Single nucleic RNA sequencing after mouse spinal cord injury: Effects of athlete-derived extracellular vesicles

Spinal cord injury (SCI), a disease with a very poor prognosis, poses a serious social hazard and therapeutic burden due to its high morbidity, disability, and mortality. Despite the availability of existing treatments including surgery and drugs, their limited efficacy necessitates the development of innovative therapeutic approaches. As a new form of cell death, ferroptosis plays an important role in the context of SCI. Therapies targeting ferroptosis could offer novel approaches to improve neuronal survival after SCI. Additionally, as an important intercellular communication mediator, extracellular vesicles show potential therapeutic effects after SCI. In this study, we conducted single nucleic RNA sequencing (snRNA-seq) on mouse spinal cords after SCI followed by injection of athlete plasma extracellular vesicles (AEVs) or PBS to clarify multiple cell type-specific transcriptional responses, especially neuronal ferroptosis. Unbiased clustering identified 8 distinct cell clusters in the mouse spinal cords according to their expression of known cell-type-specific markers at day 7 post injury. AEVs treatment increased the frequency of viable neurons after SCI and significant alterations in ferroptosis-related gene expression within neuronal populations was identified. These data reveal the important role of AEVs in regulating neuronal ferroptosis after SCI at single-cell resolution. Overall design: Six C57BL/6 mice (6-8 weeks old) were randomly assigned to two groups (n=3 per group): 1. PBS group 2. AEVs group. Mice underwent T10 spinal cord contusion injury and then received daily tail-vein injections of AEVs (containing 100 µg of total protein from EVs in 100 µL of PBS) or PBS alone as a control (100 µL) for five consecutive days. Mice were sacrificed at 7 days post-injury, and the spinal cord was dissected for scRNA-seq. One sample corresponds to three individual animal.