Amelioration of nicotinamide adenine dinucleotide phosphate–oxidase mediated stress reduces cell death after blast-induced traumatic brain injury

Abstract

A total of 1.7 million traumatic brain injuries (TBIs) occur each year in the United States, but available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to neuronal apoptosis and subsequent behavioral changes. Using a clinically relevant and validated rodent blast model, we investigated how nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression and associated oxidative stress contribute to cellular apoptosis after single and repeat blast injuries. Nox4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Using immunohistochemical-staining methods, we found a visible increase in Nox4 after single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in postmortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy. Nox4 activity correlated with an increase in superoxide formation. Alpha-lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely and increased antiapoptotic markers B-cell lymphoma 2 (t = 3.079, P < 0.05) and heme oxygenase 1 (t = 8.169, P < 0.001) after single blast. Subacutely, alpha-lipoic acid treatment reduced proapoptotic markers Bax (t = 4.483, P < 0.05), caspase 12 (t = 6.157, P < 0.001), and caspase 3 (t = 4.573, P < 0.01) after repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and paired helical filament staining. Alpha-lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t = 3.573, P < 0.05) compared with blast exposed animals without treatment. TBI can cause debilitating symptoms and psychiatric disorders. Oxidative stress is an ideal target for neuropharmacologic intervention, and alpha-lipoic acid warrants further investigation as a therapeutic for prevention of chronic neurodegeneration.

INTRODUCTION

Traumatic brain injury (TBI) remains an immense public health burden in the United States and continues to be a leading cause of morbidity and mortality in individuals younger than the age of 45 years.¹ Mild TBI can lead to progressive motor, cognitive, and behavioral decline.² These long-term outcomes result in enormous societal costs.³ In particular, blast-induced TBI has been classified as the “signature injury” of the recent wars in Iraq and Afghanistan. National and international emphasis has been placed on how acute neurotrauma leads to chronic neurodegeneration over time. The progression depends on both severity and number of exposures. To investigate the pathologic process that contributes to neurodegeneration, scaled injury in animal models must be ideally used. A blast wave injury has 3 primary effects: wave transport through tissue, acceleration-deceleration causing axonal shearing, and disruption of the blood brain barrier (BBB). Typically, the primary damage of TBI is followed by secondary injury, which can develop at a later date. The initial insult can lead to major brain damage and loss of functional outcome. Secondary injury, on the other hand, triggers a cascade of biochemical events leading to neuroinflammation, brain edema, and delayed neuronal cell death and neurodegeneration.⁴

Delayed neuronal cell death is carefully mediated through the process of apoptosis. When the cell is damaged beyond repair, a signaling cascade is initiated, which can lead to cell destruction. Many pathways contribute to apoptosis activation in the cell, but one of the main methods is the development and propagation of free radical oxygen species (ROS). The creation of ROS has been linked to the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a noninjury state, NADPH oxidase (Nox) participates in cellular host defense through cytokine signaling, gene expression regulation, post-translational processing of protein, endoplasmic reticulum stress response, and tissue homeostasis.⁵ Only after injury or infection does the Nox pathway contribute to ROS formation. Inhibiting Nox2 with apocynin after TBI has provided beneficial behavioral and histopathologic results.^6,7 Lipoic acid is another readily available supplement that can inhibit the formation of ROS but does not cross mitochondrial membranes, thereby targeting plasma membrane NADPH activity.⁸ Although the mechanism is not fully known, alpha-lipoic acid can reduce toxic free radicals and decrease Nox activity.⁹ We sought to investigate the effect of lipoic acid on the less well-studied Nox pathway, Nox4, which is found at neuronal plasma membranes (Supplementary Fig 1).

The Nox4 pathway can lead to the generation of the superoxide radical (O₂⁻).⁴ The superoxide radical is produced when an oxygen molecule gains a single electron from another substance. Excess O₂⁻ can form the toxic hydroxyl radical (OH⁻) through interaction with hydrogen peroxide (H₂O₂). The hydroxyl radical can eventually combine with nitric oxide to form peroxynitrite (ONO₂⁻).⁴ Peroxynitrite has devastating effects on cellular membranes. Historically, mitochondria dysfunction has been postulated as the primary source of free radical species generation, but recent evidence has shown that Nox can also contribute to reactive species development. Nox is a composite membrane enzyme made up of Nox and phox subunits that associate with a series of supporting proteins.^10,11 The Nox subunits come in 1 of 5 isoforms, and each isoform is heavily concentrated in a specific organ. Nox4 is one of the isoforms most heavily concentrated within the central nervous system. Nox4 is constitutively active and produces large amount of superoxide after injury. Through interaction with cellular components, Nox4 generates hydrogen peroxide and subsequently the potent hydroxyl radical.⁵ Nox4 has not been previously studied for its role in ROS formation after blast injury, therefore opening an important avenue for investigating a novel and potentially important therapeutic target.

Oxidative stress induction and the consequential damage that occurs as a result of blast exposure are likely profound, but the full extent of injury has yet to be elucidated. In this work, we investigate the effects of Nox, specifically Nox4 activity, in the progression toward apoptosis and tau hyperphosphorylation after a moderate blast injury 24 hours after blast exposure and 2 weeks after repetitive blast exposure. We selectively targeted the Nox4 response at the neuronal plasma membrane using the antioxidant alpha-lipoic acid. Alpha-lipoic acid is a natural occurring compound found in multiple cell types that acts as a cofactor in oxidative metabolism reactions.¹² It is thioctic acid with a disulfide bond, chirality, and a dithiolane ring.¹³ The bioavailability is 30%, and it is metabolized to 5 metabolites by the liver, which is then cleared primarily by catabolism.¹⁴ It acts as a free radical scavenger when administered peripherally and readily passes through the BBB and neuronal membranes but not through mitochondrial membranes.¹⁵ In particular, it has been shown to be a carbonyl scavenging compound after TBI.¹⁶ Supplementation in humans after TBI has provided reduction in adverse symptoms.¹⁷ It reduces susceptibility for seizures and excitotoxicity.¹⁸

We investigate its role in modulation of Nox4 for the prevention of the chronic sequelae associated with neurotrauma.

MATERIALS AND METHODS

The article conforms to all relevant guidelines for human and animal research.

Animals.

