OPIOIDS AND GLIA: INVESTIGATING THE MECHANISMS THROUGH WHICH ULTRA-LOW DOSE OPIOID ANTAGONISTS MODULATE OPIOID TOLERANCE AND HYPERALGESIA. by Theresa Alexandra Mattioli A thesis submitted to the Graduate Program in Pharmacology & Toxicology in the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Doctor of Philosophy Queen’s University Kingston, Ontario, Canada (April, 2013) Copyright © Theresa Alexandra Mattioli, 2013 Abstract Ultra-low doses (ULD) of the opioid receptor antagonists, naloxone and naltrexone, augment the analgesic actions of morphine, block the induction of tolerance, and reverse established tolerance by an unknown mechanism. Preclinical studies demonstrate that chronic morphine administration induces spinal gliosis and that inhibition of gliosis prevents the development of analgesic tolerance to opioids. Thus, this thesis investigated the inhibition of spinal gliosis as a mechanism by which ULD antagonists attenuate analgesic tolerance and opioid-induced hyperalgesia. Immune cell activation is implicated in the etiology of morphine tolerance and intrathecal catheterization, a technique commonly used to study the spinal effects of drugs, causes profound gliosis. Thus, the first study investigated the effects of catheter-induced gliosis on acute and chronic morphine analgesic tolerance. Catheterization-induced gliosis did not alter antinociceptive responses to acute intrathecal morphine; however, tolerance to chronic morphine was exacerbated in catheterized rats compared to sham and surgery-naïve controls. The potentiation of analgesic tolerance to chronic morphine by spinal gliosis provided evidence that glia modulate opioid analgesia; therefore, inhibition of opioid-induced activation of glia was explored as a potential mechanism by which ULD antagonists prevent tolerance. The second series of experiments reported morphine-induced activation of spinal microglia and astrocytes was blocked by co-administering ULD naltrexone with morphine. These findings prompted us to elucidate the specific molecular target through which ULD antagonists attenuate opioid analgesia. Activation of glial Toll-like receptor 4 (TLR4) induces gliosis and may contribute to analgesic tolerance and/or morphine-induced hyperalgesia (MIH). Antagonism of TLR4 by the opioid receptor-inactive (+) stereoisomer of naloxone was identified as a potential mechanism by which ULD antagonists modulate opioid analgesia. Tolerance and MIH developed in mice ii expressing non-functional TLR4 and in wildtype controls. Analgesic tolerance was stereoselectively blocked by ULD (-)naloxone, whereas MIH was blocked by both naloxone enantiomers. Collectively, these studies demonstrate analgesic tolerance and MIH occur through distinct mechanisms. ULD naloxone attenuates analgesic tolerance likely via an opioid receptor- mediated mechanism that is TLR4-independent. ULD antagonists do not attenuate tolerance via inhibition of spinal gliosis as hypothesized. In contrast, ULD antagonists prevent MIH by inhibiting opioid-induced gliosis in an opioid receptor- and TLR4-independent manner. iii Co-Authorship The research upon which this thesis is based was conducted by Theresa Alexandra Mattioli under the supervision of Dr. Catherine M. Cahill and Dr. Brian Milne. Dr. Catherine M. Cahill performed the implantation of intrathecal catheters used in Chapter 2 and Ms. Maaja Sutak provided spinal tissue used to in the immunohistochemical assay in Chapter 2. Theresa Alexandra Mattioli contributed immunohistochemical (Fig. 8c) and behavioural data (Fig. 8k) and analyzed this data in Chapter 4. Dr. Francesco Ferrini and Dr. Tuan Trang were responsible for the conduct and analysis of all other experiments in Chapter 4 and preparation of the final manuscript based on data in this chapter. Graham Skelhorne-Gross isolated RNA and performed qRT-PCR analysis of spinal tissue in Chapter 5. Ms. Lihua Xue provided technical support throughout the thesis project. iv Acknowledgements I would like to thank the many people who have supported me throughout this journey. Firstly, to my parents, I would like to thank you for always believing in me and encouraging me to follow my dreams. Your unwavering love and support, and of course the numerous donations to my ‘grocery fund,’ have helped me through a number of challenges. Thank you for teaching me to persevere through the hard times and celebrate my accomplishments. To my siblings, Justin and Jackie, you have always been a source of balance in my life. Justin, you never fail to put a smile on my face even on the toughest days. I am inspired by your spontaneous and carefree spirit, and have learned that the only limitations are those we impose on ourselves. Jax, you’re my best friend, my confidant, and my role