Cell-free DNA methylation profiling reveals injury types and severities in swine rotational and contusional models of traumatic brain injury

Abstract Early diagnosis of traumatic brain injury (TBI) is crucial to guide treatment and improve recovery. Yet neuroimaging—the current gold standard for detection—cannot detect subtle biochemical changes and is insufficient to diagnose many TBI cases. Consequently, there is an urgent need for early, brain-associated biomarkers of TBI. Following injury, brain cells release cell-free DNA (cfDNA) into peripheral blood due to neurovascular disruption. DNA methylation patterns, which inform tissue specificity, could serve as biomarkers for TBI severity and progression. To test this hypothesis, we analyzed cfDNA from peripheral blood of swine models of TBI having injuries of differing severity and type: mild and moderate contusional, and mild and moderate rotational injuries. Using whole genome bisulfite sequencing, we identified distinct brain-associated cfDNA epigenetic signatures of affected brain regions, and the underlying biological processes associated with each injury type and severity. In these brain regions, proton magnetic resonance spectroscopic imaging ( 1 H MRSI) independently confirmed changes in biomarkers of brain health and function (choline and N-acetylaspartate metabolites). Droplet digital PCR validated that cfDNA differentially methylated regions (DMRs) tracked TBI progression. Our findings provide evidence that brain-associated cfDNA methylome signatures, combined with 1 H MRSI, can characterize the neurobiological processes at sites of TBI in a preclinical model. These results support further investigation of cfDNA as a non-invasive biomarker for TBI detection and monitoring, pending validation in larger and clinically diverse cohorts.