Abstract
The complex pathophysiology of post-traumatic brain damage might need a polypharmacological strategy with a combination of drugs that target multiple, synergistic mechanisms. We currently tested a combination of apocynin (curtails formation of reactive oxygen species), tert-butylhydroquinone (promotes disposal of reactive oxygen species), and salubrinal (prevents endoplasmic reticulum stress) following a moderate traumatic brain injury (TBI) induced by controlled cortical impact in adult mice. Adult mice of both sexes treated with the above tri-combo showed alleviated motor and cognitive deficits, attenuated secondary lesion volume, and decreased oxidative DNA damage. Concomitantly, tri-combo treatment regulated post-TBI inflammatory response by decreasing the infiltration of T cells and neutrophils and activation of microglia in both sexes. Interestingly, sexual dimorphism was seen in the case of TBI-induced microgliosis and infiltration of macrophages in the tri-combo–treated mice. Moreover, the tri-combo treatment prevented TBI-induced white matter volume loss in both sexes. The beneficial effects of tri-combo treatment were long-lasting and were also seen in aged mice. Thus, the present study supports the tri-combo treatment to curtail oxidative stress and endoplasmic reticulum stress concomitantly as a therapeutic strategy to improve TBI outcomes.
SIGNIFICANCE STATEMENT Of the several mechanisms that contribute to TBI pathophysiology, oxidative stress, endoplasmic reticulum stress, and inflammation play a major role. The present study shows the therapeutic potential of a combination of apocynin, tert-butylhydroquinone, and salubrinal to prevent oxidative stress and endoplasmic reticulum stress and the interrelated inflammatory response in mice subjected to TBI. The beneficial effects of the tri-combo include alleviation of TBI-induced motor and cognitive deficits and lesion volume. The neuroprotective effects of the tri-combo are also linked to its ability to prevent TBI-induced white matter damage. Importantly, neuroprotection by the tri-combo treatment was observed to be not dependent on sex or age. Our data demonstrate that a polypharmacological strategy is efficacious after TBI.
Introduction
Traumatic brain injury (TBI) continues to be a consequential medical and socioeconomic challenge worldwide because of the lack of effective therapy (Maas et al., 2017). The complexity of the brain combined with the heterogeneity of processes contributing to the TBI pathophysiology makes devising a clinically effective neuroprotective agent difficult (Schouten, 2007; Eastman et al., 2020). A combination therapy that can target multiple pathologic processes might be more appropriate than monotherapies to protect the brain after TBI. Of the various processes that contribute to the secondary brain damage after TBI, oxidative stress, endoplasmic reticulum (ER) stress, and inflammation potentiate each other and thus play a major role (Chaudhari et al., 2014; Eastman et al., 2020). Following an injury, oxidative stress is mediated by increased production of reactive oxygen species (ROS) and decreased functionality of antioxidant enzymes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a major producer of ROS in the brain, while nuclear factor-erythroid factor 2-related factor 2 (Nrf2) is the transcription factor that induces the expression of several antioxidant enzymes (Kim and Ki, 2017; Tarafdar and Pula, 2018). Apocynin (an active ingredient of the roots of Picrorhiza kurroa) is a known inhibitor of NADPH oxidase (Hwang et al., 2019). Apocynin was shown to exhibit anti-inflammatory activity in chronic inflammatory diseases, such as atherosclerosis and colitis (Kinkade et al., 2013; Marín et al., 2013; Hwang et al., 2019), whereas tert-butylhydroquinone (tBHQ), which is a food preservative, promotes Nrf2-induced antioxidant defense by inhibiting Keap1-dependent ubiquitination of Nrf2 (Zhang and Hannink, 2003; Zagorski et al., 2013). Thus, it enables the binding of Nrf2 to the promoters of antioxidant genes, such as superoxide dismutase, catalase, and glutathione peroxidase, inducing their expression (Ma, 2013). Cellular damage following TBI leads to ER dysfunction that overloads the ubiquitin-proteasomal system leading to the accumulation of unfolded proteins that induces unfolded protein response. The unfolded protein response is propagated downstream by three major pathways, namely, inositol-requiring enzyme 1, activating transcription factor 6 (ATF6), and pancreatic ER kinase (PKR)-like ER kinase (PERK) (Kaufman, 1999). While inositol-requiring enzyme 1 and ATF6 activation induces protein chaperones leading to increased cell survival, PERK activation can be beneficial if limited as it prevents dephosphorylation of the eukaryotic initiation factor 2α leading to shutdown of protein synthesis, and thus reduces the unfolded protein load in the injured brain. However, brain injury leads to prolonged activation of PERK that is derogatory as it selectively induces the downstream pro-apoptotic transcription factors CHOP and ATF4 leading to neuronal death (Galehdar et al., 2010). Salubrinal prevents p-eukaryotic initiation factor 2α dephosphorylation, and thus limits global protein synthesis and dampens the ER stress (Boyce et al., 2005). Salubrinal also reduces inflammation by inhibiting the NF-kB pathway (Huang et al., 2012; Logsdon et al., 2016). We currently tested a tri-combo strategy with apocynin (to reduce ROS formation), tBHQ (to increase ROS disposal), and salubrinal (to minimize PERK-mediated ER stress) in a mouse model of controlled cortical impact (CCI) brain injury. This proof-of-concept study shows that simultaneous targeting of oxidative stress and ER stress could be beneficial in TBI treatment.
