Andy Y. Shih, Ph.D. - US grants

Project summary. Vascular cognitive impairment (VCI) is an insidious disease that progressively destroys memory and cognitive function with age. With a growing elderly population in the US, VCI will become a significant healthcare burden within the coming decades. Cerebral microinfarcts are small brain lesions (0.1 to 3 mm in diameter) that arise from obstruction of small cerebral vessels. They are estimated to be twice as prevalent in the demented brain. Recent advances have demonstrated the feasibility of detecting microinfarcts with 7T magnetic resonance imaging (MRI), supporting their potential as imaging biomarkers of VCI. However, a critical barrier for current progress is an inability to link these anomalous signals with microinfarcts of a specific age, size, and cellular pathology. Our long-term goal is to elucidate microinfarct-based tissue pathologies that generate signal contrast in MRI methods such as diffusion-weighted imaging (DWI), in order to optimize their detection non- invasively. Our approach is to longitudinally image microinfarcts in mice using in vivo two-photon laser- scanning microscopy (TPLSM), a method that can repeatedly probe cellular structure in the intact brain with micrometer precision. By combining TPLSM and MRI of the same animals, we will be able to link specific pathological events at the cellular level with respective DWI signals as they evolve over time. We will use a novel mouse model of microinfarction that we have developed, which provides exquisite control over lesion size, location, and timing of onset. The objective of this proposal, which is in pursuit of our long-term goal, is to determine how the reactivity of glial cells can influence DWI signal contrast. Our central hypothesis is that dramatic tissue density change caused by microglial infiltration and astroglial proliferation during microinfarction is a principle source of DWI signal change. This hypothesis is formulated based on our past histological work, which revealed that rodent microinfarcts, like human microinfarcts, are densely packed with GFAP-positive astrocytes and CD68-positive macrophages in the sub-acute period of days to weeks. We will test the hypothesis with two Aims. In Aim 1, we plan to characterize the time-course of microglial and astroglial reactivity during microinfarct growth using TPLSM in two transgenic mouse lines that specifically express fluorescent proteins in those cells. In Aim 2, we will examine the correlation between microinfarct-specific DWI signal and the spatiotemporal change in glial reactivity measured by TPLSM from the same animals. Finally, we will test whether a new DWI technique, diffusional kurtosis imaging (DKI) can improve the sensitivity of microinfarct detection, compared to conventional DWI. Our approach is innovative because it uses a completely in vivo strategy to link cellular pathology with MRI signals during microinfarction by combining two powerful imaging modalities: TPLSM and 7T MRI. The proposed research is significant because the results can immediately impact the interpretation of small anomalous signals seen by DWI, and may therefore improve detection of VCI in aged individuals before the onset of cognitive impairment.

Acute; Address; Affect; angiogenesis; Area; Axon; base; Behavioral; Biological; Biological Assay; Blood; Blood flow; Blood Vessels; Brain; Brain region; Cells; Chronic; Chronic Phase; clinically relevant; Data; Dendritic Spines; density; disability; Electric Stimulation; Energy Supply; Erythrocytes; Goals; Image; improved; in vivo; in vivo imaging; indexing; Individual; Infarction; Inflammatory; Ischemia; Knowledge; Methods; Microscopy; Microvascular Dysfunction; microvascular pathology; Motor; mouse model; Mus; neuronal growth; Neuronal Plasticity; Neurons; novel; Pathology; post stroke; preclinical study; Process; Recovery; Recovery of Function; Rehabilitation therapy; repaired; Research; research study; Resolution; Role; Sensory; South Carolina; Staging; stroke; stroke recovery; Structure; Synapses; synaptogenesis; Testing; therapeutic target; thrombolysis; Tissues; Training; two-photon; vasoconstriction; Vibrissae; Work;

