The Kim and Chung [9] method was used to create the neuropathic pain models by ligating the spinal nerves. Rats were anesthetized using pentobarbital sodium (50 mg/kg i.p.). A dorsal midline incision was made from L3 to S2. Under the microscopic guidance, the left L6 transverse process was partially resected to visualize the L5 and L6 spinal nerves. These nerves were tied up with 5-0 black silk. The wound was sutured again with a 4-0 soluble Vicryl suture, and the wound was closed using wound clips. Before the behavioral testing, the rats were housed in groups for 7 days. Among the animals, those exhibiting a foot withdrawal response to 8 von Frey filaments (0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g; Stoelting, USA) with an applied bending force of 4 g or less were considered neuropathic, and they were used in this study [10]. The rats were excluded from this experiment if they either showed the motor deficiency or did not exhibit subsequent mechanical allodynia. The mechanical withdrawal threshold test was performed for 1 week, daily after drug administration based on each group’s designation. Changes in the levels of pERK1/2, and pCREB signaling proteins and mRNA levels of proinflammatory cytokines determined using immunoblotting or reverse-transciption polymerase chain reaction (RT-PCR) techniques on tissues harvested from the spinal cord were compared among the three groups. All tests and analyses were performed by observers blinded to the assignment of animals in the treatment groups. All drug treatments were also blinded. The drug was administered intraperitoneally to neuropathic rats on the basis of their assigned treatment group of saline and amitriptyline (10 mg/kg). In the NP + Ami + MRS group, amitriptyline was administered immediately after the subcutaneous injection of MRS-1191 (1 mg/kg) 7 days after SNL [5,7,11]. The doses of amitriptyline and MRS-1191 were determined on the basis of pilot experiments conducted in our laboratory, in which the doses selected in the current study were found to have no effect on normal and pain behaviors. After an initial observation period, the rats were housed with their cage mates in an enriched environment during the experiment. Experiments for evaluating static tactile mechanical thresholds were carried out by examiners blinded to the assigned treatment groups.

After assessing the static tactile mechanical thresholds for 7 days, by using oxygen and 5% sevoflurane, the rats were anesthetized. The third lumbar vertebra and the sacrum were cut almost simultaneously, and the spinal cord from the lumbar enlargement was harvested by rapidly injecting saline into the spinal canals and pushing the spinal cord toward the lumbar vertebra. The harvested spinal cord tissues were stored at −80°C after quick-freezing with liquid nitrogen. Spinal cord tissue was dissected on ice and immediately placed in ice-cold radioimmunoprecipitation assay buffer (150 mmol NaCl, 50 mmol Tris-HCl, 1 mmol EDTA, and 1% Triton-X100 [pH 7.4]) and homogenized for 10 s on ice. Tissue was super-centrifuged (32,500 g, at 4°C, and in a vacuum) for 1 h. For the immunoblotting described above, the supernatant was collected. Bovine serum albumin was used as a standard solution and analyzed using the Bradford assay (BioRad, USA), and the sample was stabilized using the Laemmli buffer. The protein bands, obtained by electrophoresing each protein in 10% sodium dodecyl sulfate polyacrylamide gel for 4 h at 80 V, were transferred to a polyvinylidene fluoride membrane. The membranes were washed with Tris-Buffered Saline Tween-20 (TBST) solution (0.1% Tween 20 in 1 × TBS) and coated with a blocking solution (5% dry milk in TBST). The membranes were incubated with primary phospho-ERK1/2 (pERK1/2) and phospho-CREB (pCREB) antibodies (Cell Signaling Technology, USA) for 16 h. After washing with TBST solution, the membranes were incubated with the secondary antibody (goat anti-rabbit or anti-mouse IgG, Santa Cruz Biotechnology, USA) for 1 h, and developed using electrochemiluminescence reagents (Amersham, USA) and a UVP Bio-imaging System (Upland, USA).

The inflammation in the harvested spinal cord was also determined by measuring mRNA encoding markers of inflammation, including tumor necrosis factor α (TNF-α), intercellular adhesion molecule 1 (ICAM-1), macrophage inflammatory protein 2 (MIP-2), and monocyte chemoattractant protein 1 (MCP-1) [11]. Primers were designed on the basis of published Gen-Bank sequences (Table 1). A 1-ml aliquot of Trizol reagent (MRC, USA) was added to the spinal cord tissues of the rats, which were then homogenized and left for 5 min. A 0.2-ml aliquot of 1-bromo-3-chloropropane (Sigma, USA) was added, followed by centrifugation at 14,000 g for 15 min. The supernatant was then separated and centrifuged at 14,000 g for 10 min after adding the same amount of phenol:chloroform (5:1, pH 4.7) (Sigma, USA). After separating the supernatant, 100% ethanol was added and reacted at −20°C for 1 h, followed by centrifugation at 14,000 g for 10 min. The supernatant was discarded, and the RNA precipitate was washed with 75% ethanol. Diethylpyrocarbonate was added and reacted at 60°C for 5 min and stored in a refrigerator (−70°C). The RNA concentration was determined using a UVP Bio-imaging System (Upland, USA) at an absorbance of 260 nm. cDNA (50 μl) was constructed using the extracted RNA (0.5 μg) as a template and using avian marrow astrovirus reverse transcriptase (Promega, USA). The PCR was performed by generating 10 μl of the final reaction solution containing dNTPs (2.5 mM/L each) and 1 unit I Start Taq DNA polymerase (Intron, Korea), and each primer of 20 μM/L using composed cDNA of 2 μl. The PCR included 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension for 1 min at 72°C, and the reaction was terminated at 72°C for 10 min [12]. A 228-bp region of the β-actin gene was used as an internal control to determine whether appropriate mRNA was separated from the cells. After electrophoresis on 2.0% agarose gel and staining with ethidium bromide, the obtained PCR products were quantified using Gel Doc^TM XR (BioRad, USA).

Data are expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., USA). Data were analyzed using one-way ANOVA, Dunnett’s test, and Kruskal-Wallis nonparametric test for comparing of the three groups. A P value < 0.050 was considered statistically significant.