Materials and Methods
CCI
Adult (12 ± 1 weeks old) and aged (68 ± 4 weeks old) male (n = 158) and female (n = 134) C57BL/6 mice (The Jackson Laboratory) were subjected to a moderate grade TBI using a CCI device as described previously (Chandran et al., 2018). All behavioral and surgical procedures were approved by the University of Wisconsin Research Animal Resources and Care Committee, and animals were cared for in accordance with the Guide for the care and use of laboratory animals, U.S. Department of Health and Human Services Publication Number 86-23 (revised). Experiments were conducted in compliance with the Animal Research: Reporting of In Vivo Experiments guidelines (Percie du Sert et al., 2020). Animals were randomly assigned to study groups, and outcome parameters were evaluated blindly. The number of animals used for each experiment is given in Extended Data Figure 1-1. Figure 1a shows the overall schedule for the induction of TBI, drug administration, and measurement of outcome parameters.
Briefly, mice were anesthetized with isoflurane (2%), and a 10 mm midline incision was made over the skull to expose the left parietal bone. A 3 mm craniotomy was made (1 mm lateral and 1 mm caudal to bregma) keeping the dura intact. A 1-mm-deep deformation on the cortex was made using a 2.5-mm-diameter impactor with a velocity of 3.5 m/s and a 400 ms dwell time. After CCI, the bone flap was replaced and covered with Surgicel (Ethicon), the incision was closed with 6–0 silk sutures, anesthesia terminated, and 0.5% bupivacaine (100 µl) was applied along the incision. During the surgery and the recovery, body temperature was maintained at 37°C using a heating pad. Sham animals underwent the same procedure without receiving the impact.
Drug administration
A combination of apocynin (10 mg/kg; Millipore Sigma) and tBHQ (25 mg/kg; Millipore Sigma) or a combination of apocynin (5 or 10 mg/kg), tBHQ (12.5 or 25 mg/kg), and salubrinal (1.5 or 3 mg/kg; Millipore Sigma) dissolved in 1% DMSO or vehicle (1% DMSO only) were injected intraperitoneally at 5 min or 3, 24, and 48 h after TBI as described previously (Chandran et al., 2018).
Motor function analysis
Post-TBI motor function was evaluated by rotarod (time remained on an accelerating cylinder from 4 to 40 rpm, Harvard Apparatus) and beam walk (number of foot faults while crossing a 5 mm × 120-cm-long beam) tests as described previously (Chandran et al., 2018). Mice were trained for 3 d before CCI and tested between days 1 and 7 after TBI.
Cognitive function
Spatial learning and memory were assessed using the Morris water maze on days 21-24 after TBI, as described previously with slight modifications (Hanscom et al., 2021). Testing included spatial acquisition, learning to navigate based on distal cues, and reference memory, making a preference for the platform quadrant even when the platform is absent (Vorhees and Williams, 2006). During spatial acquisition, mice were gently placed in one of the quadrants of a white circular tank filled with water and trained to locate a submerged platform based on visual cues. Each trial lasted until the animal either located the hidden platform or reached the end of the 1 min trial. The time taken (escape latency) and the path followed to locate the platform were recorded. Similarly, each animal was allowed to start from all quadrants randomly, making one block of testing. Two blocks of testing were conducted per day for 4 consecutive days. On day 4, reference memory was tested by probe trial with the platform removed. All the animals were allowed to swim for 1 min from the same quadrant, and time spent on the platform quadrant was noted.
