Temporal Alterations in Cerebrovascular Glycocalyx and Cerebral Blood Flow after Exposure to a High-Intensity Blast in Rats.

The glycocalyx is a proteoglycan-glycoprotein structure lining the luminal surface of the vascular endothelium and is susceptible to damage due to blast overpressure (BOP) exposure. The glycocalyx is essential in maintaining the structural and functional integrity of the vasculature and regulation of cerebral blood flow (CBF). Assessment of alterations in the density of the glycocalyx; its components (heparan sulphate proteoglycan (HSPG/syndecan-2), heparan sulphate (HS), and chondroitin sulphate (CS)); CBF; and the effect of hypercapnia on CBF was conducted at 2-3 h, 1, 3, 14, and 28 days after a high-intensity (18.9 PSI/131 kPa peak pressure, 10.95 ms duration, and 70.26 PSI·ms/484.42 kPa·ms impulse) BOP exposure in rats. A significant reduction in the density of the glycocalyx was observed 2-3 h, 1-, and 3 days after the blast exposure. The glycocalyx recovered by 28 days after exposure and was associated with an increase in HS (14 and 28 days) and in HSPG/syndecan-2 and CS (28 days) in the frontal cortex. In separate experiments, we observed significant decreases in CBF and a diminished response to hypercapnia at all time points with some recovery at 3 days. Given the role of the glycocalyx in regulating physiological function of the cerebral vasculature, damage to the glycocalyx after BOP exposure may result in the onset of pathogenesis and progression of cerebrovascular dysfunction leading to neuropathology.

Exposure to Low-Intensity Blast Increases Clearance of Brain Amyloid Beta.

The long-term effects of exposure to blast overpressure are an important health concern in military personnel. Increase in amyloid beta (Aß) has been documented after non-blast traumatic brain injury (TBI) and may contribute to neuropathology and an increased risk for Alzheimer's disease. We have shown that Aß levels decrease following exposure to a low-intensity blast overpressure event. To further explore this observation, we examined the effects of a single 37 kPa (5.4 psi) blast exposure on brain Aß levels, production, and clearance mechanisms in the acute (24 h) and delayed (28 days) phases post-blast exposure in an experimental rat model. Aß and, notably, the highly neurotoxic detergent soluble Aß42 form, was reduced at 24 h but not 28 days after blast exposure. This reduction was not associated with changes in the levels of Aß oligomers, expression levels of amyloid precursor protein (APP), or increase in enzymes involved in the amyloidogenic cleavage of APP, the ß- and ϒ-secretases BACE1 and presenilin-1, respectively. The levels of ADAM17 α-secretase (also known as tumor necrosis factor α-converting enzyme) decreased, concomitant with the reduction in brain Aß. Additionally, significant increases in brain levels of the endothelial transporter, low-density related protein 1 (LRP1), and enhancement in co-localization of aquaporin-4 (AQP4) to perivascular astrocytic end-feet were observed 24 h after blast exposure. These findings suggest that exposure to low-intensity blast may enhance endothelial clearance of Aß by LRP1-mediated transcytosis and alter AQP4-aided glymphatic clearance. Collectively, the data demonstrate that low-intensity blast alters enzymatic, transvascular, and perivascular clearance of Aß.

Metabotropic Glutamate Receptor 2 Expression Is Chronically Elevated in Male Rats With Post-Traumatic Stress Disorder Related Behavioral Traits Following Repetitive Low-Level Blast Exposure.