All procedures involving live animals were approved by the Institutional Animal Care and Use Committee of West Virginia University and were performed according to the principles of the Guide for the Care and Use of Laboratory Animals (NIH). This work used forty 350 g male Sprague Dawley rats acquired from Hilltop Lab Animals (Hilltop Lab Animals, Inc). The method used to calculate the number of animals needed for a given study was determined using information from the book, Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. The number of animals was based on effect size, standard deviation, power, and significance. The equation to be used to calculate sample size is n = 1 + (2C [s/d]²), where C is a value dependent on the a and b values chosen. For our studies, a = 0.05 and b = 0.2 for a power of 1–b = 0.8. C_{(0.05, 0.8)} = 7.85 based on known constants. Effect size and standard deviation expected are variable per assay and were calculated based on numbers from previous work. Animals were acclimated for 1 week before experimental use and were housed under 12-hour light/12-hour dark conditions with food and water available ad libitum.

Human brain sections.

Postmortem formalin-fixed brain sections were obtained from the entorhinal cortex of a retired professional football player and professional wrestler as well as an age- and sex-matched control. Descriptions of the chronic traumatic encephalopathy (CTE) cases have been previously described.^19,20 Briefly, the professional wrestler committed suicide at age 40 years. No atrophy or necrosis was observed in the brain. Neurofibrillary tau tangles were frequently scattered, and he had the apolipoprotein E3/E3 allele. The professional football player died from traumatic injury at age 36 years. The brain was grossly intact with widely spread tauopathy. No other neurodegenerative disease diagnostic features were found in either case. The age- and sex-matched control did not have a known history of neurotrauma and no evidence of tau pathology.

Blast exposure.

Rats were exposed to TBI via a moderate blast wave exposure characterized previously by our laboratory.²¹ Briefly, the animals were anesthetized with 4% isoflurane before blast exposure. Animals were then oriented with the long axis of the animal perpendicular to the blast front. The blast was delivered perpendicular to the head with the wave encountering the right side of the head (directly) before passing through the skull and exiting the left side (indirectly). The thorax and abdomen were protected using rigid shielding. The design allows for acceleration and deceleration to occur freely because the head is not fixed, similar to battlefield exposure. Animals were divided into the following groups for the 24-hour study: (1) control (n = 4); (2) lipoic acid only (n = 4); (3) moderate blast exposure only (n = 4), and (4) moderate blast exposure along with lipoic acid treatment (n = 4). Immediately after blast, the animals were returned to a holding cage with a homeothermic heating blanket with rectal thermometer to maintain body temperature at 37°C. Once reflexes were restored, animals were returned to the home cage.

For repetitive blast exposure, animals received blast every other day for a total of 6 blast exposures over a 2-week period. The groups used for this study were control (n = 8), repeat blast (n = 8), and repeat blast along with lipoic acid treatment (n = 8). The animals were exposed to the same intensity blast wave as the animals from the 24-hour experimental group. Animals were returned to a holding cage for monitoring before being placed in their home cage. Animals were anesthetized with 4% isoflurane before every blast exposure. Animals were killed 2 weeks after the final blast exposure.

Alpha-lipoic acid treatment(s).

Alpha-lipoic acid was obtained from Sigma Aldrich. It was dissolved in sterile saline solution with 10% ethanol (0.1 mL/10 g body weight; Teknova, Fisher Scientific, Pittsburgh, Pennsylvania). For animals that were part of the drug treatment group, the animals were administered 10 mg/kg alpha-lipoic acid for 5 minutes after each blast exposure via intraperitoneal injection. Animals receiving blast injury only or controls received an intraperitoneal injection of saline after each blast. Animals were closely monitored and on full recovery were returned to the home cage.

Elevated plus maze.

The elevated plus maze (EPM) was elevated 60 cm from the floor. The 2 open arms intersected perpendicular to the 2 closed arms. Each arm was 50 cm by 10 cm. The closed arms were encased by black wood siding 30 cm tall on all exposed sides. EPM assessment was conducted 7 days after repetitive blast exposure according to procedures outlined by Walf and Frye.²² The rats were placed in the center of the maze facing an open arm. Each animal was recorded for a total of 5 minutes with open access to every arm. ANY-maze version 4.63 video tracking software (Stoelting Co) was used to record the number of entries into the open arm, time spent in the open arm, and distance traveled in the open arm. The ANY-maze software settings were as follows: test duration 300 seconds and percentage of body to be considered in arm 90%.

Immunoblotting.

Protein was isolated from each respective sample by sonication in 500 μL of hot (85°C–95°C) 1% sodium dodecyl sulfate as previously described.²³ Samples were run using 50 μg of protein/well using Tris-Glycine 10% self-poured 10-well gels in combination with 5× Laemmli sample buffer. Gels were run using a Mini-PROTEAN system (Bio-Rad) and transferred to polyvinylidene fluoride membranes (Bio-Rad) using wet electrophoretic transfer cells (Bio-Rad). Primary antibodies used were anti–B-cell lymphoma 2 (Bcl-2; rabbit) and anti–heme oxygenase 1(HO-1; goat) at 1:200 (Santa Cruz Biotechnology, Santa Cruz, California). An HRP-conjugated β-actin rabbit monoclonal antibody (Cell Signaling) was used as an endogenous control for all samples at a concentration of 1:10,000. Molecular weight determination was conducted using SeeBlue Plus2 (Life Technologies, Carlsbad, California). LI-COR secondary antibodies, IRDye 800CW (goat antirabbit) and IRDye 680RD (goat antimouse; LI-COR, Lincoln, Nebraska), were used with an Odyssey fluorescent scanner at wavelengths 800 or 700, intensity 6.0, and 84 resolution with high image quality. Images were converted to gray scale, analyzed after background subtraction, and normalized to β-actin to give relative overall intensity.

Samples used for carbonyl detection were isolated and sonicated in 6% sodium dodecyl sulfate. Control samples were mixed with derivation control solution, and treatment samples were mixed with 1× DNPH solution. The samples were incubated for 15 minutes followed by mixing with a neutralization solution. The samples were loaded 20 μL per well and ran on a 10% gel as previously outlined. The primary antibody and standards were from an OxyBlot Protein Oxidation Detection Kit (Millipore).

Reactive oxygen species detection.

Brain tissue was harvested from each treatment group including control, single blast, and single blast with lipoic acid and prepared in accordance to procedures outlined in the ROS Detection Kit manual. Briefly, tissue was homogenized, and cells were isolated by incubation in collagenase at 2 mg/mL for approximately 30 minutes. Cells were then separated by enzyme digestion and manual disruption with repeated pipetting. Cells were strained through a 70-nm nylon cell strainer followed by centrifugation at 400 × g for 5 minutes. The pellet was then resuspended to a concentration of 0.5 × 10⁶–1.0 × 10⁶ cells/mL in Dulbecco’s Modified Eagle medium.

