{"Affiliation":[{"label":"Affiliation","value":"Medicine, Faculty of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."}],"AggregatedSourceRepository":[{"label":"Aggregated Source Repository","value":"DSpace","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","classmap":"ore:Aggregation","property":"edm:dataProvider"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","explain":"A Europeana Data Model Property; The name or identifier of the organization who contributes data indirectly to an aggregation service (e.g. Europeana)"}],"Campus":[{"label":"Campus","value":"UBCV","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","classmap":"oc:ThesisDescription","property":"oc:degreeCampus"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","explain":"UBC Open Collections Metadata Components; Local Field; Identifies the name of the campus from which the graduate completed their degree."}],"Creator":[{"label":"Creator","value":"Manesh, Sohrab","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."}],"DateAvailable":[{"label":"Date Available","value":"2021-10-31T07:00:00Z","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"edm:WebResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"DateIssued":[{"label":"Date Issued","value":"2020","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"oc:SourceResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"Degree":[{"label":"Degree (Theses)","value":"Doctor of Philosophy - PhD","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","classmap":"vivo:ThesisDegree","property":"vivo:relatedDegree"},"iri":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","explain":"VIVO-ISF Ontology V1.6 Property; The thesis degree; Extended Property specified by UBC, as per https:\/\/wiki.duraspace.org\/display\/VIVO\/Ontology+Editor%27s+Guide"}],"DegreeGrantor":[{"label":"Degree Grantor","value":"University of British Columbia","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","classmap":"oc:ThesisDescription","property":"oc:degreeGrantor"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","explain":"UBC Open Collections Metadata Components; Local Field; Indicates the institution where thesis was granted."}],"Description":[{"label":"Description","value":"Remyelination occurs after spinal cord injury (SCI) but its functional relevance is\r\nunclear. We assessed the necessity of myelin regulatory factor (Myrf) in remyelination after\r\ncontusive SCI by deleting the gene from platelet-derived growth factor receptor alpha positive\r\n(PDGFR\u03b1-positive) oligodendrocyte precursor cells (OPCs) in mice prior to SCI. While OPC\r\nproliferation and density were not altered by Myrf inducible knockout after SCI, the\r\naccumulation of new oligodendrocytes was prevented. This greatly inhibited myelin regeneration\r\nresulting in a loss of myelinated axons at the lesion epicenter. However, spontaneous locomotor\r\nrecovery after SCI was not altered by remyelination failure. In controls with functional MYRF,\r\nlocomotor recovery preceded the onset of substantial oligodendrocyte myelin regeneration. We\r\nnext assessed locomotor recovery in a severe model of SCI where fewer axons were spared. Here\r\nanimals were still able to recover despite the inhibition of remyelination. We noticed that ion\r\nchannels were redistributed in demyelinated axons. Further testing showed knockout animals\r\nwere able to show conduction properties similar to that of control animals. Collectively, these\r\ndata demonstrate that MYRF expression in PDGFR\u03b1-positive cell derived oligodendrocytes is\r\nindispensable for oligodendrocyte myelin regeneration following contusive SCI, that\r\nremyelination is not required for spontaneous recovery of stepping in moderate or severe\r\ninjuries, and that demyelinated axons redistribute voltage gated ion channels and conduction\r\nproperties similar to that of unmyelinated axons.","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/description","classmap":"dpla:SourceResource","property":"dcterms:description"},"iri":"http:\/\/purl.org\/dc\/terms\/description","explain":"A Dublin Core Terms Property; An account of the resource.; Description may include but is not limited to: an abstract, a table of contents, a graphical representation, or a free-text account of the resource."}],"DigitalResourceOriginalRecord":[{"label":"Digital Resource Original Record","value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/76383?expand=metadata","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","classmap":"ore:Aggregation","property":"edm:aggregatedCHO"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","explain":"A Europeana Data Model Property; The identifier of the source object, e.g. the Mona Lisa itself. This could be a full linked open date URI or an internal identifier"}],"FullText":[{"label":"Full Text","value":"THE ROLE OF OLIGODENDROCYTE REMYELINATION IN LOCOMOTOR RECOVERY AFTER TRAUMATIC SPINAL CORD INJURY  by SOHRAB MANESH B.Sc., University of British Columbia, 2013   A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Neuroscience)    THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)         October 2020      \u00a9 Sohrab Manesh, 2020  ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: The role of oligodendrocyte remyelination in locomotor recovery after traumatic spinal cord injury  submitted by Sohrab Manesh  in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience  Examining Committee: Dr. Wolfram Tetzlaff, Zoology Co-supervisor Dr. Tim O\u2019Connor, Department of Cellular & Physiological Sciences Supervisory Committee Member Dr. Cornelia Laule, Pathology University Examiner Dr. Jean-S\u00e9bastien Blouin, School of Kinesiology University Examiner   Additional Supervisory Committee Members: Dr. Matt Ramer, Zoology Co-supervisor  Dr. Fabio Rossi, Medical Genetics Supervisory Committee Member    iii ABSTRACT Remyelination occurs after spinal cord injury (SCI) but its functional relevance is unclear. We assessed the necessity of myelin regulatory factor (Myrf) in remyelination after contusive SCI by deleting the gene from platelet-derived growth factor receptor alpha positive (PDGFR\u03b1-positive) oligodendrocyte precursor cells (OPCs) in mice prior to SCI. While OPC proliferation and density were not altered by Myrf inducible knockout after SCI, the accumulation of new oligodendrocytes was prevented. This greatly inhibited myelin regeneration resulting in a loss of myelinated axons at the lesion epicenter. However, spontaneous locomotor recovery after SCI was not altered by remyelination failure. In controls with functional MYRF, locomotor recovery preceded the onset of substantial oligodendrocyte myelin regeneration. We next assessed locomotor recovery in a severe model of SCI where fewer axons were spared. Here animals were still able to recover despite the inhibition of remyelination. We noticed that ion channels were redistributed in demyelinated axons. Further testing showed knockout animals were able to show conduction properties similar to that of control animals. Collectively, these data demonstrate that MYRF expression in PDGFR\u03b1-positive cell derived oligodendrocytes is indispensable for oligodendrocyte myelin regeneration following contusive SCI, that remyelination is not required for spontaneous recovery of stepping in moderate or severe injuries, and that demyelinated axons redistribute voltage gated ion channels and conduction properties similar to that of unmyelinated axons.      iv LAY SUMMARY Spinal cord injury (SCI) is a debilitating affliction that effects many Canadians. Currently there is no cure for SCI, however efforts are being made to reduce further damage to the spine; especially in the first few weeks after injury. In the spinal cord, there is a protective sheath (myelin) that surrounds nerves. These myelin sheaths are lost in the few weeks after injury. The body remakes the myelin; however, we do not know if this repair improves the ability to walk. Here we use an animal model to stop this repair process, which allows us to test whether recovery after spinal cord injury is connected to myelin sheath repair. We measure the animal\u2019s ability to walk and how that is connected to the nerves on a cellular level. Understanding how the body\u2019s own mechanisms work, we can tailor therapies to intervene early after injury and slow down the damaging processes.    v  PREFACE A version of Chapter 2 has been published:  Manesh, S. B*., Duncan, G. J*., Hilton, B. J., Assinck, P., Liu, J., Moulson, A., Plemel J.R., Tetzlaff, W. (2018). Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination. Nature Communications, 9(1), 3066.  *equal contribution A version of certain parts in Chapter 1 have been published:  Manesh S.B.*, Duncan G.J.*, Hilton B.J., Assinck P., Plemel J.R., Tetzlaff W. (2020) The fate and function of oligodendrocyte progenitor cells after traumatic spinal cord injury. Glia, 68(2):227-245.  *equal contribution In chapter 2, I conducted or was a major contributor in all the in vivo experiments. I was in charge of the breeding for the animals used in this study. The ICORD animal care staff helped with general animal husbandry and also as a second eye for maintain animal health. Surgeries for all contusions were done Dr. Jie Liu and I. I also completed the majority of cryostat sectioning and ultramicrotome sectioning for electron microscopy. I did all of the imaging for histological analyses and the histological analyses. Jason Plemel helped to design the experiment, initially ordered and maintained the transgenic mouse lines used in these experiments. Greg Duncan was also part of the design team, he also helped perform behavioral analyses and with the very first round of surgeries and helped with perfusions of mice. Greg Duncan was also a co-first author on the work that is published as he spearheaded the beginning of this project. Peggy Assinck helped with perfusions of mice and did a blinded analysis on OPC density and proliferation.  vi Brett Hilton helped with behavioural analyses and helped edit the manuscript. Aaron Moulson helped with perfusions of mice and also created figure 2.7 at my direction. Wolfram Tetzlaff supervised the project and edited Chapter 2. In chapter 3, I conducted or was a major contributor in all the in vivo experiments. I was in charge of the breeding for the animals used in this study. The ICORD animal care staff helped with general animal husbandry and also as a second eye for maintain animal health. Surgeries for all contusions were done by Dr. Jie Liu and I. I also completed the majority of cryostat sectioning and ultramicrotome sectioning for electron microscopy. Chloe Chernoff helped with behavioural testing of the animals and with one of the histological analysis at my direction. Sarah Wheeler helped with behavioral testing with the 3D kinematics and with the perfusions of the animals. Kathleen Kolehmainen and Katelyn Hudak both helped with some animal care and behavioural testing. Dr. Zhang, Dr. Jie Liu, and Dr. Matt Ramer were critical for setting up a method for recording electrophysiological data. Wolfram Tetzlaff supervised the project and edited Chapter 3. Certificate of Approval  The animal studies presented in this thesis were performed with ethics approval from the University of British Columbia Animal Care Committee (certificate #A18-0015 and A18-0081).       vii Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................ iv Preface ............................................................................................................................................. v Table of Contents ......................................................................................................................... vii List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................... xiv Acknowledgments ..................................................................................................................... xviii Chapter 1: Background Review ..................................................................................................... 1 1.1 Introduction ....................................................................................................................................... 1 1.2 Brief history of myelinating glial cells ............................................................................................. 1 1.3 Myelin ................................................................................................................................................. 2 1.3.1 Myelin evolution ............................................................................................................. 2 1.3.2 Physical morphology of myelin ...................................................................................... 4 1.3.3 Lipids of Myelin .............................................................................................................. 5 1.3.4 Myelin Proteins ............................................................................................................... 7 1.3.5 transcriptional regulators ............................................................................................... 11 1.4 Generating myelin ........................................................................................................................... 16 1.4.1 Oligodendrocyte progenitor cells .................................................................................. 16 1.4.2 OPCs in the adult CNS .................................................................................................. 17 1.4.3 Choosing an Axon. ........................................................................................................ 18  viii 1.4.4 Growing Myelin Sheaths ............................................................................................... 19 1.5 Myelin \u2013 Axon relationship. ............................................................................................................ 20 1.5.1 Oligodendrocytes and conduction ................................................................................. 21 1.5.2 Oligodendrocytes and trophic support .......................................................................... 21 1.6 Spinal cord injury ............................................................................................................................ 24 1.6.1 Pathophysiology of SCI ................................................................................................ 24 1.6.2 Secondary Injury. .......................................................................................................... 25 1.6.3 Demyelination after SCI ................................................................................................ 27 1.6.4 Remyelination after SCI ................................................................................................ 29 1.7 Locomotor recovery after spinal cord injury ................................................................................ 32 1.7.1 Spontaneous locomotor recovery occurs after SCI ....................................................... 32 1.8 Therapies for SCI ............................................................................................................................ 35 1.8.1 Transplanting cells to promote remyelination ............................................................... 36 1.8.2 Schwann cells to remyelinate after SCI ........................................................................ 37 1.8.3 Other therapies promoting remyelination after SCI ...................................................... 39 1.9 Transgenic mice as a research model  ........................................................................................... 40 1.10 Research Questions ........................................................................................................................ 41 Chapter 2: Oligodendrocyte Myelin does not contribute to locomotor recovery ....................... 42 2.1 Introduction ..................................................................................................................................... 42 2.2 Materials and Methods ................................................................................................................... 45 2.2.1 Transgenic mice and experimental design .................................................................... 45 2.2.2 Spinal Cord Injury and Animal Care ............................................................................. 47  ix 2.2.3 Tamoxifen and EdU Administration ............................................................................. 48 2.2.4 Perfusion and Tissue Processing ................................................................................... 48 2.2.5 Immunohistochemistry .................................................................................................. 49 2.2.6 Cell Counting and Tissue Analysis ............................................................................... 50 2.2.7 Electron Microscopy and Toluidine Blue Staining ....................................................... 51 2.2.8 Behavioural Assessments .............................................................................................. 52 2.2.9 Statistical Analysis ........................................................................................................ 54 2.3 Results ............................................................................................................................................... 55 2.3.1 Myrf ICKO mice have effective recombination in OPCs and fewer new oligodendrocytes expressing MYRF after SCI ...................................................................... 55 2.3.2 Myrf ICKO inhibits the accumulation of new oligodendrocytes following thoracic spinal cord contusion .............................................................................................................. 61 2.3.3 Myrf ICKO prevents oligodendrocyte remyelination by recombined cells but does not alter Schwann cell myelination after SCI ............................................................................... 65 2.3.4 Myrf ICKO results in chronic demyelination after SCI ................................................ 73 2.3.5 Hindlimb motor recovery occurs in the absence of oligodendrocyte remyelination .... 75 2.4 Discussion ......................................................................................................................................... 81 Chapter 3: In the absence of remyelination, locomotor recovery after SCI is mediated by conduction along spared demyelinated axons. ........................................................................... 90 3.1 Introduction ..................................................................................................................................... 90 3.2 Materials and Methods ................................................................................................................... 92 3.3.1 Transgenic mice and experimental design .................................................................... 92 3.2.2 spinal cord injuries ........................................................................................................ 93  x 3.2.3 Tissue preparation ......................................................................................................... 94 3.2.4 Immunohistochemistry .................................................................................................. 95 3.2.5 Confocal Imaging and ion channel stereology .............................................................. 96 3.2.6 Electrophysiology .......................................................................................................... 96 3.2.7 Behavioural studies ....................................................................................................... 97 3.2.8 Kinematics analysis ....................................................................................................... 99 3.2.9 Statistics ........................................................................................................................ 99 3.3 Results ............................................................................................................................................. 100 3.3.1 Experimental design and transgenic mice efficacy ................................................. 100 3.3.2 Larger spinal cord lesions and fewer myelin sheaths are observed after severe spinal cord injuries .......................................................................................................................... 104 3.3.3 Hindlimb locomotor recovery occurs even with severe spinal cord injury and over a long period of recovery ........................................................................................................ 107 3.3.4 Myrf ICKO causes ion channel expression along demyelinated axons ...................... 112 3.3.5 No changes to hindlimb locomotor recovery using 3D kinematics assessment ......... 115 3.3.6 Conduction along demyelinated fibres may be driving hindlimb locomotor recovery after SCI. .............................................................................................................................. 124 3.4 Discussion ....................................................................................................................................... 127 Chapter 4: Conclusions and Future thoughts .......................................................................... 133 Question 1: Does inhibiting new oligodendrocyte myelination after moderate thoracic SCI prevent hindlimb locomotor recover in mice? .................................................................................. 134 Hypothesis 1: Oligodendrocyte remyelination following a moderate thoracic spinal contusion is a major contributor to spontaneous locomotor recovery. ................................ 134  xi Question 2: How do mice recover locomotor function if remyelination is halted and does not contribute to locomotor recovery? ..................................................................................................... 137 Hypothesis 2: Spared myelinated fibres at the injury rim are capable of restoring locomotor function regardless of demyelination ................................................................................... 137 Hypothesis 3: In the absence of oligodendrocyte remyelination, locomotor recovery following thoracic SCI is mediated by demyelinated axons. ............................................... 138 Final remarks ....................................................................................................................................... 140 Bibliography ............................................................................................................................... 142             xii LIST OF FIGURES  Figure 1.1 \u2013 Comparison of select myelin proteins between CNS and PNS myelin\u2026\u2026\u2026\u2026\u2026.11 Figure 1.2: Transcriptional regulators of oligodendrocyte differentiation\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026....16 Figure 2.1 - Myrf ICKO mice have effective recombination in OPCs and do not have altered injury dynamics or tissue sparing following moderate thoracic SCI. ........................................... 58 Figure 2.2 - Myrf ICKO mice are unable to generate new oligodendrocytes in response to SCI. 64 Figure 2.3 - Myrf ICKO blocks oligodendrocyte remyelination in recombined cells after SCI. . 67 Figure 2.4 - Myrf ICKO does not alter Schwann cell myelination following SCI. ...................... 72 Figure 2.5 - Chronic demyelination of spared axons in Myrf ICKO following SCI. ................... 75 Figure 2.6 - Myrf deletion from OPCs does not impair motor recovery following moderate thoracic contusive SCI. ................................................................................................................. 77 Figure 2.7 - The location and extent of new oligodendrocyte and Schwann cell myelination after SCI and its relationship to locomotor recovery. ........................................................................... 80 Supplementary Figure 1 - Additional cohort of control and Myrf ICKO mice demonstrate no differences in recovery of hindlimb motor function following moderate thoracic contusive SCI\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026 88 Supplementary Figure 2 - Compiled data from both cohorts reveals no difference in locomotor recovery following thoracic SCI in Myrf ICKO mice\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026.\u2026 89 Figure 3.1 - Experimental design and transgenic mice efficacy\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026. 102 Figure 3.2 - Larger spinal cord lesions and fewer myelin sheaths are observed after severe spinal cord injuries\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026 105  xiii Figure 3.3 - Hindlimb locomotor recovery occurs even with severe spinal cord injury\u2026\u2026\u2026 108 Figure 3.4 - Hindlimb locomotor recovery occurs in severe spinal cord injury over a chronic time point\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026 111 Figure 3.5 - Myrf ICKO causes ion channel expression along demyelinated axons\u2026\u2026\u2026\u2026.  113 Figure 3.6 - Experimental design and BMS behavioural scores of moderately injured animals.116 Figure 3.7 - Three-dimensional analysis of mouse step cycles\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026...120 Figure 3.8 - Three-dimensional analysis of mouse joint angles\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026..123 Figure 3.9 - Conduction along demyelinated fibres may be driving hindlimb locomotor recovery after SCI\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026\u2026.. 126          xiv LIST OF ABBREVIATIONS   AIS  Impairment Scale  AMPAR  \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ANOVA  Analysis of Variance ASIA   American Spinal Injury Association ATP   Adenosine Triphosphate BMP   Bone Morphogenetic Protein BMS  Basso Mouse Scale BSB  Blood Spinal cord Barrier CNS  Central Nervous System DNA   Deoxyribonucleic Acid EAE  Experimental Allergic Encephalomyelitis EdU    5-ethynyl-2\u2019-deoxyuridine ECM  Extracellular Matrix EM   Electron Microscopy GFAP   Glial Fibrillary Acidic Protein  xv GFP   Green Fluorescent Protein GLUT1 Glucose Transporter 1 ID proteins Inhibitors of DNA-binding proteins Ig  Immunoglobulin IL   Interleukin IPL  Intraperiod Line  LINGO1  Leucine-rich Repeat and Ig-domain-containing 1  MAG  Myelin Associated Glycopro tein MBP  Myelin Basic Protein MCT  Monocarboxylate Transporter MDL  Major Dense Line mG   Membrane-Tethered Green Fluorescent Protein MN  Motor Neuron MOBP  Myelin-Associated oligodendrocytic protein MOG  Myelin Oligodendrocyte Glycoprotein MS   Multiple Sclerosis MSC  Mesenchymal Stem Cell  xvi mT   Membrane-Tethered Tomato Myrf  Myelin Regulatory Factor NF200  Neurofilament 200 NG2    Neural\/Glial antigen 2 NMDA  N-methyl-D-aspartate receptor Nrg-1  Neuregulin-1  NSPC  Neural Stem and Progenitor Cells O-  Super Oxide OCT   Optimal Cutting Temperature Compound OEC  Olfactory Ensheathing Cell OH-  Hydroxyl Radical OPC  Oligodendrocyte Precursor Cell P0  Protein Zero PBS   Phosphate-Buffered Saline  PDGFRa Platelet-Derived Growth Factor Receptor Alpha PFA  Paraformaldehyde PLP  Proteolipid Protein  xvii pMN  Motor Neuron Progenitors PNS  Peripheral Nervous System PSA-NCAM Polysialylated Neural Cell Adhesion Molecule ROS   Reactive Oxygen Species RST  Rubrospinal Tract SCI  Spinal Cord Injury TNF   Tumour Necrosis Factor UEMS  Upper Extremity Motor Score VLFA   Very Long Chain Fatty Acids WNT  Wingless And Integration Site WPI  Weeks Post Injury YFP   Yellow Fluoresecent Protein       xviii ACKNOWLEDGMENTS   The journey that I have taken to bring me here has been a difficult but extremely positive one. I would have to first off express my supervisor Dr. Wolfram Tetzlaff for the support and mentorship that he has given me over the years. I originally met Wolf in Biol 204 and through a small conversation, he had invited me to his lab to help with whatever I could. Since then we have worked together for almost 9 years. As a supervisor, he guided and taught the scientific process at first, but also allowed in my later years to create my own ideas and pursue them at my own discretion. His attitude towards mentorship has changed me not as just a student, but as a person as well. The times we have spent travelling to conferences, giving lectures, and sampling local cuisine and wine will always be with me for ever. For all the years, I cannot thank you enough Wolf.   I would also like to thank my committee members Matt Ramer, Tim O\u2019Connor, and Fabio Rossi. They were always happy to help, even when I would sometimes ask for too much. Matt especially has also been a great collaborator and co-supervisor. I had the great pleasure of working one on one with you near the end of my degree and learnt so much from you. Thank you for always having your door open and teaching me all that I have asked for.   None of this research would be possible without the generous funding support from the Canadian Institutes of Health Research (CIHR), the Wings for Life (WFL) foundation, and the Multiple Sclerosis society of Canada. The MS society provided me with a studentship, so I was able to focus on my work. The CIHR and WFL have generously provided funding for many of the projects that have been presented in this work.  Also, I would like to thank ICORD and the  xix Rick Hansen Man in Motion Foundation for the funding support over the years and for providing such an amazing research building.  I would also like to take this time and thank the wonderful members of the Tetzlaff lab for being such excellent coworkers, but most importantly like a second family. Nicole, Jie, Ward, Oscar, Behnia, Liz, Aaron, Kathleen, Nima, Katelyn, Sarah, Fraser, Peter T, Peter F, Dough, Jason, Greg, Peggy, Brett, Clarrie, Joe, Yuen, Ming, Chloe, Fatemeh, Mohammad and all the students that I had the opportunity to meet through my years at ICORD, thank you for the excellent decade. Jason Plemel, you were my first mentor and my introduction to science. When I volunteered in second year, you put me to work right away. More importantly, immediately included me into your life. It did not feel like a hierarchy, but a friendship. Your warm heart and bear hugs will forever be in my heart. Greg Duncan, Peggy Assinck, and Brett Hilton, you 3 were the senior students when I started my degree. Each and every one of you took time out of your very busy schedules to make sure I was able to learn. I would also like to thank the current Tetzlaff lab, Sarah, Kathleen, Oscar, Katelyn, Doug, Aaron, Nima, Behnia, Jie, Ming, Chloe, and Fatimeh you have all been great scientists and collaborators. Thank you for all you have done for me during my time with you. Nicole, your support has been so extraordinary.   