Male Sprague-Dawley rats weighing 200-250 g (Kyudo Co., Saga, Japan) were used in the present study. Animals were housed in groups of four to five per cage, with lights on from 7:00 to 19:00 h. Animals had free access to food and water in their home cages. All experiments were approved by the Experimental Animal Care and Use Committee of Kyushu University according to the National Institutes of Health guidelines, and we followed International Association for the Study of Pain (IASP) Committee for Research and Ethical Issues guidelines for animal research .
Oxaliplatin (Elplat®) was obtained from Yakult Co., Ltd. (Tokyo, Japan). KN-93, Ro 25-6981 hydrochloride hydrate and trifluoperazine dihydrochloride were purchased from Sigma-Aldrich (Missouri, USA). KN-92 was purchased from Calbiochem (California, USA). Oxaliplatin was dissolved in 5% glucose solution. The vehicle-treated rats were injected with 5% glucose solution. KN-93, KN-92 and Ro 25-6981 were dissolved in 100% dimethyl sulfoxide (DMSO). Trifluoperazine was dissolved in distilled water. The doses of these drugs were chosen based on previous reports [2, 3, 7].
Production of neuropathy
Mechanical allodynia and cold hyperalgesia were induced according to the method described previously . Oxaliplatin (4 mg/kg) or vehicle (5% glucose solution) was administered i.p. twice a week for 4 weeks (on days 1, 2, 8, 9, 15, 16, 22 and 23). The volume of vehicle or drug solution injected was 1 mL/kg for all drugs.
Behavioral test was performed blindly with respect to drug administration.
von Frey test for mechanical allodynia
The mechanical allodynia was assessed by von Frey test. Each rat was placed in a clear plastic box (20 × 17 × 13 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. von Frey filaments (The Touch Test Sensory Evaluator Set; Linton Instrumentation, Norfolk, UK) ranging from 1- to 15-g bending force were applied to the midplantar skin of each hind paw six times, with each application held for 6 s. The paw withdrawal threshold was determined by a modified up-down method .
Acetone test for cold hyperalgesia
The cold hyperalgesia was assessed by acetone test. Each rat was placed in a clear plastic box (20 × 17 × 13 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. Fifty microliters of acetone (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was sprayed onto the plantar skin of each hind paw 3 times, and the number of withdrawal responses was counted for 40 s from the start of the acetone spray.
Rota-rod test for motor coordination
The rota-rod test was performed to investigate the change of motor coordination. Rats were placed on a rotating rod (Muromachi Kikai Co., Ltd., Tokyo, Japan) and the latency to falling was measured for up to 2 min according to the method described previously . The test was performed three times, and the rotating speed was 10 rpm.
Effects of KN-93, KN-92 and trifluoperazine on Oxaliplatin-induced mechanical allodynia
We confirmed the incidence of mechanical allodynia in the von Frey test on day 24. We carried out the drug evaluation on the next day. KN-93 (10-50 nmol) or KN-92 (50 nmol) was administered i.t. injection by direct lumbar puncture in a volume of 50 µL. The von Frey test was performed immediately before (0 min) and at 30, 60, 90 and 120 min after administration of the drugs. Trifluoperazine (0.05-0.3 mg/kg) was administered p.o. The von Frey test was performed immediately before (0 min) and at 30, 120 and 240 min after oral administration of trifluoperazine.
Effect of KN-93 on Oxaliplatin-induced cold hyperalgesia
We confirmed the incidence of cold hyperalgesia in the acetone test on day 5. KN-93 (10-50 nmol) was administered i.t. injection by direct lumbar puncture in a volume of 50 µL. The acetone test was performed immediately before (0 min) and at 30, 60, 90 and 120 min after administration of the drug.
Effect of trifluoperazine on mechanical nociceptive threshold
We investigated the effect of trifluoperazine on the mechanical nociceptive threshold in the von Frey test. Trifluoperazine (0.3 mg/kg) was administered p.o. in intact rats. The von Frey test was performed immediately before (0 min) and at 30, 120 and 240 min after oral administration of trifluoperazine.
Effect of trifluoperazine on motor coordination
We investigated the effect of trifluoperazine on the motor coordination in the rota-rod test. Trifluoperazine (0.3 mg/kg) was administered p.o. in intact and oxaliplatin-treated rats. The rota-rod test was performed immediately before (0 min) and at 30 min after oral administration of trifluoperazine.
Western blotting analysis
The lumbar sections (L4-6) of the spinal cord were quickly removed at 30 min after administration of KN-93 (50 nmol, i.t.), Ro 25-6981 (300 nmol, i.t.) or trifluoperazine (0.3 mg/kg, p.o.) on day 25. The tissues were homogenized in a solubilization buffer containing 20 mM Tris-HCl (pH 7.4, 2 mM EDTA, 0.5 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 0.32 M Sucrose, 2 mg/ml aprotinine, 2 mg/ml leupeptin), and the homogenates were subjected to 12.5% SDS-PAGE, and proteins were transferred electrophoretically to PVDF membranes. The membranes were blocked in Tris-buffered saline Tween-20 (TBST) containing 5% BSA (Sigma-Aldrich) for an additional 1 h at room temperature with agitation. The membrane was incubated overnight at 4°C with mouse polyclonal anti-CaMKIIα antibody or rabbit polyclonal anti-(Thr286)pCaMKII (1:5000; Santa Cruz Biotechnology, California, USA) and then incubated for 1 h with corresponding horseradish peroxidase conjugate secondary antibodies (1:5000; Jackson Immuno Research Laboratories, Inc., PA, USA). The immunoreactivity was detected using Enhanced Chemiluminescence (Perkin Elmer, Massachusetts, USA). Ratios of the optical densities of pCaMKII to those of CaMKII were calculated for each sample.
Values were expressed as the means ± SEM. The values were analyzed by the Student's t-test or one-way analysis of variance (ANOVA) followed by the Tukey-Kramer post-hoc test (StatView; Abacus Concepts, Berkely, CA, USA) to determine differences among the groups. A p value of less than 0.05 is considered as statistically significant.