Adult male C57BL/6 mice, 10 weeks old (weight 20–25 g), purchased from the Experimental Animal Center of Fujian Medical University, were raised in a clean temperature-controlled environment (23 ± 2 °C), with a 12 h light/dark cycle and with food and drink available ad libitum. The experimental protocol of this study, including all surgical operations and animal use, was approved by the Experimental Animal Ethics Committee of Fujian Medical University and carried out in accordance with the National Institutes of Health (Bethesda, MD, USA) "Guidelines for the Care and Use of Laboratory Animals".
The mice were randomly divided into a Sham group, TBI group, TBI+Vehicle group, and TBI+NADP group (n = 24), and each group was divided into four subgroups (1-, 3-, 7-, and 14-day time points). The controlled cortical impact (CCI) injury model used in here was established as previously described [31, 32]. Anesthesia was administered in a Perspex container using 3% isoflurane delivered by a animal anesthetic machine (RWD Life Science Co., Shenzhen, China). Mice were then subjected to an impact using a 2 mm metal flat-tip impactor (Brain and Spinal Cord Impactor, 68099H, RWD Life Science). The center of impact was bregma 2.5 mm and lateral 2.5 mm. The velocity was 5 m/s, depth 3 mm, and impact speed 100 ms. In the sham operation group, the surgery was performed but the impact was omitted. Mice in the TBI+NADP group were injected with NADP (300 mg/kg/day; Sigma–Aldrich, St. Louis, MO, USA) 0.5 h after the surgery , those in the TBI+Vehicle group were given an equal volume of the vehicle dimethyl sulfoxide as a negative control.
Neurological impairment score
Mice were subjected to exercise (muscular phenotype and abnormal action), sensation (visual, tactile, and balance), and reflex examinations and assigned a modified neurological severity score (mNSS). A score was recorded when the mice failed to complete the task or showed no corresponding reflexes. The mNSS score ranged from 0 to 18 points, where a total score of 18 points indicated severe neurological deficits and a score of 0 indicated normal performance. Researchers, blinded to the experimental groups, measured the neurological function of mice at different time points.
Measurement of brain water content and blood brain barrier (BBB) permeability
The wet weight-dry weight method was used to calculate the brain water content8. The animals were sacrificed after neurological assessment, and the cerebral cortex was excised at the edge of the bone window. Filter paper was used to remove excess blood and cerebrospinal fluid. The wet weight was measured and the brains were dried in an oven for 24 h at 100 °C until a constant weight was achieved, at which point the dry weight was measured. The percentage of brain water content was calculated as (wet weight − dry weight)/wet weight × 100%.
BBB permeability was investigated by measuring the extravasation of Evans blue dye (2% in saline; 4 mL/kg; Sigma-Aldrich), which was injected intravenously 2 h prior to sacrifice on the third day after injury. Following sacrifice, the mice were transcardially perfused with PBS followed by PBS containing 4% paraformaldehyde. Each tissue sample was immediately weighed, homogenized in 1 mL of 50% trichloroacetic acid, and centrifuged. Then, the absorption of the supernatant was measured with a spectrophotometer (UV-1800 ENG 240V; Shimadzu Corpomiceion, Kyoto, Japan) at a wavelength of 620 nm. The quantity of Evans blue dye was calculated using a standard curve and expressed as µg/g of brain tissue.
The formaldehyde-fixed specimens were embedded in paraffin, cut into 4 μm thick sections, deparaffinized with xylene, and rehydrated in a graded series of alcohol. After being treated with Nissl staining solution for 5 min, the damaged neurons were atrophied or contained vacuoles, while the cells of normal neurons were larger and fuller with larger nuclei. Five areas were randomly selected for microscopic examination by a researcher who was blinded to the to the experimental groups.
Cell culture and treatment
Mouse microglia and BV2 cells were provided by Basic Medical Sciences Chinese Academy of Medical Sciences (Beijing, China) and cultured at 37 °C, 95% O2, 5% CO2, in Dulbecco's Modified Eagle's Medium (KeyGEN Biotech,Nan jing, China) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (KeyGEN Biotech). BV2 cells were stimulated with 10 μg/ml LPS (Meilune, China) for 2 h to induce inflammation. The ADAM17 inhibitor TAPI-1 (1 μM: MCE, China) was applied to the BV2 cell groups for downstream study of the FTO/m6A/ADAM17 signaling pathway-mediated polarization.
