Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation.

BACKGROUND:Since their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stabchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical 'synaptic stripping' but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI).METHODS:Rats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells.RESULTS:We identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68.CONCLUSION:Diffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells / these observations entice further investigation for impact on neurological outcome.

The prostaglandins comprise a group of bioactive lipids generated from arachidonic acid by cyclooxygenases and cell type-specific prostaglandin and thromboxane synthases. Prostaglandins mediate local cell signaling interactions by activation of G-protein coupled prostanoid receptors. Because the prostaglandins and their receptors are active in all tissues, they have an extraordinarily broad spectrum of physiological and pathophysiological functions that have hampered the development of safe prostanoid-based medications. This situation has emphasized the importance of understanding the functional properties of the prostanoid receptors and developing selective ligands capable of being used in patient care.The aims of this project were to identify novel regulatory functions of endogenous EP and FP prostanoid receptors in cultured human cells. Our results show that activation of EP₂ receptors in human microglia and astrocytes led to increased secretion of BDNF, a growth factor that regulates the survival of neurons. In the same cell lines, FP receptors regulate the induction of TNF-α gene expression through a classic G_q-PKC pathway. In microglia these FP receptors also stimulate a novel signaling crosstalk mechanism involving the up-regulation of TCF transcriptional function by Raf kinases, which culminates in the expression of the angiogenic inducer Cyr61. FP receptors also regulate the induction of angiogenic immediate early genes in cultured ciliary muscle cells, which may constitute the early steps in a mechanism by which commercial FP agonists reduce intraocular pressure in glaucoma therapy.The up-regulation of BDNF through glial EP₂ receptors constitutes a mechanism by which elevated PGE₂ in the inflamed brain might elicit either healing processes in the brain or neuronal apoptosis. On the other hand, induction of TNF-α and Cyr61 by glial FP receptors may mediate neuroinflammation and may also contribute to glioma tumor growth. Stimulation of FP receptors in the ciliary muscle leads to the induction of immediate early genes capable of coordinating tissue remodeling processes that have been previously documented. The results of these studies suggest novel regulatory functions of the prostanoid receptors in the brain and eye. Furthermore, these findings provide insight on how the selective modulation of the EP₂ and FP receptors might be therapeutically advantageous.

<p>The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.</p><p>There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.</p><p>The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.</p> / Dissertation

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

No description available.

Gestational diabetes mellitus (GDM) is a common complication of pregnancy and population health studies have linked it to impaired cognitive performance in the offspring. GDM triggers inflammatory responses, which can critically affect development of neuronal circuitry. We hypothesized that GDM promotes inflammatory responses in the fetus that can disturb fine-tuning of neuronal networks during early development, resulting in lifelong impaired cognitive functions. The cognitive performance of 15 week old offspring exposed to diet induced GDM were assessed. The brain tissue of the 15 week old and neonatal (E20) offspring were analyzed by immunohistochemistry, western blot, and cytokine assay. Cultured microglial responses to elevated glucose and/or fatty acid levels mimicking GDM associated diabetic conditions were analyzed. Our data reveals chronic neuroinflammation in GDM offspring, which combined with deregulation of microglial functions may explain hippocampal CA1 layer neuronal derangement and synaptic degradation that correlates with impaired cognitive performance of GDM offspring. / October 2016

Multiple sclerosis (MS) is a neurodegenerative disorder characterized by CNS inflammation and axonal demyelination. In addition, axonal pathology has also been reported in MS and may be responsible for the functional deficits associated with this disease. Based on preliminary data from our laboratory, we propose that a specific domain of the neuron, known as the axon initial segment (AIS), is targeted in MS. Consistent with our work from the human tissue, we have also observed disruption of AIS integrity in a murine CNS inflammatory model and observations strongly implicate reactive microglia as mediators of AIS disruption. In contrast, a murine model of demyelination did not exhibit AIS pathology but reactive microglia were prevalent. Since we propose that reactive microglia drive AIS disruption in our inflammatory CNS model but observe no AIS pathology following demyelination even in the presence of reactive microglia, we propose that reactive microglia in these models exhibit different interactions and molecular profiles. To test this hypothesis, we employed immunofluorescence labeling combined with confocal microscopy to quantify microglia reactivity and microglia-AIS interaction. Additionally, we conducted a microarray using RNA isolated from microglia from both the inflammatory and demyelinating models. Our findings show that microglia are reactive prior to pathology in both models and that the extent of AIS-microglial contact is similar between the models but significantly increased as compared to naïve mice. Our microarray data reveal a substantial difference in gene expression indicating functional differences between the reactive microglia in the inflammatory and demyelinating models. Finally, following functional enrichment analysis of microarray data, the complement pathway emerged as a potential contributor to the AIS pathology observed in EAE.

Epidemiological studies report that infarct size is decreased and stroke outcomes are improved in young females when compared to males. However, mechanistic insight is lacking. We posit that sex-specific differences in glial cell functions occurring immediately after ischemic stroke are a source of dichotomous outcomes. In this study we assessed astrocyte Ca2+ dynamics, aquaporin 4 (AQP4) polarity, Sloop expression pattern, as well as, microglia morphology and phagocytic marker CD11b in male and female mice following 60 min of middle cerebral artery (MCA) occlusion. We reveal sex differences in the frequency of intracellular astrocyte Ca2+ elevations (F-(1,F-86) = 8.19, P = 0.005) and microglia volume (F-(1,F-40) = 12.47, P = 0.009) immediately following MCA occlusion in acute brain slices. Measured in fixed tissue, AQP4 polarity was disrupted (F-(5,F-86) = 3.30, P = 0.009) and the area of non-S100 beta immunoreactivity increased in ipsilateral brain regions after 60 min of MCA occlusion (F-(5,F-86) = 4.72, P = 0.007). However, astrocyte changes were robust in male mice when compared to females. Additional sex differences were discovered regarding microglia phagocytic receptor CD11b. In sham mice, constitutively high CD11b immunofluorescence was observed in females when compared to males (P = 0.03). When compared to sham, only male mice exhibited an increase in CD11b immunoreactivity after MCA occlusion (P = 0.006). We posit that a sex difference in the presence of constitutive CD11b has a role in determining male and female microglia phagocytic responses to ischemia. Taken together, these findings are critical to understanding potential sex differences in glial physiology as well as stroke pathobiology which are foundational for the development of future sex-specific stroke therapies. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.