All the animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Anhui Medical University (No. LLSC20160052). C57BL/6J female mice at 6–8 weeks of age were purchased from the Experimental Animal Center of Anhui Medical University. The mice were housed in a temperature- and humidity-controlled room with a 12:12-light/dark cycle and allowed free access to food and water.
2.2 SCI model
All the surgical procedures were performed under pentobarbital anesthesia. The skin around the injury site was shaved and disinfected using iodophor. Subsequently, the T10 spinal cord was exposed via a dorsal laminectomy, and moderately severe crush SCIs were made using No. 5 Dumont forceps (Fine Science Tools, 11252-20, Heidelberg, Germany) ground down to a tip with a width of 0.5 mm by compressing the cord laterally from both sides for 5 s . Then, twitching of the hind limbs and movement of the tail were observed, which indicated that the SCI model was successfully established. Finally, the wound was sutured with 3 − 0 silk threads. The mice with SCI were examined daily to monitor their recovery, and their bladders were expressed manually three times a day until the return of reflexive bladder control. The sham group were subjected to laminectomy alone. The mice were sacrificed at 3, 7 and 14 days after SCI.
2.3 Microglia depletion.
To eliminate microglia, mice were administered PLX5622 (MedChemExpress, HY-114153) at 130 mg/kg by oral gavage once a day for 17 consecutive days. PLX5622 was diluted in 5% DMSO, 40% polyethylene glycol 300, 5% polysorbate 80, and 50% saline, according to the manufacturer’s instructions. An equal volume of vehicle was used as the control. SCI was established on the third day after gavage.
2.4 Preparation of myelin debris
Myelin debris was isolated as previously described . Briefly, 6- to 8-week-old mice were euthanized, and their brain tissues were harvested and homogenized in ice-cold 0.32 M sucrose. Myelin debris was isolated from the brain tissues by sucrose density gradient centrifugation. The endotoxin concentration of the myelin debris was below the limit of detection of the Limulus Amebocyte Lysate assay (Lonza, Switzerland). Myelin debris was added to cells at a final concentration of 1 mg/mL in all the experiments.
2.5 Cell culture and transfection.
The BV-2 microglial cell line was obtained from the American Type Culture Collection (CRL-3265, ATCC, Manassas, VA, USA) and cultured and maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, SH30021) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270106), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco, Grand Island, NY, USA). The cells were incubated in a humidified chamber at 37°C in a 95% O2 and 5% CO2 atmosphere. Small interfering RNA (siRNA) was transfected into these cells using jetPRIME (Polyplus Transfection, 114 − 15), according to the manufacturer's instructions. siRNA targeting mouse Fascin-1 (siRNA: 5’- GAUGCCAACCGUUCCAGUUTT − 3’) and nonspecific control siRNA (NC) were purchased from GenePharma (Shanghai, China).
2.6 Microglial polarization
BV-2 cells were plated in poly-d-lysine (PDL, Sigma, P7280)-coated 6-well plates at a density of 1×106 cells/ml and cultured overnight. After serum deprivation for 24 h, the BV-2 cells were polarized toward the M1 phenotype by treatment with lipopolysaccharide (LPS; 100 ng/ml, Beyotime Biotechnology, ST1470, Shanghai, China) and IFNγ (20 ng/ml, Beyotime Biotechnology, P6137) or toward the M2 phenotype by treatment with IL-4 (20 ng/ml, Beyotime Biotechnology, P5916), and the cells were cultured for 24 h .
2.7 Tissue processing
For Western blot analysis, the mice were anesthetized and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) to remove the blood. Spinal cord tissues of 5 mm centered at the injury site were harvested. For histological analysis, once the blood was removed, the mice were transcardially perfused with 4% paraformaldehyde (PFA), and a 5-mm portion of the spinal cord encompassing the injury epicenter was extracted and embedded in paraffin. Then, the tissues were sagittally sectioned at thicknesses of 6 µm on a microtome (Leica RM2235). Every tenth section was collected and mounted onto a series of slides.
