Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Michael J. Petris. Includes bibliographical references.

Prion diseases are a group of fatal neurodegenerative protein-misfolding diseases. Microglia, the resident myeloid cells found within the brain, have been shown to demonstrate a reactive morphology during the disease process with conflicting evidence for both a neurotoxic and neuroprotective role. The studies presented here aimed to investigate the role of microglia activation using transcriptomic and morphological analysis of prion disease in mice. Initially, the host immune response to prion disease was explored using a publically available mouse prion disease dataset. Re-analysis of this dataset was performed using BioLayout Express3D; a novel software tool that supports the visualisation and clustering of correlation networks. Disease-associated genes up-regulated during the later stages of infection were present in two main clusters. The cellular origin of these genes was explored by examining their expression in a dataset comprised of pure populations of cells. This demonstrated that the primary cluster of up-regulated transcripts encompassed genes expressed mainly by microglia and to a lesser extent astrocytes and neurons. The secondary cluster comprised almost exclusively of interferon response genes. The conclusions of these analyses were different from those of the original study that suggested disease-associated genes were primarily neuronal in origin. Mouse models of prion disease were established by infecting a novel line of BALB/cJ inbred mice, expressing EGFP under control of a myeloid specific Csf1r promoter, with the 79A prion strain. Quantification of the morphological changes of EGFP expressing microglia suggested the cells accumulated in the medulla at sites of early misfolded protein deposition with minimal change in their overall appearance. An activated microglia morphology was not observed until protein deposition was extensive. Isolation of EGFP expressing microglia was performed for transcriptome analysis. The vast majority of disease associated genes demonstrated increased expression at the onset of clinical symptoms. The gene list was found to be highly enriched for genes associated with an innate immune response regulated by the NFκB signalling cascade. Also highly enriched were processes associated with protein translation, energy production and stress response. These data suggest a high metabolic load is burdened by proliferating microglia; and as part of a response which is strikingly more pro-inflammatory in nature than has previously been attributed to the microglia phenotype within prion disease. As an active contributor to normal homeostasis, microglia are more than just innate immune surveillance and are now considered an integral component in both the healthy and diseased brain. The ramifications of activation toward the microglia phenotype shown here will have direct and potentially cytotoxic influence on neighbouring microglia and other brain cell types implying microglia as major contributors to the neurotoxic environment found within the CNS during prion disease. Furthermore the identification of genes associated with metabolism offer many intriguing possibilities for manipulating the activity of microglia in pre-clinical therapeutic intervention.

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Indiana University-Purdue University Indianapolis (IUPUI) / The glial cells of the retina aid in function and maintenance of the retina. The macroglia, Muller cells and the retinal astrocytes, become reactive following injury or disease in the retina, a response that is characterized by hypertrophy, dedifferentiation, loss of functionality, proliferation, and remodeling of tissue and extracellular matrix (ECM). The microglia which are the resident macrophages, also respond to injury/disease becoming activated, undergoing characteristic molecular and morphological changes, which include regulation of secreted factors, changes in inflammatory response and increased phagocytosis. Reactivity in Muller glia is thought to be the result of secreted signals, such as epidermal growth factor, ciliary neurotrophic factor, and broblast growth factor, which are released at the injury site to interact with quiescent glial cells. Furthermore, microglia and macroglia have been shown by some studies to interact following activation. While BMPs are known to be upregulated following injury in the CNS, little information is available concerning their role in reactive gliosis in the retina. We hypothesize that BMP7 indirectly triggers Muller gliosis by activating microglia. Using RT-qPCR, immunofluorescence and western blot, we assessed changes in gliosis markers in the mouse retinal glia following treatment with BMP. Our results showed that BMP7 was able to trigger Muller cell gliosis in the retina in vitro and in vivo. Furthermore, ablation of microglia lead to a subdued gliosis response in the mouse retina following BMP7 exposure. Thus, BMP7 triggers activation of retinal microglia in addition to the Muller glia. IFN-gamma and IL6 could play a role in microglia mediated activation of Muller glia, following exposure to BMP7. We also assessed the role of the Hippo/YAP pathway in the regulation of gliosis in the retina. We demonstrated that YAP was localized to the nucleus of the Muller cells of the retina and was upregulated in IFN-gamma induced gliosis in the mouse retina.

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Bei der vorliegenden Arbeit handelt es sich um eine experimentell durchgeführte Charakterisierung von CD11c-positiven Zellen des Gehirns im direkten Vergleich zu CD11c-positiven Zellen aus Lunge, Leber und Milz. Mittels Konfokal- und Fluoreszenzmikroskopie wurde die Existenz von intraparenchymalen Zellen nachgewiesen, welche den Zellmarker für Dendritische Zellen CD11c exprimieren. Durch die Etablierung einer einheitlichen Isolierungsmethode von CD11c-positiven mononukleären Zellen aus dem Gehirn, Milz, Lunge und Leber, war es uns möglich, diese mittels Durchflusszytometrie, auf die Expression wichtiger Marker für mononukleäre Zellen zu untersuchen und phänotypisch miteinander zu vergleichen. Durch diese Zellanalysen zeigten wir, dass CD11c-positive Zellen des Gehirns sowohl aufgrund ihrer spezifischen CD45-Expression, als auch durch die Expression von CD11b einen Mikrogliaphänotyp aufwiesen. Dabei konnten wir beobachten, dass CD11c-positive Zellen aus dem Gehirn einzigartig in der Eigenschaft ihrer geringen Major Histocompatibility Complex (MHC)-II-Expression sind. Mit Hilfe einer transgenen Mauslinie, welche unter dem Promotor von MHC-II das grün-fluoreszierende Protein (GFP) exprimiert, konnten wir nachweisen, dass Mikroglia selbst in der Umgebung von MHC-II-positiven Zellen, kultiviert auf Schnittkulturen der Milz, ihre MHC-II-Negativität behalten. Im Vergleich dazu adaptierten sich MHC-II-positive Splenozyten, kultiviert auf Schnittkulturen vom Hippocampus, an die neue Umgebung und verringerten die Expression von MHC-II. Unsere Daten lassen also die Schlussfolgerung zu, dass sich CD11c-positive Mikroglia hinsichtlich ihrer Expression von MHC-II intrinsisch von CD11c-positiven Zellen anderer Organe unterscheiden. Ebenso scheinen auch lokale Faktoren im Gehirn dazu beizutragen, die Expression von MHC-II unter physiologischen Bedingungen wirkungsvoll zu unterdrücken.

P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.