Pharmacology and Pathophysiology of the Control of Breathing

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PHARMACOLOGY AND PATHOPHYSIOLOGY OF THE CONTROL OF BREATHING


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LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball 20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg


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21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva 26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland 27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant’Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders


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44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay 56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray 60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J.-P. Martin, and R. Masse


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69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer 70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M.-C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O’Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos 86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium–Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology • Pathogenesis • Clinical Manifestations • Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman 92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J.-P. Derenne, W. A. Whitelaw, and T. Similowski


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93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey 95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse


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118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky 119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck 120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S.-E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind 124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma’s Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin


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141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield 145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus 148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill 153. Environmental Asthma, edited by R. K. Bush 154. Asthma and Respiratory Infections, edited by D. P. Skoner 155. Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, S. Rak, and R. A. Pauwels 156. Genetic Models in Cardiorespiratory Biology, edited by G. G. Haddad and T. Xu 157. Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder 158. Ventilator Management Strategies for Critical Care, edited by N. S. Hill and M. M. Levy 159. Severe Asthma: Pathogenesis and Clinical Management, Second Edition, Revised and Expanded, edited by S. J. Szefler and D. Y. M. Leung 160. Gravity and the Lung: Lessons from Microgravity, edited by G. K. Prisk, M. Paiva, and J. B. West 161. High Altitude: An Exploration of Human Adaptation, edited by T. F. Hornbein and R. B. Schoene 162. Drug Delivery to the Lung, edited by H. Bisgaard, C. O’Callaghan, and G. C. Smaldone 163. Inhaled Steroids in Asthma: Optimizing Effects in the Airways, edited by R. P. Schleimer, P. M. O’Byrne, S. J. Szefler, and R. Brattsand


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164. IgE and Anti-IgE Therapy in Asthma and Allergic Disease, edited by R. B. Fick, Jr., and P. M. Jardieu 165. Clinical Management of Chronic Obstructive Pulmonary Disease, edited by T. Similowski, W. A. Whitelaw, and J.-P. Derenne 166. Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, edited by A. I. Pack 167. Biotherapeutic Approaches to Asthma, edited by J. Agosti and A. L. Sheffer 168. Proteoglycans in Lung Disease, edited by H. G. Garg, P. J. Roughley, and C. A. Hales 169. Gene Therapy in Lung Disease, edited by S. M. Albelda 170. Disease Markers in Exhaled Breath, edited by N. Marczin, S. A. Kharitonov, M. H. Yacoub, and P. J. Barnes 171. Sleep-Related Breathing Disorders: Experimental Models and Therapeutic Potential, edited by D. W. Carley and M. Radulovacki 172. Chemokines in the Lung, edited by R. M. Strieter, S. L. Kunkel, and T. J. Standiford 173. Respiratory Control and Disorders in the Newborn, edited by O. P. Mathew 174. The Immunological Basis of Asthma, edited by B. N. Lambrecht, H. C. Hoogsteden, and Z. Diamant 175. Oxygen Sensing: Responses and Adaptation to Hypoxia, edited by S. Lahiri, G. L. Semenza, and N. R. Prabhakar 176. Non-Neoplastic Advanced Lung Disease, edited by J. R. Maurer 177. Therapeutic Targets in Airway Inflammation, edited by N. T. Eissa and D. P. Huston 178. Respiratory Infections in Allergy and Asthma, edited by S. L. Johnston and N. G. Papadopoulos 179. Acute Respiratory Distress Syndrome, edited by M. A. Matthay 180. Venous Thromboembolism, edited by J. E. Dalen 181. Upper and Lower Respiratory Disease, edited by J. Corren, A. Togias, and J. Bousquet 182. Pharmacotherapy in Chronic Obstructive Pulmonary Disease, edited by B. R. Celli 183. Acute Exacerbations of Chronic Obstructive Pulmonary Disease, edited by N. M. Siafakas, N. R. Anthonisen, and D. Georgopoulos 184. Lung Volume Reduction Surgery for Emphysema, edited by H. E. Fessler, J. J. Reilly, Jr., and D. J. Sugarbaker 185. Idiopathic Pulmonary Fibrosis, edited by J. P. Lynch III 186. Pleural Disease, edited by D. Bouros


