A staggering number of injury related disabilities and deaths are connected to traumatic brain injury (TBI) worldwide. Traumatic brain injury (TBI) involves an intricate and multifaceted cascade of events, starting with an initial mechanical impact followed by secondary injury brought on by numerous physiological changes that involve significant dysfunction at the cellular and molecular level. One major predictor of severe TBI outcome is the extent of blood-brain barrier (BBB) disruption, which under normal conditions prevents the passage of bacteria, neurotoxins, and macromolecules from entering the brain. Disruption of the BBB is linked to worse clinical outcomes in patients in both the acute, subacute, and chronic phases. However, the principal mechanisms responsible for regulating BBB permeability, where, and for how long that permeability occurs following TBI remains to be elucidated. Previous research has shown increased mRNA and protein expression of ephrin receptor A4 (EphA4), a well-established axon guidance molecule, within hours and days following TBI. This study is the first comprehensive investigation of the role of endothelial cell-specific EphA4 in TBI on regulating the BBB using advanced techniques like single-cell RNA and spatial transcriptomic sequencing, in addition to our newly established dual dye-labeling system. The central hypothesis is that endothelial cell-specific deletion of EphA4 enhances BBB integrity, characterized by changes in single cell gene expression consistent with improved barrier function, altered cellular metabolism, and reduced neuroinflammation within the BBB niche. This hypothesis will be tested by leveraging spatial sequencing to identify upregulation of genes associated with BBB stability and neuroprotection and utilizing a novel approach for assessing BBB permeability that addresses the limitations of traditional Evans Blue Dye (EBD) assays, including lack of spatial resolution, enabling precise analysis of molecular weight-dependent extravasation patterns. / Doctor of Philosophy / Traumatic brain injury (TBI) is a major global cause of disability and death, involving a complex series of events that lead to both immediate and ongoing brain damage. One key aspect of brain injury is the disruption of the blood-brain barrier (BBB), a protective shield that controls the flow of blood into the brain and prevents harmful substances from entering. When the BBB is compromised, it can worsen the damage caused by TBI. Despite its importance, how and why the BBB breaks down after TBI remains poorly understood. This study focused on a specific protein, EphA4, which is found in the cells that make up the blood vessels in the brain. We wanted to see if deleting EphA4 from these cells would help maintain BBB integrity and reduce brain inflammation after injury. To do this, we used advanced techniques, including single-cell RNA sequencing (which looks at gene expression in individual cells) and new methods to measure the extent of BBB disruption. The study also aimed to improve traditional methods of assessing BBB breakdown, which previously lacked the ability to measure the BBB's spatial and temporal changes in detail. We found that deleting EphA4 from the blood vessels in mice helped maintain the BBB and reduce inflammation in the brain after injury. We also developed a new method to measure BBB disruption more accurately, showing that smaller molecules leaked into the brain tissue more than larger ones. Overall, this research suggests that EphA4 plays an important role in regulating the BBB after TBI and that targeting it could offer new ways to protect the brain from injury in the future.

Strokes are a leading cause of death and disability in the United States, predominantly caused by ischemic events. Ischemic strokes occur when a clot or other obstruction lodges in a blood vessel of the brain, restricting the movement of blood. Subsequent rapid cell death occurs and often leads to long term neurological deficits. Pial collaterals are a well-established determinant of patient outcome due to their unique ability to remodel into conductance arteries that can reroute blood back to the ischemic tissue. During development, pial collaterals arise within the pia mater and establish connections between distant arterioles of cerebral arteries. Under healthy conditions, these vessels are exposed to bidirectional blood flow, keeping them small and dormant. Following vascular obstruction, pial collaterals are exposed to unidirectional blood flow, triggering them to expand through an adaptive process termed, arteriogenesis, allowing for retrograde perfusion into the obstructed artery and its affected tissue. However, hyperacute arteriogenesis following ischemic stroke has been poorly investigated. The following dissertation aims to address this research gap and leverage the findings to develop therapeutics that enhance arteriogenesis. Previous research has revealed EphA4 restricts arteriogenesis through the Tie2 signaling axis, therefore this work sought to evaluate the endothelial cell (EC) specific role of the EphA4/Tie2 axis in acute arteriogenesis. EC-specific EphA4 KO mice displayed increased pial collateral size from 4.5 to 24-hours post-injury, which was associated with reduced tissue damage, improved cerebral blood flow, and enhanced motor function. Additionally, pharmaceutical stimulation of the Tie2 axis using Vasculotide, an angiopoietin-1 memetic peptide, replicates these findings. Administration of 3ug/kg Vasculotide to wildtype mice immediately after permanent middle cerebral artery occlusion leads to significantly larger pial collateral diameters, correlating with reduced tissue damage and improved functional recovery. Unlike Vasculotide, device stimulation using low intensity focused ultrasound failed to increase collateral diameter, despite resulting in