OVERALL PROGRAM: Elucidating the Role of Increased Neuroinflammation and Related Structural and Functional Neurological Sequelae After Exposure to Repetitive Blast

Exposure to explosive blast shock waves has been a pervasive feature of combat-related injuries experienced by operational personnel. Traumatic brain injury (TBI) caused by exposure to blast dramatically increased from 60% in 2008 to 74% in 2009 in the U.S. military. In addition to combat-related blast TBI, concern exists that repetitive exposures to low-level blasts during day-to-day training and operations may be linked to long-term neurological problems. These concerns have fueled research to explore potential injury mechanisms following repetitive blast exposure. A major focus of these research efforts has been to assess the effects of repeated low-level blast exposure in military operational personnel with repetitive exposures throughout their military career. A study performed by our group explored the neurological changes in the brains of career Breachers, who are exposed to repetitive low-level blast as a routine part of their jobs. The study showed that Experienced Breachers had significant alterations in brain structure and function. Experienced Breachers also had higher levels of markers of brain inflammation (known as brain-derived exosomes) in their blood. Specifically, alterations in the inflammation markers tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and IL-10 were noted, signaling increased inflammation in the brains of experienced Breachers. Subsequently, a pilot study was initiated by our group in partnership with U.S. Special Operations Command (USSOCOM) to explore whether brain inflammation is seen within special operators with a history of prior blast exposure. Brain inflammation was assessed using positron emission tomography (PET) imaging, a technique that uses a radioactive tracer to observe and measure the function of cells in an organ, the brain in this case. The radioactive inflammation tracer [18F]DPA-714 was used in the PET pilot study. Additionally, inflammatory biomarkers were assessed in brain-derived exosomes. An elevation in TNF-alpha was seen within brain-derived exosomes in special operators exposed to blast compared with controls who do not have a history of blast exposure. No differences between groups were observed in TNF-alpha in the blood, confirming the brain-specific nature of these findings. These observations in the pilot PET study were similar to our observations in the Experienced Breachers. The proposed focused program effort will examine the role of brain inflammation in response to chronic, repetitive blast exposure in active-duty military populations, Veterans, and in an established animal model. The program will use multiple blood-based tests as well as brain imaging, which may be used in the future for detection and treatment of neurological injury in individuals with repetitive blast exposure. This program will address Focus Area #1 “Understand,” to develop an understanding of the biological factors contributing to an individual’s response following a brain injury and Focus Area #2 “Prevent,” which includes identification and validation of biomarkers or other objective markers for diagnosis of brain injuries, repetitive exposures, and associated sequelae and potential physiological targets for intervention. The four projects outlined below will explore specific facets of the role of inflammation in long-term changes in the brain following blast exposure.

Project 1 will explore inflammation through brain imaging of the [18F] DPA-714 PET inflammation tracer and assessment of brain-derived exosomes and, specifically the activity of brain immune cells known as microglia, in both active-duty and retired military personnel who have been exposed to repetitive blast during training and deployments. This study will expand upon the recently completed USSOCOM pilot study to examine inflammation across a continuum of blast exposure and to understand the secondary effects of brain inflammation. Project 2 will explore inflammation in active-duty Service Members with clinically diagnosed TBI and history of blast exposure. Project 3 will examine inflammation in Veterans with clinically diagnosed TBI and prior blast exposure. Project 4 will explore underlying biological features of brain inflammation, and specifically one of the TNF-a producing pathways, and how it relates to changes in the function of brain blood vessel in an established animal model of blast overpressure exposure. Projects 1-3 will provide a comprehensive characterization of the role of brain inflammation in clinically diagnosed personnel exposed to blast in both active-duty and retired populations. Importantly, execution of these studies within a unified program will allow for alignment of methods and analyses, improving our ability to compare the data across the different populations. Building upon preliminary data demonstrating increases in TNF-a within blood brain-derived exosomes of blast-exposed Service Members, Project 4 will provide key insights into the role of TNF-a following blast exposure, which may enable the design of novel diagnostic, therapeutic, and mitigation strategies for these populations. Together, it is anticipated these projects will lead to a significantly enhanced understanding of the role of brain inflammation in brain injury caused by exposure to blast and how the degree of prior blast exposure relates to brain inflammation. These findings hold the potential for identifying physiological targets to inhibit or block increased inflammation in the brain and the potential adverse consequences that result.

