Mild traumatic brain injury (mTBI) accounts for approximately 73-83% of all traumatic brain injuries (TBI) and continues to be a serious clinical challenge [1]. The role of injury mechanism in TBI has been widely debated, and it is believed that although there are differences between diffuse and focal TBI, the resulting injury is not influenced by the way in which it was acquired [1], [2]. It is known that TBIs can cause cognitive impairments that are often due to injury experienced in the hippocampus [2]. In response to insult, quiescent neural stem cell (NSC) populations within the dentate gyrus region of the hippocampus become activated. Stem cell differentiation following injury is hypothesized to be unique for diffuse and impact TBIs, primarily due to the differences in mechanotransduction pathways triggered by each respective injury. By quantifying the lineage of stem cells through immunohistochemistry, this study examined the dentate gyrus following mTBI in a rodent model, and the contribution that injury mechanism plays in mTBI outcomes. Additionally, the behavioral effects of mTBI were assessed through open field testing at 72 hours and four weeks following injury. Overall, these findings indicated that after four weeks following mTBI, there are not significant differences between impact and blast both from an immunohistochemical and behavioral standpoint. Despite there being few differences between injury groups, these findings help clarify the role of injury mechanism not only in the context of neurogenesis, but they also inform future studies addressing preventative and treatment strategies for mTBI. / Master of Science / Mild traumatic brain injury (mTBI) accounts for approximately 73-83% of all traumatic brain injuries (TBI) [1]. There are two main ways in which a mTBI can occur: through diffuse or focal injury. A diffuse injury is due to the brain experiencing a force that does not physically come into contact with the head, such as a shockwave from an explosion. These types of injuries typically affect the entire head. Impact injuries on the other hand, are caused by the head encountering an object at a force that causes injury to the brain. These injuries tend to be focal, as the entire head rarely comes into contact with an object. Both diffuse and focal injuries can cause mTBI, and there is a current debate questioning if the mode of injury has an impact on the damage experienced by the brain [1], [2]. However, it is also known that mTBI can cause cognitive impairments such as changes in behavior, memory, and even mental health, which can occur in the hippocampus of the brain [2]. Within the hippocampus, there is a small subset of cells referred to as neural stem cells (NSC) that become active following injury. The activation of these cells is believed to be in response to injury in the brain. Furthermore, NSCs have the ability to differentiate into various cell types within the brain, including astrocytes, oligodendrocytes, and neurons. Each of these cell types perform an integral role in the function of the brain. It is hypothesized that the response of NSCs in the hippocampus is unique depending on if an injury was acquired through diffuse or impact mechanisms. To investigate this, the lineage of NSCs was quantified within the hippocampus following blast and impact mTBI in a rodent model. Additionally, the behavioral effects of diffuse and impact injury were investigated at 72 hours and four weeks following injury. Despite there being no significant differences in outcomes between injury groups, these findings help clarify the role of injury mechanism not only in the context of NSC response, but they also inform future studies addressing preventative and treatment strategies for mTBI.

Blast-induced traumatic brain injury (bTBI) remains a significant problem among military populations. When an explosion occurs, a high magnitude positive pressure rapidly propagates away from the detonation source. Upon contact, biological tissues throughout the body undergo deformation at high strain rates and then return to equilibrium following a brief negative pressure phase. This mechanical disruption of the tissue is known to cause oxidative stress and neuroinflammation in the brain, which can lead to neurodegeneration and consequently poor cognitive and behavioral outcomes. Further, these clinical outcomes, which can include chronic headaches, problems with balance, light and noise sensitivity, anxiety, and depression, may be sustained years following blast exposure and there are currently no effective treatments. Thus, there is a need to investigate the acute molecular responses following bTBI in order to motivate the development of effective therapeutic strategies and ultimately improve or prevent long-term patient outcomes. It is important to not only understand the acute molecular response, but how the brain tissue mechanics drive these metabolic changes. The objective of this work was to identify the interplay between the tissue-level biomechanics and the acute bTBI pathophysiology. In a rodent bTBI model, using adult rats, intracranial pressure was mapped throughout the brain during blast exposure where frequency contributions from skull flexure and wave dynamics were significantly altered between brain regions and were largely dependent on blast magnitude. These findings informed the subsequent spatial and temporal changes in neurometabolism. Amino acid molecular precursor concentrations decreased at four hours post-blast in the cortex and hippocampus regions. This motivates further investigation of amino acids as therapeutic targets aimed to reduce oxidative stress and prevent prolonged injury cascades. However, neurochemical changes were not consistent