Animals

Adult male Sprague‒Dawley (SD) rats (250 g-300 g; specific pathogen-free) and pregnant rats provided by Chongqing Medical University were used for this work. All procedures were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were kept at room temperature (24 ± 1 °C) on a 12-h light/12-h dark cycle. No limitations for food and water accession. All rats were acclimated for 30 min before each experiment. The rats were grouped randomly except for those with an abnormal baseline pain threshold.

Materials

All antibodies used are listed in Table 1. L-glutamine, deoxyribonuclease (DNase), poly-L-lysine (PLL), bradykinin, histamine, serotonin, and prostaglandin E2 were provided by Sigma‒Aldrich (Missouri, USA). Fluo-4 AM was purchased from Beyotime (Beijing, China). Fetal bovine serum, neurobasal medium, DMEM-HG medium, and B27 were purchased from Thermo (Waltham, USA). LY294002, sulpiride, NASPM, PP2, and 740YP were obtained from MCE (Shanghai, China), and quinpirole was purchased from Sigma‒Aldrich (Missouri, USA). Quinpirole, LY294002, PP2, sulpiride, NASPM, and 740YP were dissolved in 5% DMSO, and the inflammatory soup (IS) was produced with bradykinin (1 mM), histamine (1 mM), serotonin (1 mM), and prostaglandin E2(0.1 mM) (all from Sigma‒Aldrich, Missouri, USA), which were mixed in phosphate-buffered saline (PBS) [34, 35].

Table 1 Information on antibodiesSurgery for establishing the CM model

The surgical procedure was performed as described previously [36]. First, rats under anesthetized with sevoflurane were put on a stereoscopic rack (Stoelting Co, Chicago, USA). A notch was made above the skull and the periosteum was removed to uncover the bregma. A hole with an approximate diameter of 1-mm was made over the left dura anterior fontanelle using a cranial drill (-1.0 mm posterior and + 1.5 mm lateral to the bregma). Next, a sterile catheter was placed at the eyelet with dental cement. After suturing, the rats were positioned on a warm mat until consciousness was restored. Thereafter, the rats were allowed to recover for at least 1 week to assure that their pain thresholds returned to baseline levels. During this time, the surgical area was disinfected daily with iodophor. CM rats were treated with IS (5 μL) daily for 7 days, while the IS was replaced with PBS in the Sham group.

Animal grouping and drug delivery

As needed for experiments, rats were assigned to the following groups: Sham group, Sham + DMSO group, Sham + quinpirole group, Sham + sulpiride group, Sham + LY294002 group, Sham + PP2 group, Sham + NASPM group, CM group, CM + DMSO group, CM + quinpirole group, CM + sulpiride group, CM + LY294002 group, CM + PP2 group, CM + NASPM group, CM + quinpirole + 740YP group, CM + quinpirole + DMSO group, CM + quinpirole + sulpiride group. For the CM + quinpirole group, CM + sulpiride group, CM + PP2 group, CM + NASPM group, and CM + LY294002 group, all drugs were administered on the 7th day after IS infusion. For the CM + quinpirole + 740YP group, on Day 7 after IS injection, quinpirole was administered first, followed by 740YP 30 min later; the vehicle control was 5% DMSO. The doses of quinpirole, LY294002, PP2, sulpiride, NASPM, and 740YP were determined according to previous studies [19, 25, 33, 37, 38]. All doses of the drugs were administered by intracerebroventricular injection.

Pain behavior test

All behavioral trials were conducted under light conditions between 09:00 and 18:00. Before the tests, the rats were acclimated to the environment for at least 30 min. Baseline testing of pain thresholds was performed before the infusion of IS or PBS. Subsequently, pain thresholds were tested daily after IS injection or after drug administration on Day 7.

First, rats were staged on a test device, and a von Frey monofilament (ranging from 1 to 26 g) was then applied vertically to the hind paw or the periorbital region (rostral area on the left or right side of the face) of each rat using an up-and-down approach to measure the mechanical threshold as described previously [34, 36].

The paw twitch latency (PWL) is thought to represent the thermal pain threshold [39]. Briefly, rats were positioned in the cage, then the radiant heat was applied from the bottom to the plantar surface of the hind paw. The time at which the rat responded to the stimulus was recorded and considered as the PWL. The maximum time was set to 25 s to protect the rats.

All tests were repeated 3 times at 5-min intervals, and the average thresholds were calculated. Throughout the experiment, the experimenter was blinded to the experimental group.

Quantitative real-time polymerase chain reaction (qRT‒PCR)

Briefly, RNA was acquired from fresh TNC tissue using RNAiso reagent (TaKaRa, Tokyo, Japan). A PrimeScript RT Kit (TaKaRa, Tokyo, Japan) was used for reverse transcription. Then the qRT‒PCR was conducted on a thermocycler (Bio-Rad, USA) using SYBR Premix Ex Taq TM II (TaKaRa, Tokyo, Japan) to evaluate DRD1 and DRD2 mRNA expression levels. Specific primers were provided by Sangon Biotech (Shanghai, China) and are listed in Table 2. Gene expression was analyzed by the standard 2 − ΔΔCT method.