model. You’ve always been there for me, whether the voice of reason, getting me out of trouble, or joining me in mischief, I could not ask for a better sister. Thank you for always seeing the best in me and helping me to see obstacles as challenges to be embraced rather than impossibilities. To my closest friends, Alana, Julie, Maaike and Megan, your support over the years has been instrumental to my achievements. Thank you for enabling me to keep perspective, to share a laugh or a tear, and always bringing joy to my life. Many thanks to my thesis committee and the members of the Pharmacology and Toxicology program for their ongoing support and encouragement. Special thanks to Dr. Khem Jhamandas for your valuable insight and guidance throughout this project. Your willingness to share your passion and wisdom has been both infectious and engaging. I would also like to thank the other members of the Cahill lab for all of the laughs, dance parties, and assistance throughout the years. I am very grateful to all of you and will cherish the memories of our years together. To my supervisors, Dr. Catherine Cahill and Dr. Brian Milne, I would like to express my sincere gratitude for your leadership and direction throughout my studies. Cathy, your endless patience, encouragement and advice have been invaluable. You are not only an outstanding academic mentor but also an exceptional role model. You have taught me that it is possible to have it all. Brian, your optimism and fresh perspective were always appreciated, particularly during overwhelming and challenging times. I am excited to start the next chapter of my life and feel well prepared for whatever lies ahead thanks to my supervisors who have certainly gone above and beyond. v Table of Contents Abstract ............................................................................................................................................ ii Co-Authorship ................................................................................................................................ iv Acknowledgements .......................................................................................................................... v List of Figures……………………………………………………………………………………...x List of Tables……………………………………………………………………………………..xii List of Abbreviations…………………………………………………………………………….xiii CHAPTER 1: INTRODUCTION .................................................................................................... 1 1.1.1 Social and Economic Impact of Chronic Pain ......................................................... 1 1.2 Opioid Analgesics ............................................................................................................ 2 1.3 Opioid Tolerance ............................................................................................................. 6 1.3.1 Mechanisms of Opioid Tolerance ............................................................................ 8 1.3.2 Bimodal Theory of Tolerance .................................................................................. 9 1.3.3 Involvement of Spinal Glia in Tolerance ............................................................... 11 1.3.3.1 Glial Activation .................................................................................................. 11 1.3.3.2 Opioid-Induced Glial Activation ....................................................................... 11 1.3.3.3 Gliosis and Opioid Tolerance ............................................................................ 12 1.3.3.4 Inhibition of Gliosis Augments Opioid Tolerance ............................................. 13 1.3.4 TLR4-Induced Gliosis............................................................................................ 14 1.3.4.1 TLR4 Structure & Signaling .............................................................................. 15 1.3.4.2 TLR4 and Opioids .............................................................................................. 17 1.4 ULD Antagonists Attenuate Opioid Tolerance & Hyperalgesia .................................... 18 1.4.1 ULD Antagonists and the Bimodal Theory ........................................................... 18 1.4.2 ULD Antagonists and Filamin A Hypothesis ........................................................ 18 1.4.3 ULD Antagonists and Gliosis ................................................................................ 19 1.5 Research Statement and Objectives ............................................................................... 20 CHAPTER 2: INTRATHECAL CATHETERIZATION INFLUENCES TOLERANCE TO CHRONIC MORPHINE IN RATS ..............................................................................................