Multimodal molecular profiling of the metabolic penumbra in hyperacute stroke

Background The ischemic penumbra, a metabolically compromised yet potentially salvageable region surrounding the ischemic core, is a prime target for acute stroke intervention. Yet an objective molecular definition of the penumbra, particularly during the earliest stages of ischemia, remains lacking. Methods and Results We applied principal component analysis (PCA) followed by k-means clustering to high-resolution mass spectrometry imaging data covering multiple metabolic pathways to identify a metabolically defined penumbra in a mouse model of hyperacute stroke (45 min middle cerebral artery occlusion, MCAO). Targeted spatial metabolomic profiling by matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) reveals a distinct penumbral metabolic profile, marked by relative preservation of high-energy phosphates, comparable lactate accumulation, and reduced succinate accumulation relative to the core. Spatial transcriptomics revealed selective induction of immediate-early genes, including Npas4, Fos and Junb, within the penumbra. Consistently, imaging mass cytometry shows enrichment of phospho-histone H3 (pHH3) within the penumbra, suggesting a chromatin-associated response potentially linked to immediate-early gene activation. Conclusion Together, these findings provide a multimodal molecular atlas of the hyperacute metabolically defined penumbra and reveal molecular features that facilitates its identification and inform future therapeutic strategies.