A HISTOPATHOLOGICAL AND MAGNETIC RESONANCE IMAGING ASSESSMENT OF MYELOCORTICAL MULTIPLE SCLEROSIS: A NEW PATHOLOGICAL VARIANT A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By: Megan C. Vignos May, 2016 © Copyright All rights reserved Table of Contents Table of Contents……………………………………………………………………………...ii List of Figures………………………………………………………………………………....iv List of Tables…………………………………………………………………………………..v Acknowledgements……………………………………………………………………………vi Chapters I. Introduction…………………………………………………………………....1 II. Multiple Sclerosis: General Background ………………………………...…...6 Diagnosis Criteria of MS……………………………………………………...6 Evaluating Neurological Disability……………………………………………9 Magnetic Resonance Imaging in MS Diagnosis……………………………...10 Clinical Manifestations………………………………………………………..12 MS Pathology………………………………………………………………....20 Magnetic Resonance Imaging Metrics Related to MS Pathology………….…30 Disease-Modifying Therapies for MS…………………………………….…..52 Conclusion of Review…………………………………………………….…..58 Research Questions and Hypotheses…………………………………….……59 III. Patients and Methods…………………………………………………..……...63 Subjects………………………………………………………………….…….63 Postmortem Tissue Processing……………………………………………..….67 Quantification of Macroscopic Cerebral White Matter Lesions…………….....68 Region Selection…………………………………………………………….…69 MRI Procedures……………………………………………………………..…69 Immunohistochemical Analysis………………………………………….……72 Tissue Processing and Paraffin Embedding of Hemispheric Sections………...73 Quantification of Histology…………………………………………………....74 Research Design……………………………………………………………….77 ii Data Analysis and Statistics…………………………………………………...78 IV. Results………………………………………………………………………….79 Characterization of Spinal Cord, Cortical and Deep Gray Matter Lesions in the Absence of White Matter Lesions……………………………………………..79 Characterization of Spinal Cord Demyelination of Classical and Myelocortical MS Patients…………………………………………………………………….81 Characterization of Cortical Demyelination of Classical and Myelocortical MS Patients………………………………………………..……………………….84 Characterization of Demyelination and association with meningeal inflammation on Hemispheric Sections………………………………………………………88 Characterization of Hippocampal Lesions in Classical and Myelocortical MS Patients………………………………………………..……………………….89 Characterization of Thalamic Lesions of Classical and Myelocortical MS Patients………………………………………………..……………………….91 Identification of Lesions in the Brainstem of Classical and Myelocortical MS Patients………………………………………………..……………………….92 Summary of Histological examination of CNS of Classical and Myelocortical MS Patients………………………………………………..……………………….93 Pathology Underlying Magnetic Resonance Imaging Abnormal Signals in Myelocortical MS Patients…………………………………………………….94 MRI-Defined Normal Appearing White Matter in Myelocortical MS………..95 MRI-Defined T2-Only Regions in Myelocortical MS………………………...96 MRI-Defined T2T1MTR Regions in Myelocortical MS………………….…..97 Atrophy and White Matter Lesion Volumes by MRI………………………..104 Comparing Whole Brain Atrophy Between Controls, Classical and Myelocortical MS…………………………………………………………………………….106 V. Conclusions and Discussion………………………………………………......110 Summary of Major Findings………………………………………………….110 Conclusions…………………………………………………………………...112 Importance of the Studies in Management of MS……....…………………….120 Impact Statement…………………………………………………………...…128 Limitations of the Thesis…………………………………………….………..129 Recommendations for Future Research………………………………………131 Removal of Inhibitors of Remyelination…………………………………..…131 Mechanism of Cortical Demyelination…………………………………..…..145 Closing Remarks……………………………………………………………..151 List of References…………………………………………………………………………….153 iii List of Figures Figure 1: Schematic of Multiple Sclerosis Progression Figure 2: Methods of MRI White Matter Region Selection Figure 3: Methods of Lesion Load Measurements Figure 4: Cerebral White Matter Lesion Loads Figure 5: Spinal Cord Lesion Loads Figure 6: Cortical Gray Matter Lesion Loads Figure 7: Coronal Images of Classical and Myelocortical MS Figure 8: Peripheral Inflammatory Cell Counts Figure 9: Hippocampal Demyelination Figure 10: Histology of MRI Defined White Matter Regions Figure 11: Myelin and Axonal Densities in MRI Regions Figure 12: MRI Scans from Classical and Myelocortical MS Patients Figure 13: Disease Mechanisms of the Multiple Sclerosis Spectrum Figure 14: Sox9 Staining in MS and Control Tissue Figure 15: Sox9 Staining in Cuprizone Mouse Model Figure 16: Sox9 Expression Pattern in MS White and Gray Matter Lesions Figure 17: Elimination of Cell Types Not Expressing Sox9 Figure 18: Astrocyte Labeling of Sox9 Figure 19: Cortical Myelin Density for MS and Control Patients Figure 20: Oligodendrocyte Morphometry and Internodal