Total ROS and superoxide were detected using a Total ROS/Superoxide detection kit (Enzo Life Sciences) according to manufacturer’s instructions for a fluorescent microplate assay. In brief, 100 μL of suspended cells were added to each well of a dark-walled 96 well plate with a clear bottom. The cells were incubated overnight at 37°C in Dulbecco’s Modified Eagle medium. Media was then removed the following day, and 100 μL of ROS/Superoxide detection solution, prepared using kit reagents, was added to each well and incubated in the dark for 60 minutes. Total concentrations of ROS determined by a glutathione-based reaction (green) and superoxide determined by a xanthine oxidase reaction (red/yellow) were detected at an excitation/emission of 488/520 nm and 550/610 nm, respectively, using a BioTek Synergy H1 Hybrid Reader. Data were collected using Gen5 2.01 software.

Histologic preparations.

Animals were anesthetized as described previously and perfused transcardially with cold 0.9% saline followed by 10% formalin for a total of 10 minutes. The brain was then extracted and placed into fresh 10% formalin for a minimum of 24 hours. After the fixation, the brain was blocked into sections, and paraffin was embedded as previously described.²⁴ Briefly, tissues were processed using the Tissue-Tek Q4 170 VIP 5 automatic tissue process (Sakura Finetek) and embedded in paraffin using the Tissue-Tek TEC 5 embedding system (Sakura Finetek). Tissues were sliced using a Leica RM2235 microtome (Leica Microsystems), and slices mounted on slides for staining. Standard fluorescent and DAB staining protocols were used for staining iron (Sigma Aldrich), 8-hydroxy-2-deoxyguanosine, Nox4, p22phox, and Bax (Santa Cruz); caspase 3 and caspase 12 (Cell Signaling); and paired helical filament (PHF), MCI, and CP-13 (kind gift from Dr Peter Davies).

Images were acquired from the S1BF region of the cortex (10 slides per animal). Imaging was performed using a Zeiss Axio Imager 2 for brightfield images and Zeiss Axio Observer Z1 for fluorescent images shown. For fluorescent staining, 10 cells per slide were randomly selected, outlined, and measured with ImageJ software (NIH) by an observer blinded to experimental group. Density was adjusted per mean area to give corrected total cell fluorescence normalized to background. For DAB and iron staining, an observer blinded to experimental group selected 100 random cells per slide and recorded the ratio of positive cells to total cells.

Data analysis.

Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, California). A 1-way ANOVA with Tukey’s post hoc test for multiple comparisons was used for western blot, fluorescent IHC, and EPM. Tukey’s post hoc multiple comparisons test is a pair-wised comparison between each group with every other group. The graphs show the comparisons that were statistically significant. χ² analyses were used for DAB and iron staining. A P value < 0.05 was considered statistically significant for all data analyzed.

RESULTS

An increase is seen in intraparenchymal iron, Nox4, and p22phox after single blast.

Iron staining revealed an increase in intracellular iron deposition (19/100) 24 hours after blast injury in the left prefrontal cortex compared with control (2/100; Fig 1, A). Free iron induces oxidative stress, which is toxic to neuronal plasma membranes.²⁵ Lipoic acid administered 5 minutes postblast leads to decreased intracellular accumulation of iron (11/100) compared with blast only animals with a χ² = 40.594, P < 0.0001. Nox4 is found at plasma membranes and contributes to free radical formation.²⁶ Nox4 was visibly increased (29/100) 24 hours after blast injury in the left prefrontal cortex compared with control (8/100; Fig 1, B). Lipoic acid visibly reduced Nox4 (12/100) when given 5 minutes postblast with χ² = 29.079, P < 0.0001. p22phox forms an activation complex with Nox4 to regulate downstream genetic markers. p22phox was also visibly increased (23/100) 24 hours after single blast in the left prefrontal cortex compared with control (6/100). Likewise, alpha-lipoic acid treatment 5 minutes postblast decreased p22phox (13/100) with χ² = 24.031, P < 0.0001. Therefore, iron-mediated toxicity possibly acting through Nox4 oxidative stress is mitigated by alpha-lipoic acid treatment.

Fig 1.

Increased intracellular iron and Nox in the left prefrontal cortex 24 hours postblast. Scale bar for whole images = 50 μm. Scale bar for inlay images = 20 μm. Arrows indicate area enlarged for high power image. (A) The amount of intracellular iron accumulation increased 24 hours after single blast (19/100) compared with control (2/100). When alpha-lipoic acid was given 5 minutes postblast the amount of intracellular iron decreased (11/100) compared with single blast indicating preserved plasma membrane integrity. (B) DAB staining for Nox4 shows an increase in expression in the left prefrontal cortex 24 hours after single blast (29/100) compared with control (8/100). The increased level of Nox4 suggests an elevated production of reactive oxygen species. Alpha-lipoic acid treatment decreased Nox4 back toward control levels (12/100). (C) DAB staining of p22phox, which forms an activation complex with Nox4, showed increased expression in single blast (23/100) compared with control (6/100). Alpha-lipoic acid treatment 5 minutes postblast decreased p22phox (13/100) back toward control levels. Nox, nicotinamide adenine dinucleotide phosphate oxidase.

Nox4 activity increases superoxide formation but not total ROS after single blast.

The Nox4/p22phox complex can lead to the generation of superoxide. Superoxide can damage the cell if not readily cleared. Blast only animals had a visible increase in superoxide 24-hour postblast compared with control (Fig 2, A). Lipoic acid administered 5 minutes postblast visually decreased superoxide back to control levels. Fluorescent intensity measurement revealed a significant difference between groups for superoxide (F[2, 15] = 30.93, P < 0.001). Post hoc comparison revealed that single blast exposure significantly increased superoxide compared with control (q = 9.1, P < 0.001), but lipoic acid administered 5 minutes after single blast ameliorated this response (q = 10.09, P < 0.001; Fig 2, B). No significant difference was seen between groups for total ROS (F [2, 15] = 3.568, P = 0.054) lending credence to a NADPH-specific response vs mitochondria-dependent response.

Fig 2.

Lipoic acid reduces the generation of superoxide in the left prefrontal cortex postblast. The Nox4 pathway can lead to the generation of free radicals at the plasma membrane. In particular, NADPH leads to the generation of superoxide. This radical can damage intracellular organelles, which contribute to apoptosis. (A) A visual representation of superoxide staining shows an increase after single blast that is reduced by lipoic acid administration postblast. (B) A significant increase in superoxide between single blast and control groups was detected with fluorescent scanning at 24 hours postblast (q = 9.01, P < 0.001) but was ameliorated by lipoic acid administration (q = 10.09, P < 0.001). ***P < 0.001 SB vs CTRL. ###P < 0.001 SB vs LA + SB. (C) No significant changes were reported for total reactive oxygen species. CTRL, control; LA, lipoic acid; NADPH, nicotinamide adenine dinucleotide phosphate; Nox, NADPH oxidase; ROS, free radical oxygen species; SB, single blast.

