Animals
A total of 105 adult female pathogen-free Wistar rats (160–180 g) were procured from Yosi Experimental Animal Corporation (Changchun, China) and maintained under constant conditions with a 12-h light/dark cycle and free access to food and water. Every effort was made to avoid animal suffering at all stages. All measurements were performed under double-blind conditions to avoid subjective influences.
Intrathecal intubation and tumor cell inoculation
The rats were anesthetized using sodium pentobarbital (1%, 40 mg/kg), and 14-cm intrathecal catheters made of polyethylene PE10 tubing (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) were implanted into the subarachnoid space until reaching the lumbar enlargement. The catheter’s rostral end was attached firmly to the thoracic spine within the soft tissue in the interscapular region. Animals that developed motor dysfunction after implantation were excluded. After 1 week of recovery, the rats developed bilateral lower limb paralysis within 30 s. They recovered completely within 30 min after administering 1% lidocaine (20 µL) through the catheter, confirming the placement of the catheter in the subdural space. Walker 256 breast cancer cells (107, Heilongjiang Cancer Research Institute, Harbin, China) were injected intraperitoneally into the rats. After 5 d, the ascites fluid was extracted and centrifuged (3000 rpm, 5 min), and the supernatant was removed. Cell clumps were diluted using phosphate buffered saline (PBS, pH 7.4), and penicillin (120 000 units/10 mL) was then added to avoid microbial contamination. We implanted 100 µL of tumor cells into the right hind paw (away from motor disturbance) to establish the cancer pain model, as previously described.[41]
Experimental design and drug administration
The rats were randomly divided into seven groups (n = 15 for each group) based on the treatment. The groups received subcutaneous morphine (10 mg/kg·mL) combined with an intrathecal injection of CXCL12-neutralizing antibody (CXCL12-Ab, 10 µg/10 µL; morphine + CXCL12-Ab, MAb group), exogenous CXCL12 (2 µg /10 µL, morphine + CXCL12, MC group), AMD3100 (CXCR4 antagonist, 5 µg/10 µL, morphine + AMD3100, MD group), AM1241 (0.07 µg/10 µL, morphine + AM1241, MA group), AM630 (CB2 receptor antagonist, 10 µg/10 µL), and AM1241 (0.07 µg/10 µL, 30 min after AM630; morphine + AM1241 + AM630, MAA group) or the same volume of vehicle (1:1 ratio of DMSO:PBS, morphine + vehicle, MV group). Drug doses were selected based on previous studies.[15, 46] The control rats received subcutaneous saline and intrathecal administration of the same volume of vehicle [saline + vehicle, SV (control) group]. Five days after tumor inoculation, drugs were administered once daily (8:00–14:00) for eight consecutive days to establish a model of morphine tolerance, as previously described.[15] AMD3100 was administered two h before morphine, CXCL12 and CXCL12-Ab 1.5 h before morphine, AM630 30 min before AM1241, and AM1241 30 min before morphine. Behavioral indicators were measured on the day before the first administration (day 0) and 30 min after the daily morphine injection during days 1–8.
Mechanical pain withdrawal threshold
PWT was measured using the von Frey method. The animals (n = 15) were placed in a glass chamber with a steel wire mesh at the bottom and acclimatized to room temperature for 20 min. A series of standardized von Frey fibers (Stoelting, Wood Dale, IL, USA; 1.0–60 g) was used to stimulate the plantar area for 6 to 8 s, and the degree of foot contraction was recorded. Triplicate measurements were randomly conducted on the right hind paw with 15-s intervals. A fast foot contraction or filament removal was recorded as a positive reaction during the stimulation time. The threshold for mechanical foot contraction was replaced by the average value of three degrees.
Thermal pain withdrawal latency
PWL was measured using a hot-plate (52°C) apparatus in a plastic cylinder (n = 15; Technology & Market Corporation, Chengdu, China). The incubation period for hyperalgesia was defined as the interval between contact with the plate and licking the soles of the feet or jumping. The plate was heated for 30 s to prevent tissue damage. Three latency measurements were performed at at least 10 min intervals, and the average was used as the measure of hyperthermia.
Sampling and storage
Following the day-8 behavioral assessment, the rats were anesthetized using sodium pentobarbital (60 mg/kg, administered intraperitoneally), followed by transcardial perfusion of cold saline and 4% paraformaldehyde in 0.1 M PBS (pH 7.2–7.4, 4°C) in succession. The rats were then decapitated, the lumbar L3/L4/L5 spinal cord and DRG segments ipsilateral to the tumor cell injections were collected, and the samples were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) and immunofluorescence (IF). Tissues obtained without transcardial perfusion were rapidly frozen in liquid nitrogen and stored at − 80°C for WB and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses.