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187. Oxygen/Nitrogen Radicals: Lung Injury and Disease, edited by V. Vallyathan, V. Castranova, and X. Shi 188. Therapy for Mucus-Clearance Disorders, edited by B. K. Rubin and C. P. van der Schans 189. Interventional Pulmonary Medicine, edited by J. F. Beamis, Jr., P. N. Mathur, and A. C. Mehta 190. Lung Development and Regeneration, edited by D. J. Massaro, G. Massaro, and P. Chambon 191. Long-Term Intervention in Chronic Obstructive Pulmonary Disease, edited by R. Pauwels, D. S. Postma, and S. T. Weiss 192. Sleep Deprivation: Basic Science, Physiology, and Behavior, edited by Clete A. Kushida 193. Sleep Deprivation: Clinical Issues, Pharmacology, and Sleep Loss Effects, edited by Clete A. Kushida 194. Pneumocystis Pneumonia: Third Edition, Revised and Expanded, edited by P. D. Walzer and M. Cushion 195. Asthma Prevention, edited by William W. Busse and Robert F. Lemanske, Jr. 196. Lung Injury: Mechanisms, Pathophysiology, and Therapy, edited by Robert H. Notter, Jacob Finkelstein, and Bruce Holm 197. Ion Channels in the Pulmonary Vasculature, edited by Jason X.-J. Yuan 198. Chronic Obstuctive Pulmonary Disease: Cellular and Molecular Mechanisms, edited by Peter J. Barnes 199. Pediatric Nasal and Sinus Disorders, edited by Tania Sih and Peter A. R. Clement 200. Functional Lung Imaging, edited by David Lipson and Edwin van Beek 201. Lung Surfactant Function and Disorder, edited by Kaushik Nag 202. Pharmacology and Pathophysiology of the Control of Breathing, edited by Denham S. Ward, Albert Dahan and Luc J. Teppema 203. Molecular Imaging of the Lungs, edited by Daniel Schuster and Timothy Blackwell 204. Air Pollutants and the Respiratory Tract: Second Edition, edited by W. Michael Foster and Daniel L. Costa

The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.


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PHARMACOLOGY AND PATHOPHYSIOLOGY OF THE CONTROL OF BREATHING

Edited by

Denham S. Ward University of Rochester Medical Center Rochester, New York, U.S.A.

Albert Dahan Leiden University Medical Center Leiden, Netherlands

Luc J. Teppema Leiden University Medical Center Leiden, Netherlands

Boca Raton London New York Singapore

DK1337_Discl.fm Page 1 Wednesday, April 13, 2005 3:16 PM

Published in 2005 by Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2005 by Taylor & Francis Group, LLC No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-5890-0 (Hardcover) International Standard Book Number-13: 978-0-8247-5890-5 (Hardcover) Library of Congress Card Number 2005043947 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data Ward, Denham. Pharmacology and pathophysiology of the control of breathing / Denham Ward. p. cm. Includes bibliographical references and index. ISBN 0-8247-5890-0 1. Respiration--Regulation. 2. Lungs--Pathophysiology. 3. Pulmonary pharmacology. 4. Inhalation anesthesia. I. Title. QP123.W37 2005 612.2--dc22

2005043947

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INTRODUCTION

‘‘What is the use of breathing? That it is not a trifling use is clear from our inability to survive for even the shortest time after it has stopped. Hence also it is obvious that its importance is not for any particular and partial activity, but for life itself.’’ De Usu Respirationis Galen (c.120–c.200) In a second writing titled De Causis Respirationis, Galen went on to say: ‘‘It is impossible either to confirm the hypothesis of breathing or to put it right if it is impeded or completely stopped, without knowing its causes.’’ Galen’s issues are, in fact, the subject of this latest addition to the series Lung Biology in Health and Disease—Pharmacology and Pathophysiology of the Control of Breathing. This topic has fascinated generations of fundamental and clinical researchers, and their contributions have played an essential role in our current understanding of the control of breathing in health and diseases. And today the names of many of these scientific giants grace the virtual walls of the Biomedicine Hall of Fame! A turning point in the evolution of this work was the presentation of the Silliman Lectures given in 1916 by J.S. Haldane at Yale University. That was followed in 1921 by the publication of Respiration by J.S. Haldane and J.G. Priestley. In the preface to this monumental book, the authors acknowledged that ‘‘many gaps remain to be filled’’ but ‘‘the observations and experiments required [to fill them] are not yet available.’’ Today, almost a century later, many of the gaps have been filled thanks to the brilliant and dedicated researchers who have worked in this field. Nonetheless, the road to understanding the control of breathing has vii

