Severe traumatic brain injury temporally affects cerebral blood flow, endothelial cell phenotype, and cilia

Abstract

BackgroundPrevious clinical work suggested that altered cerebral blood flow (CBF) in severe traumatic brain injury (sTBI) correlates with poor executive function and clinical outcome. However, the molecular consequences of altered CBF on endothelial cells (ECs) and their blood flow-sensor organelle called cilia are not known. MethodsWe performed laser speckle contrast imaging, single cell isolation, and single cell RNA sequencing (scRNAseq) after sTBI in a closed skull, linear impact mouse model. Validation of select ciliary target protein changes was performed using flow cytometry. Additionally, in vitro experiments modeled the post-injury hypoxic environment to evaluate the effect on cilia protein ARL13B in human brain microvascular ECs. ResultsWe detected immediate reductions in CBF that were sustained for at least 100 minutes in both impacted and non-impacted sides of the brain. Our scRNAseq data detected heterogeneity in the brain cortex-derived EC cluster and demonstrated that two of five unique EC sub-clusters changed their relative proportions post-sTBI. Consistent with flow changes, we identified multiple genes associated with the fluid shear stress pathway that were significantly differentially expressed in brain ECs post-injury. Also, ECs displayed activation of ischemic pathway as early as day 1 post-injury, and enrichment of hypoxia pathway at day 7 and 28 post- injury. Arl13b ciliary gene expression was lost on day 1 in ECs cluster and remained lost for the entire course of the injury. We validated the loss of cilia protein ARL13B specifically from brain ECs as early as day 1 post-injury and detected the protein in the peripheral blood of the injured mice. We also determined that hypoxia could induce loss of ARL13B protein from cultured ECs. ConclusionsIn severe TBI, blood flow is disrupted in both impacted and non-impacted regions of the brain, creating a hypoxic environment that may influence ciliary gene and protein expression on ECs.