CCA CTT TGC CAC TG-3′. The condition of PCR amplifications was set at 95 °C for 30 s, and then 40 cycles (5s for 95 °C and 30 s for 60 °C). The PCR data were analyzed with StopOne Software v2.3 to show graph. Melt curves were used to judge the specificity of PCR products. Quantification was performed by normalizing cycle threshold (Ct) values with GAPDH Ct and analyzed with the 2−ΔΔCt method.
Immunofluorescence
After deeply anesthetized with the isoflurane, the mice were transcardially perfused with 0.01 M PBS, followed by 4% paraformaldehyde in 0.01 M PBS. The TG was dissected carefully and postfixed in the fixative solution overnight, and then was placed in 30% sucrose in 0.01 M PBS for 2 d. The TG was embedded in OCT solution and then cut into 15 mm sections in a cryostat (Lecia, Germany). These sections were processed for immunofluorescence as we described previously [16]. In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, these sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), CGRP (mouse, 1:1000, Sigma-Aldrich), NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight. Following three rinses with 0.01 M PBS, these sections were further incubated with the secondary antibodies DAR-CY3 (1:1000, Jackson ImmunoResearch) and DAM-488 (1:1000, Jackson ImmunoResearch) or IB4-FITC (1:200, Sigma-Aldrich). The fluorescence signals were checked and captured with fluorescence microscope (Nikon Eclipse Ni-E, Japan). The fluorescence images were analyzed with Image J software (NIH, USA).
Western blot
The TG tissue in WT and Tlr8-/- mice was collected, and homogenized in a lysis buffer containing protease and phosphatase inhibitors (Sigma). The protein concentration was checked using BCA Protein Assay (Pierce Biotechnology, USA). The 30 μg protein was loaded in each lane of SDS-PAGE gel, and then transferred to Polyvinylidene-Fluoride (PVDF) membrane. After blocked by 5% skim milk, the PVDF membrane was incubated with the primary antibody against pERK (rabbit, 1:500, Cell Signaling Technology), ERK (rabbit, 1:500, Cell Signaling Technology), p-p38 (rabbit, 1:500, Cell Signaling Technology), p38 (rabbit, 1:500, Cell Signaling Technology). The PVDF membrane was further incubated with IRDye 800CW secondary antibody (goat-anti-rabbit, 1:10000, LI-COR) and captured the images with Odyssey CLx system (Odyssey, USA). The size of bands was evaluated by the prestained protein marker (Thermo Fisher Scientific), and the intensity of bands was calculated by the Image J software (NIH, USA).
HEK293 Cell Culture and Transfection
HEK293 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Bio Whittaker Europe, Vervier, Belgium) with 10% (vol/vol) foetal calf serum (PAA, Linz, Austria) and 0.5% penicillin/streptomycin at 37 °C in a humidity-controlled incubator with 5% (vol/vol) CO2. Cells were transfected with the TLR8 or control plasmids using Lipofectamine 2000 (Invitrogen, Netherlands) at 80% confluence. Transfected cells were cultured in the same growth medium for 36 h before calcium measurements.
TG Neuron Culture
After anesthesia using isoflurane, the mouse cranium and brain (4-6 weeks old) were quickly removed, and then the TGs were rapidly collected [26]. The meninges and connective tissues on the TG were carefully stripped in an ice-cold oxygenated balanced salt solution (BSS: 125 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 10 mM glucose, 2.4 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES, pH 7.2 and osmolarity: 300 mOsm), and each TG was shredded with scissors. These TG tissues were kept at 37 °C for 90 min in the oxygenated aCSF which contained collagenase (3.0 mg/ml, Roche) and dispase-II (2.4 units/ml, Roche), and then were washed with standard aCSF twice. TG Neurons were separated mechanically by polished glass pipettes, and cultivated on the adhesive coverslips (diameter, 13mm) in the 24-well plates. The dissociated TG neurons in each well were incubated in the aCSF at 37 °C (humidified 95% O2 and 5% CO2). After 24 h incubation in the carbon dioxide cell incubator chamber, TG neurons were used for calcium imaging measurements.
Ca2+ Imaging
HEK 293 cells or TG neurons were loaded with Fura-2AM (2 µM, Molecular Probes) mixed with 0.02% pluronic (Life technologies) for 90 min at RT in a dark circumstance. Coverslips with HEK 293 cells or TG neurons were washed with BSS, and then placed on an inverted fluorescence microscope (Olympus IX73, Japan). The fluorescence emission at 510 nm with excitation at 340 nm and 380 nm was detected at 2 s intervals using a computer-controlled F4500 fluorescence spectrophotometer (Hitachi, Japan). Wavelength selection and the timing of excitation and acquisition of images were controlled using the Metafluor program (Molecular Devices). Digital images were stored on hard disk for off-line analysis. The ratio of fluorescence intensities (λ340/λ380) at these two wavelengths was recorded as a relative level of intracellular Ca2+.
Quantification and statistics
All results were showed as mean ± SEM. The behavioral data were analyzed by two-way repeated measures (RM) ANOVA followed by Bonferroni test as the post-hoc multiple comparison analysis. For the analysis of TLR8+ neurons in the TG, the immunofluorescent images of TG were captured, and then the number of TG neurons was counted using a computer-assisted imaging analysis system (Image J). For western blot, the density of specific bands was measured with Image J, and the intensity of the background was subtracted in each lane. The levels of pERK, pp38 were normalized to total ERK and p38, respectively. Differences between groups were compared using one-way ANOVA followed by Bonferroni test or using Student’s t-test if only 2 groups were applied. The criterion for statistical significance was set at P < 0.05.