2.1. Animals
All experimental procedures and protocols used in this study were approved by the Animal Use and Care Committee of Hubei University of Medicine (Hubei, China) and were conducted in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals and with the ethics guidelines of the International Association for the Study of Pain. Adult female Sprague-Dawley (SD) rats weighing 180–220 g and newborn rats within 24 h were provided by the Institute of Laboratory Animal Science, Hubei University of Medicine (Hubei, China). Rats were housed at 22 ± 2°C with a 12-h light-dark cycle and free access to food and water.
2.2. Preparation of Walker 256 carcinoma cells
We injected 0.5–1 mL (3 × 107 cells/mL) of Walker 256 rat mammary gland carcinoma cells into the abdominal cavity of a female SD rat weighing 80–100 g. Cells were harvested by differential centrifugation from ascitic fluid 6–7 days later, diluted to achieve a final concentration of 106 cells/mL and kept on ice until surgery. For the sham group, Walker 256 cells were prepared in the same way but were boiled for 20 min prior to injection.
2.3. BCP model establishment
The rat BCP model was established by injection of Walker 256 rat mammary gland carcinoma cells into the bone marrow cavity of the right tibia of SD rats according to a previous report [15].
2.4. Lentivirus construction and microinjection
A lentiviral vector containing green fluorescent protein (gcGFP) was employed in this study to stably overexpress NL1 or NL3. To this end, the vector GV492 (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin; Shanghai GeneChem Co. LTD, Shanghai, China) was recombined with the gene sequences of NL1 (NM_053868; NL1-LV) or NL3 (NM_134336; NL3-LV). The same vector framework, carrying gcGFP without the target gene sequences, was used as a negative lentivirus control (NC1-LV or NC3-LV). The lentiviral vector GV320 (Ubi-MCS-3FLAG-SV40-Cherry; Shanghai GeneChem Co. LTD, Shanghai, China) containing Cherry was recombined with the NL2 gene sequence (NM_053512; NL2-LV) to stably overexpress this NL. The same vector backbone carrying Cherry only was used as negative control (NC2-LV). The viral titers of NL1-LV, NL2-LV, and NL3-LV were 1.0 × 109 TU/mL, 1.0 × 109 TU/mL, and 2.0 × 109 TU/mL, respectively. Following carcinoma implantation, the L4−L6 lumbar spinal cord was exposed carefully by laminectomy. A glass capillary (35 ± 5 mm diameter) containing a viral suspension (5 µM) was injected into the right spinal dorsal horn. The injection point was at 0.4 mm from the midline in the right dorsal horn, and the glass capillary was lowered into the parenchyma to 0.5 mm below the pia mater to reach the dorsal horn. Finally, the incision was dusted with penicillin powder and the skin was sutured.
2.5. Pain behavior assessment
Mechanical allodynia of rats was assessed by means of the paw withdrawal threshold (PWT) using a dynamic plantar esthesiometer (Ugo Basile, Stoelting, IL, USA) on days 0 (before surgery), 3, 6, 9, 12, 15, 18, and 21. The measurements were conducted from 8:30 to11:30 a.m. Rats were placed in separate transparent boxes on an elevated wire mesh platform. After 20 min of acclimation, mechanical allodynia was measured using the hind paw withdrawal response to von Frey filament stimulation. The filament (0.5 mm diameter) was placed beneath the hind paw until it touched the plantar surface of the paw and began to exert an upward force. The forces were recorded in grams when the withdrawal response was elicited, up to a maximum of 50 g. Each rat was assessed at least three times at intervals of 3 min.
2.6. Real-time polymerase chain reaction analysis
RNA isolation, cDNA synthesis, and real-time polymerase chain reaction (RT-PCR) were performed as previously described [16]. The gene names and forward (F) and reverse (R) primers are listed in Supplementary Table 1.
2.7. Protein extraction and Western blot analysis
Proteins from rat L4 to L6 lumbar spinal cord were extracted using radioimmune precipitation lysis buffer (CST, America). Total protein concentrations were assayed using a bicinchoninic acid protein assay kit (N3219; TIAN GEN, China). Samples were calibrated to 30 µg/µL with loading buffer solution and phosphate-buffered saline (PBS) and heated for 10 min at 95°C. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% skim milk in tris-buffered saline (TBS) containing 0.1% Tween (TBST) for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. After three washes in TBST, they were incubated with horseradish peroxidase-linked secondary antibodies for 40 min at room temperature. Finally, the membranes were washed in TBST three times, processed using the ECL Plus kit and exposed to MP-ECL film.
