Experiments were performed on adult (weighing 20–25 g) male C57BL/6 J mice. Animals were obtained from Experimental Animal Center of Zhejiang University and were on a 12:12 light–dark cycle with a controlled room temperature (23-24°C, 60-70% relative humidity), and received food and water ad libitum. All experiments were performed in according with the guidelines of the International Association for the Study of Pain  and were approved by the Animal Research Committee of Zhejiang University.
Morphine hydrochloride and 5-aza-2′-deoxycytidine (5-aza-dC) were purchased from Sigma (St. Louis, MO) were dissolved in physiological saline. Saline was used for control injections. Intraplantar injections (i.pl.) were given using a Hamilton microsyringe connected to polyethylene tubing with a 30-gauge hypodermic needle at the tip. The volume of injection was 20 μl in thermal paw withdrawal tests. The intrathecal injections (i.t.) were performed freehand between spinal L5 and L6 segments according to the method of Hylden and Wilcox . The exact placement of the drug substances was checked by a quick flicking motion of the mouse’s tail upon entry of needle. The Intracerebroventricular injections (i.c.v.) were carried out into the left lateral ventricle of mice. Injections were performed using a Hamilton microsyringe fitted with a 26-gauge i.c.v. needle according to the method of Haley and McCormick . The site of injection was 2 mm caudal and 2 mm lateral to the bregma and 3 mm in depth from the skull surface. Both i.t. and i.c.v. injections were given in a volume of 5 μl. The mice received the subcutaneous injections (s.c.) in a volume of 0.1 ml/10 g of body weight. All doses of drugs are based on the results of preliminary experiments.
Chronic constrictive injury model
Chronic constrictive injury (CCI) model was performed following the method of Bennett and Xie. In brief, mice were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal injection). Left sciatic nerve was exposed at midthigh level through a small incision, and a unilateral constriction injury just proximal to the trifurcation was performed with three loose ligatures using a 5–0 silk thread (spaced at a 1-mm interval). In sham-operated animals, the nerve was exposed but not ligated. The incision was closed in layers, and the wound was treated with antibiotics.
Measurement of thermal hyperalgesia
Thermal hyperalgesia was measured using the thermal-withdrawal latency (TWL) according to the method described previously . In brief, mice were placed in clear plastic chambers (7-9-11 cm) and allowed to acclimatize to the environment for 1 h before drug application. The heat source was focused on a portion of the hindpaw, which was flush against the glass, and a radiant thermal stimulus was delivered to that site. The nociceptive endpoints in the radiant heat test were the characteristic lifting or licking of the hind paw. The time to the endpoint was considered the TWL. The radiant heat intensity was adjusted to obtain basal TWL of 10–12 s. An automatic 20 s cutoff was used to prevent tissue damage. After 1 h of adaptation, morphine was injected s.c., i.t., i.c.v., or i.pl., and the thermal withdrawal latencies were measured at every 10-min interval until 60 min. AUC analgesic was measured by deducting the area under the time-response curve of saline (AUC-saline) from the area under the time-response curve of morphine (AUC-morphine). In some experiments, the paw withdrawal latencies at 10 or 30 min after drug administration were measured. The behavioral testing was performed by an investigator blinded to the treatment.
Real-time quantitative RT-PCR (qRT-PCR)
Under deep anesthesia (sodium pentobarbital, 60 mg/kg, intraperitoneal injection), L4-L5 DRGs and L4-L5 spinal cord segment was quickly removed. Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was then synthesized using a Thermo Scientific Verso cDNA synthesis kit (ABgene, Thermo Scientific) with oligo(dT) primer. Quantitative RT-PCR (qRT-PCR) was performed with a DyNAmo Flash SYBR Green qPCR kit (Thermo Scientific). The thermal cycle conditions used to assess the expression of MOR was: 95°C for 3 minutes; 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds and finally 30 min at 72°C with the primers: MOR (qRT-PCR), forward TCCTGGTCATGTATGTGATTGT A AGA and reverse CGTGCTAGTGGCTAAGGCATCT; GAPDH, forward TATGACTCCACTCACGGCAAAT and reverse GGGTCTCGCTCCTGGAAGAT. Relative mRNA levels were calculated using the 2 - ΔΔCT method.
Western blot analysis
Under deep anesthesia, the L4-L5 DRGs and L4-L5 spinal cord segment, sciatic nerves and plantar surface of hindpaw skin of mice were quickly extracted and stored in liquid nitrogen. Tissue samples were homogenized in lysis buffer (12.5 μl/mg tissue) containing a mixture of protease inhibitors (Roche) and PMSF (Sigma). After incubating in ice for 30 min, samples were centrifuged at 10,000 rpm for 15 min at 4°C. The supernatants were used for Western blotting. Equal amount of protein (~30 μg) was loaded and separated in 10% Tris-Tricine SDS-PAGE gel. The resolved proteins were transferred onto polyvinilidene difluoride (PVDF) membranes (Amersham Bioscience). The membranes were blocked in 5% non-fat milk for 1 h at room temperature (RT), and incubated overnight at 4°C with rabbit anti-MOR antibody (Neuromics, 1:1,000) or anti-GAPDH antibody (Cell Signaling Technology, 1:2000) primary antibody. The blots were then incubated with the secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (1:1000, Cell Signaling Technology), for 2 h at room temperature. Signals were finally visualized using enhanced chemiluminescence (ECL, Pierce) and the blots were exposed by The ChemiDoc™ XRS + image system (Bio-Rad; Hercules). All Western blot analysis was performed at least three times, and consistent results were obtained. Western blot densitometry analysis of signal intensity was performed using Quantity One 4.6.2 (Bio-Rad; Hercules) and levels of MOR from densitometry were normalized to GAPDH. The blot density from control groups was set as 100%.
