Animals

Male C57BL/6J mice (6–23 weeks old) (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan) used in the present study were allowed access to food and water ad libitum and maintained on a 12 h:12 h light/dark cycle (light on at 8 a.m.) in a temperature- and humidity-controlled room (24 ± 1 °C, 55 ± 5%). All mice were housed in groups of 2–6 mice. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Hoshi University School of Pharmacy and Pharmaceutical Sciences, which is accredited by the Ministry of Education, Culture, Sports and Technology of Japan.

Production of a model of neuropathic pain by sciatic nerve ligation

Under 3% isoflurane anesthesia (FUJIFILM Wako Pure Chemical Co. LTD, Osaka, Japan), tight ligation of the sciatic nerve in the right hind limb of mice was performed by using 8-0 silk suture around approximately one-half of the diameter of the nerve, as previously described [16, 17]. In sham-operated mice, the sciatic nerve was just exposed but not ligated. To prevent dehydration after surgery, water and food were available ad libitum.

Creation of a model of persistent postsurgical pain by electrocautery

Under isoflurane anesthesia, a 3-mm longitudinal incision was made in the skin and fascia of the right hind paw of a mouse using a No. 23 scalpel blade from 3 mm from the proximal end of the heel to the toe. Next, with the use of a monopolar electrosurgery unit (at 50 W; Vetroson® V-10; Summit Hill Laboratories, NJ, USA) with a dispersive electrode pad placed under the mouse's body, a 3-mm longitudinal incision was made on the plantaris muscle, as previously described [18]. Electrocautery was performed while maintaining coagulation and hemostasis of the incision during dissection. The skin was stitched with two mattress sutures of 7-0 nylon. In sham operated mice, the plantaris muscle was exposed without the incision, and the skin was stitched with a simple interrupted suture of 7-0 nylon.

Chemogenetic manipulation of sensory neurons

Under isoflurane anesthesia, a 1–2 cm longitudinal incision was made in the skin and the connective tissue between the gluteus superficialis and biceps femoris muscles to expose the sciatic nerve in the right hind limb (ipsilateral side) of a mouse, as previously described [19]. Next, an internal cannula (Eicom Co., Kyoto, Japan) was inserted into the sciatic nerve and adeno-associated virus (AAV) vector was microinjected at 1 µL/min for 4 min (4 µL total volume) with a glass micropipette and an air pressure injector system (Micro-syringe Pump-Model ESP-32; Eicom Co.). After the surgical procedures, mice were intraperitoneally injected with clozapine N-oxide (CNO) (3 mg/kg, t.i.d., abcam, Cambridge, UK) for 2 weeks. The following viruses were used in this study: AAV6-hSyn-hM3Dq-mCherry, AAV6-hSyn-hM4Di-mCherry, AAV6-hSyn-EGFP and AAV6-hSyn-mCherry. AAV6-hSyn-EGFP and AAV6-hSyn-mCherry were used as a control vector. All of the viruses used in this study were kindly provided by Dr. Akihiro Yamanaka (Nagoya University, Nagoya, Japan).

Immunohistochemistry

Immunohistochemistry was performed according to a previously reported protocol [17]. Over 2 weeks after AAV injection, mice were anesthetized with 3% isoflurane and intracardially perfused with 4% paraformaldehyde in 0.1 M PBS (pH 7.4; PFA). After perfusion, the lumbar spinal cord and the lumbar dorsal root ganglion (DRG) were quickly removed and post-fixed with PFA and cryoprotected in 20–30 (w/v) % sucrose (FUJIFILM Wako). The tissue sections were embedded in an O.C.T. compound (Sakura Finetec USA. Inc., CA, USA), and the sections were cut on a cryostat (15 μm for spinal cord, 8–10 μm for DRG) (CM1860; Leica Microsystems, Heidelberg, Germany). The slices were blocked in 3% normal goat serum (NGS: Vector laboratories, Inc., CA, USA) or normal horse serum (Vector laboratories)/0.1–0.2% Triton X-100 in 0.01 M PBS or 5% NGS for 1 h at RT. Then incubated with the primary antibodies used were as follows: rabbit anti-mCherry (1:1000, abcam), chicken anti-mCherry (1:2500, abcam), sheep anti-CGRP (1:1000, Enzo Life Sciences, PA, USA), rabbit anti-substance P (1:1000, ImmunoStar, WI, USA), goat anti-peripherin (1:50, Santa Cruz Biotechnology, TX, USA.) for 48 h at 4 °C or 24 h at RT. Following washes, they were incubated in goat anti-rabbit Alexa 546 (1:10,000; Thermo Fisher Scientific Inc., MA, USA), goat anti-chicken Alexa 546 (1:10,000; Thermo Fisher Scientific), donkey anti-sheep Alexa 488 (1:750; Thermo Fisher Scientific), chicken anti-rabbit Alexa 488 (1:700; Thermo Fisher Scientific), donkey anti-goat Alexa 488 (1:400; Thermo Fisher Scientific) conjugated secondary antibodies for 2 h at RT. Immunofluorescence was detected by a light microscope (BX53, Olympus, Tokyo, Japan) and captured using a high-sensitivity digital CCD camera (MD-695; Molecular Devices, CA, USA). Imaging analysis was performed using Metamorph 7.8 software (Molecular Devices).

