Blast overpressure (BOP) exposure has become a subject of increasing concern in the Department of Defense due to increased use of breaching operations and heavy weapons systems in both combat and in training, in which military personnel are exposed to repeated low-level BOP (Tate et al., 2013). The symptomatology reported by these individuals is not typically clinically diagnosed as a concussion, due to the current lack of either sophisticated diagnostic techniques or observation by appropriately trained medical personnel (DeKosky et al., 2010; Tate et al., 2013). Transient symptoms include headache, slowed thinking, and memory deficits, and repeated exposures throughout the Warfighters’ careers may lead to chronic problems such as performing military tasks requiring critical thinking, cognitive issues, behavioral changes, and mood disorders (Tate et al., 2013; Carr et al., 2015, 2016; Kamimori et al., 2018; Sajja et al., 2019). Although blast and impact injuries are biomechanically different, some neurological symptoms of moderately-severe BOP exposure are similar to those in impact traumatic brain injury (TBI) but have been shown to have different pathophysiological profiles (Shively et al., 2016; Yamamoto et al., 2018).

Contrary to impact TBI, BOP exposure causes complex, whole-body injury, with air-filled organs such as the lungs and ears being particularly vulnerable (Sajja et al., 2020). Several studies have shown brain perturbations in the form of the disrupted blood-brain barrier (BBB) and increased brain permeability following BOP exposure (Rubovitch et al., 2012; Elder et al., 2015). In particular, tight junction proteins such as claudin-5 and occludin are altered in Warfighters’ blood-based clinical assessments following repeated BOP exposure (Duckworth, 2018). Also, glial fibrillary acidic protein (GFAP), an astrocytic marker, and vascular endothelial growth factor (VEGF), a mediator of angiogenesis in the brain, were shown to be affected following BOP exposure (Sajja et al., 2012a; Kamnaksh et al., 2014; Duckworth, 2018; Eonta et al., 2019). Similar to previous clinical and preclinical findings, we observed that exposure to BOP intensities above 13 psi for up to four exposures triggered alterations in the tight junction proteins claudin-5 and occludin, as well as alterations in neurodegeneration-related proteins and in the mechanosensitive protein Piezo2 (Heyburn et al., 2019a). The breakdown of the border separating cerebral blood flow from the brain parenchyma leaves the brain susceptible to infiltrating cells and brain-toxic elements that can promote neuroinflammation and cell death (Shetty et al., 2014).

Neuroinflammation is a major component of the injury cascade of TBI and has been the focus of many preclinical blast TBI (bTBI) studies (Ziebell and Morganti-Kossmann, 2010; Elder et al., 2015). Animal studies employing a variety of BOP exposure techniques demonstrated brain inflammation, including gliosis, neutrophil activation, and increased cytokine production [interleukin (IL)-6, interferon-γ, MCP-1, tumor necrosis factor-α (TNFα; Cernak et al., 2011; Kovesdi et al., 2011; Sajja et al., 2012b; Cho et al., 2013; Valiyaveettil et al., 2013; Kamnaksh et al., 2014; Simard et al., 2014]. Peripheral expression of pro-inflammatory cytokines, including IL-6 and TNF-α is increased in humans following moderate BOP exposure (Gill et al., 2017a, b).

A BOP wave is essentially an overpressure compression wave that can cause skull flexure, leading to increased intracranial pressure (ICP; Bolander et al., 2011). This increased ICP can potentially cause cellular and molecular perturbations, including mechanotransduction, which together may lead to subsequent deficits and symptoms (Heyburn et al., 2019a). Piezo2 is a component of mechanosensitive ion channels and acts as a pressure sensor in the brain and body, influencing neuronal function, signal transduction, and somatosensory function (Schrenk-Siemens et al., 2014; Szczot et al., 2017). Increased levels of Piezo2 in the brain could lead to increased sensitivity to pressure alterations produced by BOP exposure, lowering the threshold for deleterious signal transduction cascades and making the brain more vulnerable to low-intensity BOP (Heyburn et al., 2019a; Zhang et al., 2019).

