A spatiotemporal immune atlas of subarachnoid hemorrhage from single-cell and spatial transcriptomics

Abstract Background and Purpose Subarachnoid hemorrhage (SAH) triggers a complex immune response that critically influences early brain injury (EBI) and long-term outcomes. However, the precise spatiotemporal dynamics and heterogeneity of immune cell infiltration and microglial reprogramming remain poorly understood. We aimed to construct a high-resolution immune atlas to delineate cell states, lineage trajectories, and spatial niches following SAH. Methods We integrated single-cell RNA sequencing (scRNA-seq) of CD45+ immune cells with spatial transcriptomics (ST) in a murine endovascular perforation SAH model. Immune landscapes were profiled at 24 hours (acute phase) and 72 hours (subacute phase) post-injury, compared with sham controls. Advanced bioinformatics integrated transcriptional signatures with spatial localization to map macrophage, neutrophil, and microglial dynamics. Results Our atlas reveals a coordinated immune transition from acute inflammation to reparative processing. We identified five macrophage, four neutrophil, and eight microglial subsets with distinct spatiotemporal patterns. Notably, we discovered a SAH-specific inflammatory microglial population (MG_03; Spp1+ / Lpl+ ) that clusters at the rupture site during the acute phase. This subset is transcriptionally distinct from disease-associated microglia (DAM) in other neurodegenerative conditions. Trajectory analysis suggests MG_03 acts as a signaling hub for immune recruitment before transitioning toward proliferative and reparative states (MG_06–08) that disperse into the parenchyma by 72 hours. Conclusions This study provides the first comprehensive spatiotemporal immune atlas of SAH, highlighting the distinct role of the Spp1+ MG_03 subpopulation in early injury sensing. These findings offer a roadmap for identifying precise therapeutic windows and targeting specific immune subsets to mitigate EBI. Graphical Abstract Experimental workflow for single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) in a mouse SAH model induced by endovascular perforation. Brain tissue from the ipsilateral (injured) hemisphere was collected from sham and at 24 h and 72 h post-SAH. For scRNA-seq, CD45⁺ immune cells were isolated prior to library preparation.