A NET-competent neutrophil subpopulation drives early brain injury after subarachnoid hemorrhage

Introduction Early brain injury (EBI) following subarachnoid hemorrhage (SAH) critically determines neurological outcomes, yet the cellular drivers remain unclear. Methods To systematically dissect acute pathogenic programs, we performed single-cell RNAsequencing of CD45 + immune cells isolated from a mouse model of SAH, coupled with weighted gene co-expression network analysis (WGCNA). Results This revealed a 24-h-specific injury module enriched for inflammatory and myeloid-related pathways. Mapping this module onto the single-cell landscape identified neutrophils as the principal cellular mediators. High-resolution subclustering further defined a distinct neutrophil subset, N_04, as the primary carrier of the 24-h pathogenic signature. N_04 is transcriptionally primed for Neutrophil Extracellular Trap (NET) formation, with key effectors including Padi4, Mmp8, and Lcn2, and spatial mapping localized N_04 to regions adjacent to the puncture site. Transcriptional regulatory analysis identified Padi4, Nfe2, and Cebpe as central nodes sustaining N_04 effector identity, while in vivo Padi4 deficiency or NET degradation markedly alleviated neurological deficits. Pseudotime and computational perturbation analyses indicated that N_04 represents a terminal effector state orchestrating hyper-inflammatory, degranulation, and stress-response programs. Intercellular communication modeling further positioned N_04 as a signaling hub, mediating bidirectional crosstalk with macrophages via Cxcl, Ccl, and APP pathways. Discussion Collectively, these findings define a NET-competent neutrophil subpopulation as a central driver of acute post-SAH brain injury and nominate Padi4-dependent NETosis as a potential therapeutic target.