Spatiotemporal multi-omics profiling of secondary brain injury after intracerebral hemorrhage in an optimized autologous blood-induced mouse model with human tissue validation.

Abstract

BACKGROUND: Secondary brain injury (SBI) following intracerebral hemorrhage (ICH) involves complex molecular events such as oxidative stress, inflammation, and immune cell infiltration. Understanding their spatiotemporal patterns is essential for identifying therapeutic targets and optimal intervention windows. METHODS: We optimized an autologous blood-induced ICH mouse model and performed neuroimaging, behavioral testing, histological evaluation, and transcriptomic and proteomic analyses across multiple time points (Days 1, 3, 5, 8, and 12). Time-resolved analyses focused on dynamic changes in SBI-related pathways, including oxidative stress, ferroptosis, leukocyte migration, neutrophil extracellular trap (NET) formation, and phagocytic-lysosomal activity, with spatial validation by immunofluorescence and histology. Model refinements reduced blood coagulation and leakage, improving procedural success and reproducibility. In addition, imaging and perihematomal tissue from a patient 66 h after ICH onset were examined for comparison. RESULTS: Principal component and clustering analyses revealed a time-dependent molecular trajectory, with rapid early transcriptional changes followed by delayed but sustained protein-level responses. Markers of oxidative stress and ferroptosis (HMOX1, FTH1), adhesion molecules (VCAM1, CD11b), and phagocytic activity (CD68, CTSD) displayed distinct temporal and spatial expression patterns. NET formation peaked between Days 3 and 5 and then gradually declined. Human ICH tissue demonstrated similar activation of oxidative, inflammatory, and phagocytic pathways. CONCLUSIONS: This study delineates the spatiotemporal dynamics of key pathways and molecules involved in secondary brain injury after ICH, revealing stage-specific molecular features and potential therapeutic windows. The optimized autologous blood-induced ICH model exhibits good stability, reproducibility, and relevance to human pathology.