The direct endothelial contact with adjacent astrocytic end-feet is believed to establish blood-brain barrier (BBB) typical characteristics in endothelial cells of the central nervous system (CNS). However, this contact is only present in capillary vessels of the brain parenchyma and absent in larger veins, arteries and vessels within the meninges. To investigate a potential impact of direct endothelial interactions with adjacent astrocytic end-feet on the molecular tight junction (TJ) composition and ultrastructure, we performed a systematic analysis of endothelial cell contacts within the vascular tree of parenchymal and leptomeningeal vessels. Immunofluorescence labeling for claudin-3, claudin-5, zonula occludens-1 and occludin was used to compare the molecular composition, without showing significant differences in their distribution along the vascular tree of parenchymal and leptomeningeal vessels. Furthermore, electron microscopy in combination with quantitative analyses was performed to investigate the endothelial ultrastructure revealing significant differences within the length of endothelial overlaps between the different vessel types. Here, parenchymal arteries exhibit noticeably longer cell contacts compared to capillaries, which could not be observed in leptomeningeal vessels. It was also observed that arterial vessels regularly contain a higher density of endothelial vesicles throughout the parenchyma and meninges as a sign for transendothelial traffic. Hence, endothelial expression of blood-brain barrier typical TJs is not limited to capillary vessels with an intimate contact to surrounding astrocytes, but is also observed in arteries and veins of the brain parenchyma as well as the meninges, the latter of which are lacking a direct astrocyte-endothelial interaction. These vessel-specific characteristics can now be used to address and compare alterations of the BBB in different settings of CNS pathologies.:Table of Content 1. INTRODUCTION 4 1.1 THE BLOOD-BRAIN BARRIER 4 1.2 HISTORY 5 1.3 STRUCTURE AND COMPOSITION 6 1.4 THE ROLE OF THE MICROENVIRONMENT 8 1.4.1 ASTROCYTES 8 1.4.2 PERICYTES 9 1.5 BLOOD BRAIN BARRIER FUNCTION 10 1.5.1 PHYSIOLOGIC CONDITIONS 10 1. 5.2 PATHOLOGIC CONDITIONS 11 2. OPEN QUESTIONS AND SCIENTIFIC APPROACH 12 3. PUBLICATIONS 13 3.1 DIFFERENT SEGMENTS OF THE CEREBRAL VASCULATURE REVEAL SPECIFIC ENDOTHELIAL SPECIFICATIONS, WHILE TIGHT JUNCTION PROTEINS APPEAR EQUALLY DISTRIBUTED 13 3.2 THE BLOOD-BRAIN BARRIER 28 4. SUMMARY 40 5. REFERENCES 43 6. PROOF OF SIGNIFICANT CONTRIBUTION 48 7. DECLARATION OF ACADEMIC HONESTY 49 8. ACKNOWLEDGMENT 50 9. CURRICULUM VITAE 51

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Central nervous system (CNS) tissues are highly metabolically active which makes them particularly susceptible to vascular injury. Disruption to the supply of oxygen and nutrients by damaged vasculature can result in neurodegeneration in both the eye and brain. The retina is an accessible part of the CNS that can be taken advantage of to study neurovascular diseases through live, non-invasive visualization of vascular and neuronal conditions upon injury. Retinal vein occlusion (RVO) is a common neurovascular disease of the eye and is the second leading cause of blindness in working age adults. While pathophysiology is well described and can be determined by retinal edema, breakdown of the blood-retina-barrier (BRB), inflammation, and neurodegeneration, the underlying signaling pathways behind the pathology is not well understood. To understand the mechanism of disease in RVO, the Troy lab has employed a mouse model to investigate pathways. Previous studies in the lab determined that as early as 1 hour post RVO, there was a large induction of caspase-9, a known cell death protease, in endothelial cells. When further investigated, it was confirmed that these cells were not dying despite the high expression of caspase-9, implying a non-apoptotic role. Deletion of endothelial caspase-9 was sufficient to protect against the development of retinal edema, capillary ischemia, and neuronal death, indicating caspase-9 is a key player in the mechanism of disease. This thesis work aims to investigate which signaling events drive non-apoptotic endothelial caspase-9 signaling by investigating upstream and downstream mechanisms of endothelial caspase-9. To interrogate this question, the mouse model of RVO was optimized, limiting the variability previously observed to ensure accurate and reproducible results. Then, we used a tamoxifen inducible endothelial cell Apaf-1 (apoptosis protease activating factor-1) knock out (Apaf-1 iECKO) mouse line in order to investigate the contribution of upstream activation of non-apoptotic endothelial caspase-9 signaling. Apaf-1 iECKO mice and WT littermates were subjected to RVO. Then, expression of caspase-9 and -7, retinal edema, capillary ischemia, neuronal death, vision dysfunction, and BRB integrity were measured. The deletion of endothelial Apaf-1 resulted in reduced expression of cl-caspase-9 and caspase-7, indicating endothelial caspase-9 was activated by Apaf-1. Apaf-1 deletion also resulted in protection against some of the pathologies seen after RVO including retinal edema, capillary ischemia, and neurodegeneration. Lastly, in order to elucidate the signaling pathway further, experiments using endothelial cell-specific AAVs (adeno-associated virus) packaged with a downstream caspase-7 inhibitor were proposed and described. In sum, this thesis work reveals that endothelial caspase-9 is canonically activated by Apaf-1, but still leads to non-apoptotic signaling, indicating downstream caspase-9 substrates could be the source for non-apoptotic function within endothelial cells.

