University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-01-07 Caffeine Exacerbates Postictal Hypoxia Phillips, Thomas James Phillips, T. J. (2019). Caffeine Exacerbates Postictal Hypoxia (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/109459 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Caffeine Exacerbates Postictal Hypoxia by Thomas James Phillips A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN NEUROSCIENCE CALGARY, ALBERTA JANUARY, 2019 © Thomas James Phillips 2019 i Abstract Background: Postictal hypoxia (PIH) is a stroke-like event that follows seizures and may be responsible for the postictal state. PIH may also be a contributing factor to the development of seizure-induced brain abnormalities and behavioral dysfunction associated with epilepsy. Caffeine is the world’s most popular drug with ~85% of people in the US consuming it daily. Thus, persons with epilepsy are likely to have caffeine in their system during seizures. This pre- clinical study investigated the acute and chronic effects of caffeine on tissue oxygenation pre and post seizure. Methods: We utilized the electrical kindling model in rats. A stimulating/recording electrode was placed into the rat ventral hippocampus (CA3), and an oxygen measuring optrode in dorsal hippocampus (CA1). Rats were administered vehicle (saline) or caffeine (5.0, 10.0, or 15.0 mg/kg) intraperitoneally, 30 minutes prior to an elicited seizure. Hippocampal oxygen levels were continually measured post-injection, and post-seizure, until returning to the normoxic range. Further, rats were administered various agonists and antagonists to determine adenosine receptors role in PIH. Lastly, rats were administered a chronic regime of caffeine to determine the long-term effects of caffeine in relation to PIH. Results: Caffeine at high (15. 0mg/kg) doses, caused a significant drop in pre-seizure hippocampal pO2. Following a seizure, caffeine at 10.0mg/kg and 15.0mg/kg, increased the time below the severe hypoxic threshold (10mmHg). Caffeine’s metabolites, paraxanthine, theobromine, and theophylline also increased the time below the severe hypoxic threshold. Adenosine A1 receptor agonist n6-Cyclopentyladenosine caused a significant drop in pre-seizure mean pO2 and increased the area below severe hypoxic threshold. A2A receptor antagonist SCH- i 58261 caused a significant drop in pre-seizure mean pO2. The A2B receptor, appeared to have no effect on hippocampal pO2 or PIH. Chronic caffeine consumption resulted in an increase in the area below the severe hypoxic threshold on day 15. Rats became tolerant to the drop-in pre- seizure pO2 via chronic consumption but became sensitized to the postictal severe hypoxia effects. Conclusions: Caffeine exacerbates postictal hypoxia; thus, this research hopes to guide future clinical investigations into the effect of caffeine on postictal blood flow and postictal symptomology in persons with epilepsy. ii Acknowledgements I would like to thank Dr. Cam Teskey for being a superb supervisor; Cam has offered excellent guidance over the past two years, and his advice has proven to be invaluable. Throughout my neuroscience masters, I had the privilege of working with some great people. Thank you to my incredible colleagues, with special thanks to Marshal, Roberto and Jordan for teaching me all the specialized techniques required to complete my thesis. Finally, I would like to thank all my family and friends. I would not have been able to accomplish what I have, without you. Mum, Dad, Ryan, your encouragement means a lot. I’m truly thankful to have such a great family. I would also like to thank my girlfriend Claire; your unconditional love and support has allowed me to make it through the past two years. I am unable to name everyone, however, a lot of you have been there for me, and I thank you. Another special thanks to the three cats throughout my life, all amazing – please see the figure below. Figure: The tripartite of felines throughout my life. iii Table of Contents Abstract ...........................................................................................................................................i Acknowledgements .......................................................................................................................iii Table of Contents ...........................................................................................................................iv List of Tables .................................................................................................................................vi List of Figures and Illustrations.....................................................................................................vii List of Symbols, Abbreviations and Nomenclature .....................................................................viii Chapter One: General introduction 1.1 Introduction to the postictal period…..