Experimental animals and grouping
A total of 160 specific-pathogen-free C57BL/6j male mice (45–56 days old) were purchased from Three Gorges University. Mice were randomly divided into sham, model, mmu-miR-27a-5p, mmu-miR-27a-5p-negative control (NC) groups, with 40 mice in each group. The animal study was approved by the ethics committee of our hospital.
Preparation of mmu-miR-27a-5p mimics
mmu-miR-27a-5p mimics was synthesized by Wuhan Biofavor Biotech Services Co., Ltd. (Wuhan Biofavor Biotech Services Co., Ltd., Wuhan, China), an unrelated universal sequence was as a negative control (NC). RAW 264.7 macrophages were seeded into 6-well plates and cultured with mmu-miR-27a-5p mimics and mmu-miR-27a-5p-NC, respectively, at 37 °C overnight. Lentivirus vector solution (1 × 109TU/ml) were mixed and diluted with medium at a multiplicity of infection of 100, 10, 1. The old medium was aspirated from the 6-well plate, and replaced with the virus gradient dilutions. After 24 h of culture at 37 °C, culture medium was replaced with fresh media. After 48 hours of culture, transfection efficiency was observed under a fluorescent microscope. The optimal transfection condition was chosen and used for subsequent experiments.
SCI modeling
After 3 days of acclimation, all mice were anesthetized intraperitoneally with 4% chloral hydrate. After hair removal and skin disinfection in the surgical field, a longitudinal incision was made centered on T10 spinous process, then the T9, T10 spinous processes and lamina were removed to expose the dura mater. In the model, mmu-miR-27a-5p, and mmu-miR-27a-5p-NC groups, SCI at T9-10 levels was produced by freely dropping a 10 g weight from a height of 5 cm using a weight drop device onto the T9-10 spinal segment [17]. The signs of successful model establishment included spastic swinging of the tail and retraction-like flutter and flaccid paralysis of the hind limbs immediately after injury. In the sham group, T9-10 spinous processes and lamina were removed without damaging the spinal cord. After surgery, the mice were kept warm. And after recovery from anaesthesia, mice were moved to the animal room. All mice were fed normally. 20,000 units of penicillin were injected intraperitoneally into mice once a day within 3 days after surgery, and the bladder of the mice was squeezed manually twice daily. If mice died during surgery, new mice was added and animal model was established again.
Treatment
All mice in each group were treated at day 3 after SCI model was successfully established. Mice in sham group received no treatment, mice in model group were treated with 10 µl saline. Mice in mmu-miR-27a-5p group and mmu-miR-27a-5p-NC group received 10 µl of mmu-miR-27a-5p suspension and mmu-miR-27a-5p-NC suspension, respectively. Before administration, mmu-miR-27a-5p suspension and mmu-miR-27a-5p-NC suspension were co-cultured with BrdU (10 µmol/L) for 72 hours. Multi-point injections were slowly given for 5 minutes at the injured spinal cord, the needle was left in place for additional 5 min before removal, then feeding was continued.
Behavioral testing
Before surgery and at 1, 3, 7, and 14 days after surgery, open field test was perfromed [5], mice were placed in an open field, allowed to freely explore fo for 5 min. Hindlimb and trunk motor function were assessed using Basso Mouse scale (BMS) by a examiner who did not participate in the study design.
Spinal cord tissue acquisition
After successful established of SCI model, 10 mice of each group were sacrificed at 1, 3, 7, and 14 days after SCI to collect their spinal cord samples. Mice were deeply anesthetized with ketamine/xylazine, and perfused through the heart with saline, followed by 40 g/L paraformaldehyde. Approximately 1.5 cm length of spinal cord tissue centered at the injury site was taken, placed in paraformaldehyde overnight at 4 °C, dehydrated in 30% sucrose solution at 4 °C until sinking. Then the tissue was embedded in an optimal cutting temperature, 20-µm sections were cut, and frozen at − 80 °C.
Detection of macrophage markers using western blot analysis
The collected spinal cord tissues were homogenized in RIPA lysis buffer (Beyotime Biotechnology, China) at 4 °C for 5 min to obtain 10% homogenate. The homogenates were lysed in RIPA buffer at 4 °C for 45 min, centrifuged at 12000r/min for 5 min, then the supernatant were collected. The BCA protein assay kit was used to measure total protein concentration according to the manufacturer's protocol. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in TBS and Tween-20 for 1.5 h, incubated with primary antibodies against M1 markers IL-1β, TNF-α and M2 markers IL-10, Arginase-1. Immunoblots were visualized using the ECL western blotting detection system
Detection of macrophage markers using immunofluorescence staining
Sections were permeabilized and blocked in 0.2% Triton X-100 in PBS, and incubated with primary antibodies against Arginase-1 (1:100, WACO, Osaka, Japan), IL-10 (1:200, WACO, Osaka, Japan), TNF-α (1:30,, WACO, Osaka, Japan), IL-1β (1:200, WACO, Osaka, Japan) overnight at 4 °C. After the sections were rinsed, the sections were incubated for 2 h at room temperature with species-specific secondary antibodies, including CY3 Goat Anti-Rabbit IgG Antibody (1:100 dilution), Alexa-594 (red)-conjugated TNF-αand IL-1βantibodies (1:250, Invitrogen, USA), Alexa Fiuor-488 (green)-conjugated Arginase-1, IL-10 antibodies (1:250, Invitrogen, USA). Finally, sections were washed, mounted with DAPI, and visualized using fluorescent microscope (DM3000) or a confocal laser scanning microscope (TCS SP5 II; both Leica Microsystems, Inc., Germany). Three non-overlapping fields at the injury site were randomly selected in each slice at 400 magnification. Positively stained cells were counted from the nine randomly selected microscopic fields by using the Image-Pro Plus (IPP 6.0) software and the mean values were calculated.
Detection of macrophage markers using flow cytometry
1 ml of PBS containing 0.5% bovine serum albumin and 1 × 106 cells were added to each flow tube, and incubated with PE-conjugated CD16/CD32 monoclonal antibody (clone 93, 0.125 µg per test, BD Biosciences, USA), and APC-conjugated Arginase-1 monoclonal antibody (A1exF5, 1.0 µg per test) at 4 °C in the dark for 30 min. After centrifugation at 15000r/mim for 5 min, the supernatant was discarded, then cells were washed once with 1 ml PBS, and centrifuged at 15000r/mim for 3 min. The supernatant was removed and the cells were resuspended in 0.2 ml PBS containing 0.5% BSA. Data were analyzed using flow cytometer (Attune NxT, Thermo Fisher Scientific, Inc. USA).
Detection of macrophage markers using real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using the Trizol reagent (Thermo Fisher Scientific, Inc. USA). Reverse transcription for DNA was synthesized using PrimeScript™ reverse transcription kit (Takara, Japan). RT-qPCR was performed in a 10 µL reaction system using UltraSYBR premixture (CWbio Co., Ltd., Beijing, China). RT-qPCR conditions included 10 min of predenaturation at 95 °C, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing/ elongation at 60 °C for 1 min. The dissolution curve conditions were 95 °C for 15 S, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 15 s. The relative quantification was calculated using the 2−ΔΔCT method with reference genes β-actin.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.01 software. Measurement data were expressed as mean ± standard deviation (mean ± SD). Differences in the results from RT-PCR and immunofluorescence staining were analyzed using t test, and differences in the results of behavioral scoring were analyzed using repeated measures Analysis of Variance (ANOVA). A difference with a P-value of < 0.05 was considered statistically significant.