Many military veterans who experienced blast-related traumatic brain injuries in the conflicts in Iraq and Afghanistan currently suffer from chronic cognitive and mental health problems that include depression and post-traumatic stress disorder (PTSD). Male rats exposed to repetitive low-level blast develop cognitive and PTSD-related behavioral traits that are present for more than 1 year after exposure. We previously reported that a group II metabotropic receptor (mGluR2/3) antagonist reversed blast-induced behavioral traits. In this report, we explored mGluR2/3 expression following blast exposure in male rats. Western blotting revealed that mGluR2 protein (but not mGluR3) was increased in all brain regions studied (anterior cortex, hippocampus, and amygdala) at 43 or 52 weeks after blast exposure but not at 2 weeks or 6 weeks. mGluR2 RNA was elevated at 52 weeks while mGluR3 was not. Immunohistochemical staining revealed no changes in the principally presynaptic localization of mGluR2 by blast exposure. Administering the mGluR2/3 antagonist LY341495 after behavioral traits had emerged rapidly reversed blast-induced effects on novel object recognition and cued fear responses 10 months following blast exposure. These studies support alterations in mGluR2 receptors as a key pathophysiological event following blast exposure and provide further support for group II metabotropic receptors as therapeutic targets in the neurobehavioral effects that follow blast injury.

(2R,6R)-Hydroxynorketamine Treatment of Rats Exposed to Repetitive Low-Level Blast Injury.

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Male rats subjected to repetitive low-level blast exposure develop chronic cognitive and PTSD-related traits that develop in a delayed manner. Ketamine has received attention as a treatment for refractory depression and PTSD. (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a ketamine metabolite that exerts rapid antidepressant actions. (2R,6R)-HNK has become of clinical interest because of its favorable side-effect profile, low abuse potential, and oral route of administration. We treated three cohorts of blast-exposed rats with (2R,6R)-HNK, beginning 7-11 months after blast exposure, a time when the behavioral phenotype is established. Each cohort consisted of groups (n = 10-13/group) as follows: 1) Sham-exposed treated with saline, 2) blast-exposed treated with saline, and 3) blast-exposed treated with a single dose of 20 mg/kg of (2R,6R)-HNK. (2R,6R)-HNK rescued blast-induced deficits in novel object recognition (NOR) and anxiety-related features in the elevated zero maze (EZM) in all three cohorts. Exaggerated acoustic startle was reversed in cohort 1, but not in cohort 3. (2R,6R)-HNK effects were still present in the EZM 12 days after administration in cohort 1 and 27 days after administration in NOR testing of cohorts 2 and 3. (2R,6R)-HNK may be beneficial for the neurobehavioral syndromes that follow blast exposure in military veterans. Additional studies will be needed to determine whether higher doses or more extended treatment regimens may be more effective.

Progressive Transcriptional Changes in the Amygdala Implicate Neuroinflammation in the Effects of Repetitive Low-Level Blast Exposure in Male Rats.

Chronic mental health problems are common among military veterans who sustained blast-related traumatic brain injuries. The reasons for this association remain unexplained. Male rats exposed to repetitive low-level blast overpressure (BOP) exposures exhibit chronic cognitive and post-traumatic stress disorder (PTSD)-related traits that develop in a delayed fashion. We examined blast-induced alterations on the transcriptome in four brain areas (anterior cortex, hippocampus, amygdala, and cerebellum) across the time frame over which the PTSD-related behavioral phenotype develops. When analyzed at 6 weeks or 12 months after blast exposure, relatively few differentially expressed genes (DEGs) were found. However, longitudinal analysis of amygdala, hippocampus, and anterior cortex between 6 weeks and 12 months revealed blast-specific DEG patterns. Six DEGs (hyaluronan and proteoglycan link protein 1 [Hapln1], glutamate metabotropic receptor 2 [Grm2], purinergic receptor P2y12 [P2ry12], C-C chemokine receptor type 5 [Ccr5], phenazine biosynthesis-like protein domain containing 1 [Pbld1], and cadherin related 23 [Cdh23]) were found altered in all three brain regions in blast-exposed animals. Pathway enrichment analysis using all DEGs or those uniquely changed revealed different transcription patterns in blast versus sham. In particular, the amygdala in blast-exposed animals had a unique set of enriched pathways related to stress responses, oxidative phosphorylation, and mitochondrial dysfunction. Upstream analysis implicated tumor necrosis factor (TNF)α signaling in blast-related effects in amygdala and anterior cortex. Eukaryotic initiating factor eIF4E (EIF4e), an upstream regulator of P2ry12 and Ccr5, was predicted to be activated in the amygdala. Quantitative polymerase chain reaction (qPCR) validated longitudinal changes in two TNFα regulated genes (cathepsin B [Ctsb], Hapln1), P2ry12, and Grm2. These studies have implications for understanding how blast injury damages the brain and implicates inflammation as a potential therapeutic target.