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been long and tortuous. First, researchers observed changes in breathing patterns that occur with environmental variations, especially altitude, in an effort to uncover the mechanisms of breathing control. Then, they turned attention to the impact of pathology on this process, and breathing disruptions caused by disease became a fertile topic of investigation. In many ways, the 15 volumes on (or related to) control of breathing that we have published illustrate the purpose of this series when it was conceived more than three decades ago — namely, to report about and stimulate areas of biology and medicine, especially those that are so amazingly dynamic. This volume takes us to a new level by filling many of the gaps that Haldane and Priestley identified. In addition, as Thomas Hornbein says in his Foreword, ‘‘Within this volume are many enticing next steps.’’ The series of monographs Lung Biology in Health and Disease is most pleased to present this volume to its readership and I, personally, owe a great debt of gratitude to the editors, Drs. Denham S. Ward, Albert Dahan, and Luc Teppema, and the many authors for the privilege of introducing this important new contribution. Claude Lenfant, MD Gaithersburg, Maryland

PREFACE

We would be seriously remiss if we did not first acknowledge the work of the ‘‘fourth editor’’ of this volume, Debra L. Lipscomb. Without her careful scrutiny of every chapter and her abilities to pick out problems in meaning, grammar, spelling and references, the quality of this volume would have been severely compromised. Previous volumes in this series have dealt with the regulation of breathing and it was the volume edited by Dr. Thomas Hornbein in 1981 that has served as an essential reference for us during our careers. We are particularly gratified that he has provided a Foreword to this volume. Subsequent volumes in this series dealing with the control of breathing, as well as the two volumes in the American Physiological Society’s Handbook of Physiology have provided up-to-date summaries of the field in the face of a rapid increase in knowledge. Pharmacology has long played an important role in the control of breathing, both in providing tools for physiological experiments and in understanding the ventilatory effects of pharmacological agents. The increase in knowledge in both physiology and pharmacology has greatly extended our knowledge of the cellular and subcellular elements involved in controlling ventilation. Drug effects on control of breathing were covered as a single chapter in both Hornbein’s original volume as well as in the Handbook of Physiology. However, the increase in our understanding of the mechanisms of action of pharmacological agents on the control of breathing now warrants a separate volume. We have attempted to organize this knowledge by first reviewing the relevant physiology from a perspective of the substrate for pharmacological action and also by reviewing pharmacology principles as they can be applied to the control of breathing. We then have selected topics ix

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of pathophysiology and pharmacology that are relevant to clinical practice. It is hoped that this organization, rather than organizing by drug, will provide a more useful reference for both clinicians and research scientists. There are many clinical problems both in respiratory side effects of drugs and in finding drugs that will treat abnormalities in the control of breathing that remain to be solved. The increased understanding of the specific ion channels, receptors and neurotransmitters involved in respiratory control provides important information for pharmacological research. We hope this volume will set the stage for future research and provide as much motivation for beginning reseachers as Dr. Hornbein’s volume did for us.

FOREWORD

When I was young I fell in love with the carotid body. Though I did not know it at the time, this infatuation was kindled when my prepubertal proclivity for climbing things—trees and houses—bumped into a teenage discovery: mountains were better. With medical school my addiction to the reading of mountaineering literature was supplanted by exploration of the literature about human adaptation to high altitude. One paper, in particular, became seminal. I found in a report by Hugo Chiodi, a Peruvian clinician-investigator, that when permanent residents of high altitude were compared to lowlanders acclimatized for a time to the same altitude, the highlanders appeared to exhibit less ventilation (higher alveolar PCO2 ) and greater polycythemia than the lowlanders [1]. Was there a connection, I wondered, between the two? Did the hypoventilation provoke the greater polycythemia? Or might it be the other way around, that polycythemia somehow enables less ventilation? Or might both possibilities coexist in a kind of positive feedback system? During my final year in medical school, I seized upon a six-week elective period to embark upon my first research project, to study the effect of polycythemia on breathing. With the oversight of Albert Roos, who would become the mentor for my subsequent research training, I transfused myself with five units of blood to elevate my hematocrit from 45% to 60%. Pedaling away on a cycle ergometer while breathing oxygen mixtures to simulate different altitudes, Dr. Roos and I compared my breathing at high hematocrit with that at normal hematocrit. Not surprisingly, ventilation was less at the higher hematocrit. I cannot say it was significantly less, for this paper, ‘Effect of Polycythemia on Respiration’ was published in spite of being performed on only one subject (other volunteers were hard to find) and hence had no statistical analysis [2]. xi