To develop BOP injury risk criteria for brain and lungs, our group has evaluated poly-organ trauma as evidenced by lung and brain injuries and established that 8.5 psi is the threshold for lung contusion for up to 30 daily exposures and that multiple daily exposures to 10 psi BOP cause around 1% lung injury (Heyburn et al., 2019a; Sajja et al., 2020). To expand on these previous findings and to further develop head injury risk criteria associated with repeated low-level BOP exposure, several markers known to be altered in the brain from relatively high-intensity BOP exposure were evaluated in the absence of lung injury. The current study focused on exposures of 8.5 and 10 psi to define the pathophysiological response and determined how repeated primary BOP alone, with little or no injury to the lung, affects the brain. In this study, an advanced blast simulator (ABS), which closely mimics “free-field” BOP, was used to expose rats to repeated low-level BOP, with one daily exposure for 14 days. Brains were evaluated 24 h after the last blast exposure for acute alterations in markers of inflammation, BBB integrity, neurodegeneration, and pressure sensitivity to study short-term brain effects that could contribute to the pathophysiology of neurodegeneration. This information will contribute to our understanding of the consequences of repeated BOP exposure in various military training environments and the development of health hazard assessment for repeated BOP injury.

All the results reported are from samples collected 24 h following final BOP exposure.

A variety of acute changes in the brain that relate to processes of brain injury and neurodegeneration were observed following repeated low-level BOP exposure. The BBB unit is an especially important focus here and the observed changes in levels of tight junction proteins, mediators of angiogenesis (VEGF), and GFAP-positive astrocytes could help in our understanding of the injury risk of BOP to the brain. Piezo2, a mechanosensitive ion channel protein, which was shown to actively support cerebrovascular integrity by regulation of endothelial cells and angiogenesis could similarly play an important role in our understanding of blast pathophysiology (Ferrari et al., 2015; Yang et al., 2016; Zhang et al., 2017; Heyburn et al., 2019a). Piezo2 leads to cation channel activation and signal transduction in neurons and other cell types (Coste et al., 2010), and therefore we predict that increases in Piezo2 expression may cause hypersensitivity of cells in the brain in the response to pressure changes, leading to altered signal transduction. Daily exposure to either 8.5 or 10 psi BOP was shown to produce alterations in regulators of the BBB, reduction in levels of inflammatory markers, and decreased levels of neurodegeneration-related proteins. Additionally, increased levels of astrocyte-specific GFAP and increased pressure sensitivity (Piezo2) in the brain was observed. Together, these data indicate that repeated daily exposure to low-level BOP has significant effects on the brain in the short term, and these effects may contribute to long-term deficits reported by Warfighters. Importantly, the low intensities of BOP studied here do not cause gross lung injury (Sajja et al., 2020), so these effects are likely due to the direct effects of BOP on the brain rather than as the indirect result of systemic injuries.

Inflammation is a major component of TBI and likely plays a role in the pathogenesis of blast-related brain injury (Readnower et al., 2010; Corps et al., 2015). In this study, two cytokines (IL-4 and IL-6) were significantly altered after repeated low-level BOP exposure. Though many studies of BOP exposure using animal models have demonstrated an increase in pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα in the brain (Kovesdi et al., 2011; Cho et al., 2013; Perez-Polo et al., 2014; Elder et al., 2015), most of the inflammatory markers we measured were reduced, though not significantly, compared to shams (Supplementary Figure 1). These differences across studies might be attributed to higher exposure pressures along with experimental methodologies, specifically the use of cylindrical blast simulators in the previous studies, which impart high dynamic pressure (i.e., blast wind), plateaued peaks, and long duration blast waves (Sajja et al., 2018). In the current study, the anti-inflammatory cytokine IL-4 and pro-inflammatory cytokine IL-6 were significantly reduced in the brain following repeated exposure to 10 psi BOP (Figure 2), whereas Gill et al. (2017a, 2017b) found increased peripheral IL-6 in blood samples of human subjects participating in breaching training, though this increase could be a contribution from peripheral organs affected by the BOP exposure (Gill et al., 2017a). In addition to perhaps reflecting bidirectional changes in the brain and the circulation, it is also possible that brain changes are limited and restricted to discrete neuroanatomical locations that were obscured in our larger tissue measurements of the whole cerebrum. On the other hand, a recent study comparing pre-and post-deployment peripheral cytokine levels in populations experiencing mild bTBI showed decreased levels of IL-4 and IL-6 after deployment, which is a finding seemingly more consistent with our observations in the current study (Rusiecki et al., 2020). Overall reductions in inflammatory markers, whether pro- or anti-inflammatory, indicates that there is no strong immune response following repeated low-level BOP, contrary to what might be expected following more severe injury. These molecules, along with other proteins, may also have been cleared from the brain via the glymphatic system, which is responsible for clearing the brain of proteins (Iliff et al., 2012). The differences between the two intensity groups may be due to differing time course of pathogenesis, or involvement of different signaling cascades due to poly-organ injury following 14 × 10 psi BOP exposure.