Lastly, I would like to thank my family. My mother and father, Mitra and Amir. You both sacrificed everything for me. You pushed me to be the best that I could and have continued to push me. My loving Wife, Naomi. This PhD was just as much work for you as it was for me. I truly would not have made it without your unconditional support. Thank you all.   1 CHAPTER 1: BACKGROUND REVIEW  1.1 INTRODUCTION    In this Chapter, I aim to provide a general literature review of myelin biology and spinal cord injury (SCI).  First, I will outline the biological properties of myelin and the role that oligodendrocytes play in the developing and adult body. Then I will discuss the intricate relationship between axons and myelin, focusing on how myelin supports axonal function. As well, I will discuss how locomotion is driven by axonal conductance and what other contributors drive hindlimb locomotion. Next, I will discuss SCI, focussing on how myelin and axonal damage occurs and looking into the gaps that are left in the field. Lastly, I will outline the questions set forth for this thesis and the hypotheses that I will be testing in Chapter 2 and Chapter 3.  1.2 BRIEF HISTORY OF MYELINATING GLIAL CELLS    Myelin as we know it today was not discovered in one experiment, but incrementally formulated and understood over time through the work of many scientists. As early as the 16th century, distinctions between white matter and grey matter were made by court physician Andreas Vesalius (Vesalius et al., 1543). As glass lens technology developed through the 17th century, cylindrical \u201ctubes\u201d were described by Antoni van Leeuwenhoek when observing teased nerves (Leeuwenhoek, 1719). Leeuwenhoek took the secret of his lenses to his grave, so these theories were not widely corroborated until Robert Remak in the 19th century. The leading theory  2 until Remak\u2019s was that nerves were hollow tubes transporting the animal spirit, while Remak theorized a solid structure existed within the tube; essentially describing the axon (Remak, 1837). Soon after, Theodor Schwann, noticing nuclei within the outer membrane, showed that the inner nerve and outer sheath were distinct. He coined them the \u201csheaths of Schwann\u201d which is still used to describe Schwann cells (Schwann, 1839). The current terminology was not used until used until Rudolf Ludwig Virchow\u2019s works coined the term \u201cmyelin\u201d from the Greek \u201cMyelos\u201d to describe the fatty substance as it resembled the darker colour and texture of bone marrow (Virchow, 1854). His popular work gave the word \u201cmyelin\u201d wide exposure, even though he did not use it to specifically describe the nervous system myelin but more of a general term for substances of similar colour and texture. After the discovery of using osmium for tissue staining in 1865 by Max Schultze, myelin could be observed with high contrast. This gave the histological tools necessary to describe myelin more accurately, resulting in discoveries such as the incisures of Schmidt and Lanterman (Schmidt, 1874) to the nodes of Ranvier by Louis-Antoine Ranvier (Ranvier, 1872). Perhaps the latest historically relevant development is the discovery that myelin is created by oligodendroglia. Although debated, P\u00edo del R\u00edo-Hortega showed the most convincing evidence that myelin membranes were connected to oligodendrocyte cell bodies using the silver carbonate method of staining. He also suggested that Schwann cells and oligodendroglia serve similar functions (el R\u00edo-Hortega, 1922).  The work of these early pioneers has allowed for a large scientific community to build off these discoveries and develop further theories. The contribution of these scientist should continue to be recognized and celebrated. 1.3 MYELIN 1.3.1 MYELIN EVOLUTION   3  Understanding the evolutionary origins of myelin can give insight to its role in injury and disease. The animal in which myelin was first seen is the jawed fish placoderm; deduced from fossil records and dated to the Devonian era, some 400 million years ago (Zalc et al., 2008). These 9-meter-long prehistoric hunters dominated the seas for millions of years, at least partially due to the advantages of myelin. Yet even before the time of the placoderm, glia-neuron relationships had developed. Primitive neurons and primitive glia most likely co-evolved from epithelial cells (Rey et al., 2020). As organisms grew in size, communication between cells was necessary over longer distances and at more rapid speeds. Without myelin, axons must increase diameter to have increased conduction speed, creating a ceiling effect to how big an animal can get. Additionally, metabolically providing for large axons can be difficult, thus providing a driver towards a supportive cell that could provide both.   The evolutionary precursor to myelin is likely seen in ensheathing cells. A modern example of these cells can be seen in crustaceans like tiger prawns (genus: Penaeus) (Heuser & Doggenweiler, 1966). The axons of the tiger prawn are wrapped only once by an ensheathing glia that also facilitate node formation at their edges, much like myelin in vertebrates. These nodes combined with an inter-axonal space between the axon and the ensheathing glia provides a low-resistance ion pathway leading to one of the fastest conduction speeds in the animal kingdom of ~210m\/s (Heuser & Doggenweiler, 1966). However, these cells do not form multiple lipid layers in contrast to those found in the earthworm (genus: Lumbricus)(Xu & Terakawa, 1999). Earthworm ensheathing glia wrap axons with 60-200 lipid layers, but do not contain defined nodes yet still reach fast action potentials (Roots & Lane, 1983). Ensheathing glia in Drosophilla wrap peripheral nervous system (PNS) axons much like Remak bundles in mammals (Freeman & Rowitch, 2013). These cells also have functions similar to astrocytes, suggesting  4 diverse roles in early glia (Freeman & Rowitch, 2013). These examples show how myelin may have developed in stages, while alternatives to its function also exist in the animal kingdom. Also, research looking into the transcriptional regulatory systems in species with \u201cmyelin-like\u201d structures can greatly benefit our understanding of mammalian myelin. This topic has been reviewed by Li & Richardson (Li & Richardson, 2016).  1.3.2 PHYSICAL MORPHOLOGY OF MYELIN  Classically, the morphological details of myelin have come from work using electron microscopy and recently from live in vivo imaging as well. In short, central nervous system (CNS) myelin consists of many layers of continuous lipids wrapped tightly around an axon and attached to the axon membrane at the paranodal domains. If unraveled and laid flat, one can imagine a large rectangular sheet with one edge extending from the oligodendrocyte cell body and the other adjacent to the axon membrane. The edge opposite the oligodendrocyte is the closest to the axonal surface. The growth of myelin sheaths occurs middle-out and the leading growth edge of the sheet is the most interior layer. This has eloquently been shown using live imaging of zebrafish alongside electron microscopy (Snaidero et al., 2014).   In cross-sections of myelinated axons, there is an evident pattern of alternating electron-dense and electron-light bands. These bands are 1) the slightly thicker intraperiod line (IPL) which represent extracellular space and 2) the slightly narrower major dense line (MDL) which represent the intracellular space of the oligodendrocytes, for review (Poitelon et al., 2020). The IPL and MDL have structural differences in both the composition of lipids and their protein content (discussed further in this chapter). Longitudinally, adjacent myelin sheaths meet to form Nodes of Ranvier, with a variety of internodal distances. While an uninjured mouse spinal cord  5 will have internodal distances of ~200-400 microns in the spinal cord white matter, injury can reduce the internodal distances, thus creating more nodes per axon (Powers et al., 2012). This can affect conduction and will be discussed further in this chapter.  Nodes of Ranvier are composed of the node, the paranode, and the juxtaparanode. The node is a 0.8 to 1.1 \u00b5m long gap between adjacent myelin sheaths and here the axon constricts to ~30% to 50% of its diameter (Stadelmann et al., 2019). The paranode is the very edge of the myelin and is made of multiple septate-like structures that repeat and attach the myelin to the axon (Einheber et al., 1997; Gumbiner & Louvard, 1985; Rosenbluth, 1976). The paranode flanks the node on both sides and covers 3 to 4 times the area (Hildebrand et al., 1993; Stadelmann et al., 2019). The juxtaparanode is located adjacent to the paranodes and within the periaxonal space. All these components are integral to saltatory conduction and will be discussed in the section 1.5.1. Along the length of the myelin sheath also exist cytoplasmic channels that allow for the free diffusion molecules between the inner and outer myelin sheaths (Velumian et al., 2011). Lastly, gap junctions connect oligodendrocyte myelin to the periaxonal space, the axon, and surrounding astrocytes and pericytes (Scherer et al., 1995). Overall, the morphology of myelin reveals great detail about how it works and in combination with lipidomic, proteomic, and genetic studies we can attempt to understand its role in human disease and injury.  1.3.3 LIPIDS OF MYELIN   Compared to most cells in the body, myelin contains a much higher percentage of lipids. While an average cell contains about a 50% lipid to 50% protein ratio, myelin exhibits a lipid composition of about 70%-85% and a protein composition of about 30%-15% (Morell, 1999). As with most cell membranes, three main classes of lipids comprise the myelin membranes;  6 phospholipids, glycolipids and cholesterol. Notably, the ratio of these classes in myelin is distinct with a 40%:20%:40% (phospholipids, glycolipids, cholesterol) compared to a 65%:10%:25% in most other biological membranes (O'Brien, 1965; Poitelon et al., 2020). Phospholipids within myelin take on multiple forms such as plasmalogen, lecithin, and sphingomyelin, while glycolipids are commonly found in the form of galactosylceramide and sulfatides (Poitelon et al., 2020). Unlike some myelin proteins, these lipids are not specific to myelin, but instead their collective behaviour has a unique dynamic that gives myelin its properties.   Lipid membranes are not a solid mass, but a fluid and changing structure. To give more stability to myelin, there is a higher presence of saturated very long chain fatty acids (VLFA) (Sastry, 1985). Long hydrocarbons lacking double bonds pack very tightly next to one another and create a more dense and rigid membrane. Reducing VLFAs within galactosylceramides and sulfatides with a knockout of ceramide synthase 2 gene reduces myelin stability and disrupts myelin compactness (Imgrund et al., 2009). The biophysical properties of myelin lipids are necessary for ion permeability, as high membrane fluidity using ceramide galactosyltransferase knockout disrupts saltatory conduction (Bosio et al., 1998). Myelin also contains lipid domains in its external leaflets known as lipid rafts that are composed of cholesterol and other lipids like galactosylceramide and phosphatidylcholine (Gielen et al., 2006). Lipid rafts are essential for the formation of myelin through protein trafficking (like myelin proteolipid protein (PLP)), the survival of oligodendrocytes through integrin-mediated growth factor signalling, and facilitate axon-glia interactions (Decker & ffrench-Constant, 2004; Simons et al., 2000). Lipid rafts have been more difficult to study compared to proteins for many years, but as more creative methods of experimentation are being developed, more insight will be made into the diverse roles that lipid rafts play in myelin (Boyanapalli et al., 2005; Vinson et al., 2003).   7  Both developmental myelination and adult remyelination are metabolically expensive processes. De Novo synthesis of fatty acids and cholesterol help provide the molecular building blocks needed to create such large lipid membranes. Fatty acid synthesis by oligodendrocytes (Dimas et al., 2019) and by astrocytes (Camargo et al., 2017) are both essential for the formation of developmental myelin, but in both cases can be compensated for with fatty acid uptake from external sources, albeit with delayed myelination. Similarly, impairment of galactosylceramide synthesis does not prevent myelination, but detrimentally effects myelin maintenance in the long term (Saadat et al., 2010). This again goes back to how lipids function as a collective and that the absence of individual lipids can be compensated for (Schmitt et al., 2015). However, during remyelination all efforts should be taken to reduce potential bottlenecks in the synthesis of myelin. For example, taurine supplementation provides the serine pools necessary for glycosphingolipid biosynthesis and drastically enhances the effects miconazole on oligodendrocyte differentiation and myelination (Beyer et al., 2018). As well, when done in moderation, cholesterol consumption can improve myelination (Berghoff et al., 2017; Saher et al., 2012; Zhou et al., 2019). Overall, lipids serve a structural, signalling, and maintenance role for myelin and are essential to its function.  1.3.4 MYELIN PROTEINS  Myelin specific proteins have a vital function in myelin structure and function. Although less diverse compared to cytoplasmic proteins, myelin specific proteins are needed in large quantities. Some proteins like Proteolipid protein (PLP) make up as high as 45% of total myelin proteins (Jahn et al., 2009). There is noticeable diversity between CNS and PNS myelin proteins. Though they share some proteins (eg. Myelin Basic Protein), distinct myelin specific proteins exist in the CNS only (eg. PLP) and PNS only (eg. Protein Zero). The transcriptional control of  8 these proteins is also important in disease and injury. A complete review of myelin proteomics and genomics is beyond the scope of this section, instead this section will focus on key proteins of CNS myelin and a summary of their transcriptional control.  1.3.4.1 Myelin Basic Protein  Myelin basic protein (MBP) is found in CNS and PNS myelin. The protein has a very high net charge and an isoelectric point of ~10, making it a very basic protein (Harauz et al., 2009). MBP has a primary role of maintaining the long-term stability of lipid layers and myelin compaction (Suresh et al., 2010). Within the MDL, the net negative charge of phospholipid head interacts with the positive charge of MBP and help attract and fold the protein (Nawaz et al., 2009; Raasakka et al., 2017; Widder et al., 2018). This sandwiches MBP between lipid layers and the protein acts as a positively charged \u201cglue\u201d to keep the negatively charged membranes held together. The ablation of the mbp gene was studied many years ago in the shiverer mouse which produces myelin with fewer loops, visible cytoplasm between lamellae, and a severe seizure-like phenotype resulting in a greatly reduced lifespan (Chernoff, 1981; Rosenbluth, 1980). MBP can be spliced into multiple isomers and has roles outside of myelin compaction as well (Vassall et al., 2015). For example, MBP plays a role in oligodendrocyte differentiation by interacting with Fyn Kinase during development (De Avila et al., 2014; Smith et al., 2012a; Smith et al., 2012b). Also, interactions with actin and calmodulin shows certain isomers of MBP are involved in oligodendrocyte processes outgrowth. These interactions provide insight into the multifaceted roles that single protein genes can have because of alternative splicing, however, the dominant role of MBP remains concentrated on myelin maintenance and compaction.   9 It should be briefly mentioned that Myelin-Associated oligodendrocytic protein (MOBP) is also a basic protein that is abundantly found in CNS myelin (Yamamoto et al., 1994). However, its function is not as clear. Homozygous knockouts of MOBP do not produce any overt changes to myelin phenotypes, yet it can be used to induce experimental allergic encephalomyelitis (EAE) in mice, showing it may have a role in MS pathology (Montague et al., 2006; Yool et al., 2002). 1.3.4.2 Proteolipid Protein   Proteolipid protein (PLP) is the most abundant protein found in CNS myelin sheaths (Jahn et al., 2009). This highly hydrophobic protein is found nested within the plasma membrane as a dimer (Daffu et al., 2012; Greer & Lees, 2002). There is also an alternate splice form termed DM20 with 35 fewer residues that is found in large quantities in CNS myelin. Mutations in the PLP1 gene in humans is the most likely cause of Pelizaeus-Merzbacher disease (Garbern, 2007; Woodward, 2008). PLP-null mice are capable of forming myelin but display abnormities in MDL and IPL sizes and subtle losses of compaction in the outer lamellae when observed with electron microscopy (Rosenbluth et al., 2006; Rosenbluth et al., 1996). These mice are more sensitive to osmolarity changes, suggesting PLP plays a role in myelin stability. Likewise, overexpression of PLP can lead to dysmyelination (Karim et al., 2007; Rosenbluth et al., 2009). A likely mechanism could be that changes to lamellar compaction from altered PLP expression can affect the recruitment of other important proteins like MBP (Karim et al., 2007). Recently, altered expression of PLP has also been linked to defects in mitochondria and increased oxidative stress in oligodendrocytes and axons, a potential cause of axonal degeneration (Appikatla et al., 2014; Huttemann et al., 2009; Ruiz et al., 2018).  10 1.3.4.3 Immunoglobulin family proteins  Immunoglobulin (IG) family proteins are cell adhesion molecules that play a critical role in myelin. They contain an extracellular domain, a transmembrane domain, and an intracellular domain. One member of IG myelin proteins is myelin associated glycoprotein (MAG). MAG is expressed in PNS and CNS and is found in the noncompact regions of myelin and on the innermost layer, contacting the axon (Raasakka & Kursula, 2020). Two different isoforms exist; S-MAG and L-MAG. S-MAG binds to microtubules and is thought to help stabilize myelin (Kursula et al., 2001; Kursula et al., 1999). L-MAG dimerizes and binds the axon using gangliosides, forming a defined 9-12nm periaxonal space. The cytoplasmic domains of L-MAG activate Fyn kinase, a process necessary for the initiation of myelination (Marta et al., 2004; Yamauchi et al., 2012).   A CNS specific IG protein is myelin oligodendrocyte glycoprotein (MOG). MOG is most notable for its autoantigen properties and is the most commonly used peptide in autoimmune encephalomyelitis (EAE), a rodent model of MS (Mayer & Meinl, 2012). The function of MOG is still being elucidated, but the protein forms a dimer and could serve as an adhesion molecule between adjacent oligodendrocyte myelin sheaths, a trait not seen in PNS myelin where MOG is not found.  The most common Ig myelin protein is protein zero (P0), found normally in PNS myelin. There it helps facilitate myelin compaction by forming antiparallel dimers using its extracellular domain between lipid layers (Raasakka et al., 2019). Although not normally found in CNS myelin, after a contusion SCI immunohistochemistry shows many P0 positive myelin sheaths are present (covered in more detail in section 1.8.2).  11  Figure 1.1 \u2013 Comparison of select myelin proteins between CNS and PNS myelin. Top illustration represents a Schwann cell and bottom a single oligodendrocyte myelin sheath. Figure showing proteins that overlap between the two types of myelin and also specific proteins to CNS and PNS myelin. This is not a complete list, but highlights the proteins used in this thesis and a select few important proteins. Legend: P0 (myelin protein zero), P2 (peripheral myelin protein 2), PMP22 (peripheral myelin protein 22), PRX (periaxin), EC (epithelial cadherin), MBP (myelin basic protein), MAG (myelin-associated glycoprotein), Cxs (connexin), PLP (Proteolipid protein), MOG (Myelin\/oligodendrocyte glycoprotein), MOBP (Myelin-associated oligodendrocytic basic protein), OSP (oligodendrocyte-specific protein\/claudin 11), Jux (juxtanodin), CNPase (2 ,3 -cyclic nucleotide 3 -phosphodiesterase)   1.3.5 TRANSCRIPTIONAL REGULATORS   The transition from oligodendrocyte precursor to a mature myelinating oligodendrocyte requires key transcription factors (Li & Richardson, 2016). Although the proteins discussed thus far give myelin functional properties, key transcriptional regulators guide myelin formation and oligodendrocyte differentiation. A delicate balance must be struck between regulators that promote a proliferative state (eg. Hes5, Id, SoxD) and those that promote a differentiative or  12 myelinating state (eg. Olig2, Nkx2.2, Sox10, Myrf) for proper myelination and remyelination. Here some of these applicable players will be briefly discussed, but for the sake of length, many regulators are left out. 1.3.5.1 Proliferative Factors  The proliferation of oligodendrocyte progenitor cells (OPCs) is an intricate cascade of factors that regulate the process. Here I will only provide a brief overview of some factors, for a more detailed review see (Bergles & Richardson, 2015). The transcription factor Hes5 (hairy and enhancer of split 5) gets its name from the drosophila gene responsible for neurodevelopment (Nakao & Campos-Ortega, 1996). The Hes family of genes bind promoter regions, specifically N-box and E-box regions, of key genes and recruit histone deacetylases to repress expression (Akazawa et al., 1992; Grbavec & Stifani, 1996). In OPCs , Hes5 is upregulated to maintain a proliferative state by repressing myelin gene expression (Liu et al., 2006). It also works by sequestering Sox10, thereby preventing its bioactivity (Liu et al., 2006).   A series of structurally related genes, (Sox3, Sox6, and Sox 13) are collectively known as SoxD proteins. These genes help promote a proliferative OPC state. Deletion of SoxD transcription factors from OPCs results in differentiation into oligodendrocytes (Stolt et al., 2006). SoxD proteins also have a role in OPC migration directly promoting PDGFRa expression in conjunction with Sox10 (Baroti et al., 2016). Interestingly, Sox10 is involved in SoxD protein expression, thus suggesting a complex regulatory control system guides these functions (Dugas et al., 2010; Reiprich et al., 2017; Sock & Wegner, 2019; Stolt et al., 2006).  Lastly, Id (Inhibitors of DNA-binding) proteins also promote a proliferative state in OPCs. Both Id2 and Id4 inhibit oligodendrocyte differentiation, working as downstream  13 effectors of BMP signalling. The factors outlined here show a small window of the complex regulatory system involved in oligodendrocyte lineage cell regulation. Many of these interactions are still being uncovered and their study can lead to a stronger understanding of remyelination failure in diseases and injuries.  1.3.5.2 Olig2  The Olig family of genes (Olig1, Olig2, Olig3, Olig4) are from a sub-group of the helix-loop-helix family of genes that share a common ancestral origin and also include inhibitors of DNA-binding (ID) and Hes 5\/6 (Kuspert & Wegner, 2016). Of interest here, Olig1 and Olig2 are the factors associated with promoting myelination. Early on during development, Olig2 is expressed in the motor neuron progenitor (pMN) domain where the majority of astrocytes and oligodendrocytes lineage cells originate (Li et al., 2011). Olig2 is also expressed in cells that become motor neurons (MN), as an Olig2 knockout in mice results in a lack of spinal motor neurons and thus failure to survive past birth (Lu et al., 2002; Takebayashi et al., 2002; Zhou & Anderson, 2002). However, Olig2 only defines the fate of MNs, its forced continual expression actually inhibits their differentiation (Lee et al., 2005). Importantly, Olig2 also specifies OPC fate and is expressed in these cells prior to defining OPC markers such as PDGFRa and NG2 (Lu et al., 2002; Takebayashi et al., 2002; Zhou & Anderson, 2002). It has been proposed that the diverse functions of Olig2 during development comes from specific phosphorylation of certain amino acid residues.    After OPC specification, Olig2 continues to play an important role in oligodendrocyte lineage cells. Overexpression of Olig2 in Nestin-positive progenitors results in enhanced OPC generation (Maire et al., 2010) and overexpression of Olig2 in OPC results in greater  14 oligodendrocyte differentiation in response to focal demyelinating lesions (Wegener et al., 2015). Olig 2 is essential for myelin production and is expressed in oligodendrocytes; from OPC to mature myelinating oligodendrocytes. Olig1 is also important for the maintenance of myelin and is involved in the production of MBP (Li et al., 2007). However, whether Olig1 is essential for developmental myelin is disputed as Olig1 is absent in birds, despite the presence of myelin (Li & Richardson, 2016; Paes de Faria et al., 2014).  1.3.5.3 Sox 10   Sox10 is found in the oligodendrocyte lineage as well as Schwann cells, although in the CNS Sox9 and Sox8 are also expressed. The 3 structurally similar paralogs are collectively termed SoxE proteins and their similarities provide some redundancy between them (Stolt et al., 2004; Stolt et al., 2003; Stolt et al., 2002). Sox9 is expressed first during development and is responsible for the maintenance of neural stem cells (Scott et al., 2010) as well as the specification of glial fate (Stolt et al., 2003). Further in development, Sox9 and Olig2 guide the expression of Sox8 and Sox10 as OPC specification begins (Kang et al., 2012). Sox10 and Sox8 are crucial for oligodendrocyte differentiation and myelination as a double knockout halts myelination altogether (Hornig et al., 2013). Sox8 cannot rescue a Sox10 knockout, even though some residual myelin genes are still expressed in Sox10 null mice (Hornig et al., 2013). In OPCs, Sox10 drives a survival and migratory state by activating PDGFRa expression (Finzsch et al., 2008). Sox10 also has binding affinity and participates in the regulation of NG2 expression (Gotoh et al., 2018). Therefore, it seems Sox10 plays both a role in maintaining an OPC state as well as being essential in differentiation and myelination. It is the interaction of Sox10 with Myrf that redirect it away from OPC genes towards oligodendrocyte differentiation (Aprato et al., 2020; Emery, 2013).   15 1.3.5.4 Myelin regulatory factor  Unlike Olig2 and Sox10, myelin regulatory factor (Myrf) is only expressed in mature and maturing oligodendrocytes. Myrf is a membrane-tethered transcription factor which undergoes autoproteocleavage to form 2 parts, the N-terminus which binds DNA and a C-terminus that remains in the endoplasmic reticulum (Bujalka et al., 2013; Li et al., 2013). This autoproteocleavage is preceded by a trimerization of MYRF in a process that is mediated by intramolecular chaperone domains within the protein, similar to that of bacteriophage tailspike proteins (Bujalka et al., 2013; Li et al., 2013; Schwarzer et al., 2007). The N-terminus of Myrf is then released into the nucleus still as a trimer with three identical binding domains, a morphology that is required for proper binding of DNA (Aprato et al., 2020). Within the nucleus Myrf interacts not only with the DNA, but other transcription factors as well.  During OPC differentiation, Sox10 induces the expression of Myrf (Hornig et al., 2013). However, once Myrf is in the nucleus it binds Sox10, stopping Sox10 from binding OPC-specific regions and instead guiding it to genes associated with differentiation and myelination (Aprato et al., 2020; Hornig et al., 2013). Myrf\u2019s interaction with Olig2 is less known. Olig2 can bind the enhancer regions of Myrf with cooperative effects of Brg1\/Brm, a chromatin remodeling protein (Bischof et al., 2015; Yu et al., 2013). The studies mentioned so far put Sox10, Olig2, and Myrf at the beginning of the regulatory cascade, making them key regulatory proteins in myelination and great targets for studying demyelination and remyelination.  16  Figure 1.2: Transcriptional regulators of oligodendrocyte differentiation. Pointing arrows on the top dictate those genes that promote differentiation. T-arrows on the bottom show factors that inhibit differentiation or promote proliferation of OPCs. This is not a complete list but highlights those genes talked about in this thesis and some other important factors. Myrf ICKO (used in this study) inhibits the transition of Pre-myelinating oligodendrocytes to myelinating oligodendrocytes.  1.4 GENERATING MYELIN  Myelination of the CNS occurs during post-natal development and the process is highly controlled. Not all axons will end up being myelinated and those that are, may not stay myelinated throughout life. The process is affected by chemical, physical, and activity-dependent cues, culminating in a highly tuned network of myelinated and unmyelinated axons that remain plastic into and throughout adulthood. Here we will discuss the myelination process, beginning with oligodendrocyte progenitor cells.  1.4.1 OLIGODENDROCYTE PROGENITOR CELLS The CNS houses a cell with stellate morphology that is found in grey and white matter known as oligodendrocyte progenitor cells or OPCs. Developmentally, these cells (expressing markers of PDGFRa, NG2) are first seen in the ventricular germinal zones of the brain and spinal cord at embryonic day 12.5 (E12.5)(Pringle & Richardson, 1993; Timsit et al., 1995; Warf  17 et al., 1991) while a second wave of these cells are observed in the dorsal cord at E15.5 (Cai et al., 2005; Fogarty et al., 2005; Tripathi et al., 2011; Vallstedt et al., 2005). Ultimately, these two populations of OPCs display near identical properties, yet still populate their respective areas of the spinal cord in adults (Tripathi et al., 2011). The embryonic origins and plasticity of embryonic OPCs has been thoroughly reviewed and beyond the scope of this introduction (Bergles & Richardson, 2015). 1.4.2 OPCS IN THE ADULT CNS In adult mice, OPCs cover the entire CNS through a mechanism of self-repulsion (Hughes et al., 2013). This allows them to claim a territory devoid of other OPCs and constantly scan their environment with branching filopodia. In healthy adults, if an OPC is lost or differentiates, a neighbouring OPC quickly senses this, proliferates, and replaces it (Hughes et al., 2013). OPCs form the myelinating cell of the CNS, the oligodendrocyte, not only during development but throughout adulthood as well (Hill et al., 2018; Hughes et al., 2018; Young et al., 2013), possibly to fine-tune conduction in neural circuits (Chang et al., 2016; Fields, 2015; Purger et al., 2016). Their ability to produce myelinating oligodendrocytes in adults makes them a primary candidate in studies of learning and myelin plasticity as well (Hughes et al., 2018). There has been many studies showing myelination accompanies the learning of skilled tasks (Hughes et al., 2018; Keiner et al., 2017; Xiao et al., 2016; Young et al., 2013).  