Repetitive Low-Level Blast Exposure Improves Behavioral Deficits and Chronically Lowers Aß42 in an Alzheimer Disease Transgenic Mouse Model.

Public awareness of traumatic brain injury (TBI) in the military increased recently because of the conflicts in Iraq and Afghanistan where blast injury was the most common mechanism of injury. Besides overt injuries, concerns also exist over the potential adverse consequences of subclinical blast exposures, which are common for many service members. A TBI is a risk factor for the later development of neurodegenerative diseases, including Alzheimer disease (AD)-like disorders. Studies of acute TBI in humans and animals have suggested that increased processing of the amyloid precursor protein (APP) toward the amyloid beta protein (Aß) may explain the epidemiological associations with AD. In a previous study, however, we found in both rat and mouse models of blast overpressure exposure that rather than increasing, rodent brain Aß42 levels were decreased after acute blast exposure. Here we subjected APP/presenilin 1 transgenic mice (APP/PS1 Tg) to an extended sequence of repetitive low-level blast exposures (34.5 kPa) administered three times per week over eight weeks. If initiated at 20 weeks of age, these repetitive exposures, which were designed to mimic human subclinical blast exposures, reduced anxiety and improved cognition as well as social interactions in APP/PS1 Tg mice, returning many behavioral parameters in APP/PS1 Tg mice to levels of non-transgenic wild type mice. Repetitive low-level blast exposure was less effective at improving behavioral deficits in APP/PS1 Tg mice when begun at 36 weeks of age. While amyloid plaque loads were unchanged, Aß 42 levels and Aß oligomers were reduced in the brain of mice exposed to repetitive low-level blast exposures initiated at 20 weeks of age, although levels did not directly correlate with behavioral parameters in individual animals. These results have implications for understanding the nature of blast effects on the brain and their relationship to human neurodegenerative diseases.

Blast-induced temporal alterations in blood-brain barrier properties in a rodent model.

The consequences of blast-induced traumatic brain injury (bTBI) on the blood-brain barrier (BBB) and components of the neurovascular unit are an area of active research. In this study we assessed the time course of BBB integrity in anesthetized rats exposed to a single blast overpressure of 130 kPa (18.9 PSI). BBB permeability was measured in vivo via intravital microscopy by imaging extravasation of fluorescently labeled tracers (40 kDa and 70 kDa molecular weight) through the pial microvasculature into brain parenchyma at 2-3 h, 1, 3, 14, or 28 days after the blast exposure. BBB structural changes were assessed by immunostaining and molecular assays. At 2-3 h and 1 day after blast exposure, significant increases in the extravasation of the 40 kDa but not the 70 kDa tracers were observed, along with differential reductions in the expression of tight junction proteins (occludin, claudin-5, zona occluden-1) and increase in the levels of the astrocytic water channel protein, AQP-4, and matrix metalloprotease, MMP-9. Nearly all of these measures were normalized by day 3 and maintained up to 28 days post exposure. These data demonstrate that blast-induced changes in BBB permeability are closely coupled to structural and functional components of the BBB.

Development of a Blast Exposure Estimator from a Department of Defense-Wide Survey Study on Military Service Members.

