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.