FTO siRNAs and plasmids were utilized to knock down (siFTO group) and overexpress FTO in BV2 cells (oeFTO group), to explore the function of FTO in regulating the microglial polarization. BV2 cells were transfected with FTO siRNA and pcDNA3.1-Flag-FTO, respectively. Mouse FTO siRNA and negative control were chemically synthesized by RiboBio (Guangzhou, China). The mouse pcDNA3.1-Flag-FTO plasmid and vector were obtained from Honorgene (Changsha, China). The sequence for the control siRNA was as follows: 5′-AAGGCUCUAUGAAGAGGCUTG-3′. The three sequences for the FTO siRNAs were as follows (FTO siRNA01-siRNA03): 5′-AGACCUUCCUAAAGCUCAAUG-3′, 5′-GCACCUACAAGUACUUGAACA-3′ and 5′-CCAAAGAUGAUGAGUUCUAUC-3′. The FTO siRNA and FTO plasmid, and its corresponding negative control, were transiently transfected into BV2 cells with Lipofectamine 2000 for 24 h at 37 ℃, according to the manufacturer’s instructions and previous work [34, 35]. After, the transfected BV2 cells were treated with the ADAM17 inhibitor, TAPI-1 (1 μM), for another 48 h at 37 °C for signaling pathway assessment.
RNA isolation and RT-PCR
Total RNA from the tissues or the cultured samples was purified using TRIzol (Invitrogen, ThermoFisher Scientific) and reverse transcribed using the ABI reverse transcriptase (ABI, ThermoFisher Scientific), oligo (dT) primers, or specific RT primers. Template (1 µL) was amplified by real-time PCR using the primers listed in Suppl. Table 4, Supporting Information (Integrated DNA Technologies). Each sample was run in triplicate in a 10 µl reaction with 100 nm forward and reverse primers, 2µl of SYBR Green mix (ABI, ThermoFisher Scientific), and 10 ng cDNA. The PCR reactions were carried out using a STEP-ONE 96 real-time PCR system. GAPDH was used as an internal control for normalization. Ratios of mRNA levels from the treated groups or mRNA at levels different time points compared with the mRNA level of the normal control group were calculated using the ΔCt method (2−ΔΔCt). All data were normalized to GAPDH.
RNA m6A quantification
Total RNA was isolated with TRIzol (Invitrogen, ThermoFisher Scientific) according to the manufacturer’s instructions and RNA quality was measured by using a NanoDrop3000. The m6A RNA methylation quantification kit (Abcam, UK) was used to measure the m6A content of the RNA. Briefly, 200 ng of RNA was detected in each well. The capture antibody solution and detection antibody solution were then added to assay wells separately in a suitable diluted concentration following the manufacturer’s instructions. The m6A levels were quantified calorimetrically by reading the OD450 absorbance of each well and calculations were performed based on the standard curve.
RNA m6A sequence and m6A-RNA immunoprecipitation assay
The chemically fragmented RNA (100 nucleotides) was incubated with the m6A antibody and immunoprecipitation was performed according to the standard protocol of the Magna methylated RNA immune-precipitation (MeRIP) m6A Kit (Merck Millipore, USA). Enrichment of m6A containing mRNA was analyzed by qRT-PCR using the primers listed in Suppl. Table 5. For high-throughput sequencing, the RNA fragments purified from m6A-MeRIP were used for library construction with the NEBNext® Ultra™ RNA Library Prep Kit (NEB, USA) and sequenced with an Illumina SE50. Sequencing reads were aligned to the mouse genome mm9/mm10 by Bowtie2, and the m6A peaks were detected by magnetic cell sorting as described .
Peak annotation and motif identification
MACS2 (version 184.108.40.20651222) was employed to identify m6A peaks, after which Hommer (version: 4.8) was used to annotate the m6A peaks. The nucleotides in m6A peak regions were used for detection of the consensus m6A motif by DREME (version: 4.11.1) and MEME (version: 4.11.1). Motif central enrichment was performed by CentriMo (version: 4.11.1). Differential methylation was determined by Pyicoenrich (version: 2.0.7).
Luciferase reporter assays and mutagenesis assay
The dual-luciferase vector pmiGLO (Promega, USA) was used to identify the m6A functional site of ADAM7. The CDS and 3’UTR regions of ADAM17 (NM_009615.6) were amplified by PCR using the genomic DNA from BV2 cells as a wild type template. Three putative m6A recognition sites were identified in the CDS and 3’UTR. A QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, USA) was used to generate a point mutation (alanine to thymine) according to the manufacturer’s instructions. Luciferase activity was measured with a Dual Luciferase Reporter Gene Assay (Beyotime, China) on an MD SpectraMax M3 plate reader. Experiments were performed in triplicate. Data are presented as mean values ( ±SD).
