Adult female Wistar rats (180 ± 20 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China, Permission Number: SCXK (Jing)—2016—0011) and housed for 1 week to acclimate to the environment and until their weight was up to 200–220 g. The experimental animal protocols were approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Tianjin, China, IRM-DWLI-2019-008) and were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (7th Edition, National Academy Press, Washington DC).

All surgical procedures were carried out under aseptic conditions. The rats were initially anesthetized with 5% isoflurane (RWD life science, Shenzhen, China) and then maintained with 2–2.5% isoflurane (Venniro et al., 2018). A 1-cm midline incision was made over the thoracic vertebrae, and laminectomy on T10 and the caudal half of T9 vertebrae was performed. Spinal cord contusion injury was conducted by NYU Impactor Model III (W.M. Keck Center for Collaborative Neuroscience Rutgers, The State University of New Jersey, United States) using a 10-g node dropping freely from a height of 2.5 cm (Constantini and Young, 1994) and muscles and skin sutured in layers. Sham controls underwent laminectomy without the contusion. To prevent infection at the incision, cefuroxime sodium was applied for 3 days after injury. The bladders were emptied manually twice daily in the first week after injury.

The rats were randomly divided into three groups: the sham group, the SCI-vehicle group, and the SCI-EDV group. After SCI, the SCI-EDV group received edaravone (Sigma-Aldrich, MO, United States, 5 mg/kg). This dose was chosen according to a previous study on SCI rats (Ohta et al., 2005). Edaravone was dissolved in ethyl alcohol at 10 mg/ml and then diluted with sterile saline, with a final concentration of 1 mg/ml of edaravone and 10% ethyl alcohol. The rats were injected intraperitoneally with 5 mg/kg edaravone 30 min post injury and then once a day for 7 consecutive days after SCI.

0.5-cm spinal cord tissues around the lesion center were harvested with PBS perfusion and homogenized on ice in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) to quantify ferroptosis-related proteins. 20 μg of the protein in each well was loaded on 12% SDS-PAGE and transferred onto a PDVF membrane, which was blocked with 5% skimmed milk at room temperature for 1.5 h followed by overnight incubation with primary antibodies against glutathione peroxidase 4 (GPX4, 1:5000, ab125066, and Abcam), System Xc⁻ light chain (xCT, 1:2000, ab175186, and Abcam), Acyl-CoA synthetase long-chain family member 4 (ACSL4, 1:5000, ab155282, and Abcam), 5-lipoxygenase (5-LOX, 1:1000, ab169755, and Abcam), and IBA-1 (1:1000, ab178846, and Abcam) at 4°C. After rinsing with Tris-buffered saline and Tween (TBST), the membrane was incubated with secondary antibody (Anti-mouse IgG, HRP-linked Antibody, 1:1000, 7076, CST; Anti-rabbit IgG, HRP-linked antibody, 1:1000, 7074, CST; Danvers, MA, United States) at room temperature for 1 h. Chemiluminescence was detected and quantified using ImageJ software. β-actin (1:5000, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and GAPDH (1:1000, ab8245, and Abcam) were used as the internal standard. The mean and SD of relative protein levels across at least three independent experiments were used for statistical comparisons.

