Animal Groups and SCI Model
Female Sprague-Dawley (SD) rats (Anhui Medical University) were chosen as the experimental animal in the present study (8-10 weeks old, weighing 230-260 g). All rats were housed under a 12 h light/dark cycle pathogen-free condition with a controlled temperature of 24 ± 2 °C and 60 ± 5% humidity and free access to food and water. All experiments and procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The study protocols regarding the animals were approved by the Ethics Committee of Anhui Medical University of China (No.LLSC 20201135). All rats were divided into three groups: Sham, SCI_Day 3 and SCI_Day 14.
The SCI model was induced via modified version of Allen’s method as described previously. Animals were anesthetized by 10% chloral hydrate (3 ml/kg, intraperitoneal injection). When the corneal reflex disappeared, T10 laminectomy was performed to expose the spinal cord under an aseptic condition. Subsequently, the rats in SCI group underwent a surgery using an impactor (10 g weight × 5 cm height) to induce T10 spinal cord injury and the rats in sham group were accepted the T10 laminectomy only. The bladder was evacuated manually twice a day until bladder function was recovered.
Behavioral Assessment for SCI
The classical Basso, Beattie and Bresnahan (BBB) scale was used here for the behavioral assessment. BBB scale ranges from 0 to 21 points, which means complete hindlimb disability and normal locomotor function. Rats were assessed to evaluate locomotor function 30 min preoperatively and 1, 3, 7 and 14 days postoperatively. To avoid experimental errors, the rats should be allowed to move freely for 5 minutes in the open filed before every evaluation. To avoid bias results, assessments were executed by three researchers that were blinded to animal groups.
Hematoxylin-Eosin, Nissl and Perls-Blue Staining
Necrosis cavity, number of motor neurons and iron accumulation was observed via HE, nissl and perls-blue staining severally. The rats were sacrificed and one centimeter of the SCI tissue was removed, 5mm to the rostral and caudal centered around epicenter lesion. Then the specimens went through the steps that included fixation, dehydration, embedded, section (horizontal sections, thickness 4 μm) and incubation (65℃ for 20 minutes). After incubation, slices were stained by hematoxylin-eosin, nissl staining solution with cresyl violet (G1430, Solarbio) and prussian blue iron stain kit with nuclear fast red solution (G1422, Solarbio) respectively.
Transmission Electron Microscope
Rats were perfused with transmission electron microscope (TEM) fixation liquid (Paraformaldehyde 2% - Glytaraldehyde 2.5% in PBS, P885738, Macklin). Epicenter lesion samples were collected from removed spinal cords cut into sections (70-90 nm). After post-fixed in 2% osmium tetroxide, dehydrated in ethanol and then embedded in eponate, sections were placed on a copper mesh and stained with 2% uranyl acetate and 0.04% lead citrate. These sections were observed and imaged with TEM (Talos L120C G2, Thermo Scientific).
Iron and MDA Concentration Test
Iron accumulation in SCI was tested using the tissue iron assay kit (A039-2-1, Nanjing Jiancheng Bioengineering Institute). Malondialdehyde (MDA), as a natural product of lipid peroxidation, was quantified using Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime). The protocols of iron and MDA concentration tests were performed according to the menufacturer’s instructions and the absorbance was measured at 532 nm and 520 nm respectively.
ROS production was determined by chemiluninescence using the fluorescent probe 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, WanleiBio). After anesthetization, rats were perfused transcardially with precooled PBS and the spinal cords were digested by pancreatin to obtain single-cell suspensions. Cells were incubated with DCFH-DA (10 μM) at 37℃ for 20 minutes. Later, the cells were observed and pictured via the inverted fluorescence microscope (Axio Observer 3, Germany). The ROS fluorescence intensity was calculated by software Image J.
Protein Extraction and Western Blot Test
The isolated spinal cords were homogenated with RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor (Beyotime Biotechnology, Shanghai, China) for 30 minutes on ice. The homogenates were centrifuged at 12000 g for 25 minutes at 4°C and supernatants were collected. NanoDrop 2000C (Thermo Scientific, USA) was then used for protein concentrations determination. The homogenates and loading buffer 5× (Beyotime Biotechnology, Shanghai, China) were mixed at a ratio of 4: 1, denatured in the metal bath (100°C, 8~10 mins), cooled to room temperature and loaded. Protein samples were subjected to 12.5% SDS-PAGE (Epizyme Biotech, Shanghai, China) and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in protein free rapid blocking buffer (Epizyme Biotech, Shanghai, China) for 15 minutes at room temperature. After blocking, membranes were incubated with the primary antibodies against ATF3 (DF6660, Affinity Biosciences), XBP1 (WL00708, WanleiBio), HMOX1 (WL02400, WanleiBio), DDIT3 (GADD153, Proteintech), CHAC1 (DF9353, Affinity Biosciences) and GAPDH (AF7021, Affinity Biosciences) overnight at 4°C. Membranes were incubated with the secondary antibody at room temperature for 1.5 h. Protein bands were captured using an ECL chemiluminescence system (Epizyme Biotech, Shanghai, China).