Long-term, repeated exposure to low-intensity blast overpressure is a potential causal factor of lasting outcomes reminiscent of post-concussion syndrome. Wearable blast sensor engineers are exploring elements of blast that are associated with outcomes. Currently, however, there are no devices that can truly record all blasts experienced by an individual. Military service members (n = 984) were surveyed about their lifelong exposure and behavioral health. Using heavy-arms-associated target outcomes, we calculated a generalized blast exposure value (GBEV) for each participant. A threshold of 200,000 GBEV units was established at which a participant was likely to report more intense symptomology. If repetitive, low-intensity blast exposure has even a subtle effect over time, operational readiness could be negatively impacted. A threshold of exposure can inform decisions about how to reduce detrimental exposure. The GBEV can be used to track ongoing exposure and potentially identify those who may be at risk for developing blast-related outcomes.

Pulmonary injury risk curves and behavioral changes from blast overpressure exposures of varying frequency and intensity in rats.

At present, there are no set guidelines establishing cumulative limits for blast exposure numbers and intensities in military personnel, in combat or training operations. The objective of the current study was to define lung injury, pathology, and associated behavioral changes from primary repeated blast lung injury under appropriate exposure conditions and combinations (i.e. blast overpressure (BOP) intensity and exposure frequency) using an advanced blast simulator. Male Sprague Dawley rats were exposed to BOP frontally and laterally at a pressure range of ~ 8.5-19 psi, for up to 30 daily exposures. The extent of lung injury was identified at 24 h following BOP by assessing the extent of surface hemorrhage/contusion, Hematoxylin and Eosin staining, and behavioral deficits with open field activity. Lung injury was mathematically modeled to define the military standard 1% lung injury threshold. Significant levels of histiocytosis and inflammation were observed in pressures ≥ 10 psi and orientation effects were observed at pressures ≥ 13 psi. Experimental data demonstrated ~ 8.5 psi is the threshold for hemorrhage/contusion, up to 30 exposures. Modeling the data predicted injury risk up to 50 exposures with intensity thresholds at 8 psi for front exposure and 6psi for side exposures, which needs to be validated further.

Blast exposure results in tau and neurofilament light chain changes in peripheral blood.

OBJECTIVES: To evaluate how blast exposure impacts peripheral biomarkers.in military personnel enrolled in 10-day blast training. METHODS: On day 7, 21 military personnel experienced peak overpressure <2 pounds per square inch (psi); while 29 military personnel experienced peak overpressure ≥5 psi. Blood samples were collected each day to measure changes in amyloid beta (Aß), neurofilament light chain (NFL), and tau concentrations. RESULTS: Within 24 hours following exposure ≥5 psi, the ≥5 psi group had lower Aß42 (p = .004) and NFL (p < .001) compared to the <2 psi group and lower Aß42 (9.35%) and NFL (22.01%) compared to baseline. Twenty-four hours after ≥5 psi exposure, the ≥5 psi group had lower tau (p < .001) and NFL (p < .001) compared to the <2 psi group and baseline. Seventy-two hours after exposure ≥5 psi, tau increased in the ≥5 psi group compared to the <2 psi group (p = .02) and baseline. The tau:Aß42 ratio 24 hours after blast (p = .012), and the Aß40:Aß42 ratio 48 hours after blast (p = .04) differed in the ≥5 psi group compared to the <2 psi group. CONCLUSIONS: These findings provide an initial report of acute alterations in biomarker concentrations following blast exposure.

Neuroendocrine function and associated mental health outcomes following mild traumatic brain injury in OEF-deployed service members.