Lesion volume and white matter damage determination
On day 28 or 98, animals were killed by transcardiac 4% PB PFA perfusion. Each brain was postfixed, cryoprotected, and sectioned (coronal; 30 μm thickness). Five serial sections from each brain covering the cortical lesion (from −1.0 to −3.0 mm from bregma) were stained with cresyl violet, scanned, and analyzed using the National Institutes of Health ImageJ software (Schneider et al., 2012). The volume of the lesion was computed by the numeric integration of data from the five serial sections with respect to the sectional interval as described previously (Yi et al., 2008). To determine white matter damage, sections were stained with Luxol fast blue and counterstained with cresyl violet, and the thickness of the medial corpus callosum (CC) and area of the external capsule (EC) on the ipsilateral side were measured. The EC area was determined by measuring the area of white matter tracts lateral to putamen starting from the divergence of the internal capsule.
Immunostaining
Mice were transcardially perfused with 4% PFA at 3 d after TBI. Thirty-micrometer coronal sections were washed with TBS with 0.1% Tween 20, treated with sodium citrate buffer, and blocked in SuperBlock (Thermo Fisher Scientific). Sections were incubated in primary antibodies against goat anti-8-OHdG (1:200; Novus Biologicals) and goat anti-Iba1 (1:300; Novus Biologicals) overnight at 4°C followed by appropriate secondary antibodies for 1 h at room temperature and mounted on slides with DAPI containing mounting medium (Vector Laboratories). Images were taken at two randomly selected cortical fields adjacent to the injury using a fluorescence microscope (Keyence). Fluorescence intensity was measured using National Institutes of Health ImageJ software and normalized with the number of cells in the selected field.
Single-cell suspension and flow cytometry
Flow cytometry was conducted as described previously (Hsu et al., 2019). Brains were minced and digested in 1 mg/ml of collagenase/dispase (Roche) in RPMI supplemented with 10% heat-inactivated FBS for 20 min at 37°C. The digested tissue was then mechanically dissociated by passing the tissue through an 18-gauge needle, quenched with RPMI, and spun down at 270 × g. Pelleted cells were resuspended in 70% Percoll, and 30% Percoll was overlaid over top and spun down at 540 × g at 4°C for 30 min without brake. Myelin was removed from the top layer, and cells at the interface between the two layers were isolated, washed with RPMI, suspended in FACS buffer (pH 7.4, 0.1 m PBS, 1 mm EDTA, and 1% BSA), washed twice in FACS buffer, and immunolabeled with the appropriate conjugated antibodies for 30 min at 4°C. Antibodies used for flow cytometry were as follows: rat anti-CD3 conjugated to Pacific Blue (BioLegend), rat anti-F4/80 conjugated to AlexaFluor-488 (BioLegend), rat anti-Ly6G conjugated to PE (BioLegend), rat anti-CD11b conjugated to PerCP/Cy5.5 (BioLegend), and rat anti-CD45 conjugated to APC-eFluor780 (Thermo Fisher Scientific). Data were acquired using Cytek's 3-laser Northern Lights and analyzed using FlowJo software.
Statistical analysis
All analyses were performed in Prism 9 (GraphPad). The Shapiro–Wilk normality test was performed on all datasets (Extended Data Fig. 1-1). Student's t test (normal distribution) or Mann–Whitney U test (non-normal distribution) was used for comparing two groups, and one-way ANOVA with Tukey's post hoc test was used for multiple groups. Two-way repeated-measures ANOVA with Sidak's post hoc test was used for motor function data collected repeatedly from the same set of subjects at different time points. Statistically significant (p < 0.05) outliers were identified by using Grubb's test and excluded from the analysis.
