CCL2- and Notch2-mediated Central Sensitization in a Rat Chronic Pelvic Pain Model

Materials and Methods

Animal model establishment. This study was approved by the Ethics Committee of Tongji Hospital (KYSB-2018-[088]). Adult female Sprague–Dawley rats, aged 8 weeks and weighing 280-320 g, were purchased from Shanghai Siple-Bikai Laboratory Animal Co (Shanghai, PR China). All rats were housed and maintained under specific pathogen-free conditions and allowed to accommodate to the environment for 1 week. The animal model of CPP was established by pelvic nerve ligation. In brief, 30 rats were randomly assigned to the CPP group and the control group at a ratio of 1:1. On day 0, rats were intraperitoneally anesthetized with volatile isoflurane, and the pelvic nerve was exposed through a large sciatic foramen incision in the buttock. In the CPP group, the pelvic nerve was ligated at four sites with nonabsorbable surgical sutures at an interval of 3-5 mm. In the control group, the left pelvic nerve was exposed without ligation. On day 42, behavioral tests were performed. Behavioral tests were performed again after the rats were injected with recombinant CCL2 (control group) or a CCR2 antagonist (CPP group). On day 44, the serum, vascular endothelial cells and CSF were collected for further tests. The rats were euthanized immediately at pre-designated time points.

Chronic pelvic pain assessment by behavioral testing. Rats were subjected to behavioral test 1 day before and 42 days after operation. Tests were performed in an individual transparent plastic box with a stainless-steel wire grid floor. They were allowed to accommodate to the environment for 20 min before testing. The hyperalgesia and tactile allodynia were tested using von Frey filaments with forces of 1, 4, 8, 15 and 26 g (18). Each filament was applied for 1-2 seconds with an interstimulus interval of 5 s, testing was done a total of 10 times, and the filaments were tested in ascending order of force. Stimulation was confined to the lower abdominal area in the general vicinity of the pelvic cavity, and care was taken to stimulate different areas within this region to avoid desensitization or “windup” effects. An investigator blind to the grouping assessed the behavioral responses of animals. The behaviors were graded as positive responses to filament stimulation: 1) sharp retraction of the abdomen, 2) immediate licking or scratching the area of filament stimulation, or 3) jumping (19).

Extraction and detection of exosomes. Rats were anaesthetized with 1% pentobarbital (35 mg/kg) intraperitoneally. After laparotomy, blood was collected via the inferior vena cava. Blood samples were allowed to stay for 15 min at room temperature and then centrifuged at 3,500 r/min for 15 min. The serum was harvested for exosome extraction. It was centrifuged at 4°C for 5 min at 2,000 g. The supernatant was removed, followed by centrifugation at 4°C for 15-20 min. The supernatant was removed again, followed by centrifugation at 4°C for 30-40 min at 10,000 g. The resultant sediment was collected, transferred to an ultra-high-speed centrifuge tube, and was re-suspended in 1× phosphate-buffered saline (PBS). After centrifugation, the supernatant was removed, and the precipitate was resuspended in 1× PBS, which was filtered through a 0.22-μm sterile filter. The filtrate was transferred to a centrifuge tube, followed by centrifugation at 120,000×g for 70 min at 4°C. After centrifugation, the supernatant was removed and the sediment at the bottom were the exosomes. Then, 100 μl of 1×PBS was added, which was then transferred to a centrifuge tube (0.5 ml). For the assay, 10 μl of exosome suspension was collected, stained with 2% uranium dioxyacetate solution, fixed for 1 min, blotted, and air dried at room temperature. The morphology of the exosomes was examined under a transmission electron microscope (TEM). The size and distribution of the exosomes were measured using the NanoSight NS300 nanoparticle tracking analysis (NTA). The expression of CD63 and TSG101, the characteristic proteins of exosomes, was measured by Western blotting.

Detection of CCL-2 expression in exosomes by quantitative polymerase chain reaction. Quantitative real-time polymerase chain reaction (PCR) was used to detect the mRNA expression of CCL2 in serum exosomes. In brief, RNA was extracted from serum exosomes using an Exosome RNA Isolation Kit (Norgen Biotek, #NGB-58000, AmyJet Scientific Inc, Wuhan, PR China). The cDNA was reverse transcribed using a Titan One Tube RT-PCR kit (Roche, #11939823001, Yubo Biological, Shanghai, PR China) according to the manufacturer’s instructions. PCR was performed as follows: 95°C for 5 min, 95°C for 10 s, 60°C for 30 s, 72°C for 10 s (for a total of 40 cycles). The experiment was done in triplicate. The mRNA expression of CCL2 in the exosomes was analyzed using the 2^−ΔΔCt method and GAPDH was used as an internal control.

