Single-cell analysis reveals temporal heterogeneity of neutrophil-lineage states after spinal cord injury.

Spinal cord injury (SCI) elicits a complex immune response that evolves from acute inflammation to chronic tissue remodeling, but the temporal organization of neutrophil-lineage cells remains incompletely defined. Here, we reanalyzed three public SCI single-cell RNA sequencing datasets, including immune-cell-enriched and whole-lesion datasets. We combined neutrophil-state annotation, maturation assessment, differential expression, functional module scoring and ligand-receptor inference with immunofluorescence validation in a C57BL/6 mouse contusion model. Neutrophil-lineage cells were detected beyond the acute phase and exhibited temporal variation in the composition of reference-associated G0-G5 states. Cells assigned to G3-G5 displayed predominantly post-mitotic transcriptional profiles and were analyzed together as neutrophils to characterize temporal expression patterns. Inflammatory and chemotactic expression was most prominent early after injury, whereas lipid-associated expression increased at selected later stages alongside persistent inflammatory features. These broad patterns were observed across datasets, with differences in state composition and response timing. Cell-cell communication analysis predicted interactions involving chemokines, thrombospondin and osteopontin between neutrophils and other immune populations. Immunofluorescence showed a higher APOE+ fraction and a lower IL1B+ fraction among Ly6G+ cells at 28 days than at 1 day after injury. These findings reveal sustained, temporally heterogeneous neutrophil-lineage responses and provide a basis for investigating their contributions to inflammation and lesion remodeling after SCI.