Brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure.

Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [18F]AV1451 (flortaucipir) tau ligand, we found that five of 10 veterans exhibited excessive retention of [18F]AV1451 at the white/gray matter junction in frontal, parietal, and temporal brain regions, a typical localization of CTE tauopathy. We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [18F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI.

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.

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.

An exploratory investigation of brain-selective estrogen treatment in males using a mouse model of Alzheimer's disease.

Estrogens are neuroprotective, and studies suggest that they may mitigate the pathology and symptoms of Alzheimer's disease (AD) in female models. However, central estrogen effects have not been examined in males in the context of AD. The purpose of this follow-up study was to assess the benefits of a brain-selective 17ß-estradiol estrogen prodrug, 10ß,17ß-hydroxyestra-1,4-dien-3-one (DHED), also in the male APPswe/PS1dE9 double-transgenic mouse model of the disease. After continuously exposing 6-month old animals to DHED for two months, their brains showed decreased amyloid precursor and amyloid-ß protein levels. The DHED-treated APPswe/PS1dE9 double transgenic subjects also exhibited enhanced performance in a cognitive task, while 17ß-estradiol treatment did not reach statistical significance. Taken together, data presented here suggest that DHED may also have therapeutic benefit in males and warrant further investigations to fully elucidate the potential of targeted estrogen therapy for a gender-independent treatment of early-stage AD.

A comparative evaluation of treatments with 17ß-estradiol and its brain-selective prodrug in a double-transgenic mouse model of Alzheimer's disease.

Estrogens are neuroprotective and, thus, potentially useful for the therapy of Alzheimer's disease; however, clinical use of hormone therapy remains controversial due to adverse peripheral effects. The goal of this study was to investigate the benefits of treatment with 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective prodrug of 17ß-estradiol, in comparison with the parent hormone using APPswe/PS1dE9 double transgenic mice to model the pathology of the disease. Ovariectomized and intact females were continuously treated with vehicle, 17ß-estradiol, or DHED via subcutaneous osmotic pumps from 6 to 8months of age. We confirmed that this prolonged treatment with DHED did not stimulate uterine tissue, whereas 17ß-estradiol treatment increased uterine weight. Amyloid precursor protein decreased in both treatment groups of intact, but not in ovariectomized double transgenic females in which ovariectomy already decreased the expression of this protein significantly. However, reduced brain amyloid-ß peptide levels could be observed for both treatments. Consequently, double-transgenic ovariectomized and intact mice had higher cognitive performance compared to untreated control animals in response to both estradiol and DHED administrations. Overall, the tested brain-selective 17ß-estradiol prodrug proved to be an effective early-stage intervention in an Alzheimer's disease-relevant mouse model without showing systemic impact and, thus, warrants further evaluation as a potential therapeutic candidate.

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.

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.

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.

Blast-Related Mild TBI Alters Anxiety-Like Behavior and Transcriptional Signatures in the Rat Amygdala.

The short and long-term neurological and psychological consequences of traumatic brain injury (TBI), and especially mild TBI (mTBI) are of immense interest to the Veteran community. mTBI is a common and detrimental result of combat exposure and results in various deleterious outcomes, including mood and anxiety disorders, cognitive deficits, and post-traumatic stress disorder (PTSD). In the current study, we aimed to further define the behavioral and molecular effects of blast-related mTBI using a well-established (3 × 75 kPa, one per day on three consecutive days) repeated blast overpressure (rBOP) model in rats. We exposed adult male rats to the rBOP procedure and conducted behavioral tests for anxiety and fear conditioning at 1-1.5 months (sub-acute) or 12-13 months (chronic) following blast exposure. We also used next-generation sequencing to measure transcriptome-wide gene expression in the amygdala of sham and blast-exposed animals at the sub-acute and chronic time points. Results showed that blast-exposed animals exhibited an anxiety-like phenotype at the sub-acute timepoint but this phenotype was diminished by the chronic time point. Conversely, gene expression analysis at both sub-acute and chronic timepoints demonstrated a large treatment by timepoint interaction such that the most differentially expressed genes were present in the blast-exposed animals at the chronic time point, which also corresponded to a Bdnf-centric gene network. Overall, the current study identified changes in the amygdalar transcriptome and anxiety-related phenotypic outcomes dependent on both blast exposure and aging, which may play a role in the long-term pathological consequences of mTBI.

Blast exposure interacts with genetic variant 5HTTLPR to predict posttraumatic stress symptoms in military explosives personnel.