The mice were transcardially perfused with PBS followed by perfusion with 4% paraformaldehyde, and tissues were fixed with 4% paraformaldehyde for 24 h. After being dehydrated by 30% sucrose, the spinal cord tissues were embedded in OCT and were cut at 10-μm thickness. The tissue sections were fixed with acetone for 30 min and then washed in phosphate-buffered saline (PBS). The sections were blocked with 5% goat serum at room temperature for 1 h and incubated overnight with the primary antibodies: glutathione peroxidase 4 (GPX4, 1:100, ab125066, Abcam, MA, United States), acyl-CoA synthetase long-chain family member 4 (ACSL4, 1:100, ab155282, Abcam, MA, United States), 5-lipoxygenase (5-LOX, 1:50, ab169755, Abcam, MA, United States), NeuN (1:200, ab104224, Abcam, MA, United States), ChAT (1:200,MA5-31382, Invitrogen, California, United States), CC-1 (1:200, OP80, Millipore, MA, United States), and GFAP (1:200, ab7260, Abcam, MA, United States) antibodies at 4°C, washed in phosphate-buffered saline with Tween (PBST), and incubated with secondary antibody (Goat Anti-Rabbit IgG H&L Alexa Fluor 488, 1:200, ab15008, and Goat Anti-Mouse IgG H&L Alexa Fluor 555), 1:200, ab150118; Abcam, MA, United States) for 1 h at room temperature. Immunofluorescent images were taken by a fluorescence microscope (Leica DMi8) (Figure 6) and confocal microscope (Figures 3, 4) (ZEISS LSM 900, Germany)/Vectra Polaris Automated Quantitative Imaging System (Vectra Polaris, America) (Figures 3, 4). The intensity was quantified by ImageJ software (NIH, Bethesda, MD, United States). All quantification was performed using ImageJ. Three images per animal were obtained from coronal spinal cord sections of the same epicenter and caudal position, and n = 3 animals in each group. The average mean density values are expressed as mean ± sd.

According to the following method, the spinal cord epicenter tissues at 1 and 3 d post injury were collected and underwent a rat cytokine array (61 cytokines, G-series rat cytokine array, RayBiotech, Norcross, GA, United States). Chemiluminescence signals were detected using a ChemiDocMP System (Bio-Rad, Hercules, United States) and analyzed using ImageLab software (NIH, United States). The heat map was generated using TBtools (http://cj-chen.github.io/TBtools/) (Chen et al., 2020).

HE staining was used to evaluate the injured spinal cord tissue after contusion. At 4 weeks post SCI, the rats were perfused intracardially with cold PBS and then with cold 4% paraformaldehyde. At the injury epicenter, a 1-cm spinal cord tissue was obtained and fixed for 24 h in 4% paraformaldehyde at 4°C. The sample was then embedded in paraffin and cut into 10-μm-thick cross-sections for HE staining, which was performed with a HE Staining kit (Beyotime, C0105, Shanghai, China) according to the manufacturer’s instructions.

Basso, Beattie, and Bresnahan (BBB) scores are the hindlimb movement’s open-field locomotion test (Basso et al., 1995). BBB scores were evaluated weekly for up to 8 weeks. Briefly, rats in the open field were allowed to move freely for 5 min. Two independent blinded observers scored the rats on the hindlimb movement. For the objective assessment of locomotor coordination, the Catwalk gait analysis system (Noldus Information Technology B.V, Netherlands) was applied at 8 weeks after SCI as previously described (Hamers et al., 2006). Briefly, the rats were placed on the glass floor of the catwalk system in complete darkness, and all the rats were allowed to walk freely from left to right in the walkway. Paw prints were recorded by a high-speed video camera under the walkway. Catwalk software 10.6 was used to label each paw print during each run and analyze the gait parameters of the rats. The regularity index (%) is a common and comprehensive parameter to measure the degree of limb coordination.

The rats were anesthetized at 8 weeks after injury with pentobarbital. Motor evoked potential (MEP) was detected at 8 weeks post SCI by electrophysiological devices (YRKJ-G2008; Zhuhai Yiruikeji Co, Ltd, Guangdong, China) after SCI as previously described (Yao et al., 2021). The backup skin of rats was disinfected after anesthesia, and the electrodes were placed. The reference electrodes were placed under the skin between the ears. The recording electrode was placed in the gastrocnemius muscle of the lower extremity, and the stimulation electrode was placed behind the head and neck. Single 5-mA stimulation was administered to stimulate the motor region of the cerebral cortex.