Length Figure 21: Myelin Protein Degeneration iv List of Tables Table 1: Patient Demographics Table 2: Disease Modifying Therapies Table 3: Magnetic Resonance Imaging Guided Histopathological Analysis of Myelocortical Multiple Sclerosis White Matter Table 4: Magnetic Resonance Imaging Characteristics of Myelocortical Multiple Sclerosis and Classical Multiple Sclerosis Patients v Special Recognitions I must extend my overwhelming gratitude to those that have been on this journey with me. I first wish to thank my mother and father for instilling in me a work ethic and a thirst for knowledge that have suited me well in this expedition for my doctorate. Next, I need to thank my husband for the love, support and encouragement he has given me over these years and never doubted that I could and would complete this work. I want to thank my friends that have been beside me through the highs and the lows of a science career. Lastly, I want to thank my two nieces for reminding me to laugh, smile and just enjoy life. vi Further Acknowledgements With regards to the work in the following thesis, I want to thank: Dr. Bruce Trapp: For supervising the entirety of this work and advising all aspects of my doctoral thesis. Dr. Ansi Chang: For providing training of immunohistochemical staining, coordinating the Rapid MS Autopsy Program, advice and other technical support. Dr. Zhihong Chen: For providing several analysis techniques, constant feedback and meaningful advice and discussions over the years. Dr. Elizabeth Fisher: For her extensive contributions to my thesis work, her insightful discussions and mentorship. Drs. Jennifer McDonough, Robert Dorman, MaryAnn Raghanti, and Marilyn Norconk: For serving on my PhD guidance committee, teaching me numerous lessons, and for giving countless pieces of advice on my thesis work. Drs. Susan Staugaitis and Robert Fox: For their involvement in the Rapid MS Autopsy Program and for providing helpful suggestions on the direction of my thesis work and manuscript. Dr. Sverre Mork: For teaching me the techniques for hemispheric sectioning, and for his stimulating discussions. Dr. Jacqueline Chen: For teaching me repeatedly about the inner workings of MRI, fascinating discussions, singing 80’s songs with me in the lab, and for much needed support and encouragement. vii Dr. Chris Nelson: For editing manuscripts, my prospectus and my thesis as well as providing advice and support. Drs. Ranjan Dutta and Grahame Kidd: For their discussions on presentations, image processing, data analysis, and advice on manuscripts. Trapp Lab members, of which there have been many over the years: For their support, monkey business, camaraderie, and hard work in the autopsy program. Lastly, I would like to thank the MS patient donors and their families, for without their generous and significant contributions this work would never have come to fruition. viii Chapter 1 Section 1: Introduction Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease with underlying neurodegeneration of the human central nervous system, which affects over 2.3 million individuals worldwide and currently has no cure. This disease affects 2 times more females than males and is the major cause of non-traumatic neurological disability in young adults, with onset occurring often in the third decade of life (average age 25-33, (1), although pediatric MS also occurs in those 18 years old and younger). The etiology of MS involves a complex relationship between environment and genetic susceptibility. The environmental factors that have been proposed to play a role in MS etiology and risk range from (A) sunlight exposure which is also highly connected to vitamin D status, with an increased prevalence of MS for individuals residing further from the equator; (B) smoking; and (C) infections from viruses like Epstein - Barr virus and human endogenous retrovirus-W. Although these factors seem to play a role in the risk for developing MS, how they actually affect different aspects of the disease, including the onset or course of the disease, is largely unknown. Genetics also play a role in susceptibility to this disease, as demonstrated by the fact that the risk of developing MS increases in those individuals with a family history of the disease, particularly in immediate family members (2). Twin studies have demonstrated an increased concordance rate of 31% in monozygotic twins, compared to 5% for dizygotic twins (3). A specific allele has been identified 1 as carrying a risk of 16-60% chance of developing MS (4). The gene is for the Human Leucocyte Antigen (HLA) Class II locus, which is part of the major histocompatibility complex located on chromosome 6p21 and has been shown to have a dose effect of