Cold atmospheric plasma-activated saline alleviates secondary injury post-SCI by inhibiting extracellular matrix remodeling and infiltration of proinflammatory macrophages

Abstract

Background

Cold atmospheric plasma (CAP) has been shown to improve the recovery of transected peripheral nerves. We determined the protective role of CAP-activated saline (CAP-AS) treatment in the acute and subacute stages of spinal cord injury (SCI) in mice.

Methods

C57BL/6 SCI mice were treated with CAP-AS for 14 days. Injury recovery was assessed weekly for four weeks by conducting motor function tests, including the Basso Mouse Scale (BMS) and footprint test. Transcriptome analysis was conducted on day 14 to elucidate potential mechanisms, which were further validated through immunofluorescence examinations of the injured spinal cord tissues on day 28 and the levels of proinflammatory cytokines produced by macrophages in vitro.

Results

Compared to the SCI group, the CAP-AS-treated groups presented significantly better hindlimb motor function after four weeks. The downregulated (SCI vs. SCI + CAP-AS, with CAP-AS activated for 20 min) differentially expressed genes (DEGs) were enriched in the extracellular region, extracellular matrix (ECM), and ECM-receptor interaction. In contrast, the upregulated DEGs were enriched in immune response-associated pathways. Histological changes in the CAP-AS-treated groups were observed to further validate the predicted mechanisms 28 days post-injury. The alleviation of secondary injury was confirmed by an increase in GFAP-positive and NFH-positive areas, and enhanced outgrowth of 5-HT-positive fibers. Inhibited ECM remodeling was confirmed by a decrease in the areas positive for PDGFRβ, fibronectin, and laminin. A decrease in the infiltration of macrophages and activation of microglia was determined by a decrease in CD68-positive and F4/80-positive areas. The inhibitory effect of CAP-AS on inflammation was further supported by a decrease in the levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in CAP-AS-treated M1 macrophages.

Conclusion

CAP-AS can alleviate secondary injury in SCI model mice by inhibiting ECM remodeling in injured tissues and reducing the infiltration or activation of proinflammatory macrophages/microglia.