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I REGENERATIVE MEDICINE APPROACHES to SPINAL CORD REGENERATIVE MEDICINE APPROACHES TO SPINAL CORD INJURY A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Ashley Elizabeth Mohrman March 2017 i ABSTRACT Hundreds of thousands of people suffer from spinal cord injuries in the U.S.A. alone, with very few patients ever experiencing complete recovery. Complexity of the tissue and inflammatory response contribute to this lack of recovery, as the proper function of the central nervous system relies on its highly specific structural and spatial organization. The overall goal of this dissertation project is to study the central nervous system in the healthy and injured state so as to devise appropriate strategies to recover tissue homeostasis, and ultimately function, from an injured state. A specific spinal cord injury model, syringomyelia, was studied; this condition presents as a fluid filled cyst within the spinal cord. Molecular evaluation at three and six weeks post-injury revealed a large inflammatory response including leukocyte invasion, losses in neuronal transmission and signaling, and upregulation in important osmoregulators. These included osmotic stress regulating metabolites betaine and taurine, as well as the betaine/GABA transporter (BGT-1), potassium chloride transporter (KCC4), and water transporter aquaporin 1 (AQP1). To study cellular behavior in native tissue, adult neural stem cells from the subventricular niche were differentiated in vitro. These cells were tested under various culture conditions for cell phenotype preferences. A mostly pure (>80%) population of neural stem cells could be specified using soft, hydrogel substrates with a laminin coating and interferon-γ supplementation. To guide and possibly recruit native stem cells, as well as reduce injury in the spinal cord, an injectable delivery iii strategy is necessary. An in situ cross-linking hydrogel could increase latency and localization of treatments. In this project, a chitosan/PEG based hydrogel was tailored for CNS tissues with low swelling post-gelation, a low elastic modulus (0.37 kPa), and very low cytotoxicity. When injected into the spinal cord parenchyma, the hydrogel elicited close to the same response as the saline injected surgical sham group. Overall, these platforms can be used to manufacture future strategies to locally deliver therapeutics that combat spinal cord injury. iv ACKNOWLEDGEMENTS Graduate school has been a long and bumpy road. I definitely would not have made it to the end in one piece without many of wonderful people encouraging, helping, and sometimes down right pushing me along the way. I would like to thank Dr. Leipzig, for taking me into his lab and teaching me to do research when I had no real experience. I’m sure there were some frustrating times along the way (writing the mini-book comes to mind!), but you stuck with me and made me into a stronger person. I appreciate all the times you came into the lab to teach us techniques and you open door policy. Getting so much face time with your advisor can be a rare thing, and I am happy to have received direction “from the source.” Thank you for encouraging a sense of family in the lab, from semi-annual lab outings to sharing your garden produce and dahlia tubers. A big thank you to my committee, Drs. Chase, Cheng, Joy, Willits, and the newly joined Dr. Monty! Your comments, critiques, and questions have helped to mold this project. I am grateful that I got to learn from you in and out of the classroom, you all greatly inspire me. A special thank you to Dr. Monty and Dr. Willits for talks and advice, I strive to someday be the role-model to future graduate students the way you were to me. To all the students I have had the pleasure of working with, graduate and undergraduate, I am extremely grateful for research discussions, non-research talks, pick- me-ups after failed experiments, and all around brightening my day. I believe that great research comes from a collaborative effort, where minds from different backgrounds and v areas of expertise really meld to make something special. Thank you to Andrew, Hannah, Mahmoud, Pritam, Shahrzad, and Trevor for answering my incessant questions, being excellent sounding-boards, suggesting solutions to problems big and small, and just for being amazing scientists and surrounding me with positivity. The early (read: glory) years would not have been the same without my previous labmates, who are now very dear friends. Hang, Aleesha, Natalie, and Liza: I miss you every day and wish each of you were still in my daily life! I must also thank and spread the appreciation to friends and sources of knowledge outside of my own lab. Frank, you helped me immensely in and out of the machine shop! Not only did you make amazing things that greatly enhanced my research, but you kept me sane with all the trips to the rec and introduced me to new friends through crazy walleyball games. Mary Beth, I definitely would not have finished everything up without our running (a.k.a. therapy) sessions towards the end of my project. Thank you for being a great friend and a kick- butt colleague. You all were so supportive and integral to my success as a graduate student. I look up to each one of you, and I hope we continue to stay in contact and remain friends. Saving the best for last, I must acknowledge my family, without whom I certainly would not have achieved so much in my life. I am eternally grateful that I get to call you all family (be it by blood or by choice), you are my rock, my lifeline, my cheerleaders, my angry mob, my everything. Also, thank you for keeping me grounded in the fact that there is life going on OUTSIDE of a laboratory. I cannot name everyone here, I do like trees, but need to say a few special thanks to Anthony, Caitlin, and Kent, and to Leah and Amanda, my oldest friends. I must officially thank my husband, Brian, you should be vi nominated for saint-hood. I cannot wait to see what else life has in store for us (should be easy compared to these last couple years)! I would not have even started this journey if not for the constant love, support, and encouragement of my parents and my brother, all of whom have helped make me the person I am today. Thank you for teaching me by example, the person that I want to be. vii TABLE OF CONTENTS Page LIST OF FIGURES ......................................................................................................... xiv CHAPTER I. INTRODUCTION ........................................................................................................... 1 II. CENTRAL NERVOUS SYSTEM GROSS ANATOMY, CELL TYPES, AND MATERIAL BACKGROUND ........................................................................................... 7 Introduction ..................................................................................................................... 7 Anatomy of the CNS & Progress of Neurological Damage ........................................... 9 Anatomy & Physiology of the CNS............................................................................ 9 Loss of Neural Function............................................................................................ 18 Neurodegenerative Diseases ..................................................................................... 25 Role of Glia in Degeneration & Regeneration of CNS Axons ................................. 26 Biomaterials for Scaffold Preparation .......................................................................... 28 Definition of Biomaterial & Requirements for Neural TE Scaffolds ....................... 28 Biodegradable Scaffolds ........................................................................................... 29 Sample of current Biomaterials in CNS TE .............................................................. 33 Cell Sources for CNS TE .............................................................................................. 38 viii Primary Cell Treatment of CNS Injury: Glial Cells ................................................. 40 Pluripotent Stem Cells: Embryonic Stem Cells ........................................................ 42 Induced Stem Cells ................................................................................................... 46 Adult Stem Cells: Endogenous Stem Cells in the Brain & Spinal Cord .................. 48 Mesenchymal Stem Cells .......................................................................................... 52 Stimulation & Guidance ............................................................................................... 55 Physical Cues ............................................................................................................ 56 Chemical Cues .......................................................................................................... 63 Electrical Stimulation................................................................................................ 73 Concluding Remarks ..................................................................................................... 75 III. MOLECULAR LEVEL INVESTIGATION OF SPINAL CORD INJURY MODEL ........................................................................................................................................... 77 Summary ....................................................................................................................... 77 Introduction ..................................................................................................................
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