OPCs are also mechanosensitive. In vitro studies show they differentiate preferentially in softer environments versus stiffer ones (Jagielska et al., 2012; Urbanski et al., 2016). Although CNS mechanical properties change with age and in some neurological diseases (Antonovaite et al., 2018; Arani et al., 2015; ElSheikh et al., 2017), their correlation with OPC function is  18 understudied. After SCI, the mechanical stiffness of the spinal cord is increased by the glial scar (Cooper et al., 2020; Saxena et al., 2012). A combination of microglia, extracellular matrix (ECM), reactive astrocytes, and molecules such as chondroitin sulfate proteoglycans (CSPG) form the glial scar are contributors to inhibition of regeneration. (Busch & Silver, 2007; Horner & Gage, 2000; Silver & Miller, 2004). The ECM particularly contributes the physical stiffness of the injury area (Cooper et al., 2020; Saxena et al., 2012)and is an important direction for future research as it pertains to OPC mechanosensitivity after SCI and potentially remyelination.  1.4.3 CHOOSING AN AXON.   Choosing which axon to ensheathe is an important part of myelination. Oligodendrocytes have intrinsic mechanisms that guide this process. This was tested with paraformaldehyde fixed axons which obviously mimic axonal diameter accurately, although there is potential for surface markers to still be present and functional (Rosenberg et al., 2008). However, even with engineered carbonfibre (Althaus et al., 1987), glass (Bullock & Rome, 1990), vicryl (Howe, 2006), or polystyrene nanofibers (Lee et al., 2012a), oligodendrocytes ensheathe and begin myelination. It also seems that the ensheathment of manmade fibres, occurs before the production of myelin proteins like MBP (Lee et al., 2012a), suggesting physical cues are important early in myelination. This phenomenon has also been observed in this study (fig 2.3.4 d). Furthermore, oligodendrocytes display region specific responses. For instance, when cultured on nanofibres, oligodendrocytes from the spinal cord make myelin with longer internodes than oligodendrocytes of the cortex, mimicking in vivo internodes and showing intrinsic regional identity without the existence of external cues (Bechler et al., 2015).   19  So why then do oligodendrocytes not myelinate every fibre-like structure in the body? One reason (as mentioned in the nanofiber studies above) is that even though axonal size can influence myelination, a variety of other cues are also present in the CNS. Negative cues such as polysialylated neural cell adhesion molecule (PSA-NCAM) exist on immature neurons that inhibit myelination (Charles et al., 2000). Other negative signals like leucine-rich repeat and Ig-domain-containing 1 (LINGO1) on neurons and OPCs, or the secreted factor semaphorin3A found in MS lesions play a role in inhibiting myelination (Lee et al., 2007). Axonal activity is a substantial factor in myelin plasticity and can regulate myelin thickness (Gibson et al., 2014; Kirby et al., 2006; Mitew et al., 2018; Ortiz et al., 2019). Even the density and microenvironment of OPCs can affect their fate (Rosenberg et al., 2008). These examples only show a sliver of the complex cues that regulate myelination and leads us into to exploring the events after myelination has begun.  1.4.4 GROWING MYELIN SHEATHS   Myelin sheath growth in the CNS is different than that of the PNS. Schwann cell myelin first grows along the length of the axon and then begins to wrap (Bunge et al., 1961). This means that along the length of the sheath, the same number of layers and amount of thickness is observed during the myelination process. The growing edge of the sheath is the most inner layer that is touching the axon, a commonality between CNS and PNS myelination. A combination of in vivo imaging in zebrafish and high-pressure freeze fracture microscopy has shed light into how CNS myelin grows.   The growing myelin sheath of oligodendrocytes can be imagined as similar to a croissant. The growing tongue begins encircling the axon from the middle, adding layers as it loops while  20 also growing laterally (Snaidero et al., 2014). This has been observed using high-pressure freezing methods that preserve myelin cytostructure. This method also highlights cytoplasmic channels within the myelin sheath, presumably to transport the essential building blocks of myelin from the cell body to the leading tongue. Although these channels close after development, they can be re-opened using elevated phosphatidylinositol-(3,4,5)-triphosphate that reinitiate myelin growth (Goebbels et al., 2010; Snaidero et al., 2014).   Due to the transparency and size of juvenile zebrafish, they make an excellent model to visualize the growth of myelin as the entire animal can be imaged while alive. In vivo time-lapse videos of zebrafish myelination show that oligodendrocytes establish myelin length in about 3 days and are able to maintain it throughout the growth of the animal\u2019s body size (Auer et al., 2018). Ablating single myelin sheaths results in the restoration of the myelin back to pre-ablation levels (Auer et al., 2018; Kirby et al., 2006). An oligodendrocyte will at first over-produce myelin sheaths, then prune back 10-20% of these sheaths as myelin elongates and matures (Czopka et al., 2013). A single oligodendrocyte can myelinate large and small axons simultaneously (Almeida et al., 2011), suggesting myelin sheaths are tailored to axons and oligodendrocytes are adaptable. After myelination is complete, myelin stays relatively stable throughout adult life (Yeung et al., 2014).   1.5 MYELIN \u2013 AXON RELATIONSHIP.   Understanding the relationship between myelin and axons can help us understand the pathobiology of myelin diseases and injury. Myelin helps axons serve their function by speeding up impulses while also providing axons with trophic support, consequently helping in the survival of axons. Here we will briefly cover the relationship between axons and myelin.   21 1.5.1 OLIGODENDROCYTES AND CONDUCTION  Myelin facilitates rapid signal conduction in axons. As a multilamellar membrane, myelin increases the resistance and lowers the capacitance of the nerve fibre. These insulating properties allow for rapid impulse propagation, with current being reinitiated at the unmyelinated nodes of Ranvier in a process called \u2018saltatory conductance\u2019 (Lillie, 1925). This permits the rapid propagation of action potentials in even relatively small caliber axons, minimizing the space necessary for fast conduction of signals. Subtle myelin changes can also fine tune impulse propagation. Factors such as internodal length, myelin thickness or partial myelination of single axons can be modulated to alter conduction velocity throughout life (Etxeberria et al., 2016; Ford et al., 2015; Hill et al., 2018; Hughes et al., 2018). After SCI, demyelination may silence or slow action potential propagation (Blight, 1983; Hains et al., 2004; James et al., 2011; Nashmi & Fehlings, 2001) and potentially contribute to functional deficits.   1.5.2 OLIGODENDROCYTES AND TROPHIC SUPPORT  Axons project long distances from the neuronal cell body, which presents logistical challenges for energy production. Within the axon, the areas of highest energy demand are at the nodes of Ranvier, where mitochondria accumulate, particularly during periods of high electrical activity or axonal stress (Misgeld et al., 2007; Ohno et al., 2011). Although myelin reduces the energy requirements for repeated action potentials, the overall metabolic requirement of myelinated axons is higher than developmentally unmyelinated axons when factoring in myelin genesis and maintenance of oligodendrocyte resting potential (Harris & Attwell, 2012).  22 Demyelination results in the redistribution and upregulation of ion channels along the axon (England et al., 1991; England et al., 1990). More specifically, Nav1.2, Nav 1.6, Na\/Ca2+ exchanger, Kv1.1, and Kv1.2 expression is upregulated and redistributed on demyelinated axons (Craner et al., 2004; Rasband et al., 1998). Although normally confined to nodes of Ranvier and juxtaparanode, these ion channels spread along the axon length following demyelination and increase the energy expenditure necessary to repolarize the axolemma. Demyelination therefore shifts energy demands to the axons rather than spreading the ATP usage between separate cells; demyelinated axons use more energy per unit length than myelinated axons (Waxman, 2006b).  The increased energy demands of the demyelinated axon may cause an energy crisis ultimately leading to their degeneration. If ATP demands are not met, Na+\/K+ ATPases will not effectively remove Na+ ions thus reversing the Na+\/ Ca2+ exchanger and causing an influx of Ca2+ ions (Stys et al., 1991). This overload of intracellular Ca2+ ions activates calcium dependent systems such as calpains, which in turn can lead to degradation of the cytoskeleton, compromising axonal structure (Liu et al., 2014; Posmantur et al., 1997). Increases to intracellular Ca2+ can trigger a mitochondrial permeability transition that leads to depolarization of mitochondrial membranes, impairing mitochondrial function and contributing to axonal degeneration (Bernardi, 1992; Lehninger et al., 1978). Since many white matter axons are frequently active, being forced to maintain and replenish an ion gradient over such a large surface area can lead to ATP depletion and a state of \u201cvirtual hypoxia\u201d that eventually leads to axonal death (see review by (Trapp & Stys, 2009)). Given the slow movement of molecules within the cytoplasm (Oblinger et al., 1988) and that oligodendrocyte myelin physically separates axons from vasculature, they must receive metabolites locally, at least during times of high energy demand (Nave, 2010).    23 The idea of glia-axon energy transfer was first proposed by way of lactate shuttling through monocarboxylate transporters (MCTs) from astrocytes to neurons (Brown et al., 2004; Brown et al., 2003; Pellerin & Magistretti, 1994). Although astrocytes do provide contact with axons, their interaction is limited to neuronal cell bodies, synapses, and the nodes of Ranvier, whereas oligodendrocytes make considerably more contact with energetically demanding white matter axons. This puts them in a favorable position to provide metabolic support to axons. Given than myelin cuts axons off from vasculature, axons rely on the transport of monocarboxylic acids like lactate, pyruvate, or ketone bodies from oligodendrocytes to maintain a high state of activity. MCT1, a transporter of lactate, is highly enriched in oligodendrocytes and seems to be a necessary for axons as its disruption causes axonal damage (Lee et al., 2012b). More so, glycolytic oligodendrocytes (done with a COX mutation) are fully capable of supporting themselves and axons through lactate alone (Funfschilling et al., 2012). However, in the corpus callosum, it has recently been shown that oligodendrocytes are capable of supporting axons through glucose delivery when MCT1 is inhibited but fail when glucose transporter 1 (GLUT1) is also inhibited (Meyer et al., 2018).  These studies outline mechanisms which oligodendrocytes can support axons, yet there are many variabilities between axons of various caliber, location, and activity. This regional heterogeneity may explain why oligodendrocytes have evolved to have NMDA receptors which play an important role in adapting to the need of axons (Saab et al., 2016). Although their contact with axons is much less than oligodendrocytes, astrocytes still play a major role in CNS energy metabolism where they serve to uptake glucose, store glycogen, and pass along energy substrates to oligodendrocytes through gap junctions and axons near nodes of Ranvier (Brown & Ransom,  24 2007; Maglione et al., 2010; Nave & Werner, 2014). It has become evident in recent years that axons depend on oligodendrocytes for their function and health.  However, it remains unclear if demyelination and oligodendrocyte loss is driving axonal degeneration after SCI. Whether or not demyelination persists for long enough after SCI or whether the extent of demyelination crosses this threshold to induce axon loss is unclear. If demyelination persists in more chronic settings, mitochondrial DNA damage and free radical production creates a vicious cycle increasing the likelihood of axonal death due to a metabolic deficit (Campbell et al., 2014; Criste et al., 2014). Collectively, it is clear that axons depend on oligodendrocytes for their function and health, and oligodendrocyte deficiency may leave axons vulnerable to loss.   1.6 SPINAL CORD INJURY  In Canada, there are approximately 80,000 individuals with a spinal cord injury (SCI), half of those are considered traumatic SCIs and an estimated 1500-2000 new cases occur every year (Noonan et al., 2012). Worldwide, these numbers can be up to 500,000 new cases every year (Lee et al., 2014). A traumatic SCI can leave an individual with chronic impairment and pain, life-long changes to their day-to-day activities, and introduce significant financial burdens. Understanding the pathophysiology of SCI can lead to treatments that help alleviate these hardships.   1.6.1 PATHOPHYSIOLOGY OF SCI  25    The pathophysiology of traumatic SCI can be broken down to two phases; primary injury and secondary injury. Primary injury is caused by the physical forces put onto the spinal cord at the time of injury. Often the direct damage comes from the surround tissue of the spinal cord. Dislocated vertebrae or burst fracture are common injuries and involve shearing and compression forces to the spinal cord respectively (Mattucci et al., 2019; Myers & Winkelstein, 1995). The magnitude of force, the velocity, and the displacement of the spinal cord will dictate the severity of the initial injury (Mattucci et al., 2019; Tator, 1983). The majority of the SCIs in humans are anatomically incomplete (Biering-Sorensen et al., 2011; Bunge et al., 1993; Young, 2002). This means tissue is spared at the injury cite and only partial motor and sensory outputs are lost. Although, when combined with the heterogeneity of the population and the complexity of the spinal cord; every injury can be unique. Primary forces and tissue displacement kill a portion of axons, oligodendrocytes, vasculature, and other cells of the spinal cord. The immediate damage occurs quickly, but the violent death of these cells causes cascading damage to the adjacent spinal cord tissue that survived the initial impact.  1.6.2 SECONDARY INJURY.   The cascade of damage after primary injury defines the secondary injury. This process begins minutes after injury and can extend for weeks. Hemorrhaging, ischemia, excitotoxicity, oxidative stress, inflammation are all contributors of secondary injury (Oyinbo, 2011)   Vasculature damage coincides with injury severity and can be seen within minutes after injury and is followed by low blood flow to the affected regions of the injury for up to 24 hours  26 (Choo et al., 2007; Noyes, 1987; Rivlin & Tator, 1978). Even if larger vessels remain intact, the smaller network of capillaries rupture, breaking the blood-spinal cord-barrier (BSB) (Tator & Koyanagi, 1997). Within days after injury, this breakdown of the BSB can spread to adjacent areas of the spinal cord that were not damaged during the primary insult (Popovich et al., 1996). Although perhaps highly dependent on the injury, re-establishment of the BSB can take up to 2 months after SCI (Bartanusz et al., 2011; Cohen et al., 2009), leaving the spinal cord vulnerable to blood-borne pathogens, inflammatory cells and serum components not normally found in the spinal cord. Ischemia reduces metabolic provisions (oxygen and glucose) needed for ATP production (Dirnagl et al., 1999), while high ATP demand needed to maintain membrane potentials and, in the case of oligodendrocytes, to maintain myelin lipid and protein turnover, leads to cellular death (Harris & Attwell, 2012; Szydlowska & Tymianski, 2010).   Another contributor of secondary injury is excitotoxicity. Neurotransmitters like glutamate accumulate in the injury cite and can lead to not only neuronal damage but oligodendrocyte death as well (Gottlieb & Matute, 1997; Karadottir & Attwell, 2007; Vanzulli & Butt, 2015). Glutamate induced excitotoxicity, consequent kinase, NMDA, and AMPA receptor activation leads to increased intracellular Ca2+ concentrations (Faden & Simon, 1988; Li & Stys, 2001; Wada et al., 1999). This leads to mitochondrial dysfunction (see section 1.5.2), the release of pro-apoptotic factors and activation of caspases (Li & Stys, 2000) (Matute et al., 2007). Excitotoxicity is exasperated by the length and intensity of glutamate exposure, correlating it to injury severity (Matute et al., 2007). Since oligodendrocytes express AMPA receptors on their cell bodies and NMDA receptors on myelin sheaths, they are especially vulnerable to excitotoxicity (Karadottir et al., 2005; McDonald et al., 1998; Micu et al., 2006).   27 Oxidative stress is another contributor of secondary damage (Carrico et al., 2009; Fleming et al., 2008). Oligodendrocytes are especially susceptible to oxidative stress (Husain & Juurlink, 1995; Merrill et al., 1993). Oxygen species such as super oxide (O-) (Liu et al., 1998)and hydroxyl radicals (OH-) (Bao & Liu, 2004; Liu et al., 2004) are generated after SCI. Nitrogen species like peroxynitrite can be destructive to oligodendrocytes as well (Scott et al., 1999; Xiong et al., 2007). Both of these are short-lived intermediary products that are highly reactive, thus damaging cells via lipid, protein, and DNA peroxidation (Aksenova et al., 2002; Demopoulos et al., 1982; Xiong et al., 2007). The abundance of myelin lipids and importance of myelin proteins can make oligodendrocytes especially vulnerable to oxidative damage, while OPC\u2019s high iron levels lower their capacity to scavenge reactive oxygen and nitrogen species.   Other contributors of secondary injury like elevated extracellular ATP levels contribute to oligodendrocyte death (Wang et al., 2004). Pro-inflammatory cytokines like tumour necrosis factor-\u03b1 (TNF-\u03b1) and interleukin (IL)-1\u03b2 have also been shown to induce oligodendrocyte death (Sherwin & Fern, 2005; Steelman & Li, 2011). Both of these contributors have peak concentrations around 24 hours after injury (Kwon et al., 2010; Stammers et al., 2012; Wang et al., 2004). The mechanisms stated here show only a fraction of the complexity of the secondary injury process, yet for the focus of this introduction the consequence is cell death and demyelination after spinal cord injury. 1.6.3 DEMYELINATION AFTER SCI   Demyelination is the process of losing myelin sheaths after SCI and was first observed almost 50 years ago using electron microscopy (Gledhill et al., 1973a). Using a weight-drop model of SCI in cats, Blight (1985) observed rapid destruction of axons and myelin in the first 2  28 days of injury. However, he also observed continuous demyelination of axons that had survived the primary injury from 2-7 days after injury alongside massive invasion of macrophages and membranous debris (Blight, 1985). He suggested that such secondary mechanisms could be the culprit.   Studies following this provided more diverse evidence for demyelination after SCI. Apoptotic cells are found hours to weeks after SCI in the white matter tract of rat spinal cords (Crowe et al., 1997). This corroborated (Blight, 1985) that cellular death could be a primary mechanism for demyelination. Reduced levels of myelin protein transcripts are observed after injury in studies using in-situ mRNA hybridization (Wrathall et al., 1998). Lowered gene expression of myelin proteins could suggest altered expression of surviving cells but could also be indicative of demyelination after injury. Ex vivo measurements of compound action potentials after SCI shows altered conduction suggestive of demyelination (Fehlings & Nashmi, 1995). The breakup of nodes of Ranvier and the distribution of voltage gated ion channels on axons within the lesion cite suggest not only demyelination and also adaptations to demyelination by the axons (Arroyo et al., 2002; Karimi-Abdolrezaee et al., 2004). Notably, merely the presence of remyelination (seen even early on in the field (Gledhill et al., 1973a, 1973b)) suggests that demyelination occurs after SCI. As more genetic and imaging tools became available, more accurate evidence of the timing and extent of demyelination was discovered.   Earlier work suggested that chronic demyelination may be present for months to years after injury. Looking in rats 12 months after SCI, suggestions were made of at least 30% of axons remain demyelinated (Salgado-Ceballos et al., 1998). Others had similar conclusions, showing at least some demyelinated axons even up to 450 days after injury (Totoiu & Keirstead, 2005), suggesting a failure to completely remyelinated but also that demyelinated axons had  29 survived at the lesion cites for this extended period of time. However, many had also reported that no chronic demyelination remains and there have been conflicting reports in the past of whether demyelination is found in chronic SCI. Even in human studies, there are reports of both chronic demyelination as well as no demyelinated axons (Bunge et al., 1993; Guest et al., 2005; Kakulas, 1999; Norenberg et al., 2004) . Studies of mouse rubrospinal tract (RST) and corticospinal tract (CST) after injury showed that by 12 weeks no demyelinated axons were found (Lasiene et al., 2008; Powers et al., 2012). The myelination status of axons also varies depending on the relative location to the injury cavity seen in rats (James et al., 2011). There is little doubt that demyelination occurs after SCI.  1.6.4 REMYELINATION AFTER SCI   Remyelination is the re-establishment of myelin sheaths on axons that were previously myelinated. Newly generated myelin exhibits certain characteristics that distinguish it from more \u201cmature\u201d myelin. Many of the studies mentioned above showing demyelination, have also shown remyelination (Blight, 1985; Gledhill et al., 1973a; Griffiths & McCulloch, 1983). Early evidence for remyelination came from electron microscopy. When observed in cross-sections using transmission electron microscopy, remyelinated axons appear thinner (having a higher g-ratio). This was the only available methodology of distinguishing new myelin for many years but has a downside of misinterpreting normally thin myelin from new myelin, such as those found on smaller caliber axons of the corpus callosum. Also, as this is done in cross-sections, after injury it is very difficult to distinguish whether the axons are spared or remnants of proximal segments of transected axons. Lastly, recent evidence shows that new myelin can be made by existing adult oligodendrocytes (Bacmeister et al., 2020; Duncan et al., 2017b; Duncan et al., 2018b).  30 Myelin sheaths made this way will appear thin and can be misinterpreted as new myelin (Duncan et al., 2018b). Nonetheless, the sheer abundance of thin myelin after SCI gives evidence that remyelination occurs after SCI.   By tracing mouse RST axons using actively transported dextrans, remyelination was observed on spared axons after contusion SCI (Lasiene et al., 2008). This study showed that internode lengths were much shorter in remyelinated axons, illustrating characteristics of oligodendrocyte myelin formation after SCI in mice. The same group later showed a similar finding in rats, while also showing that severed axons remain within the lesion and could remain demyelinated (Powers et al., 2012). Severed axons would be visually similar to demyelinated intact axons in cross-sections, potentially a source of false positive accounts of demyelination. The presence of punctate Caspr staining, found exclusively at paranodes (Peles et al., 1997), was used as a surrogate measure of intact myelin (Arancibia-Carcamo et al., 2017; Etxeberria et al., 2016; Pedraza et al., 2009). Additionally, shorter internodes, characteristic of adult-derived myelin and remyelination (Young et al., 2013), near the lesion area in the RST suggest extensive remyelination (Powers et al., 2012). No intact axons had evidence of Caspr or potassium channel spreading, typical of demyelination, and contrasted with transected axons (Powers et al., 2012). Together, these data indicate that spared axons are most likely not chronically demyelinated and implies they are instead effectively remyelinated.  Cellular fate-mapping of oligodendrocytes and OPCs has shed light on remyelination after SCI. Early work utilized chemical demyelination or irradiated diet to produce myelin vacuolation and subsequent demyelination in the spinal cord to measure remyelination (Duncan et al., 2009; Jeffery & Blakemore, 1997). However, these methods have the negative side of producing debris and an inflammatory response that could skew results. Modern genetic tools,  31 such as tamoxifen inducible CreER-activated GFP expression in PDGFRa+ cells, can labels OPCs in the CNS (Assinck et al., 2017b; Hesp et al., 2015; Kang et al., 2010), a model also used in this dissertation. Since the timing of GFP expression is controlled via tamoxifen injections, detailed chronology of OPC proliferation, migration, and oligodendrocyte differentiation has been mapped (Hesp et al., 2015). Both new and total amounts of oligodendrocytes increase significantly after SCI induced demyelination, the majority of which occurs within 4 weeks of injury (Hesp et al., 2015). More extensive fate-mapping of OPCs shows upwards of 28% of the axons at the lesion epicenter being wrapped by new myelin at 12 weeks post SCI, some of which by Schwann cells (Assinck et al., 2017a). Schwann cell remyelination has been observed after SCI previously (Felts & Smith, 1987; Honmou et al., 1996), and their role in functional recovery will be discussed below.   Since such detailed tools do not exist in human studies, it is difficult to ascertain the quantity and timing of remyelination in patients with SCI. Humans often have poor remyelination efficiency in diseases like multiple sclerosis (MS) (Goldschmidt et al., 2009; Patrikios et al., 2006; Yeung et al., 2019), and thus it is plausible that remyelination efficiency might be decreased after SCI relative to rodents. Most studies are done post-mortem and show some demyelinated axons, albeit a relatively low number (Guest et al., 2005; Kakulas, 2004; Kakulas & Kaelan, 2015; Norenberg et al., 2004). Perhaps the presence of demyelinated axons could be explained by the low rate of proliferation and differentiation of human OPCs vs rodent OPCs (Ehrlich et al., 2017; Windrem et al., 2004; Yeung et al., 2014). However, merely an absence of demyelinated axons years after injury could be explained by demyelination induced axonal death (Buss et al., 2004; Nave & Werner, 2014) just as easily as efficient remyelination. Post mortem evidence of remyelination relies on the presence of thin myelin sheaths seen with  32 TEM and immunohistochemistry of Schwann cell myelin which is normally not found within the CNS, both of which have been observed (Bunge et al., 1993,Guest, 2005 #215; Kakulas, 2004; Kakulas & Kaelan, 2015). Other animal models like canines (Jeffery et al., 2006) or primates (Lachapelle et al., 2005) also display effective spontaneous remyelination. So, it seems remyelination does occur in humans, but more research tools are necessary to determine the kinetics of this process. Advances in myelin water imaging (MRI) has made progress in detecting myelin changes in MS patients (Laule & Moore, 2018) and may be the ideal route for SCI studies in humans, although we may yet be years from this.    1.7 LOCOMOTOR RECOVERY AFTER SPINAL CORD INJURY   Spinal cord injuries are life changing to patients and lead to a drastic loss in quality of life for most people. A systematic review of multiple studies that directly surveyed people with SCI showed upper extremity function and walking consistently came in the top 25% of life domain priorities for patients, similar with bowel movements and sexual function (Simpson et al., 2012). Understanding how people with SCI prioritize their needs will help guide research to better serve the community. Here we will focus on the biology of lower limb locomotion and how it may be possible to give back some of the lost function to SCI patients. 1.7.1 SPONTANEOUS LOCOMOTOR RECOVERY OCCURS AFTER SCI   As mentioned previously, the majority of human SCIs are anatomically incomplete (Bunge et al., 1993). This means that many people with SCIs have some amount of locomotor  33 function (Biering-Sorensen et al., 2011; Raineteau & Schwab, 2001; Young, 2002), which are assessed using clinical tests such as the American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade or the upper extremity motor score (UEMS).  Yet within the first year after injury, about 70% of patients spontaneously recover at least one motor score level regardless of their initial motor level (Steeves et al., 2011). This spontaneous recovery typically occurs in the early months after injury and may continue for up to 18 months, suggesting some plasticity exists (Fawcett et al., 2007; Roth et al., 1991).  This type of locomotor recovery without any intervention is also seen in animal models of SCI (Gensel et al., 2006; Hilton et al., 2016). Following a unilateral cervical SCI in non-human primates, significant recovery of forelimb and hand function occurs without treatment (Nout et al., 2012; Salegio et al., 2016). More widely used hindlimb scoring systems for rat SCI shows that spontaneous locomotor function is partially restored in rats as well (Basso et al., 1995; Manohar et al., 2017).  Mice, used exclusively in the experiments of this dissertation, also show locomotor recovery of hindlimb function within 2-3 weeks after injury despite no intervention (Basso et al., 2006; Duncan et al., 2018a). The question is, where does this \u201cspontaneous\u201d recovery come from and is it an appropriate candidate for intervention?  