across blast magnitudes, which may be explained by the disparities in biomechanics at lower blast pressures. Lastly, we investigated the acute changes in metabolic regulators influencing excitotoxicity where it was found that astrocytes maintained normal clearance of excitatory and inhibitory neurotransmitters prior to astrocyte reactivity. Outcomes of this work provide improved understanding of blast mechanics and associated acute pathophysiology and inform future therapeutic and diagnostic approaches following bTBI. / Doctor of Philosophy / Blast-induced traumatic brain injury (bTBI) remains a significant problem among military populations. When an explosion occurs, a high magnitude positive pressure wave rapidly propagates away from the detonation source. Upon contact, biological tissues throughout the body undergo deformation that can cause injury. This mechanical disruption of the tissue is known to trigger negative biological processes that lead to persistent cognitive and behavioral deficits. Further, these clinical outcomes, which can include chronic headaches, problems with balance, light and noise sensitivity, anxiety, and depression, may be sustained years following blast exposure. There are currently no effective treatments that can help those afflicted, and biomarkers for injury diagnostics are limited. Thus, there is a great need to investigate the early biological responses following bTBI in order to motivate the development of effective therapeutic strategies and ultimately improve or prevent long-term patient outcomes. It is important to not only understand the immediate responses, but also how the brain tissue mechanics drive these metabolic changes. The objective of this work was to identify the interplay between the brain biomechanics and the acute bTBI pathophysiology. Using a translational animal model, pressure inside the brain was measured with pressure sensors during blast exposure. Subsequent spatial and temporal changes in neurochemical concentrations were quantified. The results showed (1) significant disparities in the pressure dynamics inside the brain and it varied across brain regions, (2) neurochemical precursors may have therapeutic potential post-injury, and (3) biomechanical and neurochemical responses were dependent on blast severity. Outcomes of this work provide improved understanding of blast mechanics and associated pathophysiology and inform future therapeutic and diagnostic approaches to prevent prolonged injury cascades.

Across almost all types of neurological pathophysiology, inflammation and corresponding breakdown of the Blood Brain Barrier (BBB) are hallmarks of injury/disease progression. In fact, BBB disruption can occur early during neuropathophysiological development, in many cases even before neurological and cognitive impairments become apparent. Whether as an early causative factor, a side effect, or both as it pertains to neurological injury/disease, BBB breakdown and dysfunction represents a novel and under investigated target for therapeutic development, especially for neurological pathologies with unmet therapeutic needs. Toward this goal, the endocannabinoid system (ECS) has emerged as a promising biological target for drug discovery efforts. Particularly, the Cannabinoid 2 Receptor (CB2R) has been proposed as a druggable target due to its anti-inflammatory effects and since it is not associated with the neurological side effect profile representative of Cannabinoid 1 Receptor (CB1R) drugs. Interestingly, neuroinflammatory conditions promote upregulation of CB2R on brain microvascular endothelial cells (BMVECs) suggesting a possible role toward resolution of inflammation in this cell type. Moreover, previous research has shown promising effects of CB2R agonists on cerebrovascular function, although these effects cannot be directly attributed to endothelial CB2R. The central hypothesis of this research is that endothelial CB2R activation confers effects which are vascular protective and that promote BBB repair, (irrespective of the effects of CB2R in other central nervous system (CNS) cell types). To address this hypothesis, endothelial CB2R expression dynamics were assessed following experimental Traumatic Brain Injury (TBI) followed by a series of assays to assess the therapeutic potential of a novel chromenopyrazole based CB2R agonist, PM289. Results of these experiments demonstrated upregulation of CNR2, the gene which encodes CB2R, following in vivo experimental TBI and in vitro cytokine induced inflammation. Moreover, PM289 exhibited robust CB2R-dependent therapeutic potential by partially restoring TNFa-induced physical barrier disruption, attenuating TNFa-induced ICAM1 upregulation, and promoting rapid monolayer repair following electrolytic wound. Mechanistically, these effects may be explained via CB2R-dependent inhibition of NFkB/P65 signaling. Overall, these results are supportive of the notion that CB2R in BMVECs could aid in vascular protection and promote BBB function in the context of neuroinflammation. Future studies are warranted to understand the in vivo therapeutic efficacy of PM289 in a variety of injury/disease models. Additionally, alternative cell signaling mechanisms should be considered including a comprehensive examination of potential interplay between ECS components and candidates that fall under the umbrella of the endocannabionoidome (ECBome). / Biomedical Sciences