profound neuroprotection. Taken together, this dissertation work demonstrates that the EphA4/Tie2 signaling pathway can be pharmacologically targeted to improve arteriogenesis following ischemic stroke. / Ph.D. / Worldwide, strokes are a leading cause of death and long-term disability with many cases being ischemic strokes, where a blood clot blocks blood flow to the brain. Without the critical oxygen and nutrients that the blood provides, cells in the affected region of the brain begin to rapidly die, leading to neurological deficits. While current treatments focus on removing the clot, it does not guarantee the restoration of blood flow to the damaged area. In contrast, our research focuses on pre-existing blood vessels in the brain, called pial collaterals, that can ease the loss of blood flow after stroke. These vessels, although relatively inactive under normal conditions, can enlarge after a stroke to reroute blood flow to the injured tissue. Thus, pial collateral growth is a critical process in the initial hours after stroke when this blood flow can prevent brain cells from dying. Previous work has shown EphA4, a receptor known for its role in nervous system development, restricts pial collateral size by inhibiting the Tie2 signaling pathway. Loss of EphA4 in endothelial cells allows for Tie2 receptor activation, increased pial collateral size, and decreased tissue damage. To explore therapeutic enhancement of pial collaterals, we administered Vasculotide, a drug that activates the Tie2 receptor, to wildtype mice expressing EphA4 after a stroke. The mice treated with Vasculotide displayed significantly larger pial collateral vessels one day after the stroke, compared to control mice. Moreover, Vasculotide-treated mice exhibited reduced tissue damage and performed better on behavioral assessments. In addition to pharmaceutical stimulation with Vasculotide, we also investigated the effects of low-intensity focused ultrasound (LIFU) on collateral size. LIFU treatment resulted in decreased tissue damage compared to untreated controls; however, it did not impact collateral size. These findings suggest that inhibiting EphA4 or stimulating Tie2 could serve as novel therapeutic targets to promote the expansion of pial collateral blood vessels, thereby restoring critical blood flow to injured areas of the brain.

Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. Differences in the magnitude and characteristics of the peripheral-derived immune cell response to TBI are key contributors to the secondary cascades of damage following brain trauma, and means of modifying this response to improve clinical outcome are a current area of active research. Our work elucidated the peripheral immune response to TBI by characterizing the transcriptomic profile of juvenile vs adult peripheral immune cells following TBI as well as discovering a novel role for the tyrosine kinase receptor EphA4 in the peripheral-derived immune response to brain trauma. Previous work has demonstrated significant differences in recovery from TBI in young vs adult animals, and some studies have indicated that the immune response contributes to these differences. We utilized next-generation sequencing to compare gene expression profiles of blood cell fraction samples in juvenile and adult mice. Our work demonstrated that juvenile peripheral immune cells show a more dynamic response to TBI than adult and that pattern recognition receptor signaling is a cornerstone of these differences. To assess the specific mechanisms involved in the peripheral response to TBI, we utilized a bone marrow chimeric mouse model lacking EphA4 in the hematopoietic compartment. These studies found decreased lesion infiltration of peripheral immune cells, specifically activated macrophages, in the absence of EphA4. We also showed that EphA4 interacts with the Tie2/Angiopoietin signaling axis to regulate macrophage phenotype on the M1/2 continuum. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the peripheral-derived immune cell response to TBI. / Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. While the blood-brain barrier (BBB) excludes immune cells in the blood from entering the healthy brain, brain trauma compromises BBB integrity and allows massive infiltration of peripheral neutrophils, macrophages, and other immune cells. This circulating immune cell response to TBI contributes to damage following brain trauma, and means of modifying this response to improve recovery are a current area of active research. Our work explored the circulating immune cell response to TBI by comparing the gene expression profile of young vs adult circulating immune cells following TBI as well as discovering a novel role for the EphA4 protein in the circulating immune cell response to brain trauma. Previous work has found significant differences in recovery from TBI in young vs adult animals and that the immune response contributes to these differences. To explore this, we compared gene expression profiles of blood immune cells in young and adult mice and found that young immune cells show a more dynamic response to TBI than adult. To assess the specific pathways involved in the circulating immune cell response, we used a mouse model lacking EphA4 in these cells. Our studies found decreased numbers of immune cells, specifically macrophages, entering the injury area in the absence of EphA4. We also showed that EphA4 interacts with the Tie2 protein and its Angiopoietin protein binding partners. Originally studied as an important contributor to blood vessel function, Tie2 has recently been found to play a role in the function of macrophages. Our work demonstrated that EphA4 interacts with Tie2 to regulate pro-recovery vs proinflammatory characteristics in macrophages. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the circulating immune cell response to TBI.

Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke. / Doctor of Philosophy / Stroke is the fifth leading cause of death in the United States. Ischemic stroke is the most common type of stroke and occurs when blood flow to part of the brain is impeded. Lack of blood results in cell death and tissue damage in the brain. In an effort to restore blood flow, specialized blood vessels in the brain called collaterals remodel and become larger to allow re-routed blood to the blood-deprived region of the brain. The duration it takes to remodel these remarkable blood vessels and re-route blood varies in humans, and sometimes is not able to prevent adequate tissue damage. The current work explores novel therapeutic targets to accelerate collateral remodeling in an effort to reduce tissue loss after stroke. We present studies which show that a protein called EphA4, found on endothelial cells restricts remodeling, and when inhibited in the brain can increase collateral remodeling and reduced adverse effects after ischemic stroke.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Traumatic Brain Injury (TBI) mainly affects young persons in traffic accidents and the elderly in fall accidents. Improvements in the clinical management have significantly improved the outcome following TBI but survivors still suffer from depression, memory problems, personality changes, epilepsy and fatigue. The initial injury starts a series of events that give rise to a secondary injury process and despite several clinical trials there is no drug available for clinical use that targets secondary brain injury mechanisms. Some recovery of function is seen during the first months following injury but is usually limited and there are no drugs that stimulate the recovery of lost function. Some of the recovery is attributed to plasticity, the brains ability to adapt to new circumstances, and enhancing plasticity via increased axonal growth has the potential to partly restore lost function. In this thesis mice were subjected to the controlled cortical impact model of TBI and functional outcome was evaluated using Morris water maze, the cylinder test and the rotarod. Brain tissue loss was measured in all Papers but the additional histological analyses differ among the Papers. Attempts to increase axonal growth were made by interfering with Nogo receptor function in Paper I and by conditional knockout of ephA4 in Paper II. Contrary to the hypothesis cognition was impaired in Paper I but otherwise no effects of treatment were detected in Paper I and II. Much is still unknown about plasticity and despite the discouraging results of Papers I and II this treatment approach is still worth further exploration. It is firmly established that TBI results in an inflammatory response and some aspects of it may damage brain tissue. In Papers III and IV the inflammatory response was attenuated using an IL-1β directed antibody which resulted in reduced tissue loss and edema while improving cognitive function. The results from Papers III and IV are encouraging and the possibility to find a treatment based on IL-1β inhibition appears promising.

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Stroke, a life-threatening condition, primarily resulting from ischemic events often caused by occlusion of the middle cerebral artery (MCA). Pre-existing leptomeningeal collateral (LMC) vessels connect MCA branches to anterior or posterior arteries, situated along the brain's cortical surface or meninges, under healthy conditions these vessels remain dormant due to their small diameters and relatively low flow velocity. LMCs serve as vascular redundancies that retrogradely re-supply blood to help salvage the penumbra following cerebral vascular occlusion. Their outward growth or remodeling (arteriogenesis) is essential for promoting cerebral reperfusion and preventing tissue damage after ischemic stroke. Increased fluid shear stress on collateral vessel wall activates arteriogenesis result in the activation of the endothelium and subsequent recruitment of peripheral-derived immune cells (PDICs), which have been shown to aid this unique adaptive process in other organ systems, however their role and mechanism(s) involved in LMC remodeling in stroke has not previously been evaluated. Initial findings suggest the EphA4, a well-established axonal growth and guidance receptors, plays a novel role in LMC arteriogenesis. This dissertation examined PDIC-specific functions of EphA4 using GFP labeled bone marrow chimeric mice subjected to permanent middle cerebral artery occlusion (pMCAO). We assessed immune cell population changes, infarct volume, functional recovery, characterized subtypes of infiltrated immune cell, and measured collateral vessel diameters. Additionally, we explored the Tie2-mediated PI3K signaling pathway in peripheral-derived monocyte/macrophages (PDM) treated with soluble Tie2-Fc and a PI3K p110α inhibitor. The results from this dissertation show that loss of PDIC-specific EphA4 led to increased collateral remodeling, associated with decreased infarct volume, improved cerebral blood flow, and functional recovery within 24 hours post-pMCAO. The crosstalk between EphA4-Tie2 signaling in PDMs, regulated through PI3K/Akt axis, inhibited pial collateral remodeling. In conclusion, our findings highlight the negative regulatory role of PDM-specific EphA4 in collateral growth and remodeling by inhibiting Tie2 function via the PI3K regulated pathway. Peripheral myeloid-derived EphA4 emerges as a new regulator of cerebral vascular injury and neuroinflammation following acute ischemic stroke. / Doctor of Philosophy / Stroke, a life-threatening condition, occurs when blood flow to part of the brain is disrupted due to the vascular occlusion of a major brain artery, such as the MCA. Within protective layers of our brain, there are pre-existing pial collateral vessels that act as backup connections. These vessels play an important role in increasing cerebral reperfusion and preventing tissue damage after stroke. One fascinating aspect of stroke recovery involves PDICs. These immune cells migrate into the blood hypo-perfused region of the brain and regulate the growth of collateral vessels. However, the specific functions of PDICs, particularly a receptor called EphA4, has remained unclear. Our research delved into the immune response following ischemic stroke using genetically modified mice. We examined immune cell populations, infarct volume (the damaged brain tissue), functional recovery, and collateral vessel diameters. Notably, we discovered that deletion of PDIC-specific EphA4 enhanced collateral vessel remodeling. This led to decreased infarct volume, better blood flow, and improved functional recovery within 24 hours after stroke. Furthermore, we explored a signaling pathway involving Tie2 and PI3K in PDM. This crosstalk between EphA4 and Tie2, mediated through PI3K regulation, played a critical role in suppressing collateral vessel remodeling. In summary, understanding how immune cells contribute to stroke recovery may pave the way for novel therapeutic approaches to enhance outcomes for stroke patients.