Results
Tri-combo treatment improved functional outcomes and attenuated lesion volume after TBI
In mice, CCI injury leads to severe motor and cognitive functional deficits (Fox et al., 1998). We previously showed that an antioxidant di-combo (apocynin and tBHQ) promotes better functional recovery after CCI injury (Chandran et al., 2018). We now analyzed whether adding an anti-ER-stress compound (salubrinal) to this promotes better outcomes than di-combo after TBI. The tri-combo (apocynin, tBHQ, and salubrinal) treated adult male mice showed significantly better motor function recovery estimated by rotarod test (Fig. 1b) and beam walk test (Fig. 1c), better cognitive function estimated by the Morris water maze test (Fig. 1d), and smaller lesion size (Fig. 1e), compared with either vehicle-treated control or di-combo treated cohort. As tri-combo treatment was proven to confer better neuroprotection than di-combo treatment following TBI, tri-combo treatment alone was tested in the rest of the studies. The tri-combo treatment promoted significantly better motor function recovery (Fig. 1f,g) and cognitive function recovery (Fig. 1h), and decreased the lesion size (Fig. 1i) in adult female mice subjected to TBI. This indicates that the neuroprotection offered by the tri-combo treatment is not sex-specific.
Tri-combo treatment protected young male and female mice after TB1. Cohorts of mice were subjected to CCI injury and treated with di-combo (male only) or tri-combo or vehicle (i.p.) on 3 d and subjected to motor function testing (rotarod test and beam walk test) on days 1, 3, 5, and 7 after TB1 (a). Mice were trained in both tasks for 3 d before TB1. Cognitive testing (Morris water maze [MWM]) between days 21 and 24 was followed by death on day 28 after TB1 (a). The number of animals used and animal exclusion details for each experiment are given in Extended Data Figure 1-1. Brains were sectioned and stained with cresyl violet to estimate lesion volume (outlined). In adult males, the tri-combo group remained more time on the rotarod (b), had fewer foot faults (c), showed improved cognitive function (d), and reduced lesion volume (e) compared with the vehicle and di-combo groups. Tri-combo–treated female mice also showed improved motor function (f,g), cognitive function (h), and reduced lesion volume (i) compared with vehicle control. Values are mean ± SD; n = 8-10/vehicle; n = 8-12/di-combo; n = 9-12/tri-combo (male) and n = 8/vehicle and n = 7 or 8/tri-combo (female). *p < 0.05 compared with respective vehicle group. #p < 0.05 compared with di-combo by two-way repeated-measures ANOVA with Sidak's post hoc test (multiple time points) or one-way ANOVA followed by Tukey's post hoc test (more than two groups) or Student's t test (two groups). The Morris water maze trace maps from probe trials (d,h), and the cresyl violet-stained serial sections (e,i) are from representative mice of each group.
Figure 1-1
Summary of animals used and data distribution. The table shows the number of animals in each group before TBI induction, the number of excluded animals and the type of data distribution for each experiment. Download Figure 1-1, DOCX file.
Tri-combo treatment prevented TBI-induced oxidative DNA damage
TBI is known to promote oxidative DNA damage, especially the nucleobase modifications, such as the formation of 8-OHdG (Davis and Vemuganti, 2021). Neuronal, as well as non-neuronal cells, exhibited oxidative DNA damage after TBI. However, the majority of the 8-OHdG reactivity in the perilesional area was neuronal (Fig. 2). Brain sections from both male and female mice treated with tri-combo after CCI injury showed significantly curtailed 8-OHdG staining in the ipsilateral cerebral cortex, compared with sex-matched vehicle-treated cohorts (Fig. 2).
Tri-combo treatment reduced TB1-induced oxidative DNA damage. Compared with the vehicle-treated group, the tri-combo–treated group showed decreased levels of 8-OHdG immunostaining in the perilesional cortex (orange rectangle) of male and female mice at 3 d after TB1. Data are mean ± SD; n = 5/group. *p < 0.05 compared with the vehicle group by Student's t test. Scale bar, 20 μm.
Tri-combo treatment modulated TBI-induced inflammation
Both brain-resident immune cells and peripheral immune cells participate in the inflammatory response following TBI (Shi et al., 2019). Although inflammatory response after TBI is crucial for recovery, it can exacerbate secondary injury if not regulated (Simon et al., 2017). Flow cytometric analysis of the peri-injury cerebral cortex with the depicted gating strategy (Fig. 3a) showed increased numbers of reactive microglia, macrophages, neutrophils, and T cells at 3 d following CCI injury in both males (Fig. 3b) and females (Fig. 3c) treated with vehicle. In males, tri-combo treatment after TBI significantly reduced the number of reactive microglia, neutrophils, and T cells compared with the vehicle control (Fig. 3b), whereas in females, tri-combo treatment after TBI significantly reduced the number of reactive microglia, macrophages, and T cells compared with vehicle control (Fig. 3c). In support of the flow cytometry results, immunofluorescence staining of brain sections with Iba1 antibodies showed a ∼30% reduction in the number of microglia/macrophages in the perilesional cortex of both male and female mice treated with tri-combo compared with the sex-matched vehicle-treated cohorts at 3 d after TBI (Fig. 4). Moreover, tri-combo treatment reduced the post-TBI ameboid microglia/macrophages (reactive) in both sexes compared with respective vehicle controls (Fig. 4).