Statistical analysis and graphs were both performed by GraphPad Prism 8.0 software. BBB scores comparisons were analyzed by LSD t-test. Iron content, MDA content and ROS fluorescence intensity comparisons were analyzed using one-way analysis of variance (ANOVA). P < 0.05 indicated that the difference was statistically significant.
The microarray expression profiling dataset GSE45006 was download from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The gene sets of ferroptosis markers and regulators were download from FerrDb (http://www.zhounan.org/ferrdb/). GSE45006 was based on GPL1355 [Rat230_2] Affymetrix Rat Genome 230 2.0 Array. GSE45006 contained 24 samples consisting of 4 intact spinal cords and 20 injured spinal cords that were from different time points. We grouped the injured spinal cords by 1day, 3days, 7 days, 14 days and 56 days and the 4 intact spinal cords were defined as control group.
Analysis of DEGs
We used GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) to compare samples of control group and 5 injured groups in order to identify genes that are differentially expressed at different time point after SCI. Genes obtained from GEO2R were screened by following criteria: (1) adjusted P-value < 0.05 and (2) |logFC| ≥ 0.5. The intersecting part of screened genes and ferroptosis makers was identified using the Venn diagram webtool (http://bioinformatics.psb.ugent.be/webtools/Venn/) and this part was considered as DEGs.
GO and KEGG Enrichment Analysis of DEGs
The Database for Annotation, Visualization and Integrated Discovery (DAVID v6.8, https://david.ncifcrf.gov/home.jsp) was used in this step to carry the Gene Ontology (GO) functional enrichment analysis on DEGs. The GO annotation analysis was focused on biological process (BP), molecular function (MF), and cellular component (CC). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichmennt analysis was performed using the KOBAS online tool (http://kobas.cbi.pku.edu.cn/). Adjusted P-value < 0.05 was considered as the cutoff criteria. Bar charts of GO enrichment analysis were visualized by “ggplot2” package of R software, GOChord plot and the bubble plot for KEGG enrichment analysis was produced by http://www.bioinformatics.com.cn, a free online platform for data analysis and visualization.
Gene Set Enrichement Analysis
Gene set enrichment analysis (GSEA) was performed for all time points to prevent loss of ferroptotic genes that were out of threshold we set but crucial to SCI. All the ferroptosis marker genes of each time point were defined as the target gene set. Background gene set was combination of KEGG and GO (BP, MF, CC) gene sets. An absolute value of the standardized enrichment score (NES) > 1 and p-value < 0.05 was taken as the cutoff criteria of statistical significance[19, 20, 21]. Packages “org. Rn.eg.db”, “ClusterProfiler”, “enrichplot”, “gseaplot2” of R software were used for GSEA analysis and visualization.
PPI Network Analysis and Hub Gene Identification
STRING (https://string-db.org/) is a database of known and predicted protein-protein interactions that include direct (physical) and indirect (functional) associations. We used the STRING to construct the PPI network of DEGs under the condition that combined score of PPI pairs was greater than 0.4. Visualization of PPI network was completed via Cytoscape software v3.7.2 (www.cytoscape.org). CytoHubba, a plugin in Cytoscape software, can predict and explore important nodes and subnetworks in a given network by several topological algorithms. We used CytoHubba to calculate the MCC score of each protein node and top six genes were identified as hub genes.
Selection of Key Genes for Ferroptosis Regulation in SCI
We focused on the links between differentially expression and ferroptosis regulation of hub genes obtained from 1 day, 3 days, 7 days, 14 days and 56 days. The hub genes that exist in acute phase, sub-acute phase and chronic phase of SCI were identified as the key genes. The regulating effect of the key genes in the process of ferroptosis were checked in the Ferroptosis driver, suppressor and marker dataset downloaded from FerrDb. The function of key genes were annotated by the GeneCard database (https://www.genecards.org/).