Lipoic acid prevents formation of oxidative stress byproducts.

Previous studies have shown that byproducts of oxidative stress are significantly increased after TBI.²⁷ In particular, 8-hydroxy-2-deoxyguanosine and carbonyls are elevated after injury.²⁸ We found a significant difference between groups for 8-hydroxy-2-deoxyguanosine in the cortex 24 hours postblast (F [2, 27] = 28.18, P < 0.001; Fig 3, A). Tukey’s post hoc comparison showed a significant difference between control and blast (q = 10.53, P < 0.001) and blast and blast + lipoic acid (q = 4.074, P < 0.05). We found a significant difference between groups for carbonyls (F[3, 16] = 10.63, P < 0.001; Fig 3, B). Tukey’s post hoc comparison showed a significant difference between control and blast (q = 6.429, P < 0.01), lipoic acid and blast (q = 4.226, P < 0.05), and blast and blast + lipoic acid (q = 7.302, P < 0.001). Lipoic acid significantly reduced oxidative stress byproducts.

Fig 3.

Lipoic acid significantly reduces oxidative stress byproducts. 8-hydroxy-2-deoxyguanosine and carbonyls have been showing to be increased after TBI. (A) A significant increase in 8-hydroxy-2-deoxyguanosine was seen 24 hours postblast compared with control (q = 10.53, P < 0.001). Lipoic acid administration prevented this increase (q = 4.074, P < 0.05). (B) A significant increase in carbonyls was seen between control and blast (q = 6.429, P < 0.01). Other significant comparisons were seen between the lipoic acid and blast groups (q = 4.226, P < 0.05) and the blast group and blast + lipoic acid group (q = 7.302, P < 0.001). **P < 0.01, ***P < 0.001, #P < 0.05, ###P < 0.001, !P < 0.05. LA, lipoic acid; SB, single blast; TBI, traumatic brain injury.

A decrease in antiapoptotic markers is seen 24 hours postblast, but lipoic acid restores toward baseline.

Western blot investigation showed significant changes to antiapoptotic makers in the left prefrontal cortex after a single blast exposure. Bcl-2 is an important antiapoptotic oncogene that provides neuroprotection when increased.²⁹ A significant difference was seen between groups 24 hours postblast (F[3, 12] = 4.637, P < 0.05). Post hoc comparison revealed that single blast exposure significantly decreased Bcl-2 compared with lipoic acid only (q = 3.079, P < 0.05; Fig 4, A). The multiple comparisons between other groups showed no significant difference. HO-1 is an inducible enzyme that degrades toxic heme after oxidative stress. Pharmacologic upregulation of HO-1 after TBI leads to improved neuronal survival by preventing apoptosis.³⁰ A significant difference was seen between groups for HO-1 24 hours postblast (F[3, 12] = 11.33, P < 0.001). Post hoc comparison revealed that lipoic acid only increased the level of HO-1 expression when compared with control (q = 4.826, P < 0.05; Fig 4, B). Post hoc comparison also showed that the expression of HO-1 decreased significantly between the lipoic acid only and single blast (q = 8.169, P < 0.001) animals. The level of expression also increased significantly between single blast and single blast treated with lipoic acid groups (q = 4.417, P < 0.05) back toward control levels. The overall increase in antiapoptotic markers after lipoic acid treatment suggests a protective mechanism limiting Nox4-induced oxidative stress damage.

Fig 4.

Antiapoptotic markers from left prefrontal cortex 24 hours after injury. (A) A decrease in Bcl-2 expression was seen in single blast compared with lipoic acid only (q = 3.079, P < 0.05). No significant difference is seen when comparing other experimental groups. *P < 0.05 LA vs SB. (B) Significant differences were seen between groups when probing for HO-1. An increase in HO-1 was seen between control compared with lipoic acid only (q = 4.826, P < 0.05). A decrease was seen between lipoic acid only and single blast (q = 8.169, P < 0.001), and an increase was seen between single blast compared with single blast treated with lipoic acid (q = 4.417, P < 0.05). No significant difference was seen when comparing the other experimental groups. *P < 0.05 CTRL vs. LA. ###P < 0.001 LA vs SB. !P < 0.05 SB vs LA + SB. Bcl-2, B-cell lymphoma 2; CTRL, control; HO-1, heme oxygenase 1; LA, lipoic acid; SB, single blast.

Mild changes in proapoptotic markers were seen 24 hours postblast.

Fluorescent immunohistochemistry of the left prefrontal cortex showed mild changes of proapoptotic markers 24 hours after single blast exposure. Bax is the proapoptotic regulator of Bcl-2 that is often increased after TBI.³¹ Not surprisingly, a significant difference between groups was seen for Bax (F[2, 27] = 3.604, P < 0.05). Post hoc comparison revealed a significant difference between animals exposed to a single blast compared with control (q = 3.724, P < 0.05; Fig 5, A). No other significant expression changes were seen between the other experimental groups. Caspase 12 is a proapoptotic marker that can lead to the activation of caspase 3. A significant difference was seen between groups (F[2, 27] = 4.325, P < 0.05). Post hoc comparison revealed a decrease in the expression of caspase 12 in animals exposed to a single blast with alpha-lipoic acid treatment compared with control (q = 4.056, P < 0.05; Fig 5, B). No other changes in expression were seen between any other groups. Caspase 3 is an important downstream regulator of apoptosis. If caspase 3 is active, apoptosis will occur. No significant change in caspase 3 expression was seen between experimental groups (F[2, 33] = 1.985, P = 0.1535; Fig 5, C).

Fig 5.

Immunohistochemistry of proapoptotic markers in the left prefrontal cortex at 24 hours postblast. Scale bar = 100 μm. Arrows represent area enlarged for high-power image. (A) Bax expression is increased in animals exposed to single blast compared with control (t = 3.724, P < 0.05). No change is seen between the other groups. *P < 0.05 CTRL vs. SB. (B) A decrease is seen in the expression of caspase 12 in animals treated with alpha-lipoic acid after blast compared with control (t = 4.056, P < 0.05). No change is seen between other experimental groups. *P < 0.05 CTRL vs SB + LA. (C) Caspase 3 expression shows no change between experimental groups. CTRL, control; LA, lipoic acid; SB, single blast.

