Adult male Sprague-Dawley rats (160-200 g) and C57BL/6 mice (20-26 g) were obtained from the Institute of Experimental Animals, Sun Yat-Sen University, China (License number SCXK (yue) 2008-0002). All procedures were approved by the Animal Care Committee of Sun Yat-Sen University, and were consistent with the National Institutes of Health animal care and ethics guidelines for experimental pain investigation. Animals were housed in separate cages with controlled humidity (50-60%), temperature (24 ℃), and 12-hour light/dark cycle (06:00-18:00 h). Food and water were available ad libitum.
Spared nerve injury (SNI) of sciatic nerve
SNI surgery was performed as described previously . Briefly, animals were anaesthetized intraperitoneally (i.p.) with 0.4% sodium pentobarbital (40 mg/kg, Sigma-Aldrich). The skin on the lateral surface of the left thigh was incised and then sectioned to expose the sciatic nerve and its three terminal branches: sural, common peroneal and tibial nerves. The common peroneal and tibial nerves were ligated and sectioned (removal of a 2 mm length), leaving the sural nerve intact. In the sham operated group, an identical operation was performed to expose the nerves without being injured.
L5 ventral root transection (L5-VRT)
L5-VRT surgery was performed as described by Li et al . Animals were anesthetized similarly to SNI model. Briefly, a left L5 hemi-laminectomy was performed to expose the left L5 nerve root. The ventral root was pulled out with fine forceps. The dissection was then performed 2-3 mm proximal to the DRG, and a small portion (2 mm) of the root was removed. In the sham group, the ventral root was only exposed .
Mechanical allodynia test
The up-and-down method with von Frey hairs was used to measure the mechanical allodynia in the hind paws of rats and mice . Briefly, animals were placed in separate plexiglass chambers positioned on a mesh table. The allodynia test was performed after 15 min of habituation. Starting with a dose of 2.04 g (rat) and 0.40 g (mouse), von Frey hairs of logarithmically incremental stiffness (rat: 0.41, 0.70, 1.20, 2.04, 3.63, 5.50, 8.51, 15.14 g; mouse: 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0 g) were applied bilaterally to hindpaws. Fifty percent paw withdrawal thresholds were recorded and the response to mechanical stimuli was evaluated.
Immunohistochemistry and immunofluorescence
As described previously , animals were anesthetized and perfused through the ascending aorta with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Following perfusion brains were removed and postfixed for 5 h, then dehydrated in 30% sucrose for 5 days. After that, brain tissue containing ACC (bregma +3.0 ~ +1.7mm) was coronally sliced (25 µm thickness) with a freezing microtome (LEICA CM3050S, Germany).
For immunohistochemistry, sections were first blocked with 5% donkey serum for 1 h at room temperature, then incubated with mouse anti-c-Fos antibody (1:200, Millipore) or rabbit anti-TNF-α antibody (1:100; Bioworld) for overnight at 4 °C. After three PBS washes, 10 min/each, the sections were incubated with a Cy3-conjugated goat anti-mouse (or anti-rabbit) secondary antibody (1:400; Jackson Immuno Research, USA) for 1 h at room temperature, and then washed with PBS.
For double immunofluorescence staining, the brain sections were incubated with a mixture of sodium channel voltage-gated type VIII alpha subunit antibody (anti-Nav1.6 antibody; 1:100; Alomone) plus either anti-TNF-α antibody (1:100; Bioworld), anti-IL-6 antibody (1:200; CST),, monoclonal neuronal-specific nuclear protein (mouse anti-NeuN; neuronal marker, 1:200; Chemicon, USA), glial fibrillary acidic protein (mouse anti-GFAP, astrocyte marker, 1:400; CST, USA) or goat anti-Iba1 (microglia marker, 1:200; Abcam) for overnight at 4 °C. Double-immunofluorescence staining of anti-IL-6 antibody with anti-NeuN， anti-GFAP or anti-Iba1 and of fractalkine chemokine domain antibody (anti-CX3CL1 antibody; 1:100; R&D systems) with anti-NeuN， anti-GFAP or anti-Iba1 in the brain sections were also made.After three rinses with PBS, 10 min/each, sections were incubated with fluorescein isothiocyanate (FITC)- and Cy3-conjugated secondary antibodies (1:400; Jackson Immuno Research, USA) for 1 h at room temperature, followed by PBS washes.
For imaging, sections were mounted on gelatin-coated slides and air-dried. Images were obtained using a fluorescence microscope attached to a CCD spot camera (LEICA DFC350FX/DMIRB, Germany) and processed with LEICA IM50 software (Germany). To verify specificity of the immunostaining and primary antibodies, negative control sections were processed in parallel without primary antibodies (data not shown).
