Forty-five male Sprague-Dawley rats (aged between 8 and 12 weeks; body weight: 229.1 ± 22.1 g) were purchased from the experimental Animal Center of Hubei University of Medicine, China. The rats were housed under controlled conditions: temperature (22 ± 1 °C), with a 12-h light/dark cycle and free access to food and water. The experimental protocols were approved by the Animal Use and Care Committee of Hubei University of Medicine and were consistent with the Ethical Guidelines of the International Association for pain research.
Paclitaxel (Bristol-Myers Squibb, Paris, France) was dissolved in cremophor EL: ethanol (1:1, Sigma), and diluted further with 0.9 % saline. 1 ml of a 6 mg/ml solution was administrated intraperitoneally (i.p.) at dose of 2 mg/kg on days 0, 2, 4 and 6. This dose was previously reported to induce mechanical allodynia/hyperalgesia in rats (Kawakami et al. 2012). Control rats received an equivalent volume of vehicle (cremophor EL:ethanol, 1:1) diluted with 0.9 % saline.
Evaluation of mechanical allodynia
Mechanical allodynia was measured by recording the maximum pressure required to trigger hind paw withdrawal on day 0 (before paclitaxel administration, as baseline) and on days 7, 14 and 21 after first paclitaxel administration. The rats were allowed to habituate the testing chambers (22.0 × 15.0 × 12.5 cm) for 20 min. Mechanical allodynia was assessed using electronic von Frey anesthesiometer (0–90 g, electronic von Frey anesthesiometer, IITC Inc., Life Science Instruments, Woodland Hills, CA, USA) using a rigid tip, following a protocol adapted from that described by Vivancos et al. (2004). The pressure-meter consisted of a hand-held force transducer fitted with a 0.8 mm diameter polypropylene tip. The investigator applied the rigid tip perpendicularly to the mid-plantar surface of the hind paw with a gradual increase in pressure. A tilted mirror below the grid provided a clear view of the animal’s hind paw. The tests consisted of poking the hind paw to provoke a flexion reflex, followed by a clear flinch response after paw withdrawal. With the electronic pressure-meter, the maximum pressure of the stimulus was automatically recorded when the paw was withdrawn, as the pain threshold. The stimulation of the paw was repeated until the animal responded similarly (within 10 g) three times.
Measurement of Nav1.7 expression in DRG
Five rats in each group were sacrificed by deep anesthesia with pentobarbital (50 mg/kg, i.p.) on days 7, 14 and 21 after first administration of paclitaxel or vehicle. DRGs (L4–6) were collected and processed for real-time RT-PCR and Western blotting, as described below.
Quantitative real-time RT-PCR
RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Reverse transcription and real-time PCR were performed as previously described (Wu et al. 2010). The rat β-actin primer sequences were as follows: sense: 5′-CGTTGACATCCGTAAAGACCTC-3′; anti-sense: 5′-TAGGAGCCAGGGCAGTAATCT-3′. The Nav1.7 primer sequences of were as follows: sense 5′-CGATGGGTCACGATTTCCTAC-3′; anti-sense 5′-CGTGAAGAATGAGCCGAAGAT-3′. In all cases, amplification was confirmed by the presence of a single peak in the melting temperature analysis and linear amplification throughout the PCR cycles. 2−ΔΔCt was calculated to represent the relative mRNA expression of target genes. β-actin was used as an internal control.
Western blot analysis
Protein was extracted from the DRG samples using a homogenizer in an ice-cold denaturing lysis buffer (25 mmol/L Tris–HCl, PH 7.5, 150 mmol/L Nacl, 5 mmol/L ethylenedianinetetraacetic acid, 1 % Triton X-100, 1 mmol/L PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin), then the homogenate was centrifuged at 12,000×g for 20 min at 4 °C. The supernatant was collected, and protein concentration of the supernatant was determined via BCA Protein Assay Kit (Pierce, Rockford, USA). We loaded 40 μg of protein on each lane of 5 % SDS–PAGE gel, and transferred separated proteins to the polyvinylidene fluoride membrane (Millipore, Billerica, USA).
The membrane was blocked with 5 % non-fat dry milk in TBS-T (50 mmol/L Tris–HCl, pH 7.5, 140 mmol/L NaCl, 0.1 % Tween 20) overnight at room temperature (RT). The membranes were incubated with rabbit anti rat Nav1.7 primary antibody (1:500, Cell signaling Technology, MA, USA, #14573) for 3 h at RT, then incubated with HRP-anti-rabbit secondary antibody (1:4000, KPL,074-1506) for 1 h at RT. Blots were developed in ECL solution (Pierce, Rockford, USA) for 3 min, and exposed onto Kodak X-OMAT BT Film (Eastman Kodak, Rochester, USA) for 2 min. Densitometric analysis was performed using AlphaEaseFC software (Alpha Innotech, San Leandro, CA). Expression of Nav1.7 was normalized to the level of β-actin (1:10,000, TDY, Beijing, TDY051) in each sample.
Dorsal root ganglionic injection of Nav1.7 antibody
The Nav1.7 antibody (57 μL of 0.7 μg/μL,Millipore, AB5390) was neutralized by incubation with 40 μg lyophilized antigen (Purified rat PN1 peptide, amino acids 446–460, Accession AAB50403, Millipore) for 1 h at RT according to the manufacturer’s instructions. The neutralization efficiency was assessed by Western blot, with protein extracted from the rat ganglion tissues.
We performed microinjection of Nav1.7 antibody into the DRGs using a previously described direct injection method (Fischer et al. 2011). Briefly, animals were anesthetized with pentobarbital (50 mg/kg, i.p.). To expose the DRG, an incision of approximately 3 cm was made in the skin just to the right of the dorsal midline, starting from the superior iliac crest. The fascia and muscles were separated, exposing the lateral aspect of the fourth and fifth lumbar (L4 and 5). The L4 and L5 spinal nerves, and the intervertebral foramina, from which they emerge, were exposed. Accessory processes that descend from the base of the transverse process were removed using a rongeur. The distal fourth and fifth DRGs were then exposed. The injections were performed using a pulled glass capillary injection tip with a diameter of 40–60 μm. Five rats received injection to the right side L4 and L5 DRGs, as previously described by Bi et al. (2013), with 7 μL (5 μg) Nav1.7 antibody (Millipore, AB5390). Five rats underwent the same treatment with neutralized Nav1.7 antibody, and five rats underwent surgical exposure but no injection (sham surgery). After the injection was completed, the wound was closed in layers.
The paclitaxel was intraperitoneally injected at dose of 2 mg/kg, 2 h after DRG microinjection on days 0, 2, 4 and 6. The PWT was measured at 1 day before-injection (baseline) and 7 days after first paclitaxel administration.
Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA). All data are presented as mean ± SEM. All data of rat behavioral experiments were performed by two-way repeated-measure ANOVA with least significant difference (LSD) test for post hoc analysis. Other data were analyzed using independent sample t test, paired sample t test or one-way ANOVA with LSD test for post hoc analysis, as appropriate. Correlations between Nav1.7 protein levels with individual animal/time changes in von Frey PWT responses after paclitaxel administration were analyzed using the Pearson correlation test. A value of P < 0.05 was considered statistically significant.