Macrophage Accumulation Near Injured Neuronal Cell Bodies Is Necessary And
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MACROPHAGE ACCUMULATION NEAR INJURED NEURONAL CELL BODIES IS NECESSARY AND SUFFICIENT FOR PERIPHERAL AXON REGENERATION by JON PAUL NIEMI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Richard E. Zigmond Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY January 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jon Paul Niemi candidate for the degree of Doctor of Philosophy *. Committee Chair Dr. Heather Broihier Committee Member Dr. Richard E. Zigmond Committee Member Dr. Gary Landreth Committee Member Dr. Ruth Siegel Date of Defense July 11th, 2016 *We also certify that written approval has been obtained for any proprietary material contained there in Dedication To my wife, my son, and my parents, thank you for your love and support. i Table of Contents DEDICATION ....................................................................................................................... iI TABLE OF CONTENTS ..........................................................................................................ii I LIST OF FIGURES ................................................................................................................ ivV ACKNOWLEDGMENTS ........................................................................................................ vIiI CHAPTER 1: GENERAL INTRODUCTION ............................................................... 1 1.1 PERIPHERAL AND CENTRAL NERVOUS SYSTEM INJURY ............................................. 2 1.2 EXTRINSIC MECHANISMS SUPPORTING PNS REGENERATION .................................... 3 1.3 SLOW WALLERIAN DEGENERATION MOUSE (WLDS) .................................................. 5 1.4 INTRINSIC MECHANISMS SUPPORTING PNS REGENERATION .................................... 10 1.5 MACROPHAGE RESPONSE TO INJURY ........................................................................ 16 1.6 MACROPHAGE POLARIZATION .................................................................................. 20 1.7 ROLE OF MACROPHAGES IN AXONAL REGENERATION ............................................. 23 1.8 FOCUS OF THESIS ..................................................................................................... 28 CHAPTER 2: A CRITICAL ROLE FOR MACROPHAGES NEAR AXOTOMIZED CELL BODIES IN STIMULATING NERVE REGENERATION ........................................................................................................................................... 29 2.1 ACKNOWLEDGEMENTS ............................................................................................. 30 2.2 ABSTRACT ............................................................................................................... 31 2.3 INTRODUCTION ........................................................................................................ 32 2.4 MATERIALS AND METHODS ..................................................................................... 33 2.5 RESULTS .................................................................................................................. 39 ii 2.6 DISCUSSION ............................................................................................................. 45 CHAPTER 3: OVEREXPRESSION OF THE MONOCYTE CHEMOKINE CCL2 IN DORSAL ROOT GANGLION NEURONS CAUSES A CONDITIONING-LIKE INCREASE IN NEURITE OUTGROWTH AND DOES SO VIA A STAT3 DEPEDNDENT MECHANISM ................................................................................... 70 3.1 ACKNOWLEDGEMENTS ............................................................................................. 71 3.2 ABSTRACT ............................................................................................................... 72 3.3 INTRODUCTION ........................................................................................................ 73 3.4 MATERIALS AND METHODS ..................................................................................... 75 3.5 RESULTS .................................................................................................................. 82 3.6 DISCUSSION .............................................................................................................. 89 CHAPTER 4: DISCUSSION ...................................................................................... 110 4.1 CCL2: MORE THAN JUST A CHEMOKINE ................................................................. 111 4.2 MACROPHAGES AND REGENERATION: NEW SITE OF ACTION ................................. 115 4.3 PRO-REGENERATIVE MACROPHAGE MECHANISMS ................................................ 