In the motor system, recovery arises as a result of plasticity in the motor cortex, in descending pathways and in local changes within the spinal cord itself (Barbeau et al., 1999; Brown & Martinez, 2019; Isa, 2017). Topographical maps of the motor cortex correlate with limb movements and can be studied non-invasively in humans using transcranial magnetic stimulation (Hallett, 2007). Cortical motor representations change following learning of motor tasks in human and animal models (Kleim et al., 1998; Pascual-Leone et al., 1995), but not after unskilled tasks that do not require learning (Plautz et al., 2000) (Kleim et al., 2004). After SCI  34 cortical maps reorganize, reducing some motor areas and expanding others to reflect patients\u2019 motor functions. In animal models, rehabilitation after SCI can significantly influence motor maps while improving function (Girgis et al., 2007; Martinez et al., 2009b) . For example after lateral hemisection of the spinal cord in rats, the motor maps are lost, but then reorganize to represent the new spontaneously gained function (Fouad et al., 2001; Martinez et al., 2010). The motor cortex is highly plastic and capable of reorganizing to compensate for lost function, even after unilateral pyramidotomy the contralateral cortex can take on some of the motor roles that were lost. The mechanisms guiding this plasticity are complex and beyond this introduction (reviewed (Fink & Cafferty, 2016; Serradj et al., 2017))  Recovery can also be mediated by descending pathways that form \u201crelay\u201d connections. A T12 lateral hemisection injury will abolish stepping on one side of the animal, but given time, connections are made from supraspinal fibres to non-injured ipsilateral fibres and restore function to the affected limb (Courtine et al., 2008). These relays can occur multiple times following consecutive injuries and are abolished with NMDA-mediated ablation of gray matter spinal cord neurons above the lesion (Courtine et al., 2008). Both corticospinal and reticulospinal neurons have a capacity to form relay connections with propriospinal neurons in incomplete models of SCI, while also showing significant spontaneous behavioural recovery (Bareyre et al., 2004; Filli et al., 2014). When combined with rehabilitation, relay connections are reinforced, and the effects are more pronounce (Asboth et al., 2018). These relays likely present in more severe injuries and contribute in some way to spontaneous recovery so long as some fibres are spared and bypassing the lesion.   Lastly, local changes in spinal cord central pattern generators (CPGs) can contribute to spontaneous locomotor recovery. It has been known for decades that oscillating and rhythmic  35 firing of spinal neurons can generate gait with zero input from the brain (Forssberg et al., 1980a; Forssberg et al., 1980b; Grillner & Zangger, 1979). This plays a role in walking, running, swimming, and other processes like breathing. Following a complete spinal cord transection in cats, spontaneous coordinated walking can occur if weight support is provided (Forssberg et al., 1980a; Forssberg et al., 1980b). Kinematics of fully transected mice show walking is limited after injury, but animals spontaneously re-express hindlimb locomotion within 14 days after spinalization. This locomotor recovery is maintained even after a second transection (Leblond et al., 2003), showing that indeed circuits below the injury are responsible.  Ultimately for incomplete injuries, all of these mechanisms likely contribute in some way to recovery. Importantly, much of this relies on spared fibres that pass the injury site. It is therefore important to minimize secondary damage to these fibres and enable them to function at their peak capacity. This means understanding the role that myelin plays on spared axonal fibres after SCI can help maximize recovery for patients of SCI.   1.8 THERAPIES FOR SCI   Now that we have discussed how most injuries leave spared axons that cross the lesion and that demyelination occurs after injury, it is not surprising that promoting remyelination after SCI has been an attractive target for therapies. This idea is logical and leans heavily on characteristics discussed so far. Those being that 1) prolonged demyelination can lead to axonal degeneration and that 2) Myelin facilitates conduction. Accordingly, many have hypothesized that myelin regeneration is a plausible therapeutic target to promote signal conduction and  36 protect axons from secondary damage (Alizadeh et al., 2015; Myers et al., 2016; Papastefanaki & Matsas, 2015; Plemel et al., 2014). However, there is no direct evidence that oligodendrocyte remyelination contributes to the limited functional recovery after SCI. Despite this, transplantation of myelin-forming cells is\/has been used in several clinical trials of SCI (https:\/\/clinicaltrials.gov\/). Moreover, oligodendrocyte lineage cells have diverse roles following traumatic SCI beyond oligodendrocyte remyelination. They are capable of producing remyelinating Schwann cells, contribute to glial scaring where they alter the growth of axons and may even regulate immune responses. Here, I will discuss some approaches that have been attempted while highlighting the necessity for the basic understanding of the role myelin plays after SCI.  1.8.1 TRANSPLANTING CELLS TO PROMOTE REMYELINATION    Transplantation of cells into the lesions of SCIs has been an approach many have pursued (Myers et al., 2016). Cells that are commonly used in transplantation studies are neural stem and progenitor cells (NSPCs), OPCs, olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs), for review (Assinck et al., 2017a; Tetzlaff et al., 2011). Any cell put into the spinal cord with the intention of promoting remyelination will be in competition with endogenous OPCs, Schwann cells, and oligodendrocytes that are very effective at remyelinating the spinal cord after injury (Hesp et al., 2015; Lasiene et al., 2008; Powers et al., 2012). With this in mind, the window in which enhancing remyelination could be useful is likely early after injury. Various studies have addressed this, with many transplanting cells in the first few weeks after injury to show remyelination of the lesion and some locomotor recovery (Biernaskie et al., 2007; Cao et al., 2010; Karimi-Abdolrezaee et al., 2006; Pearse et al., 2004). These studies also show  37 remyelination using methods that clearly identify new myelin like myelin thickness measurements using electron microscopy or co-labelling of transplanted cells with myelin protein antibodies. Yet merely the presence of myelin does not mean that remyelination is the source of recovery after transplantation and many mechanisms exist that could be attributing to recovery (Assinck et al., 2017a; Tetzlaff et al., 2011), especially as transplanted NPCs and OPCs can differentiate into multiple cell types.   The best evidence for transplantation promoting myelination and locomotor recovery comes from studies using the shiverer mouse line (Chernoff, 1981; Rosenbluth, 1980), which contains NPCs unable to produce myelin. NPCs from shiverer mice were transplanted into injured spinal cords and compared to transplantations of wildtype NPCs (Hawryluk et al., 2014; Yasuda et al., 2011). In both studies shiverer NPCs failed to produce myelin in wildtype injured animals while also correlating with less functional recovery compared to wildtype NPCs. However, these cells were ensheathing axons and thereby most likely preventing any endogenous remyelination from occurring. This then provides evidence for a potential negative effect. The complexity of cell transplantation makes it difficult to pinpoint the exact mechanisms of locomotor recovery and therefore requires additional controlled basic biological experiments. 1.8.2 SCHWANN CELLS TO REMYELINATE AFTER SCI   As mentioned previously, Schwann cells are not normally found in the CNS and only myelinate the PNS. However, after SCI much of the dorsal spinal cord is myelinated by Schwann cells (Assinck et al., 2017a; Biernaskie et al., 2007; Bunge et al., 1993). Schwann cell myelin is seen in the CNS after induced demyelination as well (Hampton et al., 2012; Zawadzka et al., 2010). While, the majority of these Schwann cells are derived from PDGFR\u237a-positive OPCs and  38 are myelinating axons in the spinal cord, both FOXJ1-expressing non-myelinating and P0+ myelinating Schwann cells from the PNS form remyelinating Schwann cells within the CNS (Assinck et al., 2017a; Ma et al., 2018; Zawadzka et al., 2010). Although the mechanism guiding a fate-switch in OPCs to differentiate into Schwann cells is still not understood, Schwann cells are almost exclusively found where there are fewer astrocytic processes (Assinck et al., 2017b; Talbott et al., 2006; Zawadzka et al., 2010). It has been shown that a BMP\/Wnt rich environment devoid of astrocytes guides OPCs into a Schwann cell fate, while at the same time inhibiting them from forming oligodendrocytes (Ulanska-Poutanen et al., 2018). Schwann cell differentiation from OPCs is dependent on the injury environment with contusive injuries being very conducive to producing an environment capable of supporting considerable Schwann cell myelination. In the PNS, Schwann cell remyelination is capable of restoring conduction as well (Felts & Smith, 1992; Honmou et al., 1996). Therefore, there is potential for this form of remyelination to be contributing to functional recovery after SCI.  The gene Neuregulin-1 (Nrg-1) is expressed in Schwann cells and experimenting with its expression levels lead to hypomyelination or hypermyelination axons (Michailov et al., 2004). Inducible deleting Nrg-1 correlates with less remyelination in the spinal cord after SCI and lower locomotor functional scores (Bartus et al., 2016). Unfortunately, the tamoxifen inducible CAG-creER mice used in this experiment removed Nrg-1 from all cells (Hayashi & McMahon, 2002). Nrg-1 is important in many other cellular functions like inflammation, astrogliosis, and oligodendrocyte differentiation, and therefore could be guiding recovery by other means (Alizadeh et al., 2017; Gauthier et al., 2013). More recently, an excellent model was used to inhibit OPC derived Schwann cells specifically knocking out ErbB3\/4 receptors from PDGFRa-positive cells (Bartus et al., 2019). This resulted small BMS subscore changes and deficits in  39 beam-walking tests. The authors propose that remyelination of a small number of sensory fibres affects the proprioceptive inputs for functional locomotor recovery, but their data do not rule out other indirect mechanisms resulting from ErbB3\/4 deletion.  The methods in which remyelination effects recovery after SCI are still being uncovered.    1.8.3 OTHER THERAPIES PROMOTING REMYELINATION AFTER SCI    One last method that will be discussed is the cellular manipulation of OPCs to induce remyelination after SCI. As OPCs are the resident myelinating cells of the CNS and highly prolific, it seems logical to pursue means of forcing differentiation and myelination as a means of testing locomotor recovery. After SCI, OPC differentiation is inhibited with bone morphogen protein (BMP) signalling released from astrocytes in the lesion environment and blocking BMP signalling promotes oligodendrocyte differentiation (Hart et al., 2020; Sellers et al., 2009; Wang et al., 2011). Another inhibitor of oligodendrocyte differentiation (Kotter et al., 2006; Plemel et al., 2013) and axonal regeneration (Boyanapalli et al., 2005; McKerracher et al., 1994; Wang et al., 2002) is myelin itself. As myelin debris and myelin regeneration is plentiful after injury, this can be an important factor in recovery after injury. As for improving locomotor recovery after SCI, recombinant Nrg-1-b1 treatment can result in significantly increased number of oligodendrocytes while reducing astrocyte differentiation along with causing open-field locomotor improvements (Gauthier et al., 2013; Kataria et al., 2018). Although alongside promoting remyelination, this also reduces glial scarring and alters inflammation (Gauthier et al., 2013). It is difficult to attribute functional gains from treatments such as Nrg-1 or BMP  40 inhibitors like noggin to just myelin regeneration, however this route of treatment should continue to be explored for therapeutic purposes. 1.9 TRANSGENIC MICE AS A RESEARCH MODEL    Many of the therapies mentioned above and the basic science that has led up to their use were validated using animal models. The most common animals used to study SCI are rats and mice, with primates, pigs, cats, dogs, and other animals making a smaller portion of the studies (Sharif-Alhoseini et al., 2017). Rodents have the advantage of being relatively inexpensive, leading to studies with a large power, are highly validated through years of being used, and have similar electrophysiology, functional, and morphological characteristics to humans (Cheriyan et al., 2014; Kwon et al., 2002; Metz et al., 2000). Non-human primates have the advantage of modeling more complex tasks to assess recovery and being genetically and physiologically more similar to humans, however, their advanced cognition, slower life cycle, and higher overall expense makes them difficult to use in large numbers (Cheriyan et al., 2014; Iwanami et al., 2005; Kwon et al., 2002). Larger animals such as pigs can model the physical attributes of human spinal cords very well, making the murine model excellent for translation studies involving transplantation (Gao et al., 2019; Kwon et al., 2002; Schomberg et al., 2017).  For this dissertation, we use the mouse as a model for all the experiments. Mice have the advantage of being inexpensive and having a short life cycle, allowing for in house breeding and ultimately, large experimental cohorts that lead to higher powered experiments. However, the biggest advantage mice bring is their transgenic potential. Being able to remove one specific gene, from one specific type of cell type allows for the designing of very specific hypotheses and experiments. In this dissertation, we are able to halt an entire process (oligodendrocyte  41 remyelination) by genetically removing one gene (myrf), allowing us to ask questions that can help uncover the pathology of SCI. Overall, there is no one perfect model for SCI, but the experimental design and cost factors all play a role in deciding which model fits best.    1.10 RESEARCH QUESTIONS  So far, we have covered that myelination is an ongoing process that occurs well into adulthood. Myelin provides support for axons by creating efficient conduction and providing metabolic support. After incomplete SCI, some locomotor function is lost but then regained spontaneously. Similarly after SCI, some myelin is lost, but then regained naturally. However, the relevance of myelin production in functional recovery after SCI is still unknown. Over the course of this thesis, I aim to address the following questions: 1. Question 1: Does inhibiting new oligodendrocyte myelination after moderate thoracic SCI prevent hindlimb locomotor recover in mice? a. Hypothesis 1: Oligodendrocyte remyelination following a moderate thoracic spinal contusion is a major contributor to spontaneous locomotor recovery. 2. Question 2: How do mice recover locomotor function if remyelination is halted and does not contribute to locomotor recovery? a. Hypothesis 2: Spared myelinated fibres at the injury rim are capable of restoring locomotor function regardless of demyelination  b. Hypothesis 3: In the absence of oligodendrocyte remyelination, locomotor recovery following thoracic SCI is in part mediated by demyelinated axons.   42 CHAPTER 2: OLIGODENDROCYTE MYELIN DOES NOT CONTRIBUTE TO LOCOMOTOR RECOVERY1  2.1 INTRODUCTION  SCI can lead to severe and permanent motor, sensory and autonomic dysfunction due to the adult mammalian spinal cord\u2019s inability to regenerate lost neurons and their connections (Hilton & Bradke, 2017). Most SCIs in humans do not result in the complete transection of the spinal cord but instead axons are spared at the lesion epicentre (Norenberg et al., 2004) and a period of limited functional improvement commences soon after SCI despite axon regeneration failure(Fawcett et al., 2007; Steeves, 2015). Enhancing the functional connectivity of the spared circuitry may be a viable means of promoting functional improvements following SCI (Hilton et al., 2016). However, chronic oligodendrocyte death (Crowe et al., 1997) and demyelination of spared axons are characteristic after SCI (Blight, 1985; Bresnahan et al., 1976; Lasiene et al., 2008; Totoiu & Keirstead, 2005) and could diminish connectivity of spared circuits. Demyelination impairs the amplitude and speed of electrical conductance (Blight, 1983; James et al., 2011; Nashmi & Fehlings, 2001) and oligodendrocyte loss may leave axons vulnerable to degeneration (Funfschilling et al., 2012; Lee et al., 2012b). For these reasons, strategies to enhance oligodendrocyte remyelination of spared axons have been hypothesized to promote functional improvements following SCI (Alizadeh et al., 2015; Myers et al., 2016; Papastefanaki & Matsas, 2015; Plemel et al., 2014).   1 This chapter has been published. (doi: 10.1038\/s41467-018-05473-1)  43 Myelin regeneration is a spontaneous process: new oligodendrocytes and Schwann cells regenerate lost myelin in the absence of therapeutic intervention (Assinck et al., 2017a; Barnabe-Heider et al., 2010; Bartus et al., 2016; Hesp et al., 2015; Lytle & Wrathall, 2007; Powers et al., 2013; Sellers et al., 2009; Tripathi & McTigue, 2007). PDGFRa+ OPCs produce new oligodendrocytes after SCI (Assinck et al., 2017a; Barnabe-Heider et al., 2010; Hesp et al., 2015). Ependymal cells can also contribute to oligodendrocyte production, albeit minimally (Barnabe-Heider et al., 2010; Meletis et al., 2008). Intricate transcriptional regulation is required for OPCs to differentiate into new myelinating oligodendrocytes. During both development and myelin regeneration, the transcription factor myelin regulatory factor (Myrf) is essential for OPC differentiation and myelin protein expression (Duncan et al., 2017a; Emery et al., 2009). Nevertheless, the role of MYRF has not been elucidated after SCI, nor whether PDGFR\u03b1+ OPCs constitute an indispensable source of remyelinating oligodendrocytes. The functional relevance of oligodendrocyte remyelination after SCI is also unclear. Remyelination in the spinal cord is correlated with improvements in locomotion following chemical demyelination and after the consumption of an irradiated diet (Duncan et al., 2009; Jeffery & Blakemore, 1997). Transplantation of cells capable of forming new oligodendrocytes after SCI is coupled with functional improvements when the overall extent of remyelination is increased (Cao et al., 2010; Karimi-Abdolrezaee et al., 2006; Keirstead et al., 2005; Plemel et al., 2011). Endogenous myelin regeneration is an efficient process after SCI, as indicated by the presence of numerous thinly myelinated axons (Blight, 1985; Gledhill et al., 1973a; Harrison & McDonald, 1977; James et al., 2011), shorter internodes (Lasiene et al., 2008; Powers et al., 2012; Powers et al., 2013) and by fluorescently labeling new myelin in transgenic mice (Assinck et al., 2017a; Hesp et al., 2015). Given the extent of endogenous oligodendrocyte remyelination,  44 it is plausible that remyelination contributes to the limited level of locomotor recovery after SCI. However, axons are capable of conducting through short segments of demyelination in vivo (Felts et al., 1997) and the extent of demyelination amongst intact axons may not be sufficient to contribute to detectable functional decline. Despite this, myelin regeneration is the mechanistic basis of several ongoing clinical trials and has become an important (Alizadeh et al., 2015; Papastefanaki & Matsas, 2015), yet contentious (Assinck et al., 2017a; Myers et al., 2016; Plemel et al., 2014) therapeutic target.  To ascertain the role of myelin regeneration in locomotor recovery, we used transgenic mice which permit the selective ablation of oligodendrocyte remyelination. Oligodendrocyte remyelination requires the differentiation of OPCs into new oligodendrocytes (Kang et al., 2010; Keirstead & Blakemore, 1997), so we deleted Myrf, crucial for OPC differentiation (Duncan et al., 2017a; Emery et al., 2009), prior to moderate thoracic spinal cord contusion injury in mice. We found that MYRF was essential for both the accumulation of new oligodendrocytes and for remyelination. This demonstrates that effective remyelination requires local PDGFR\u03b1+ precursors to differentiate and express MYRF after SCI to generate new oligodendrocytes. Surprisingly, the recovery of hindlimb motor function assessed on open field testing, the horizontal ladder and Catwalk gait analysis was unaltered by the deletion of Myrf from OPCs. Further, by labeling new myelin, we demonstrate that nearly all new oligodendrocyte myelin forms after the initial recovery of hindlimb stepping in mice with functional MYRF. These data indicate that while spontaneous remyelination is extensive following SCI, it is not associated with improvements in hindlimb motor function during spontaneous recovery in this model.    45     2.2 MATERIALS AND METHODS  2.2.1 TRANSGENIC MICE AND EXPERIMENTAL DESIGN   Procedures involving live animals were approved by the University of British Columbia, in accordance with guidelines from the Canadian Council on Animal Care (A13-0328). Experiments were initiated in 8-10 week old mice that were group housed, fed a standard chow ad libitum and maintained on a 12 hour reverse dark\/light cycle for the experiment. Myrffl\/fl mice (Emery et al., 2009)(Jackson Laboratory stock # 010607), which express LoxP sites around exon 8 of Myrf, were crossed with PDGFR\u03b1-CreERT2 (Kang et al., 2010)(Jackson Laboratory stock # 018280) mice to ultimately produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice, hereafter referred to as Myrf ICKO. Exon 8 of Myrf contains the putative DNA binding domain and its deletion results in a truncated, non-functional protein (Duncan et al., 2017a; Emery et al., 2009; McKenzie et al., 2014). Littermate Myrffl\/fl animals lacking Cre recombinase were used as controls. Mice were on a mixed strain background comprised of C57bl\/6 and SJL. A total of n=76 mice were used in two cohorts. The larger cohort was used for histological analysis and behavioural scores were reported for this cohort in the results. Cohorts were not combined as injuries were done on two different IH impactors and resulted in slightly different levels of recovery between the control  46 groups beginning 3 weeks post injury (WPI) until 6 WPI (F (1, 21) = 9.850, two-way repeated measures ANOVA, P=0.005). Behavioural data from the second cohort was reported in Supplementary Fig. 1, and combined data in Supplementary Fig. 2. Group sizes were determined prior to the experiment by conducting a power analysis from data generated in a pilot experiment with mice on the same genetic background to determine the group size which is required to detect a 1 BMS difference ( n=14 per group) given the variability in our data (\u03b1<0.05, Power = 0.80). To ensure we had sufficient power, a total of n=60 Myrf ICKO and control mice received spinal cord injuries. Animal grouping could not be randomized as it was dependent on genotype. An additional, n=16 (8 Myrf ICKO and 8 controls, split between males and females) aged-matched animals without an injury were examined to determine if Myrf ICKO was sufficient to induce demyelination or behavioural deficits during this timeframe without an injury. Injured animals (n=4 Myrf ICKO, 5 controls) were excluded due to subsequent health issues including digit autotomy (1 mouse), hernia (1 mouse), bladder infections\/complications (5 mice), or surgical deaths (2 mice). Additionally, mice were excluded after the experiment if they were statistical outliers (below lower quartile - 1.5x the interquartile range or above the upper quartile + 1.5x interquartile range) on displacement relative to their experimental grouping (2 mice) or if they demonstrated evidence of plantar stepping immediately post injury (3 mice) (BMS score \u2265 4), both a priori exclusion criteria. The remainder of the mice (n=46) were used in behavioural and histological analyses and there were n=23 Myrf ICKO and n=23 controls, with n=12 males in the control group and n=10 males in the Myrf ICKO group. Mice were perfused for use in either immunohistochemistical or electron microscopic analysis. Mice used for electron microscopy were grouped so they did not statistically differ in their BMS scores from those used for immunohistochemistry. Genotyping was performed on ear clippings and DNA was extracted  47 using the REDExtract-N-AMP Tissue Kit (Sigma, St. Louis, MO, R4775) and amplified with primers specific for the transgenes (Emery et al., 2009; Kang et al., 2010; Muzumdar et al., 2007). Genotypes were visualized by running PCR solutions on a 1.5% agarose (Invitrogen, Carlsbad, CA, 16500) gel. Following the experiment, all animals were genotyped again to ensure the fidelity of the groups.   To determine the extent of new myelin produced by OPCs, Myrffl\/fl PDGFR\u03b1-CreERT2 or Myrfwt\/wt PDGFR\u03b1-CreERT2 mice were crossed with Rosa26-mGFP (mT\/mG) mice (JAX # 007576)(Muzumdar et al., 2007) which induces GFP expression that is tethered to the membrane following tamoxifen induced Cre-mediated recombination (mGFP). When labeled OPCs differentiate and form new myelinating oligodendrocytes this approach allows for the visualization of new myelin (Duncan et al., 2017a; Kang et al., 2010; Powers et al., 2013). A total of n=23 mice were used in the study and n=1 animal died during surgery bringing the total animals to n=12 perfused at 6 weeks post injury (WPI) and n=10 perfused two weeks post injury.   2.2.2 SPINAL CORD INJURY AND ANIMAL CARE  Prior to surgery, mice were anaesthetized for three minutes with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1mL of Ringer\u2019s solution (Braun, Montreal, Canada, L7500) and buprenorphine (0.05mg\/kg) (Reckitt-Benckiser Slough, Toronto, Canada) analgesic prior to surgery. The back was shaved and then disinfected using successive betadine (Purdue Pharma, 41731) and 70% alcohol washes. Upon entering surgery, the mice were placed on a heatpad controlled by a rectal temperature probe set to 37oC. An incision and  48 separation of the erector trunci muscles from the spine followed by a dorsal laminectomy of T9-10 was performed. The vertebral column was stabilized by clamping the exposed T8 and T10 vertebrae with forceps prior to positioning the animal under the Infinite Horizons Impactor(Basso et al., 2006) (Precision Systems). The IH impactor tip was lowered until it just contacted the exposed spinal cord, raised 1 cm, and set to deliver 70 kilodynes of force. Following surgery, the skin and overlying musculature were sutured with 6-0 nylon sutures (Ethicon, San Lorenzo, Peurto Rico, 667G) and the mice were placed into a temperature and humidity-controlled incubator at 32 \u00b0C until they awoke. Mice were administered buprenorphine twice daily for the following two days and Ringer\u2019s solution daily for five days or longer if needed. Bladders were also expressed twice daily until spontaneous micturition was achieved. 2.2.3 TAMOXIFEN AND EDU ADMINISTRATION    Tamoxifen was dissolved in corn oil (Sigma, St. Louis, MO, C8267) at 20mg\/ml before administration. All mice received 100mg\/kg\/day intraperitoneal injections of tamoxifen (Sigma, St. Louis, MO, T5648) beginning 9 days prior to SCI and continuing for five consecutive days. For the first two days after SCI, 5-ethynyl-2\u2019-deoxyuridine (EdU) (Invitrogen, Eugene, OR A10044) was dissolved in sterile PBS and administered by intraperitoneal injection (5mg\/kg). After two days, EdU (Carbosynth, San Diego, CA, 61135-33-9) was dissolved in drinking water (0.2mg\/mL) with 1% D-glucose to encourage consumption(Young et al., 2013). EdU water was changed every two days and the mice were administered EdU in their water until 4 WPI. 2.2.4 PERFUSION AND TISSUE PROCESSING   49  To collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20mL of PBS followed by 40mL of freshly prepared 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at 2 or 6 WPI. The injury site was identified, then 1 cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 hours, then incubated in ascending sucrose solutions (12%, 18% and 24%) for cryoprotection, all at 4\u00b0C. Tissue was submerged in OCT compound (Tissue-Tek, Torrance, CA 4583) frozen on dry ice and stored at -80\u00b0C. All spinal cords were sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, HM-525) into 20\u00b5m thick cross-sections, which were mounted in series on 10 slides making each individual section on a slide 200\u00b5m apart.  Spinal cords were collected for electron microscopy at 6 WPI. Mice were transcardially perfused with 20 mL of 0.01M PBS followed by 40mL of 4% PFA with 1% glutaraldehyde chilled to 4\u00b0C (Electron Microscopy Sciences, Hatfield, PA, 16220). The injury site was identified, then segments of the spinal cord were removed at, and adjacent to, the lesion epicenter. The epicenter and adjacent sections were dissected into 1mm blocks and fixed in 2% glutaraldehyde for two hours before being washed three times in 0.1M cacodylate buffer with 5.3mM CaCl2, and then incubated with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, 19190) with 1.5% potassium ferrocyanide (BDH, Toronto, Canada) for 1.5 hours. Once fixed, the tissue went through ascending alcohol washes before being washed with propylene oxide (Electron Microscopy Sciences, Hatfield, PA 20401) and embedded in Spurr\u2019s resin (Electron Microscopy Sciences, Hatfield, PA 14300).  2.2.5 IMMUNOHISTOCHEMISTRY   50  To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 minutes. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc) for 2 hours, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), Olig2 (1:500, Millipore, AB9610), MYRF (1:300, N-terminus, generously provided by Dr. Michael Wegner), GFP (1:4000, Abcam, ab13970), GFAP (1:1000, Sigma, G3893), PDGFR\u03b1 (1:200, R and D Systems, AF-307-NA), NF200 (1:1000, Sigma, N0142), SMI312 (1:1000, Covance, SMI-312R-100)  and P0 (1:100, Aveslabs, PZO). 2.2.6 CELL COUNTING AND TISSUE ANALYSIS   All analyses were performed blinded to animal genotypes. A Zeiss Axio-Observer M1 inverted confocal microscope with a Yokogawa spinning disk and Zen 2 software (Zeiss) was used for imaging. For analysis of the area of spared tissue, images of whole spinal cord cross sections stained with GFAP were taken at 100x magnification (10x lens, numerical aperture (NA) 0.45) and analyzed in ImageJ (NIH). The intact area was determined by manually circling the lesion border indicated by GFAP+ immunoreactivity with spared cytoarchitecture then calculating the total area for each section.   51 For analysis of cell densities, we imaged the epicenter of injury and the next two sections 200 and 400 \u00b5m rostral and caudal for each animal for a total of five sections per mouse. We performed systematic random sampling within each section(West, 2012) by overlying a grid (individual grid size 103 \u00b5m x 108 \u00b5m) onto a low magnification preview image of a cross section of spinal cord. One counting square for every 3 x 3 grid area was imaged at 400x magnification (40x objective NA 1.3). Z-stacks were imaged through the entire depth of the 20\u00b5m thick section with 1 \u00b5m spacing between optical sections and cells were counted in three dimensional space within a 100 x 100 \u00b5m optical dissector. We analyzed approximately 10-15 Z-stacks per spinal cord section, depending on the size of the cord after injury. The number of cells within the specified volume of the sampling box were averaged per section, giving the density of cells per mm3.  