Cerebral aneurysms occur due to the ballooning of blood vessels in the brain. Rupture of aneurysms can cause a subarachnoid hemorrhage, which, if not fatal, can cause permanent neurologic deficits. Minimally invasive neurovascular devices, such as embolization coils and flow diverters, are methods of treatment utilized to prevent aneurysm rupture. The rapidly growing market for neurovascular devices necessitates the development of accurate aneurysm models for preclinical testing. In vivo models, such as the rabbit elastase model, are commonly chosen for preclinical device testing; however, these studies are expensive, and aneurysm geometries are difficult to control and often do not replicate the variety of geometries found in clinical cases. A promising alternative for preclinical testing of neurovascular devices is an aneurysm blood vessel mimic (aBVM), which is an in vitro tissue-engineered model of a human blood vessel composed of an electrospun scaffold with an aneurysm geometry and human vascular cells. Previous work in the Cal Poly Tissue Engineering Lab has established a process for creating different aneurysm scaffolds based on the shape of different geometries, and this work aimed to further advance these aneurysm geometries in order to enhance the versatility of the in vitro model. The overall goal of this thesis was to customize the aBVM model through variations of different dimensions and to validate the scaffold variations for neurovascular device testing. First, a literature review was performed to identify critical ranges of aneurysm neck diameters and heights that are commonly seen in rabbit elastase models and in human clinical settings in order to set a foundation for creating new geometries. Based on the results, aneurysm geometries with varying neck sizes and heights were modeled and molded, and scaffolds were fabricated through electrospinning. Methods were developed to characterize scaffolds with internal measurements through imaging techniques using a scanning electron microscope. To validate these scaffolds for use as aBVMs for neurovascular device testing, constructs were created by dual-sodding human endothelial cells and smooth muscle cells into scaffolds with varying neck sizes. Finally, flow diverters were deployed in constructs with varying neck sizes in order to evaluate feasibility and initial healing. Customized aneurysm scaffolds can eventually be used with a variety of device studies for screening of neurovascular devices or as a predecessor for in vivo preclinical testing.

Astrocytes comprise the most abundant cell population in human brain (1). First described by Virchow as being 'glue' of the brain (2), modern research has truly extended our knowledge and understanding regarding the vast array of roles these cells execute under normal physiological conditions. Examples include neurotransmitter reuptake at the synapse (3), the regulation of blood flow at capillaries to meet neuronal energy demand (4), and maintenance/repair of the blood-brain barrier (BBB) (5), which is comprised, in part, of tight junction proteins such zonula-occludens-1 (ZO1) (6) and Claudin-5 (7). Underlying the execution of these processes is the morphological and spatial arrangement of astrocytes between neurons and endothelial cells comprising blood vessels, where comprehensively speaking, these cells form what is known as the gliovascular unit (8). Astrocytes extend large processes called endfeet that intimately associate with and enwrap up to 99% of the cerebrovascular surface (9). Disruptions to this association can occur in the form of retracted endfeet, and this has been characterized in several disease states such as major depressive disorder (10-12), ischemia (13-15), and normal biological aging (16-18). Disruption can also take the form of cellular/protein aggregate intercalation, which our lab previously characterized in a human-derived glioma model (19) and vascular amyloidosis human Amyloid Precursor Protein J20 (hAPPJ20) animal model (20). In both models, focal astrocyte-vascular disruptions coincided with perturbations to astrocyte control of blood flow, with deficits in BBB integrity present in the glioma model as well. These findings lead to the preliminary work in this dissertation where we aimed to extend BBB findings in the glioma model to the hAPPJ20 vascular amyloidosis model. Immunohistochemical analysis in two-year old hAPPJ20 animal arterioles revealed that indeed in locations of vascular amyloid buildup and endfoot separation, there was a significant reduction in a tight junction protein critical for BBB maintenance, ZO1. This reduction in ZO1 expression was accompanied by extravasation of 70kDa FITC and the ~1kDa Cadaverine, suggesting that BBB integrity was compromised. These findings led to the objective of this dissertation, which was to determine if focal ablation of an astrocyte is sufficient to disrupt BBB integrity. By utilizing the in vivo 2Phatal single-cell apoptosis induction method (21), we found that 1) focal loss of astrocyte-vascular coverage does not result in barrier deficits, but rather induces a plasticity response whereby surrounding astrocytes extend processes to reinnervate vascular vacancies no longer occupied by previously ablated astrocytes. 2) Replacement astrocytes are capable of inducing vasocontractile responses in blood vessels, and that 3) aging significantly attenuates the kinetics of this process. We then tested the hypothesis that focal loss of astrocyte-vascular coverage leads to a gliovascular structural plasticity response, in part, through the phosphorylation of signal transducer and activator of transcription 3 (STAT3) by Janus Kinase 2 (JAK2). This dissertation found that 4), this was indeed the case, and finally, 5) we determined that gliovascular structural plasticity occurs after reperfusion post-focal photothrombotic stroke. Together, the work presented in this dissertation sheds light on a novel plasticity response whereby astrocytes maintain continual cerebrovascular coverage and therefore physiological control. Future studies should aim to determine if 1) astrocytes also replace the synaptic contacts with neighboring neurons once held by a previous astrocyte, and 2) what therapeutic opportunity gliovascular structural plasticity may present regarding BBB repair following stroke. / Doctor of Philosophy / Astrocytes are the most abundant cell type in the brain. Their anatomical relationship to neurons and endothelial cells allows them to execute many vital brain functions, and comprehensively speaking, these cells form what is known as the gliovascular unit. Important for maintaining the expression of proteins preventing vascular leakage in the brain are molecules released from astrocytes processes called endfeet. These endfeet intimately enwrap blood vessels, and disruptions to endfeet-vascular coverage often coincide with vascular leakage in the brain. This dissertation therefore aimed to determine if astrocyte-vascular coverage is necessary in preventing vascular leakage. State-of-the art imaging in live animals determined this not to be the case, and rather found that focal loss of astrocyte-vascular coverage induces a plasticity response wherein neighboring astrocytes extend new endfeet to reinnervate vascular vacancies. Furthermore, we found that the kinetics of endfoot replacement are significantly reduced in aging, and that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) is a critical arbiter underlying this response. Finally, given that we found endfoot replacement to occur in locations of lost astrocyte-vascular contact following reperfusion post-focal photothrombotic stroke, these findings may have implications regarding repair of the blood-brain barrier following CNS insults such as stroke.

