Animals and pain models
C57Bl/6 background WT mice were purchased and bred in the Animal Facility of the Fourth Military Medical University. Young mice (4-6 weeks old, C57Bl/6) were used for electrophysiological studies in spinal cord slices. All the animal procedures were approved by the Animal Care Committee of the Fourth Military Medical university.
Intrathecal administration
C57BL/6 mice were anaesthetized by 1% pentobarbital sodium. A midline incision was made along L2 to L4 vertebral plate and the muscle attached to spinous process removed[17]. With the tip of the sharp scissor, a 1-mm hole on the left vertebra was made until dura and clean CSF was exposed. An intrathecal catheter (polyethylene-10 tubing) was inserted from L3 and passed rostrally into the subarachnoid space until it reached L1/T13. After a flush with 10 μl saline, the exterior end of catheter was sealed by heat. Penicillin antibiotics were used to prevent infection at the end of intrathecal catheterization. The mice were allowed to recover for 3 days. Any mouse showing motor deficits would be excluded. CCL2(5 μl) diluted by normal saline was intrathecally applied 3 day after intrathecal catheter implantation.
DRG injection of RS504393
C57Bl/6 mice were anaesthetized with 2 % isoflurane. The procedure for intervertebral foramen injection of RS504393 was the same as described previously[18]. In brief, the left sciatic nerve was exposed at midthigh level and RS504393 (20 μg) or vehicle was injected into the DRG using a pulled micropipette. RS504393 was suspended in a 1% DMSO/saline mix. The bilateral iliac spines were exposed to locate the L3 and L4 vertebrae of mice. The 26-gauge needle mated to a Hamilton syringe (Hamilton, Reno, NV) was inserted at a 45° angle at the intersection of the lower edge of the ipsilateral L3 and L4 vertebrae. There was a sense of restriction when the needle entered the transverse foramen, and the paw retraction reaction of mice was the sign of the needle entering the transverse foramen.
Behavioral analysis
Animals were habituated in the testing environment for at least 2 d before baseline testing. Thermal hyperalgesia and mechanical allodynia were tested as previously described[14, 19]. The experimenters were blinded to treatments.
Spinal cord slice preparation and patch-clamp recordings
As we previously reported[14], the Krebs’ solution contains (in mM): 127 NaCl, 3.6 KCl, 2.4 CaCl2, 1.3 MgCl2, 1.2 NaH2PO4, 26 NaHCO3, and 15 glucose. After establishing the whole-cell configuration, neurons were held at the potential of +40 mV to record NMDAR evoked EPSC (NMDAR-eEPSCs) by stimulating the dorsal root entry zone via a concentric bipolar electrode using an isolated current stimulator[19]. The internal solution contains (in mM): 110 Cs2SO4, 2 KCl, 0.1 CaCl2, 2 MgCl2, 1.1 EGTA, 10 HEPES, 5 ATP-Mg. QX-314 (5 mmol/L) was added to the pipette solution to prevent discharge of action potentials. Signals were filtered at 2 kHz and digitized at 5 kHz. Data were stored and analyzed with a personal computer using pCLAMP10 software (Molecular Devices).
Immunofluorescence labelling
Immunohistochemistry was performed according to standard protocols, and the following primary antibodies were used: Isolectin B4 antibody (Biotinylated GRIFFONIA, 1:200, vector laboratories, B-1205), CGRP antibody (Goat, 1:300, Abcam, ab36001), NF200 antibody (mouse, 1:200, Sigma-Aldrich, N5389), CCR2 antibody (rabbit, 1:300, NOVOUS, NBP1-48337), Alexa Fluor® 594 (donkey anti-rabbit IgG, 1:1000, Abcam, ab150132), Alexa Fluor® 488 (donkey anti-mouse IgG, 1:1000, Abcam, ab150105; donkey anti-goat IgG, 1:1000, Abcam, ab150129). Alexa Fluor 488-conjugated streptavidin to visualize the riboprobes (1:1000, Invitrogen, E13345).