Assessments of the density of newly generated myelin sheathes were examined within the spared tissue at epicenter and 200\u00b5m rostral and caudal. Similar to the quantifications for cell densities, we overlaid a grid (individual grids 68.5 \u00b5m x 69.0 \u00b5m) across a cross section of the spinal cord then used systematic random to image a Z-stack in 1 out of every 9 3 x 3 grid boxes at 630x magnification (63x objective lens NA 1.4) through its entire depth. We imaged approximately 15 to 20 images per spinal cord cross section, depending on the size of the cord after injury. Images were quantified in the middle optical section of the in focus Z stacks within an optical dissector of 4047.4 \u00b5m2. New myelin was defined as co-localization of GFP fluorescence and with myelin fluorescence (MBP or P0) that fully surrounded an axon (SMI312 or NF200-positive). The total number of myelin sheaths were divided by the area of the counting squares and averaged between all the sample squares to give sheathes density. 2.2.7 ELECTRON MICROSCOPY AND TOLUIDINE BLUE STAINING  52    Spinal cords embedded in resin were sectioned to 1\u00b5m thickness on an ultramicrotome (Ultracut E, Reichert-Jung). Ultra and semi-thin sections were collected every 20\u00b5m and semi-thin sections were viewed under a light microscope to find the injury epicenter, defined by the lowest number of myelinated axons by a rater blinded to genotype. Myelin was visualized in semi-thin sections by brief staining with 1% toluidine blue and 2% borax solution then coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ, SP15). The imaging of toluidine blue semi-thins was performed on a Zeiss, Axio Imager.M2 microscope at 630x magnificaiton (63x objective, NA 1.4). The entire cross section at epicenter was imaged. A grid with box dimensions of 50\u00b5m x 50\u00b5m was overlaid on top of the merged image. We employed systematic random sampling, counting one in every seven grids. This was done over the extent of the spared ventro-lateral white matter at the injury epicenter. Spared tissue was indicated by intact cytoarchitecture and the presence of myelin sheaths. Between 1500-2500 myelin sheaths were counter per animal. For transmission electron microscopy, ultrathin sections of 100nm thickness at the lesion epicenter were stained with Reynold\u2019s lead citrate and uranyl acetate to enhance contrast, and imaged at 5000x primary magnification on a Zeiss EM910 equipped with a digital camera.  2.2.8 BEHAVIOURAL ASSESSMENTS  All behavioural assessments were performed during the dark cycle to increase activity. The raters were blinded to animal genotype while running behavioral tests and during subsequent analyses. Behavioural assessements were run in mice lacking the mT\/mG reporter to avoid any confounding effects of the expression of fluorescent proteins on myelin compaction or  53 behavioural function.  Three different motor behavioural assessments were conducted: open field testing using the Basso mouse scale (BMS) (Basso et al., 2006), regular horizontal ladder (Cummings et al., 2007) and Catwalk gait analysis (Hamers et al., 2006). These tests are all sensitive measures of hindlimb recovery following thoracic contusive SCI (Basso et al., 2006; Cummings et al., 2007; Hamers et al., 2006). Mice were handled repeatedly and pretrained on the Catwalk and horizontal ladder tacks by running the mice three times per day for five consecutive days prior to baseline testing. Mice were familiarized with BMS open field box with cagemates, then alone prior to testing. All animals were then tested before and after tamoxifen induction to establish baseline values.  During open field BMS testing mice were placed into a 150 cm by 90 cm clear plexiglass box with 30cm high sides and observed by two blinded raters. The BMS assessed hindlimb function, tail position, trunk stability and coordination. A score of 0-2 on the BMS demonstrated varying degrees of motion in the ankle joint, a score of 3-5 ranges from the capacity to plantar place the hindpaw to frequent plantar stepping but lacking coordination. A score of 6-8 is given based on the degree of coordination, the rotation of paws and the stability of the trunk. A score of 9 is indicative of no impairment in hindlimb or tail function. Scores were averaged between limbs. The BMS subscore, a cumulative score based off the frequency of stepping, paw position, level of coordination, trunk stability and tail position, was also recorded(Basso et al., 2006). For horizontal regular ladder analysis, mice were videotaped with a high definition camera (Sony, HDR-XR200) crossing of 30 rungs spaced 1.3 cm apart at 30 cm height. Each mouse had five complete runs recorded and analyzed per time point. Mice were rerun if they paused for more than several seconds, reared or reversed course. At least fifteen minutes were given per mouse between runs to reduce fatigue. The home cage was placed on the opposite side  54 of the ladder to encourage mice to complete the ladder task. Analysis reported the number of success (plantar or toe placements on the rung or skipped rungs) as well as errors (slips, misses, and drags) and scored by a blinded observer (Cummings et al., 2007). Three mice were excluded from analysis (n=2 controls and n=1 Myrf ICKO). These mice dragged more than half the run during the 2 WPI time point and were strong outliers for percent error (three times the interquartile range from the first or third quartile) at two or more consecutive time points after injury. Gait analysis was performed using the Noldus Catwalk. The base of support, stride length, relative paw position, duty cycle and the percentage of time individual paws were on the platform were shown. These are outcome measures sensitive to motor dysfunction following SCI (Hamers et al., 2006). The camera was placed 20 cm below the runway and mice run through a 5 cm wide darkened tunnel. At least 5 uninterrupted crossings with continuous movement were recorded. The runs were averaged for each animal. Only runs which had three consistently sped step cycles were analyzed (at least four per animal per timepoint). The settings on the Catwalk were contrast 3990, brightness -140mV and analyzed at gain of 10. During week three and five, the catwalk brightness was increased to -80mV and analyzed at gain of 16 to better resolve footprints in animals with poor gait. As this could affect the intensity score and paw position these analyses were not compared between time points.  2.2.9 STATISTICAL ANALYSIS  Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) or Graphpad 6.0 (Prism). Individual data points were displayed when possible and represent a single mouse. However, bar graphs were plotted for lesion size, the contribution of  55 PDGFR\u03b1-cell derived myelin to total myelin and BMS scores to increase clarity of the data. All data in graphs portray the mean \u00b1 the standard error of the mean (SEM). If data met assumptions for normality, tested with the Shapiro-Wilk test, t-tests were run with or without Welch\u2019s correction depending on the homogeneity of variance (tested with Levene\u2019s test). Comparisons of the density of recombined oligodendrocytes or lesion area were compared using a two-way ANOVA with Tukey\u2019s post hoc test to detect individual differences. For behavioural analyses and analyses, a two-way repeated measures ANOVA was conducted with comparisons using Tukey\u2019s or \u0160id\u00e1k post hoc to compare individual groups. Comparisons were two-tailed and considered statistically significant if P < 0.05.  2.3 RESULTS   2.3.1 MYRF ICKO MICE HAVE EFFECTIVE RECOMBINATION IN OPCS AND FEWER NEW OLIGODENDROCYTES EXPRESSING MYRF AFTER SCI     The cellular mechanisms that drive locomotor improvements following SCI are poorly understood. Genetic fate mapping reveals extensive remyelination by resident OPCs differentiating into new oligodendrocytes in response to SCI (Assinck et al., 2017b), however the extent to which it contributes to spontaneous motor improvements is unknown. Removing a gene, like Myrf, essential for OPC differentiation should halt remyelination in response to SCI (Duncan et al., 2017a). This would enable an assessment of the role of endogenous remyelination in functional improvements. We crossed mice carrying LoxP sites flanking exon 8 of the Myrf gene (Myrffl\/fl) with a mice expressing a tamoxifen-inducible Cre recombinase under the PDGFR\u03b1 reporter to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice (Myrf ICKO). When tamoxifen is administered, recombination occurs in PDGFR\u03b1+ OPCs resulting excision of exon 8 of the  56 Myrf gene in Myrf ICKO mice (Figure 2.1A), thereby rendering this critical transcription factor non-functional (Duncan et al., 2017a; Emery et al., 2009; McKenzie et al., 2014). Control mice were littermate Myrffl\/fl mice which lacked PDGFRa-CreERT2, so that when tamoxifen is administered exon 8 is not removed and the gene remains functional (Figure 2.1a). Adult mice were pretrained on behavioural tasks then dosed with tamoxifen prior to injury (Figure 2.1b). Mice were injured with a moderate contusive injury known to induce demyelination of spared axons (Bartus et al., 2016; James et al., 2011; Lasiene et al., 2008). Like most human injuries, moderate contusions have axon sparing and demonstrate limited locomotor improvement. There were no differences in injury force or displacement applied by the Infinite Horizon (IH) impactor between Myrf ICKO and controls (Figure 2.1c, d).  We examined the effectivess of tamoxifen to induce recombination in the spinal cord of both Myrfwt\/wt and Myrffl\/fl mice crossed with the PDGFR\u03b1-CreERT2 and with Rosa26 mGFP (mT\/mG) mice after injury. These mice expressed membrane tethered fluorescence in response to Cre-mediated recombination permitting morphological and phenotypical assessment of these recombined cells (Figure 2.1e). Recombination within OPCs (defined as Olig2+PDGFR\u03b1+ double-positive cells) at 6 WPI resulted in mGFP expression in 88.3 \u00b1 2.9% of control and 89.4 \u00b1 1.6% of Myrf ICKO mice OPCs (Figure 2.1f).  Thus, recombination was highly efficacious and labeled the majority of OPCs. As OPCs differentiate during remyelination they begin to express MYRF(Duncan et al., 2017a). Accordingly, we found that MYRF was expressed only in cells CC1+OLIG2+ oligodendrocytes and was not expressed in oligodendrocyte lineage cells which had not differentiated (Olig2+CC1-negative) ( Figure 2.1g). EdU was administered after SCI and effectively labels proliferative cells including OPCs and can be used to distinguish newly differentiated oligodendrocytes(Young et al., 2013). In Myrf ICKO mice, MYRF was  57 absent from new oligodendrocytes labelled with CC1+ and EdU, in contrast to controls where CC1 was synonymous with MYRF expression (Figure 2.1h). Myrf deletion from OPCs did not alter the extent of spared tissue indicated by GFAP staining (Figure 2.1i) at any point examined between 800 \u00b5m rostral to caudal of lesion epicenter following thoracic SCI (Figure 2.1j). Collectively, these data demonstrated that Myrf ICKO mice can be used to effectively induce recombination in OPCs thereby reducing MYRF expression in new oligodendrocytes in response to SCI, but did not alter injury dynamics or tissue sparing.   58  FIGURE 2.1 - MYRF ICKO MICE HAVE EFFECTIVE RECOMBINATION IN OPCS AND DO NOT HAVE ALTERED INJURY DYNAMICS OR TISSUE SPARING FOLLOWING MODERATE THORACIC SCI.   (a) Illustration of transgenes used in experiment. Myrf ICKO mice were generated by crossing mice with exon 8 of Myrf floxed with mice with the PDGFR\u03b1-CreERT2 transgene to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice. Control mice lacked the PDGFR\u03b1-CreERT2 transgene. (b) Illustration of experimental timeline. (c) Impact force (kilodynes) imparted on the spinal cord during SCI indicates no difference between groups (df=25, t=0.103, P=0.912, Student\u2019s t-test). (d) Displacement (\u03bcm) of the impactor tip upon contact with the spinal cord during thoracic contusion shows no statistical difference between groups (df=25, t=0.037, P=0.971, Student\u2019s t- test).    59 (e) Overview images from the ventrolateral white matter adjacent to the lesion epicenter in control and Myrf ICKO mice crossed with a tamoxifen inducible reporter that tethers GFP to the membrane (mT\/mG). The majority of PDGFR\u03b1+Olig2+ cells are recombined (GFP expression, yellow arrowheads) but there is ocassional non-recombined PDGFR\u03b1+ are observed (PDGFR\u03b1+ GFP-, blue arrowheads). (e\u2019) Inlays of single optical sections demonstrating colabeling of PDGFR\u03b1 with GFP in Olig2+ cells.    60  61 (f) Quantification of the recombination efficiency in OPCs at 6 WPI. There is no difference in recombination between control and Myrf ICKO mice (df=10, t=0.368, P= 0.627, Student\u2019s t-test). (g) Single optical confocal section micrographs demonstrating colabeling of MYRF in CC1+Olig2+ oligodendrocytes (yellow arrowheads). Olig2+ cells lacking CC1 do not have MYRF expression in either group (blue arrowheads). (h) Single optical section from control or Myrf ICKO mice demonstrating colabeling of MYRF in CC1+EdU\u00b1 oligodendrocytes in control mice, but not Myrf ICKO. (i) Spinal cord cross section of the lesion epicenter stained with GFAP+ 6 WPI in Myrf ICKO and control mice. (j) Quantification of GFAP+ spared tissue at different distances from lesion epicenter. There is no significant difference between Myrf ICKO and control mice at any given distance from lesion epicenter (multiple Student's t-test with Holm-\u0160\u00edd\u00e1k correction, epicenter t=1.095 P=0.291) ns = non significant. Scalebars = 50 \u03bcm (e), 10 \u03bcm (g), 100 \u03bcm (i).   2.3.2 MYRF ICKO INHIBITS THE ACCUMULATION OF NEW OLIGODENDROCYTES FOLLOWING THORACIC SPINAL CORD CONTUSION    We next determined if Myrf ICKO was effective at inhibiting the accumulation of new oligodendrocytes in response to SCI. Oligodendrocyte lineage cells (Olig2+) were mostly lost within the lesion, but present in the spared white matter (Figure 2.2 a, b). To identify we new oligodendrocytes we examined EdU expression in Olig2+CC1+ cells. (Figure 2.2c, d). There was over an 11-fold reduction in new oligodendrocytes (EdU+CC1+Olig2+) in Myrf ICKO mice compared to the controls (1286 \u00b1 250 mm3 to 14315 \u00b1 1308 mm3), indicating that Myrf ICKO almost completely prevented the accumulation of new oligodendrocytes following SCI (Figure 2.2 e). Myrf ICKO mice also had fewer total oligodendrocytes (CC1+Olig2+) than control mice, with a mean of 14760 \u00b1 2233 cells\/mm3 in Myrf ICKO mice compared to 34034 \u00b1 2585 cells\/mm3 in control mice (Figure 2.2 e). Additionally, the mean difference in total oligodendrocyte densities (19,274 cells\/mm3) was mostly accounted for by the lack of new oligodendrogenesis in Myrf ICKO, as nearly this many EdU+ oligodendrocytes (14315 \u00b1 1308 cells\/mm3) were produced in wildtype mice after SCI. Myrf ICKO was successful at preventing the accumulation of new oligodendrocytes at all distances examined from lesion epicenter  62 (Figure 2.2f). OPC density (PDGFR\u03b1+Olig2+) did not differ between Myrf ICKO and controls, nor did the density of OPCs which have proliferated (EdU+PDGFR\u03b1+Olig2+) (Figure 2.2g). Therefore, MYRF is not required for the proliferation or recruitment of OPCs after SCI but inhibits the accruement of new oligodendrocytes.   63    64 FIGURE 2.2 - MYRF ICKO MICE ARE UNABLE TO GENERATE NEW OLIGODENDROCYTES IN RESPONSE TO SCI. Overview of Olig2 staining at injury epicenter in (a) control and (b) Myrf ICKO mice 6 WPI. Boxes are approximate areas where (c) and (d) were imaged. (c-d) Example high magnification representative images of the ventrolateral white matter stained with pdgfr\u03b1, Olig2+, CC1+, and edu in control and Myrf ICKO mice. Single channel images are displayed separately on the right. Yellow arrows indicate oligodendrocytes lacking edu (OLIG2+ CC1+ edu-negative), which are likely spared oligodendrocytes, while white arrows indicate new oligodendrocytes (OLIG2+CC1+edu+) and blue arrows indicate opcs (OLIG2+pdgfr\u03b1+edu+\/-). There are very few new oligodendrocytes following SCI in Myrf ICKO.    65 (e) Quantification demonstrates control mice have a higher density of new oligodendrocytes (CC1+Olig2+ EdU+) (df=15, t=9.224, P<0.001, Student\u2019s t-test) and total oligodendrocytes (CC1+Olig2+) (df=15, t=5.570, P<0.001, Student\u2019s t-test) compared to Myrf ICKO animals. (f) Quantification of the distribution of newly generated cells at different distances from lesion epicenter (OLIG2+CC1+EdU+). At all distances, control mice have more new oligodendrocytes relative to Myrf ICKO mice (multiple Student's t-test with Holm-\u0160\u00edd\u00e1k correction, epicenter t=4.100, P=0.001, all others distances P<0.001) (g) Quantification of the density of OPCs that have proliferated (PDGFR\u03b1+Olig2+EdU+) and total density of OPCs (PDGFR\u03b1+Olig2+) indicate there is no statistical difference between Myrf ICKO and controls (total OPC density: df=15, t=1.535, P=0.146; proliferative OPC density: df=15, t=1.267, P=0.225, Student\u2019s t-tests). ** = P\u22640.01 *** = P\u22640.001. Scale bars = 100 \u03bcm (a, b), 20 \u03bcm (c, d).   2.3.3 MYRF ICKO PREVENTS OLIGODENDROCYTE REMYELINATION BY RECOMBINED CELLS BUT DOES NOT ALTER SCHWANN CELL MYELINATION AFTER SCI    We next determined whether Myrf knockout from resident OPCs was sufficient to halt new myelination in response to SCI. Chronically after a moderate thoracic contusion injury, there was sparing of some ventrolateral white matter at the lesion epicenter (Figure 2.1g), which contains both undamaged and regenerated myelin. New oligodendrocyte myelin cannot be conclusively determined by myelin thickness alone after SCI (Powers et al., 2013), so to unequivocally differentiate newly-generated myelin from surviving myelin we crossed Myrf ICKO mice with Rosa26mGFP (mT\/mG) mice. When administered tamoxifen, OPCs with active Cre recombinase express a membrane-anchored GFP that can be visualized within new myelin produced by oligodendrocytes which have differentiated from OPCs (Figure 2.3a, b)(Assinck et al., 2017a; Duncan et al., 2017a; Kang et al., 2010; Powers et al., 2013).  By 6 WPI, in the ventrolateral white matter, control mice had numerous new myelin sheaths which were indicated by GFP+ colabeling within MBP+ sheaths around NF-200\/SMI312 positive axons (Figure 2.3c, c\u2019). Conversely, in Myrf ICKO mT\/mG mice, GFP processes wrapped axons, but were almost always negative for MBP (Figure 2.3d, d\u2019). After six weeks, the Myrf ICKO mice had generated  66 only 248 \u00b1 56 new myelin sheaths per mm2 in contrast to control mice which had 4664 \u00b1 674 sheaths\/mm2 (Figure 2.3e). Overall 1.7 \u00b1 0.4 % of the myelinated axons at the lesion epicenter in Myrf ICKO had new myelin as compared to 28.4 \u00b1 3.0 % in control mice (Figure 2.3f). There was also a small population of axons that were wrapped by GFP+ processes, but did not express MBP in both control and Myrf ICKO mice and this may represent an early stage of axon ensheathment by oligodendrocyte lineage cells (Figure  2.3d, d\u2019) and did not differ between groups (Figure 2.3g). At 2 WPI, there were very few GFP+ processes that colabeled with MBP+ myelin sheaths in both control (Figure 2.3h) and Myrf ICKO mice (Figure 2.3i) indicating little remyelination occurred in the first 2 WPI (Figure 2.3e). Taken together, endogenous oligodendrocyte remyelination after SCI occurs largely after 2 WPI and this was almost completely prevented by removing Myrf from OPCs.  67  FIGURE 2.3 - MYRF ICKO BLOCKS OLIGODENDROCYTE REMYELINATION IN RECOMBINED CELLS AFTER SCI. (a) Illustration of transgenic mice used. Myrf ICKO and control mice were crossed with a mouse line that has a Rosa26mGFP (mT\/mG) membrane-tethered GFP reporter that is Cre inducible. (b) Overview of injury epicenter at 6 WPI showing MBP, GFP, and NF200\/SMI312 labeling. Representative areas from boxes are shown at higher magnification in (c-d). (c) Single optical confocal sections stained with GFP for recombined cells, MBP to label myelin and NF-200\/SMI312 to label a wide range of axons in control mice. (c\u2019) Individual oligodendrocytes processes wrapping around NF-200\/SMI312+ axons and colabeling with MBP in control mice (yellow arrows). (d) In Myrf ICKO mice, there are few MBP+GFP+ sheaths in the ventrolateral white matter. (d\u2019) Myrf ICKO mice have processes that wrap NF-200\/SMI312+ but these processes typically do not express MBP (blue arrow).   68   69  (e) Quantification of the density of newly generated myelin sheaths (mGFP+MBP+ around NF200\/SMI312+ axons) in spared tissue at 2 and 6 WPI. Control mice and Myrf ICKO animals do not significantly differ at 2 WPI in their newly generated myelin sheath densities, but at 6 WPI control mice have a higher density of newly generated myelin sheaths compared to the Myrf ICKO animals (F(1, 18)= 37.77 two-way repeated measures ANOVA, P<0.001; 2 WPI Myrf ICKO vs Control: P=0.812 | 6 WPI Myrf ICKO vs Control: P<0.001 Tukey\u2019s post hoc test). In control mice, there is a statistically higher density of myelin sheaths at 6 WPI when compared to 2 WPI animals (F(1, 18)= 55.07 two-way repeated measures ANOVA, P<0.001; 2 WPI vs 6 WPI: P<0.001 Tukey\u2019s post hoc test). (f) Quantification of the percentage of MBP+ sheaths around axons that are GFP+ (new myelin) at 6 WPI at the lesion epicenter. There are more new myelin sheaths in control mice relative Myrf ICKO (df=10, t=10.69, P<0.001, Student\u2019s t-test). (g) Quantification of GFP+ processes which completely wrap axons but fail to express detectable MBP and likely represent ensheathment by oligodendrocyte lineage cells reveals no statistical differences at 6 WPI (df=10, t=1.665, P=0.127, Student\u2019s t-test). (h-i) The ventrolateral white matter at 2 WPI showing few GFP+MBP+ myelin sheaths in both control animals and MYRF ICKO mice. *** = P\u22640.001, ns = non significant. Scale bar = 10 \u03bcm (c-f).   The majority of new (GFP+) myelin sheaths in Myrf ICKO mice were found in the dorsal column, a location of extensive Schwann cell myelination following dorsal SCI(Assinck et al., 2017a; Bartus et al., 2016). Given that MBP is not only expressed in oligodendrocyte myelin but also Schwann cell myelin(Kirschner & Ganser, 1980), so we determined whether  the new myelin produced in Myrf ICKO mice were derived from Schwann cells. The myelin protein zero (P0) is a Schwann cell-specific myelin marker   and can be used to distinguish Schwann cell myelin from oligodendrocyte myelin(Assinck et al., 2017b; Bartus et al., 2016). At both 2 and 6 WPI, axons were wrapped by P0+ myelin in both controls and Myrf ICKO mice, some of which was produced by recombined cells (mGFP+) ( Figure 2.4a). The presence of GFP+P0+ myelin supports our previous findings that PDGFR\u03b1+ cells produce Schwann cells after SCI(Assinck et al., 2017b). Using higher magnification confocal microscopy we find clear colabeling of P0 with GFP in the dorsal column in control. (Figure 2.4b) and Myrf ICKO mice (Figure 2.4d), but  70 virtually no P0 in the ventralolateral white matter of the spinal cord in either group (Figure 2.4c, e). Myrf ICKO mice had no difference in the density of P0+ Schwann cell myelin relative to controls at 2 or 6 WPI (Figure 2.4f). Similarly, the density of PDGFR\u03b1-derived Schwann cell myelin (GFP+P0+) was not different in Myrf ICKO mice relative to control mice at 2 or 6 WPI (Figure 2.4g), nor was the percentage of P0+ myelinated axons derived from PDGFR\u03b1+ cells (2 WPI: 17.5% \u00b1 4.0% for controls, 13.6% \u00b1 5.1% for Myrf ICKO at 6 WPI: 23.8% \u00b1 9.9% for controls, 20.5% \u00b1 3.0% for Myrf ICKO)(Figure 2.4h), demonstrating that MYRF was not required for Schwann cell myelination from PDGFR\u03b1+ cells. While we previously found the majority of Schwann cells were PDGFR\u03b1+ cell derived, the percentage of PDGFR\u03b1-derived Schwann cell myelin increases over time and the quantification in this study was undertaken at an early time point than previous studies (Assinck et al., 2017b). In Myrf ICKO mice by 6 WPI, the total density of myelin produced by recombined cells (GFP+MBP+, 248 \u00b1 56 sheathes\/mm2)(Figure 2.3d) could be almost entirely accounted for by the amount of Schwann cell myelination (GFP+P0+, 240 \u00b1 39 myelin sheathes\/mm2)(Figure 2.4f). Therefore, MYRF is dispensable for Schwann cell myelination in the CNS after SCI and impairing oligodendrocyte remyelination does not cause a compensatory increase in Schwann cell myelination.   71   72 FIGURE 2.4 - MYRF ICKO DOES NOT ALTER SCHWANN CELL MYELINATION FOLLOWING SCI.  (a) Overview images of a spinal cord cross sections from control and Myrf ICKO mT\/mG mice stained with GFP, the Schwann cell myelin marker P0 and NF- 200\/SMI312 to label axons. In both Myrf ICKO and controls, P0+ staining is mostly confined to the dorsal column. (b-e) Single optical confocal micrographs in either the dorsal or ventral white matter of control and Myrf ICKO mice with the mT\/mG reporter. In the dorsal column of control and Myrf ICKO mice there is a mix of P0+ sheaths around NF200\/SMI312+ axons, some of which colabel with GFP. There are typically very few P0+ sheaths in either the ventral white matter of control or Myrf ICKO mice.     73 (f) Quantification of the total density of P0 myelin sheaths (P0+) demonstrates there is no difference between groups at 2 WPI (df=8, t=0.128, P=0.901, Student\u2019s t-test) or at 6 WPI (df=10, t=0.154, P=0.880, Student\u2019s t-test). (g) Quantification of the density of newly generated P0 myelin sheaths (P0+ mGFP+) demonstrates there is no difference between groups at 2WPI (df=8, t=0.218, P=0.883, Student\u2019s t-test) or at 6WPI (df=10, t=0.001, P=0.999. Student\u2019s t-test). (h) Graph demonstrating the percentage of P0+ myelin sheaths which are derived from PDGFR\u03b1+ cells relative to the total P0+ myelin sheaths do not differ between knockouts and controls at 2 WPI (df=8, t=0.621, P=0.552. Student\u2019s t-test) or at 6 WPI (df=10, t=0.364, P=0.724, Student\u2019s t-test). ns = not significant. Scale bar = 100 \u03bcm (a), 10 \u03bcm (b-e).   2.3.4 MYRF ICKO RESULTS IN CHRONIC DEMYELINATION AFTER SCI  Genetic fate mapping revealed that recombined cells in Myrf ICKO mice are nearly unable to produce new oligodendrocyte myelin following SCI. However, de novo myelination does not require overt demyelination (Young et al., 2013), nor does it reveal differences in the total level of myelination between groups. We visualized resin-embedded sections at the lesion epicenter of Myrf ICKO and control mice to determine the degree of myelination. Following staining with Toluidine blue, the core of the lesion was filled with phagocytes and nearly devoid of myelinated axons at 6 WPI in both groups (Figure 2.5a). However, in the spared ventrolateral white matter, an increasing gradient of myelinated axons radiated outwards from the lesion epicenter to the most lateral portions of the white matter (Figure 2.5b). The area of spared tissue within the ventrolateral white matter at the lesion epicenter was the same in each group (control 0.398 \u00b1 0.056 versus Myrf ICKO 0.396 \u00b1 0.039 mm2, df=8, t=0.028, P= 0.978 Student\u2019s-t test). Control mice had 27239 \u00b1 4587 myelinated axons relative to 15200 \u00b1 1616 myelinated axons in Myrf ICKO mice consistent with an impaired capacity to form new oligodendrocyte myelin (Figure 2.5c).  This reveals that Myrf ICKO mice had a nearly 44% decline in the number  of myelinated axons resulting in  myelin sheaths compared to control mice and suggests that upwards of 12000 axons are typically remyelinated by oligodendrocytes within the ventrolateral  74 white matter of mice with functional Myrf after moderate contusive SCI. Electron micrographs of the lesion epicenter of Myrf ICKO and control mice demonstrated thinly myelinated large caliber axons in controls while in Myrf ICKO mice axons of similar size (> 1 \u00b5m) were unmyelinated, indicative of profound chronic demyelination at 6 WPI (Figure 2.5d). Together, these data demonstrate that Myrf ICKO was effective at reducing total myelination even chronically after SCI, and in the presence of Myrf there was extensive remyelination of spared axons.   75 FIGURE 2.5 - CHRONIC DEMYELINATION OF SPARED AXONS IN MYRF ICKO FOLLOWING SCI.  (a) Whole cross sections of control and Myrf ICKO spinal cords at lesion epicenter stained with Toluidine blue at 6 WPI. The majority of myelin is found in the ventrolateral white matter. (b) High magnification images of box inset from (a) in Myrf ICKO and control animals. (c) Quantification of myelinated axons in spared white matter. Myrf ICKO animals had significantly lower myelinated axons when compared to the control animals (df=8, t=2.475, P=0.038 Student\u2019s t-test). (d) Example transmission electron micrographs of the injured mouse lesion epicenters. Blue shading depicts thinly myelinated axons, pink shading depicts axons devoid of myelin, and green shading depicts axons with thick myelin sheaths. Many thinly myelinated axons are found in control mice whereas Myrf ICKO mice are almost completely devoid of thinly myelinated large caliber axons, and instead have demyelinated axons greater than 1 \u03bcm in size at 6 WPI. * = P\u22640.05, Scale bars = 5\u03bcm.   2.3.5 HINDLIMB MOTOR RECOVERY OCCURS IN THE ABSENCE OF OLIGODENDROCYTE REMYELINATION  Given the large amount of oligodendrogenesis and remyelination that occurred in control mice after moderate thoracic SCI, we wanted to understand if oligodendrocyte remyelination was causative in locomotor recovery. In contrast to mice with functional Myrf, Myrf ICKO mice were almost completely unable to produce new oligodendrocyte myelin resulting in profound chronic demyelination of spared axons at the lesion epicenter. Thus, Myrf ICKO mice provide the necessary contrast to understand the contribution of remyelination to locomotor recovery. To our surprise, we found that impaired remyelination in Myrf ICKO mice was not associated with a difference in functional recovery using open field testing at any time point following SCI (Figure 2.6a, b). Both Myrf ICKO and controls resulted in locomotor scores of between five and a six on the BMS by 6 WPI, indicative of the recovery of hindlimb stepping in most animals. We also measured fine differences in locomotion using a regular horizontal ladder task, which is known to have higher discriminative capacity for mice with a BMS score from 5-7 than the BMS alone(Cummings et al., 2007). Both controls and Myrf ICKO mice showed an increased number  76 of errors after injury on the horizontal ladder, however again, there was no difference between either group at 2, 4 or 6 WPI (Figure 2.6c).  Mice also underwent footprint analysis using the Catwalk apparatus, which quantifies numerous aspects of gait and is capable of detecting subtle differences in locomotion(Matyas et al., 2017) (Figure 2.6d, e, f). In control mice and Myrf ICKO, injury induced profound impairments in stride length (Figure 2.6g), base of support (Figure 2.6h), and an increase in relative bilateral paw position (Figure 2.6i), but did not alter hindlimb duty cycle in mice (time standing \/ time standing + time in swing) ( Figure 2.6j).  