? DESCRIPTION (provided by applicant): Ischemic blood-brain barrier (BBB) disruption is a major contributor to tissue injury during stroke. Understanding the mechanisms that regulate this process will lead to new approaches to mitigate brain damage and lengthen the window for thrombolytic treatment. Pericytes are essential for development and maintenance of the BBB. However, little is known about their impact on BBB integrity during ischemic injury in the adult brain. Using in vivo two-photon microscopy, we have found that pericytes elicit punctate BBB disruptions specifically at their somata. Pericyte somata cover only 7% of the total capillary surface, while their fine and extensive processes cover the rest. However, harmful blood-borne molecules can diffuse far beyond the point of extravasation, strengthening our need to understand this uncharacterized leakage route. Our central hypothesis is that pericytes rapidly upregulate matrix metalloproteinase activity, leading to local disassembly of endothelial tight junctions (paracellular leakage). This hypothesis will be tested using state-of- the-art approaches. In Aim 1, we will use in vivo two-photon microscopy to directly visualize MMP9 activity using a fluorescent gelatin probe, following stroke induction in pericyte-labeled mice. In vivo pharmacological and pericyte-specific deletion experiments will be performed to test putative signaling cascades that can lead to rapid MMP9 activation. Ex vivo biochemical studies will be performed to confirm the role of these signaling cascades. In Aim 2, we will use 3-D serial block-face electron microscopy to examine the nature of endothelial disruption at pericyte somata. Our findings will be compared with neighboring capillary regions not covered by somata. The proposed research is significant because it is expected to define pericytes as inducers of BBB injury during ischemia, which contrasts their emerging role as nurturers of BBB integrity during development and normal brain function.

Project Summary. Numerous clinical studies have shown that cerebral microinfarcts are likely contributors to vascular cognitive impairment and dementia (VCID). However, the mechanism by which these small, but prevalent lesions lead to brain-wide neural dysfunction remains unknown. Our central hypothesis is that microinfarct injury leads to neural impairments that extend well beyond the restricted lesion cores seen during histological and radiological examination. These remote effects, when accumulated, are a mechanism by which microinfarcts cause large- scale disruption of brain function and cognitive decline. The rationale of the proposed research is to use a mouse model where the timing and location of microinfarcts can be controlled in order to better understand how they cause brain dysfunction. We plan to examine: i) the spatial extent and chronicity of functional impairments induced by individual microinfarcts, ii) the cumulative effects of multiple microinfarcts, and iii) the cellular/molecular changes that underlie their remote effects. Our model uses state-of-the-art methods for controlled optical occlusion of targeted cortical penetrating arterioles, individually and in multiples, to precisely and non-invasively form small regions of ischemic injury that mimic human microinfarcts. The associated injury processes can then be studied in vivo over time using parallel high-resolution two-photon fluorescence calcium imaging and 7T MRI to reveal detailed aspects of brain pathophysiology that are potentially invisible to MRI or histopathology. We further use behavioral paradigms that are sensitive to microinfarcts to uncover their effects on sensory perception and cognitive function. Aim 1 of the project tests the hypothesis that cortical microinfarcts induce sustained neuronal deficits beyond their lesion core following their strategic induction within the mouse vibrissa sensory system. It further examines whether aberrant change in excitatory-inhibitory balance contributes to these deficits. Aim 2 of the project tests the hypothesis that the accumulation of multiple microinfarcts, spatially distributed throughout the cortices of both cerebral hemispheres, is sufficient to cause subcortical white matter degeneration (assessed in vivo with diffusion MRI tractography and ex vivo with histology) and impairment in cognitive tasks. This work will complement clinical research on VCID in several ways. First, it will provide detailed mechanistic information on how, and to what extent, microinfarcts impair remote brain tissues. Second, it will clarify what aspects of microinfarct injury are visible or invisible to MRI, the primary means to detect these lesions during life. Third, it will provide unique in vivo MRI-ex vivo histopathology comparisons to reveal the underlying biological processes that cause MRI signal change during gray and white matter injury. Fourth, it will establish a first-of-its-kind in vivo experimental platform to study mechanisms of microinfarct-induced pathology and to gauge the utility of new therapeutic agents.