Tri-combo treatment reduced post-TB1 peripheral immune cell infiltration and activation of resident microglia. Perilesional cortical tissue from tri-combo and vehicle-treated male and female cohorts at 3 d after TB1 was analyzed by flow cytometry using the gating strategy shown (a). T cells (CD45high CD3+), macrophages (CD45high F4/80+ CD11b+), neutrophils (CD45high CD11b+ Ly6G+), and microglia (CD45inter F4/80+ CD11b+) were sorted and plotted as the percentage of the total frequency of singlets in males (b) and females (c). Data are mean ± SD; n = 4-7/group. *p < 0.05 compared with vehicle group. #p < 0.05 compared with sham by one-way ANOVA followed by Tukey's post hoc test.
Tri-combo treatment decreased post-TB1 inflammatory response in both males and females. At 3 d after TB1, total (Iba1+) and reactive (ameboid shaped) microglia/macrophages in the perilesional cortex (orange rectangle) decreased significantly in the tri-combo group compared with the vehicle group. Data are mean ± SD; n = 5/group. *p < 0.05 compared with the vehicle group by Student's t test. Scale bars: 50 μm; magnified images, 10 μm.
Tri-combo treatment promoted a long-term reduction in lesion volume and attenuated CC atrophy after TBI
We allowed a cohort of mice to survive up to 98 d after TBI to estimate long-term gray and white matter damage. In both males and females treated with tri-combo after TBI, the lesion volume remained significantly lower (by ∼40%) compared with the vehicle group even at 98 d after TBI (Fig. 5a,b). TBI is known to promote macrostructural volume loss in the CC in both rodents and humans that is linked to cognitive and motor function deficits (Jokinen et al., 2007; Wu et al., 2010; Frederiksen et al., 2011; Goldman et al., 2017; Leconte et al., 2020). The tri-combo–treated male mice showed a significantly smaller reduction in EC area (∼25%) and CC thickness (∼11%) compared with the vehicle-treated group at 98 d after TBI (Fig. 5c). Similarly, female mice treated with tri-combo exhibited better preservation of EC area compared with the vehicle-treated group; however, CC thickness was not significantly different at 98 d after TBI (Fig. 5d). Survival of the white matter might be one of the contributing factors for better motor and cognitive recovery after TBI and this is importantly not sex-specific.
Persistent protection after tri-combo treatment following TB1. Cohorts of male and female mice subjected to CCI injury and treated with tri-combo (i.p.) or vehicle on days 1, 2, and 3 after injury were killed on day 98 after TB1. Brains were sectioned and stained with cresyl violet, and lesion volume (outlined area) was estimated. In both sexes, the lesion volume was significantly lower in the tri-combo cohorts compared with vehicle control cohorts. The tri-combo–treated mice of both sexes showed a significantly smaller percentage reduction (over sham) in the EC area (traced in red) and CC width (red line) compared with the sex-matched vehicle cohorts (c,d). Data are mean ± SD; n = 8/vehicle; n = 7 or 8/tri-combo; n = 4/sham (male) and n = 7 or 8/vehicle; n = 7-9/tri-combo; n = 6/sham (female). *p < 0.05 compared with respective vehicle group by Student's t test. Cresyl violet-stained serial sections (a,b) and Luxol fast blue-stained magnified images (c,d) are from representative mice of each group. Scale bar, 100 μm.