Mice were deeply anesthetized and undergone sternotomy, intracardially perfuse with 20 ml saline followed by 4% ice-cold paraformaldehyde in 0.1 mol/l phosphate buffer (PB). L4-L5 DRGs, L4-L5 spinal cord segments, sciatic nerves, and plantar surface of hindpaw skin were removed, post-fixed in 4% paraformaldehyde for 3 h, and subsequently allowed to equilibrate in 30% sucrose in PB overnight at 4°C. DRG and transverse spinal cord sections (15 μm), sciatic nerve sections (10 μm) and skin sections (40 μm) were cut on a cryostat and every fifth section was collected in PB. After washing in phosphate buffer saline (PBS), the tissue sections were incubated in PBS containing 5% normal goat serum and 0.3% TritonX-100 at room temperature for 30 min. The sections were incubated overnight at 4°C with rabbit anti-MOR antibody (Neuromics, 1:500) followed by Rhodamine Red™-X goat anti-rabbit IgG (Invitrogen, 1:500) for 2 h at room temperature. Nonspecific staining was determined by excluding the primary antibodies. Sections were rinsed, mounted, and cover-slipped with glycerol containing 2.5% of anti-fading agent DABCO (Sigma) and stored at -20°C in the dark. Images were captured using a fluorescent microscope (DMIRB, Leica, Germany). The number of immunoreactive neuronal profiles was counted in a blinded fashion. Twenty sections were selected from four mice in each group. For DRG sections, Neuronal cell bodies were identified by the presence of a nucleus. The density threshold for positive staining was determined by averaging three cell bodies in each section that were judged to be minimally positive. All neurons for which the mean density exceeded the threshold were counted as positive, and the positive cells were expressed as a percentage of total counted neurons.
Genomic DNA from the DRG was isolated using the Wizard Genomic DNA Purification Kit (Qiagen) and linearized with the restriction enzyme EcoRV. Bisulfite treatment of DNA was carried out according to the manufacturer’s recommendations (EZ DNA Methylation-Gold Kit; Zymo Research). The resulting bisulfite-modified DNA was amplified by PCR. The PCR conditions were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 min, and finally 30 min at 72°C. After PCR amplification, the PCR products were purified using a gel extraction kit (QIAGEN) and cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer’s instructions. Twenty clones containing an insert of the correct size from each mouse were randomly chosen for DNA sequencing. The primers specific for methylated (methylation-specific PCR [MSP]) MOR DNA were designed as follows: MOR (PP), forward GTGGGTAAAGGATAATATTAATAATTTT and reverse CAACTTACAAAAACTAAAAAATCAAAAC; MOR (DP), forward GAGAAGAAGATTGAGGTAAAGTAGTA T and reverse CATACCAAATCTACTCTCCTAAACCTAC.
In vitro methylation of reporter plasmid and reporter gene assays
In vitro methylation of reporter plasmids was carried out as reported previously . Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs). Complete methylation was determined by digesting the DNA constructs with the methylation-sensitive restriction enzyme Bstu1, HpaI, HpaII (New England Biolabs) respectly and running the products on an agarose gel. Only DNA that was completely methylated was used. The construction of all luciferase fusion plasmids (pL450, pLup, and pL1.3 k) used in this study has been described previously . SH-SY5Y cells were plated 24 h prior to transfection at a density of 3×105 cells/well in six-well culture plates. Transfection was carried out using the effectene transfection reagent (QIAGEN) as described by the manufacturer. Cells were washed and lysed with lysis buffer (Promega) 48 h after transfection. To correct differences in transfection efficiency, a one-fifth molar ratio of a pCH110 plasmid (Amersham Biosciences) containing the β-galactosidase gene under the simian virus 40 promoter was included in the transfection to normalize values. The luciferase and galactosidase activities of each lysate were determined as described by the manufacturers (Promega and Tropix, respectively). Each normalized value represents the average of at least three independent determinations.
Data are expressed as mean±SEM. Statistical analysis between two samples was performed using Student t test. Statistical comparison of more than two groups was performed using one-way ANOVA followed by a Tukey post hoc test. The significance of any differences in thermal latency in behavior test was assessed using two-way ANOVA. “Time” was treated as “within subjects” factor and “treatment” was treated as a “between subjects” factor. The area under the pain threshold change versus time curve was calculated by GraphPAD Prism5 (Graph Pad Software, Inc., San Diego, CA) in some behavioral test. Statistical analyses of data were generated using GraphPAD Prism 5. All p values given are based on two-tailed tests. A value of p less than 0.05 was considered as statistically significant.