Drugs and administration
The mitogen-activated protein kinase (MEK1) inhibitor, PD98059 (Cell Signaling technology, EMD Millipore, Billerica, MA, USA); CCR2 antagonist, RS504393 (Tocris Bioscience, Bristol, UK); CCL2 (R & D Systems, Minneapolis, MN, USA) were used in this study.
Injection of AAV virions in DRG in vivo
Virus injection in DRG was performed as described previously[20]. Briefly, mice were anesthetized with isoflurane and L3/L4 DRGs exposed by removal of the lateral processes of the vertebrae. The epineurium over the DRG was opened, and the glass pipette with fine tip was inserted into the ganglion, to a depth of 100-150 mm from the surface of the exposed ganglion. After waiting 2 min to allow sealing of the tissue around the pipette tip, 700 nl of virus solution was injected at a rate of 0.1 ml/min using microprocessor-controlled minipump (RWD). The pipette was removed after a further delay of 5 min. The muscles overlying the spinal cord was carefully sutured and mice allowed to recover at 37°C warming blanket. Mice were allowed to recover for 4 weeks before commencing various tests.
Calcium imaging with GCaMP6s in presynaptic terminals of nociceptors
As described above, rAAV-Ef1a-DIO-GCaMP6s-WPRE-pA was injected into L3/L4 DRGs of SNS-Cre mice, a mouse line expressing Cre recombinase under control of the Nav1.8 promoter. Four weeks after virus expression, transverse 350-450 mm- thick spinal cord slices with dorsal roots attached were obtained. GCaMP6s signal in the presynaptic terminal of nociceptors in the superficial spinal dorsal horn was visualized using an upright super-resolution Olympus FV1200 confocal microscope (Olympus, Japan). Images were acquired at 1 Hz. Fluorescence intensity of each puncta was measured. Bath application of CCL2 (100ng/ml) was used to activate the Ca2+ signal in the presynaptic terminals of nociceptors.
Fluorescence imaging with iGluSnFR in presynaptic terminals of nociceptors
rAAV-EF1a-DIO-iGluSnFR(A184S)-WPRE-hGH-pA was injected into L3/L4 DRGs of SNS-Cre mice as described above. 4 weeks after virus expression, transverse 350-450 mm thick spinal cord slices with dorsal roots attached were obtained. iGluSnFR signal in the presynaptic terminal of nociceptors in the superficial spinal dorsal horn was visualized using an upright super-resolution Olympus FV1200 confocal microscope (Olympus, Japan). Images were acquired at 1 Hz. Fluorescence intensity of each puncta was measured. Bath application of CCL2 (100ng/ml) was used to activate the fluorescence signal intensity in the presynaptic terminals of nociceptors.
Pre-embedding immunogold-silver cytochemistry
The protocol was adapted from the previous investigation[21]. Briefly, mice were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M PB. The spinal cords were removed and postfixed in the same fixative for 4 h at 4 °C. Serial coronal sections at 100 μm thicknesses (VS1000s, Leica, Heidelberger, Germany). The 0.05% Triton X-100 was used for CCR2 immunogold-silver cytochemistry. After rinsing, the sections were postfixed in 2% glutaraldehyde in PBS for 45 min. Signals of CCR2 immunoreactivity were detected by silver enhancement kit in the dark (HQ Silver Kit, Nanoprobes). Sections were postfixed in 0.5% osmium tetroxide in 0.1 M PB for 2h. They were dehydrated with graded ethanol, replaced with propylene oxide, and finally embedded in Epon 812 between plastic sheets. Flat-embedded sections were examined under the light microscope. The sections containing CCR2 immunoreactivity in spinal dorsal horn were cut with a diamond knife (Diatome, Hatfield, PA). After counterstaining with uranyl acetate, ultrathin sections were examined under the JEM-1230 electron microscope (JEOL LTD, Tokyo, Japan). Postsynaptic and presynaptic membrane, synaptic vesicles were measured using ImageJ (NIH) by observers blinded to the genotype of the samples.
Statistical analysis
Differences between groups were compared using 1-way ANOVA or 2-way repeated measures ANOVA followed by Bonferroni’s test or by Student’s t test (2-tailed) if only 2 groups were applied. The criterion for statistical significance was P < 0.05.