Tri-combo treatment after TBI is also neuroprotective in aged mice
With age, the brain becomes more vulnerable to secondary injury and recovery is less efficient, compared with the younger population (Gardner et al., 2018). In 64-72 weeks old mice subjected to CCI injury, tri-combo treatment promoted better motor function recovery (rotarod test) at 5 and 7 d in males and at 7 d in females, compared with the sex-matched vehicle-treated controls (Fig. 6a,b). The secondary lesion volume was also significantly smaller by ∼55% in both aged males and females treated with tri-combo after TBI, compared with the sex-matched vehicle-treated controls (Fig. 6c,d).
Tri-combo treatment protected the brains of aged male and female mice after TB1. When 68 ± 4 weeks old male and female mice subjected to TB1 were treated with tri-combo, they remained longer on the rotarod on day 5 (males only) and day 7 (both sexes) compared with the sex-matched vehicle-treated controls (a,b). Both sexes also showed significantly reduced lesion volume (outlined area) in tri-combo cohorts compared with the sex-matched vehicle cohorts (c,d). Data are mean ± SD; n = 9/group for both vehicle and tri-combo (male) and n = 12 or 13/vehicle and n = 10 or 11/tri-combo (female). *p < 0.05 compared with vehicle group by two-way repeated-measures ANOVA with Sidak's post hoc test (rotarod test) or Mann–Whitney U test (male lesion volume) or Student's t test (female lesion volume).
A lower dose or delayed tri-combo treatment is neuroprotective following TBI in male mice, but not in females
When adult male mice subjected to TBI were given tri-combo at half of the original dose (apocynin: 5 mg/kg; tBHQ: 12.5 mg/kg; salubrinal: 1.5 mg/kg) at 5 min, 24 h, and 48 h after the injury, there was a significant improvement in the time on the rotarod and reduction in the number of foot faults on post-TBI days 5 and 7 compared with vehicle control (Fig. 7a). This half-dose–treated cohort also showed improved cognitive recovery (Fig. 7b) and decreased lesion volume (Fig. 7c) compared with vehicle control. Conversely, a half-dose treatment was not sufficient in female mice to ameliorate TBI-induced motor and cognitive function deficits and to reduce the lesion volume (Fig. 8a–c). When the first dose was delayed for 3 h after TBI (second and third doses at 24 and 48 h), the tri-combo administered male mice showed significantly improved recovery of motor function (Fig. 7d) and cognitive function (Fig. 7e), and significantly decreased lesion volume (Fig. 7f) compared with the vehicle-treated group. Similarly, a delayed treatment improved motor function in female mice (Fig. 8d); however, it did not alter the cognitive function and lesion volume (Fig. 8e,f).
Tri-combo retained its beneficial effects in male mice even when given at half-dose or in a delayed fashion after TB1. a–c, Adult male mice subjected to TB1 and injected with half-dose of tri-combo (apocynin: 5 mg/kg; tBHQ: 12.5 mg/kg; salubrinal: 1.5 mg/kg) showed significantly improved motor function (a) and cognitive function (b), and reduced lesion volume (outlined area) (c). d–f, Adult male mice subjected to TB1 and injected with tri-combo at a delay of 3, 24, and 48 h after TB1 also showed significantly improved motor function (d) and cognitive function (e), and reduced lesion volume (f). Data are mean ± SD; n = 9 or 10/group for both vehicle and tri-combo (half-dose) and n = 8/vehicle; n = 9/tri-combo (delay). *p < 0.05 compared with vehicle group by two-way repeated-measures ANOVA with Sidak's post hoc test (multiple time points) or Student's t test (two groups) or Mann–Whitney U test (delay platform quadrant).
Delayed or half-dose tri-combo treatment lost most of its beneficial effects in female mice subjected to CCI. a–c, Adult female mice subjected to TB1 and injected with half-dose of tri-combo (apocynin: 5 mg/kg; tBHQ: 12.5 mg/kg; salubrinal: 1.5 mg/kg) showed no significant improvement in motor function (a) and cognitive function (b), and reduction in lesion volume (outlined area) (c). d–f, Adult female mice subjected to TB1 and injected with tri-combo at a delay of 3, 24, and 48 h after TB1 showed significant improvement in motor function (d), but had no significant effect on cognitive function (e), and lesion volume (f). Data are mean ± SD; n = 8/group for both vehicle and tri-combo (half-dose) and n = 7 or 8/vehicle; n = 8 or 9/tri-combo (delay). *p < 0.05 compared with vehicle group by two-way repeated-measures ANOVA with Sidak's post hoc test (multiple time points).