Table 2 Information on primersWestern blot (WB)

In brief, TNCs were removed after rats were euthanized and were then lysed with RIPA buffer containing PMSF (Beyotime, Beijing, China) to obtain total protein. A Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, USA) was applied to isolate the plasma membrane fraction. SDS-polyacrylamide gels were prepared to separate the proteins. After that, the proteins were transferred onto PVDF membranes, which were then incubated with specific primary antibodies overnight at 4 ℃ after 2 hours of blocking with 5% nonfat milk. After being incubated with secondary antibodies, the membranes were then developed by an imaging system (Fusion, Germany) using ECL reagents (Beyotime, Shanghai, China) to detect signals. Finally, the immunoblots were analyzed with ImageJ software, and the levels of the target proteins were normalized to the corresponding GAPDH level.

Immunofluorescence staining (IF)

Rats under deep anesthesia were subjected to cardiac perfusion with PBS followed by 4% paraformaldehyde. The intact TNC was then immediately isolated and postfixed with 4% paraformaldehyde at 4 °C. After dehydration using graded concentrations of sucrose solution (20% and 30%), the tissues were sectioned using a cryostat (Leica, Japan) into 15-μm slices, which were placed on carrier slides. Antigen repair with sodium citrate (Beyotime, Beijing, China) and blocking with 10% goat serum (Boster, Beijing, China) were then executed. For fluorescence colabeling experiments, the primary antibodies were mixed and diluted with 1% PBS and were subsequently incubated with the sections at 4 °C overnight. After incubation with the fluorescent secondary antibody and counterstaining of the nuclei with 4’,6-diamidino-2-phenylindole (DAPI), a confocal laser scanning fluorescence microscope (Zeiss, Germany) was used to acquire images, which were analyzed with ImageJ.

Golgi-Cox staining

An FD Rapid Golgi Staining Kit TM (NeuroTechnologies, USA) was utilized to observe the dendritic spines in the TNC. After rats were sacrificed, TNCs were harvested and then washed quickly with double-distilled water and immersed in a premixed solution (liquid A and liquid B at a ratio of 1:1) which was refreshed once in 24 h and then kept for 2 weeks (in a dark environment). Then the tissues were transferred to liquid C and incubated for 3 days (in a dark environment). A vibratome (Leica VT 1200S, Japan) was used to generate 150-μm-thick tissue slices, which were then stained in a mixture of liquid D, liquid E, and double-distilled water at a ratio of 1:1:2 for 10 min. Next, after being dehydrated with increasing concentrations of ethanol (50%, 75%, 95%, and 100%) and permeating with xylene, the slices were sealed with neutral resin. Images were acquired using a microscope (Axio Imager A2) and were analyzed with ImageJ. All steps were carried out at room temperature.

Transmission Electron Microscopy (TEM)

The relevant steps can be found in our previous work [40]. Briefly, rats were anesthetized and then subjected to cardiac perfusion with PBS followed by 2.5% glutaraldehyde. TNCs were separated and cut into 1 m3 pieces using a blade. Next, the pieces were soaked in 4% glutaraldehyde at 4 °C and then post-treated at Chongqing Medical University. Finally, images were acquired using a JEM-1400 PLUS transmission electron microscope at 50,000 X or 30,000 X magnification and then statistically analyzed with Image-Pro 6.0.

Culture of TNC primary neurons

TNCs were removed from embryos on Day 18 of pregnancy sterilely, and then the meninges and blood were removed as described previously [41]. Next, the tissues were digested with papain (2 mg/ml) for 25 min. The tissues were gently and repeatedly disrupted by pipetting until the large cell clumps disappeared at the end of the digestion step. After centrifugation, the cells were resuspended in DMEM-HG medium. Finally, the cells were plated (2.5 X 105 cells/dish) on confocal dishes that were precoated with poly-L-lysine (PLL) at 37 °C and 5% CO2. About 4–6 h later, the DMEM-HG was replaced with neurobasal containing B27(2%) and L-glutamine (0.5 mM/L), followed by half volume changes every 3 days. Cells were incubated until Day 7 for subsequent experiments.

Determination of intracellular Ca2+ concentrations

The primary neurons were pretreated with quinpirole (1 μM) [19] for 12 h and LY294002 (20 μM) [42] for 1 h at 37 °C and 5% CO2, and were then incubated with 4 μM Fluo-4-AM (150 μl/well, diluted in PBS buffer) for 30 min at 37 °C. Then, 900 μl of HBSS was left in each well after washing three times with warm (37 °C) HBSS solution. Then, 100 μl of 10X NMDA was added at a specific time (the final concentration is 50 μM), and the changes in the intracellular calcium concentration were determined using the confocal microscope described above (Zeiss, Germany) at a detection wavelength of 488 nm over a total of 500 s for each group. The fluorescence intensity in the collected images was analyzed by ZEN software [32].

Statistical analysis

All data were analyzed, and graphs were generated using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Data in this paper are expressed as mean ± SEM. The Kolmogorov‒Smirnov test was used to check for normality. Significant differences for two-group and multiple-group comparisons were analyzed by the independent‒sample t-test and one-way ANOVA followed by Dunnett’s multiple comparison test, respectively. Two-factor analysis followed by the Bonferroni post hoc test was used to analyze the behavioral data. P values < 0.05 were thought to be statistically significant.