Traumatic brain injury (TBI) continues to be a widespread problem in the United States with approximately 1.7 million occurrences annually [1]. Current automotive crash test standards use the Head Injury Criterion (HIC) [2] to assess head injury potential, but this metric does not relate an impact to underlying damage. For an injury metric to effectively predict TBI, it is crucial to relate level of impact to resulting injury. The research presented in this dissertation explains the development and repeatability of two novel injury devices, impact response characterization over the course of 24 hours in the Gottingen minipig and the relationships between metabolite changes, underlying disruption, and impact kinematics, and the characterization of impact response over the course of 72 hours. The translation-input and combined translation and rotation-input injury devices were shown to be repeatable, minimizing the number of animals needed for testing. Impact response over the course of 24 hours showed axonal disruption through immunostaining and proton magnetic resonance spectroscopy. The translation-input injury group metabolite analyses revealed the initial stages of glutamate excitotoxicity while the combined-input injury group showed a clear pathway for glutamate excitotoxicity. Numerous correlative relationships and potential underlying disruption predictors were found between metabolites, immunostaining, and kinematics. The most promising predictor combination for the translation-input injury device was N-acetylaspartylglutamate/Scyllo at 24 hours compared to 1 hour and linear speed for predicting underlying light neurofilament disruption. For the combined-input injury device, the strongest predictor combination was Glutamine/N-acetylaspartylglutamate at 24 hours compared to baseline and angular acceleration for predicting underlying light neurofilament disruption. Statistically significant predictors were found between Glutamate+Glutamine/Total Creatine at 24 hours compared to baseline and all kinematics and injury metrics with an angular component for predicting heavy neurofilament disruption. Analyses over the course of 72 hours revealed persistent axonal disruption and metabolite perturbations. Overall, this dissertation and the complementary parts of this project have many societal implications. Due to the high incidence of traumatic brain injury, there is a need for prevention, mitigation, and treatment strategies. Developing a new injury metric will help improve prevention strategies, especially in the automotive, sporting, and military environments. 1 Faul, M., Xu, L., Wald, M. M., and Coronado, V. G. (2010). Traumatic Brain Injury in the United States. Atlanta, GA: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. 2 Versace, J. (1971). A Review of the Severity Index. SAE Technical Paper. No. 710881 / Ph. D.

Traumatic brain injury (TBI) elicits an immediate neuroinflammatory response that involves resident glia and infiltrating peripheral immune cells that coordinate tissue damage and functional deficits. The activation of resident microglial has been associated with a change in their morphology from a branched-like ramified cell to an ameboid state. This activation is thought to initiate a pro-inflammatory response leading to the release of neurotoxic, immune chemoattractant, and antigen-presenting signals. Subsequently, peripheral-derived immune cells (PICs), such as neutrophils and monocytes, travel to the site of injury and help coordinate this response. However, little is known regarding whether PICs influence the progressive activation state of microglia in the acute and chronic phases of injury. Overactivation of microglia can lead to neuroinflammation-mediated tissue damage and death or dysfunction of healthy neurons. Therefore, understanding how microenvironmental cues may regulate the microglial response may aid in strategies to retool their activation state in the brain. EphA4 receptor tyrosine kinase has been identified as a potential cell-to-cell contact protein on PICs that could be involved in the inflammatory changes following TBI. While microglial activation changes have been described in TBI models, the mechanistic role of infiltrating peripheral-derived immune cell (PIC) recruitment on microglial fate and function is not well understood. The purpose of my project is to gain a better understating of the temporospatial influence that EphA4-expressing PICs, specifically monocyte/macrophages, have on microglial proliferation, survival, activation phenotype, and debris clean-up using bone marrow GFP chimeric mice and the cortical contusion injury TBI model. / Doctor of Philosophy / Traumatic brain injury (TBI) triggers an immediate response from the brain's immune system, involving both local glial cells and immune cells from outside the brain. These cells work together to mediate the initial injury but, in some cases, cause development of a secondary injury. Microglia, the brain's resident immune cell, change their shape and behavior when activated by a TBI, becoming more aggressive and releasing inflammatory proteins. At the same time, immune cells from the bloodstream, like neutrophils and monocytes, rush to the injury site to assist. Yet, it's unclear how these immune cells affect microglia over time during the injury's acute and chronic phases. If microglia become too active, they can cause further damage to brain tissue and harm healthy neurons. Therefore, understanding the signals that control microglial activity could help us develop therapies to manage brain inflammation. One protein of interest in this process is the EphA4 receptor found on immune cells, which might play a crucial role in inflammation following TBI. While we know that microglia change post-TBI, we don't fully understand how the recruitment of immune cells from outside the brain affects them. My research aims to clarify how EphA4-expressing immune cells, especially monocytes/macrophages, influence microglia in terms of growth, behavior, and their ability to mediate a TBI.