After injury, there was also a decrease in the duration of time a mouse had one or two paws placed on the walkway (Figure 2.6k) and an increase in the time in which three or four paws were simultaneously in contact with the walkway (Figure 2.6l). However, we did not find at any time point a difference between between Myrf ICKO and controls on any of these or other parameters. These same analyses were run on an additional cohort of Myrf ICKO and control mice with the same protocol (Supplementary Figure 1) and again, we found no difference in hindlimb locomotion between Myrf ICKO and controls using open field testing (Supplementary Fig. 1a, b), the horizontal ladder (Supplementary Fig. 1c) and on the Catwalk (Supplementary Fig. 1d-i). Combining, these separate cohorts did not result in differences between controls and Myrf ICKO following SCI on the BMS (Supplementary Fig. 2a), BMS subscore (Supplementary Fig. 2b), or horizontal ladder (Supplementary Fig. 2c). Importantly, when the rate of remyelination is compared to locomotor improvements, we find the majority of hindlimb recovery following moderate thoracic SCI occurs during the first two weeks, when little oligodendrocyte remyelination is present (Fig. 3g relative to Fig. 6a, and summarized in Fig. 7). Collectively, these data demonstrate that the initial recovery of hindlimb locomotion transpires independently of oligodendrocyte remyelination following thoracic contusive SCI.   77  FIGURE 2.6 - MYRF DELETION FROM OPCS DOES NOT IMPAIR MOTOR RECOVERY FOLLOWING MODERATE THORACIC CONTUSIVE SCI.  (a) Time course of recovery evaluated by the open field BMS. While Myrf ICKO and controls did not differ after SCI (F(3, 39)=286.0, P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.518 | uninjured Myrf ICKO vs uninjured control P>0.999, Tukey\u2019s post hoc test), they were statistically different from uninjured controls at all time points post-injury (P<0.001, Tukey\u2019s post hoc test). (b) On the BMS subscore there is no difference between Myrf ICKO and controls (F(3, 39)=388.0, P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.966, Tukey\u2019s post hoc test). (c) The percentage of hindlimb errors (% = error \/ error + success) on the regular horizontal ladder task. Injured mice had increased number of errors (F(3, 38)=25.86, P<0.001, two-way repeated measures ANOVA; P<0.001, Tukey\u2019s post hoc test for injured to non-injured groups) at all time points following injury compared to uninjured mice, but there was no difference between Myrf ICKO and controls after injury (P=0.942, Tukey\u2019s post hoc test).   78   (d) An illustration of paw recordings from the Catwalk along with parameters in (g- i) used to assess gait. LH = left hindlimb, LF = left forelimb, RF = right forelimb, RH = right hindlimb. (e) Example of the time course in which a paw is in contact with platform (coloured boxes). (f) Example recordings of three full step cycles from the Catwalk prior to injury and tamoxifen dosing, and at 3 WPI, and 6 WPI. Both Myrf ICKO and control mice with an SCI have an obvious decrease in stride length, base of support and increase in paw position post- injury.   79  (G-L) No differences in gait were observed between Myrf ICKO and controls either with or without an injury on (G) hindlimb stride length (F(3, 37)=44.13, P<0.001 two-way repeated  80 measures ANOVA; injured Myrf ICKO vs control P=0.977, Tukey\u2019s post hoc test) (H) hindlimb base of support (F(3, 37)=48.09, P<0.001 two-way repeated measure ANOVA; injured Myrf ICKO vs control P=0.630, Tukey\u2019s post hoc test), (I) combined paw position (F(3, 37)= 52.74 two-way repeated measures ANOVA, P<0.001 injured Myrf ICKO vs control P=0.983 Tukey\u2019s post hoc test) (J) hindlimb duty cycle (F(3, 37)=0.933, P=0.435, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.738, Tukey\u2019s post hoc test) (K) percent of run with 1 or 2 paws on the platform (F(3, 37)=17.47, P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.651, Tukey\u2019s post hoc) or (L) 3 or 4 paws on the platform (F(3, 37) 15.46. P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.934, Tukey\u2019s post hoc). Groups were compared at all post-injury time points. All statistical comparisons were made using a two-way repeated measures ANOVA, and a Tukey\u2019s post hoc for individual group differences.  FIGURE 2.7 - THE LOCATION AND EXTENT OF NEW OLIGODENDROCYTE AND SCHWANN CELL MYELINATION AFTER SCI AND ITS RELATIONSHIP TO LOCOMOTOR RECOVERY.  (a) Schematic of the uninjured and injured spinal cord 2 and 6 WPI following moderate dorsal thoracic contusion. In the uninjured spinal cord, axons are myelinated solely by oligodendrocyte myelin and peripheral nerves are myelinated by Schwann cells. By 2 WPI, the lesion epicenter is ringed by a glial scar and mostly devoid of axons. Demyelinated axons are seen in an increasing  81 gradient towards the medial spinal cord. At 6 WPI, extensive oligodendrocyte remyelination is observed throughout the ventrolateral white matter in control mice with functional MYRF, but Myrf ICKO mice fail to produce new oligodendrocyte myelin. Schwann cell myelination is generally confined to the dorsal column. The degree of Schwann cell myelination does not differ in the injured spinal cord between Myrf ICKO and control mice. (b) Diagram illustrating the relative amount and rate of open field hindlimb motor performance compared to the extent of oligodendrocyte and Schwann cell myelin after injury in the spinal cord. Graph has only the 2 wpi and 6 wpi data points for myelination and the curve is based on an educated guess. After thoracic SCI, there is a decline in both hindlimb motor performance and number of myelinated axons in the CNS. The majority of recovery of hindlimb locomotor function on open field testing occurs within the first two weeks in both Myrf ICKO and controls. In contrast, the vast majority oligodendrocyte remyelination does not occur until after two weeks post injury. Therefore, the relative time course of oligodendrocyte remyelination is not associated with hindlimb motor recovery after SCI. Schwann cell myelination occurs within the first two weeks after SCI and occurs at a relatively steady rate. The height of the lines is approximately proportional to the extent of loss and subsequent recovery after SCI.   2.4 DISCUSSION   Myelin regeneration is considered a key therapeutic target to enhance function following SCI but the transcriptional control and functional relevance of this process are unknown. We used a loss-of-function approach to ascertain the role of endogenous oligodendrocyte remyelination in locomotor improvements following SCI. By removing Myrf from PDGFR\u03b1+ OPCs, the accumulation of new oligodendrocytes was inhibited while OPC proliferation and recruitment in response to SCI were preserved. Myrf ICKO blocked oligodendrocyte remyelination from recombined cells, resulting in a 44% decline in the number of myelinated axons at the lesion epicenter in Myrf ICKO mice. Surprisingly, despite chronic demyelination, knockout of Myrf from OPCs did not alter the amount or rate of hindlimb motor recovery in this model. Further, by genetically fate mapping new myelin formation we demonstrated that the vast majority of oligodendrocyte remyelination occurs after the recovery of hindlimb stepping.  82 Therefore, oligodendrocyte remyelination is not a crucial component to recovery of hindlimb stepping following moderate thoracic SCI in mice.  We demonstrate the expression of MYRF in PDGFR\u03b1+ cell-derived oligodendrocytes is essential for effective remyelination of the spinal cord following traumatic injury. Myrf deletion from OPCs did not affect OPC recruitment or proliferation. Microarray and immunohistochemical stains demonstrate that MYRF is not typically expressed in OPCs (Duncan et al., 2017a; Emery et al., 2009), so it is not surprising their density or proliferation is not altered after SCI. Emerging evidence suggests OPCs may be crucial for mediating inflammation (Kang et al., 2013) and may entrap dystrophic axon tips within the glial scar (Filous et al., 2014), so altering OPC numbers would confound an interpretation of the role of oligodendrocyte remyelination in locomotor recovery. Reduced oligodendrogenesis in Myrf ICKO mice were likely a result of a failure of OPCs to fully differentiate\/mature and subsequently being more vulnerable to apoptosis (Duncan et al., 2017a). Oligodendrocyte genesis by resident PDGFR\u03b1+ OPCs cannot be compensated for by other cell sources like ependymal cells or Schwann cells, even when resident differentiation is blocked following SCI. Blocking the accumulation of new oligodendrocytes reveals a large number of axons (~12000) are normally receptive to oligodendrocyte remyelination after moderate thoracic SCI in the spared tissue consistent with extensive early demyelination after SCI (Lasiene et al., 2008; Totoiu & Keirstead, 2005). However, when mice have functional Myrf, these axons are remyelinated by six weeks post SCI. Therefore, we demonstrate that PDGFR\u03b1+ OPCs require MYRF expression upon differentiation and have an indispensable role in generating new oligodendrocytes to regenerate myelin after SCI.   83 There is some evidence that myelin changes are involved in learning, but also myelin loss is associated with the absence of learning or stimuli (Hughes et al., 2018; Keiner et al., 2017; Xiao et al., 2016; Young et al., 2013). However, it is unclear exactly how myelin changes affect learning and memory. Neuronal activity is shown to promote oligodendrogenesis and myelination. (Gibson et al., 2014; Liu et al., 2012; Makinodan et al., 2012). Also, many axons of the cortex display partial myelination, suggesting room to add more myelin sheaths or possibly remove myelin sheaths (Tomassy et al., 2014). Tasks such as playing the piano or training with an abacus are correlated with larger white matter regions, especially for children during development (Bengtsson et al., 2005; Hu et al., 2011; Scholz et al., 2009). Yet adult oligodendrocytes can also contribute to myelination (Bacmeister et al., 2020; Duncan et al., 2018b), suggesting the possibility that myelination status of axons can be modulated throughout life. A study using the same Myrf ICKO showed that knockout mice performed worst in learning tasks within hours of the knockout (McKenzie et al., 2014), but the timing suggests a process other than myelination (Xiao et al., 2016). Future work looking at various learning tasks and long-term adult learning can help us truly identify the role myelin plays in learning, However, for these reasons we made sure to test locomotion in tests that did not require any prior learning in case Myrf ICKO affected learning in any way.  Given the high level of endogenous remyelination that occurs following SCI, it\u2019s surprising that hindlimb motor recovery is not affected when oligodendrocyte remyelination is ablated. One possibility is that myelin formed in response to SCI fails to restore conduction. While we cannot directly discount this possibility, the increased conduction velocity seen with the onset of remyelination at two weeks (James et al., 2011), and computer modeling indicating very thin myelin is sufficient to improve conduction (Hines & Shrager, 1991; Koles &  84 Rasminsky, 1972) argues against such conduction failure. In cats fed an irradiated diet during gestation myelin vacuolation and severe demyelination are observed (Duncan et al., 2009). When these cats are returned to a normal diet they generated thin myelin that is restorative for both function (Duncan et al., 2009), indicating that thinly remyelinated axons in the spinal cord are functional, at least in this case. A second, and in our view more plausible alternative, is the limited rostral-caudal extent of demyelination along axons may not be sufficient to block conduction long-term. Segmentally demyelinated spinal cord axons can conduct in vivo through demyelinated lengths of at least 2.5mm (Felts et al., 1997) and following SCI, demyelination of spared axons has been shown to be reasonably focal to the lesion epicenter (Powers et al., 2012). Labeling of descending rubrospinal tract axons (RST) after contusion injury indicates that 80% of the abnormally short internodes (<100\u00b5m), suggestive of new adult-generated myelin, are found within 1 mm rostral and caudal of the lesion (Powers et al., 2012). Conduction can be restored in demyelinated axons by the redistribution of sodium channels along the demyelinated axolemma (England et al., 1990), a process that may take time, but could explain the partial restoration of conductance at 1 and 2 WPI prior to extensive oligodendrocyte remyelination (James et al., 2011). Thus, perhaps over short distances of demyelination like those observed in rodent SCI, remyelination is not required to activate residual neural circuitry. Locomotor recovery following incomplete SCI relies on the reorganization of descending circuits to deprived spinal segments (Courtine et al., 2008; Takeoka et al., 2014) and to changes in cellular and circuit properties within the central pattern generator (Husch et al., 2012; Rossignol & Frigon, 2011) and motor neurons (Murray et al., 2010) below the level of injury. In cervical models of SCI, very few corticospinal neurons can mediate forelimb motor improvements (Hilton et al., 2016; Weidner et al., 2001) and sparing of less than 20% of the  85 ventrolateral funiculus is associated with locomotor recovery following thoracic SCI (Blight & Decrescito, 1986; Schucht et al., 2002). As such, relatively few descending circuits may be necessary to re-establish the excitatory input required for locomotor recovery (Blight & Decrescito, 1986; Hilton et al., 2016; Schucht et al., 2002). Given this, remyelination of descending axons may not be functionally relevant except in cases where very few axons persist (Kloos et al., 2005; Schucht et al., 2002) or more sustained\/extensive demyelination is observed.  Schwann cell myelination was observed within the first two weeks following SCI when the majority of functional recovery occurs. Schwann cell myelin within the CNS is sufficient to improve conductance following CNS demyelination (Felts & Smith, 1992) and transplantation of Schwann cells into the injured spinal cord has been reported to confer functional benefits (Assinck et al., 2017b). Consistent with the possibility of Schwann cells potentially driving a portion of recovery is a study demonstrating that the inducible knockout of neuregulin-1, which prevents Schwann cell myelination following moderate thoracic SCI, is correlated with diminished functional locomotor recovery (Bartus et al., 2016). Interestingly, we found that ablation of oligodendrocyte remyelination did not induce a compensatory increase in Schwann cell myelination, which was still primarily confined to the dorsal column. This raises the intriguing possibility, that the injury environment leaves different CNS axon populations selectively permissible to either Schwann cell or oligodendrocyte remyelination following SCI. The size of the axon (Powers et al., 2013), the proximity to peripheral roots (Franklin & Blakemore, 1993), absence or reactivity of astrocytes (Zawadzka et al., 2010), and BMP signaling(Talbott et al., 2006) could contribute to producing a milieu permissible for OPCs to differentiate into Schwann cells rather than oligodendrocytes. Importantly, Schwann cell myelination occurs early enough after injury to potentially mediate recovery. However,  86 determining the role of  Schwann cell myelination during recovery after SCI still requires future cell-specific knockout experiments. Remyelination has been considered a promising target for SCI (Alizadeh et al., 2015; Myers et al., 2016; Papastefanaki & Matsas, 2015; Plemel et al., 2014). Oligodendrocyte remyelination is extensive by 6 WPI, but nearly absent at 2 WPI when hindlimb stepping has typically recovered following moderate thoracic SCI. It is conceivable that an acceleration of oligodendrocyte remyelination within the first two weeks after SCI could speed or increase the extent of locomotor recovery. In accordance, cell transplants targeting remyelination injected within the first two weeks were associated with more remyelination and improved locomotor recovery whereas more chronic transplants did not alter remyelination or subsequent locomotor recovery (Karimi-Abdolrezaee et al., 2006; Keirstead et al., 2005). However, both the time course of oligodendrocyte remyelination, and the unimpaired recovery relative to control mice in the absence of oligodendrocyte remyelination question both the role of remyelination in this model to drive recovery and the viability of this model to test remyelinating therapies. Ultimately, this study raises doubts whether remyelination is a validated target for clinical translation following moderate spinal cord contusion.         87 SUPPLEMENTARY FIGURE 1 - ADDITIONAL COHORT OF CONTROL AND MYRF ICKO MICE DEMONSTRATE NO DIFFERENCES IN RECOVERY OF HINDLIMB MOTOR FUNCTION FOLLOWING MODERATE THORACIC CONTUSIVE SCI.  (a) Open field function of a cohort of mice run in replication of the original study was assessed using the BMS and scored by two blinded observers. There was no difference between Myrf ICKO and control on the BMS following thoracic SCI (F (1, 17)=0.017, P=0.899) (b) A graph of the BMS subscore also demonstrate no differences between Myrf ICKO and controls (F(1, 17)=0.003, P=0.960). (c) Graph demonstrating the percentage of hindlimb errors (% = error \/ error + success) on the regular horizontal ladder task. Following injury, there is an increase in errors in both groups but no statistical difference between groups (F(1, 15) = 1.772, P=0.203). (d-i) Catwalk analysis was used to determine differences in various parameters of gait following thoracic SCI. No difference between Myrf ICKO and controls was detected in (d) stride length (F (1, 17)=0.415, P=0.528), (e) hindlimb base of support (F(1, 17)=2.262, P=0.151), (f) paw position (F(1, 17)=0.556, P=0.466) (G) hindlimb duty cycle (F(1, 17)= 0.921, P=0.351) (h) percent of run with 1 or 2 paws on the ground (F(1, 17)= 0.679, P=0.421), and (i) percent of run with 3 or 4 paws on the ground (F(1, 17)=0.439, P=0.517). Groups were compared at all time points post injury. All statistical comparisons were made using a two-way repeated measures ANOVA. Individual time points were compared with \u0160\u00edd\u00e1k post hoc test. Bars are mean \u00b1 SEM. ns = non-significant.      88       a b cd e fg h jSupplementary Fig. 1 Additional cohort of control and Myrf ICKO mice demonstrate no differences in recovery of hindlimb motor function following moderate thoracic contusive SCI. (a) Open field function of a cohort of mice run in replication of the original study was assessed using the BMS and scored by two blinded observers. There was no difference between Myrf ICKO and control on the BMS following thoracic SCI (F (1, 17)=0.017, P=0.899) (b) A graph of the BMS subscore also demonstrate no differences between Myrf ICKO and controls (F(1, 17)=0.003, P=0.960). (c) Graph demonstrating the percentage of hindlimb errors (% = error \/ error + success) on the regular horizontal ladder task. Following injury, there is an increase in errors in both groups but no statistical difference between groups (F(1, 15) = 1.772, P=0.203). (d-i) Catwalk analysis was used to determine differences in various parameters of gait following thoracic SCI. No difference between Myrf ICKO and controls was detected in (d) stride length (F (1, 17)=0.415, P=0.528), (e) hindlimb base of support (F(1, 17)=2.262, P=0.151), (f) paw position (F(1, 17)=0.556, P=0.466) (G) hindlimb duty cycle (F(1, 17)= 0.921, P=0.351) (h) percent of run with 1 or 2 paws on the ground (F(1, 17)= 0.679, P=0.421), and (i) percent of run with 3 or 4 paws on the ground (F(1, 17)=0.439, P=0.517). Groups were compared at all time points post injury. All statistical comparisons were made using a two-way repeated measures ANOVA. Individual time points were compared with \u0160\u00edd\u00e1k post hoc test. Bars are mean \u00b1 SEM. ns = non-significant.  89 SUPPLEMENTARY FIGURE 2 - COMPILED DATA FROM BOTH COHORTS REVEALS NO DIFFERENCE IN LOCOMOTOR RECOVERY FOLLOWING THORACIC SCI IN MYRF ICKO MICE.  (a) Graph of open field assessment of BMS score reveals no differences between both cohorts of Myrf ICKO and control mice with an injury (F (8, 464) = 80.47 P<0.001; two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.783, Tukey\u2019s post hoc test) or on (B) BMS subscore (F(3, 58)=148.7 P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.988, Tukey\u2019s post hoc test). (C) Graph of the assessment of horizontal regular ladder errors (% = error \/ error + success) reveals no differences (F (3, 55)=24.76, P<0.001 two- way repeated measures ANOVA; injured Myrf ICKO vs control, P=0.999, Tukey\u2019s post hoc test). Groups were compared post injury. All statistical comparisons were made using a two-way repeated measures ANOVA, and compared with a Tukey\u2019s post hoc for individual group differences.     a b cSupplementary Fig. 2 Compiled data from both cohorts reveals no difference in locomotor recovery following thoracic SCI in Myrf ICKO mice. (a) Graph of open field assessment of BMS score reveals no differences between both cohorts of Myrf ICKO and control mice with an injury (F (8, 464) = 80.47 P<0.001; two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.783, Tukey\u2019s post hoc test) or on (B) BMS subscore (F(3, 58)=148.7 P<0.001, two-way repeated measures ANOVA; injured Myrf ICKO vs control P=0.988, Tukey\u2019s post hoc test). (C) Graph of the assessment of horizontal regular ladder errors (% = error \/ error + success) reveals no differences (F (3, 55)=24.76, P<0.001 two-way repeated measures ANOVA; injured Myrf ICKO vs control, P=0.999, Tukey\u2019s post hoc test). Groups were compared post injury. All statistical comparisons were made using a two-way repeated measures ANOVA, and compared with a Tukey\u2019s post hoc for individual group differences.   90 CHAPTER 3: IN THE ABSENCE OF REMYELINATION, LOCOMOTOR RECOVERY AFTER SCI IS MEDIATED BY CONDUCTION ALONG SPARED DEMYELINATED AXONS.   3.1 INTRODUCTION  So far, we have shown in mice that an inducible and conditional knockout (ICKO) of myrf from OPCs results in their inability to differentiate into myelinating oligodendrocytes. We then showed that after a moderate contusion SCI, the Myrf ICKO mice had around 44% fewer myelin sheaths than control mice. We also found that despite inhibiting remyelination and an overall reduction in myelin sheaths at the injury site, both the Myrf ICKO and the control mice performed identically in tests of locomotor function (open field, horizontal ladder, gait). We concluded that myelination by OPC-derived oligodendrocytes after SCI is not contributing to spontaneous locomotor recovery in moderate thoracic SCI contusion. Given that spontaneous locomotor recovery occurs, we next need to uncover what is driving spontaneous locomotor recovery if not oligodendrocyte remyelination.  Deciphering the source of spontaneous locomotor recovery can lead to therapies targeting important contributors of function. Understanding the contribution of remyelination to behavioral recovery is particularly relevant in the context of preclinical testing of neural stem cells and oligodendrocyte precursors.  Although many explanations were proposed (section 2.4) and all of which are appropriate paths to pursue, we prioritized 2 hypotheses that we deemed were best for this dissertation. 1)  91 Spared myelinated fibres at the injury \u201crim\u201d are capable of restoring locomotor function regardless of demyelination and 2) spared but demyelinated axons are capable of conducting across the lesion site to restore locomotor function.  Even though Myrf ICKO very effectively impaired remyelination, ~56% of the axons at the lesion epicenter were never demyelinated following secondary injury. This population of spared, myelinated axons could potentially provide enough descending signals to mask the losses brought about by injury induced demyelination. Much of locomotor recovery after an incomplete SCI can be mediated by the reorganization of descending connections without input from the brain or any regeneration of axons (Courtine et al., 2008; Murray et al., 2010; Rossignol & Frigon, 2011; Takeoka et al., 2014). Notably, after thoracic injuries in rats, sparring of only 20% of the ventrolateral funiculus was needed to observe locomotor recovery (Schucht et al., 2002). Similarly, increasing the severity of contusion injuries showed that weight supported stepping could return with as little as 10% spared white rim (Basso et al., 2006). Therefore, remyelination may play a larger role after SCI if fewer axons were spared and as a corollary, by increasing the severity of injury the role of remyelination should be more readily apparent.   Another explanation for MYRF ICKO mice exhibiting recovery without remyelination may be that demyelinated axons remain functional despite losing their myelin at the injury epicenter. Axons are known to be electrically active when demyelinated (Felts et al., 1997), albeit with a slower conduction speed. Since the demyelination is restricted to the area around the injury (Blight et al., 1983; Totoiu et al., 2005) and myelin sheaths are between 250 to 500 \u00b5m in length (Powers et al., 2012), it means an individual axon may only lose 2 to 4 myelin sheaths along its entire length. It is therefore plausible through sodium channel production and spreading along the axons (England et al., 1990) that these demyelinated axons are still functional. By  92 performing electrophysiological analyses of spared axons, we can assess whether demyelinated axons in the MYRF ICKO mice are capable of firing action potentials and if there are delays in the conduction speed of these axons. From these experiments, we can uncover the plasticity of axons in response to demyelination and if the focal demyelination that is observed after SCI is detrimentally affecting axons. Together these experiments will explain the detrimental effects of demyelination after SCI and uncover how compensation occurs within the spinal cord lesion environment.  3.2 MATERIALS AND METHODS  3.3.1 TRANSGENIC MICE AND EXPERIMENTAL DESIGN  Procedures involving live animals were approved by the University of British Columbia, in accordance with guidelines from the Canadian Council on Animal Care (A13-0328). Experiments were initiated in 8-10 week old mice that were group housed, fed a standard chow and maintained on a 12 hour reverse dark\/light cycle for the experiment. As before, Myrffl\/fl mice (Emery et al., 2009)(Jackson Laboratory stock # 010607), which express LoxP sites around exon 8 of Myrf, were crossed with PDGFR\u03b1-CreERT2 (Kang et al., 2010)(Jackson Laboratory stock # 018280) mice to ultimately produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice, referred to as Myrf ICKO. Exon 8 of Myrf contains the putative DNA binding domain and its deletion results in a truncated, non-functional protein (Duncan et al., 2017a; Emery et al., 2009; McKenzie et al., 2014). Littermate Myrffl\/fl lacking Cre recombinase were used as controls. The difference between these mice and the ones in chapter 2 is that they are bred back to C57bl\/6 for 8 generations while the previous mice were a mixed C57bl\/6 and SJL.   93 Mice receive tamoxifen to recombine DNA. Tamoxifen was dissolved in corn oil (Sigma, St. Louis, MO, C8267) at 20mg\/ml before administration. All mice received 100mg\/kg\/day intraperitoneal injections of tamoxifen (Sigma, St. Louis, MO, T5648) beginning 9 days prior to SCI and continuing for five consecutive days. They are given 4 days between the last injection and the surgery.  A total of n=36 mice were used in the severe injury experiments and a total of n=36 mice were used in the followup experiment for kinematics. In the severe injury experiment, the cohort was split into 3 groups, one used for histological analysis of ion channel spreading, one third for electron microscopy, and one third put aside for the long-term study. For the kinematics analysis, all animals received the same injury and behavioral experiments. Histology on these animals will be performed by future students.  3.2.2 SPINAL CORD INJURIES   As mentioned before, prior to surgery, mice were anaesthetized for three minutes with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602) to oxygen mixture. Anesthesia was maintained at 1.5-2% isofluorane as needed during surgery. Each animal received 1mL of Ringer\u2019s solution (Braun, Montreal, Canada, L7500) and buprenorphine (0.05mg\/kg) (Reckitt-Benckiser Slough, Toronto, Canada) analgesic prior to surgery. The back was shaved and then disinfected using successive betadine (Purdue Pharma, 41731) and 70% alcohol washes. Upon entering surgery, the mice were placed on a heatpad controlled by a rectal temperature probe set to 37oC. An incision and separation of the erector trunci muscles from the spine followed by a dorsal laminectomy of T9-10 was performed. The vertebral column was stabilized by clamping the exposed T8 and T10 vertebrae with forceps prior to positioning the animal under the Infinite  94 Horizons Impactor(Basso et al., 2006) (Precision Systems). The injuries of mice shown in the kinematics study received the same injuries as section 2.2.2. For severe injuries, the IH impactor tip was lowered until it just contacted the exposed spinal cord, raised 1 cm, and set to deliver 70 kilodynes of force and a 1 second dwell. Following surgery, the skin and overlying musculature were sutured with 6-0 nylon sutures (Ethicon, San Lorenzo, Peurto Rico, 667G) and the mice were placed into a temperature and humidity-controlled incubator at 32 \u00b0C until they awoke. Mice were administered buprenorphine twice daily for the following two days and Ringer\u2019s solution daily for five days or longer if needed. Bladders were also expressed twice daily until spontaneous micturition was achieved. 3.2.3 TISSUE PREPARATION   As before, to collect spinal cords for immunohistochemical analysis, mice were transcardially perfused with 20mL of PBS followed by 40mL of freshly hydrolysed 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA A11313) at 6 WPI and 36 WPI. The injury site was identified, then 1 cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 hours, then incubated in ascending sucrose solutions (12%, 18% and 24% in PBS) for cryoprotection, all at 4\u00b0C. Tissue was submerged in OCT compound (Tissue-Tek, Torrance, CA 4583) frozen on dry ice and stored at -80\u00b0C. In comparison to Chapter 2\u2019s cross-sections, tissue was instead sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, HM-525) into 1cm long and 20\u00b5m thick longitudinal sections, which were mounted in series on 10 slides making each individual section on a slide 200\u00b5m apart.  