Isolation and culture of primary cells
One-day-old mice were sacrificed in 75% ethanol, the brains removed to precooled Dulbecco’s modified Eagle medium (DMEM; KeyGEN, Nanjing, China) and the meninges and blood vessels removed. The cerebral cortex was then isolated, placed in new DMEM, cut into pieces and 2 mg/mL papainase (Sigma-Aldrich, St. Louis, MO, USA) added. The cortical tissue was then shaken on a shaker for 10 min at a constant temperature of 37 °C. After centrifugation at 1200 g for 5 min, the supernatant was removed and cells were resuspended and incubated in DMEM containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C. For neuronal isolation, the cells were resuspended in neurobasal medium (Gibco) after 4 h cultured, then the medium was changed every 2 days. For microglia isolation, the medium was changed every 3 days. After 14 days, microglial cultures were shaken on a shaker for 4 h at a constant temperature of 37 °C and the cells collected in 6-well plates (Corning, NY, USA) containing DMEM containing 10% FBS. Microglia were pretreated with the p38 MAPK inhibitor BIRB 796 (500 nM, Axon Medchem, Groningen, The Netherlands) for 6 h or the NLRP3 inhibitor glyburide (50 mM, Sigma-Aldrich) for 2 h, after which LPS (1 μg/mL, Sigma-Aldrich) was administered to activate microglial inflammation for 24 h.
Co-culture of microglia and neurons
To mimic the effect of microglial inflammation on neuronal apoptosis in vitro, a co-culture model was established as shown in Fig. 3A. Neurons (in 500 μL of neurobasal medium) were incubated on the bottom of the 24-well plates (Corning) for 24 h, after which cell inserts (Corning) were placed in each well and the treated microglia, suspended in 200 μL medium, were seeded on the top of the inserts and cultured for 72 h.
SCI model
C57BL/6 J adult mice, (males, average weight of 20 g, 8 weeks of age) purchased from Charles River (Beijing, China), were anesthetized with ketamine (80 mg/kg). The SCI protocol used here was the same as that in our previous study [21]. Briefly, the skin was cut and the fascia muscle separated on the back, after which the lamina at T10 segment was resected. A moderate contusion (5 g × 5 cm) was created on the spinal cord of the mouse using a spinal cord impactor (RWD, Shenzhen, China). The SCI mice were treated with artificial micturition twice per day until their cystospasm was eliminated. The sham mice received a T10 laminectomy only.
Regulation of FANCC expression
The FANCC overexpression (OE-FANCC) and shRNA targeting FANCC for knockdown (KD-FANCC) were loaded in plasmids, which were constructed from BIOG CO., LTD (Changzhou, China), and respectively transfected into primary microglial cultures using RFect Plasmid Transfection Reagent (BIOG). The shRNA-FANCC adeno-associated virus (AAV) vector, which was injected into the tail veins of mice at 1 week before SCI modeling, was obtained from Genechem CO., LTD (Shanghai, China).
Real-time quantitative reverse-transcription PCR (qRT-PCR)
Total RNA from microglia and spinal cords was extracted using TRIzol reagent (YiFeiXue Biotechnology, Nanjing, China) according to the manufacturer's instructions. Reverse transcription was performed using the Yfx 1st Strand cDNA Synthesis Kit (YiFeiXue Biotechnology). Quantitative analysis of RNA was accomplished using the Roche LightCycler 480 (Roche, Basel, Switzerland) with a qPCR Kit (YiFeiXue Biotechnology). The primer sequences were as follows: FANCC: forward: GAGACAGGACTTAACTCGTGGA; reverse: AGCCATCCGACTTTGAGTGC; GAPDH: forward: TGACCTCAACTACATGGTCTACA; reverse: CTTCCCATTCTCGGCCTTG.