The first of its kind, this study determined whether blast exposure interacts with genetic variant 5HTTLPR to predict posttraumatic stress (PTS) symptoms in 78 military explosives operators. In all models, blast-exposed 5HTTLPR S carriers registered definitively higher PTS symptoms in comparison to non-exposed S carriers, as well as exposed and non-exposed LL carriers (all p < 0.01). All findings were robust to confounding influences of age and traumatic brain injury diagnosis. Not only is blast exposure prevalent in EOD personnel, but it also interacts with genetic predisposition to predict trauma symptoms in this unique, at-risk military population.

Blast-induced "PTSD": Evidence from an animal model.

A striking observation among veterans returning from the recent conflicts in Iraq and Afghanistan has been the co-occurrence of blast-related mild traumatic brain injury (mTBI) and post-traumatic stress disorder (PTSD). PTSD and mTBI might coexist due to additive effects of independent psychological and physical traumas experienced in a war zone. Alternatively blast injury might induce PTSD-related traits or damage brain structures that mediate responses to psychological stressors, increasing the likelihood that PTSD will develop following a subsequent psychological stressor. Rats exposed to repetitive low-level blasts consisting of three 74.5 kPa exposures delivered once daily for three consecutive days develop a variety of anxiety and PTSD-related behavioral traits that are present for at least 9 months after blast exposure. A single predator scent challenge delivered 8 months after the last blast exposure induces additional anxiety-related changes that are still present 45 days later. Because the blast injuries occur under general anesthesia, it appears that blast exposure in the absence of a psychological stressor can induce chronic PTSD-related traits. The reaction to a predator scent challenge delivered many months after blast exposure suggests that blast exposure in addition sensitizes the brain to react abnormally to subsequent psychological stressors. The development of PTSD-related behavioral traits in the absence of a psychological stressor suggests the existence of blast-induced "PTSD". Findings that PTSD-related behavioral traits can be reversed by BCI-838, a group II metabotropic glutamate receptor antagonist offers insight into pathogenesis and possible treatment options for blast-related brain injury. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

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.

Low-level blast exposure disrupts gliovascular and neurovascular connections and induces a chronic vascular pathology in rat brain.

Much concern exists over the role of blast-induced traumatic brain injury (TBI) in the chronic cognitive and mental health problems that develop in veterans and active duty military personnel. The brain vasculature is particularly sensitive to blast injury. The aim of this study was to characterize the evolving molecular and histologic alterations in the neurovascular unit induced by three repetitive low-energy blast exposures (3 × 74.5 kPa) in a rat model mimicking human mild TBI or subclinical blast exposure. High-resolution two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of purified brain vascular fractions from blast-exposed animals 6 weeks post-exposure showed decreased levels of vascular-associated glial fibrillary acidic protein (GFAP) and several neuronal intermediate filament proteins (α-internexin and the low, middle, and high molecular weight neurofilament subunits). Loss of these proteins suggested that blast exposure disrupts gliovascular and neurovascular interactions. Electron microscopy confirmed blast-induced effects on perivascular astrocytes including swelling and degeneration of astrocytic endfeet in the brain cortical vasculature. Because the astrocyte is a major sensor of neuronal activity and regulator of cerebral blood flow, structural disruption of gliovascular integrity within the neurovascular unit should impair cerebral autoregulation. Disrupted neurovascular connections to pial and parenchymal blood vessels might also affect brain circulation. Blast exposures also induced structural and functional alterations in the arterial smooth muscle layer. Interestingly, by 8 months after blast exposure, GFAP and neuronal intermediate filament expression had recovered to control levels in isolated brain vascular fractions. However, despite this recovery, a widespread vascular pathology was still apparent at 10 months after blast exposure histologically and on micro-computed tomography scanning. Thus, low-level blast exposure disrupts gliovascular and neurovascular connections while inducing a chronic vascular pathology.

Recovery from Mild Traumatic Brain Injury Following Uncomplicated Mounted and Dismounted Blast: A Natural History Approach.

