Investigating the Central Role of Astrocytes in Mediating Postanesthetic Memory Deficits by Kirusanthy Kaneshwaran A thesis submitted in conformity with the requirements for the degree of Master in Science Department of Physiology University of Toronto © Copyright by Kirusanthy Kaneshwaran 2017 Investigating the Central Role of Astrocytes in Mediating Postanesthetic Memory Deficits Kirusanthy Kaneshwaran Master of Science Department of Physiology University of Toronto 2017 Abstract Anesthetics cause postanesthetic memory deficits in animal models, and similar deficits may contribute to postoperative delirium and cognitive dysfunction in patients. We previously showed using a mouse model that etomidate-induced persistent memory deficits result from increased tonic GABAA receptor-mediated inhibitory current in the hippocampus, through an astrocyte-mediated mechanism. However, the underlying mechanisms are uncertain. The aims of this study were to: 1) determine whether widely used anesthetic and sedative agents persistently increase tonic GABAA current; 2) determine whether human astrocytes mediate the increase, and 3) identify the underlying mechanisms. The results show that several GABAergic drugs persistently increase tonic current and human astrocytes release soluble factors that mediate etomidate’s effect on tonic current. Furthermore, etomidate and sevoflurane act through astrocytic GABAA receptors to trigger a pro-inflammatory signaling pathway, which underlies the persistent increase in tonic current. These results increase the mechanistic understanding of postanesthetic memory deficits and delineate treatment targets. ii Acknowledgements During the past two years I have undergone immense growth as a researcher and more importantly, as an individual. I have been privileged enough to be surrounded by wonderful people who have supported me and encouraged me in all my endeavors, research-related and otherwise. I would like to thank my supervisor, Dr. Beverley Orser for your immense support and guidance. You have offered me pearls of wisdom pertaining to not only research, but also life. I strive to be as strong and inspiring a woman as you are in my future. Until then “I will fake it, till I become it”. Thank you for giving me this opportunity to work with amazing individuals and hone my research skills. I also thank the members of my Advisory Committee, Dr. Rob Bonin and Dr. Richard Horner for your time, support, excellent advice (and reference letters upon short notice!). Your advice has really helped me develop my ideas and realize that there is still so much to learn. You have helped me develop a love for learning through example. I would also like to thank Dr. Richard Horner for providing me with an important learning experience as a TA. I would like to convey my sincerest gratitude to the entire Orser Lab. Thank you Dr. Sinziana Avramescu for being my “laboratory mom”: giving me hugs when I needed them most, listening to my worries, sharing my happiness, supporting me even when I was at my worst, and so much more. My gratitude cannot be put in to words. You were everything I needed: a caring friend, an incredible mentor, an inspiring researcher, etc.. Thank you Dr. Dian-Shi Wang, for being one of the best teachers I have had in my life! Your guidance has really helped me improve my critical thinking, organizational, and even technical and math skills! Thank you Dr. Irene Lecker for giving me some of the best advice I have ever received and for supporting me throughout this phase of my life. Thank you Fariya Mostafa for being an incredible friend. I have become a better person through our friendship, and I hope to one day be as kind and caring an individual as you are. I am extremely grateful that you were with me iii through the majority of these past two years: as a fellow laboratory mate to discuss experiments and results, as a friend to share both sad and happy things alike, and as an older sister who gave me incredible advice and support. Thank you Dr. Shelly Au, for being a great friend, listening to my worries and teaching me about life. Thank you Shahin for being my rock, inspiring me to have a love for science and helping me out whenever I needed it. Thank you Nathan Chan for being my ray of sunshine! Thank you Agnes Crnic for being you! Your great humor, Microsoft Word and PowerPoint skills, and friendship have made my life significantly more enjoyable! Also, thank you to everyone who has been a part of the lab: Michael, Alejandro, Dr. Gang Lei, Dr. Junhui Wang, Dr. Yu Feng Xia, Sean Haffey and Dr. Agnes Zurek. I am extremely privileged to have worked with you in the last two years. I would also like to thank all my fellow Physiology graduate students and GASP (Melanie, Ankur, Hanna, Susmita, Farwah, Vivian, Feiya…), who have given me valuable experiences and tolerated my craziness. Last but not least, I would like to thank my family, who has provided me with immense support during the past two years. Thank you for tolerating me when I was at my worst, keeping me humble when I was at my best, and supporting and encouraging me whenever I needed it. Thank you Appa for listening to me talk about science and life during our car rides and giving me your opinion and advice. Thank you Amma for listening to me ramble and vent and supporting me in all that I do. I can never repay you for all that you do for me, and I sincerely value every little thing that you do to make my life better. Thank you Guhaverl and Lokeesan for letting me burrow your stuff and take up your time. These two years have been the best years of my life thanks to you, and I will cherish my relationship with all of you and the experiences we have had together as precious memories for the rest of my life! iv List of Contributions Kirusanthy Kaneshwaran produced all of the results and content of this thesis except those listed below. The electrophysiological data presented in Figure 4.1 were collected with the help of Ms. Fariya Mostafa, Dr. Sinziana Avramescu, Dr. Irene Lecker, and Dr. Dianshi Wang. They contributed to the recording of tonic current in neurons using different anesthetics. Also, in Chapter 5, Dr. Sinziana Avramescua and Dr. Irene Lecker contributed recordings from neurons for data presented in Figure 5.1C, and Mr. Sean Haffey provided data for Figures 5.3B and 5.4A. This work was completed with the financial support from a Kirk Weber Graduate Award provided by the Department of Anesthesia, Sunnybrook Health Sciences Centre and an Ontario Graduate Scholarship. v Table of Contents Abstract ............................................................................................................................................. ii Acknowledgements .......................................................................................................................... iii List of Contributions .......................................................................................................................... v Table of Contents ............................................................................................................................. vi List of Figures ................................................................................................................................. viii List of Abbreviations ........................................................................................................................ ix Chapter 1. Thesis overview ............................................................................................................... 1 1.1. Rationale ................................................................................................................... 1 1.2. Hypothesis and specific aims .................................................................................... 6 1.3. Thesis structure ......................................................................................................... 9 Chapter 2. General Introduction ..................................................................................................... 11 2.1. Postoperative delirium and cognitive dysfunction ................................................. 11 2.1.1. Defining postoperative delirium and POCD ................................................ 11 2.1.2. Incidence ............................................................................................. 12 2.1.3. Effects on patient outcomes ....................................................................... 14 2.1.4. Risk factors and potential causes of postoperative delirium and POCD in patients ............................................................................................. 15 2.2. Cognitive deficits after anesthesia in animal models ............................................. 19 2.2.1. Evidence in the literature ............................................................................ 19 2.2.2. Proposed mechanisms of postanesthetic cognitive deficits in animals ...... 20 2.3. GABA and GABAA receptors-mediated inhibition ................................................... 24 2.3.1. GABA: synthesis, release, transport and metabolism ................................. 24 2.3.2. Overview of GABA receptors ...................................................................... 26 2.3.3. GABAA receptors mediated inhibition ........................................................ 27 2.3.4. GABAA receptors antagonists (bicuculline) ................................................. 31 2.3.5. Subunit composition