Animal model of bone cancer pain and drug delivery
Adult female Wistar rats (weighing 200–250 g) were purchased from the Institutional Center of Experiment Animals, and were housed in the standard lab conditions (22 ± 1°C and 12:12 h light cycle) with unrestricted free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee in China Academy of Chinese Medical Sciences, and were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgery was performed under appropriate anesthesia, and all efforts were made to minimize suffering in the animals.
The rat model of bone cancer pain was established by implantation of Walker 256 rat mammary gland carcinoma cells into the right tibias, as previously reported
. Briefly, suspensions of tumor cells in PBS was prepared as previously described
. Tumor cell implantation was achieved by injecting the tumor cells (1 × 105 cells/μl, 5 μL) into the intramedullary space of the right tibia to induce bone cancer in rats under the anesthesia with pentobarbital (45 mg/kg). Boiled cells were injected with a similar procedure in the control group. The investigators who performed behavioral tests, immunoblotting, and electrophysiological recording were blinded to this procedure.
Intrathecal cannulation was performed as described previously
. Intrathecal catheters (PE-10 polyethylene tubing) were implanted 1 week before any procedures. The catheters were advanced 8 cm caudally through an incision in the cisternal membrane and secured to the musculature at the incision site. Only animals with no evidence of neurological deficits after catheter insertion were studied. TrkB-Fc chimera (1.5 μg, R&D systems cat#: 688-TK-100)
, FSLLRY-NH2 (10 μg, Tocris cat#: 4751)
, pyrrolidine dithiocarbamate (PDTC, 1 μg, Sigma cat#: P8765)
, or vehicle was administered in a volume of 10 μl followed by a 10-μl flush with normal saline. The drugs were given daily from day 3 to day 15 after carcinoma cell inoculation.
Mechanical allodynia and thermal hyperalgesia were assessed in rats to evaluate painful behavior induced by tumor cell implantation. Mechanical sensitivity was assessed by using a series of von Frey filaments with logarithmic incremental stiffness (Stoelting Co., Wood Dale, IL), and 50% probability paw withdrawal thresholds were calculated with the up-down method as previously described
. In brief, the animals were placed individually beneath an inverted ventilated cage with a metal-mesh floor, and allowed to habituation for 30 minutes before testing. The filaments were applied to the plantar surface of the hindpaws for approximately 6 seconds in an ascending or descending order after a negative or positive withdrawal response, respectively. Six consecutive responses after the first change in response were used to calculate the paw withdrawal threshold (in grams). The maximum pressure was set as 16 g.
Thermal nociceptive thresholds were measured using radiant heat
. Briefly, rats were placed into individual plastic cubicles mounted on a glass surface maintained at 30°C and allowed an hour habituation period. A thermal stimulus (a radiant heat source) was then delivered to the plantar surface of the hindpaws. The time for the rat to remove the paw from the thermal stimulus was electronically recorded as the paw withdrawal latency (PWL). The stimulus shut off automatically when the hind paw moved or after 16 seconds to prevent tissue damage. The intensity of the heat stimulus was maintained constant throughout all experiments.
Protein extraction and immunoblotting
After behavioral testing, the rats were deeply anesthetized with pentobarbital sodium (50 mg/kg), and the ipsilateral L4-L5 segments were quickly removed and homogenized in the lysis buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM NaVO3, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin). The lysates were centrifuged at 14,000 rpm for 10 min at 4°C, and the protein concentrations in the supernatant were measured by using the Bio-Rad protein assay kit. The samples (containing 20 μg proteins) were separated on a 15% (for BDNF) or 7.5% (for other proteins) SDS-polyacrylamide gel, and blotted to a nitrocellulose membrane. The blots were incubated overnight at 4°C with a rabbit polyclonal anti-BDNF primary antibody (1:250; Santa Cruz Biotechnology cat#: sc-546), monoclonal anti-PAR2 antibody (1:1000; Millipore cat#: MABF243), polyclonal anti-NF-κB p65 (1:1000, Abcam cat#: ab7970), anti-phosphorylated NF-κB p65 (1:1000, Abcam cat#: ab106129), or monoclonal anti-β-actin antibody (1:2000; Santa Cruz Biotechnology, cat#: sc-81178). The membranes were washed extensively with Tris-buffered saline and incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibody (1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit; Amersham Biosciences). The intensity of the bands was captured digitally and analyzed quantitatively with Image J software. The immunoreactivities of target proteins were normalized to that of β-actin.
Spinal cord slice preparation and electrophysiological recording
The rats were deeply anesthetized with inhalation of halothane, and the lumbar segment of the spinal cord was removed through laminectomy. The spinal tissue was immediately placed in ice-cold artificial cerebrospinal fluid containing (in mM): sucrose 230, KCl 3.5, MgCl2 1.5, CaCl2 2.0, NaH2PO4 1.2, glucose 12, and NaHCO3 25. Transverse spinal cord slices (400 μm) were cut with a vibratome (Technical Products International, St. Louis, MO), and incubated in Krebs’ solution (containing: 117 mM NaCl, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 11 mM glucose, and 25 mM NaHCO3, bubbled with 95% O2 and 5% CO2) at 35°C for at least 1 h before the recording was performed.
Whole-cell recording on the spinal cord slices (L4-L5) was performed as described previously
. The neurons in lamina II of the ipsilateral dorsal horn were visualized using an upright microscope with infrared illumination (BX50WI; Olympus, Tokyo). Whole-cell voltage-clamp recordings were performed using an Axopatch 200B amplifier (Molecular Devices) with 3–5 MΩ glass electrodes containing the following internal solution (in mM): K-gluconate, 126; NaCl, 5; MgCl2 1.2; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; guanosine 5-O-(2-thiodiphosphate) 1; lidocaine N-ethyl bromide (QX314), 10; pH 7.3; 290 – 300 mOsmol. A seal resistance of ≥ 2 GΩ and an access resistance of 15 – 20 MΩ were considered acceptable. The series resistance was optimally compensated by ≥70% and constantly monitored throughout the experiments. The membrane potential was held at -60 mV throughout the experiment. Excitatory postsynaptic currents (EPSCs) in ipsilateral lamina II neurons were evoked by electrical stimulation (0.25 ms, 0.05 – 0.3 mA) of the dorsal root in the presence of strychnine (2 μM) and bicuculline (10 μM). The evoked EPSCs were filtered at 2 kHz, digitized at 10 kHz, and acquired and analyzed using pCLAMP 9.2 software (Molecular Devices).
All data were presented as means ± SEM. For analysis of immunoblotting data, differences between groups were compared by Student’s t-test or ANOVA followed by Fisher’s PLSD post hoc analysis. The electrophysiological and behavioral data were compared with two-way ANOVA with repeated measurements. The criterion for statistical significance was P < 0.05. Statistical tests were performed with SPSS 13.0 (SPSS, USA).