Immunohistochemistry
We prepared 4-µm-thick frozen sections using a cryostat (Eppendorf, Germany). We prepared three slides of the L5 spinal cord and DRG segments, each containing three sections (nine sections per spinal cord and DRG segment). The tissues were dewaxed with xylene, dehydrated using an ethanol gradient, and rehydrated. Citric acid was used for antigen retrieval, and the sections were incubated with 3% H2O2 for 25 min, rinsed in PBS, and blocked with 10% normal goat serum for 30 min at 26–28°C. The sections were incubated with primary antibodies overnight at 4°C. The primary antibodies used were rabbit anti-MOR (1:100, Abcam), rabbit anti-CXCR4 (1:200, Wanleibio, Shenyang, China), and rabbit anti-CXCL12 (1:200, Wanleibio). The sections were then washed and incubated with horse radish peroxidase (HRP)- and fluorescent molecule-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch, West Grove, PA, USA) for 50 min at room temperature. The sections were washed, embedded, and examined under a confocal fluorescence microscope (Eclipse Ci-L, Nikon, Tokyo, Japan). The images were processed and quantified using ImageJ software version 7 (National Institutes of Health, Bethesda, MD, USA), and the optical densities (OD) of MOR-, CXCL12-, and CXCR4-positive cells relative to the total area of positive cells were calculated for each section per rat, thus yielding the AOD.
qRT-PCR
Total RNA was isolated using 1 mL Trizol (Invitrogen). A Transcriptor first-strand cDNA synthesis kit (Roche, Basel, Switzerland) was used to reverse-transcribe the cDNA according to the manufacturer’s instructions. The cDNA samples were stored at − 20°C before amplification using Fast Start Universal SYBR Green master mix (Roche). Relative mRNA levels were determined using the 2−ΔΔCt method and were normalized to GAPDH levels (n = 5 per group). The following oligonucleotide primers were used: Rat GAPDH: forward 5′-ATGCCGCCTGGAGAAACC-3′, reverse 5′-GCATCAAAGGTGGAAGAATGG-3′; MOR: forward 5′-CCAGTTCTTTACGCCTTCC-3′, reverse 5′-TAGTGTTCTGACGGACTCG-3′; CXCL12: forward 5′-CTTATATTCATCCGTGCCCTCG-3′, reverse 5′-AGCAAACCGAATACAGACC-3′; and CXCR4: forward 5′-AGGAGCATGACAGACAAGTACCG-3′, reverse 5′-TGGCGTGGACAATGGCAAG-3′. The amplification steps (95°C 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min) were managed using an ABI 7500 fast real-time PCR system (Applied Biosystems, Waltham, MA, USA).
Western Blotting
WB assays were conducted to examine MOR (45 kDa), CXCL12 (15 kDa), and CXCR4 (39 kDa) protein expression (n = 5 per group). The tissues were homogenized, and total protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes. After blocking, the membranes were successively incubated with the primary antibodies (4°C, overnight) and secondary antibodies (37°C, 45 min). The primary antibodies used were anti-MOR (1:2,000, Abcam), anti-CXCL12 (1:500, Wanleibio), anti-CXCR4 (1:500, Wanleibio), and anti-GAPDH (36 kDa, 1:5,000, loading control), while the secondary antibody was IgG-HRP (1:5,000; Wanleibio). The immunoreactivity of each target protein was detected using electrochemiluminescence (ECL) using a Plus chemiluminescence detection system (Amersham ECL Hyper film), visualized by exposure to an autoradiographic film for 1–60 min. The band intensities were quantified using densitometry (Bio-Rad Laboratories, Hercules, CA, USA), and absorbance values were determined using ImageJ (NIH).
Immunofluorescence
The sections were treated in a similar manner as for IHC, blocked, and incubated with the following primary antibodies: rabbit anti-MOR (1:100, Abcam) + mouse anti-CXCR4 (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-CB2 (1:100, Abcam) + mouse anti-CXCR4 (1:100, Santa Cruz Biotechnology). The following day, the sections were rewarmed to room temperature for 30 min, washed, and incubated with Cy3-labeled goat anti-rabbit IgG (1:200, red, Proteintech, Wuhan, China) and FITC-labeled goat anti-mouse IgG (1:200, green, Proteintech) at room temperature in the dark for 60 min. The nuclei were counterstained using 4',6-diamidino-2-phenylindole (DAPI, 0.5 µg/mL, Aladdin, Wuhan, China) for 8 min, and the sections were sealed using an anti-fluorescence quenching agent before examination (Eclipse Ci-L, Nikon) and imaging (Nikon DS-F12). Between four and six L5-DRG sections were randomly selected at each time point, and the intensities of the red (MOR, CB2) and green fluorescence (CXCR4), DAPI (cell nucleus, blue), and merged fluorescence were analyzed using ImageJ. The numbers and AOD of cells with MOR + CXCR4 and CB2 + CXCR4 colocalizations were calculated for each section.
Statistical analysis
Data were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Data are represented as the mean ± standard deviation (SD). A power analysis was performed based on preliminary behavioral testing and molecular biological parameters. Behavioral data were tested using a two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. The protein and mRNA levels of MOR, CXCL12, and CXCR4 were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test. Statistical significance was set at P < 0.05.
Animal Ethics declaration
The study was performed in accordance with the International Association for the Study of Pain, and the protocol was authorized by the Animal Care and Use Committee of the Harbin Medical University Cancer Hospital (Harbin, China). The study was in accordance with ARRIVE guidelines.