A Single Primary Blast-Induced Traumatic Brain Injury in a Rodent Model Causes Cell-Type Dependent Increase in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Isoforms in Vulnerable Brain Regions

Abstract

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.

Keywords: 4-hydroxynonenal; NADPH oxidase; astrocytes; blast injury; microglia; neuron; oxidative stress; traumatic brain injury.

FIG. 1.

Schematic depiction of the shock tube. (A) Schematic of 9 × 9 inch square, 30 feet long shock tube with section I-Breech with high pressure helium gas separated from section II by different thickness of mylar sheets that generate pure shockwave in section III where the specimens are located. Section IV is past the section and is a design requirement; the pressure-time cycle is identical to live fire tests with actual C-4 (or trinitrotoluene equivalent) explosives at specified standoff distance. (B) Composite of actual experimental profiles that generate 180 kPa with only about five kPa variation in peak pressure and less than a millisecond in duration. The front of the pressure rise indicates shock-wave conditions. (C) Schematic of rodent model in prone position facing the shock front. The shock travels in the rostral-caudal direction traversing the pre-frontal cortex, striatum, hippocampus, thalamus, visual cortex, and cerebellum within a period of a millisecond with minimal attenuation of pressure loading. Color image is available online at www.liebertpub.com.neu

FIG. 2.

Blast increases the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms. Immunoblots of NOX1 and NOX2 isoforms in the cerebral hemisphere and cerebellum 4 h after blast at 180 kPa blast over pressure. There was a significant increase (80%) in NOX1 in both cerebral hemispheres and cerebellum, while NOX2 increased by 83% in cerebral hemispheres and 38% in cerebellum. n = 3, *p < 0.05.

FIG. 3.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms show a differential increase in different brain regions. Fluorescence intensities (red) of NOX1 and NOX2 in the hippocampus and cerebellum from control and blast-injury animals. Quantification of fluorescence intensities in different brain regions show a striking increase in NOX1 in the hippocampus of blast-injured animals compared with controls. Intensities of NOX2 display a higher increase in the frontal cortex compared with other regions. n = 4. *p < 0.01–0.05. Fluorescent intensities NOX1 in the cerebellum display a striking increase (96%) compared with NOX2 (38%). Color image is available online at www.liebertpub.com.neu

FIG. 4.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX)1 shows greater co-localization in neurons. Representative merged images showing the co-localization of NOX1 with NeuN, GFAP and Iba1 in the hippocampus indicating neuronal, astrocytic, and microglia localization, respectively, of NOX1 in control and blast-injured animals. Majority of increase in NOX1 with respect to corresponding controls is in neurons compared with astrocytes and microglia. Quantification of florescence intensities in different brain regions show a striking increase in NOX1 fluorescence in neurons in the hippocampus of blast-injured animals compared with controls. n = 4, *p < 0.01–0.05. Color image is available online at www.liebertpub.com.neu

FIG. 5.

Neurons show the highest increase in nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 expression. Quantification of florescence intensities in different brain regions show a striking increase in NOX2 fluorescence in the hippocampus and thalamus of blast-injured animals compared with other brain regions. n = 4, *p < 0.01–0.05.

FIG. 6.

Cerebellum displays an increased nicotinamide adenine dinucleotide phosphate oxidase (NOX)1 expression in neurons compared with astrocytes and microglia. Representative merged images showing the co-localization of NOX1 with NeuN, GFAP, and Iba1 in the cerebellum indicating neuronal, astrocytic, and microglia localization, respectively, of NOX1 in control and blast-injured animals. n = 4, *p < 0.01–0.05. Color image is available online at www.liebertpub.com.neu

FIG. 7.

Nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX)2 displays a greater increase in neurons compared with astrocytes and microglia. Similar to NOX1, the majority of NOX2 is localized in neurons compared with astrocytes and microglia. n = 4, *p < 0.01–0.05.

FIG. 8.

Primary blast increases superoxide levels in different brain regions. Representative fluorescent intensities (red) of dihydroethidium (DHE, dye that recognizes superoxide production) in hippocampus, thalamus and cerebellum in control, Blast-injured and blast+ apocynin. Quantification of fluorescence intensities in different brain regions shows a striking increase in DHE fluorescence in the hippocampus of blast-injured animals compared with controls indicating high levels of superoxide production in the hippocampus. Note that a pre-treatment with apocynin (APO), an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, completely blocks the DHE fluorescence increase indicating that the superoxide increase is mediated by activation of NOX. n = 5 *p < 0.01–0.05. Color image is available online at www.liebertpub.com.neu

FIG. 9.

Primary blast displays a strong tendency to increase 4HNE protein adducts. Immunoblot analysis of 4HNE in lysates from the hippocampus, thalamus, and cerebellum 4 h after blast at 180 kPa blast overpressure is shown. Two proteins of 100 and 70 kDa show a strong tendency to increase in the hippocampus and thalamus. n = 3.

FIG. 10.

Schematic of experimental blast injury model and effective loading and tissue-specific response. (A) The top panel shows the blast overpressure-time pulse applied to the rostral to caudal to rostral regions of the rodent brain travels with minimal change because of very short duration of less than a millisecond. The bottom panel shows different regions S1 to S4 analyzed in this study (B) A given brain volume comprises the six components (neurons, astrocytes, microglia, oligodendrocyte, vasculature, and interstitial/cerebrospinal fluid) that can vary from region to region. The mechanical properties of the representative volume given in B3 indicate that the effective mechanical stresses in the volume and hence the neural components will be determined by the differential volume fraction of that cell type. The different nicotinamide adenine dinucleotide phosphate oxidase expressions seen in this work are hypothesized to be driven by this differential cell volume fraction. Color image is available online at www.liebertpub.com.neu