2.8. Immunohistochemistry
For immunohistochemical (IHC) staining, rats were deeply anesthetized with 3% isoflurane and transcardially perfused with cold PBS followed by 4% paraformaldehyde (PFA). After perfusion, the lumbar (L4−L6) spinal cord was harvested, post-fixed in 4% PFA at 4°C for 2 h and cryoprotected in 30% sucrose in PBS at 4°C for 24 h. The frozen sections (15 µm) were blocked using 10% donkey serum at room temperature for 10 min and then incubated with primary antibodies diluted in blocking solution at 4°C overnight. For immunofluorescence detection, the slices were incubated with corresponding fluorescent secondary antibodies at room temperature in the dark for 1 h. Following washes with PBS, the stained sections were mounted by coverslips with 50% glycerinum. The sections were examined, and immunostaining images were obtained with the laser scanning confocal microscope Leica TCS SP8 (Leica, Wetzlar, Germany). The primary antibodies used are listed in Supplementary Table 2.
2.9. Electrophysiology
Primary neurons were cultured as described previously. Briefly, the neocortex of newborn rats was harvested from postnatal pups and cut into small pieces within 1 mm3, papain-digested (10 min, 37°C), and then passed through a 400-µm mesh strainer. The dissociated cells were plated on poly-L-lysine (P7405; Sigma, America)-coated glass coverslips in 12-well plates (1.5 × 105 cells/well). The cultures were maintained in Dulbecco’s Minimum Essential Medium (Gibco, America) supplemented with 10% fetal calf serum (Gibco). After 4−6 h, the culture medium was changed to Neurobasal A medium (10888-022; Gibco) supplemented with 2% B27 (17504044; Gibco) and 1% GlutaMAX (Gibco) and incubated at 37°C in 5% CO2. Neurons were transfected with lentivirus and recorded on day 10.
In vitro whole-cell patch-clamp recordings in cultured primary neurons were performed at room temperature. The extracellular solution contained the following (in mM): 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES; pH 7.4, 300 mOsm). Borosilicate glass recording pipettes (resistance: 5−8 MΩ) were filled with intracellular solution, which contained the following (in mM): 135 K-gluconate, 2 MgCl2, 10 HEPES, 1 ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), and 5 MgATP, Na3GTP (pH 7.3, 290 mOsm). Data were collected using a Multiclamp 700B (Molecular Devices, Sunnyvale, CA, USA), filtered at 2 kHz and digitized at 10 kHz by a Digidata 1550A and Clampex 10.5 software (Molecular Devices). To elicit action potentials (APs), increasing amounts of depolarizing currents from 0 pA to 220 pA with 20 pA increments were used. The parameters of the first evoked AP were used for analysis. The evoked spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded by holding a potential of −70 mV and 0 mV, respectively.
In vivo whole-cell patch-clamp recordings were made in superficial spinal dorsal horn neurons from adult rats. Briefly, rats were deeply anesthetized and transcardially perfused with 60 mL ice-cold cutting solution containing the following (in mM): 93 N-methyl-D-glucamine, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 25 glucose, 20 HEPES, 5 Na-ascorbat, 3 Na-pyruvate, 2 thiourea, 10 MgSO4, and 0.5 CaCl2. Laminectomy was performed, and the lumbar enlargement was quickly removed to the ice-cold cutting solution with 95% O2 and 5% CO2. Sagittal slices (400 µm) were prepared with a Leica VT1000S vibratome. Subsequently, the slices were transferred into artificial cerebro-spinal fluid (containing, in mM: 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl2) and incubated for 30 min at room temperature. Under a dissecting microscope with transmitted illumination, the substantia gelatinosa was clearly visible as a relatively translucent band across the dorsal horn. Patch pipettes (3−5 MΩ) were filled with internal solution, which contained (in mM): 135 potassium gluconate, 0.5 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 0.5 Na3GTP, and 5 MgATP. All experiments were performed at room temperature. The series resistance was 10−30 MΩ and was monitored during the process. Data were collected as described above. To elicit APs, increasing amounts of depolarizing currents were injected for 20 ms in a stepwise manner from −60 pA to 220 pA in 20 pA increments. The parameters of APs were determined from the first AP evoked at threshold. To identify the firing pattern, a 150-pA current was applied for 150 ms. The membrane potential was held at −70 mV for sEPSCs and 0 mV for sIPSCs. APs were analyzed using Clampfit 10.5 and spontaneous postsynaptic currents (sPSCs) were analyzed with Mini Analysis (Synaptosoft, America).
2.10. Statistical analysis
Data were analyzed using SPSS 22.0 software (IBM, Armonk, NY, USA) and results are presented as the mean ± SEM. Comparisons among groups were analyzed using one-way analysis of variance (ANOVA) and, where appropriate, followed by the Student-Newman-Keuls test. A value of P < 0.05 was considered to be statistically significant.