Spinal cords were collected for electron microscopy at 6 WPI. Mice were transcardially perfused with 20 mL of 0.01M PBS followed by 40mL of 4% PFA with 1% glutaraldehyde  95 chilled to 4\u00b0C (Electron Microscopy Sciences, Hatfield, PA, 16220). The injury site was identified, then segments of the spinal cord were removed at, and adjacent to, the lesion epicenter. The epicenter and adjacent sections were dissected into 1mm blocks and fixed in 2% glutaraldehyde for two hours before being washed three times in 0.1M cacodylate buffer with 5.3mM CaCl2, and then post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, 19190) with 1.5% potassium ferrocyanide (BDH, Toronto, Canada) for 1.5 hours. Once fixed, the tissue went through ascending alcohol dehydration before being placed into propylene oxide (Electron Microscopy Sciences, Hatfield, PA 20401) and embedded in Spurr\u2019s resin (Electron Microscopy Sciences, Hatfield, PA 14300).  3.2.4 IMMUNOHISTOCHEMISTRY  To prepare for antibody staining, cryostat sections collected on slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, the myelin lipids were removed by dipping the slides through ascending, then descending ethanol dilutions (50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 minutes. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc) for 2 hours, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), Olig2 (1:500, Millipore, AB9610), GFP (1:4000, Abcam, ab13970), GFAP (1:1000, Sigma, G3893), PDGFR\u03b1 (1:200, R and D Systems,  96 AF-307-NA), NF200 (1:1000, Sigma, N0142), SMI312 (1:1000, Covance, SMI-312R-100), Nav1.2 (1:200, Alamone, ASC-002), Nav1.6 (1:200, Alamone, ASC-008), and Kv1.2 (1:200, Alamone, APC-009) 3.2.5 CONFOCAL IMAGING AND ION CHANNEL STEREOLOGY  All analyses were performed blinded to animal genotypes. A Zeiss Axio-Observer M1 inverted confocal microscope with a Yokogawa spinning disk and Zen 2 software (Zeiss) was used for imaging. 1 cm long sections of the spinal cords were cut longitudinally into 20 \u00b5m thick sections with the injury epicentre in the middle and stained with antibodies as explained above. Tiled images were taken spanning 1mm rostrocaudal, centered around the lesion. Z-stacks were imaged through the entire depth of the 20\u00b5m thick section with 1 \u00b5m spacing between optical sections. Images were taken from the left and right side of the spinal cord starting from the most ventral section and moving up until no spared white matter was observed. The number of axons within the specified volume of the sampling box were counted throughout the Z-stacks and averaged per section, giving the density of axons per mm3.  3.2.6 ELECTROPHYSIOLOGY  All procedures were approved by the University of British Columbia, in accordance with guidelines from the Canadian Council on Animal Care. All procedures were terminal. Mice were anaesthetized for 3\u2009min with a 3% isofluorane (Fresenius Kabi, Toronto, Canada, CPO40602) to oxygen mixture. Anesthesia was maintained at 1.5\u20132% isofluorane during the whole procedure. Animals were then intubated by inserting a tracheal catheter through an incision between tracheal rings. The catheter is tightly secured with suture thread. At this point, the tracheal  97 catheter is already connected to a ventilator (Inspira asv by Harvard Apparatus) that is used for the rest of the experiment to supply oxygen and isofluorane to the mouse. The animal is then given a dose of the muscle relaxant gallamine triethiodide. This ensures the animal does not move when stimulated and is why the animal needed to be ventilated. The animal is then turned to a prone position. The C4-C6 and the T13-L2 vertebrae are exposed. The animal is secured with earbars, a spine clamp, then suspended to reduce ventilation movement from the chest. Single tungsten wires are used to stimulate and to record. The coordinates are as follows: Dorsal Column: 50-150\u00b5m penetration from the immediate left or right of the posterior spinal vein. Ventral Funiculus: 1000-1200 \u00b5m penetration from the immediate left or right of the posterior spinal vein. Lateral Funiculus: 400-600 \u00b5m penetration from the immediate lateral border of the dorsal horn, which was always visible under magnification and strong light. Stimulations were repeated with a 1 second interval, an intensity of 1mA, and a pulse duration of 100\u00b5s. Pulses were controlled using ISO-Flex stimulus isolator (A.M.P.I) and MASTER-9 stimulator (A.M.P.I). To filter out signals made from noise all recordings are an average of 180 consecutive recordings spaced 1 second apart (Powerlab 16\/30; ADinstruments). After a high thoracic transection, all signals reported here were lost.  3.2.7 BEHAVIOURAL STUDIES   All behavioural assessments were performed during the dark cycle to increase activity. The raters were blinded to animal genotype while running behavioral tests and during subsequent analyses. As mice were more severely injured and had limited recovery, these groups received a different set of behavioural testing than before. Two different motor behavioural assessments were conducted: open field testing using the BMS (Basso et al., 2006) and the inclined plane test  98 (Rivlin & Tator, 1977). Inclined plane tests were done on 3 separate textures of ramps, one with horizontal grooves, vertical grooves, and a homogenous texture with no grooves. All ramps were made in house. Mice were handled repeatedly and acclimatized to the testing rooms and equipment by running the mice three times per day for five consecutive days prior to baseline testing. All animals were then tested before and after tamoxifen induction to establish baseline values. For kinematics analysis, animals were tested for gait changes after injury using 3D motion capture system (Vicon Motion Systems). The testing was done on variable-speed rodent treadmill (Maze Engineers). Animals had their gait and coordination analyzed through a quantitative assessment of locomotion. Pre-trained animals were filmed by infrared cameras while walking across a moving treadmill at 8 metres\/min. Animals were detected via the infrared cameras through small, domed, reflective stickers (4mmx4mm) placed onto the mouse. Mice had their hair removed from the locations of the stickers (top of the head, mid-spine, sacrum, right\/left: hip, knee and foot) through firstly shaving the mouse, followed by NairTM hair removal to ensure the skin is clear of hair follicles and proper adhesion of reflective stickers. The mice were anesthetized by 2% Isoflurane and oxygen mixture for the entirety of this process. Onto the naked skin a small amount of eyelash glue was placed, and the sticker placed onto the mouse. After recovering from anesthesia for 30-45 minutes, mice were moved to the treadmill for 10 to 15 runs of approximately 15 seconds; treats were given to motivate the mouse to move along the belt. Mice were allowed to rest for 2-3 minutes between runs to avoid exhaustion. Video captured of the animal was recorded and analyzed using Nexus 2 and ProCalc software (Vicon Motion Systems). Animals were sedated and the same experimenter was used for marker placement to insure consistency. Animals were run on a treadmill at 8 meters\/minute (0.5 km\/hr)  99 for 2 weeks and at least 6 practice runs before recordings to become acclimatized to the treadmill. No negative reinforcement was used. The animal\u2019s cage was placed at the end of the treadmill and provided positive motivation to run along the treadmill. The animals were given seeds as a reward after completing their day of runs.  3.2.8 KINEMATICS ANALYSIS     All of the analysis was done in house using code written by myself. All representations of the \u201c% of step cycles\u201d was calculated by applying cubic-spline interpolation to the original data. This obtained exactly 100 data points per step, regardless of small variations between step lengths. Mathematical normalization was done using custom Matlab code (The Mathwork Inc., Natick, MA). Step cycles were divided into swing phase and plantar phase. The direction of the y-velocity vector for the animal\u2019s toes determined the boundaries between these two phases. This was possible because animals were on a treadmill and plantar placement resulted in negative velocities. During liftoff and as the animal moved its feet forward and the y-velocity was positive. Toe height was calculated using the x-displacement of the toe marker. Joint angles were calculated in 3D by creating vectors between markers and calculating angles between those vectors. Angles were taken for Hip, Knee, and Ankle.  3.2.9 STATISTICS  Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) or Graphpad 6.0 (Prism). Individual data points were displayed when possible and represent a single mouse. All data in graphs portray the mean \u00b1 the standard error of the mean (SEM). If data met assumptions for normality, tested with the Shapiro-Wilk test, t-tests were run  100 with or without Welch\u2019s correction depending on the homogeneity of variance (tested with Levene\u2019s test). Comparisons of the density of recombined oligodendrocytes or lesion area were compared using a two-way ANOVA with Tukey\u2019s post hoc test to detect individual differences. For behavioural analyses and analyses, a two-way repeated measures ANOVA was conducted with comparisons using Tukey\u2019s or \u0160id\u00e1k post hoc to compare individual groups. Comparisons were two-tailed and considered statistically significant if P < 0.05.    3.3 RESULTS  3.3.1 EXPERIMENTAL DESIGN AND TRANSGENIC MICE EFFICACY   To test whether spared-myelinated fibres at the injury \u201crim\u201d are capable of restoring locomotor function irrespective of demyelination, more severe injuries were performed on Myrf ICKO mice and control mice. In rats it has been previously reported that after thoracic injuries, sparring of only 20% of the ventrolateral funiculus was needed to observe locomotor recovery (Schucht et al., 2002). Therefore, we opted for a more severe thoracic contusion injury in the form of a 70 kilodyne force with an addition of a 1 second dwell at the T9\/T10 level. All other parameters were kept as described in chapter 2. The Myrf ICKO mice used in this study were again a cross between mice carrying LoxP sites flanking exon 8 of the Myrf gene (Myrffl\/fl) with a mice expressing a tamoxifen-inducible Cre recombinase under the PDGFR\u03b1 reporter to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice (Figure 3.1A). However, these mice were back-crossed to C57bl\/6 mice for 8+ generations, giving a more homogenous background. Adult mice were pretrained on behavioural tasks then dosed with tamoxifen prior to injury (Figure 3.1B). The  101 experimental endpoints were 6 WPI and 36 WPI (Figure 3.1B).  Recordings of injury force and displacement applied by the Infinite Horizon (IH) impactor between Myrf ICKO and controls shows no differences between groups (Figure 3.1C, D).   The injury parameters of 70 kilodynes plus a 1 second dwell was chosen to test our hypothesis. Other injuries were tested, and although increasing the force to 90 kilodynes with no dwell time resulted in similar BMS scores, animals had much higher health problems post-surgery (Figure 3.1 E). Also, increasing the injury severities further by increasing dwell time to 2 seconds produces very little locomotor recovery and results in excessive animal health problems and poor survival by 2 WPI (Figure 3.1 E). A portion of the mice also had YFP expression in PDGFRa + cells through the addition of a third transgene (Gt(ROSA)26Sor tm1(EYFP)Cos\/J, JAX:006148). When given tamoxifen, not only was Myrf conditionally removed form PDGFRa+ cells, but now also expressed YFP in their cytoplasm. This allowed us to compare PDGFRa+YFP+ cells to PDGFRa+YFP- cells and calculate the recombination efficiency (Figure 3.1 F). We calculated a recombination efficiency of 88% and a total OPC density of around 5397 cells\/mm3 (Figure 3.1 G ,H). This is consistent with our previous findings of recombination efficiency in the Myrf ICKO (Figure 2.1) and of OPC densities (Duncan et al. 2018; Assinck et al. 2017).   102  FIGURE 3.1 - EXPERIMENTAL DESIGN AND TRANSGENIC MICE EFFICACY. A) Illustration of transgenes used in experiment. Myrf ICKO mice were generated by crossing mice with exon 8 of Myrf floxed with mice with the PDGFR\u03b1-CreERT2 transgene to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice. Control mice lacked the PDGFR\u03b1-CreERT2 transgene. (B) Illustration of experimental timeline. (C) Displacement (\u03bcm) of the impactor tip upon contact with the spinal cord during thoracic contusion shows no statistical difference between groups (df=29, t=1.195, P=0.2418, Student\u2019s t- test) (D) Impact force (kilodynes) imparted on the spinal cord during SCI indicates no difference between groups (df=27, t=0.6448, P=0.5245, Student\u2019s t-test).   103  (E) BMS analysis of mice subjected to SCI using 3 different injury parameters. 70kd +1 second dwell was chosen as the parameters used for further severe experiments (F) Overview images from the white matter adjacent to the lesion epicenter PDGFRaCreER mice crossed with a tamoxifen inducible reporter that tethers GFP to the membrane (mT\/mG). An example of a PDGFR\u03b1+ cells that is also GFP+. (G) Cell densities of PDGFRa+\/GFP+ cells (recombined cells) alongside all PDGFRa+ (all positive cells) showing densities calculated to mm2 at 6 WPI . (H) Quantification of the recombination efficiency in OPCs at 6 WPI. There is no difference in recombination between control and Myrf ICKO mice (df=10, t=0.368, P= 0.627, Student\u2019s t-test).     104 3.3.2 LARGER SPINAL CORD LESIONS AND FEWER MYELIN SHEATHS ARE OBSERVED AFTER SEVERE SPINAL CORD INJURIES   We have already shown that Myrf ICKO prevents remyelination and results in fewer myelinated fibres at 6 WPI. However, a spared white matter \u201crim\u201d containing myelinated axons still remained in Myrf ICKO mice (Figure 2.5 A, B). These fibres can compensate for locomotor losses through various methods (described in section 1.7). Here, we visualized resin-embedded  sections at the lesion epicenter of Myrf ICKO and control mice to determine the degree of myelination after a more severe injury. Staining 1 micron thin sections of resin embedded spinal cords with toluidine blue reveals a similar phagocytic-like lesion cavity and a spared, albeit smaller, myelinated rim (Figure 3.2 A, B). Higher magnification images of the spared white matter rim show a lower density of myelinated fibres in the Myrf ICKO when compared to the Control animals, consistent with earlier findings (Figure 3.2 C, D). The density of myelinated fibres in Myrf ICKO mice was 25904 sheaths\/mm2 and the density of myelinated fibres in Control animals was 34497 sheaths\/mm2 (Figure 3.2 E). This is a difference of 25% between Myrf ICKO and Control animals in severe injuries as supposed to the 44% seen with moderate injuries. We also observed a lateral variability in myelin densities within the spinal cord. The myelin density in Myrf ICKO animals was significantly more on the right side of the spinal cord, as compared to the left, while the Control animals had only slight variabilities between sides (Figure 3.2 F, G). These data show that severe injuries resulted in fewer myelin sheaths but did not completely eliminate the spared rim in Myrf ICKO mice. It also shows the difficulties of producing consistent injuries with more severe parameters.   105  FIGURE 3.2 - LARGER SPINAL CORD LESIONS AND FEWER MYELIN SHEATHS ARE OBSERVED AFTER SEVERE SPINAL CORD INJURIES  (A,B) Whole cross sections of control and Myrf ICKO spinal cords at lesion epicenter stained with Toluidine blue at 6 WPI. (C, D) High magnification images of box inset from (A, B) in Myrf ICKO and control animals.      106  (E) Quantification of myelinated axon densities in spared white matter. Myrf ICKO animals had significantly lower myelinated axons when compared to the control animals (df=8, t=2.475, P=0.0438 Student\u2019s t-test). (F,G) Quantifications of myelin densities separated to show the left and right side of the spinal cord. Myrf ICKO mice had significantly more myelinated axons on the right side of the spinal cord than the left side of the spinal cord (df = 6, P=0.028; Student\u2019s t-test). Control mice myelinated axon densities were not significantly different from the left to the right side of the spinal cord. (df=8, P=0.0936; Student\u2019s t-test).    107 3.3.3 HINDLIMB LOCOMOTOR RECOVERY OCCURS EVEN WITH SEVERE SPINAL CORD INJURY AND OVER A LONG PERIOD OF RECOVERY   Earlier we reported that despite inhibiting oligodendrocyte remyelination after SCI, locomotor recovery is unaffected. Here we reduced the amount of spared myelin after injury using a severe spinal cord injury to test whether remyelination plays a more important role in locomotor recovery if fewer fibres are spared. Like before, we found no differences in BMS scores between Myrf ICKO and control mice (Figure 3.3 A). Also, as the animals did not recovery plantar stepping, the also had no BMS subscore by 6 WPI (Figure 3.3 B). However, the average BMS score of the mice was 3.5 for severe injuries (plantar weight support, but no plantar stepping and sometimes observed dorsal stepping), while the moderate injury showed an average BMS score of around 6 (often plantar stepping with little coordination). Oddly, our severe spinal cord contusions resulted in more myelin sparring on the right side of the animals (Figure 3.2 F, G). Although the mean BMS scores on the right side of the animals were slightly higher, there were no statistically significant differences between recovery on the right versus the left hindlimbs of the animals (Figure 3.3 C, D).  Due to the increased severity of the injury most animals did not recover plantar hindlimb stepping. This means that the other hindlimb locomotor tests used in Chapter 2 would not provide meaningful data and therefore we opted for the more classic incline plane method for measuring hindlimb recovery (Rivlin & Tator, 1977). Here the mice use their forelimbs to grasp onto the ramp while their hindlimb ankle strength and range of motion determines the maximum angle that they can maintain for at least 5 seconds (Figure 3.3. E).  Three different textures were used for the inclined planes to account for various levels of friction; horizontal grooves, vertical grooves, and homogenous texture. The horizontal grooves provided the easiest hold as animals  108 could reach around 90 degrees when uninjured, but after SCI, both groups could not hold past 55 degrees after 6 WPI while the Vertical grooves were most difficult to grip and only allowed a maximum of 40 degrees for Control and 45 degrees for Myrf ICKO at  6 WPI (Figure 3.3. F, G, H). No statistically significant differences were observed between Myrf ICKO and Control animals, except for in one time point, 4 WPI, where in fact the Myrf ICKO animals outperformed the Control animals in the inclined plane test (Figure 3.3 F, G, H).  Notably, no statistical differences were observed between 1 WPI and 6 WPI in any of the tests (Figure 3.3 F, G, H).  FIGURE 3.3 - HINDLIMB LOCOMOTOR RECOVERY OCCURS EVEN WITH SEVERE SPINAL CORD INJURY  109  (A) Time course of recovery evaluated by the open field BMS. Myrf ICKO and controls did not differ after SCI at any time points (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; minimum P>0.727) (B) On the BMS subscore there is no difference between Myrf ICKO and controls (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P>0.999). (C, D) BMS scores at 6 WPI separated to show the left and right hindlimb. There was no significant difference between the left and right side of the spinal cord in Control or Myrf ICKO imce (Myrf ICKO; df = 32, P=0.265; Student\u2019s t-test | Control; df = 30, P = 0.208).    (E) Photograph of injured mouse displaying the experimental set up for the inclined plane test. (F, G, H) Time course of recovery evaluated by Inclined plane test on horizonal, homogeneous, and vertical textured ramps respectively. Myrf ICKO and control mice did not  110 show any differences at any time point in any of the tests (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; minimum P>0.717) except for at 4 WPI in the vertical groove test where Myrf ICKO mice reached a greater angle than Control mice (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P=0.0417)   To test whether Myrf ICKO causes detrimental effects to hindlimb locomotor function in a chronic setting, behavioural scores were taken for a subset of animals to 36 WPI. Animals in both the Myrf ICKO and the Control groups showed consistent behavioural results with no statistical changes or differences between groups for the duration of the 36 WPI in the BMS test (Figure 3.4 A). The inclined plane test also showed no statistical differences between groups over the 36 WPI in all 3 different ramp textures (Figure 3.4 B, C, D). Together these data show that Myrf ICKO does not affect locomotor recovery in severe thoracic spinal cord contusions as measured by BMS and the inclined plane test. It also shows that behavioural recovery is maintained in Myrf ICKO and Control mice for at least 36 weeks.   111  FIGURE 3.4 -  HINDLIMB LOCOMOTOR RECOVERY OCCURS IN SEVERE SPINAL CORD INJURY OVER A CHRONIC TIME POINT. (A) Time course of recovery evaluated by the open field BMS over a 36-week period after injury. Myrf ICKO and controls did not differ after SCI at any time points (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test). (B, C, D) Time course of recovery evaluated by Inclined plane test on horizonal, homogeneous, and vertical textured ramps respectively over a 36-week period after injury. Myrf ICKO and control mice did not show any differences at any time point in any of the tests (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test).  112 3.3.4 MYRF ICKO CAUSES ION CHANNEL EXPRESSION ALONG DEMYELINATED AXONS  Demyelination results in the redistribution and upregulation of ion channels along the axon (England et al., 1991; England et al., 1990; Waxman, 2006a). Nav1.2 is not normally found in white matter tracts or on fully formed nodes of Ranvier but is expressed on unmyelinated axons, demyelinated axons, and also in early node formation (Boiko et al., 2001; Craner et al., 2003; Kaplan et al., 2001; Rasband et al., 2003). Ion channel spreading is also a mathematically plausible method of conduction along unmyelinated axons (Akaishi, 2017) and demyelinated axons have been shown to conduct (Felts et al., 1997). To test whether this is also happening after SCI, antibody stains for voltage gated sodium channels (Nav1.2) and voltage gated potassium channels (Kv1.2) were done alongside axonal marker NF200. An overview of Kv1.2 and Nav1.2 co-expression Shows densely packed ion channels on NF200 positive axons (Figure 3.5 A, B). The expression pattern is markedly different from normal nodal expression since Nav1.2 is completely absent from the nodes of Ranvier and Kv1.2 has a distinct expression in the juxtaparanode (Figure 3.5 C). We measured the length of Kv1.2 expression across the entire flanking portions of the node of Ranvier to be ~31 \u00b5m in Myrf ICKO and Control mice (Figure 3.5 D). In order to not accidentally count normal nodes, we set a minimum continuous expression distance for Kv1.2 of 50\u00b5m and any expression below that was not counted. Co-expression of Kv1.2, Nav1.2, and NF200 was observed in Myrf ICKO (2701 axons\/mm3) and rarely seen in Control mice (111 axons\/mm3) (Figure 3.5 E). A large portion of NF200+ axons were also Kv1.2 positive, but negative for Nav1.2 (Figure 3.5 F). This population of axons was found in Myrf ICKO (1511 axons\/mm3) and Control mice (1628 axons\/mm3). An average of  113 300-500 axons were counted per animal. Overall, Myrf ICKO causes ion channel redistribution that is unlike what is normally found in Control animals after SCI.    114 FIGURE 3.5 - MYRF ICKO CAUSES ION CHANNEL EXPRESSION ALONG DEMYELINATED AXONS  (A) Overview images of ventral white matter regions adjacent to the injury epicenter of mice with SCI at 6WPI. Antibody markers are Kv1.2 (white), Nav1.2 (red), NF200 (green). The left window shows all 3 markers concurrently. Image is one Z-stack. (B) A zoomed in image of an axon expressing NF200, Kv1.2, and Nav1.2. It appears short as the image is showing only one Z-stack.     115 (C) An example image of normal Kv 1.2 expression on NF200+ axon. The red line illustrates how kv1.2 expression was measured to obtain a minimum length criteria (D) Quantification of the average length of Kv1.2 expression in Myrf ICKO and control mice. There was no statistical differences between Myrf ICKO and Control mice in their expression of Kv1.2 on normal nodes of Ranvier. (df=6, t=0.432, P=0.6811; Student\u2019s t-test). (E) Quantification of NF200+, Kv1.2+, Nav1.2+ axons in Myrf ICKO and Control mice. Myrf ICKO had significantly more of these axons than control mice (df = 6, t=9.72, P<0.0001; Student\u2019s t-test). (F) Quantification of NF200+, Kv1.2+, Nav1.2- axons in Myrf ICKO and Control mice. There was no statistical difference between groups in this quantification (df =6, t=0.258, P=0.805; Student\u2019s t-test)  3.3.5 NO CHANGES TO HINDLIMB LOCOMOTOR RECOVERY USING 3D KINEMATICS ASSESSMENT   In order to test whether demyelinated axons in Myrf ICKO mice are functional, animals were again subjected to a moderate contusion SCI and performed more rigorous behavioural testing. This experiment used the same injury parameters as Chapter 2 where mice receive a T9\/T10 midline contusion using a 70 kilodyne force-controlled impactor known to induce demyelination of spared axons(Bartus et al., 2016; James et al., 2011; Lasiene et al., 2008). Again, the Myrf ICKO mice used in this study were a cross between mice carrying LoxP sites flanking exon 8 of the Myrf gene (Myrffl\/fl) with a mice expressing a tamoxifen-inducible Cre recombinase under the PDGFR\u03b1 reporter to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice (Figure 3.6A). Adult mice were pretrained on behavioural tasks then dosed with tamoxifen prior to injury (Figure 3.6 B). BMS was performed at regular intervals, while kinematics assessments were performed pre-injury and at 6 WPI (Figure 3.6 B).  There were no differences in injury force or displacement applied by the Infinite Horizon (IH) impactor between Myrf ICKO and controls (Figure 3C, D). Both Myrf ICKO and controls resulted in locomotor scores of between five and a six on the BMS and between 3 and 4 on the BMS subscore by 6 WPI, indicative of the recovery  116 of hindlimb stepping in most animals (Figure 3.6 E). Like before, there was no statistical difference in the hindlimb motor scores of these two groups of animals.   FIGURE 3.6 - EXPERIMENTAL DESIGN AND BMS BEHAVIOURAL SCORES OF MODERATELY INJURED ANIMALS.  117 (A) Illustration of transgenes used in experiment. Myrf ICKO mice were generated by crossing mice with exon 8 of Myrf floxed with mice with the PDGFR\u03b1-CreERT2 transgene to produce Myrffl\/fl PDGFR\u03b1-CreERT2 mice. Control mice lacked the PDGFR\u03b1-CreERT2 transgene. (B) Illustration of experimental timeline. (C) Impact force (kilodynes) imparted on the spinal cord during SCI indicates no difference between groups (df=26, t=0.215, P=0.832, Student\u2019s t-test). (D) Displacement (\u03bcm) of the impactor tip upon contact with the spinal cord during thoracic contusion shows no statistical difference between groups (df=26, t=0.4967, P=0.624, Student\u2019s t- test)     118 (E) Time course of recovery evaluated by the open field BMS. Myrf ICKO and controls did not differ after SCI at any time points (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; minimum P>0.413) (F) On the BMS subscore there is no difference between Myrf ICKO and controls (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; minimum P>0.344).    We next sought to measure hindlimb locomotion using 3D kinematics. As the cameras recorded at 320 frames\/second, small differences could be recorded that the naked eye could miss in tests such as BMS, inclined plane, horizontal ladder. Three markers were placed along the spine of the animal; at the base of the tail, the arch of the back, and the base of the neck (Figure 3.7 A; yellow markers). Further markers were placed on the hip, knee, ankle, and metatarsal of the mouse hindlimbs (Figure 3.7 A; green and red markers). Vectors were created between these markers (Figure 3.7 A; green and red lines). The step cycle of the animals did not differ from Pre-injury to 6 WPI, nor did it differ between Myrf ICKO and Control animals at either time point (Figure 3.7 B) The average step cycle length was 0.42 seconds for Pre injury and 0.43 seconds for 6 WPI. However, the animals at 6 WPI displayed a longer plantar phase during their stepping (0.36 seconds in 6WPI vs 0.23 seconds in pre-injury) while the swing phase was longer in the pre-injury group (0.08 seconds in 6 WPI vs 0.19 seconds in the pre-injury) (Figure 3.7 C, D). This meant the injured animals spent roughly 80% of their step cycle in the plantar phase while the uninjured animals spent roughly 55% of their step cycle in the plantar phase. However, there were no statistical differences between Myrf ICKO and Control in both time points for either the swing phase or the plantar phase of the step cycle at either time point (Figure 3.7 C, D). There was also no statistical difference between Myrf ICKO and Control at either time points when comparing peak toe clearance (Figure 3.7 E). Next, the vectors generated by the markers placed on the joints were used to calculate joint angles in 3D. The ankle, knee,  119 and hip angles were calculated across the entire step cycle (Figure 3.8 A, B, C). Although there were no statistically significant differences between Myrf ICKO and control animals, the peak ankle angle (Figure 3.8 A) was a 15 degrees difference (40o in Myrf ICKO and 55o in control animals; P=0.583) near the end of the swing phase between Myrf ICKO and control animals. Overall these data suggest that inhibition of remyelination through a Myrf ICKO does not prevent locomotor recovery but may cause small gait changes.  120  FIGURE 3.7 - THREE-DIMENSIONAL ANALYSIS OF MOUSE STEP CYCLES.  (A) 3D reconstruction of mouse hindlimbs and body. Yellow markers were placed along the spine of the animal; base of the tail, arch of back, base of neck. Red markers represent the left leg  121 and green marker represent the right leg. (B) Quantification of the time the mice take to do one full step cycle. No statistical differences over time or between groups (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; minimum P>0.999). (C) Quantification of the time the mice spend in the plantar phase of their step. There are no differences between Myrf ICKO and control, but there are differences between Pre-injury and 6WPI in all comparisons (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P>0.999 | P<0.001 respectively) (D) Quantification of the time the mice spend in the swing phase of their step. There are no differences between Myrf ICKO and control, but there are differences between Pre-injury and 6WPI in all comparisons (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P>0.999 | P<0.001 respectively).     