While there has been ample research examining the relationship between an acute concussion on immediate neuropsychological performance, very little research has examined the relationship between lifetime concussion history with current neuropsychological performance. We collected preseason neuropsychological test performance (ImPACT) and a detailed lifetime concussion history questionnaire from 71 UCF football players. Stepwise linear regressions were conducted for each of the five ImPACT domain scores for the 18 participants that reported at least one lifetime concussion. The regressions used the following four concussion history predictors: total number of lifetime concussions, length of time between last concussion and lmPACT testing, severity of worst concussion, and severity of most recent concussion. Results revealed that only one ImpACT domain score had at least one predictor enter the model. For the domain of visual memory, the predictor of length of time between last concussion and ImPACT testing entered the model (and only that predictor),P = 4.07, t(l7) = 2.78,p = .01, R1 = .33, as a shorter length of time between the last concussion and the preseason testing related to lower performance on the visual memory tests. Many athletes and clinicians assume that the cognitive effects of a concussion are relatively brief in duration. However, the results of this study suggest that, at least for visual memory, these effects may last for several years following a concussion. The correlational design of this study precludes drawing conclusions about the causal direction of this relationship, but future longitudinal research may be able to clarify this important preliminary finding.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among young children and adults. This primary injury initiates an inflammatory response that may lead to a secondary brain injury. Nursing care in the critical care setting supports prevention or reduction of secondary injury through control of intracranial pressure (ICP), mean arterial pressure (MAP), and the subsequent cerebral perfusion pressure (CPP). While secondary injury may be preventable, some nursing interventions may contribute to increased ICP and decreased CPP. Patients with increased ICP or decreased CPP are at risk for poor clinical outcomes. This literature review examined the effort of routine nursing care interventions on outcomes of TBI patients in the critical care setting. Eleven research articles studying head of bed elevation, head and neck positioning, turning, and spacing of patient care activities were the focus of the analysis. Results typically showed positive outcomes by elevating the head of the bed to thirty degrees. CPP was also maintained at thirty degrees, but showed varied results. ICP and CPP are best controlled with the head and neck in a neutral position. Turning patients is a routine nursing intervention that contributes to increased ICP in some positions in some patients. Most studies suggest ICP is lowest in the supine position and highest in the left lateral position, but differences in findings were noted. Providing basic nursing care interventions in close succession also may contribute to increases in ICP in some patients. Results from this review provide evidence to support the importance of assessing and planning care for each TBI patient individually. It is hoped that findings from this review will provide guidance for bedside nurses to improve clinical practice and drive future research to support best practices for care of patients who suffer TBI.

An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future. / Ph.D. / Traumatic brain injuries (TBIs) impact millions of individuals each year in the United States, making it a significant cause of death and disability. Furthermore, TBI has been linked to other comorbidities such as Alzheimers Disease, mood disorders, and epilepsy. Since the primary impact of a TBI cannot be predicted or prevented, research focuses on the secondary injury response as a therapeutic target to improve the outcomes following brain insult. Blood brain barrier (BBB) disruption is a well described consequence of TBI and has been correlated to a worse prognosis. The BBB normally provides a barrier between the circulating blood and the brain as protection and to maintain homeostasis. It is understood that decreased BBB integrity leads to subsequent edema, inflammatory response, and glial excitotoxicity, however, the mechanisms regulating this response remain to be investigated. Recent focus has been on a family of receptor tyrosine kinases, Eph receptors, that are unregulated following brain injury. Utilizing a mouse model, we can manipulate the temporal and spatial expression of Eph receptors to understand their role in the secondary injury cascade. Findings indicated that ablation of Eph receptors specifically on endothelial cells (ECs) resulted in preservation of BBB integrity at 1-, 4-, and 7- days following injury. Based on these results, we hypothesize that Eph receptor signaling on ECs negatively mediates BBB function and recovery following TBI. To test this hypothesis, we performed a comparative analysis between wild type (WT) and knockout (KO) mice on the expression of genes integral to BBB integrity, functional motor deficits, and loss of tissue in the lesion site following injury. We discovered significant decreases in lesion volume correlating with improvements in motor function in the KO mice compared to the WT. Moreover, KO mice showed increased expression of genes important for BBB maintenance such as Occludin and Tie2. To further discern the mechanism for these effects, we blocked Tie2 in the KO mice and observed similar negative prognostic indicators as in the WT. Future studies are warranted to understand the downstream signaling of Eph receptors on the Tie2 pathway. This data provides evidence that Eph signaling influences the BBB negatively following TBI through the Tie2 pathway and may be exploited for therapeutic means in the future.

