Edaravone Modulates Neuronal GPX4/ACSL4/5-LOX to Promote Recovery After Spinal Cord Injury

Abstract

The FDA-approved drug edaravone has a neuroprotective effect on spinal cord injury (SCI) and many other central nervous system diseases. However, its molecular mechanism remains unclear. Since edaravone is a lipid peroxidation scavenger, we hypothesize that edaravone exerts its neuroprotective effect by inhibiting ferroptosis in SCI. Edaravone treatment after SCI upregulates glutathione peroxidase 4 (GPX4) and system Xc-light chain (xCT), which are anti-ferroptosis proteins. It downregulates pro-ferroptosis proteins Acyl-CoA synthetase long-chain family member 4 (ACSL4) and 5-lipoxygenase (5-LOX). The most significant changes in edaravone treatment occur in the acute phase, two days post injury. Edaravone modulates neuronal GPX4/ACSL4/5-LOX in the spinal segment below the lesion, which is critical for maintaining locomotion. Moreover, the GPX4/ACSL4/5-LOX in motor neuron is also modulated by edaravone in the spinal cord. Therefore, secondary injury below the lesion site is reversed by edaravone via ferroptosis inhibition. The cytokine array revealed that edaravone upregulated some anti-inflammatory cytokines such as IL-10, IL-13, and adiponectin. Edaravone reduced microgliosis and astrogliosis, indicating reduced neuroinflammation. Edaravone has a long-term effect on neuronal survival, spinal cord tissue sparing, and motor function recovery. In summary, we revealed a novel mechanism of edaravone in inhibiting neuronal ferroptosis in SCI. This mechanism may be generalizable to other neurological diseases.

Keywords: edaravone; ferroptosis; neuroinflammation; neuroprotection; spinal cord injury.

FIGURE 1

Edaravone modulates key proteins of the ferroptosis pathway in the spinal cord. (A) Schematic diagram of the experimental timeline. Wistar rats were intraperitoneally injected with 5 mg/kg edaravone daily during the first 7 days. At 2 and 7 days post SCI, tissues from spinal cord lesion epicenter were collected for western blot. At 2 days post injury the lesion epicenter and caudal spinal cord were observed from the immunofluorescence. (B,D,F,H) Representative western blot images of xCT (B), GPX4 (D), ACSL4 (F) and 5-LOX (H). (C,E,G,I) Quantifications of three western blot band of xCT (C), GPX4 (E), ACSL4 (G) and 5-LOX (I). Experiments were repeated three times. Data are expressed asmean ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *p < 0.05, n = 3. (J) Diagram depicts that the ferroptosis pathway of SCI is modulated by edaravone.

FIGURE 2

Edaravone upregulates GPX4 after SCI (A) Upper, schematic diagram of GPX4 detected injured epicenter and caudal spinal cord ventral horn (3 mm to the lesion) at 2 days post SCI, Lower, immunofluorescence images of GPX4 (green) in the lesion epicenter and caudal spinal cord section. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (B) Quantification of the GPX4 intensity in the lesion and caudal segment. (C,D) Representative immunofluorescence images of GPX4 (green)/NeuN (red) (C) and GPX4 (green)/ChAT (red) (D) in the caudal spinal cord at 3.0 mm to the lesion at 2 days post SCI. Scale bar,100 μm. (E,F) Quantification of Neuronal (NeuN positive) GPX4 (E) and Motor neuronal (ChAT positive) GPX4 (F). (G) Representative immunofluorescence images of GPX4 (green)/CC1 (red) in both lesion epicenter and in the caudal spinal cord at 3.0 mm to the lesion at 2 days post SCI. Scale bar,100 μm. (H) Quantification of oligodendrocyte (CC1 positive) GPX4. Data are expressed as mean ± standard deviation. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (B,H) or one-way ANOVA with Tukey’s post hoc test (E,F). *p < 0.05, **p < 0.01, and ***p < 0.001, n = 3, bar = 100 μm.