Objective: The purpose of this study is to utilize a natural history approach to describe and understand symptom recovery in personnel diagnosed with a blast-related mild traumatic brain injury (mTBI) resulting from an improvised explosive device blast. Participants and Design: The population included military personnel who experienced a blast mTBI while mounted (vehicle; n = 176) or dismounted (on foot; n = 37) (N = 213). Patients had no co-morbid psychiatric or muscle-skeletal issues and were treated within 72 h of injury. Prevalence and duration of self-reported symptoms were separately analyzed by injury context (mounted vs dismounted). Results: Headache was prominently reported in both mounted (85%) and dismounted (75%) populations. The mean time from injury to return to full duty was between 7.8 d (mounted) and 8.5 d (dismounted). The dismounted population reported visual changes that lasted 0.74 d longer. Conclusion: Our analysis implicates that headache is a common and acutely persistent symptom in mTBI regardless of injury context. Additionally, patients in mounted vs dismounted injury did not report significant differences in symptom prevalence. Although knowing the injury context (i.e., dismounted vs mounted) may be beneficial for providers to understand symptom presentations and deliver accurate anticipatory guidance for patients with blast-related mTBI, no significant differences were observed in this population. This may be due to the population characteristic as the trajectory of recovery may vary for patients who were not able to return to full duty within 30 d or required higher levels of care.

Embryonic effects of an environmentally relevant PCB mixture in the domestic chicken.

Studies were conducted to develop methods to assess the effects of a complex mixture of polychlorinated biphenyls (PCBs) in the domestic chicken (Gallus domesticus). Treatments were administered by egg injection to compare embryonic effects of an environmentally relevant PCB congener mixture in the domestic chicken over a range of doses. Chicken eggs were injected with the PCB mixture with a profile similar to that found in avian eggs collected on the upper Hudson River, New York, USA, at doses that spanned 0 to 98 µg/g egg. Eggs were hatched in the laboratory to ascertain hatching success. In the domestic chicken, the median lethal dose was 0.3 µg/g. These data demonstrate adverse effects of an environmentally relevant PCB mixture and provide the basis for further work using in vitro and other models to characterize the potential risk to avian populations. Environ Toxicol Chem 2018;37:2513-2522. © 2018 SETAC.

Applications of Transductive Spectral Clustering Methods in a Military Medical Concussion Database.

Traumatic brain injury (TBI) is one of the most common forms of neurotrauma that has affected more than 250,000 military service members over the last decade alone. While in battle, service members who experience TBI are at significant risk for the development of normal TBI symptoms, as well as risk for the development of psychological disorders such as Post-Traumatic Stress Disorder (PTSD). As such, these service members often require intense bouts of medication and therapy in order to resume full return-to-duty status. The primary aim of this study is to identify the relationship between the administration of specific medications and reductions in symptomology such as headaches, dizziness, or light-headedness. Service members diagnosed with mTBI and seen at the Concussion Restoration Care Center (CRCC) in Afghanistan were analyzed according to prescribed medications and symptomology. Here, we demonstrate that in such situations with sparse labels and small feature sets, classic analytic techniques such as logistic regression, support vector machines, naïve Bayes, random forest, decision trees, and k-nearest neighbor are not well suited for the prediction of outcomes. We attribute our findings to several issues inherent to this problem setting and discuss several advantages of spectral graph methods.

Lack of chronic neuroinflammation in the absence of focal hemorrhage in a rat model of low-energy blast-induced TBI.

Blast-related traumatic brain injury (TBI) has been a common cause of injury in the recent conflicts in Iraq and Afghanistan. Blast waves can damage blood vessels, neurons, and glial cells within the brain. Acutely, depending on the blast energy, blast wave duration, and number of exposures, blast waves disrupt the blood-brain barrier, triggering microglial activation and neuroinflammation. Recently, there has been much interest in the role that ongoing neuroinflammation may play in the chronic effects of TBI. Here, we investigated whether chronic neuroinflammation is present in a rat model of repetitive low-energy blast exposure. Six weeks after three 74.5-kPa blast exposures, and in the absence of hemorrhage, no significant alteration in the level of microglia activation was found. At 6 weeks after blast exposure, plasma levels of fractalkine, interleukin-1ß, lipopolysaccharide-inducible CXC chemokine, macrophage inflammatory protein 1α, and vascular endothelial growth factor were decreased. However, no differences in cytokine levels were detected between blast-exposed and control rats at 40 weeks. In brain, isolated changes were seen in levels of selected cytokines at 6 weeks following blast exposure, but none of these changes was found in both hemispheres or at 40 weeks after blast exposure. Notably, one animal with a focal hemorrhagic tear showed chronic microglial activation around the lesion 16 weeks post-blast exposure. These findings suggest that focal hemorrhage can trigger chronic focal neuroinflammation following blast-induced TBI, but that in the absence of hemorrhage, chronic neuroinflammation is not a general feature of low-level blast injury.