Project summary. Cerebral pericytes are specialized mural cells that line the entire cerebrovascular capillary bed. The concept that pericytes are contractile and have the capacity control capillary flow dates back to their discovery in the 1890s. Yet, due to a lack of methods to target and manipulate pericytes in vivo, this facet of pericyte biology has remained highly understudied. It is imperative to understand pericyte contractility because many human and animal studies have demonstrated insufficiency in capillary flow during stroke, with aberrant constriction of capillaries being a likely contributor. Modulating the contractile ability of pericytes may represent a valuable therapeutic approach to improve cerebral blood supply during ischemia, and a means to further improve the efficacy of existing clot-removal treatments. Currently, little is known about the signaling pathways involved in pericyte contraction. Recent studies have shown that the vast majority of pericytes that line the brain capillary bed are negative for ?- smooth muscle actin (?-SMA), which is central to actomyosin-based contraction of smooth muscle cells of arterioles. Yet, our preliminary data suggest that these ?-SMA-negative pericytes retain the ability to contract in vivo and can impede capillary flow, pointing to an alternative contractile mechanism. Our central hypothesis is that brain capillary pericytes can contract through dynamic actin cytoskeleton reorganization, rather than actomyosin cross-bridge cycling. We test this hypothesis by combining pharmacology with a novel optogenetic assay to activate individual capillary pericytes in a ?cause and effect? manner both in vivo and ex vivo. In Aim 1, we will test whether drugs that inhibit or promote actin polymerization can alter optogenetically-induced pericyte contraction in the brains of live mice. We will further test these drugs on pericyte contractility in an ex vivo, pressurized arteriole-to- capillary preparation to exclude indirect actions from non-vascular brain cells. In Aim 2, we directly visualize actin polymerization in capillary pericytes in the normal and ischemic brain in vivo. We will express Lifeact-GFP, a novel fluorescent probe for F-actin, specifically in vascular mural cells and examine whether F-actin content increases in pericytes prior to pathological capillary constriction. This project will shed light on capillary pericyte cytoskeletal dynamics and its relation to capillary flow, an aspect of pericyte biology that is highly understudied in the brain microvasculature in vivo. If successful, our findings will help to establish the rationale, methodologies, and mouse models for further investigations of how pericyte cytoskeletal dynamics are involved in capillary flow impairment during stroke and related brain pathologies.

Project Summary Brain capillaries are composed of a single layer of endothelial cells covered by specialized mural cells called pericytes. Communication between pericytes and endothelial cells is essential for brain capillary health. Recent studies indicate that Alzheimer?s disease and vascular dementia involve increased death or degeneration of brain pericytes. This is thought to contribute to the impairment of both blood-brain barrier integrity and cerebral blood flow, which subsequently exacerbates neurodegeneration. Therefore, strategies to mitigate or compensate for loss of pericyte coverage may help to preserved vascular function in these neurological diseases. We recently discovered that brain pericytes have the ability to structurally remodel in the adult brain. In response to focal ablation of single pericytes in vivo, we observed the robust extension of processes from neighboring pericytes, which could reach over large stretches of capillary bed to regain contact with the exposed endothelium. In young healthy mice, the transient loss of pericyte coverage led to persistent capillary dilation and abnormally high blood cell flux, until pericyte contact was regained. These findings suggest that pericyte remodeling is a reparative mechanism to compensate for pericyte loss. We hypothesize that this capacity is diminished with age and further impaired with amyloid deposition during cerebral amyloid angiopathy, a frequent small vessel disease in Alzheimer?s. To address this hypothesis, we plan to use in vivo two-photon microscopy to directly observe pericytes dynamics in normal mice and mice with cerebral amyloid angiopathy. In Aim 1, we will test whether remodeling capacity is reduced in young and aged Tg-SwDI mice, which exhibit a unique enrichment of capillary amyloid deposits over time. In Aim 2, we will use a novel oxygen-sensitive probe designed for two-photon imaging to better understand the consequence of pericyte loss on blood flow and local tissue oxygenation. This project will shed light on a largely unstudied facet of pericyte biology that may lead to novel approaches to augment pericyte-endothelial contact and preserve brain capillary health in Alzheimer?s disease. It will advance the field by: 1) Characterizing the dynamics of pericyte remodeling in detail using in vivo optical imaging, 2) providing insight on how small vessel disease impairs the reparative capacity of brain capillaries, and 3) promoting the development of new tools to study pericytes with unprecedented specificity in the living mouse brain.

