A Single Cell and Spatial Transcriptomic Investigation of Traumatic Brain Injury: Novel insights into endothelial-derived Eph signaling

A staggering number of injury related disabilities and deaths are connected to traumatic brain injury (TBI) worldwide. Traumatic brain injury (TBI) involves an intricate and multifaceted cascade of events, starting with an initial mechanical impact followed by secondary injury brought on by numerous physiological changes that involve significant dysfunction at the cellular and molecular level. One major predictor of severe TBI outcome is the extent of blood-brain barrier (BBB) disruption, which under normal conditions prevents the passage of bacteria, neurotoxins, and macromolecules from entering the brain. Disruption of the BBB is linked to worse clinical outcomes in patients in both the acute, subacute, and chronic phases. However, the principal mechanisms responsible for regulating BBB permeability, where, and for how long that permeability occurs following TBI remains to be elucidated. Previous research has shown increased mRNA and protein expression of ephrin receptor A4 (EphA4), a well-established axon guidance molecule, within hours and days following TBI. This study is the first comprehensive investigation of the role of endothelial cell-specific EphA4 in TBI on regulating the BBB using advanced techniques like single-cell RNA and spatial transcriptomic sequencing, in addition to our newly established dual dye-labeling system. The central hypothesis is that endothelial cell-specific deletion of EphA4 enhances BBB integrity, characterized by changes in single cell gene expression consistent with improved barrier function, altered cellular metabolism, and reduced neuroinflammation within the BBB niche. This hypothesis will be tested by leveraging spatial sequencing to identify upregulation of genes associated with BBB stability and neuroprotection and utilizing a novel approach for assessing BBB permeability that addresses the limitations of traditional Evans Blue Dye (EBD) assays, including lack of spatial resolution, enabling precise analysis of molecular weight-dependent extravasation patterns.