PROJECT 1: Exploring the Role of Neuroinflammation in Blast-Exposed Operational Personnel

Traumatic brain injury (TBI) is a known risk to military Service Members and may occur following events such as motor vehicle accidents, falls, or exposure to high explosives. Recent work suggests that exposure to low levels of blast over time may also cause changes to the brain that may have an adverse effect on brain health. Observations in Service Members who routinely work with low-level blast in training and operations have shown changes in the structure as well as the function of the brain. Little is known about how these changes occur or what level of blast over time may cause these changes. However, research is now providing information that suggests that the immune system may play a role in the processes that are associated with these changes. The immune system is an important part of the brain’s process of maintaining brain health. In addition to helping to keep the brain clear of infection, the immune system is also involved in helping to clear away damaged materials when the brain is injured and to help restore the healthy environment within the brain. However, the immune system is known to become active in a way that may be damaging to brain health over time, participating in cycles of activation, brain cell death, and clearance, which over time can cause changes in the brain that can lead to loss of brain tissue and function. These cycles are known to occur in a variety of brain diseases such as Alzheimer’s disease and Parkinson’s disease. Recent research that shows that brain structure and function is changing over time in Service Members exposed to repeated blasts along with work that shows that the immune system is also active in these Service Members must be expanded upon. Our study will broaden previous work by examining both active-duty and Veteran Service Members without a clinical diagnosis of TBI but with prior history of repeated blast exposure. This project will address Focused Program Award (FPA) Focus Area #1 “Understand,” to develop an understanding of the biological factors contributing to an individual’s response following a brain injury and FPA Focus Area #2 “Prevent,” which includes identification and validation of biomarkers or other objective markers for diagnosis of brain injuries, repetitive exposures, and associated sequelae and potential physiological targets for intervention. We will ensure that a full spectrum of blast exposure is assessed across these groups and that potentially susceptible communities such as special operators, Breachers, and Explosive Ordnance Disposal (EOD) personnel are included within the study. We will collect information from the blood that helps us to understand whether activation of the brain immune system is present. We will also perform imaging of the brain using approaches that allow us to directly see whether brain immune system activation is occurring. We will use other brain imaging methods to look at the structure and function of the brain and will ask about prior history of blast along with other questions that will help us to understand important information about topics such as medical history and brain performance. All of this information will be considered together to help us to understand the role of the brain immune system in potentially susceptible Service Members with a history of prior blast exposure. This information will be made available to decision-makers across these communities to help ensure the safety of Service Members. The information will also be made available to medical and research communities to help with the design of new treatments and strategies to prevent injuries to help support the brain health of Service Members exposed to blast over a career.

PROJECT 2: Neuroinflammation in Active-Duty Service Members with Clinically Diagnosed TBI

It is hard to predict the recovery period for Service Members (SMs) who sustain a traumatic brain injury (TBI). While most SMs recover within weeks, about a third will continue to have symptoms. Our group’s research focuses on finding out which SMs are at higher risk for having long-term symptoms, and ways to reduce these risks. This project will address Focused Program Award (FPA) Focus Area #1 “Understand,” to develop an understanding of the biological factors contributing to an individual’s response following a brain injury and FPA Focus Area #2 “Prevent,” which includes identification and validation of biomarkers or other objective markers for diagnosis of brain injuries, repetitive exposures, and associated sequelae and potential physiological targets for intervention. In this project, we will look at levels of proteins that relate to neuronal risks and inflammation. To understand brain-initiated processes, we will examine brain-derived exosomal vesicle (BDEV) levels of these same proteins. This type of study will provide insights into the relationship between neuronal damage and chronic symptoms that are associated with TBIs. It may allow us also to determine those SMs who are at risk of poor outcomes. Exosomes are vesicles secreted by neurons, with several characteristics making them potential key biomarkers of injury and recovery after TBI. First, neuronal exosomes contain cytokines used in neuron-to-neuron communication and potentiate neuroinflammatory processes. Second, neuronal exosomes assist in removal of proteins from the brain, including tau, which can aggregate and be changed so that is can no longer able to leave the brain easily, which is found in Alzheimer’s disease. Third, exosomes cross the blood-brain barrier and can be isolated from peripheral blood. Here we propose to measure BDEV levels of neuronal proteins (i.e., tau, phosphorylated tau [p-tau], and neurofilament light chain [NfL]), and inflammatory biomarkers (i.e., interleukin [IL]- IL-1ß, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-alpha [TNF-a], vascular endothelial growth factor [VEGF]) in BDEV and also microglia derived exosomal vesicles (MDEVs). In our current and past studies, we have observed higher immune activity in neuronal exosomes and specifically in SMs with high career exposures to blasts. To further understand this, we have developed a novel way to isolate and examine microglial exosomes. Microglia are the central regulators of immune function, and overactivity is linked to greater inflammation and is an established link to Alzheimer’s disease. We assert that neuronal and microglial exosomal biomarkers reflect brain activities that contribute to the development of chronic symptoms and deficits following a TBI in SMs and that previous blast exposure will increase this risk.

This project will examine changes in inflammatory and neurodegenerative biomarkers in neuronal and microglial exosomes acutely following a mild TBI, and then again at 1, 3, 6, and 12 months following injury, allowing for identification of biomarkers that are predictive of poor recovery. We will also be able to determine the impact of previous blast exposures. Project 2 will develop a better understanding of how TBIs result in long-term symptoms following a TBI. It also will determine the role of previous blast in biomarker changes. Specifically, we will use brain-derived and microglia exosomes as a way to examine central (brain-derived) processes using peripheral samples of blood. This will possibly advance our ability to understand central key processes and how they relate to risk for non-optimal recovery from TBIs. Understanding these relationships will help determine the nature and extent of the role of neuronal inflammation and markers of degeneration in recovery from TBIs. These findings may lead to new treatments and more personalized approaches to reduce these risks.

