Examining the Regulation of Inflammation through CD200 and CD200R Following Spinal Cord Injury Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Graduate School of The Ohio State University By Bryan Andrew Brautigam, B.S. Biomedical Sciences Graduate Program The Ohio State University 2013 Thesis Committee: Lyn B. Jakeman, Ph.D., Advisor Phillip Popovich, Ph.D. Virginia Sanders, Ph.D. Amy Lovett-Racke, Ph.D. Copyright by Bryan Andrew Brautigam 2013 Abstract Spinal cord injury (SCI) is a debilitating condition resulting in permanent loss of sensation and paralysis with a lifetime estimated health cost ranging from one million to four million dollars. After SCI, a complex cellular response exacerbates the initial damage and the spinal cord fails to regenerate damaged neurons. Various immune cells are recruited to the site of injury, including resident microglia and macrophages, which are known for their dual and conflicting role of facilitating neural growth/regeneration and pathogenesis. Targeting the inflammatory immune response could serve to reduce secondary damage and create a permissive environment for axonal regeneration. One mechanism for regulating myeloid cells is through the neuroimmune regulatory proteins, CD200 and CD200R, which contribute to neuron-mediated inhibition of myeloid cell activation and pro-inflammatory signaling cascades. CD200 is expressed on a wide variety of cells throughout the body, including neurons, while CD200R is primarily expressed on microglia and macrophages. While a regulatory role of CD200/CD200R is evident in many central nervous system (CNS) disease models, it is not known if this mechanism contributes to neuroprotection following trauma to the CNS. By understanding how CD200 regulates myeloid cells after SCI, we hope to reduce inflammation with the goal of creating an environment conducive for axonal regeneration. The overall hypothesis is that CD200 is downregulated after SCI resulting in decreased CD200R activation on macrophages and microglia causing increased inflammation. The goal is to determine if delivery of a soluble CD200R agonist after SCI ii will reduce inflammation and secondary damage. We determined that CD200 is highly expressed within the gray matter and to some extent within the white matter, while CD200R is expressed at extremely low levels on resting microglia. After lesioning the dorsal roots, sciatic nerve and corticospinal tract, we determined that CD200 expression is maintained after injury or not expressed by these specific neurons. Injection of CD200R antagonist into the naïve spinal cord resulted in microglial activation. Following contusion SCI, CD200 expression is decreased within the lesion, while CD200R increases with the time course of macrophage migration into the injured spinal cord. In the crush injury model, CD200 expression is maintained in the spared gray matter rostral and caudal to the lesion and within the lesion, while the glial scar does not express CD200. Administration of CD20Fc to bone marrow derived macrophages does not reduce LPS induced increases in nitric oxide production. Based on our results, CD200 does not appear to play a significant role in reversing myeloid cell activation under the specific conditions tested. While antagonizing CD200R in the spinal cord resulted in microglial activation, the presence of CD200 or CD200R agonists was not sufficient to overcome activation in the presence of activating stimuli. We believe that CD200R agonists would not be sufficient to overcome the excessive number of inflammatory stimuli present after SCI, therefore targeting CD200R for treatment in locations with fewer inflammatory stimuli or before macrophage activation may prove successful. iii Dedication Dedicated to my wonderful wife, Megan. iv Acknowledgements First and foremost, I would like to thank my advisor, Lyn Jakeman, who spent countless hours proof-reading my papers, helping me troubleshoot experiments and supporting me through this research project and my graduate career. I would also like to thank my thesis committee, Doctors Phillip Popovich, Virginia Sanders and Amy Lovett-Racke for guiding me in various ways throughout this process. I would like to recognize Richa Manglorkar for her assistance characterizing CD200 expression and glial reactivity following select nervous system injuries, Feng Qin Yin for performing surgeries and providing support, as well as, Kent Williams for teaching me various laboratory techniques. I would also like to thank the Popovich and McTigue labs for assistance during my laboratory rotation and for teaching me various new techniques that were crucial to my development as a scientist. Specifically, thank you to Jodie Hall and Kristina Kigerl for help in experimental design and cell culture techniques. A special thank you to Jodie Hall for proof reading this document. I could not have performed these experiments or written this document without all of your help over the last two years. Finally, I owe all of this to my God and Savior, Jesus Christ. Without His direction and guidance, I would not be the man, scientist or teacher that I am today. Its not about me, its about Jesus, so I give Him all the glory. v Vita 2007………………..........................................Grace Christian School 2011…………………………………………………….B.S. Biology and Chemistry, Westmont College 2011-2013…………………………………………….Graduate Research Associate, Department of Physiology and Cell Biology, The Ohio State University Publications Shaum K, Tylski E, Alamillo J, Brautigam B, Cochran K, Mudder P, Nishimura A. Evaporation Rates of Alkanes and Alcohols on a Glass Surface as Observed by Optical Interference. Chemical Educator. 2011 Sep 6;16:214–6. Fields of Study Major Field: Biomedical Sciences Emphasis: Neurological Disorders vi Table of Contents Abstract ................................................................................................................................ ii Dedication ............................................................................................................................iv Acknowledgements ...............................................................................................................v Vita .......................................................................................................................................vi List of Tables .........................................................................................................................x List of Figures.......................................................................................................................xi Introduction .......................................................................................................................... 1 Chapter 1: Regulation of Microglia and Macrophages with CD200 ....................................3 1.1: Microglia Actively Survey and Protect the Central Nervous System.......................3 1.2: Neuroimmune Regulatory Elements .......................................................................4 1.3: CD200-CD200R.......................................................................................................5 Chapter 2: Spinal Cord Injury ............................................................................................ 11 2.1: Injury Models ......................................................................................................... 11 2.1.1: Contusion Injury Models ............................................................................. 12 2.1.2: Crush and Compression Injury Models ...................................................... 12 2.1.3: Transection and Hemisection Injury Models ............................................. 13 2.2: Primary Damage and Cell Death ........................................................................... 13 2.3: Secondary Inflammatory Damage......................................................................... 15 2.3.1: Neutrophils .................................................................................................. 15 2.3.2: B and T Cells................................................................................................ 17 vii 2.3.3: Microglia and Macrophages........................................................................19 2.4: Astrocytes and the Glial Scar................................................................................ 22 2.4.1: Beneficial Aspects of the Glial Scar.............................................................23 2.4.2: Negative Aspects of the Glial Scar ............................................................. 24 2.4.3: Macrophages Modify the Glial Scar............................................................25 Chapter 3: Hypothesis ....................................................................................................... 28 Chapter 4: Materials and Methods .................................................................................... 30 4.1: Surgical Procedures and Animal Care .................................................................. 30 4.1.1: CD200 and CD200R Distribution Study.................................................... 30 4.1.2: Altered Expression of CD200 and CD200R Following Select Nervous System Injuries...................................................................................................... 31 4.1.3: CD200R Antagonist Intraspinal Microinjections....................................... 31