It has been well-established in the literature that a history of concussion makes individuals more susceptible to sustaining subsequent concussions. However, there is little neuropsychological evidence of how sustaining multiple lifetime concussions affects cognitive functioning in the general adult population. It is known from previous traumatic brain injury and single concussion literature that impairments in cognitive performance across domains often follow the initial injury, and exploratory studies have shown that multiple concussions can have a measurable impact on cognition. However, existing research is often limited by its reliance on archival data and abbreviated neuropsychological batteries. Therefore, the current study aimed to fill this gap in the literature to help inform clinical prognoses and treatment considerations. Analyses of attention and memory outcomes revealed significant associations between concussion history and performance, but they were directionally opposite from expectations. When discrepancy scores were used to account for participants’ estimated intellectual functioning, these associations were no longer significant. Executive functioning was also not significantly associated with concussion history, either with or without accounting for intelligence, contrary to expectations. On language and spatial measures, outcomes were unrelated to concussion history, as expected. Together, results from the present study emphasized the multifaceted nature of concussions and highlighted the many necessary considerations when investigating long-term outcomes, particularly when multiple concussions are involved. Future research would likely benefit from continuing to explore the neurocognitive impact of sustaining multiple concussions in the general adult population and expanding the current research with larger, more representative samples, neuroimaging, and baseline data, as available.

Traumatic brain injury (TBI) is a growing health concern worldwide that affects a broad range of the population. As TBI is the leading cause of disability and mortality in children, several pre-clinical models have been developed using rodents at a variety of different ages; however, key brain maturation events are overlooked that leave some age groups more or less vulnerable to injury. Thus, there has been a large emphasis on producing relevant animal models to elucidate molecular pathways that could be of therapeutic potential to help limit neuronal injury and improve behavioral outcome. TBI involves a host of different biochemical events, including disruption of the cerebral vasculature and breakdown of the blood brain barrier (BBB) that exacerbate secondary injuries. A better of understanding of the mechanism(s) underlying cerebral vascular regulation will aid in establishing more effective treatment strategies aimed at improving cerebral blood flow restoration and preventing further neuronal loss. Our studies reveal an age-at- injury dependence on the Angiopoetin-Tie2 axis, which mediates neuroprotection in a model of juvenile TBI following cortical controlled impact (CCI) that is not seen in adult mice. The protection observed was mediated, in part, by the microvascular response to CCI injury and prompted further detailed analysis of the larger arteriole network across several mouse strains and models of TBI. Our second study revealed both a model and species dependent effect on a specialized network of arteriole vessels, called collaterals after trauma. We demonstrated that a repetitive mild TBI (rmTBI) can induce collateral remodeling in C57BL/6 but not CD1 mice; however, CCI injury had no effect on collateral changes in either strain. Together, these findings demonstrate an age-dependent and species/model dependent effect on vascular remodeling that highlights the importance of individualized therapeutics to TBI. / Ph. D. / Traumatic brain injury (TBI) is the most frequent cause of death in adults and children in the developed world and children are at the greatest risk of injury. In the United States alone there is a reported incidence of 1.7 million injuries a year and about half of these injuries are to children. Patients that survive TBI experience long-term neurological disabilities as there is not an effective treatment available. While the initial brain trauma cannot be treated, preventing further damage of delayed secondary responses of injury has garnered attention from researchers to better understand how the injury progresses. In order to mimic TBI in the lab, scientist use animal models of TBI to better understand the mechanism(s) involved in injury with the purpose of creating pharmacological targets to mitigate the effects of further tissue damage. While there are many cell types within the brain that are affected after TBI, our studies focus on endothelial cells that line the vascular system and allow for the circulatory function of blood to supply energy to the neurons of the brain. Our mouse model mimics the effects of sustaining a focal brain injury and we are interested in how the juvenile brain responds to this injury. We have found that juvenile mice are better protected after brain injury as they have less tissue damage compared to adult mice and we attribute this protection to better blood vessel numbers and function. While we observed changes to the vascular network in the juvenile model, this prompted studies to focus on other models of TBI to understand how blood vessels respond to a concussive-like injury. In these studies, we found that a particular species of mouse and the less severe injury prompted a special type of blood vessels to increase their diameter that was not seen in the more severe model of TBI. Taken together our findings demonstrate an age-dependent and species/model dependent effect on blood vessel remodeling.