Breakdown of the BBB, which is a common outcome of BOP exposure in animal models, is likely a pivotal contributor to the pathophysiology of BOP insults to the brain (Readnower et al., 2010; Yeoh et al., 2013; Shetty et al., 2014; Hue et al., 2016; Kawoos et al., 2016). We previously reported disruption in tight junction proteins and VEGF following higher intensity BOP exposure (Heyburn et al., 2019a). In the current study, we found that VEGF and the tight junction proteins claudin-5 and occludin are decreased following repeated low-level BOP exposure, with a significant reduction in occludin after repeated 8.5 psi BOP and a significant reduction in VEGF after repeated 10 psi BOP exposure (Figure 3). Similar disruptions in tight junction proteins have been reported in rats following higher intensity BOP exposure (Abdul-Muneer et al., 2013) and in humans undergoing heavy weapons training (Duckworth, 2018), indicating that BBB disruption may be a common effect of BOP exposure across a range of intensities. Though we observed a reduction in VEGF levels, presumably indicating an absence of angiogenesis, other groups have found that BOP exposure leads to elevated VEGF levels (Sajja et al., 2012a; Kamnaksh et al., 2014; Hubbard et al., 2017; Gama Sosa et al., 2017). It is important to note that these studies used higher intensity BOP than was used in this study, which could have resulted in poly-organ injury. Overall, tight junction proteins appear to be vulnerable to BOP at varying intensities, whereas VEGF was increased following higher intensity BOP (Heyburn et al., 2019a) but decreased following repeated low-intensity BOP (Figure 4), a result that could help distinguish among pathophysiological responses to BOP.

GFAP, an astrocyte-specific protein that is increased during astrogliosis in response to injury, has been recognized as a serum biomarker to predict mild-to-moderate TBI, astrocyte injury, and BBB damage (Papa et al., 2012; Eonta et al., 2019). Astrocytes are involved in inflammatory and repair processes in the brain, and are also a major component of the BBB, with astrocytic end feet encircling blood vessels in the brain microvasculature and assisting in the maintenance of tight junction integrity (Shetty et al., 2014). In this study, we found that GFAP is significantly increased in the brain following exposure to repeated 10 psi BOP (Figure 5). The measured increase in GFAP may be part of a compensatory or regenerative mechanism elicited to repair the damaged BBB by increasing or restoring astrocytic endfeet at the blood vessels. To determine the cause or consequence of the increase in GFAP, future experiments employing immunohistochemical analysis will be required to distinguish the neuroanatomical localization of GFAP responses in the brain. It is speculated that both inflammatory and BBB restoration mechanisms are activated in tandem to protect the brain against cellular loss and to maintain cellular homeostasis.

It has been shown that BOP can cause large ICP changes, and the degree of response to these changes in pressure is speculated to correlate with the severity of the injury, such that altered mechanosensitivity in the brain could make it more vulnerable to injury from subsequent BOP exposure (Coste et al., 2010; Sajja et al., 2018). Piezo2, when activated by pressure changes such as BOP, leads to mechanotransduction, which may play a role in cellular injury response to brain injury (Hemphill et al., 2015; Szczot et al., 2017; Shin et al., 2019). Our group previously reported that Piezo2 is increased in the brain after high-intensity BOP exposure and that its levels correlated with BOP intensity (Heyburn et al., 2019a). In the current study, we again observed that Piezo2 is significantly increased in the brain following repeated exposures to 10 psi, but not 8.5 psi, BOP. Together, these findings indicate that high intensity and repeated low-intensity BOP exposure lead to increased levels of Piezo2, which may, in turn, lead to increased sensitivity to subsequent changes in pressure in the brain. Also, Piezo2 is involved in vascular endothelial cell-dependent mechanotransduction (Ferrari et al., 2015), pointing to a role for Piezo2 in vascular regulation that could contribute to BBB homeostasis. The increased Piezo2 following 10 psi BOP exposures complements the observed changes in GFAP and could potentially reflect roles for each in BBB structure and function.