Project Summary Much of our understanding of brain microcirculation comes from studies on arteriolar perfusion. Blood efflux through venules plays an equally important role in determining blood flow through the brain, since all blood entering the brain must exit via venules. The structure and function of cerebral venules can change dramatically during cerebrovascular disease. Preclinical and clinical studies have demonstrated marked alterations in venule tortuosity and vascular wall composition during Alzheimer?s disease and Alzheimer?s disease-related dementias. Compared to arterioles, the slower flow and distinct endothelial biology of venules makes them more susceptible to hemostasis, thrombosis, and immune cell adhesion during disease. Collectively, these factors point to venules as a site of vulnerability in cerebral perfusion that remains highly understudied. This project focuses on principal cortical venules (PCVs), a subset of venules that descend from the brain surface into the deepest layers of cortex and underlying white matter. Although PCVs are less common compared to smaller cortical venules, they extend massive, horizontally projecting branches in deeper tissues, suggesting a critical role in perfusion of deep cortex and adjacent white matter tracts. However, there exists almost no information on the structure, physiology and perfusion territories of PCVs. Cerebral white matter is particularly sensitive to blood flow deficit and degenerates in early stages of Alzheimer?s disease and Alzheimer?s disease-related dementias. Understanding the regulation of perfusion in and near white matter tracts will be critical in understanding the basis of this white matter degeneration. Our central hypothesis is that PCVs are the main drainage system for deep cortical layers and the underlying white matter. In Aim 1, we will test this hypothesis by using emergent deep in vivo two-photon imaging and three-photon imaging to measure how capillary flow is drained in cortical layer 6 and its adjacent white matter tract in the mouse brain, respectively. These activities will be performed in adult (3-9 months) and aged mice (18-24 months) to test a secondary hypothesis that age is associated with deterioration in PCV structure and function. In Aim 2, we will we will quantify the radius of cortical tissue dependent upon PCV drainage by measuring how photothrombotic occlusion of a single PCV affects flow into the cortex through neighboring penetrating arterioles. We will further use histology to assess the volume of hypoxic tissue in gray and white matter created by occlusion of single PCVs. This project is significant because it addresses the understudied topic of venular perfusion in white matter using novel in vivo imaging approaches. It further establishes an experimental foundation needed for future research on venular dysfunction as a mechanism of impaired cerebral blood flow and white matter degeneration in Alzheimer?s disease and Alzheimer?s disease-related dementias.

Project Summary Pericytes are specialized mural cells in the basement membrane of brain capillaries. Their contact and communication with the endothelium is critical for multiple aspects of vascular function, including control of microvascular blood flow and blood-brain barrier integrity. There is significant evidence that increased loss of pericytes occurs during Alzheimer's disease and Alzheimer's-related dementias, and that this loss causes accelerated degradation of microvascular integrity, leading to neuronal dysfunction. Preserving pericyte- endothelial contact may therefore improve cerebrovascular function in these neurodegenerative diseases. However, there remain fundamental gaps in knowledge on how the adult brain responds to and recovers from pericyte loss in vivo. We recently discovered that pericytes of the brain undergo a repair strategy to maintain coverage of the endothelium in the event of pericyte loss (Berthiaume et al. Cell Reports, 2018, 22(1):8-16). Pericytes can structurally remodel their far-reaching processes to invade endothelial regions that lack pericyte contact. The goal of this project is to investigate this novel facet of brain pericyte biology and its role in maintenance of capillary function. Our innovative approach will assess the effect of pericyte loss and repair in a completely physiological setting. We will use high-resolution, in vivo two-photon microscopy to image and selectively ablate pericytes, while assessing capillary hemodynamics, tissue oxygenation, and neural synaptic activity. This approach provides an exceptionally clear view of how the brain responds to pericyte loss, and the reparative responses that are mounted over days. In Aim 1, we will determine how the pericyte remodeling mechanism manages graded increases in severity of pericyte loss. We will examine the physiological consequence of this pericyte loss on capillary flow, structure and integrity, and determine whether the repair capacity is diminished with increasing age. In Aim 2, we will examine how pericyte loss alters the microstructure of tissue oxygen distribution and neuronal synaptic function using novel imaging probes. In Aim 3, we will determine whether pericyte remodeling is altered by activation or inhibition of PDGF-B/PDGFR?, a key signaling pathway for developmental recruitment of pericytes to their peri-endothelial niche. If successful, our aims will establish whether it is useful to restore pericyte coverage in conditions such as Alzheimer's disease and related dementias. We will obtain information on how selective pericyte loss in adult and aged brain affects the dynamics of capillary function. Finally, we will establish novel methods to quantify and manipulate pericyte remodeling, allowing the phenomenon to be studied broadly in other models of cerebrovascular disease.