No appreciable difference in antiapoptotic markers were observed 2 weeks postrepeat blast.

Repeat injury is a growing concern in the field of neurotrauma. Repetitive concussions have been linked to CTE and other tauopathies.³² An area in need of further investigation is the development of treatment options to prevent chronic neurodegeneration after acute TBI. We sought to investigate the beneficial effects of lipoic acid administration given 5 minutes after each blast (6 blasts total every other day for 2 weeks). Western blot analysis of the left prefrontal cortex was performed to determine expression changes in antiapoptotic markers 2 weeks after repetitive blast exposure. No significant difference between groups was observed for Bcl-2 (F[3, 12] = 2.538, P = 0.1057; Fig 6, A). Likewise, no significant difference was observed between groups for HO-1 (F[3, 12] = 0.2129, P = 0.8855; Fig 6, B). Expression of all markers was shown to remain consistent across all treatment groups. The data support that no changes in antiapoptotic markers were seen subacutely after repeat injury.

Fig 6.

Immunoblot of antiapoptotic markers in the left prefrontal cortex 2 weeks after repetitive blast exposure. Animals were exposed to 6 blasts total over the time course of 2 weeks. No significant differences between groups were observed for (A) Bcl-2 or (B) HO-1. Bcl-2, B-cell lymphoma 2; CTRL, control; HO-1, heme oxygenase 1; LA, lipoic acid; LA + RB, lipoic acid treatment 5 minutes after each blast; RB, repeat blast.

Lipoic acid reduced proapoptotic markers 2 weeks after repetitive blast.

Apoptosis is an important indicator of neurodegeneration.³³ We examined 3 proapoptotic markers in the left prefrontal cortex 2 weeks after repetitive blast. A significant difference was seen in the expression of Bax between groups (F[2, 27] = 7.93, P < 0.01). Post hoc comparison showed an increase in Bax with repetitive blast animals compared with control (q = 5.194, P < 0.01), but the increase was ameliorated when lipoic acid was given after each blast (q = 4.483, P < 0.05; Fig 7, A). No significant difference existed between the control and repetitive blast treated with alpha-lipoic acid groups. A significant difference between groups was noted for caspase 12 (F[2, 27] = 11.96, P < 0.001). Post hoc comparison revealed a significant increase in expression between repetitive blast animals compared with control (q = 5.809, P < 0.001), but lipoic acid administration after each blast negated this response (q = 6.157, P < 0.001; Fig 7, B). No significant difference existed between the control and repetitive blast treated with alpha-lipoic acid groups. A significant difference in caspase 3 expression was noted between groups (F[2, 27] = 12.6, P < 0.001). Post hoc comparison revealed a significant increase in caspase 3 for animals exposed to repetitive blast compared with control animals (q = 6.989, P < 0.001), but repetitive blast animals treated with alpha-lipoic acid had a reduced response (q = 4.573, P < 0.01; Fig 7, C). Lipoic acid effectively decreases expression of proapoptotic markers at subacute time points (2 weeks) after repetitive blast.

Fig 7.

Immunohistochemistry of proapoptotic markers 2 weeks after repetitive blast exposure. Scale bar = 100 μm. Arrows represent area enlarged for high-power image. (A) A significant increase in Bax expression is seen between animals exposed to repetitive blast compared with control (t = 5.194, P < 0.01). Lipoic acid treatment administered 5 minutes after each blast reduced Bax expression back toward control levels compared with the repeat blast group (t = 4.483, P < 0.05). No change is seen between the other groups. **P < 0.01 CTRL vs RB. #P < 0.05 RB vs RB + LA. (B) An increase in the expression of caspase 12 was seen for animals exposed to repetitive blast compared with control (t = 5.809, P < 0.001). Alpha-lipoic acid treatment reduced caspase 12 levels back toward control compared with the repeat blast group (t = 6.157, P < 0.001). No change is seen between the control and the repetitive blast with alpha-lipoic acid treatment groups. ***P < 0.001 CTRL vs RB. ###P < 0.001 RB vs RB + LA. (C) An increase is seen in the expression of caspase 3 in the repetitive blast group compared with the control group (t = 6.989, P < 0.001). A significant reduction was noted when alpha-lipoic acid was administered after blast compared with the repetitive blast group (t = 5.459, P < 0.01). No change is seen between the control and repetitive blast treated with lipoic acid groups. ***P < 0.01 CTRL vs RB. ##P < 0.01 RB vs. RB + LA. CTRL, control; LA, lipoic acid; and RB, repeat blast.

Nox markers and proapoptotic markers are increased in human CTE samples.

We examined the NADPH markers, Nox4 and p22phox, in human CTE samples obtained from a professional football player and professional wrestler that were then compared with an age- and sex-matched control. A visible increase in Nox4 was observed in the football player (32/100) and wrestler (27/100) samples compared with control (6/100) with χ² = 26.527, P < 0.0001 (Fig 8, A). Likewise, a visible increase in p22phox was observed for the football player (17/100) and wrestler (24/100) samples compared with control (3/100) with χ² = 34.585, P < 0.0001 (Fig 8, B). The visual evidence suggests that concussive injuries lead to an increase in the expression of NADPH markers at extended time points. This increase is likely associated with the production of free radicals that can lead to permanent tissue damage. The response to tissue damage in a diseased brain is often impaired. A significant difference between groups was observed for the antiapoptotic marker Bcl-2 (F[2, 27] = 23.8, P < 0.001). Post hoc comparison was significant between football player and control (q = 7.302, P < 0.001) and between wrestler and control (q = 9.255, P < 0.001; Fig 9, A). Neurofibrillary tangles have been proposed to spread between intact cells. These cells must be able to avoid apoptosis when injured. A significant difference was also seen between groups for the proapoptotic marker Bax (F[2, 27] = 23.38, P < 0.001). Post hoc comparison was significant between football player and control (q = 5.424, P < 0.01), between wrestler and control (q = 4.221, P < 0.05), and between football player and wrestler (q = 9.645, P < 0.001; Fig 9, B). The football player case was a less severe form of CTE indicating that apoptosis may play a role in the early disease stages. The wrestler case had severe CTE pathology and showed that apoptosis maybe less likely to play a role in late disease stages.

Fig 8.