Rats were euthanized at designated time points and tissue samples of ACC were quickly dissected from brain slices (bregma +3.0 ~ +0.5 mm) using an anatomical microscope (red area in Fig. 1B). Tissue samples were centrifuged at 12,000 rpm for 20 min at 4 ℃ and proteins were quantified. Proteins were separated by gel electrophoresis (SDS-PAGE) and electro-transferred to a PVDF membrane (Millipore). After blocking with 5% nonfat milk (containing Tris-phosphate buffer, 0.05% T-ween 20) for 1 h at room temperature, the membrane was incubated overnight at 4 ℃ with mouse anti-c-Fos antibody (1:200; Millipore), polyclonal rabbit TNF-α antibody (1:1000; Bioworld), anti-IL-6 antibody (1:1000; Abcam), anti-CX3CL1 antibody (1:1000; Abcam), anti-Nav1.6 antibody (1:200; Alomone), anti-CD11b antibody (1:1000; Bioss） or anti-Iba1 (1:1000; Abcam). β-actin was used (1:1000, Boster) as a loading control. The blots were washed three times with TBS-T for 10 min., and then incubated with HRP conjugated donkey anti-mouse, anti-goat or anti-rabbit secondary antibodies (1:10000; Abcam). The target protein bands were detected using enhanced chemiluminescence (Bio-Rad) and imaged using a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology). The protein level was quantified by densitometry using an imaging analysis system (KONTRON IBAS 2.0, Germany) and expressed relative to the level of β-actin.
Viral injections for the designer receptors exclusively activated by designer drugs (DREADD)
As described previously , mice were deeply anesthetized (1%-2% isoflurane) and then mounted in a stereotaxic frame with nonpuncturing ear bars. To selectively express hM4Di (Gi-coupled human M4 muscarinic receptor) or hM3Dq (Gq-coupled human M3 muscarinic receptor) in ACC excitatory pyramidal neurons, the pAOV-CaMKⅡa-hM4D(Gi)-mCherry-3Flag (hM4Di-mCherry) or pAAV-CaMKⅡa-hM3D(Gq)-mCherry (hM3Dq-mCherry) were injected into the contralateral (right) ACC of mice with or without SNI surgery, respectively. In parallel, all control mice received right injections of pAOV-CaMKⅡa-mCherry-3Flag or pAAV-CaMKⅡa-mCherry (mCherry). Viral particles (approximate titer 1.0E+12 GC/ml,Obio Technology Shanghai Corp., Ltd.) were administrated using a nanoinjector with injection micropipette (Nanoject Ⅱ Auto-Nanoliter Injector, DRUMMOND, USA) at the following coordinates: anteroposterior (AP) +1.0 mm, mediolateral (ML) 0.2 mm, dorsoventral (DV) -1.2 mm. A total volume of 500 nl was injected at the speed of 23 nl per second. After injection, incisions were stitched and mice were individually housed for 2 weeks before behavioral tests. At designated time points animals received i.p. injections of the ligand clozapine-N-oxide (CNO, Sigma-Aldrich) at a dose of 10 μl/g body weight. CNO working solution was first dissolved in dimethylsulfoxide (DMSO) and then diluted to a final concentration (5.0 mg/kg) with saline. The final concentration of DMSO was 0.5%.
Chemokines measurements with use of protein microarray
Rats were euthanized at designated time points and tissue samples of ACC were quickly dissected from brain slices (bregma + 3.0~ + 0.5 mm) using an anatomical microscope. Tissue was excised and homogenized in PBS with protease inhibitors. After homogenization, Triton X-100 was added to a final concentration of 1%. Tissue samples were centrifuged at 10,000 rpm for 5 min at 4 °C and proteins were quantified. Samples were assay immediately or stored in aliquotes at -70 ℃. Duplicate levels of 20 secreted proteins were determined using the Proteome Profiler mouse chemokine array kit according to manufacturer’s instructions (R&D systems, Proteome Profiler Rat XL Cytokine Array. Catalog Number: ARY030). Blots were imaged using enhanced chemiluminescence (Bio-Rad) and imaged using a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology). The band intensity of each blot was analyzed by densitometry using an imaging analysis system (KONTRON IBAS 2.0, Germany) and expressed relative to negative control spots or a clear area of the array.
Intra-ACC drug microinjection
As described previously , stereotaxic surgery was performed on anesthetized rats (10% chloralhydrate, 0.4 g/kg, i.p.) according to the rat brain atlas animals. A stainless-steel guide cannula with a stainless steel stylet plug was inserted into the ACC on the opposite side of the operation and secured with dental acrylic cement. The stereotaxic coordinate for ACC injection site from bregma was as follows: AP +2.0 mm, ML 0.5 mm, DV-2.5 mm. After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.
For immunohistochemistry, a density threshold above background level was first established to identify positively stained structures. For each animal, five slices were extracted from a series of consecutive ACC slices (four slice intervals) for statistical analysis. The immunofluorescence intensity per slice in the same Cg1 region of ACC (300 × 300 pixels) was measured and the mean ± SEM across animals was determined.
Changes in values over different groups were tested using one-way ANOVA followed by Dunnett's multiple comparisons test or using two-way ANOVA followed by Sidak's multiple comparisons test. For behavioral testing data, nonparametric two-way ANOVA followed by Friedman test was employed. In all cases, p< 0.05 was considered statistically significant.