Special Poster Session 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Introduction: 

Distraction spinal cord injury (DSCI) is a serious complication of spinal deformity correction surgery, caused by excessive distraction and compression. The sphingosine 1-phosphate (S1P) pathway, especially S1PR2, plays a role in neuroinflammation, but its involvement under mechanical stress is not well understood. This study aims to explore the role of S1PR2 in neuroinflammatory injury after DSCI, focusing on its nuclear translocation and DNA methylation in microglia.

Material and Methods: 

A large animal model of DSCI that mirrors clinical conditions was created, validated by a 75-100% reduction in motor evoked potentials. Immunofluorescence was used to analyze microglial activation. RNA-Seq and bioinformatics identified DSCI mechanisms and targets, confirmed by Western blotting. Our custom SD rat DSCI model was assessed for stability and treated with the S1PR2 inhibitor JTE-013. In vitro, we used BV2 microglia to study S1PR2/ERK/STAT3 and S1PR2/DNMT1/SOCS1 pathways in neuroinflammation. S1PR2 nuclear translocation and S1PR2-DNMT1 complex formation were examined by immunoprecipitation, and cytokine levels in spinal cord, cerebrospinal fluid, and serum were measured using ELISA and flow analysis.

Results: 

Compared to the Sham group, the DSCI group showed significantly reduced behavioral scores. LFB staining revealed severe demyelination, while HE and Nissl staining indicated a significant loss of neurons and increased inflammation, congestion, and edema. Fluorescence staining showed a significant rise in CD16 and CD206 in microglia/macrophages (P < 0.05), and elevated levels of IL-1β, IL-6, and TNF-α in the spinal cord and cerebrospinal fluid (P < 0.05). KEGG analysis indicated that S1PR2 was significantly upregulated at 7 days post-DSCI, while other S1PR family members remained unchanged. Our custom-designed DSCI rat model demonstrated stability and efficiency. Treatment with the S1PR2 inhibitor JTE-013 improved BBB and ramp test scores, reduced hindlimb muscle atrophy, and alleviated spinal cord edema and hemorrhage. JTE-013 also reduced white matter demyelination and improved neuron survival. S1PR2 expression increased after DSCI, with SPHK2 levels rising in tandem. Immunofluorescence revealed S1PR2 in the nucleus and elevated expression in both the membrane and nucleus. The NF-κB inflammatory pathway and levels of TNF-α and IL-6 also increased with distraction duration. Western blot and Co-IP analyses showed enhanced interactions between DNMT1 and S1PR2, and between S1PR2 and KPNB1 proteins under stress. Under 10% distraction stress, DNMT1 expression in BV2 nuclei increased after 6 hours, with decreases in SOCS1 and increases in STAT3 (P < 0.01). S1PR2 knockdown reduced total and membrane S1PR2 levels, impairing receptor activation and signaling. After 14 days of DSCI, serum levels of TNF-α, CCL3, IL-1β, and CXCL1 increased but were reduced by JTE-013.

Conclusion: 

In this study, DSCI models in Bama pigs and SD rats were established. Microglial activation, including M1/M2 and macrophages, is key in secondary neuroinflammation. S1PR2 levels rise with stress duration and damage, and it translocate to the nucleus via KPNB1, binding DNMT1 to regulate SOCS1 methylation. This activates the SOCS1/STAT3 pathway, causing neuroinflammation. JTE-013 reduces inflammation by inhibiting S1PR2 activation and nuclear translocation. These findings provide insights into DSCI inflammation mechanisms and potential drug targets.