All experimental procedures were approved by the Institutional Animal Care and Use committee review panels at Kyushu University.
Male Sprague–Dawley rats (8–11 weeks old) were obtained from Japan SLC (Hamamatsu, Japan). The rats were housed at a temperature of 22 ± 1°C with a 12-hour light–dark cycle and had ad libitum access to food and water.
Paclitaxel (LKT Laboratories, St. Paul, USA) was dissolved in a 1:1 mixture of ethanol and Cremophor EL (Sigma-Aldrich, St. Louis, USA) to make a stock solution of 12 mg/mL. Prior to administration, the paclitaxel solution was further diluted with sterile saline (1:3). Under isoflurane (2%) anesthesia, rats were administered the solution via the tail vain on days 0 and 3 after paw withdrawal threshold was measured. We used a previously characterized model of paclitaxel-induced peripheral neuropathy produced by repeated infusions of paclitaxel at a cumulative dose of 36 mg/kg (2 × 18 mg/kg, 3 days apart)
. Control rats received equivalent volumes of the Cremophor/ethanol vehicle.
For immunohistochemical experiments, rats were deeply anesthetized by pentobarbital and perfused transcardially with phosphate-buffered saline (PBS, composition in mM: NaCl 137, KCl 2.7, KH2PO4 1.5, NaH2PO4 8.1; pH 7.4) followed by ice-cold 4% paraformaldehyde/PBS. The L5 segment of the lumbar spinal cord was removed, postfixed in the same fixative, and placed in 30% sucrose solution for 24 hr at 4°C. Transverse L5 spinal cord sections (30 μm) were cut on a Leica CM 1850 cryostat (Leica Biosystems, Wetzlar, Germany) and incubated for 2 hr at room temperature in a blocking solution (3% normal goat serum), and then incubated for 48 hr at 4°C in the primary antibody for ionized calcium-binding adapter molecule 1 (Iba1, 1:2000, Wako, Osaka, Japan), a marker of microglia. Spinal sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:1000, Life Technologies Japan, Tokyo, Japan) and mounted in Vectashield containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, USA). Two to three sections from the L5 spinal cord segments of each rat were randomly selected and analyzed using an LSM510 Imaging System (Carl Zeiss Japan, Tokyo, Japan). The numbers of Iba1+ cells in the SDH (lamina I – IV) were counted.
For quantitative real-time PCR, rats were deeply anesthetized with pentobarbital, perfused transcardially with PBS, and the L5 spinal cord was removed immediately. The tissues were separated into ventral and dorsal horn. The sample was homogenized with TRIsure (Bioline, London, UK) and RNA was purified using an RNeasy mini plus kit (Qiagen, Valencia, USA). The amount of RNA was quantified using NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA). RNA was transcribed using PrimeScript Reverse Transriptase (Takara Bio, Otsu, Japan). Quantitative PCR was performed using Premix Ex Taq (Takara) together with a 7500 real-time PCR system (Life Technologies Japan, Tokyo, Japan), and the data were analyzed using 7500 System SDS Software 1.3.1 (Life Technologies Japan, Tokyo, Japan). Expression levels of genes of interest were normalized to the values for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as fold change over control rats. The sequences of TaqMan primer pairs and probes are described below: rat Iba1, 5′-GATTTGCAGGGAGGAAAAGCT-3′ (forward), 5′-AACCCCAAGTTTCTCCAGCAT-3′ (reverse), 5′-CAGGAAGAGAGGTTGGATGGGATCAA-3′ (Taqman probe); rat CCL3, 5′-CCACTGCCCTTGCTGTTCTT-3′ (forward), 5′-GCAAAGGCTGCTGGTTTCAA-3′ (reverse), 5′-CGCCATATGGAGCTGACACCCCG-3′ (Taqman probe); rat CCR1, 5′-CTAAGATGGCTAGGGCCCAAATA-3′ (forward), 5′-TCCCTGAGGGCCCGAACTGTCA-3′ (reverse), 5′-CCTGGGCTTATACAGTGAGATCTTC-3′ (Taqman probe); rat CCR5, 5′-GACCGGGTATAGACTGAGCTTACAC-3′ (forward), 5′-ACTCTTGGGATGACACACTGCTGCCTC-3′ (reverse), 5′-CAGGCAATGCAGGTGACAGA-3′ (Taqman probe); and rat purinoceptor P2XR7, 5′-CATGGAAAAGCGGACATTGA-3′ (forward), 5′-CCAGTGCCAAAAACCAGGAT-3′ (reverse), 5′-AAAGCCTTCGGCGTGCGTTTTGA-3′ (Taqman probe).
Mechanical allodynia was assessed using von Frey filaments (North Coast Medical, Gilroy, USA). Rats were placed in an aluminum cage with a wire mesh grid floor in a quiet room, 30 min before the start of testing. The von Frey filament (1.0–15.0 g) was inserted through the mesh floor bottom and was applied to the middle of the plantar surface of the hindpaw. The 50% paw withdrawal threshold (PWT) was determined using the up-down method
For intrathecal administration, under isoflurane (2%) anesthesia, rats were implanted with a 32-gauge intrathecal catheter (ReCathCo, Allison Park, USA) through the atlanto-occipital region into the lumbar enlargement of the spinal cord. Seven days after implantation, the catheter placement was verified by the observation of transient hindpaw paralysis induced by intrathecal injection of lidocaine (2%, 5 μL). Animals that failed to display paralysis following lidocaine administration were not included in the experiments. To test for possible effects on the development of paclitaxel-induced mechanical allodynia, rats were injected with a CCL3-neutralizing antibody (4 ng/10 μL; R&D Systems, Minneapolis, UAS) (or control IgG2A) and the selective P2X7R antagonist A438079 (1 μg/10 μL, Tocris Bioscience, Bristol, UK) (or PBS) once a day for 7 days starting from 1 day before the first injection of paclitaxel. Intrathecal administration of these drugs was done soon after the completion of behavioral measurement (10:00 ~ 12:00). For experiments testing their effects on maintenance of paclitaxel-induced mechanical allodynia, these agents were administered intrathecally to paclitaxel-treated rats, once, on day 7.
Data are expressed as the means ± SEM. Statistical analyses of the results were conducted with the Student’s t test, one-way ANOVA with post hoc Dunnett’s multiple comparisons, and two-way ANOVA with Bonferroni’s post hoc analysis. The threshold for statistical significance was set at a P value < 0.05.