Western blotting (WB) assay
Total protein was extracted from cells and cord tissue using the Total Protein Extraction Kit (KeyGEN) according to the manufacturer’s instructions, after which the concentration was quantified using the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Total of 60 μg proteins in each group were used for WB. Relevant primary and secondary antibodies used in WB analyses were as follows: anti-FANCC (1:1000, 22,857, Singalway Antibody, CollegePark, MD, USA), anti-p38 (1:1000, 14,451, Cell Signaling Technology, Boston, MA, USA), anti-phospho-p38 (1:1000, 4092, Cell Signaling Technology), anti-NLRP3 (1:1000, 29,125, Singalway Antibody), anti-ASC (1:1000, 40,618, Singalway Antibody), anti-GSDMD (1:1000, ab219800, Abcam, Cambridge, MA, USA), anti-GSDMD-N (1:1000, ab215203, Abcam), anti-Caspase-1 (1:1000, ab138483, Abcam), anti-cleaved-Caspase-1(1:1000, 89,332, Cell Signaling Technology), anti-IL-1β (1:1000, 12,703, Cell Signaling Technology), anti-cleaved-IL-1β (1:1000, 83,186, Cell Signaling Technology), anti-β-actin (1:10,000, HRP-60008, Proteintech, Rosemount, IL, USA), anti-β-tubulin (1:10,000, HRP-66240, Proteintech), HRP Goat-anti-Rabbit secondary antibody (1:10,000, YFSA02, YiFeiXue Biotechnology) and HRP Goat-anti-Mouse secondary antibody (1:10,000, YFSA01, YiFeiXue Biotechnology). Protein signals were captured with a Gel Document System (SYNGENE, Cambridge, UK), followed by quantitative analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Enzyme-linked immunosorbent assays (ELISAs)
Cord tissues (5 mm length) were minced into homogenate and centrifuged at 4 °C, after which the supernatants were stored at − 80 °C until further use. ELISAs were performed to test the levels of IL-1β and IL-18 in supernatants using respective ELISA Kits (YiFeiXue Biotechnology) according to the manufacturer's instructions. The absorbance was then determined at 450 nm using a microplate reader (BioTek, Vermont, USA).
Flow cytometry assay (FCA)
The degree of neuronal apoptosis was detected using the Apoptosis Detection Kit (YiFeiXue Biotechnology) according to manufacturer’s instructions. Neurons were incubated with Annexin V-FITC reagent at room temperature for 10 min, followed by incubation with Propidium Iodide reagent for 5 min. Primary microglia were incubated with F4_80-PE (565,410, BD Biosciences, Franklin Lakes, NJ, USA) and iNOS-FITC (610,330, BD Biosciences) for 30 min at 4 °C, injected into a flow cytometer (FACSVerse 8, BD) and the obtained data were analyzed using FlowJo software (Version 7.6.1; FlowJo, Treestar, OR, USA).
TUNEL staining
Neuronal and microglial death was measured using a TUNEL Staining Kit (Servicebio, Wuhan, China) according to the manufacturer’s instructions. Briefly, neurons and sections of spinal cords were incubated with antigen retrieval solution prior to permeabilization, treated with a mixture of terminal deoxynucleotidyl transferase enzyme, deoxyuridine triphosphates and buffer at ratio of 1:5:50, then were counterstained with diaminobenzidine (DAPI) to visualize the nuclei and observed under a fluorescence microscope (Leica, Oskar, Germany).
Immunofluorescence (IF) staining
After blocking with Immunol Staining Blocking Buffer (Beyotime) for 1 h, sections and cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorescent secondary antibodies in the dark for 1 h. The antibodies used were as follows: anti-FANCC (1:100, 22,857, Singalway Antibody), anti-NeuN (1:100, MAB377; Millipore, Burlington, MA, USA), anti-GFAP (1:300, 3670; Cell Signaling Technology), anti-IBA-1 (1:500, ab178847; Abcam), anti-NLRP3 (1:100, 29,125, Singalway Antibody), anti-iNOS (1:100, ab15323, Abcam), anti-Caspase-1 (1:100, ab138483, Abcam) and anti-NF200 (1:200, ab82259, Abcam). After counterstaining with DAPI, the samples were visualized under a fluorescent microscope (Leica). The fluorescence intensity in the images was analyzed using ImageJ software (National Institutes of Health).
Nissl, hematoxylin–eosin (HE) and Luxol Fast Blue (LFB) staining
To evaluate the number of neurons, morphology of the spinal cord tissue and integrity of the neuronal myelin sheath after SCI, we used Nissl Staining Reagent (Servicebio), HE Staining Reagent (Servicebio) and LFB Staining Reagent (Servicebio), respectively, following the manufacturer’s instructions. The sections were under an optical microscope (Leica).
Behavioral assessment
Locomotor function of hindlimbs was evaluated using the Basso Mouse Scale (BMS) and Louisville Swim Scale (LSS) as previously described [22, 23]. Mice in each group were tested in an open field assessed by two blinded researchists at 1, 3, 7, 14, 21 and 28 days post injury (dpi).
Statistical analysis
Data are shown as the mean ± standard deviation values and were analyzed using Prism software, version 8.3 (GraphPad, San Diego, CA, USA). Comparisons between two groups were analyzed using t-tests and among more than two groups using one-way or two-way ANOVAs followed by Tukey’s post hoc test. P-value < 0.05 were considered as significance.