Blast-induced traumatic brain injury (bTBI) is a “signature wound” in soldiers during training and in combat and has also become a major cause of morbidity in civilians due to increased insurgency. This work examines the role of blood-brain barrier (BBB) disruption as a result of both primary biomechanical and secondary biochemical injury mechanisms in bTBI. Extravasation of sodium fluorescein (NaF) and Evans blue (EB) tracers were used to demonstrate that compromise of the BBB occurs immediately following shock loading, increases in intensity up to 4 hours and returns back to normal in 24 hours. This BBB compromise occurs in multiple regions of the brain in the anterior-posterior direction of the shock wave, with maximum extravasation seen in the frontal cortex. Compromise of the BBB is confirmed by (a) extravasation of tracers into the brain, (b) quantification of tight-junction proteins (TJPs) in the brain and the blood, and (c) tracking specific blood-borne molecules into the brain and brain-specific proteins into the blood. Taken together, this work demonstrates that the BBB compromise occurs as a part of initial biomechanical loading and is a function of increasing blast overpressures.

Blast-induced traumatic brain injury (bTBI) has been recognized as the common mode of neurotrauma amongst military and civilian personnel due to an increased insurgent activity domestically and abroad. Previous studies from our laboratory have identified enhanced blood-brain barrier (BBB) permeability as a significant, sub-acute (four hours post-blast) pathological change in bTBI. We also found that NADPH oxidase (NOX)-mediated oxidative stress occurs at the same time post-blast when the BBB permeability changes. We therefore hypothesized that oxidative stress is a major causative factor in the BBB breakdown in the sub-acute stages. This work therefore examined the role of NOX1 and its downstream effects on BBB permeability in the frontal cortex (a region previously shown to be the most vulnerable) immediately and four hours post-blast exposure. Rats were injured by primary blast waves in a compressed gas-driven shock tube at 180 kPa and the BBB integrity was assessed by extravasation of Evans blue and changes in tight junction proteins (TJPs) as well as translocation of macromolecules from blood to brain and vice versa. NOX1 abundance was also assessed in neurovascular endothelial cells. Blast injury resulted in increased extravasation and reduced levels of TJPs in tissues consistent with our previous observations. NOX1 levels were significantly increased in endothelial cells followed by increased superoxide production within 4 hours of blast. Blast injury also increased the levels/activation of matrix metalloproteinase 3 and 9. To test the role of oxidative stress, rats were administered apocynin, which is known to inhibit the assembly of NOX subunits and arrests its function. We found apocynin completely inhibited dye extravasation as well as restored TJP levels to that of controls and reduced matrix metalloproteinase activation in the sub-acute stages following blast. Together these data strongly suggest that NOX-mediated oxidative stress contributes to enhanced BBB permeability in bTBI through a pathway involving increased matrix metalloproteinase activation.

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Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 (NOX1, NOX2), corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells.

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