Experimental animals
The experiments were performed on adult male C57Bl/6 mice at the age of 8–10 weeks. Animals were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. China. They were housed under a 12 h light/dark cycle at a room temperature of 23 ± 0.5 °C with free access to food and water. Prior to experimental manipulation, mice were habituated in the animal room for at least one week after delivery. The experiment was designed as a single study, approved by local ethical committee at School of Basic Medical Sciences, Fudan University, People’s Republic of China (Agreement No. 20140226-087).
Induction of CFA-induced inflammatory pain
According to the previous study [51], to establish the CFA-induced inflammatory pain model, 20 μl CFA (Sigma, F5881-10ML) was subcutaneously injected into the plantar surface of hind paw to induce an inflammatory response in mice. Normal saline with the same volume was used in the control group.
Behavioral tests: up-and-down method
All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Ethical Issues of the International Association for the Study of Pain. For all behavioral testing, the mice were exposed to the testing environment without any stimulation for 1–2 h per day for 2 days before the formal testing and the investigators were blinded to the treatment.
Mechanical allodynia was measured using a series of von Frey hairs (0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0 and 1.4 g) (Stoelting, Wood Dale, Illinois, USA) as described previously [27]. Briefly, each mouse was placed individually into a plexiglass chamber for 30-min acclimation. Then a von Frey hair was applied and held for approximately 3–4 s with a 10-min interval between applications. A trial began with the application of 0.16 g von Frey hair. The hair force was increased or decreased according to the response. A positive response was defined by a brisk withdrawal of the hind paw upon stimulation. The test contained five more stimuli after the first change in response occurred. Final score was converted to a 50% von Frey threshold using the Dixon up-and-down paradigm [52].
EA treatment
As described previously [53], during EA treatment, the mice were placed in a specially designed holder, and the trunk of mice was kept motionless while the head and four limbs were left free to move. Two pairs of stainless-steel needles (0.16 mm in diameter) were inserted into ‘Kunlun’ (BL60, at the ankle joint level and between the tip of the external malleolus and tendo calcaneus) and ‘Zusanli’ (ST36, 5 mm lateral to the anterior tubercle of tibia) at a depth of 2 mm and 3 mm, respectively. Alternating trains of dense-sparse frequencies (100 Hz and 2 Hz each for 3 alternately), 1 mA and 2 mA for 15 min, respectively, were delivered by Han’s Acupoint Nerve Stimulator (LH202, Huawei Co. Ltd., Beijing, China). EA treatment, starting on the first day after the CFA injection, was applied once a day in the EA treatment group. Mice in sham-EA group were restrained the same way and the needles were inserted into ST36 and BL60 at a depth of 3 mm and 2 mm in the muscle without electric stimulation.
Intraspinal AAV virus injections
The virus injection was applied 3 weeks before the CFA injection. Mice were anesthetized and then placed in a motorized stereotaxic frame, and the vertebral column was immobilized using a pair of spinal adaptors to expose the L4-L5 lumbar segment. The glass capillary for Nanoliter 2010 (outer diameter of 1.14 mm, inner diameter of 0.53 mm) was used to be inserted into the dorsal spinal cord at a depth of 200–300 μm. The rate of injection (60 nl/min) was controlled using a motorized perfusion system (Nanoliter 2010, World Precision Instruments). The glass capillary was left in place for 5 min after the injection. Wounds were sutured, and the animals were allowed to recover on a heat mat. AAV virus vectors used in this study are provided by OBiO Technology (Shanghai, China) and BrainVTA (Wuhan, China). Mice received 5 daily 100 μl injections of tamoxifen (1 mg, i.p.) (Sigma, T5648-1G) to induce CRE activity; tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil [54]. For GRK2 overexpression experiment, two viruses were used, one (AAV2/8 carrying hSyn-CreERT2) was the virus expressing CRE enzyme induced by tamoxifen and the other (AAV2/8 carrying CMV-DIO-GRK2-2A-EGFP) was the virus expressing GRK2 dependent on CRE enzyme. For overexpression of the neuronal GRK2 in the spinal cord, mice were injected with a 2:1 volume mixture of an AAV2/8 carrying CMV-DIO-GRK2-2A-EGFP and an AAV2/8 carrying hSyn-CreERT2 (OBiO Technology Co. Ltd., Shanghai, China). Tamoxifen was first dissolved in 95% ethanol and then diluted to its final concentration in corn oil. For downregulation of the neuronal GRK2 expression in spinal cord, mice were injected with 600 nl of an AAV2/9 carrying GRK2 shRNA with a neuronal specific promoter Syn (OBiO Technology Co. Ltd., Shanghai, China).
