Traumatic brain injury (TBI) is a serious public health concern. After TBI, elderly patients suffer from higher mortality rate and worse functional outcome compared to young patients. A further complication in the treatment of TBI occurs due to lack of clear knowledge on sex differences in TBI. The influence of biological age and sex on the outcomes of TBI is an important but understudied area of research and one of the major reasons for the limited success observed in several TBI-related clinical trials is, attributed to their failure to account for critical sex and age-related differences in disease expression. Considering this gap in knowledge, the major goal of our proposal is to conduct a pre-clinical study that focuses on the influence of age and sex in TBI. One of the most devastating clinical symptoms of TBI is the formation of cerebral edema leading to an increase in intracranial pressure that impairs cerebral perfusion and oxygenation, and contributes to additional ischemic injuries. Maintaining integrity of the blood-brain barrier (BBB), the protective barrier of the brain, and the prevention of the leakage of fluid and proteins from small blood vessels to the extravascular space (microvascular hyperpermeability) are critical to preventing and treating brain vasogenic edema. We propose a novel paradigm of age and sex dependent changes in microvascular hyperpermeability following TBI, and evaluate if an inflammatory-mediated signaling mechanism responsible for this hyperpermeability is age and sex dependent. Our overarching goal is to understand how age and sex of an individual differentially influence vascular permeability and leading to cerebral edema formation following TBI and ultimately develop new and effective therapeutics for head trauma patients of all ages/sex. In this proposal, we will investigate how age and sex differentially control microvascular permeability and molecular pathways that lead to activation of Neutrophil Extracellular Traps (NET) and NLRP3 inflammasome signaling resulting in vascular hyperpermeability, following TBI and how age and sex influence this pathway. Our central hypothesis is that age/sex differentially influence microvascular permeability, cerebral fluid dynamics and outcomes of TBI, in an NET/NLRP3 inflammasome- dependent manner. Increased NET formation that occurs following TBI promotes NLRP3 inflammasome- mediated endothelial tight junction (TJ) breakdown, hyperpermeability and cerebral edema formation in an age and sex dependent manner. Our specific aims are to: 1) Determine the influence of age and sex on microvascular hyperpermeability following TBI; 2) Determine the upstream regulators of microvascular hyperpermeability following TBI and the influence of age and sex. The proposed novel, integrated approach capitalizes on a refined and relevant mouse model of TBI combined with intravital microscopic imaging of microcirculation, thin-section and feeze-fracture electron microscopy of tight junc...