Animals and ethics statement
Eight-week-old C57BL/6 female mice (weighing approximately 20-23 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and housed in the Department of Laboratory Animals, Central South University (Changsha, China). All animals had free access to food and water and lived in a temperature-suitable room under natural light. All procedures carried out were approved by the Animal Ethics Committee of Central South University.
Establishment of chronic spinal cord injury
Thirty-eight mice were randomly divided into two groups: a control group and a CSCI group (19 mice per group). Based on previous research, we applied a water-absorbable polyurethane polymer (Guangzhou Fischer Chemical Co., Guangzhou, China) to construct the CSCI model26. In brief, the mice were anesthetized with 75 mg/kg sodium pentobarbital. The dorsal skin at the T10 level was incised, followed by fascia and muscle stripping to expose the T10 lamina. A portion of the lamina was removed, and a polyurethane polymer sheet (1.5 mm*1.0 mm*0.5 mm) was inserted into the epidural space. For the mice in the control group, after the T10 lamina was exposed, a portion of the lamina was removed without placing a polyurethane polymer sheet in the epidural space. Then, the muscles, fascia and skin were sutured sequentially. After the mice were awake, they were returned to their rearing cages. For the next three days, the mice were injected with penicillin daily to prevent infection.
Histological and immunofluorescence staining
Four weeks after injury, the mice were anesthetized and sequentially perfused from the left ventricle with heparinized saline and 4% paraformaldehyde. Subsequently, a 10 mm length of spinal cord containing the injured area was harvested and dehydrated for sectioning. For histological staining, samples were dehydrated in gradient alcohol and embedded in paraffin. Spinal cord segments were transected into 8 µm thick slices and stained with an HE staining kit (Solarbio, China) according to the manufacturer’s instructions.
For immunofluorescence staining, samples were dehydrated with sucrose solution and embedded with OCT compound (Sakura, United States). The spinal cord was sectioned longitudinally into 16 µm thick slices and incubated overnight at 4 °C with anti-Mettl14 antibody (1:100, Zen Bio, China) and anti-NeuN antibody (1:400, Abcam, United States). After washing with PBS, the slices were incubated at room temperature for 1 hour with secondary antibodies. 4',6-Diamidino-2-phenylindole (DAPI, GeneTex, United States) was used to stain the cell nuclei.
Quantitative Real-time PCR (qRT–PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, United States). Complementary DNA (cDNA) was synthesized with a reverse transcription kit (Promega, United States). GoTaq® qPCR Master Mix (Promega, United States) and primers (Sangon Biotech, China, Table S1) were added to the cDNA mixture for qRT–PCR. CT values were detected using a quantitative PCR system (ABI, United States). GAPDH was used as an internal reference, and the relative gene expression was calculated using the 2 (−ΔΔCt) method.
Western blotting
Total protein was extracted from the spinal cord using RIPA lysis buffer (Beyotime, China), and the protein concentration was detected with a BCA kit (Beyotime, China). The denatured proteins were separated via electrophoresis using 10% SDA-PAGE gels and then electrotransferred to PVDF membranes (Millipore, United States). Then, the membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with anti-Mettl14 antibody (1:1000, Zen Bio, China) and anti-β-actin antibody (1:2000, Cell Signaling Technology, United States). The next day, the membranes were washed with 0.1% TBST solution and incubated with secondary antibody for 90 minutes. Enhanced chemiluminescence (ECL) reagent was used to detect the membrane bands.
Locomotor function assessment and neuroelectrophysiological analysis
Basso mouse scale (BMS) scoring was used to evaluate locomotor function as previously described27. The BMS score ranged from 0-9 (0 indicates complete paralysis, and 9 indicates normal locomotion). BMS scores were evaluated at 1, 3, 7, 14, 21 and 28 days postinjury. Neuroelectrophysiological analysis was performed at 28 days postinjury as described in our previous research28. Stimulating electrodes were placed on the surface of the skull (caudal/lateral to the bregma: 1 mm/0.5 mm, -4 mm/0.6 mm). Recording electrodes were inserted into the tibialis anterior muscle. The reference electrode was placed under the dorsal skin. Motor evoked potentials (MEPs) were documented to assess neurological connectivity.
RNA extraction, meRIP-seq and mRNA-seq
meRIP-seq and mRNA-seq were carried out by Seqhealth Technology Co., Ltd. (Wuhan, China). Total RNA was extracted from the spinal cord area of CSCI and the same segments in the control group using TRIzol (Invitrogen, United States) and enriched with Oligo d (T) magnetic beads. A total of 150 µl of each sample was taken, and a NanodropTM OneC spectrophotometer (Thermo Fisher, United States) was used to detect the concentration and purity of the RNA. An RNA Nano 6000 Assay Kit (Agilent, United States) was applied to detect the RNA quality number (RQN). The quality of the RNA library was evaluated using agarose gel electrophoresis. Finally, the qualified RNA library was quantified using Qubit 3.0 with a QubitTM RNA Broad Range Assay kit (Life Technologies, United States). VAHTS mRNA Capture Beads (Vazyme, China) were used for polyadenylated RNA enrichment. Then, 20 mM ZnCl2 was added to the RNA to obtain fragmented RNA distributed in a range from 100 nt to 200 nt. Ninety percent of the RNA was used for m6A immunoprecipitation (IP) with anti-m6A antibody, and the rest was saved as “Input”. The RNA sequencing library was generated using a KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog no. DR08502, Wuhan Seqhealth Co., Ltd. China) and finally sequenced on a NovaSeq 6000 sequencer (Illumina).
Bioinformatics analysis
Raw reads acquired from the Illumina HiSeq sequencer were filtered using Trimmomatic (version 0.36) to remove low-quality and adaptor-contaminated beads. Clean beads were harvested through UID deduplication. Briefly, we performed cluster analysis on all sequences of the same UID to obtain different clusters. Then, sequence alignments were performed on the multiple sequences under each cluster, thereby obtaining a consensus sequence. The deduplicated clean data were mapped to the reference genome of mice using STAR software (version 2.5.3a). The specific region to which the protein binds (peak calling) was analyzed using exomePeak (version 3.8) software. After m6A peak annotation with bedtools (Version 2.25.0), the differentially methylated sites were identified with a Python script using Fisher’s test. GO and KEGG pathway enrichment analyses were carried out using KOBAS software (version: 2.1.1). The threshold of significant enrichment was set as p < 0.05.
Statistics
GraphPad Prism 8.0 was used to analyze the data. Data are presented as the mean ± standard deviation (SD). Unpaired Student’s t test was applied to compare differences between two groups, such as in qRT–PCR, western blotting, and fluorescence quantitative analyses. Repeated-measures two-way ANOVA was used to analyze BMS scores. p < 0.05 was considered to indicate a significant difference (*p < 0.05, **p < 0.01).