Ferroptosis Inhibitors Improve Early and Delayed Treatment Outcomes of Mild Spinal Cord Injury

Academic Background

Spinal cord injury (SCI) causes significant secondary damage not only in the acute period but also in the chronic period. These injuries are typically triggered by multiple factors, including oxidative stress, inflammatory response, and cell death mechanisms. Within this context, an iron-mediated non-apoptotic form of cell death known as ferroptosis has garnered increasing attention from the scientific community. In recent years, ferroptosis has been linked to various nervous system disorders, but its specific mechanisms and contributions to SCI require further investigation.

Source Introduction

This paper was collaboratively authored by several scientists, including Fari Ryan, involving research institutions such as McGill University, Charité Universitätsmedizin Berlin, Helmholtz Zentrum München, and other renowned research organizations. The article was published in the 2024 Volume 147, Page 106, issue of Acta Neuropathologica.

Research Background and Objectives

Ferroptosis, a non-apoptotic form of cell death, is induced by increased iron loading resulting from red blood cell (RBC) phagocytosis. Scientists have found that, following SCI, the release of redox-active iron can trigger ferroptosis, leading to secondary damage and functional loss. This study aims to explore the potential impact of ferroptosis in SCI and evaluate the effectiveness of ferroptosis inhibitors in SCI treatment, with the hope of providing new therapeutic avenues for SCI.

Research Methods

The study employed various experimental methods, including Western Blotting, Immunofluorescence, Capillary Electrophoresis coupled with Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS), and analyses of both mouse and human samples.

Subjects and Grouping

The research subjects were 8-week-old female C57BL/6 mice subjected to SCI using a spinal cord injury model. The mice were randomly divided into treatment and control groups, receiving injections of the ferroptosis inhibitor UAMC-3203 (15 mg/kg) or a vehicle solution (3% DMSO saline).

Procedures and Protocols

1. Establishing the SCI Model: The mice underwent partial laminectomy, followed by spinal cord contusion with varying intensities (30 and 40 kdyn) using an Infinite Horizon Impactor.
2. Behavioral Testing: Motor recovery was assessed using the Basso Mouse Scale (BMS), observing the activity levels of the treatment and control groups at different time points.
3. Molecular Biology Detection:
   • Iron Metabolism-Related Protein Detection: Expression changes in iron transport proteins (TFR1, DMT1), iron storage proteins (Ferritin), and iron release proteins (NCOA4) were detected.
   • Lipid Peroxidation and Antioxidant System Detection: Levels of lipid peroxidation products (4-HNE) and antioxidant enzymes (GPX4) and glutathione (GSH) were measured.
4. Iron Ion Content Measurement: CE-ICP-MS was used to measure the total iron content in spinal cord tissues, as well as the ratios of Fe^2+ and Fe^3+.
5. Human Sample Analysis: Cerebrospinal fluid (CSF) and serum samples from SCI patients were collected and analyzed to detect changes in ferroptosis markers.

Main Findings

Changes in Iron Metabolism

• Iron Increase and Storage: Seven days post-SCI, a significant increase in redox-active iron was observed in the spinal cords of SCI mice, accompanied by elevated expressions of iron transport proteins (DMT1, TFR1) and iron storage protein (Ferritin).
• Iron Release Mechanism: NCOA4 protein expression markedly increased post-injury, particularly in CD11b+ macrophages, suggesting a critical role in releasing iron from ferritin.
• Fe^2+/Fe^3+ Ratio Increase: CE-ICP-MS detected a significant rise in the Fe^2+/Fe^3+ ratio in SCI mouse spinal cords, which may be a key factor in inducing ferroptosis.

Insufficiency of Antioxidant System

• Glutathione System Depletion: GSH levels significantly decreased and remained low for up to 5 weeks post-SCI. The expressions of xCT and GPX4 also declined, indicating an insufficient response from the glutathione antioxidant system following SCI.
• Increased Lipid Peroxidation: Levels of the lipid peroxidation product 4-HNE significantly rose post-SCI, indicating persistent cell damage due to lipid peroxidation.

Effects of Ferroptosis Inhibition

• Motor Function Recovery: In the 30 kdyn SCI model, mice treated with the ferroptosis inhibitor UAMC-3203 showed significantly better motor recovery compared to the control group. This improvement was evident both in the early SCI phase (days 1-14) and during delayed treatment (days 28-42), as reflected by higher BMS scores.
• Reduced Secondary Damage: Treatment with the ferroptosis inhibitor significantly reduced the lesion area at the injury center, increased myelin preservation, and enhanced the distribution of the 5-HT neurotransmitter in the ventral horn, indicating that the inhibitor effectively alleviates secondary damage.

Human Sample Analysis

• In SCI patients, significant increases in CSF and serum levels of iron storage protein Ferritin, hemoglobin α, and hemopexin (which binds heme) were detected, while GSH levels significantly declined. These changes were consistent with the findings from the SCI mouse model.
• Biomarkers: Changes in these ferroptosis-related biomarkers in SCI patients provide important indicators for assessing SCI treatment efficacy and monitoring disease progression.

Significance of the Study

This study is the first to detail the mechanisms of ferroptosis in the SCI process and demonstrate the therapeutic potential of ferroptosis inhibitors. Ferroptosis prompts further damage by increasing redox-active iron, depleting the GSH antioxidant system, and promoting lipid peroxidation. Effectively inhibiting ferroptosis can mitigate acute injury and improve long-term functional recovery, offering new research directions and strategies for SCI clinical treatment.

Highlights

• Novel Mechanism: The study reveals the critical role of ferroptosis, a novel non-apoptotic cell death mechanism, in SCI.
• Cross-Species Verification: The investigation not only delves deeply into the mouse model but also validates changes in ferroptosis-related biomarkers in human SCI patients.
• Therapeutic Potential: Applying ferroptosis inhibitors shows considerable improvement in SCI functional recovery and reduces secondary damage, paving a new path for SCI treatment.

Conclusion

Ferroptosis plays a critical role in the onset and progression of spinal cord injury. Using ferroptosis inhibitors significantly improves SCI treatment outcomes. This discovery provides new insights into the pathophysiological mechanisms of SCI, offering promising prospects for future research and clinical practice. Inhibitors of ferroptosis may become essential components of SCI treatment protocols.