…………………………………..………........1 1.2 Overview of epilepsy…………………..……………………………………………...2 1.3 Living with seizure disorders………………………………..………………….…......3 1.4 Postictal behavioural disruption…………………………………………….......……..4 1.5 Postictal vasoconstriction leads to hypoperfusion/hypoxia …………….....….............5 1.6 Treatment of postictal hypoxia ……………………………………………….....……9 1.7 Three commonly consumed drugs and their effects on vasculature function………..12 1.7.1 Nicotine…………………………………………………………………….12 1.7.2 Ethanol …………………………………………………………………….13 1.7.3 Tetrahydrocannabinol …………………………………………………......13 1.8 The effects of caffeine on vasculature function……………………………………….14 1.8.1 The metabolites of caffeine………………………………………………..14 1.8.2 Adenosine………………………………………………………………….16 1.8.3 Adenosine A1 Receptors…………………………………………………...16 1.8.4 Adenosine A2 Receptors…………………………………………………...17 1.8.5 Adenosine A3 Receptors…………………………………………………...18 1.8.6 Other targets………………………………………………………………18 1.9 The kindling model of seizures……………………………………………..…...........21 1.10 Summary……………………………………………………………………............22 Chapter Two: Caffeine Exacerbates Postictal Hypoxia 2.1 Introduction…………………………………………………………………….….....23 2.2 Methods………………………………………………………………………….…...26 2.2.1 Rats………………………………………………………………………...26 2.2.2 Electrode and optrode implantation………………………………….…….26 2.2.3 Kindling and oxygen Recording……………………………………….......27 2.2.4 Pharmacology……………………………………………………………...28 2.2.5 Chronic study……………………….…………………………………….. 28 2.2.6 Statistics…………………………………………………………………....29 2.2.7 Study approval…………………….…………………………………….....31 2.3 Results………………………………………………………………………….….…31 2.3.1 Acute administration of Caffeine…………………………….……….........31 2.3.2 Caffeine’s metabolites ………………………………………………….....32 2.3.3 Adenosine receptors………………………………………………….….....36 2.3.4 Other targets in relation to PIH ……………………………………….…...37 iv 2.3.5 Chronic administration of caffeine on PIH….……….…………….………37 2.4 Discussion…………………………………………………………….……………...44 Chapter Three: General Discussion 3.1 Summary………………………………………………………………………….….50 3.2 Future Directions……………………………………………………………………..51 3.3 Conclusion…………………………………...………………………………….…....54 Appendix: Unpublished data A.1 Three commonly consumed drugs: Nicotine, Ethanol and THC in relation to PIH...55 A.2 Exogenous mixture of 5% CO2, 95% O2 fails to alleviate PIH………………..….…57 References.....................................................................................................................................59 v List of Tables 1.1 Adenosine Receptors and their expression…………………………………………………..19 2.1 Molecular targets in relation to caffeine and postictal hypoxia…….………………………..25 2.2 Summary of the investigation into mechanisms involved in caffeine and severe postictal hypoxia…………………………………………………………………………………...………41 vi List of Figures and Illustrations Figure 1.1 Local tissue oxygenation in the hippocampus of an awake, freely-moving rat…………………………………………………………………………7 Figure 1.2. Potential acute (singular) and chronic (repeated) effects of postictal hypoperfusion/hypoxia……………………………………………………………………………8 Figure 1.3 Two key molecular targets mediating postictal hypoperfusion/hypoxia…………….11 Figure 2.1 A timeline of chronic administration of caffeine……………………………….........30 Figure 2.2: Caffeine in naïve animals causes a significant reduction in hippocampal pO2……………………………………………………………………………...33 Figure 2.3: Caffeine exacerbates postictal hypoxia……………………………………………..34 Figure 2.4: Caffeine’s metabolites cause changes in hippocampal pO2, while prolonging the postictal hypoxic period……………………………………………………………………….....35 Figure 2.5: A1 receptor activation causes profound drop in hippocampal pO2………………...38 Figure 2.6: Blocking the A2A receptor causes profound drop in hippocampal pO2…………...39 Figure 2.7: Chronic caffeine administration over 15 days………………………………….......41 Figure 2.8: Caffeine naïve rats compared to chronic caffeine rats……………………………...42 Figure 2.9: Mean consumption of caffeine throughout the chronic experiment …..……...........43 Figure