PROJECT 3: Neuroinflammation in Veterans with Clinically Diagnosed, Chronic TBI

The immune system plays an important role in maintaining the health of the brain. It is responsible for protecting the brain from infection as well as helping the brain to respond to an injury by clearing out debris that would interfere with the continued normal functioning of the brain. However, the immune system may become overactivated or it may remain active for long periods in certain settings. This high or prolonged activation of the immune system can cause harm to the brain. This injury to brain tissue can result in symptoms of disease and can lead to profound loss of function in individuals experiencing these conditions. Service Members within the military are exposed to many influences in their environment, including highly compressed blast pressure waves from explosions. These pressure waves may be small and numerous, as in the case of using weapons or small explosives during training and operations. The pressure waves may also be very large and can be experienced alongside other injuries, as is the case in the setting of exposure to enemy munitions and/or roadside bombs. Information gained from recent research studies suggests that blast exposure may cause the long-term activation of the immune system within Service Members. This activation of the immune system is also seen alongside changes in the structure of the brain and function of the brain, as shown by the use of brain imaging. Little is known about the long-term effects of exposure to explosives in Service Members over a career. Additionally, little is known about how a history of blast exposure might worsen a traumatic brain injury (TBI) that is experienced alongside or separate from blast exposure events. Given the key role of the immune system in a number of brain diseases such as Alzheimer’s disease or Parkinson’s disease, it is important to better understand the role of the brain’s immune system in Service Members with blast exposure. Our study will collect information from Veterans who have been previously deployed, have a history of blast exposure, and have been diagnosed with chronic TBI. We will collect information from the blood to help us determine if activation of the brain immune system is present. We will also use brain imaging methods to help us look at the structure and function of the brain, and we will ask questions concerning any history of prior blast exposure along with other questions that will help us better understand medical history and brain performance in individuals participating in this study. This information will be considered together to help us understand the role of the brain immune system in Veterans who have been deployed, exposed to blast, and have clinically diagnosed chronic TBI. We will compare this information to another group without a history of prior blast exposure and chronic TBI. This information will be made available to decision-makers in operational communities, clinicians providing care for these individuals, and to researchers designing strategies to prevent or treat TBI in Service Members with a history of prior blast exposure. This project, along with the other projects in this overall proposal, will help to provide a comprehensive view of the brain immune system in Service Members who have been exposed to blast.

PROJECT 4: Understanding the Complex Interplay Between Neuroinflammation and Cerebrovascular Changes with Repetitive Exposure to Blast

The effects of repeated exposures to low-level explosive blast shock waves, which military personnel experience during training exercises and during their daily operational duties, may be injurious to the brain and pose a health concern for active-duty military Service Members and Veterans. Observations in Service Members as well as data from laboratory experiments in animal models of blast injury show that damage to blood vessels in the brain, which supply brain tissue with oxygen and nutrients and remove wastes, is a central mechanism of blast-related brain injury. Research conducted in our laboratory shows that changes in the function and components of blood vessels following exposure to blast overpressure are delayed, appearing weeks or months after blast exposure, and occur before damage to nerve cells in the brain (referred to as neurodegeneration) and behavioral changes, including changes in memory or the speed of performing certain mental tasks. The changes in blood vessels often occur at the same time as inflammation in the brain. Inflammation is known to be a central issue in several brain diseases, including brain injury caused by impact (for example from falls), Alzheimer’s disease, and stroke. How changes in blood vessels in the brain and inflammation after repeated exposure to blast relate to the development of brain diseases, such as Alzheimer’s disease, later in life is not known in spite of the serious impact these changes could have on the well-being and quality of lives of Service Members who sustain repeated blast-exposures over their career. The objective of our proposal is to assess in preclinical, animal studies the decrements in the function of blood vessels following exposure to repetitive blast and elucidate the mechanism underlying one of the main inflammatory responses, the response of the tumor necrosis factor alpha (TNF-alpha). Our studies will also assess the correlation of the changes in blood vessels and inflammation with changes in neurodegeneration that may increase the odds of developing serious brain diseases in the future. We hypothesize that inflammation will correlate with alterations in blood vessel function in the brain and changes in markers of neurodegeneration. We will block the production of TNF-alpha using a specific drug that targets one of the main proteins in the brain that produce TNF-alpha. Overall, our mission is to provide knowledge of the long-term effects of exposure to repeated blast on the health of blood vessels and neurons in the brain and assess how TNF-alpha inflammation affects this process. This project will address Focused Program Award (FPA) Focus Area #1 “Understand,” to develop an understanding of the biological factors contributing to an individual’s response following a brain injury and FPA Focus Area #2 “Prevent,” which includes identification and validation of biomarkers or other objective markers for diagnosis of brain injuries, repetitive exposures, and associated sequelae and potential physiological targets for intervention. The outcome of our studies will inform future studies, which may use TNF-alpha as a target for treatments or preventative measures against brain diseases that may result from exposure to repeated low-level explosive blasts. This knowledge will be critical for preventing potentially debilitating brain diseases and improving the quality life of Service Members.