University of Alberta The Effect of Caffeine on the Neurobehavioral and Neuropathological Outcome of the Newborn Rat By Omar Abu-Sa’da A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Medical Sciences Department of Paediatrics © OMAR ABU-SA’DA Spring 2010 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. 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The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author’s permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n’y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. EXAMINING COMMITTEE Dr. Jerome Y. Yager, Department of Paediatrics, Supervisor. Dr. Po-Yin Cheung, Department of Paediatrics, Co-Supervisor. Dr. Gregory D. Funk, Department of Physiology, Supervisory Committee Member. DEDICATION I dedicate my thesis to my parents who reared me to be a good person and encouraged me to be a good physician, to my loving and devoted wife, to my children who have made my life full with happiness and hope and to my teachers and colleagues. THESIS ABSTRACT Caffeine is used for the treatment of apnea of prematurity. The objective of this study was to determine the long term neuropathological and neurobehavioral effects of caffeine on the immature rat brain. Newborn rats were injected with either caffeine, or normal saline from postnatal days 3 to 7, equivalent to the human premature infant of 28-36 weeks. Behavioral tests revealed no abnormality in caffeine treated animals compared to controls. Fluro- Jade B stain of P4 rat brains showed that caffeine caused significant neuronal cell death in some areas of the brain, compared to controls, but this alteration was transient and not present at P8. Anti-NeuN stain at P21 showed significant neuronal cell loss in CA1 and hypothalamus regions in the caffeine group, but not at P160. Anti-Neurofilament M stain at P8, P21 and P160 showed no differences between the control and caffeine groups. We conclude that use of caffeine has no significant effect on the behavioral tests measured in our newborn rat pups. While caffeine caused neuronal cell death at P4, and neuronal cell loss in CA1 and hypothalamus regions at P21, there was no long-lasting effect on neuropathological outcome. However, given these latter findings, the use of caffeine in the premature infant must still be done with caution. ACKNOWLEDGEMENTS No research is a solo effort. I would like to thank Dr. Jerome Y. Yager, who has been not only a valuable adviser through this research and thesis project, but also a great role model, a caring mentor and a truly wise supervisor. My special appreciation goes to Dr. Po-Yin Cheung for being a co-supervisor, for supervising me during the course “Directed Reading in Clinical Medicine”, and for his continuous support and advice. I would also like to thank Dr. Gregory D. Funk for being a member of the supervising committee and for many varied ways of demonstrating support and advice. My thanks go also to Arlene Figley for her excellent administrative help and to Dr. Richard Schulz for his chairing the examining committee. To my colleagues in Dr.Yager's laboratory: Edward Armstrong, Dr. Alireza Karimi Pour, Keqin Shen, and Amy M. Black, I appreciate your patience, wisdom and your great contributions to my research and thesis. Thanks Ed for your advice, teaching and patience all through my research. My heartfelt thanks are due to my parents for their continuous support and praying, and to all my brothers and sisters. To my lovely daughters - Mais, Lujein and Layan- thank you for all the joy and endless light and happiness you bring to my life. Finally, my sincere thanks go to my love, my heart and my best friend: my wife; Nabeela. Thank you for the talking, listening, laughing and sharing your life with me. You are the best gift from GOD. I will continue to be grateful to you, my love, forever. TABLE OF CONTENTS PAGE Introduction:...................................................................................................... 1 References:..............................................................................................................3 Chapter 1: Apnea of Prematurity and Its Treatment:………………4 1.1. Apnea of Prematurity:………………………………………………………4 1.1.1. Background:……………………………………………………………4 1.1.2. Pathophysiologic Mechanisms of Apnea of Prematurity:........................7 1.1.3. Apnea of Prematurity and Neurodevelopmental Outcome in Very Low Birth Weight Infants:………………………………………11 1.1.4. Apnea of Prematurity and Gastroesophageal Reflux Disease in Very Low Birth Weight Infants:……………………………………14 1.1.5. Apnea of Prematurity and Feeding in Very Low Birth Weight Infants:………………………………………………………………...15 1.1.6. Apnea of Prematurity and Sudden Infant Death Syndrome in Very Low Birth Weight Infants:………………………………………16 1.2. Treatment of Apnea of Prematurity:……………………………………..17 1.2.1. Non-Pharmacological Treatment of Apnea of Prematurity:........................17 1.2.2. Pharmacological Treatment of Apnea of Prematurity:..........................21 1.2.2.1. Methylxanthines:................................................................21 1.2.2.2. Doxapram:………………………………………………..22 1.2.2.3. Carnitine:…………………………………………………23 1.2.2.4. Creatine:………………………………………………….23 References:………………………………………………………………………25 Chapter Two: Caffeine as Treatment of Apnea of Prematurity:...29 Introduction:…………………………………………………………………….29 2.1. Structure & Pharmacodynamic Properties of Caffeine:………………...29 2.2. Systemic Effects of Caffeine:………………………………………………30 2.3. Physiologic Effects of Caffeine:...…………………………………………30 2.4. Pharmacokinetic Properties of Caffeine:...……………………………….35 TABLE OF CONTENTS PAGE 2.5. Caffeine Dosage and Interactions:...………………………………………37 2.6. Therapeutic Efficacy of Caffeine:...……………………………………….39 2.7. Adenosine:...………………………………………………………………...39 2.8. The Effects of Caffeine and Adenosine on the Developing Brain:...……………………………………………………………………...41 2.8.1. Breathing:...……………………………………………………………41 2.8.2. Sleep and the Level of Arousal:...……………………………………..43 2.8.3. Anxiety and Pain:...……………………………………………………44 2.8.4. Cognition and Memory:...……………………………………………..44 2.8.5. Neuronal Protection, Injury and Maturation:...………………………..45 2.8.6. Seizure: ...…………………………………………………………….48 2.8.7. Cerebral Blood Flow:...………………………………………………..49 2.8.8. Pituitary Gland:...……………………………………………………...50 2.10. The Effects of Caffeine on Pregnancy:...………………………………...51 2.11. Caffeine and Lactation:...………………………………...........................53 2.12. Conclusion:...………………………………...............................................54 References:...……………………………….........................................................58 Chapter 3: Methodology and Experimental Protocols:...…………..68 3.1. Hypothesis:……………………………......………………………………...68 3.2. Rat Model:……………………………......………………………………...68 3.3. Study Design:……………………………......……………………………...69 3.3.1. The Period of Giving Caffeine Treatment:………………………70 3.3.2. The Dosage of Caffeine Treatment:……...………………………71 3.4. Animal Neurobehavioral Tests:…………………...………………………71 3.4.1. Early Behavioural Testing: Neonatal (P4-P9), Post-Neonatal, Infantile and Pre-Juvenile