Statistical analysis was performed with GraphPad Prism (www.graphpad.com/scientific-software/prism/, San Diego, CA). P values < 0.05 were considered statistically significant. One-way ANOVA with Tukey’s post hoc test was used if more than two groups were compared and two-way ANOVA with Sidak’s multiple comparisons test if two independent variables were compared. Statistical details for specific experiments, including animal number, precision measures, statistical tests used, and definitions of significance, are described in figure legends.

We first investigate how the ferroptosis key proteins changed upon edaravone treatment in the spinal cord contusion model in rats. At 2 and 7 days after SCI, ferroptosis key proteins of the spinal cord lesion epicenter were detected by western blot (Figure 1A). Cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) and Glutathione peroxidase 4 (GPX4), which are both ferroptosis suppressors (Koppula et al., 2021), are reduced in SCI-group at 2 and 7 days compared with the Sham group. The expression of xCT and GPX4 is rescued by edaravone treatment (p < 0.05, Figures 1B–E). Edaravone treatment in the sham group also showed elevated GPX4 level (Supplementary Figure S1). ACSL4, a biomarker to ferroptosis, (Verma et al., 2020) as well as 5-LOX, also pro-ferroptosis proteins, are both elevated at 2 days post injury, whereas edaravone downregulated the expression significantly (p < 0.05, Figures 1F–I). No significant change in the expression levels of 5-LOX and ACSL4 was detected at 7 days after SCI in any of the groups (p < 0.05, Figures 1F–I), indicating that ferroptosis mainly occurs in the acute phase. Immunofluorescence showed that GPX4 was abundant in the sham group but dramatically decreased in the SCI-vehicle group, whereas edaravone treatment rescued the GPX4 level (Supplementary Figure S2A). ACSL4 and 5-LOX increased after injury and decreased in the edaravone group (Supplementary Figure S2B,C). Altogether, the anti-ferroptosis regulators xCT and GPX4 were upregulated upon edaravone treatment, and the pro-ferroptosis regulators ACSL4 and 5-LOX were downregulated (Figure 1J), indicating the ferroptosis inhibition mechanism of edaravone.

The lesion epicenter and caudal lumbar region represent the sites where the primary and secondary injury happens, respectively (Figure 2A). Total GPX4 decreased both in the lesion and caudal region after injury but increased after edaravone treatment (Figure 2B). Moreover, the spinal cord segment below the lesion is responsible for maintaining locomotion, and whether the neurons in lesion-adjacent spinal segments are influenced by the ferroptosis pathway is worth investigating. In the normal spinal cord, the GPX4 were expressed in two patterns, in the cytosol and in the nucleus. In colocalization with the neuronal marker NeuN, it expressed in the cytosol of the neurons. Edaravone rescued GPX4 levels in the neurons (Figures 2C,E). Edaravone rescued the GPX4 level in the motor neuron as well, shown by increased GPX4 in the ChAT-positive cell (Figures 2D,F). Double-labeling immunofluorescence showed that part of the GPX4 positive cells are CC1 positive, which represent mature oligodendrocytes. And this GPX4+CC1+ cells have a typical nucleus subcellular localization. However, the treatment increased oligodendrocyte GPX4 only in the epicenter but did not change in the caudal region after SCI (Figures 2G,H). Therefore, the most significant changes may occur in the neuronal level of GPX4 upon edaravone treatment.

In the sham group, the ACSL4 level is very low, and total ACSL4 level in the SCI group increased both in the epicenter and lumbar segment below the lesion. In the lesioned spinal cord, the ACSL4 was expressed both in the cytosol and in the nucleus. Edaravone decreases the ACSL4 level in these two regions (Figures 3A,B). In the spinal cord segment below the lesion, NeuN-positive ACSL4 increased significantly in the injury group, whereas edaravone attenuated the ACSL4 level in neurons (Figures 3C,E). ChAT-positive cells (motor neurons) show a similar significant decrease in ACSL4 in the SCI-EDV group (Figures 3D,F).