Detection of CCL2 expression in pelvic vascular endothelial cell supernatants by enzyme-linked immunosorbent assay (ELISA). An incision was made bilaterally at the groin (about 1-2 cm in length). The femoral vein was separated, collected and stored in sterile PBS. The samples were washed three times with PBS and then incubated in the media containing penicillin, streptomycin, and amphotericin B solution for 10 min. A blunt syringe needle was inserted into the end of the vein, and the vein was washed with 1% PBS until transparent fluid was observed. Then, 1% type I collagenase was injected to the vascular cavity while clamping an end of the vein. The vein was incubated with 1% type I collagenase for 5, 7, and 10 min, and the fluid was harvested. Finally, the vessel was flushed with cell culture medium (M199 containing 20% fetal bovine serum). After centrifugation, the supernatant was removed, 2 ml of culture medium was added, and the cells were re-suspended, followed by staining with Trypanosoma cruzi blue. Cells were observed under an inverted phase-contrast microscope. The supernatants of vascular endothelial cells in both groups were collected and the CCL-2 content was detected using a CCL2 assay kit (ABclonal, RK00206) following the manufacturer’s instructions. The A450 was measured, and a standard curve was delineated. The content of CCL-2 was determined according to the standard curve.

Detection of CCL2 expression in CSF by ELISA. Rats were intraperitoneally anesthetized with volatile isoflurane, and the CSF was collected according to the method previously reported (19). The rat’s head was fixed in the brain fixation apparatus, and a 1-ml syringe was inserted at an angle of approximately 135°. The needle was inserted into the medullary pool of the cerebellum at a depth of approximately 0.5 cm. Then, 80-100 μl of CSF was slowly collected, and the needle was quickly withdrawn. Subsequently, 1.5 ml of CSF was transferred into a 1.5-ml EP tube and stored at – 40°C. The CCL-2 content was detected using the CCL2 Assay Kit (RK00206, ABclonal, Wuhan, PR China) according to the manufacturer’s instructions. The A450 was measured, and a standard curve was delineated. The CCL-2 content was calculated according to the standard curve.

Intrathecal injections of CCL2 or CCR2 antagonist and behavioral testing. Recombinant CCL2 (Recombinant Human CCL2/MCP-1 Protein, RP00316, ABclonal) or CCR2 antagonist (INCB3344, Shanghai Yuanye Bio-Technology Co, Shanghai, PR China) were injected for the control and CPP group, respectively, 43 days after the operation Rats were anesthetized with volatile isoflurane and an intrathecal catheter (PE-10 tube) was inserted through the gap between L4 and L5. 10 ng of recombinant CCL2 were injected into the control group and 14.43 μg (1 mM) of INCB3344 were injected into the CPP group rats, in a total volume of 25 μl. Behavioral testing was measured again after 2 h in the control group and 24 h in the CPP group by using von Frey filaments, as described previously and compared with the previous responses.

Detection of Notch2 mRNA expression in human microglia treated with recombinant CCL2. Human microglia (HMC3) were incubated with 20 ng/ml recombinant CCL2 (Recombinant Human CCL2/MCP-1 Protein, ABclonal, RP00316) for 24 h. The cDNA was reverse transcribed according to the Titan One Tube RT-PCR kit instructions, followed by qPCR. 40 cycles of 95°C for 5 min, 95°C for 10 s, 60°C for 30 s and 72°C for 10 s were performed. Three replicate wells were set up for each experiment. qPCR data were collected using the 2^−ΔΔCt method with GAPDH as the internal control.

Notch2 protein expression in human microglia treated with recombinant CCL2. Human microglia (HMC3) were incubated with 20 ng/ml recombinant CCL2 protein (ABclonal, #RP00316) for 24 h, and total protein was collected. The protein concentration was determined using a BCA kit (Beyotime, #P0012, YuanXin Bio, Shanghai, PR China). Proteins (10 μg) in each sample were mixed with loading buffer, separated by electrophoresis on a 12% SDS-PAGE gel, and transferred onto a poly(vinylidene fluoride) membrane. The membrane was then blocked with 5% non-fat milk for 1 h and incubated with primary β-actin (1:2,000) antibody (KGDN16, KeyGen BioTECH, Jiangsu, PR China) overnight at 4°C. After rinsing with Tris-buffered saline containing Tween 20, the membrane was incubated with secondary antibody (KGP1201, KeyGen BioTECH) at room temperature for 1 h. The membrane was rinsed with TBST, and then visualized with a ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). β-actin served as an internal reference.

Statistical analysis. The SPSS 27.0 statistical software (IBM, Armonk, NY, USA) was used for data analysis. Quantitative data are expressed as mean±standard deviation (SD) and compared with t-test between two groups or one-way analysis of variance among multiple groups followed by SNK-q test between two groups. A value of p<0.05 was considered statistically significant.