Poster Z14 - Attenuating Mitochondrial Dysfunction Restores Endothelial Barrier Function & Integrity Post Traumatic Brain Injury

Introduction: Traumatic brain injuries (TBIs) are life threatening and pose serious long-term consequences. Patients often experience neurological symptoms such as headaches, seizures, impaired motor skills, and an increased likelihood of developing neurodegenerative diseases later in life. The blood brain barrier (BBB) breakdown is a major initiator of the secondary traumatic brain injury cascade, associated with oxidative stress, reactive oxygen species (ROS), and inflammation of the endothelial layer of the BBB. Increased metabolic rate of the injured endothelial cells results in increased ROS generation by the mitochondria, overloading the antioxidant recovery mechanisms, and ultimately contributing to the state of oxidative stress at the BBB, leading to its eventual breakdown. BBB breakdown enables toxic and inflammatory molecules to pass into the neural micro-environment, contributing to the neuroinflammatory cascade responsible for patients’ symptoms. Currently, no treatment exists to intervene earlier in the neuroinflammatory cascade post TBI to repair and restore the integrity of the blood brain barrier and offer functional recovery. As hypermetabolism and mitochondrial dysfunction in the endothelial cells of the BBB post-TBI are a key contributor to BBB break-down, we hypothesize that attenuating mitochondrial dysfunction will restore the endothelial barrier function and integrity.

Materials and

Methods: Cell Culture: Human umbilical vascular endothelial cells (HUVECs) were maintained in tissue culture flasks at 37C and 5% CO2 in HUVEC maintenance media. HUVEC maintenance media is Large Vessel Growth Media supplemented with 2ng/mL fetal bovine serum (FBS), 1000ng/mL hydrocortisone, 10ng/mL hEGF, 3ng/mL bFGF, 10000 ng/mL heparin, and 1% penicillin/streptomycin. Media was changed every 48-72 hrs.
Barrier Integrity

Assessment: Endothelial barrier integrity was assessed by trans-endothelial electrical resistance (TEER) measurements using an EVOM2 voltmeter (Wold Precision Instruments) per manufacturers instructions.
Mitochondrial Assays: Mitochondria were labeled for live-imaging using MitoTracker Deep Red (ThermoFisher Scientific, M22426). Cells were stained with 100nM MitoTracker for 60 mins and counterstained with 0.2uL/mL of Hoechst 33342 at the same time. The cells were washed 2x with PBS after staining. Images were obtained using an Olympus IX81 microscope and Slidebook software (Olympus) using the 20x objective. Mitochondrial membrane potential was assessed using JC-1 dye (ThermoFisher Scienific, T3168). Cells were stained with 2ug/mL of JC-1 dye diluted in Large Vessel Growth Media for 30 mins at 37oC

Results, Conclusions, and Discussions: We present the development of an in vitro model of LPS induced endothelial barrier breakdown to model the breakdown of the endothelial barrier post-TBI. We also present that mitochondria specific drugs 5-(N-Ethyl-N-isopropyl)-Amiloride (EIPA) and LY344864, restore the function and integrity of an LPS-stressed in vitro endothelial barrier. These mitochondria specific drugs also attenuate endothelial IL-6 secretion. We also confirm the ability of EIPA to preserve the mitochondrial membrane potential and the ability of LY344864 to promote mitochondrial biogenesis. We demonstrate that resolving mitochondrial dysfunction restores endothelial barrier integrity and function. These results indicate that a mitochondria specific treatment may be able to restore the endothelial barrier of the BBB post-TBI.

Acknowledgements (Optional): Research reported here was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number T32 GM008339. Images created with BioRender.com.