Nox marker expression in human CTE samples. Samples obtained from the cortex of a professional football player, professional wrestler, and age- and sex-matched control. Scale bar for whole images = 50 μm. Scale bar for inlay images = 20 μm. Arrows represent area enlarged for high-power image. (A) Nox4 staining shows increased expression in tissue of the football player (32/100) and professional wrestler (27/100) when compared with the control tissue (6/100). The increase of Nox4 can lead to the formation of reactive oxygen species that cause cellular damage. (B) p22phox forms a complex with Nox4 that leads to increased Nox activity within the cell. An increase in the expression of p22phox was seen in the samples from the football player (17/100) and wrestler (24/100) compared with the control tissue (3/100). The complex formation of these 2 components suggests subsequent free radical formation. CTE, chronic traumatic encephalopathy; Nox, nicotinamide adenine dinucleotidephosphate oxidase.

Fig 9.

Proapoptotic marker expression in human CTE samples. Scale bar = 100 μm. Arrows represent area enlarged for high-power image. (A) Bcl-2 is a protective protein that is increased to prevent cellular apoptosis. In the human tissue, Bcl-2 expression is greater in football player tissue compared with control (t = 7.302, P < 0.001). Bcl-2 expression is also significantly increased between wrestler tissue compared with control (t = 9.255, P < 0.001). ***P < 0.001 CTRL vs football player and CTRL vs wrestler. The increase in Bcl-2 could be a mechanism by which neurons survive to propogate tau. (B) Bax is known to initiate the cellular apoptosis process. A significant difference was seen between the football player tissue sample and control (t = 5.424, P < 0.01). A significant difference was also seen between the wrestler tissue sample and control (t = 4.221, P < 0.05). A significant difference in Bax was also seen between the football player sample and wrestler sample (t = 9.645, P < 0.001). **P < 0.01 CTRL vs football player. *P < 0.05 CTRL vs wrestler. ###P < 0.001 football player vs wrestler. The wrestler sample had more severe CTE pathology compared with the football player sample indicating that apoptosis may play an important role in acute disease progression but not as much of a role once it has fully developed. Tauopathy progression is reliant on this disrupted apoptotic signaling. Bcl-2, B-cell lymphoma 2; CTE, chronic traumatic encephalopathy; CTRL, control; NFL, professional football player; WWE, professional wrestler.

Tau pathology increased after repetitive blast in rodents and in human CTE specimens.

After injury, tau becomes hyperphosphorylated and can undergo conformational changes leading to neurofibrillary tangles. CP-13 is a marker of pretangle tau, whereas MCI is a conformational configuration of tau seen primarily in neurodegenerative disease. PHF is a dimerized form of tau that precedes the formation of oligomers. A significant difference between groups was observed for CP-13 in blast-exposed animals (F[2, 27] = 8.15, P < 0.01). Post hoc comparison showed a significant increase in CP-13 for the repeat blast group compared with control (q = 5.54, P < 0.01), but lipoic acid ameliorated this change when administered postblast (q = 3.967, P < 0.05; Fig 10, A). A significant difference was not observed between groups for MCI in blast exposed animals (F[2, 27] = 2.492, P = .1016; Fig 10, B). A significant difference between groups was observed for PHF in blast exposed animals (F[2, 27] = 18.01, P < 0.001). Post hoc comparison showed a significant increase in PHF for the repeat blast group compared with control (q = 7.865, P < 0.001), but lipoic acid ameliorated this change when administered postblast (q = 6.703, P < 0.001; Fig 10, C). A significant difference was observed for CP-13 in human specimens (F[2, 27] = 20.31, P < 0.001). Post hoc comparison revealed a significant difference between the football player and control (q = 6.406, P < 0.001) and between the wrestler and control (q = 8.695, P < 0.001; Fig 11, A). A significant difference was observed for MCI in human specimens (F[2, 27] = 24.41, P < 0.001). Post hoc comparison revealed a significant difference between the football player and control (q = 9.88, P < 0.001) and between the wrestler and control (q = 4.838, P < 0.01; Fig 11, B). A significant difference was observed for PHF in human specimens (F[2, 27] = 51.21, P < 0.001). Post hoc comparison revealed a significant difference between the football player and control (q = 10.58, P < 0.001) and between the wrestler and control (q = 13.64, P < 0.001; Fig 11, C).

Fig 10.

Lipoic acid reduces pathologic conformational changes in tau 2 weeks after repetitive blast exposure. Scale bar = 100 μm. Arrow represents area enlarged for high-power image. CP-13 marks a form of pretangle tau, and MCI is a unique conformational configuration of tau found only in neurodegenerative disease. PHF is a paired configuration of tau marking the transition toward tau accumulation in the soma. (A) A significant increase in CP-13 was noted in the prefrontal cortex after repetitive blast exposure compared with control (q = 5.54, P < 0.01), but the response was ameliorated by lipoic acid administration postblasts (q = 3.967, P < 0.05). **P < 0.01 RB vs CTRL. #P < 0.05 RB + LA vs RB. (B) No significant differences between groups were observed for MCI. (C) A significant increase in PHF was noted in the prefrontal cortex after repetitive blast exposure compared with control (q = 7.865, P < 0.001), but was ameliorated by lipoic acid administration postblasts (q = 6.703, P < 0.001). ***P < 0.001 RB vs CTRL. ###P < 0.001 RB + LA vs RB. CTRL, control; LA, lipoic acid; PHF, paired helical filament; RB, repeat blast.

Fig 11.

Tauopathy in human chronic traumatic encephalopathy brains. Scale bar = 100 μm. Tauopathy is the characteristic finding of CTE. Hyperphosphorylated tau causes conformational changes, which facilitate the transition toward neurofibrillary tangles. (A) A significant increase in CP-13 was seen between control and football player brains (q = 6.406, P < 0.001) and between control and wrestler brains (q = 8.695, P < 0.001). ***P < 0.001. (B) A significant increase in MCI was seen between control and football player brains (q = 9.88, P < 0.001), and between control and wrestler brains (q = 4.838 = P < 0.01). ***P < 0.001. **P < 0.01. (C) A significant increase in PHF was seen between control and football player brains (q = 10.58, P < 0.001), and between control and wrestler brains (q = 13.64, P < 0.001). ***P < 0.001. CTE, chronic traumatic encephalopathy; CTRL, control; NFL, professional football player; PHF, paired helical filament; WWE, professional wrestler.

Blast injury results in an increase of impulsivity-like behavior.

