Activation of c-jun N-terminal kinase in spinal cord contributes to breast cancer induced bone pain in rats

pJNK1 and pJNK2 protein levels were detected on the ipsilateral side of L4-L5 spinal cord. We examined the expression of pJNK1/2 in either CIBP (Figure 1A) or a PBS control group (Figure 1B) at different time points after surgery. pJNK1/2 (46 kD, 52 kD) and GAPDH (36 kD) were detected in the same membrane. The levels of pJNK1/2 were not changed compared to the naïve group on day 5, day 12 or day 16 (Figure 1B, D) after the injection of PBS as a sham control. Compared to naïve rats, the pJNK1/2 protein levels were increased on the ipsilateral side of the spinal cord on day 12 and day 16 after intra-tibial inoculation with carcinoma cells (Figure 1C).

The number of pJNK positive cells was also increased by single-stained immunofluorescence on day 12 and day 16 after inoculation with carcinoma cells (Figure 2A-D). We then determined the cellular localization of pJNK1/2 in naïve and model animals (Figure 2E-M). Double immunofluorescence results showed that a small number of pJNK1/2-IR cells were double labeled with NeuN, CD11b and GFAP, indicating that pJNK1/2 was expressed in neurons, microglia and astrocytes in naïve rats (Figure 2E, H, K). A significant increase in the number of pJNK1/2-IR neurons and astrocytes was found on day 12 and day 16 in ipsilateral spinal cord after intra-tibial inoculation with carcinoma cells as compared to the naïve condition, but the number of pJNK1/2-IR microglia was not changed at any time point after intra-tibial inoculation with carcinoma cells (Figure 2N).

The CIBP rats displayed significant decreases in mechanical thresholds on day 5, day 12 and day 16 after intra-tibial inoculation with carcinoma cells as compared to naïve rats or sham control rats injected with intra-tibial PBS (Figure 3A). We sought to assess whether the activation of JNK contributed to the mechanical allodynia induced by intra-tibial inoculation with carcinoma cells. A single intrathecal injection of SP600125, which respectively inhibited JNK phosphorylation, induced an increase in paw withdrawal thresholds at 1 h; this effect lasted for 6 h (Figure 3B). Furthermore, the CIBP rats received a repeated daily intrathecal injection of SP600125 from day 10 to 14 after intra-tibial inoculation with carcinoma cells. After 3 intrathecal injections of SP600125, the analgesic effect of SP600125 was observed to last for 12 h, while there was no analgesic effect of SP600125 on 12 h after a single injection (Figure 3C). After 5 daily intrathecal injections of SP600125, the analgesic effect of SP600125 was observed to last for 24 h (Figure 3D). Intrathecal injection of 30% DMSO had no effect on mechanical allodynia at any time point throughout the experiment.

Adult female Wistar rats weighing 160–200 g were used in all experiments. All animals were kept under controlled conditions (a temperature-controlled room (22 ± 0.5°C), a 12:12 h light cycle (07:00–19:00 h light), and with unrestricted free access to food and water). All animal experiments followed the guidelines of the International Association for the Study of Pain (IASP) [38]. Efforts were made to reduce the number of animals used in the experiment.

Walker 256 rat mammary gland carcinoma cells were used in the experiment. Suspensions of 1 × 10⁸/ml tumor cells in PBS were prepared as previously described [13, 16, 17, 39]. After the animals were anesthetized with sodium pentobarbital (i.p. 50 mg/kg), 4 × 10⁵ cells in 4 μl 0.01MPBS were injected into the right tibias of female Wistar rats. Briefly, the Walker 256 carcinoma cells were obtained from an ascetic tumor-bearing rat, washed with PBS 3 times, and then diluted to 1 × 10⁸/ml during the last wash. Bilateral superficial incisions were made in the skin overlying the patella after disinfection with 70% v/v ethanol in order to expose the tibia head with minimal damage. After Walker 256 carcinoma cells were prepared, 4 μl cells followed by 4 μl of absorbable gelatin sponge dissolved in saline were slowly injected into the right tibia cavity of each rat using a 10-μl microinjection syringe. The syringe was left in place for an additional 2 min to prevent the carcinoma cells from leaking out along the injection track. The injection site was closed using bone wax while the syringe was removed to prevent tumor cells overflow. The sham group rats were treated in the same way and injected with 4 μl PBS instead of tumor cells.

The JNK inhibitor SP600125 (1,9-Pyrazoloanthrone) was purchased from Calbiochem (San Diego, California, USA). SP600125 stock solution was prepared in DMSO at a concentration 20 μg/μl and stored at -20°C until use. The concentration used for the study was 1 μg/μl, which was freshly prepared with a final DMSO concentration of 30%. Ten μg were used in the experiment, and the control group was treated with the same amount of DMSO. The dose of drug used in the experiment was chosen based on the previous research [7, 8]. Rats were anesthetized with 2% isoflurane. After the lumbar region was shaved and sterilized with 75% ethanol, animals were given a lumbar puncture at the L5-6 interspace using a 0.5-inch, 30-gauge needle. Then the drug was delivered to the CSF through the needle [40]. SP600125 was given once on day 12; for testing the addictive effect of SP600125, the drug was given daily from day 10 to day 14 after carcinoma cell inoculation.

