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University of Florida Thesis Or Dissertation Formatting EXAMINING THE NEXUS AMONGST JOINT DEGENERATION, DISABILITY, AND CHRONIC PAIN USING A RODENT MODEL OF POST-TRAUMATIC KNEE OSTEOARTHRITIS By HEIDI ELISE KLOEFKORN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016 © 2016 Heidi Elise Kloefkorn To my husband ACKNOWLEDGMENTS While a doctoral degree is only conferred to one person, the degree is earned due to the efforts of many. I would like to first thank my husband, Spencer Adams, and my parents, Randy and Leigh Ann Kloefkorn. Their support, love, and patience are invaluable. I would like to thank my chair, Kyle Allen, for seeing my potential, encouraging me to succeed, and teaching me that failure is always an option. With his guidance (and a healthy dose of snark), I have grown as a scientist and academic with a solid foundation to continue my career. I am most grateful that, when it came to his efforts to improve my writing skills, failure was NOT an option. I would also like to thank my lab mates for their comradery and expertise. They have unfailingly provided help, perspective, advice, and laughter at just the right moment. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Defining Osteoarthritis ............................................................................................ 13 Cartilage Changes in OA ........................................................................................ 14 Bony Changes in OA .............................................................................................. 16 Synovial Changes in OA ......................................................................................... 17 Pain and OA ........................................................................................................... 17 Preclinical Models of OA Degeneration and Pain ................................................... 19 Rodent Behavioral Tests ......................................................................................... 21 Rodent Gait Analysis .............................................................................................. 22 Spatiotemporal Video-Based Rodent Gait Assays .................................................. 23 Spatiotemporal Gait Parameters ............................................................................. 26 Using Histology to Assess Tissue Degeneration .................................................... 27 Early Histological Grading Schemes for OA ........................................................... 28 Modern Histological Grading Systems for OA ......................................................... 29 Aim of This Dissertation .......................................................................................... 31 2 SPATIOTEMPORAL GAIT COMPENSATIONS FOLLOWING MEDIAL COLLATERAL LIGAMENT AND MEDIAL MENISCUS INJURY IN THE RAT: CORRELATING GAIT PATTERNS TO JOINT DAMAGE ....................................... 37 Experimental Design and Animal Surgery .............................................................. 38 Spatiotemporal Gait Testing ................................................................................... 40 Mechanical Sensitivity............................................................................................. 41 Histology ................................................................................................................. 42 Statistical Analysis .................................................................................................. 43 Gait Patterns ........................................................................................................... 43 Evidence of Antalgic and Shuffling Gait Compensations ........................................ 45 Mechanical Sensitivity is Altered ............................................................................. 47 Histology Shows Progressive Degeneration ........................................................... 47 Univariate Correlations ........................................................................................... 48 Future Directions to Correlate Symptoms and Behavior ......................................... 50 5 3 AUTOMATED GAIT ANALYSIS THROUGH HUES AND AREAS (AGATHA): A METHOD TO CHARACTERIZE THE SPATIOTEMPORAL PATTERN OF RODENT GAIT ....................................................................................................... 68 Automated Gait Analysis Through Hues and Areas (AGATHA) .............................. 69 Testing Approval ..................................................................................................... 72 Gait Testing ............................................................................................................ 72 Validating AGATHA to Manual Digitization and Exploring the Effect of Video Frame Rate .......................................................................................................... 73 Validating AGATHA Using an Orthopaedic Injury Model ........................................ 74 Validating AGATHA Using a Cervical Spinal Cord Hemisection Injury Model ......... 76 Validating AGATHA Using a Cervical Spinal Cord Contusion Injury Model ............ 77 AGATHA Comparison to Manual Digitization .......................................................... 79 Orthopaedic Injury Model ........................................................................................ 80 Spinal Cord Injury Model ......................................................................................... 81 Future Directions to Improve Gait Analysis ............................................................. 81 4 A GUI FOR THE EVALUATION OF KNEE OA (GEKO); AN OPEN-SOURCE TOOL FOR RAPID GRADING OF RODENT KNEE OA ......................................... 96 A GUI for the Evaluation of Knee OA (GEKO) ........................................................ 97 Modeling OA and Preparing Histological Images .................................................. 100 Validating GEKO to Manual Grading .................................................................... 101 Exploring Grader Variation .................................................................................... 101 Exploring Grader Repeatability and Reproducibility .............................................. 101 GEKO Reproduced Comparable or Better ICCs than Manual Grading ................ 102 Multiple Graders Reproduced Manual Grading Results with High ICCs ............... 103 Inter-grader ICCs were Relatively Low ................................................................. 103 Intra-session Reproducibility was Lower than Inter-session Reproducibility ......... 104 Discussing GEKO ................................................................................................. 104 5 HISTOLOGICAL CHANGES IN THE SUBCHONDRAL BONE AND SYNOVIUM CORRELATE TO RODENT BEHAVIOR IN A MODEL OF POST-TRAUMATIC KNEE OA .............................................................................................................. 116 Experimental Design ............................................................................................. 119 Osteoarthritis Model .............................................................................................. 119 Behavioral Tests ................................................................................................... 120 Histology Results .................................................................................................. 121 Preparing Histological Images .............................................................................. 121 OARSI Grading ..................................................................................................... 121 Measuring Bony Changes ..................................................................................... 122 Measuring Trabecular Bone Area ......................................................................... 123 Quantifying Subintimal Synovium Changes .......................................................... 124 Statistical Analysis ................................................................................................ 125 Subchondral Bone Changes ................................................................................. 125 Synovial Changes ................................................................................................. 126 6 Femoral Cartilage Thickening ............................................................................... 128 Correlations to Mechanical Sensitivity .................................................................. 128 New Histology Measures Correlate with Spatiotemporal Gait ............................... 128 Future Directions in Histology ............................................................................... 129
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