Discussion
Secondary brain damage after TBI is mediated by the synergistic action of complex sequelae of pathophysiologic changes (Bains and Hall, 2012; Anighoro et al., 2014). Hence, a polypharmacological therapeutic approach might be a better option than using drugs that target a single mechanism, particularly simultaneous targeting of the interconnected mechanisms. Oxidative stress and ER stress are interrelated and potentiate each other (Cao and Kaufman, 2014). Furthermore, both these are also interconnected with inflammation following TBI (Abdul-Muneer et al., 2015; Biswas, 2016). We previously showed that NOX2 KO improves, while Nrf2 KO worsens, the motor function recovery and secondary lesion volume after TBI in mice (Chandran et al., 2018). This shows that both the formation and disposal of ROS are important to mediate oxidative stress. Hence, treatment with a combination of a NOX2 inhibitor (apocynin) and an Nrf2 activator (tBHQ) promoted better functional outcomes and smaller lesions after TBI. Moreover, previous studies indicated the neuroprotective efficacy of the ER stress inhibitor salubrinal (Logsdon et al., 2014; Wang et al., 2019). Importantly, in addition to inhibiting oxidative stress and ER stress, apocynin, tBHQ, and salubrinal were shown to prevent inflammation by inhibiting the NF-kB pathway in various disease models, including TBI (Jin et al., 2010; Huang et al., 2012; Nakajima et al., 2015; Byun et al., 2016; Choi et al., 2017; Feng et al., 2017). Interestingly, simultaneous targeting of oxidative stress and inflammation was found to be an effective therapeutic strategy in some disease conditions, such as diabetes (Adameova et al., 2009; Teodoro et al., 2019; Andrianova et al., 2020; Fabiani et al., 2021). In the present study, we showed that a combination of apocynin, tBHQ, and salubrinal that target oxidative stress and ER stress also curtails inflammation after TBI. Further, post-TBI treatment with tri-combo improved both motor and cognitive functions, and reduced lesion volume after TBI. Importantly, our studies showed that the tri-combo treatment can protect the brain and promote functional benefits in both sexes. TBI in aged subjects leads to exacerbated secondary brain damage. Encouragingly, the tri-combo treatment showed significant neuroprotection in aged male and female mice as well, indicating its promise to be efficacious regardless of age or sex.
ROS levels elevate quickly within minutes after TBI and last for days (Hall et al., 1993, 1994; Bains and Hall, 2012). Moreover, uncontrolled ROS levels can also promote inflammation and damage to biomolecules, and ultimately cell death (Hussain et al., 2016). Hence, we provided tri-combo in 3 doses starting at 5 min and ending at 2 d after TBI. This strategy efficiently protected the male and female brains after TBI. Notably, TBI-induced oxidative DNA damage was higher in males compared with females. Apart from the influence of female sex hormones, the above phenomenon could be because of paraoxonase 2-mediated sex-dependent susceptibility to oxidative stress in mice (Giordano et al., 2013; Torrens-Mas et al., 2020). Moreover, sex differences in oxidative stress were also reported in humans with cardiovascular diseases (Kander et al., 2017). Interestingly, tri-combo treatment reduced oxidative DNA damage to similar levels in both sexes. Therefore, a greater extent of protection by tri-combo was seen in male mice compared with female mice. When we delayed the first dose to 3 h after TBI, the tri-combo therapy still improved functional outcomes and reduced lesion volume, but to a lesser extent. In females, although delayed treatment preserved motor function, there was no change in cognitive function and lesion volume. This agrees with previous studies that reported a partial or complete loss of neuroprotection when an antioxidant therapy was delayed by 30 min to 6 h (Mésenge et al., 1998; Dash et al., 2009). Reducing the dose of drugs is desirable to decrease any putative toxicity. When male mice subjected to TBI were treated with half of the most efficacious doses of the three drugs (apocynin, tBHQ, and salubrinal), post-TBI motor and cognitive recovery was still observed. However, half-dose failed to improve motor and cognitive functions in female mice. This shows the importance of sexual dimorphism in the mechanisms of secondary brain damage and functional outcomes after TBI. This also warrants the need to test the therapeutic window as well as dose in both sexes in preclinical models of TBI before attempting to translate to humans.