Western blotting
Briefly, the spinal dorsal horn of the L4-L6 segments was lysed in RIPA Lysis Buffer (100 μl/g, Beyotime Biotechology, P0013B), supplemented with protease inhibitors (Beyotime Biotechology, ST506). The lysate was centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was obtained. Samples were separated with 10% acrylamide gels and then transferred onto polyvinylidene fluoride membranes by using SDS-PAGE. After blocking with 5% skim milk in tris-buffered-saline with tween (TBST) (20 mm Tris–HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween-20), the membranes were incubated with rabbit anti-GRK2 (1:300, Sc-562, Santa Cruz), rabbit anti-GAPDH (1:10,000, HRP-60004, ProteinTech) or anti-β-actin (1:10,000, HRP-60008, ProteinTech) overnight at 4 °C in a shaking incubator, then washed in TBST before incubated with appropriate HRP-conjugated secondary antibody (1:10,000) for 2 h at room temperature. The objective band were detected using an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK) and analyzed using ImageJ software (version 1.47).
Immunofluorescence staining
Mice were deeply anesthetized with 1% pentobarbital sodium and were transcranially perfused with normal saline followed by 4% formaldehyde. The spinal cord of the L4-L6 segments were immersed in 4% formaldehyde for 4 h, 20% and 30% sucrose in 0.1 M PBS overnight at 4 °C respectively. The 30 μm-thick sections of spinal cord were prepared, then blocked in Superblock Buffer (Thermo, 37580) for 1 h at room temperature, reacted with mouse anti-NeuN (1:500, MAB377, Millipore) overnight at 4 °C, and then reacted with Alexa-594-conjugated donkey anti-mouse IgG secondary antibodies (1:1000, R37115, Invitrogen) for 2 h at room temperature. Finally, the sections were patched to the slides with sealing liquid containing DAPI (SouthernBiotech, 0100-20) and were visualized under a confocal microscope (FV10i, Olympus).
Total RNA real-time PCR
Total RNA was isolated from L4-L6 spinal dorsal horn with RNSiso Plus (Takara, 9109) as per the manufacturer’s instructions. The samples were then analyzed with Nanodrop (Thermo). Reverse transcription was performed by using PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, RR047A) and SYBR Premix Ex TaqTM II (Takara, RR820A) for quantitative real-time qPCR analysis in the end. The housekeeping gene, GAPDH, was used as an internal reference for standardization of the analysis. The sequences of primers for each target mRNA are as follows: CD16, forward: 5′-AGA CCC AGC AAC TAC ATC C-3′, reverse: 5′-GAC TTC CTC CAG TAA TCC CT-3′; CD206, forward: 5′-GCT TCC GTC ACC CTG TAT G-3′, reverse: 5′-CTC CAC AAT CCC GAA CCT-3′; IL-4, forward: 5′-CCA TGA ATG AGT CCA AGT CC-3′, reverse: 5′-TGA TGC TCT TTA GGC TTT CC-3′; IL-10, forward: 5′-GGG AAG AGA AAC CAG GGA GA-3′, reverse: 5′-GGG GAT GAC AGT AGG GGA AC-3′; iNOS, forward: 5′-TTG ACG CTC GGA ACT GTA G-3′, reverse: 5′-GAC CTG ATG TTG CCA TTG T-3′; IL-1β, forward: 5′-GTA CAA GGA GAA CCA AGC AA-3′, reverse: 5′-CCG TCT TTC ATT ACA CAG GA-3′; IL-6 forward: 5′-CCA ATG CTC TCC TAA CAG AT-3′, reverse: 5′-TGT CCA CAA ACT GAT ATG CT-3′; TNF-α, forward: 5′-ACT CTG ACC CCT TTA CTC TG-3′, reverse: 5′-GAG CCA TAA TCC CCT TTC TA-3′; TGF-β, forward: 5′-GAC CGC AAC AAC GCC ATC TAT-3′, reverse: 5′-CAC TGC TTC CCG AAT GTC TGA-3′; GAPDH, forward: 5′-AAA TGG TGA AGG TCG GTG TG-3′, reverse: 5′-AGG TCA ATG AAG GGG TCG TT-3′. All primers used for PCR analysis are synthesized by Sangon Biotech (Shanghai) Co., Ltd.
Statistical analysis
All statistical analyses were carried out by using the Prism7 software packages and IBM SPSS 22.0. All data are presented as mean ± standard error of the mean (SEM). A t-test was used for direct comparisons between the two groups. For multiple comparisons within one group, one-way analysis of variance (ANOVA) was adopted. Significant differences among groups were checked by two-way ANOVA. A repeated measure ANOVA was employed to compare the differences between GRK2 overexpression group and the control group. p < 0.05 was considered statistically significant.
