Mitochondrial Mechanisms That Regulate Blood‐Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen‐Glucose Deprivation and Reoxygenation

Abstract

Ischemia‐reperfusion (IR) injury that occurs following ischemic stroke and traumatic brain injury induces blood‐brain barrier (BBB) breakdown, hyperpermeability and brain edema. Our objective was to determine the mitochondrial mechanisms that regulate BBB integrity and permeability using an oxygen‐glucose deprivation and reoxygenation (OGD‐R) in vitro model of IR injury. Rat brain microvascular endothelial cell (RBMECs) monolayers grown on Transwell inserts were exposed to OGD‐R for 1 or 3 hours alone or in presence of a caspase‐3 inhibitor Z‐DEVD or L‐ascorbic acid 1 hr prior to OGD or a caspase‐3 siRNA 48 hrs prior to OGD. Monolayer permeability was studied based on FITC‐dextran permeability. RBMECs were exposed to OGD‐R for 1 or 3 hours alone or in presence of L‐ascorbic acid. ROS was measured using DCFDA, cytosolic cytochrome c by ELISA and caspae‐3 activity fluorometrically. OGD‐R for 1 or 3 hours increased monolayer permeability, ROS, cytochrome c levels and caspase‐3 activity (p<0.05). Z‐DEVD, caspase‐3 siRNA and L‐ascorbic acid attenuated OGD‐R induced hyperpermeability (P<0.05). L‐ascorbic acid but not Z‐DEVD decreased ROS formation and cytochrome c levels (p<0.05). Immunofluorescence of ZO‐1 demonstrated disorganization of the TJs and Z‐DEVD and L‐ascorbic acid provided protection. These results suggest a major role for mitochondrial ROS in regulating OGD‐R induced BBB TJ integrity and permeability.