119 4.4 TRANSLATIONAL ASPECTS ...................................................................................... 123 4.5 FUTURE DIRECTIONS .............................................................................................. 126 4.6 CONCLUDING REMARKS ......................................................................................... 128 REFERENCES ................................................................................................................... 130 iii List of Figures CHAPTER 2 FIGURE 2.1 MACROPHAGE ACCUMULATION IN THE SCIATIC NERVE OF WLDS MICE .... 52 FIGURE 2.2 MACROPHAGE ACCUMULATION IN THE SCG AND DRG OF WLDS MICE .... 54 FIGURE 2.3 CCL2 MRNA EXPRESSION IN WLDS AND CCR2 -/- MICE ........................... 56 FIGURE 2.4 MACROPHAGE ACCUMULATION IN THE SCIATIC NERVE, DRG, AND SCG OF CCR2 -/- MICE ............................................................................................................... 58 FIGURE 2.5 MYELIN CLEARANCE IN THE SCIATIC NERVE OF WT, WLDS, AND CCR2 -/- MICE ............................................................................................................................... 60 FIGURE 2.6 SALIVARY GLAND INNERVATION IN WT, WLDS, AND CCR2 -/- MICE ......... 62 FIGURE 2.7 DRG AND SCG CONDITIONING LESION RESPONSE IN EXPLANT CULTURE FROM WT AND WLDS MICE ........................................................................................... 64 FIGURE 2.8 DRG AND SCG CONDITIONING LESION RESPONSE IN EXPLANT CULTURE FROM WT AND CCR2 -/- MICE ...................................................................................... 66 FIGURE 2.9 DRG AND SCG CONDITIONING LESION RESPONSE IN DISSOCIATED NEURONAL CELL CULTURE FROM WT, WLDS, AND CCR2 -/- MICE .............................. 68 CHAPTER 3 FIGURE 3.1 YFP LOCALIZATION AND CCL2 MRNA OVEREXPRESSION IN DRGS FOLLOWING INTRATHECAL INJECTION OF AAV5 ........................................................... 96 FIGURE 3.2 TIME-DEPENDENT INCREASE IN MACROPHAGE ACCUMULATION IN L5 DRGS FOLLOWING CCL2 OVEREXPRESSION ........................................................................... 98 FIGURE 3.3 CCL2 OVEREXPRESSION INDUCES A CONDITIONING-LIKE INCREASE IN NEURITE OUTGROWTH ................................................................................................ 100 iv FIGURE 3.4 MACROPHAGE POLARIZATION MARKERS IN THE DRG FOLLOWING CCL2 OVEREXPRESSION ......................................................................................................... 102 FIGURE 3.5 CCL2 OVEREXPRESSION IN CCR2 -/- MICE .............................................. 104 FIGURE 3.6 CCL2 OVEREXPRESSION INDUCED RAG EXPRESSION IN THE DRG .......... 106 FIGURE 3.7 PHARMACOLOGICAL STAT3 INHIBITION IN CCL2 OVEREXPRESSION DISSOCIATED CELL CULTURE ....................................................................................... 108 v Acknowledgments The work presented here would not have been possible without the help and support of many people. First, I would like to thank my advisor Dr. Richard E. Zigmond. He has truly taught me the importance of having passion for the research you are performing. Richard has shown me that perseverance through the tougher times, whether it is a failed experiment or being turned down for funding, makes the victories that much more rewarding. As a mentor, he put an emphasis on being able to effectively communicate your science and encouraged me to present my research any chance I got, which has been integral in preparing me for a future in teaching and research. I would also like to thank my committee members Dr. Heather Broihier, Dr. Gary Landreth, and Dr. Ruth Siegel for their insight, direction, and support. Our meetings helped me see the big picture while I was lost in the details. I am truly grateful to have been mentored by such excellent and respected scientists. I could not have completed this project, and had so much fun while doing it, without the help and support of the numerous members of the Zigmond Lab. Alicia DeFrancesco, our lab manager, truly keeps the lab running smoothly. She is an excellent scientist and an even better person. Jane Lindborg was always there to help with experiments, give advice on data, and keep our lab decorated for Halloween year-round. Many other members of the lab, including Dan Mandell, Lilinete Roldan-Hernandez, Madeline Howarth, and Angela Filous, contributed their time, effort, and knowledge to this project. I will forever be honored to be considered a Lablander. vi The Department of Neurosciences at Case Western Reserve University provides an excellent training environment for young scientists. I would like to thank the members of the Neurosciences Administrative Office. Nothing we do would be possible without Katie Wervey, Pam Capasso, and Narlene Brown.