Title of work: Model of cerebral focal cortical ischemia and its parametrization Work objectives: The aim of this diploma thesis was to apply modified model of focal cortical brain ischemia induced by phototrombosis and subsequently determinate its parameters. Methods: Intravenous application of photosensitive Rose Bengal dye was followed by continual illumination of green laser beam over the left sensorimotocortex for 10 minutes. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombin formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain. For histological evaluation of ischemic brain damage, animals were overdosed with urethane and transcardially perfused. Results: Histological examination of brains showed significant ischemic damage in all experimental animals. Lesion was located in left hemisphere and penetrated thought the grey matter in various extents. Size of lesion, its localization and depth has shown only a small variability in the individual groups. Noticeable differences were found right after comparing experimental groups....

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Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Cerebral autoregulation is an intrinsic physiological response that maintains a constant cerebral blood flow (CBF) despite dynamic changes in the systemic blood pressure. Autoregulation is achieved through changes in the resistance of the small blood vessels in the brain through reflexive vasodilatation and vasoconstriction. Cerebrovascular reactivity (CVR) is a measure of this response. CVR is defined as a change in CBF in response to a given vasodilatory stimulus. CVR therefore potentially reflects the vasodilatory reserve capacity of the cerebral vasculature to maintain a constant cerebral blood flow. A decrease in CVR (which is interpreted as a reduction in the vasodilatory reserve capacity) in the vascular territory downstream of a larger stenosed supply artery correlates strongly with the risk of a hemodynamic stroke. As a result, the use of CVR studies to evaluate the state of the cerebral autoregulatory capacity has clinical utility. Application of CVR studies in the clinical scenario depends on a thorough understanding of the normal response. The goal of this thesis therefore was to map CVR throughout the brain in normal healthy individuals using Blood Oxygen Level Dependant functional Magnetic Resonance Imaging (BOLD MRI) as an index to CBF and precisely controlled changes in end-tidal partial pressure of carbon dioxide (PETCO2) as the global flow stimulus.

Cerebral autoregulation is an intrinsic physiological response that maintains a constant cerebral blood flow (CBF) despite dynamic changes in the systemic blood pressure. Autoregulation is achieved through changes in the resistance of the small blood vessels in the brain through reflexive vasodilatation and vasoconstriction. Cerebrovascular reactivity (CVR) is a measure of this response. CVR is defined as a change in CBF in response to a given vasodilatory stimulus. CVR therefore potentially reflects the vasodilatory reserve capacity of the cerebral vasculature to maintain a constant cerebral blood flow. A decrease in CVR (which is interpreted as a reduction in the vasodilatory reserve capacity) in the vascular territory downstream of a larger stenosed supply artery correlates strongly with the risk of a hemodynamic stroke. As a result, the use of CVR studies to evaluate the state of the cerebral autoregulatory capacity has clinical utility. Application of CVR studies in the clinical scenario depends on a thorough understanding of the normal response. The goal of this thesis therefore was to map CVR throughout the brain in normal healthy individuals using Blood Oxygen Level Dependant functional Magnetic Resonance Imaging (BOLD MRI) as an index to CBF and precisely controlled changes in end-tidal partial pressure of carbon dioxide (PETCO2) as the global flow stimulus.