Increasingly, TBI has been associated with neurodegenerative processes and neurodegeneration-related protein abnormalities (DeKosky and Asken, 2017; Heyburn et al., 2019b). Although significant alterations in brain levels of TDP-43, a marker associated with neurodegenerative disease processes, were previously observed following moderate-intensity BOP exposure (Heyburn et al., 2019a), we did not observe a significant change in TDP-43 levels following repeated low-level BOP exposure in the current study, though it was trending upward with increased BOP intensity (Figure 6). We observed significant reductions in Alzheimer’s disease-related proteins following repeated low-level BOP, with phosphorylated tau (S396) significantly reduced after repeated 10 psi BOP exposure, and Aβ40 and Aβ42 significantly reduced after repeated 8.5 psi BOP exposure (Figure 7). Similarly, a reduction in Aβ42 was observed in human serum samples 24 h after exposure to BOP during breaching training (Edwards et al., 2020). A study of Warfighters in breaching training found that amyloid precursor protein (APP), whose cleavage products include Aβ40 and Aβ42, is reduced in the blood following training related BOP exposure at acute time points (Gill et al., 2017b), which aligns with our findings of reduced Aβ40 and Aβ42 following repeated low-level BOP exposure. In a case study of a Warfighter who sustained three BOP exposures, the brain was positive for Aβ plaques and phosphorylated tau lesions, but negative for TDP-43 inclusions (Iacono et al., 2020). While this clinical finding fits with our TDP-43 data in this study, it is inconsistent with our Aβ40, Aβ42, and pTau data (Iacono et al., 2020), which could be attributed to the severity of BOP and the chronic nature of outcomes in the clinical study. The early changes we observed in Aβ and pTau could eventually lead to an increase in these markers at a chronic time-point, which needs to be further investigated in future studies.

For several of the proteins studied (occludin, Aβ40, Aβ42), there were significant alterations in the 14 × 8.5 psi BOP group, but not in the higher intensity (14 × 10 psi) group. Similar findings were reported in a study with single low-level BOP exposure (De Gasperi et al., 2012). It was observed that Aβ40 and Aβ42 were decreased at lower pressures (either 5 or 10 psi) but not in the 17 psi group (De Gasperi et al., 2012). The higher intensity exposure may cause poly-organ injury, rather than just primary BOP injury to the brain, leading to divergent downstream cellular and molecular events. Another explanation is that at lower intensities, there is enhanced glymphatic flow, which involves clearance of molecules from the brain parenchyma via aquaporin-4 channels in astrocytes (Iliff et al., 2012). There have been studies of blast overpressure showing that AQP4 is increased following blast exposure (Gu et al., 2017; Rios et al., 2019). It is speculated that mechanisms of glymphatic flow could be associated with the decrease in some proteins and cytokines following BOP exposure. It is also possible that following a single BOP exposure, the brain is primed for subsequent blast exposure, which may result in immune tolerance and activation, which needs to be further investigated.

In this study, we identified several pathophysiological responses to repeated low-level BOP exposure, similar to that experienced by Warfighters in training environments. These exposures lead to acute changes in the brain including disruption of protein regulators of the BBB, astrogliosis, increased levels of a mechanosensitive protein, a decrease in brain cytokines, and decreased neurodegeneration-related proteins, outcomes which reflect overall perturbation of the brain by BOP forces. These changes observed in this study could potentially contribute to cumulative effects of repeated BOP exposure that could, in turn, contribute to the symptoms and behavioral deficits experienced by Warfighters. Eventually, these data can help contribute to the development of brain injury risk curves for low-level BOP and ultimately lead to the development of protective standards to prevent acute and chronic blast effects.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Walter Reed Army Institute of Research IACUC.

LH and VS designed the experiments. LH wrote the manuscript. DW performed the blast experiments. LH, RA, SG, AB, RU, JW, and PA performed the experimental procedures. LH and RA performed the data analysis. LH performed the statistical analysis. JL, SA, and VS oversaw the study. All authors contributed to the article and approved the submitted version.

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting true views of the Department of the Army, Department of the Navy, or the Department of Defense. The research was conducted under an approved animal use protocol in an AAALACi accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory.

JL and SA are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17, U.S.C., §105 provides that copyright protection under this title is not available for any work of the U.S. Government. Title 17, U.S.C., §101 defines a U.S. Government work as a work prepared by an employee of the U.S. Government as part of that person’s official duties.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.