The formaldehyde-fixed specimens were embedded in paraffin, cut into 4 μm thick sections, deparaffinized with xylene, and rehydrated in a graded series of alcohol. TAntigen retrieval was carried out by microwaving the sections in citric acid buffer. Sections were then incubated with an antibody against FTO (1:400, abcam, UK), washed, and then incubated with secondary antibody. The negative control was prepared without adding the primary antibody. Five randomly selected visual fields were analyzed as follows [3, 8, 37]: 0, no positive cells; 1, very few positive cells; 2, moderate number of positive cells; 3, many positive cells; and 4, the highest number of positive cells.
The formaldehyde-fixed specimens were embedded in paraffin, cut into 4 μm-thick sections, deparaffinized with xylene, rehydrated in a graded series of alcohol, and then the antigen was retrieved as describe above. Sections were incubated overnight at 4 °C with antibodies against ionized calcium-binding adapter molecule-1 (Iba-1; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), FTO (1:200; Santa Cruz Biotechnology), CD86, and CD206 (1:100; Boster Biotech, Wuhan, China). After washing, the sections were incubated with secondary antibodies for 1 h at room temperature, after which the cell nuclei were stained with 4',6-diamidino-2-phenylindole. Immuno-positive cells in five randomly selected fields were counted under a microscope (Leica, Wetzlar, Germany) at 400× magnification by investigators who were blinded to the experimental groups.
Fluorescent in situ hybridization (FISH) combined with immunofluorescence imaging
The Cy3-labeled probes of ADAM17 mRNA were designed and synthesized by Sangon Inc. (Shanghai), while mouse monoclonal antibody to FTO and goat anti-mouse IgG H&L were obtained from abcam (Alexa Fluor® 488, abcam, UK). FISH combined with immunofluorescence experiments were performed according to the manufacturer’s instructions. Bv2 cells were seeded in a 24-well plate on chamber cover slips and treated as described above. After the cells had reached 60%–70% confluency, they were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with pre-cooled 0.5% Triton-X-100 for 5 min at 4 °C, washed three times with PBS, and prehybridized for 30 min at 37 °C with 200 μL pre-hybridization buffer. mRNA ADAM17 FISH Probe Mix Storage solution (2.5 μL, 20 μM; mRNA FISH Probe Mix) and 100 μL hybridization buffer were added and the cells were incubated overnight at 37°C in a humidified chamber in the dark. The cells were then washed three times for 5 min each with 4× SSC and 2× SSC for 5 min and 1 × SSC for 5 min at 42 °C, followed by a 5 min wash with PBS at room temperature in the dark. Finally, glass coverslips were sealed with an anti-quenching adhesive containing DAPI and images were acquired on an IX51 inverted microscope (Olympus, Japan).
Enzyme-linked immunosorbent assay (ELISA)
Inflammatory factors in brain tissue and BV2 cell culture supernatant were detected using mouse ELISA kits (KeyGEN, China) for TNF-α, IL-1 β, IL-6, TGF-β1, and IFN-γ. The optical density at 450 nm was measured using a microplate reader (SpectraMax M3, Molecular Devices, Inc.).
Western Blotting Analysis
Samples, including brain tissues and BV2 cells, were prepared by using the nuclear and cytoplasmic proteins purification assay kit (KeyGEN Biotech, China), with modified RIPA lysis buffer (50mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). The protein concentrations of the nuclear and cytosolic lysates, respectively, were determined with a BCA kit (Beyotime, China), and approximately 25 µg of protein was loaded to each lane and transferred to polyvinylidene difluoride membranes. The membranes were then incubated with primary antibodies against B cell lymphoma-2 (Bcl-2; 1:2000), Bcl-2-associated X factor (Bax; 1:5000), cleaved caspase-3 (1:3000), Iba-1 (1:1000), CD86, CD206, IL-1β, iNOS, Arg-1 (1:2000), FTO (1:500), ADAM17 (1:1000), and TGF-β1 (1:1000) (all purchased from Abcam). Immunoreactive bands were visualized with an ECL Western Blotting Detection Kit (Millipore, Billerica, MA, USA). Grey value analysis was conducted uisng UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). The expression levels were normalized against β-actin (1:5000, Boster Biotech, China).
Data were analyzed using SPSS v.18.0 software (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate unless otherwise noted, and the results are expressed as the mean ± SD. The unpaired Student’s t test was used for comparison between groups. Multiple-group comparisons were assessed by one-way ANOVA and post hoc multiple comparisons were performed using Student-Newman-Keuls tests. P< 0.05 was considered statistically significant.