The total 5-LOX level increased both in epicenter and caudal region to the lesion after injury but decreased after edaravone treatment (Figures 4A,B). The change is more obvious in the caudal region. Neuronal colocolization with 5-LOX in the caudal region increased significantly in the injury group, whereas it was attenuated by edaravone (Figures 4C,E). ChAT-positive cells (motor neurons) show the same significant change (Figures 4D,F).

In summary, we demonstrate that the overall level of pro-ferroptosis proteins ACSL4 and 5-LOX was upregulated upon injury and downregulated after evaradone treatment. The neuron, especially the motor neuron, shows very significant downregulation of ACSL4 and 5-LOX upon edaravone treatment.

Cytokine changes are also important aspects of the microenvironment after SCI. At 1 and 3 days post injury, spinal cord tissues were collected and analyzed by anti-cytokine antibody arrays containing antibodies to 61 inflammatory mediators (Figure 5A). The heatmaps of the SCI-vehicle and SCI-EDV groups at 1 d. p.i (Figure 5B) and 3 d. p.i. (Figure 5C). are shown. Among them, the expression of IL-10 was significantly upregulated at 3 days post injury after treatment of edaravone (Figure 5D). IL-13 was significantly higher at 1-day post SCI in the EDV treatment group compared with that in the SCI-vehicle group (Figure 5E). In addition, the level of adiponectin was upregulated both at 1 and 3 days after SCI in the edaravone group (Figure 5F).

Ionized calcium-binding adapter molecule 1 (Iba-1) is a critical marker of microglia. To further observe microglia activation after treatment by edaravone, the expressions of Iba-1 in the spinal cord were detected. The result showed that the expression of Iba-1 increased 2 days post injury, and edaravone treatment inhibits the expression of Iba-1 (Figures 5G,H). Excessive astrogliosis was observed in the SCI group when compared with the sham group. However, edaravone treatment significantly inhibited astrogliosis levels compared with those in the SCI group (Figures 5I,J).

Neuronal survival is the basis of an effective functional circuit. Immunofluorescence staining for NeuN at 8 weeks post SCI decreased significantly after SCI, whereas the treatment of edaravone increased NeuN-positive fluorescence cells, reflecting more neuronal survival (Figures 6A,B). The marked square indicates the area of representative images. Representative images of HE staining of the spinal cord epicenter are shown in Figure 6C. Even after 4 weeks post injury, there is an obvious cavity in the SCI-vehicle group in the lesion epicenter, whereas edaravone treatment spared more tissue and induces a smaller cavity (Figures 6C,D).

As a final arbiter of the therapeutic efficacy of edaravone on experimental SCI, assessment of long-term locomotor and electrophysiology outcome is necessary. The functional assessments to confirm the effectiveness of edaravone on SCI are conducted up to 8 weeks post SCI (Figure 7A). BBB score is a standardized locomotor rating scale for open-field testing in rats (Basso et al., 1995). BBB scores were performed weekly till the 8th-week post injury to show the time course of recovery. The treatment of edaravone significantly elevated the BBB locomotor scores compared with those in the SCI group starting from 3 to 8 weeks after injury (Figures 7B,C). Another important assessment in recovery from SCI is the coordination of locomotion (Hamers et al., 2006). Catwalk analysis evaluates the coordination of forelimb and hindlimb. Catwalk-recorded footprint views show improved gait under the treatment of edaravone (Figure 7D). The Regularity Index (RI), a measure of step sequence patterns, as a major coordination parameter, decreased significantly at 8 weeks post SCI and was higher in the SCI-EDV group than in the SCI-vehicle group (Figure 7E). To further evaluate the neurological recovery, the changes of motor-evoked potential (MEP) electrophysiology were applied. The amplitude of MEP returned after edaravone administration (Figures 7F,G). This electrophysiology improvement correlates well with the anatomical and locomotor recovery after SCI.