Remote limb ischemic preconditioning alleviated spinal cord injury through inhibiting proinflammatory immune response and promoting neural regeneration
Authors: Yu Yan , Yan Yitong , Li Zihan , Zhang Shuangyue , Bai Fan , Jing Yingli 
Published: November 2023

Objective To investigate the protective effects of remote limb ischemic preconditionging (RLPreC) on traumatic SCI and elucidate the underlying biological pathophysiology by exploring transcriptomic changes through next-generation RNA sequencing (RNA-Seq). Methods C57/6N mice aged 8 to 9 weeks were randomly divided into three groups: the sham operation group (Sham), the spinal cord injury group (SCI), and the remote limb ischemic preconditioning group (SCI + PreC). T10 spinal cord contusion models were established. BMS score, open field test, and HomeCageScan were used to evaluate the motor function of mice in each group. We randomly selected three mice per group and extracted spinal cord tissues for next-generation RNA-Seq analysis. Bioinformatics analysis was performed for two comparisons: Sham vs. SCI and SCI vs. PreC + SCI. Immunofluorescence staining was used to observe the glial scar formation, nerve regeneration, microglia proliferation in the injured area, and the nuclear translocation of NF-κB. BioLegend's Legendplex bead-based immunoassays were used to quantitatively detect the level of inflammatory factors in the injured area. iNOS protein expression was determined by ELISA analysis. Results 1. RLPreC significantly improved BMS score, increased moving distance in the open field, and ameliorated spontaneous behaviors represented by walking, grooming, and twitching. 2. RNA-seq analysis identified a total of 5247 differentially expressed genes (DEGs) were identified (p.adj < 0.05 and fold change ≥ 2). Gene ontology (GO) analysis revealed the predominant involvement of immune signaling, leukocyte migration, cytokine signaling, neurotransmitters transport, ion channel activity, and synapse-related pathways among these DEGs. Furthermore, enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the suppression of inflammation-related pathways (such as NOD-like receptor signaling and TNF signaling) and the promotion of synapse-related pathways (such as GABAergic synapse and Glutamatergic synapse) pathways. 3. RLPreC reduced scar formation and increased neuronal axon regeneration in the injured area, suggested that RLPreC can promote nerve regeneration. 4. RLIPreC decreased the expression of pro-inflammatory cytokines IL-23, TNF-α, IL-6, IL-27, IL-1α, IFN-γ, iNOS and increased the expression of anti-inflammatory cytokine IL-10 in the injured area. 5. RLIPreC regulated the proliferation of microglial cells and nuclear translocation of NF-κB in the injured area, suggested that RLPreC can reduce the inflammatory response after spinal cord injury. Conclusion Our study highlights RLPreC as an effective therapeutic approach for alleviating motor dysfunction and pain-related symptoms in traumatic SCI. Through gene expression profiling using next-generation RNA-Seq, we identified potential therapeutic genes and pathways, providing valuable insights for future experimental and clinical investigations.