Plantar test

The latency of the hind paw withdrawal in response to nociceptive stimulation was measured by focusing a thermal stimulus on the plantar surface of the hind paw of mice using a thermal stimulus apparatus (model 33 Analgesia Meter; IITC/Life Science Instruments, CA, USA) as described previously [20]. Before the experiments, mice were habituated for 1 h in an acrylic cylinder (15 cm height and 8 cm in diameter).

Measurement of mechanical allodynia

Mechanical allodynia was assessed by a plantar electronic von Frey Anesthesiometer (ALMEMO 2450 Ahlborn; IITC/Life Science). Briefly, a chip attached to the device was applied vertically to the plantar surface of the hind paw of mice, and the pressure (g) until the hind paw was flicked off was recorded as the pain threshold. Before this assessment, mice were habituated for 1 h in an acrylic cylinder (15 cm height and 8 cm diameter) on an elevated mesh floor.

Cell culture

Lewis lung carcinoma (LLC), LLC-luc and B16 melanoma (B16) cells were cultured in Minimum Essential Medium Alpha (α-MEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). LLC-luc cells were generated by infection of LLC cells with lentiviral vector expressing ff-Luc genes. Mouse severe osteosarcoma (AXT) cells were established as previously described [21, 22]. AXT cells and AXT-luc cells were cultured under 5% CO2 at 37 °C in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% FBS.

Graft tumor growth assay

LLC, LLC-luc, B16 and AXT cells were counted after trypsinization and then resuspended in a mixture of extracellular matrix gel (Sigma-Aldrich Inc., MO, USA) and Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher Scientific) at a concentration of 5 × 105 cells/0.15 mL as appropriate, according to a previously reported protocol [23, 24]. These suspensions in a volume of 0.15 mL were inoculated subcutaneously into the right thigh close to the sciatic nerve of mice under 3% isoflurane anesthesia. Tumor size was measured using a caliper and tumor volume was calculated as (L × W2)/2, where L = length and W = width.

Cancer pain model mice

To establish tumor xenografts, AXT-luc cells (1 × 106 cells) suspended in 50 μL of IMDM were injected into the right femoral bone marrow cavity of syngeneic C57BL/6J mice, as previously described [16]. Briefly, the knee joint was flexed to 90° and the distal side of the femur was exposed by incising the skin. A 23-gauge needle was inserted into the bone marrow cavity to make a small hole, into which AXT-luc cells or medium alone were injected. All procedures were performed under inhalational anesthesia with 3% isoflurane.

In vivo imaging system (IVIS)

Approximately 10 min before imaging, the substrate luciferin was injected into the intraperitoneally at 4.5 mg/mouse (15 mg/ml, FUJIFILM Wako). Mice were anesthetized with isoflurane/oxygen and placed on the imaging stage. Dorsal images were acquired using the IVIS® Lumina Series III (PerkinElmer Inc., MA, USA).

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

RT-qPCR was performed according to our previous report [18]. For RT-qPCR analysis, total RNAs were isolated from the ipsilateral side of the mouse dorsal root ganglions (DRG) (L3–L5) and then first-strand cDNAs were synthesized. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as an internal control. Additional file 1: Table S1 represents a complete list of all primers used in this study.

Statistical analysis

The results are shown as the mean ± standard error of the mean (S.E.M.). We analyzed and described the statistical significance of differences between groups according to an unpaired t-test and one-way or two-way analysis of variance followed by the Bonferroni multiple comparisons test. The data were subjected to a comparative analysis by testing the null hypothesis for the Pearson product moment correlation. For survival analysis, the Log-rank (Mantel-Cox) test was used. All statistical analyses were performed with Prism version 9.0 (GraphPad software, CA, USA).