FIGURE 3

Edaravone downregulates ACSL4 at 2 days post SCI. (A) Upper, schematic diagram of ACSL4 detected injured epicenter and caudal spinal cord ventral horn (3 mm to the lesion) at 2 days post SCI. Lower, immunofluorescence images of ACSL4 (green) in the lesion epicenter and caudal spinal cord section. Nuclei were stained with DAPI (blue). Scale bar,100 μm. (B) Quantification of the ACSL4 intensity in the lesion and caudal segment. (C,D) Representative immunofluorescence images of of ACSL4 (green)/NeuN (red) (C) and ACSL4 (green)/ChAT (red) (D) in the caudal spinal cord at 3.0 mm to the lesion at 2 days post SCI. Scale bar,100 μm. (E,F) Quantification of neuronal (NeuN positive) ACSL4 (E) and motor neuronal (ChAT positive) ACSL4 (F). Data are expressed as mean ± standard deviation. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (B) or one-way ANOVA with Tukey’s post hoc test (E,F). *p < 0.05, **p < 0.01, and ***p < 0.001, n = 3, bar = 100 μm.

FIGURE 4

Edaravone downregulates 5-LOX at 2 days post SCI. (A) Upper, schematic diagram of 5-LOX detected injured epicenter and caudal spinal cord ventral horn (3 mm to the lesion) at 2 days post SCI, Lower, immunofluorescence images of 5-LOX (green) in the lesion epicenter and caudal spinal cord section. Nuclei were counterstained with DAPI (blue). Scale bar,100 μm. (B) Quantification of the 5-LOX intensity in the lesion and caudal segment. (C,D) Representative immunofluorescence images of 5-LOX (green)/NeuN (red) (C) and 5-LOX (green)/ChAT (red) (D) in the caudal spinal cord at 3.0 mm to the lesion at 2 days post SCI. Scale bar, 100 μm. (E,F) Quantification of neuronal (NeuN positive) 5-LOX (E) and motor neuronal (ChAT positive) 5-LOX (F). Nuclei were stained using DAPI in blue. Data are expressed as mean ± standard deviation. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (B) or one-way ANOVA with Tukey’s post hoc test (E,F). *p < 0.05, **p < 0.01, and***p < 0.001, n = 3, bar = 100 μm.

FIGURE 5

Edaravone reduced inflammation in the acute phase post SCI. (A) Timeline of the experiment about edaravone’s effect on inflammation. (B,C) Heatmap showing the changing proteins between SCI-Vehicle and SCI-EDV groups at 1d (B) and 3d (C) post SCI. (D–F) Quantifications of the relative levels of IL-10 (D), IL-13 (E) and adiponectin (F). Data are expressed as mean ± standard deviation. (G,H) Western blot analysis of Iba-1 (G) and quantification (H) at 2d post SCI are shown. (I) Spinal cord injury epicenter GFAP immunofluorescence 8 weeks post SCI. Scale bar = 100 μm. (J) Upper, the marked square in the spinal cord diagram indicates the field of microscope chosen for the representative images. Lower, quantification of GFAP. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (D–F) or one-way ANOVA with Tukey’s post hoc test (H,J). *p < 0.05, ***p < 0.001, and ****p < 0.0001, n = 5 (cytokine array at 1 d. p.i.), n = 4 (3 d. p.i.), bar = 100 μm (I).

FIGURE 6

Edaravone has a long-term effect on neuronal survival, reduced astrogliosis, and tissue sparing. (A) Representative images of immunofluorescence staining of NeuN in the injury epicenter of 8 weeks post SCI (B) Quantifications of NeuN. (C) Representative images of H&E staining of spinal cord sections at 4 weeks post injury. (D) Quantification of cavity areas made by ImageJ. Data are expressed as mean ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *p < 0.05, and ***p < 0.001, n = 3, bar = 100 μm (A), bar = 500 μm (C).

FIGURE 7

Edaravone promotes motor functional recovery after spinal cord injury. (A) Timeline of the experiment. 30 min after SCI, rats received EDV (5 mg/kg/day) continuously for 7 days. BBB scores were assessed every week for 8 weeks. In addition, Catwalk and MEP detection were performed at 8 weeks after SCI. (B) BBB locomotor scores were assessed for hindlimbs, up to 8 weeks post injury. (C) BBB scores at 8 weeks were compared. (D) Print views were recorded at 8 weeks post injury by the CatWalk XT system. (E) Regularity index (% index) is presented as an overall measure of the degree of the normal step sequence. (F) Electrophysiology of MEP at 8 weeks post spinal cord injury. (G) MEP amplitudes of each group were quantified. Data are expressed as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *p < 0.05; **p < 0.01, n = 3.

FIGURE 8

Schematic diagram of edaravone’s neuroprotective mechanism in spinal cord injury. Edaravone upregulates anti-ferroptosis protein GPX4 and xCT and downregulates pro-ferroptosis protein ACSL4 and 5-LOX. This leads to neuronal survival as well as reduced neuroinflammation, and improves recovery of SCI.