The spinal cord segments were removed and immediately placed in liquid nitrogen to freeze quickly. The ipsilateral L4-L5 segments were quickly removed and homogenized in an SDS sample buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 1 mM each, PMSF, NaF, NaVO₃, 1 μg/ml each, leupeptin, pepstatin, aprotinin), followed by centrifugation at 12000 g for 20 min. The protein concentration of the supernatant was determined by BCA Protein Assay Kit (Pierce, Rockford, USA). Thirty μg protein was boiled for 3 min at 100°C with an appropriate volume of 5× SDS-PAGE sample loading buffer (250 mM Tris–HCl, pH 6.8, 5% 2-mercaptoethanol, 10% SDS, 0.5% Bromophenol Blue, 50% glycerol). Samples were loaded into each lane of a 10% SDS-PAGE gel. The membrane was blocked by 5% bovine serum albumin in TBS-T (50 mM Tris–HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20) at 4°C overnight. Primary and secondary antibodies were also diluted in blocking solution at room temperature for 3 h. Blots were developed in ECL (Pierce, Rockford, US) solution for 3 min and exposed onto Kodak X-OMAT AR Film (Eastman Kodak, Rochester, US) for 3 min. The antibodies used were rabbit anti-phosphorylation SAPK/JNK (Thr183/Tyr185) antibody (1:1000, Cell signaling Technology, Massachusetts, US), HRP-anti-rabbit antibody(1:1000, Santa Cruz, California, US)and mouse HRP-anti-GAPDH (1:5000, Santa Cruz, California, US), which was used as a loading control in all Western blots. Densitometry analysis of pJNK1/2 bands and GAPDH bands were performed using Syngene software (GeneGnome, Syngene, Maryland, US). The same size square was drawn around each band to measure the density and subtract the background near that band. pJNK1/2 levels were normalized against GAPDH levels and expressed as fold increase, compared to the naive condition.

Four rats from each group were used in the experiment. The L4–L5 spinal segments were removed, post-fixed, frozen and cut on a freezing microtome (Leica 2000, Germany) at 30-μm thickness. The sections were washed three times and blocked with 4% donkey serum in 0.3% Triton X-100 for 1 h at 37°C and then incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 h. The primary antibodies used were rabbit anti-phosphorylation SAPK/JNK (Thr183/Tyr185) (1:400, Cell signaling Technology, Massachusetts, US), mouse anti-NeuN (neuronal marker, 1:1000, Chemicon, California, US), mouse anti-GFAP (astrocyte marker, 1:1000, Sigma-Aldrich, Missouri, US) and mouse anti-CD11b (microglia marker, 1:400, Chemicon, California, US). The secondary antibodies used were Cy3-conjugated affinity purified goat anti-mouse (1:1000, Millipore, CA) and Alexa Fluor 488-labeled donkey anti-rabbit (1:200, Invitrogen, Eugene, Oregon, USA). The stained sections were examined with a Leica fluorescence microscope. The number of pJNK-IR cells was counted in lamina I-II and lamina III-IV of the ipsilateral spinal dorsal horn that captured by using a computerized image analysis system (Leica Qwin 500). The specificity for pJNK antibody we used was confirmed by the lack of staining in the absence of primary antibody, and also specific bands on the membrane in Western blots. Based on the intensity of the staining, a threshold was chosen from the spinal cord of naïve animal to judge the signal was true or false. A signal below the threshold was considered as false positive. The backgrounds of the cell free area nearby the positive pJNK-IR and the depth lamina were subtracted. The number of pJNK-IR cells was recorded after removing the repeated count. For counting the double staining, the pJNK-IR neurons were determined by the distinct morpho-logy from glia cells and the colocalization with NeuN. The pJNK-IR glia cells were determined by the morphology and the colocalization with CD11b or GFAP. At least 4 rats from each group and each time point were analyzed. A minimum of 6 sections randomly selected from each rat were used in the experiment.

Eight rats in each group were used in the experiment. The day of carcinoma cell inoculation was referred to as day 0. Mechanical allodynia was assessed using a von Frey hair filament (Stoelting, Wood Dale, Illinois, US) as previously described [16]. An ascending series of von Frey filaments with logarithmically incremental stiffness (0.40, 0.60, 1.4, 2.0, 4.0, 6.0, 8.0 and 15.0 g) were used in the experiment. The test began with the application of the 2.0 g von Frey filament. Each plantar surface of the hind paws was stimulated individually in the experiment. Each von Frey hair was held about 1–2 s, the positive response was defined as a withdrawal of hind paw or licking. We used a lower hair when the positive response was appeared, otherwise used the higher hair. After five more stimuli counted from the first change, a score was record. The final score was gotten by using the method described by Dixon which converted to a 50% von Frey threshold. Animals were habituated to the environment daily for at least 2 days before baseline testing. To test the paw withdrawal thresholds, animals were put into the experimental environment for 30 min before stimulation. The pre-drug baseline was assessed 1 h before intrathecal injection. All of the tests were performed with researchers blinded with respect to the drugs injected.

All data were presented as mean ± standard error of the mean (SEM). The statistical significance of differences between groups was analyzed with one-way ANOVA following the Bonferroni post-test or with Student’s t-test. p < 0.05 was set as the threshold of significance.