WHY DOES MY JOINT HURT: UNDERSTANDING DISEASE PHENOTYPE AND PAIN RELATIONSHIPS USING MOUSE MODELS OF ARTHRITIS Sanaa Zaki A thesis submitted in fulfilment of requirements for the degree of Doctor of Philosophy Faculty of Medicine (Sydney Medical School) The University of Sydney November 2016 i PREFACE The research embodied in this thesis was conducted at the Raymond Purves Laboratories of Bone and Joint Research, Kolling Institute of Medical Research, University of Sydney at the Royal North Shore Hospital, St Leonards, Australia. Institutional ethics committee approval was sought and obtained prior to the commencement of all animal experiments. The research was partly funded by Arthritis Australia. I certify that the intellectual content of this thesis is the product of my own work, including the design, conduct and analysis of experiments presented in this thesis. Contributions of other researchers and all the assistance received in preparing this thesis and relevant sources have been acknowledged. This thesis has not been submitted for any degree or other purpose. Sanaa Zaki ii ACKNOWLEDGEMENTS My PhD candidature has been a very long journey and I have many colleagues, friends and family that I need to acknowledge for their contribution. Firstly, I am forever indebted to the support and mentorship of my two supervisors, Mark Connor and Chris Little. They graciously bestowed me with the freedom to pave my own path of scientific discovery, while always being there to guide and redirect me when I needed it the most. I am truly indebted to their academic brilliance, scientific rigor and boundless patience. They are truly inspirational researchers that I can only hope to, one day, emulate. Secondly, I would like to thank and acknowledge the many contributions of the Raymond Purves Laboratories team. Thank you Cindy Shu for helping me to settle in and always offering assistance when I had a technical problem in the lab. Your endless supply of delicious treats, gorgeous smile and friendship kept me going. Thank you Sue Smith for all your hard work in slide preparation and the perseverance you displayed when I was all but ready to give up after so many failed attempts at IHC. You are definitely the histology guru! Thank you Margaret Smith for showing me how to do RNA extraction and PCR when I first arrived in the lab, and for your help with the tricky statistical analysis; I wouldn’t have known where to start otherwise. Thank you Miriam Jackson for letting me borrow/steal your bench equipment and never complaining. Thank you Varshini Ravi for helping me set up my tissue culture protocol and showing me how to do a BCA assay. I really enjoyed sharing a bench with you and the many interesting conversations we had while pipetting. Thank you Kelly McKelvey for been so giving of your time and helping me with formatting my thesis. You are an excellent researcher and a kind and generous human being. iii I would also like to acknowledge the support and encouragement I received from colleagues and friends in the Veterinary Faculty, especially Merran Govendir, my special friend that I love and admire so much, and who was always willing to listen to my rants and keep me on track. “Work out what’s really important in your life and focus on that” are definitely words to live by. Finally, I’ve left the most important people to acknowledge until the end: my family, without whom I could never have gotten through this journey. Thank you to my amazing mum for her endless love and encouragement, and for always being proud of me even during my failures. Thank you to my gorgeous sister Ouafaa who was always there for me and who taught me to allow myself to enjoy the little pleasures life has to offer without feeling guilty. Thank you also for your willingness to proof read chapter after chapter without ever complaining. Thank you to my brother Jassen and my other ‘sister’ Fatima. Without you I don’t know if Aden would have survived this PhD. Thank you to my beautiful boys Ossama, Younis and Aden. The three of you are my inspiration - You are my world. Thank you for always challenging the way I view the world. Thank you for giving me space when I needed it. Thank you for putting a smile on my face after a really tough day. Thank you for making me feel like I’m amazing, even though in my world I’m pretty ordinary. Thank you for your beautiful smiles, amazing hugs and unconditional love. The last person I would like to acknowledge is my husband. Thank you Mohamed for helping me realise my dream. Thank you for your patience and support even when I wasn’t a very nice person to be around. Thank you for letting me do things my way when I needed to. Thank you for sharing the load to make my life easier. Thank you for allowing me to be more than a mother and a wife. Thank you for putting up with all the late nights and early starts. Thank you for being the amazing, kind and caring iv person that you are. Without