Although several antioxidant therapies were shown to be protective in animal models of TBI, none passed Phase III clinical trials (Hall et al., 2010; Di Pietro et al., 2020). This might be because of the synergistic action of oxidative stress with ER stress and inflammation. The tri-combo we used currently also decreased inflammatory cell infiltration into the brain, which might be one of the reasons for its efficacy. Oxidative stress starts rapidly after TBI; hence, by the time patients receive treatment, initial oxidative damage to proteins, DNA, and cell membranes might have taken place (Di Pietro et al., 2020). These might be sufficient to trigger inflammatory and apoptotic pathways after TBI (Abdul-Muneer et al., 2015). Hence, unless the early induction of inflammation and apoptosis are prevented and membranous structures, such as ER and Golgi, are protected, therapies cannot achieve the desired efficacy (Solleiro-Villavicencio and Rivas-Arancibia, 2018). In the present study, the tri-combo we tested significantly reduced oxidative DNA damage as well as inflammation following TBI.
A moderate level of inflammation after TBI is necessary to aid plasticity by clearing damaged cells (Russo and McGavern, 2016). ROS is known to exacerbate inflammatory responses, and thus precipitates cell death after an injury (Bakunina et al., 2015). Many of the antioxidants that showed neuroprotection in experimental models of TBI modulated, rather than completely suppressed, the inflammatory responses (Davis and Vemuganti, 2022). Similarly, post-TBI tri-combo treatment selectively regulated the TBI-induced inflammatory response, at least partially, by reducing oxidative stress. Previous studies also showed that individual components of tri-combo treatment ameliorated the TBI-induced inflammatory response via the NF-kB pathway (Jin et al., 2010; Huang et al., 2012; Nakajima et al., 2015; Byun et al., 2016; Choi et al., 2017; Feng et al., 2017). Previous studies showed a correlation between the extent of T cell and neutrophil infiltration and secondary brain damage after TBI (Dong et al., 2016; Liu et al., 2018). In the present study, tri-combo treatment significantly reduced the level of both T cells and neutrophils in the perilesional area that survives with proper therapy. Moreover, inflammation-mediated microglial activation is more extensive in males than females (Villa et al., 2018). Post-TBI macrophage infiltration was observed to be of similar magnitude in both sexes, but tri-combo curtailed it only in females. The post-TBI brain is known to show a time-dependent sexual dimorphism in the inflammatory response (Villapol et al., 2017). In the present study, this sexual dimorphism was visible in the reactive microglial population at 3 d after TBI.
Long-term sequelae of TBI include demyelination and white matter loss that also precipitates motor and cognitive deficits (Kinnunen et al., 2011; Farbota et al., 2012; Johnson et al., 2013; Loane et al., 2014; Bramlett and Dietrich, 2015). Apart from oxidative DNA damage-induced glial dysfunction, oxidative stress can also damage white matter by interrupting the oligodendrocyte precursor cell renewal (Miyamoto et al., 2013; Al-Mashhadi et al., 2015). Similarly, neuroinflammation plays a role in white matter damage after TBI (Johnson et al., 2013; Scott et al., 2015). In the present study, antioxidant and anti-inflammatory properties of tri-combo might have contributed to sustained protection of white matter after TBI. Thus, the efficacy of the tri-combo is promising for both short-term and longer-term neuroprotection after TBI. Unregulated oxidative stress, ER stress, and inflammation might lead to the onset and progression of neurodegenerative diseases after TBI (Cruz-Haces et al., 2017; Solleiro-Villavicencio and Rivas-Arancibia, 2018; Davis and Vemuganti, 2021). Therefore, future studies should explore the ability of tri-combo therapy to prevent TBI-induced chronic pathologies.
In conclusion, post-TBI supplementation of a combination of apocynin, tBHQ, and salubrinal to curtail oxidative stress and ER stress improved motor and cognitive functions, and attenuated lesion volume in adult and aged male and female mice. This combination therapy also regulated TBI-induced inflammatory response and prevented white matter damage.
Footnotes
This work was supported in part by Department of Veterans Affairs Merit Review Grant #I01BX004344; and Department of Neurological Surgery, University of Wisconsin-Madison. R.V. is the recipient of Department of Veterans Affairs Research Career Scientist Award #IK6BX005690.
The authors declare no competing financial interests.
- Correspondence should be addressed to Raghu Vemuganti at vemuganti{at}neurosurgery.wisc.edu
