abstract: Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective disorders. Preliminary data indicate TBI causes neuroinflammation in the hippocampus, likely due to axonal damage, and in the paraventricular nucleus of the hypothalamus (PVN), where no axonal damage is apparent. Mechanisms regulating neuroinflammation in the PVN are unknown. Furthermore, chronic stress causes HPA dysregulation and glucocorticoid receptor (GR)-mediated neuroinflammation in the PVN. The goal of this project was to evaluate neuroinflammation in the HPA axis and determine if GR levels change at 7 days post-injury (DPI). Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point. / Dissertation/Thesis / Masters Thesis Biology 2019

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付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24509号 / 医博第4951号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now the role of microglia in pruning remain under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the Purkinje cell layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in–depth histological analysis of the cerebellar cortex.:1 Introduction 6 1.1 Origin of microglia 6 1.2 Synaptic refinement 7 1.3 Fractalkine Receptor CX3CR1 9 1.4 Climbing fiber maturation and PCL development 10 1.5 Aim of the study 12 2 Materials and Methods 13 2.1 Materials 13 2.1.1 General material 13 2.1.1.1.1 Hardware 13 2.1.1.1.2 Consumable supplies 13 2.1.2 Chemicals 14 2.1.3 Solutions 14 2.1.4 Animals 14 2.1.5 Primary Antibodies 15 2.1.6 Secondary Antibodies 15 2.1.7 Software 15 2.2 Methods 15 2.2.1 Genotyping 15 2.2.2 Fixation and cryopreservation 16 2.2.3 Fluorescence Immunohistochemistry 16 2.2.4 Quantification of Microglia in the Cerebellum 16 2.2.5 Assessment of VGluT2 in the cerebellum 18 2.2.6 Statistical Analysis 19 3 Results 20 3.1 Postnatal enrichment of microglia cells in the cerebellar Purkinje cell layer 20 3.2 Microglial proximity to Climbing fibers 22 3.3 Population of the granular und molecular layer of CX3CR1 knock-out mice during early postnatal development 23 3.4 Influence of CX3CR1 knock-out on microglial morphology 25 3.5 Influence of CX3CR1 deletion on the VGluT2 expression during postnatal development 29 4 Discussion 31 4.1 Role of microglia in the developing cerebellum 31 4.2 CX3Cr1 Signaling and influence on microglial motility and morphology 32 4.3 CX3CR1 signaling and synaptic pruning 33 4.4 CX3CR1 signaling and formation of functional synapses 34 4.5 Correlation of immunohistological data with electrophysiological findings 35 5 Summary and conclusion 36 6 Zusammenfassung der Arbeit 38 7 References 41 8 Erklärung über die eigenständige Abfassung der Arbeit 49 9 Publications 50

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