Mild traumatic brain injury (mTBI) has been linked to mental health disorders (MHDs) and pituitary function alterations. Due to the complex relationship of mTBI, the neuroendocrine system, and MHDs, we propose that neuroendocrine dysfunction (NED) may play a role in negative long-term health outcomes. The goal of this study was to determine if blast-concussed service members (SMs) have a stronger likelihood of developing NED. We hypothesized that NED either pre- or post-injury is associated with poor mental and physical health outcomes. Serum samples from the Armed Forces Health Surveillance Branch were obtained from concussed (n = 59) and non-concussed (n = 72) SMs treated at the Concussion Restoration Care Center (CRCC) in Afghanistan. Serum was collected within 2 years prior to deployment and one or two times within 3 years following their CRCC visit. Samples were analyzed for luteinizing hormone (LH), testosterone, human growth hormone, cortisol, and prolactin to assess post-injury neuroendocrine function. Results indicate that SMs who incurred an mTBI exhibited long-term LH and testosterone deficiencies 3 years following injury compared to controls. Specifically, 47.6% of head-injured SMs displayed hypofunction in at least one of five hormones at 3 years post-injury. Anxiety disorders were the most common MHD observed in concussed SMs with hypopituitarism, while there was also a trend for SMs with chronic pituitary dysfunction to have MHD diagnoses. Findings indicate blast-related mTBI may be associated with long-term health outcomes following a period of incubation. Neuroendocrine screenings may increase treatment opportunities, inform rehabilitation strategies, and improve overall quality of life for patients.

Assessing a Blast-Related Biomarker in an Operational Community: Glial Fibrillary Acidic Protein in Experienced Breachers.

Mild traumatic brain injury (mTBI) is a risk for military personnel due to blast overpressures, which may result from a variety of sources, including artillery and improvised explosive devices. Much research has gone into the search for a biomarker to identify patients with a TBI. The FDA recently identified two proteins, glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1), as biomarkers to evaluate suspected brain injury. Our group previously observed changes in UCH-L1 in a military population exposed to repeated blast. In our current study we assessed GFAP protein levels in a military population exposed to repeated blast during a 2-week training protocol. We observed GFAP levels were reduced in the moderate blast cases on days 6 and 7 during the training. Specifically, moderate blast cases showed a 24.07% reduction from baseline on day 6 and a 29.61% reduction on day 7. Further, GFAP levels were negatively correlated with cumulative blast experienced during training and with duration of military service. We observed that repeated blast exposure at low levels may impact acute changes in GFAP. Additionally subacute cumulative blast exposure or duration of service was also a factor in influencing GFAP levels.

Exposure to Blast Overpressure Impairs Cerebral Microvascular Responses and Alters Vascular and Astrocytic Structure.

Exposure to blast overpressure may result in cerebrovascular impairment, including cerebral vasospasm. The mechanisms contributing to this vascular response are unclear. The aim of this study was to evaluate the relationship between blast and functional alterations of the cerebral microcirculation and to investigate potential underlying changes in vascular microstructure. Cerebrovascular responses were assessed in sham- and blast-exposed male rats at multiple time points from 2 h through 28 days after a single 130-kPa (18.9-psi) exposure. Pial microcirculation was assessed through a cranial window created in the parietal bone of anesthetized rats. Pial arteriolar reactivity was evaluated in vivo using hypercapnia, barium chloride, and serotonin. We found that exposure to blast leads to impairment of arteriolar reactivity >24 h after blast exposure, suggesting delayed injury mechanisms that are not simply attributed to direct mechanical deformation. Observed vascular impairment included a reduction in hypercapnia-induced vasodilation, increase in barium-induced constriction, and reversal of the serotonin effect from constriction to dilation. A reduction in vascular smooth muscle contractile proteins consistent with vascular wall proliferation was observed, as well as delayed reduction in nitric oxide synthase and increase in endothelin-1 B receptors, mainly in astrocytes. Collectively, the data show that exposure to blast results in delayed and prolonged alterations in cerebrovascular reactivity that are associated with changes in the microarchitecture of the vessel wall and astrocytes. These changes may contribute to long-term pathologies involving dysfunction of the neurovascular unit, including cerebral vasospasm.

Affective profiling for anxiety-like behavior in a rodent model of mTBI.