122  (E) Toe height clearance plotted against the relative position in the step cycle. Toe height is calculated using the metatarsal marker and is slightly above ground level. (F) Peak toe clearance plotted for pre-injury and 6 WPI. Statistical differences exist between pre-injury and 6WPI, but no statistical differences between Myrf and Control at each timepoint. (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P < 0.001 | P>0.999 respectively )  123   124 FIGURE 3.8 - THREE-DIMENSIONAL ANALYSIS OF MOUSE JOINT ANGLES.  (A) The angle made by the foot vector and the tibia vector plotted against the relative position in the step cycle. Dotted lines represent the transition between the plantar phase of stepping and the swing phase of stepping. Minimum angle was compared using two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test. No statistical differences were found between groups. (B) The angle made by the tibia vector and the shank vector plotted against the relative position in the step cycle. (C) The angle made by the shank vector and the body axis vector plotted against the relative position in the step cycle.    3.3.6 CONDUCTION ALONG DEMYELINATED FIBRES MAY BE DRIVING HINDLIMB LOCOMOTOR RECOVERY AFTER SCI.     Since voltage gate ion channels are spreading along axons in Myrf ICKO mice, it is likely that these axons are capable of conducting action potentials albeit at a slower speed. Action potentials would have to travel by continuous membrane depolarization instead of saltatory conduction, a process similar to that of naturally unmyelinated axons. Since demyelination spans 2-3 mm around the lesion and only a few oligodendrocyte sheaths are likely missing from axons in the lesion environment (Powers et al., 2012), we expect to see similar, albeit slightly slower conduction. To avoid measuring post-synaptic signals, stimulation and recordings were performed in the white matter lateral funiculus. The stimulating electrode was inserted in the C3\/C4 area and recordings were done in at the lumbar enlargement (Figure 3.9 A). To ensure that records were indeed from compound action potentials (CAP) and not travelling through the CSF or surrounding tissue, transection controls were done at the end of the experiment and successfully eliminated all signals except the very fast artifact seen microseconds after stimulation. (Figure 3.9 B). Both Myrf ICKO and control mice were capable of conducting at a similar pattern; initially with a prominent first peak (Figure 3.9 C, D) also observed in uninjured  125 animals (Figure 3.9 B), and second with a slightly more variable second peak (Figure 3.9 C,D). By measuring the distance between electrodes, conduction speeds could be calculated for both of these peaks. Both Myrf ICKO and control mice had a conduction velocity of 28.2 m\/s and 27.2 m\/s for their first peak (Figure 3.9 E). An uninjured animal only showed 1 peak and a conduction velocity of 37.1 m\/s (Figure 3.9 E). The second CAP peak was measured to 17.4 m\/s and 18.4 m\/s for control and Myrf ICKO mice respectively (Figure 3.9E). We have already shown that the ratio of demyelinated to spared-myelinated axons at the injury cite is roughly 1.3 spared-myelinated : 1 demyelinated (Chapter 2). Interestingly the amplitude ratio of peak1 and peak 2 of Myrf ICKO mice was 1.4 and the control animals showed a ratio of 2 (Figure 3.9 F) These two groups were not significantly different from each other however, as the variability with recordings was very high. Overall this data shows that Myrf ICKO and Control mice are likely conducting at similar velocities and may explain why we do not observe much differences in behavioural data that we have observed so far.   126  FIGURE 3.9 - CONDUCTION ALONG DEMYELINATED FIBRES MAY BE DRIVING HINDLIMB LOCOMOTOR RECOVERY AFTER SCI.  A) An illustration showing the location of the stimulating electrode and the recording electrode. Both electrodes were inserted into the lateral funiculus on either side of the lesion. (B) Voltage recordings over time showing transection of the spinal cord eliminates CAP but does not remove stimulating artifact. (C)Voltage recording over time showing a single CAP of control and Myrf ICKO mice. Two prominent peaks are seen in both groups labeled peak 1 and peak 2. (D) Voltage recording over time showing a single CAP of control and Myrf ICKO mice averaged  127 between all animals. (E) Quantification of conduction velocities at peak 1 and peak 2. There are no statistical differences between Myrf ICKO and control at peak 1 or peak 2 (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P>0.999). Conduction velocities between peak 1 and peak 2 are statistically different in Myrf ICKO and control mice (two-way repeated measures ANOVA; Sidak\u2019s multiple comparisons test; P<0.001) (F) Calculated ratio of peak 1 and peak 2. There is no statistical difference between Myrf ICKO and control mice (t= 0.739, df=9; Student\u2019s t-test; P=0.479)  3.4 DISCUSSION   Chapter 2 showed that spontaneous locomotor hindlimb recovery occurs in mice in the absence of oligodendrocyte myelination. In this chapter we sought to find the source of spontaneous locomotor recovery in Myrf ICKO mice. We used a more severe contusion SCI to reduce the number of spared-myelinated axons. However, in the severe contusion model surprisingly fewer percentage of those surviving axons (25%) seemed to be demyelinated as supposed to a more moderate injury (44%). Despite the lower total amount of myelin, spontaneous locomotor recovery still occurred in Myrf ICKO and in control mice. Even when more detailed analysis of locomotion was done in the form of 3D kinematics, mouse recovery was similar between Myrf ICKO and control mice. Histological analysis of axons showed spreading of voltage gated ion channels (Kv1.2 and Nav1.2) along the axolemma in Myrf ICKO mice, but not in control mice. When CAPs were recorded from axons spanning the lesion, both groups displayed a similar expression pattern. Lastly, a cohort of animals were kept for a 36 week period and suffered no loss of function to locomotor function after Myrf ICKO. Overall this shows that locomotor recovery in Myrf ICKO mice is likely being still supported by demyelinated axons conducting across a small demyelinated lesion. of less than 2.5 mm.  128 By blocking the formation of new oligodendrocyte myelin with Myrf ICKO, we have shown that remyelination of axons in the SCI lesion environment is halted. This is consistent with extensive early demyelination after SCI reported by other labs (Lasiene et al., 2008; Totoiu & Keirstead, 2005). Also, locomotor recovery after an incomplete SCI can be mediated by the reorganization of descending connections without input from the brain or any regeneration of axons (Courtine et al., 2008; Murray et al., 2010; Rossignol & Frigon, 2011; Takeoka et al., 2014). Notably, after thoracic injuries in rats, sparring of only 20% of the ventrolateral funiculus was needed to observe locomotor recovery (Schucht et al., 2002). So, spared-myelinated axons could be the source of locomotor recovery in the Myrf ICKO mice. Therefore, we increased the severity of the contusion injuries to reduce the number of spared-myelinated axons that are left in the lesion environment and allow remyelination to have a greater impact in functional recovery. However, in our hand increasing the severity of the injury reduced the percentage of demyelinated axons that are observed 6 WPI in Myrf ICKO from 44% in moderate injuries to 25% in severe injuries. This also coincided with spontaneous locomotor recovery in both Myrf ICKO and control mice. Most of this recovery occurs within 2 WPI in both groups. However behavioural recovery plateaued at a BMS score of 3.5 in severe injuries compared to 6 in moderate injuries, suggesting that fewer axons were left intact to contribute to function after severe injuries. One explanation for why only 25% of axons remained demyelinated after severe injuries is that the secondary injury may be too prolonged and intense to spare more vulnerable denuded axons. More cellular death during the primary injury would increase the speed and intensity at which the secondary injury begins. Factors like increased excitotoxicity (Gottlieb & Matute, 1997; Karadottir & Attwell, 2007; Vanzulli & Butt, 2015) or more vascular damage (Choo et al., 2007; Noyes, 1987; Rivlin & Tator, 1978) may take longer to return to normal and  129 demyelinated axons could be especially vulnerable to this over extended period of time. This would also occur in both the control and Myrf ICKO animals as both would have demyelinated axons early after injury. The end result is fewer axons surviving to be remyelinated in the Myrf ICKO after the secondary injury has subsided. Perhaps future work using microwave fixation EM could provide adequate resolution to test whether certain axonal populations are more susceptible to degeneration after severe injuries than others. Overall, our data show that increasing injury severity reduced the percentage of myelinated axons compared to moderate injuries and also did not inhibit hindlimb locomotor recovery in Myrf ICKO mice.  A notable consequence of prolonged demyelination is that demyelinated axons begin to express voltage gated ion channels along their axolemma (England et al., 1991; England et al., 1990; Waxman, 2006a). Kv1.2 is normally confined to the juxtaparanode, but after injury it is distributed along axons (Nashmi et al., 2000). We also found Kv1.2 spreading on axons in our control and Myrf ICKO animals, with no statistical difference between Myrf ICKO and control groups. Nav1.2 is not normally found in white matter tracts or on fully formed nodes of Ranvier but is expressed on unmyelinated axons, demyelinated axons, and also in early node formation (Boiko et al., 2001; Craner et al., 2003; Kaplan et al., 2001; Rasband et al., 2003). We found that Myrf ICKO resulted in many axons co-expressing Kv1.2 and Nav1.2 along their axons, where control animals did not. Theoretically, this could mean that these axons are capable to conduct in a mechanisms similar to that of naturally unmyelinated axons (Akaishi, 2017, 2018). Both of these ion channels are found on naturally unmyelinated axons and contribute to their conduction (Glazebrook et al., 2002; Jarnot & Corbett, 2006; Kline et al., 2005; Schild & Kunze, 2012). As colocalization was rarely seen in control animals, it appears that only demyelinated axons at 6 WPI are expressing this distribution of voltage gated ion channels. So potentially, locomotor  130 recovery could still be mediated by demyelinated axons in Myrf ICKO that are functioning like unmyelinated axons along the 2.5 mm lesion. It should be noted that these are not the only channels found on unmyelinated axons, and further testing could show if other ion channels are redistributing on demyelinated axons after SCI. In summary, inhibiting oligodendrocyte remyelination after SCI through a Myrf ICKO results in axons displaying a voltage gated ion channel composition similar to that of unmyelinated axons around the lesion environment.   Hindlimb locomotor recovery can be difficult to assess in small animals. We have used common and validated tests such as BMS, horizonal ladder, or inclined plane test to show that Myrf ICKO mice and control mice have similar locomotor hindlimb recovery profiles. Although these tests are all used widely and accepted in the field (Basso et al., 2006; Cummings et al., 2007; Rivlin & Tator, 1977), all of these tests require a semi-quantitative scoring system and are limited by the human observer. By creating a reconstructed model of the mouse gait at high (320 hz) framerates, we can observe very fine changes to gait such as joint angles and step cycles. Although the Catwalk gait analysis used in chapter 2 assess quantitative aspects of gait (Hamers et al., 2006), it still relies on the mouse choosing the walking speed it is most comfortable walking at. Here we used a treadmill so that the mice were locked at one speed to more accurately make comparisons between them. Even with all of these changes, no statistically significant differences were observed between Myrf ICKO and control mice. In the future, variables such as treadmill speed and incline\/decline walking can be manipulated to test for differences in recovery. Also, an inclusion of obstacles to the treadmill could test descending signals after SCI even better as it would circumvent reliance on CPG driven gait patters. Overall, three-dimensional kinematics did not show locomotor gait changes in animals.  131  Finally, we showed that conduction velocities between Myrf ICKO mice and control mice are very similar. Conduction velocities of CAPs for white matter axons of the lateral funiculus in uninjured animals have been reported to be around 40m\/s, however these numbers often range from as low as 17 m\/s and as high as 80 m\/s (Isu & Yokota, 1983; Tanaka et al., 2006; Tanaka et al., 2004). Our uninjured animal is in line with these previous findings, especially of those from the lateral funiculus (Tanaka et al., 2006; Tanaka et al., 2004). After SCI, we found that two distinct peaks were observed from CAP records. Likely the first peak, measured ~28m\/s, represents the spared-myelinated axons. This is slower than uninjured, but slower conduction speeds in injured animals has previously been shown that in wildtype rats where many axons remain dysfunctional and slightly slower conduction is seen for weeks after injury (James et al., 2011). The second peak in both Myrf ICKO and control animals measured ~17 m\/s. This conduction velocity is still within the previously reported range of myelinated axon CAPs (Tanaka et al., 2004). Since a second peak is absent from uninjured animals, this peak could represent either remyelinated axons in control animals that have not reached normal myelin thickness, or axons with ion channel redistribution in the case of Myrf ICKO. In both situations, demyelinating lesions are measured to only about 2-3 mm in length (Powers et al., 2012). It may be that these distances are just too small in mice to meaningfully change the conduction velocity of Myrf ICKO animals compared to controls. These similar conduction velocities also explain the similar behavioural results that have been observed in all injury models. As to whether the larger lesions of humans would act differently, will need future testing. Overall, Myrf ICKO mice are capable of conducting across chronically demyelinated SCI lesions.   132  The data from this chapter suggests that in the absence of oligodendrocyte remyelination after SCI, spared-demyelinated axons can adapt to continue their function. This means that endogenous remyelination is in fact contributing to axonal conductance, however compensatory mechanisms exist to account for this in the mouse CNS. However, it is not certain whether larger animals (with larger SCI lesions) will compensate for demyelination as mice do. In EAE models of demyelination, large portions of the spinal cord are demyelinated, and mice do exhibit functional loss (Constantinescu et al., 2011). Therefore, it is likely that compensatory mechanisms have a limit. This is highly relevant to therapeutic strategies aimed at promoting remyelination that are tested in mouse models of SCI. Or if SCI in general is producing significant demyelination to justify it as a target for therapy. Myelin and oligodendrocytes themselves have properties that inhibit axonal regeneration after SCI(Atwal et al., 2008; Caroni & Schwab, 1988; Schwab & Strittmatter, 2014). Lastly, the time-course of recovery and long-term maintenance of function all point towards remyelination as a small contributor to functional recovery. Ultimately, these data question the role of remyelination after SCI to locomotor hindlimb recovery and show that at least in the most common animal model of SCI, compensatory mechanisms exist to deal with small demyelination events.        133 CHAPTER 4: CONCLUSIONS AND FUTURE THOUGHTS   The goal of this thesis was to uncover the role remyelination has in hindlimb locomotor recovery after thoracic contusion SCI. Myelin serves axons in the CNS by not only enhancing conduction velocities, but also providing trophic support (Funfschilling et al., 2012; Lee et al., 2012b; Nave, 2010). There is little doubt that demyelination occurs after SCI (Blight, 1985; Bresnahan et al., 1976; Lasiene et al., 2008; Totoiu & Keirstead, 2005). Remyelination follows in the weeks to months after injury (Assinck et al., 2017a; Blight, 1985; Gledhill et al., 1973a; Harrison & McDonald, 1977; Hesp et al., 2015). The majority of SCIs leave some white matter intact (Biering-Sorensen et al., 2011; Bunge et al., 1993; Kakulas & Kaelan, 2015; Young, 2002) and even a small amount of spared tissue can generate locomotion (Basso et al., 2006; Schucht et al., 2002). Considering these observations, enhancing remyelination has been proposed as the mechanistical reasoning behind improvements seen in SCI clinical trials (Alizadeh et al., 2015; Myers et al., 2016; Papastefanaki & Matsas, 2015). With this thesis, I aimed to quantify the contribution of remyelination to locomotor recovery after SCI. By deleting Myrf from OPCs in an inducible and cell specific manner, their ability to generate new oligodendrocytes was halted.  This allowed measurements of locomotor recovery in the absence of oligodendrocyte remyelination after SCI. In this Chapter, I will highlight how the data from Chapter 2 and 3 answered the research questions outlined in Chapter 1. I will also discuss the future direction of this work and address these findings in the context of the research findings of others.    134 QUESTION 1: DOES INHIBITING NEW OLIGODENDROCYTE MYELINATION AFTER MODERATE THORACIC SCI PREVENT HINDLIMB LOCOMOTOR RECOVER IN MICE?  HYPOTHESIS 1: OLIGODENDROCYTE REMYELINATION FOLLOWING A MODERATE THORACIC SPINAL CONTUSION IS A MAJOR CONTRIBUTOR TO SPONTANEOUS LOCOMOTOR RECOVERY.  To address this hypothesis, we took advantage of a mouse line that utilizes viral characteristics to inhibit remyelination with few to no unwanted effects. The Myrffl\/fl mice (Emery et al., 2009) which express LoxP sites around exon 8 of Myrf, and the PDGFR\u03b1-CreERT2 (Kang et al., 2010) driving Cre-recombinase protein under the PDGFR\u03b1 promoter were combined to create Myrf ICKO mice. In Chapter 2, I showed that after recombination, these mice are unable to generate new oligodendrocytes. Since SCI causes demyelination, at 6 WPI, Myrf ICKO mice had many unmyelinated axons around the lesion, while control mice showed signs of remyelination. Using several locomotor behavioural tests, I found that despite the inability to regenerate myelin, Myrf ICKO animals were performing as well as their remyelinating counterparts. Therefore, the null hypothesis is confirmed: Oligodendrocyte remyelination following a moderate thoracic spinal contusion is not a major contributor to spontaneous locomotor recovery.   There are numerous potential explanations for this result, many of which make for compelling future studies. Both Myrf ICKO and control animals are capable of producing Schwann cell myelin within the CNS. We show that Schwann cells after SCI myelinate the dorsal column and are derived from a combination of PDGFRa+ derived cells (likely OPC) and PDGFRa- cells (likely migratory). The presence of Schwann cells after SCI has been known for decades (Bunge et al., 1993; Felts & Smith, 1987; Honmou et al., 1996). Interestingly, in the  135 absence of oligodendrocyte remyelination and with a significant number (44%) of demyelinated axons in Myrf ICKO, Schwann cell myelination did not expand to those axons left denuded. Therefore, it is unlikely that Schwann cell myelin is directly compensating for the loss of oligodendrocyte myelin. However, axons of the dorsal column could potentially be masking any changes to locomotor function lost from Myrf ICKO. Especially since our 2-week timepoint shows the presence of Schwann cells (Assinck et al., 2017b), which temporally correlates with spontaneous recovery after SCI. Previous attempts at a knockout of Schwann cell myelination have used Nrg-1 (Bartus et al., 2016), however this method introduces unwanted variability due to Nrg-1\u2019s diverse roles in recovery after SCI(Alizadeh et al., 2017; Gauthier et al., 2013). To answer this, a Nrg-1 alternative knockout of Schwann cell myelin such as PDGFRa-Cre driven krox20 deletions could piece together the significance of these mostly sensory fibres to hindlimb locomotor recovery. Also, it would be ideal to combine axonal tracing experiments to ensure those axons being myelinated in the dorsal column are indeed spared fibres and not newly sprouted axons that are guided there by the presence of Schwann cells, similar to their actions in the PNS (Cattin et al., 2015; Chen et al., 2019; Hall, 1986).   Another future direction would be to move away from hindlimb locomotion and test the importance of remyelination in forelimb recovery. Locomotion is highly influenced by interspinal factors involving CPGs (Forssberg et al., 1980a; Forssberg et al., 1980b; Grillner & Zangger, 1979). Fine movements of the forelimbs and control of digits could be greater affected by remyelination after SCI. Several studies in rodents have shown that forelimb recovery continues to improve for four to six weeks before plateauing (Gensel et al., 2006; Martinez et al., 2009a). This would be more in line with the timeline of remyelination in the CNS after SCI. Many injuries in humans also happen at the cervical level and digit control is of high value to  136 individuals with SCI (Simpson et al., 2012). There may be some difficulties recording mouse forelimbs due to their size, but this could be overcome with the advancements of artificial intelligence driven software ((Nath et al., 2019).  Lastly, the experiments included in this project do not address whether enhancing remyelination creates functional improvements. Most methods that aim to increase myelination after SCI involve cell transplantation. In some studies (but not all(Plemel et al., 2011)), injections of neural stem cells or oligodendrocyte precursors within the first two weeks were associated with increased remyelination and improved locomotor recovery whereas more chronic transplants did not alter remyelination or subsequent locomotor recovery (Karimi-Abdolrezaee et al., 2006; Keirstead et al., 2005) unless they were used in combination with co-treatments (Karimi-Abdolrezaee et al. 2015; Assinck et al. 2018 and Assinck et al 2020). However, transplanted cells can have varying effects other than myelination that provide functional improvements like neuroprotection, making it difficult to attribute gains to myelination. Yet if locomotor improvements are seen with transplants, the diverse action of cell transplantation can remain an effective route to follow for SCI therapies.  Chapter 3 focused on two other explanations for these data. The choice was made to pursue the possibility that spared-myelinated axons were capable of facilitating locomotion in the moderate contusion injury regardless of remyelination. In addition, testing was performed to determine if demyelinated axons were capable of continuous conduction across the SCI lesion. As these are part of the thesis, I will expand on their implications and future directions in the following section. Overall, by rejecting the hypothesis, these data raise questions on how or even if remyelination is a major contributing factor to locomotor recovery after a moderate contusion injury in mice.     137  QUESTION 2: HOW DO MICE RECOVER LOCOMOTOR FUNCTION IF REMYELINATION IS HALTED AND DOES NOT CONTRIBUTE TO LOCOMOTOR RECOVERY?  HYPOTHESIS 2: SPARED MYELINATED FIBRES AT THE INJURY RIM ARE CAPABLE OF RESTORING LOCOMOTOR FUNCTION REGARDLESS OF DEMYELINATION   To address this hypothesis, the number of spared-myelinated axons was reduced to diminish their contribution to locomotor recovery. This puts more emphasis on axons that are remyelinated and could bring to light locomotor differences in Myrf ICKO animals. A severe injury parameter was created by introducing a one second dwell to a 70 kilodyne contusion. Severe injuries resulted in lower behavioural scores; most animals could barely plantar step and instead relied on dorsal stepping and dragging of their feet during locomotion. This had the unwanted downside of making certain tests (Horizonal ladder, Catwalk gait analysis) impossible to perform. However, in BMS and inclined plane tests, both Myrf ICKO mice showed behavioural scores similar to that of remyelinating controls. Overall, our data show that increasing injury severity did not inhibit hindlimb locomotor recovery in Myrf ICKO animals. A smaller percentage of axons remained demyelinated in the Myrf ICKO animals with severe injuries compared to moderate injuries. A future experiment could uncover if demyelinated axons are less likely to survive in severe injuries as supposed to moderate injuries. As discussed, the milieu of the spinal cord after injury is highly toxic and factors such as increased excitotoxicity (Gottlieb & Matute, 1997; Karadottir & Attwell, 2007; Vanzulli & Butt, 2015), more vascular damage (Choo et al., 2007; Noyes, 1987; Rivlin & Tator, 1978), and  138 oxidative stress (Carrico et al., 2009; Fleming et al., 2008) are contributors of secondary damage. Increasing the severity of the primary injury intensifies the secondary injury (Oyinbo, 2011). Demyelinated axons are also missing their trophic support system and could be more vulnerable to extended exposure to secondary damage. This would occur in both the control and Myrf ICKO animals since both would have demyelinated axons early after injury. The end result is fewer axons surviving to be remyelinated after the secondary injury has subsided. Future work could utilize EM analysis of axons from tissue harvested at multiple timepoints early after injury to identify populations of axons that are more susceptible to severe contusions. Perhaps, in this scenario, initiating remyelination sooner compared to endogenous remyelination could provide a protective role. Although the short timeline in which this could be useful would be difficult for human patients due to the secondary complications that come with traumatic SCIs.  Another observation from this study is that Myrf ICKO animals co-expressed Kv1.2 and Nav1.2 on axons while remyelinating controls did not. Both of these ion channels are found on naturally unmyelinated axons and contribute to conduction (Glazebrook et al., 2002; Jarnot & Corbett, 2006; Kline et al., 2005; Schild & Kunze, 2012). Their presence in the white matter of Myrf ICKO animals after SCI suggests that demyelinated axons are capable of functioning in mechanisms similar to that of naturally unmyelinated axons (Akaishi, 2017, 2018). Since colocalization of Nav1.2 and Kv1.2 was rarely seen in control animals, it appears that only demyelinated axons at 6 WPI are expressing this distribution of voltage gated ion channels. So potentially, locomotor recovery could be driven by demyelinated axons in Myrf ICKO mice. This led to the final hypothesis: HYPOTHESIS 3: IN THE ABSENCE OF OLIGODENDROCYTE REMYELINATION, LOCOMOTOR RECOVERY FOLLOWING THORACIC SCI IS MEDIATED BY DEMYELINATED AXONS.  139 To address this hypothesis, a moderate 70 kilodyne T9\/T10 injury was used. CAPs were recorded from white matter axons spanning the SCI lesion. This showed that conduction velocities between Myrf ICKO mice and control mice are very similar. This is in line with similarities in locomotor recovery. Testing was then expanded to ensure minute changes to gait were not being overlooked by using 3D kinematics to make detailed quantifications of gait. However, still no differences in gait were observed. I can thus confirm the hypothesis that in the absence of oligodendrocyte remyelination, locomotor recovery following thoracic SCI is mediated by demyelinated axons; through the means of spreading ion channels in small demyelinated segment around the injury epicentre.   These data leads to some interesting questions about axonal biology and their relationship with oligodendrocytes. In mice, demyelination around SCI lesions are measured to only about ~2 mm in length or roughly 5-6 myelin sheath lengths (Powers et al., 2012). Given that currents take time to dissipate and that roughly 20% of an AP is needed to continue saltatory conduction, a safety factor of 5 ([current available]:[current needed] to depolarize a node) has been generally accepted for the average axon(Tasaki, 1953). This puts our demyelinated area close to the safety factor range where conduction is likely slowed but not halted (Rasminsky & Sears, 1972; Smith, 1994; Waxman & Brill, 1978). Also, considering that many axons involved in hindlimb locomotion span from the brain to the lumbar spinal cord, only 5%-10% of an individual axon is demyelinated. It then is logical that conduction velocities were not statistically changed in Myrf ICKO animals as this distance accounts for an overall small portion of the entire length that the action potential has to travel through. An interesting route for future research is to explore the limit to which axons can continue functioning before either the conduction velocities drop, or the metabolic stress becomes intolerable for the axon. Larger animals have larger spinal cords and  140 therefore larger lesions. Instead of a 2-3mm demyelinated lesion (Powers et al., 2012), a larger animal may have a 2-3 cm demyelinated lesion. Could a large animal cope with sustained demyelination the way our Myrf ICKO mice have? It is very likely that the observations we made here are restricted to small rodents. Testing larger animals could be difficult though, as genetically modified large animals are difficult to produce and rarely done. Though testing artificially longer lesions in Myrf ICKO mice could model larger lesions and still provide answers to axonal limits.   An avenue for future research could also focus on the conduction velocity changes of individual action potentials instead of CAPs. Demyelination after SCI is shown to be dispersed and not focal (Chapter 2). Therefore, CAPs in Myrf ICKO mice are a combination of myelinated and demyelinated animals. To truly see the effects of Kv1.2 and Nav1.2 channel spreading on conduction velocities, individual axons need to be recorded. However, the technical skill needed to do this in the CNS white matter is very high. An alternative method could be to instead ensure all axons are demyelinated in the Myrf ICKO animals. Although this route leans away from SCI pathobiology, it can help discover adaptive properties of axons. The findings of this section suggest axons have intrinsic adaptive systems which could be further explored and exploited for the benefit of people with SCI. FINAL REMARKS  In summary, I have shown that in the absence of oligodendrocyte myelination, mice are capable of restoring locomotor function as effectively as remyelinating control animals. 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