Single-cell transcriptomics reveals time-resolved neuronal death characteristics in traumatic brain injury

Accurate determination of the timing and progression of traumatic brain injury (TBI) is critical in forensic pathology, particularly for reconstructing injury events and estimating post-traumatic intervals. However, conventional timing approaches of TBI rely on limited pathological features and often lack sufficient accuracy. To address this limitation, we re-analyzed publicly available single-cell RNA-seq datasets from GEO (GSE269748 and GSE160763) and integrated murine cortical transcriptomes across three post-injury stages—acute (24 hours), subacute (7 days), and chronic (6 months)—to characterize time-resolved neuronal molecular changes after TBI. Neuron-focused differential expression and functional enrichment analyses revealed a progression from early stress and inflammatory-response programs toward later synaptic and neurodegeneration-associated alterations. We further curated representative gene sets for 14 regulated cell-death programs and quantified their activity using AUCell-derived AUC scoring, identifying stage-dependent shifts in death-associated transcriptional signatures, with higher necroptosis- and pyroptosis-associated signals in the acute phase and increased ferroptosis- and autophagic cell death–associated signals in the chronic phase, accompanied by transcriptional patterns consistent with altered iron handling and glutathione metabolism. This re-analysis provides a time-resolved, neuron-centered molecular framework that may support forensic estimation of injury timing and offers insight into mechanisms of secondary brain injury. • Integrated single-cell transcriptomics across TBI phases (acute/subacute/chronic; GSE160763, GSE269748) identified 672 persistently altered genes, suggesting universal TBI injury markers. • Uncovered phase-specific neuronal markers: 936 acute-specific, 95 subacute-specific, 663 chronic-specific neuronal marker genes, serving as temporal indicators for post-injury interval. • Revealed distinct TBI progression pathology: pyroptosis dominates acutely, while ferroptosis and autophagy drive chronic neuronal loss. These cell death patterns may serve as pathological indicators for time-of-injury estimation. • Summarize the temporal activation patterns of 14 distinct cell death modalities across different stages of TBI to enhance the precision of postmortem forensic pathological diagnosis