Mild traumatic brain injury is a common outcome of blast exposure, and current literature indicates high rates of comorbid posttraumatic stress disorder (PTSD) in military personnel. Blast-exposed rats display PTSD-like behavior, suggesting relationships may exist between PTSD and blast exposure. Other studies demonstrate the roles of stathmin and corticosterone associated with fear- and anxiety-like behaviors in rodent models. Furthermore, studies have observed ranges of responses to both physical and psychological trauma in animal populations (Elder 2012, Ritov 2016). This study exposed rodents to repeated blast overpressure (BOP) and analyzed behavioral responses and molecular variables at 3 weeks and 6 months after exposure. We applied a modified version of a previously reported behavioral profiling approach that separates "affected" and "unaffected" rats based on the presence of anxiety-like behaviors (Ritov, 2016). We report that "affected" 3 week animals showed higher plasma corticosterone and amygdalar stathmin levels, while "affected" 6 month animals had lower prefrontal cortex stathmin. Higher corticosterone also paralleled anxiety behavior in "affected" 3 week animals, which was not observed in 6 month animals, indicating possible negative feedback loop mechanisms. Elevated levels of amygdalar stathmin correlated with anxiety behaviors in "affected" 3 week and 6 month animals, indicating sustained molecular changes. We conclude that this unique analysis may provide more information about response to blast. This type of analysis should also be considered when treating clinical populations, since individual differences may affect behavioral and long-term outcomes. Future studies should elucidate relationships of stress and fear responses in the context of BOP.

A Novel, Inexpensive Method to Monitor, Record, and Analyze Breathing Behavior During Normobaric Hypoxia Generated by the Reduced Oxygen Breathing Device.

OBJECTIVES: Since hypoxia remains one of the most important physiological hazards the aviation environment poses, military aviators are trained to recognize symptoms of hypoxia in order to implement appropriate safety procedures and countermeasures when hypoxia occurs. A widely used commercial instrument for hypoxia training, demonstration, and research is the Reduced Oxygen Breathing Device (ROBD). Here we describe a novel, inexpensive method to use the ROBD's breathing loop pressure (BLP) to measure respiration rate, a critically important response parameter for hypoxia. METHODS: The ROBD can be controlled by a computer to export several variables including BLP, via the ROBD's RS232 port. An archived database was reanalyzed to assess the BLP data. New instrumentation added independent measures of respiration and expired oxygen and carbon dioxide; these measures were integrated with the ROBD output. RESULTS: Analysis of the archived data showed that the BLP reflected realistic breathing patterns. The new instrumentation integrated well with the ROBD, and independently supported the potential of the BLP as a valid measure of respiration. DISCUSSION: The ROBD's BLP data may provide a basis for a reliable, sensitive measure of respiration that is available at no additional cost.

Assessment of a Prototype Apache Flight Eyewear.

BACKGROUND: Apache pilots needing refractive correction are issued modified HGU-4/P aviator spectacles. However, a recently published survey found field of view (FOV) dissatisfaction with the current spectacles when sighting in with a Helmet Display Unit (HDU). A current Air Force flight frame was modified in-lab and the purpose of this study was to evaluate the FOV with the current Apache flight frame vs. the modified flight frame. METHODS: Recruited were 21 Apache pilots to assess FOV under three conditions: 1) wearing the current Apache frame; 2) wearing the modified Apache frame; and 3) wearing no frame. The main outcome measure was total FOV of four quadrants tested: superior left (45°); superior right (135°); inferior right (225°); and inferior left (315°). RESULTS: No significant differences in FOV were seen between the two frames tested while wearing the current Apache helmet (P = 0.33) and the new Apache helmet (P = 0.64). However, there were significant differences in FOV between the no frame condition and the two frame conditions tested with both helmets (P < 0.001). DISCUSSION: No significant differences in FOV were seen between the two frames tested while wearing either Apache helmet. However, with both helmets there were significant differences in FOV between the no frame condition and the two frame conditions tested. This suggests that wearing no eyewear is still optimal in integrating the HDU device. With advances in contact lens technology, future research can study the viability of the latest generation of multifocal contact lenses with Apache aviators. Walsh DV, Jurek GM, McLean WE, Statz JK, Allen RL, Riggs DW. Assessment of a prototype Apache flight eyewear. Aerosp Med Hum Perform. 2016; 87(9):800-805.