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Differential Reactivity of Microglia in Two Mouse Models of Multiple Sclerosis

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Differential Reactivity of Microglia in Two Mouse Models of Multiple Sclerosis Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2016 Differential Reactivity of Microglia in Two Mouse Models of Multiple Sclerosis Rebecca K. Hartley Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medicine and Health Sciences Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/4180 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Differential Reactivity of Microglia in Two Mouse Models of Multiple Sclerosis A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University By: Rebecca Kathleen Hartley Bachelor of Science, Indiana University of Pennsylvania, 2013 Advisor: Jeffrey L. Dupree, Ph.D. Associate Professor of Anatomy & Neurobiology Virginia Commonwealth University Richmond, Virginia May 2016 Acknowledgements I want to thank my advisor, Dr. Jeff Dupree, for providing me with knowledge and advice over the past two years, and for helping me learn what it means to be a researcher. He always urged me to learn more and move forward with a clear hypothesis in mind. His guidance was instrumental in completing my project and I cannot thank him enough for everything he has done for me. I would like to thank my other lab members for all their assistance and encouragement. I especially want to thank Kareem Clark and Savannah Benusa for providing me with their expertise and teaching me, and for their encouragement when obstacles arose. Their guidance was invaluable and I could not have completed this project without their help. I want to thank Nick George, my fellow master’s student, for providing support as we persevered through our research together. I also want to thank Brooke Sword, our laboratory technician, for helping me with my mice and our undergraduate students for their assistance in various aspects of this project. I would like to thank my committee members; Dr. Unsung Oh, Dr. Babette Fuss, and Dr. George DeVries for serving on my committee and helping to guide my research as my project incorporated new ideas and techniques. I would also like to thank my program director, Dr. Gail Christie, for her advice as I proceeded through the MBG program and Dr. Henderson and the VCU core microscopy facility for their training and guidance. Finally, I want to thank my family and friends for all of their support and encouragement. They have always encouraged me to pursue my dreams and become the best person that I could be. I want to thank them for listening to me explain my research, even when it was incomprehensible to them, and for providing moral support when I became frustrated or disheartened. They have truly motivated me to be the scientist I am today. i Table of Contents Acknowledgements ........................................................................................................................................ i List of Figures ............................................................................................................................................... iv List of Tables ................................................................................................................................................. v List of Abbreviations .................................................................................................................................... vi Abstract ........................................................................................................................................................ ix Introduction .................................................................................................................................................. 1 The Neuron ............................................................................................................................................... 1 Myelin ....................................................................................................................................................... 3 Axonal Domains ........................................................................................................................................ 5 Nodes of Ranvier ....................................................................................................................................... 6 The Axon Initial Segment .......................................................................................................................... 9 Multiple Sclerosis .................................................................................................................................... 13 Inflammation in MS................................................................................................................................. 14 Demyelination in MS ............................................................................................................................... 15 Axonal Pathology in MS .......................................................................................................................... 17 Microglia ................................................................................................................................................. 19 The Complement System of Immunity ................................................................................................... 22 Background Data ..................................................................................................................................... 25 Materials & Methods .................................................................................................................................. 31 Mice ........................................................................................................................................................ 31 Tissue Preparation .................................................................................................................................. 34 Antibodies ............................................................................................................................................... 34 Immunohistochemistry ........................................................................................................................... 35 Confocal Microscopy & Quantitation ..................................................................................................... 36 Isolation of Mouse Cortical Microglia & RNA ......................................................................................... 37 Microarray Procedure & Data Analysis ................................................................................................... 39 Western Blot Protocol............................................................................................................................. 40 Quantitative Reverse Transcriptase Polymerase Chain Reaction ........................................................... 42 ii Results ......................................................................................................................................................... 47 Contact between IBA-1+ Cells and the AIS is similar in EAE and Cuprizone ........................................... 47 Reactive Microglia from EAE and Cuprizone mice present distinct RNA profiles ................................... 50 Complement Genes are Upregulated in EAE .......................................................................................... 63 C3 Protein Expression is Upregulated in EAE .......................................................................................... 63 C3 Localizes at the AIS ............................................................................................................................. 63 Discussion.................................................................................................................................................... 71 Microglia Show Morphological Similarities in EAE and Cuprizone ......................................................... 72 Microglia Gene Expression Differs in EAE and Cuprizone ....................................................................... 72 The Complement Pathway May Mediate AIS Disruption ....................................................................... 74 References .................................................................................................................................................. 78 Vitae .......................................................................................................................................................... 106 iii List of Figures Figure 1. Schematic representation of a neuron………………………………………………………………………………………2 Figure 2. Schematic of proteins at the axon initial segment…………………………………………………………………..10 Figure 3. Diagram of the complement system……………………………………………………………………………………….23 Figure 4. Preliminary data indicated loss of axon initial segments in MS tissue……………………………………..26 Figure 5. AIS length and number were maintained in the presence of demyelination……………………………27 Figure 6. AIS length and number were altered in the absence of demyelination……………………………………28 Figure 7. AIS length increases
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