EPM has been used as a test for impulsivity.³⁴ Animals spending more time in the open arm exhibit decreased risk aversion. The closed arms represent a darkened environment, which is a preference for nocturnal rodents. A significant difference between groups was noted for time spent in the open arm at 7 days after repetitive blast (F[2, 21] = 4.327, P < 0.05). Post hoc comparison revealed that rats exposed to repetitive blast exhibited an increase in impulsive-like behavior spending more time in the open arms compared with the control group (q = 3.632, P < 0.05), and repetitive blast + lipoic acid treatment group brought the exploratory behavior back toward control levels (q = 3.573, P < 0.05; Fig 12, A). We wanted to test if animals receiving repetitive blast were overly active by measuring total distance traveled. Notably, no difference was observed between groups for overall distance traveled during the 5-minute interval within the apparatus (F[2, 21] = 0.3174, P = 0.7315; Fig 12, B).

Fig 12.

Effect of repetitive blast on impulsive-like behavior using the elevated plus maze assessment. Animals exposed to repetitive blast spent more time in the open arms compared with control (t = 3.632, P < 0.05). Time spent in open arm is a representation of impulsive-like behavior in rodents. (A) Alpha-lipoic acid treatment after blast decreased time spent in open arm compared with repetitive blast exposed animals (t = 3.573, P < 0.05). *P < 0.05 CTRL vs RB. #P < 0.05 RB vs RB + LA. (B) The groups did not differ in terms of distance traveled in the elevated plus maze. CTRL, control; EPM, elevated plus maze; LA, lipoic acid; RB, repeat blast.

DISCUSSION

We show for the first time that alpha-lipoic acid reduced Nox4 and p22phox after blast TBI. Alpha-lipoic acid acutely increased antiapoptotic markers 24 hours after injury. In addition, alpha-lipoic acid decreased proapoptotic markers both acutely and subacutely after blast exposure and decreased tau hyperphosphorylation. Animals exposed to repeat blast exposure had increased impulsive-like behavior on EPM that was decreased when alpha-lipoic acid was administered after blast. Alpha-lipoic acid, a naturally available supplement, has been used in previous neural injury studies for its role as an antioxidant and for decreasing neuroinflammation.^35–37 It has recently been suggested that lipoic acid would be a promising therapeutic for TBI treatment, but experimental preclinical studies are limited.¹⁶ We investigated for the first time the beneficial effects of alpha-lipoic acid on NADPH oxidative stress reduction after blast TBI. We hypothesize that alpha-lipoic acid reduces Nox4 complex formation with p22phox. Because lipoic acid does not cross the mitochondrial membrane, we propose it acts peripherally at the neuronal membrane to decrease superoxide formation, carbonyls, and 8-hydroxy-2-deoxyguanosine. The free radical damage is likely because of the surrounding microglia activation and peripheral immune infiltration.

The blast exposure setup used for this study has previously been shown to produce substantial neural injury to the left cortex because of acceleration/deceleration dynamics with little damage to the right cortex.²¹ We provide reasonable evidence that alpha-lipoic acid reduces the Nox (Nox4) in the contralateral left prefrontal cortex and subsequently inhibits the Nox4 complex formation with p22phox at 24 hours postblast. Nox4 has been shown to peak near contusion sites in humans 24–48 hours after TBI.³⁸ Nox4 is predominately located in neurons and is mediated by specific inducible mRNA.³⁹ The Nox4 pathway can produce toxic free radicals that may lead to cellular apoptosis.⁴⁰ We observed a significant reduction in superoxide, carbonyls, and 8-hydroxy-2-deoxyguanosine but not total ROS. Lipoic acid passes through the neuronal membrane but not the mitochondrial membrane.⁴¹ ROS generated by mitochondria are not the primary target of lipoic acid. Preventing Nox4 activation may therefore have profound effects on reducing neural injury expansion and oxidative stress byproducts over time at the plasma membrane. Inhibiting another Nox, Nox2, after TBI has already proved promising in previous studies with apocynin.^42,43 Continued investigations into the Nox4 and Nox2 pathways and their relation to TBI are warranted.

Not surprisingly, lipoic acid treatment increased expression of the antiapoptotic markers, Bcl-2 and HO-1, 24 hours after single blast injury. Enhancing Bcl-2 activity after TBI has been shown to decrease apoptosis and improve cognitive performance.⁴⁴ Likewise, increasing HO-1 activity facilitates the degradation of toxic heme and prevents plasma membrane damage and the induction of inflammatory cascades.⁴⁵ HO-1 also has been shown to reduce toxic iron-mediated damage likely accounting for the decrease in free iron observed in the lipoic acid treated group.⁴⁶ HO-1 converts toxic iron protoporphyrin into stable Fe(2+) that can be readily cleared from the brain.⁴⁷ At 2 weeks after repetitive blast (6 blasts over the time course of 2 weeks), lipoic acid treatment decreased proapoptotic markers Bax, caspase 12, and caspase 3. Bax and caspase 3 trigger rapid and sustained apoptosis after TBI.³¹ Bax deactivates Bcl-2, whereas caspase 3 triggers the final common pathway of apoptosis. Caspase 12 is readily increased when toxic free radicals damage the endoplasmic reticulum.⁴⁸ Caspase 12 can interact with proapoptotic factors released from damaged mitochondria to facilitate activation of caspase 3.⁴⁹ Reducing proapoptotic markers after TBI can decrease neurodegeneration (Supplementary Fig 1). We showed that lipoic acid reduced pathologic conformational changes of tau after repetitive blast.

The link between apoptosis and subsequent tauopathy is likely due to chronic neuroinflammation. A recent study suggests that peripheral immune cells enter the brain via a caveolin-dependent mechanism.⁵⁰ Caveolin increases ICAM-1 binding affinity for leukocytes after injury causing increased diapedesis across damaged endothelial cells.⁵¹ The peripheral immune cells that enter the brain release cytokines that trigger a robust gliosis response.⁵² The inflammatory storm can cause activation of Noxs, which further damages neurons and contributes to apoptosis.⁵³ Overtime, nuclear factor kappa B is translocated to the nucleus triggering a chronic state of inflammation.⁵⁴ This chronic inflammation can contribute to the formation of tauopathy.⁵⁵ By targeting Nox activity, lipoic acid can be used to disrupt this cycle. Further studies are warranted to investigate the long-term effects of targeting NADPH activity on chronic neuroinflammation. We provide evidence that lipoic acid improves behavior after multiple injuries.