2.8 Immunofluorescence analysis
Immunohistochemistry. Six-micrometer, paraffin-embedded spinal cord sections were dried, dewaxed, hydrated and subjected to antigen repair. Next, 10% donkey serum albumin (DSA, Solarbio, SL050) containing 0.3% Triton X-100 (Solarbio, T8200) was added and incubated at room temperature for 1 h. Then, primary antibodies were added and incubated at 4°C overnight. The primary antibodies used were as follows: mouse anti-Fascin-1 (1:50, Santa Cruz, sc-21743), rabbit anti-Fascin-1 (1:100, Abcam, ab126772), goat anti-Iba1 (1:200, Novus Biologicals, NB100-1028), rabbit anti-CX3CR1 (1:500, Abcam, ab8021), rabbit anti-GFAP (1:100, Proteintech, 16825-1-AP), mouse anti-GFAP (1:100, Proteintech, 60190-1-Ig), goat anti-PDGFRβ (1:40, R&D Systems, AF1042-SP), rabbit anti-PDGFRβ (1:200, Abcam, ab32570), mouse anti-Mac2 (1:100, GB12246, Servicebio), rabbit anti-NG2 (1:100, Proteintech, 55027-1-AP), rabbit anti-iNOS (1:100, Affinity, AF0199), rabbit anti-Arg1 (1:100, Affinity, DF6657), and rabbit anti-NeuN (1:500, Abcam, ab177487). The secondary antibodies were diluted in 1% donkey serum in PBS and incubated for 1 h at room temperature. The following secondary antibodies were used: Alexa Fluor 488 and Alexa Fluor 594 (1:500, Thermo Fisher Scientific, A-21206, A-21202, A-21203, A-21207, A-11058). The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/ml, Thermo Fisher Scientific). The fluorescence signals were obtained using an Axio Scope A1 microscope (Zeiss, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis.
Immunocytochemistry. Cells were fixed with 4% PFA for 10–15 min and blocked with 5% donkey serum in PBS for 30 min at 20–25°C. The primary antibodies (as listed above) were diluted in 1% donkey serum in PBS and incubated overnight at 4°C. The secondary antibodies (as listed above) were diluted in 1% donkey serum in PBS and incubated for 1 h at room temperature. Images were acquired as described above.
2.9 Imaging analysis and quantification
Every tenth 6-µm-thick, paraffin-embedded section was quantified, resulting in 5 analyzed slides per animal that included the entire injured spinal cord. The total number of Fascin-1+, CX3CR1+, and Fascin-1+CX3CR1+ double-positive cells in each of the sagittal sections of the spinal cord was counted under a 20× objective lens with Zeiss ZEN imaging software (Zeiss, Germany).
2.10 Western blot analysis
Tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, R0278) supplemented with protease inhibitors (Roche, 04693124001) and phosphatase inhibitors (Roche, 04906845001). The cells were washed with cold PBS, homogenized in RIPA buffer on ice for 30 min, and then centrifuged at 12,000 rpm at 4°C for 30 min. The protein extracts were quantified by using a Pierce BCA protein assay kit (Beyotime Biotechnology, P0010S). Aliquots of the protein samples containing equal protein concentrations were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and subsequently transferred to polyvinylidene (PVDF) membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.5% Tween-20 (TBST) for 1 h at room temperature and then incubated with primary antibodies, including mouse anti-GAPDH (1:2000, Proteintech, 60004-1-Ig), rabbit anti-Fascin-1 (1:5000, Abcam, ab126772), rabbit anti-iNOS (1:3000, AF0199, Affinity) and rabbit anti-CD206 (1:1000, ab64693, Abcam), at 4°C overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (1:10000, Sigma, A4416) and HRP-conjugated goat anti-rabbit secondary antibodies (1:10000, Sigma, A0545) for 1 h at room temperature. The protein band signals were obtained using an ECL detection kit (ECL, Thermol Biotech Inc., USA) and Tanon 5200 system (Tanon, Shanghai, China). ImageJ was used for quantification analysis. The intensity of the GAPDH bands was used for normalization.
2.11 Scratch assay
Cell migration was assessed by performing a scratch assay. Briefly, BV-2 cells were seeded into PDL-coated 6-well plates at a density of 2 × 105 cells/well and incubated for 24 h. The cell layers were scratched using a 200-µl pipette tip to form a wound-like gap. The cells were then maintained in DMEM with 2% FBS, and images were captured at 0 h, 24 h and 48 h after cell scratching. ImageJ was used to analyze the wound width.
2.12 Transwell assay
A 24-well plate containing a 3-µm chamber (Costar, 3415) was used to assess the migration abilities of the cells. BV-2 cells were transfected and activated in a PDL-coated 6-well plate before inoculation into the chamber. Then, the cells were suspended in serum-free media and seeded into the upper chamber (5 × 104 cells per chamber). The lower chamber contained complete media. After incubating for 12 h, the nonmigrating cells on the inside of the membrane were carefully removed with a cotton swab, and the migrating cells on the outside of the membrane were fixed with 4% PFA at room temperature for 20 min and stained with 0.1% crystal violet for 15 min. The cells were observed under a microscope and counted. At least 5 random fields were photographed, and the cells in each field were counted.
2.13 Statistical analysis
All the experiments were independently performed with at least three replicates and quantified in a blinded manner. The data are presented as the mean ± standard error of the mean (SEM). The statistical analysis was carried out with SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) software. Student’s t test to compare the difference between 2 groups and One-way analysis of variance followed by Tukey’s post hoc test was used to compare differences among multiple groups. Differences were considered statistically significant if P < 0.05.