you to share in my struggles, this achievement would mean nothing. I would like to dedicate this Thesis, and all the hard work that went into it, to the beautiful men in my life, Mohamed, Ossama, Younis and Aden. v PUBLICATIONS AND PRESENTATIONS Publications arising from this thesis 1. Kung, L. H., Zaki, S., Ravi, V., Rowley, L., Smith, M.M., Bell, K.M., . Little, C. B. (2016). Utility of circulating serum miRNAs as biomarkers of early cartilage degeneration in animal models of post-traumatic osteoarthritis and inflammatory arthritis. Osteoarthritis Cartilage. doi:10.1016/j.joca.2016.09.002 Publications related to this thesis 1. Little, C.B., & Zaki, S. (2012). What constitutes an "animal model of osteoarthritis"-the need for consensus? Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 20(4), 261-267 2. Jackson, M.T., Moradi, B., Zaki, S., Smith, M.M., McCracken, S., Smith, S.M., Jackson, C.J. & Little, C.B. (2014). Depletion of protease-activated receptor 2 but not protease-activated receptor 1 may confer protection against osteoarthritis in mice through extracartilaginous mechanisms. Arthritis & Rheumatology 66(12): 3337-3348. Conference Abstracts 1. Zaki, S., Smith, M.M., Smith, S.M., Connor, M., & Little, C.B. (2012). Distinct Pain Mechanisms in Two different Models of Arthritis. Osteoarthritis and Cartilage, 20, S252-S253. 2. Zaki, S., Smith, M.M., Smith, S., Connor, M., & Little, C.B. (2013). Contrasting Pathophysiology and Behavioural Responses Associated With Osteoarthritis and An Inflammatory Arthropathy. Arthritis and rheumatism, 65, S527-S528. 3. Zaki, S., Miller, R. E., Malfait, A. M., Smith, S., Tran, P. B., Ishihara, S., & Little, C. (2014). Characterisation of Pain-Related Behaviours In Association with Joint vi Pathology in an 8-Week Antigen-Induced Arthritis Model. Osteoarthritis and Cartilage, 22, S36-S37. 4. Miller, R. E., Zaki, S., Ishihara, S., Tran, P. B., Little, C. B., & Malfait, A. M. (2014). Establishing a Method for Measuring Primary Knee Hyperalgesia in the Murine DMM Model of Osteoarthritis. Osteoarthritis and Cartilage, 22, S419- S419. 5. Jackson, M. T., Zaki, S., Ravi, V., Moradi, B., Smith, S., Fosang, A., & Little, C. (2016). Defining the Relationship between Joint Pathology and Pain in Osteoarthritis using Genetically Modified Mice. Osteoarthritis and Cartilage, 24, S9-S10. 6. Shu, C. C., Ravi, V., Zaki, S., Smith, S. M., Schiavinato, A., Smith, M. M., & Little, C. B. (2016). The Effects of Intra-articular Injection of Mesnchymal Stem Cells versus Hyaluronan Hexadecylamide-Derivative on Post-Traumatic OA: The Relationship Between Synovial Inflammation, Structural Pathology and Pain Sensitisation Osteoarthritis and Cartilage, 24, S324-S325. vii ABSTRACT Osteoarthritis (OA), characterised by progressive joint-wide pathology, is a major health problem accounting for 48% of people living with chronic pain. There are no treatments to slow OA progression and symptom managing therapies are at best moderately effective. This failure results from a poor understanding of the mechanisms that drive OA pain. Animal models are widely used to study OA pain molecular pathways, but pre-clinical findings fail to translate into effective therapeutics for patients. In part this may be because animal models have poorly defined phenotypes not mapped to specific sub-types of human OA. This research aimed to define the relationship between joint tissue pathology, pain behaviour and gene expression in the dorsal root ganglia (DRG), over time comparing models of post-traumatic OA (DMM) and inflammatory arthritis (AIA), and identify differences in what drives pain. DMM and AIA ultimately displayed similar hallmark histopathology of OA in late stage disease. However, each model had distinct temporal patterns of pathology; associations between articular cartilage, synovium and bone pathology; and risk factors for progression. Both models displayed sensitisation (tactile allodynia, mechanical and thermal hyperalgesia) and altered gait (reduced hindlimb weight bearing, changes in stride length). However, the severity and temporal pattern of occurrence were model-specific. At each phase of OA development, DRG gene expression changes were also model-specific. It was predominantly synovium and bone pathology that were significantly associated with altered DRG gene expression and pain behavior, but differentially in the two models. The DRG expression changes associated with altered pain behaviours were also model specific. viii Combined these findings demonstrate that DMM and AIA are phenotypically unique models of OA, defined not only by initiating cause, but temporal pattern and inter- dependence of joint pathology, pain characteristics, and molecular drivers. The results suggest that the mechanisms regulating joint pain are specific to the disease pathophysiology, and confirm the importance of mapping pre-clinical findings to specific human disease phenotypes.