A behavioral alteration often seen in soldiers exposed to blast injury is impulsivity.⁵⁶ Impulsivity has been linked to brain degeneration and tauopathy in human CTE.⁵⁷ Impulsivity and aggression can lead to personal relationship and work problems.⁵⁸ In severe cases, impulsivity can lead to death because of unnecessary risk-taking behavior.⁵⁹ A behavioral measure of impulsive-like behavior in rodents is the EPM.⁶⁰ Rats with impulsive tendencies spend increased time in the open arms of the maze. We observed a significant increase in impulsive-like behavior that was ameliorated by lipoic acid administration 7 days after repetitive blast injury. Repetitive blast injury did not, however, increase overall mobility. The mechanism behind lipoic acid’s beneficial effects is likely because of its antioxidant and antiapoptotic properties. Future research will examine how lipoic acid affects cognitive function and apoptosis within the hippocampus. The current findings correlate well with the tauopathy changes we report. By decreasing apoptosis, lipoic acid can potentially mitigate the effects of tauopathy and prevent the chronic sequelae of associated neurodegenerative symptoms.

CTE is an important neurodegenerative disease of growing prevalence and importance, yet the pathophysiology is still poorly understood. A lack of prospective studies and postmortem diagnosis limits investigation into disease progression and treatment targets.⁶¹ The use of new PET scans with a radioligand, which labels both amyloid and tau, is improving diagnostic capabilities for living individuals but further work needs to be performed.⁶² We present for the first time that the Nox4/p22phox pathway may play an important role in CTE pathology. Human samples from a professional football player and wrestler had increased levels of Nox4 and p22phox in comparison to age- and gender-matched controls. In addition, the CTE samples showed disrupted apoptotic signaling mechanisms and abundant tauopathy. Bcl-2 was significantly increased in both the football player and wrestler samples. Pure tauopathies, unlike amyloid β–mediated Alzheimer’s disease, have decreased apoptosis in late stages of the disease. Hyperphosphorylated tau increases Bcl-2 allowing for effective transfer of neurofibrillary tangles between living cells.⁶³ Tauopathies also often have reduced proapoptotic markers. We observed that although Bax was increased in the football player brain, it was reduced in the more severely affected wrestler brain. The wrestler brain had neurofibrillary tangles and neuropil threads throughout the cortex, deep nuclei, and brain stem indicating the most advanced stage of CTE.²⁰ As long as tau phosphatases are still active, tau dephosphorylation can cause an increase in Bax and trigger apoptosis in injured cells.⁶⁴ At some point during the progression of CTE, tau phosphatases may decrease substantially, and the levels of Bax are markedly reduced. Nox4 fits into the picture by producing free radicals that can derange signaling pathways. These deranged pathways typically trigger apoptosis, but in neurodegenerative disease they lead to hyperphosphorylation of tau and conformational changes.⁶⁵ Hyperphosphorylated tau acts in a positive feedback loop ramping up oxidative stress.⁶⁶

Lipoic acid may therefore have multiple beneficial effects when used as a treatment for neurotrauma. Acutely, lipoic acid could decrease apoptosis by limiting oxidative stress. Chronically after CTE has developed, lipoic acid could target the positive tau feedback loop limiting excess tau hyperphosphorylation and conformational change, thereby slowing the progression of CTE. Many studies remain to be performed to investigate the beneficial properties of lipoic acid at chronic time points. Continued supplementation after injury will probably have the most successful results. To advance toward clinical trial, it will be necessary to use models that are more representative of human disease pathology. Approaches such as transgenic animals or the use of a species closely related to humans on the evolutionary tree will likely be necessary. In addition, scaling blast injury for these new species must be considered to accurately represent human injury exposure. Clinical safety studies are advancing for lipoic acid’s use in the treatment of other disorders.⁶⁷ Considerations regarding lipoic’s acid use for neurotrauma must be focused on BBB transport, effective dosing, and appropriate timing of administration.

CONCLUSION

In summary, lipoic acid provided acute neuroprotection in our clinically relevant and validated blast TBI model. It decreased Nox4 and p22phox while increasing antiapoptotic markers acutely. Subacutely it decreased proapoptotic markers, decreased tau conformational changes, and improved impulsive behavior. CTE samples from a football player and wrestler also showed signs of increased Nox4 activity. The Nox4 pathway produces hydrogen peroxide and superoxide radicals that damage mitochondria, cellular DNA, proteins, and lipid membranes.⁶⁸ Long-term consequences include neurodegenerative disease, tauopathy, and neuropsychiatric changes. Considering the limitations to diagnosis and the incomplete understanding of CTE pathophysiology, Nox4-mediated oxidative stress should be more closely investigated. Oxidative stress contributes to an important interplay with tau hyperphosphorylation. Ongoing projects conducted by our laboratory will investigate the Nox4 pathway and lipoic acid modulation at extended time points. Lipoic acid is a widely used supplement that has already begun to be evaluated in clinical trials for the treatment of other chronic disorders. Lipoic acid might be a worthwhile treatment option for TBI as well and potentially offers a breakthrough for an injury paradigm that has limited treatment options. The mechanism of lipoic acid neuroprotection is not fully known and must be further investigated going forward.

AT A GLANCE COMMENTARY.

Lucke-Wold BP, et al.

Background

Chronic traumatic encephalopathy is a long-term consequence of repeated brain traumas, typically sustained in sport and-or the battlefield. A growing clinical need exists to find therapeutics that attenuate or halt the detrimental effects of long-term neurodegeneration.

Translational Significance

This study along with comparative illustrations of preclinical and clinical examples of chronic brain injury provides compelling evidence that oxidative stress through Nox4 activation is associated with onset and progression of neurodegeneration after repeated traumatic brain injury. Understanding the sequelae of signaling pathways modulated after traumatic brain injury is paramount to developing therapeutics that benefit people who suffer long-term consequences of repeated concussive brain injuries.

Abbreviations:

CTE

chronic traumatic encephalopathy

CTRL

control

EPM

elevated plus maze

HO-1

heme oxygenase 1

LA

lipoic acid

NADPH

nicotinamide adenine dinucleotide phosphate

Nox

NADPH oxidase

NFL

professional football player

PHF

paired helical filament

RB

repeat blast

ROS

free radical oxygen species

SB

single blast

TBI

traumatic brain injury

WWE

professional wrestler

NIH

National Institutes of Health

mRNA

messenger ribonucleic acid

IHC

immunohistochemistry

DNPH

2,4-dinitrophenylhydrazine

DAB

diaminobenzadine

ANOVA

analysis of variance

PET

positron emission tomography

Std

standard

C

control

Biography

Brandon P. Lucke-Wold is an MD, PhD candidate in the Department of Neurosurgery at West Virginia University School of Medicine. His article is based on a presentation given at the Combined Annual Meeting of the Central Society for Clinical and Translational Research and the Midwestern Section American Federation for Medical Research, held in Chicago, Ill, on April 2015.