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Next came the question, how does polycythemia result in less exercise hyperpnea? Enter the carotid body and the beginnings of an affair with chemoreception and the chemical control of breathing that directed my scientific exploration for the next quarter century. At the time I climbed Mount Everest in 1963 [3], I never imagined the numbers that would be thronging to its top four decades later. Nor did I, the first time I hung the Nerve of Hering across a pair of platinum electrodes and listened to the crackling crescendo of neuronal traffic as the animal’s PO2 was lowered, anticipate the current profundity of inquiry into the ventilatory control system. The pharmacology of ventilatory control comprised a single chapter in the original volume on regulation of breathing in this series [4], as well as its second edition [5]; that pharmacology could command an entire, rich volume of its own was unimaginable not so long ago. This book (number 202 In Claude Lenfant’s series, Lung Biology in Health and Disease), Pharmacology and Pathophysiology of the Control of Breathing, has been creatively conceived and gestated by Drs. Ward, Dahan, and Teppema. The book is divided into three parts: Neuropharmacology and Physiology, Pathophysiology, and Clinical Pharmacology. The first section is, for me, the dessert, even though it comes at the beginning of the meal. This section explores the functional anatomy and physiology of the ventilatory control system from the intracellular to the integrative level, including what is coming to be known about the roles of nervous system plasticity and genes. It’s like the woods out back where one could go to explore, full of intrigue and an inexhaustible supply of new questions emerging from every answer. This first section stirred past wonderings of a time when I puzzled about why inhalational anesthetics, all of which produced anesthesia, differed in the fingerprint each left on the central nervous system with regard to side effects such as breathing. For example, diethyl ether, the founding father of general anesthesia, produced little or no ventilatory depression, as measured by the alveolar or arterial PCO2, until anesthesia became quite deep. This preservation seemed to result from an increasing tachypnea sufficient to offset the progressive diminution of tidal volume. Most other anesthetics were associated with dose-related diminutions in alveolar ventilation, some much more than others. Each anesthetic seemed to have a ventilatory identity of its own with regard to such dose-related parameters as tidal volume, ventilatory drive (Vt /Ti), relative timing of inspiration and expiration, frequency, and even the nature of inspiratory and expiratory pauses. How do we explain such differences? Do different drugs act upon different cells, or different receptors, or in different ways to account for their unique signatures? Could understanding where and how they work provide clues about how the various components of the ventilatory control

Foreword

xiii

system are put together and how the separate parts communicate with each other under physiological as well as pharmacological circumstances? My wonderings never became more than that. But one need only reflect on the potency of the tools currently available to our imaginations to realize there is world of understanding out there waiting to be explored. Within this volume are many enticing next steps. Thomas Hornbein, M.D. Professor Emeritus Departments of Anesthesiology and Physiology and Biophysics University of Washington Seattle, Washington

References 1. 2. 3. 4.

5.

Chiodi, H., Respiratory adaptations to chronic high altitude hypoxia, J. Appl. Physiol. 10, 81–87, 1957. Hornbein, T.F. and Roos, A., Effect of polycythemia on respirations, J. Appl. Physiol. 12, 86–90, 1958. Hornbein, T.F., Everest, the West Ridge, San Francisco, The Sierra Club, 1965. Hornbein, T.F., ed., Regulation of Breathing, New York, Marcel Dekker, Inc., 17, 1436 pp., 1981 (One of a series of monographs in Lung Biology in Health and Disease, edited by Lenfant, C.) Dempsey, J.D. and Pack, A., eds., Regulation of Breathing, Second edition, New York, Marcel Dekker, Inc., 79, 1219 pp., 1995 (One of a series of mongraphs in Lung Biology in Health and Disease, edited by Lenfant, C.)

CONTRIBUTORS

Khalid F. Almoosa, M.D., F.C.C.P. Clinical Instructor of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio. Peter L. Bailey, M.D. Professor, Department of Anesthesiology, University of Rochester School of Medicine and Dentistry, Rochester, New York. Ryan W. Bavis, Ph.D. Assistant Professor, Department of Biology, Bates College, Lewiston, Maine. Philip E. Bickler, M.D. Professor, Department of Anesthesiology, University of California, San Francisco, California. Dante A. Cerza University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Albert Dahan, M.D., Ph.D. Professor, Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands. Lars I. Eriksson, M.D. Professor and Academic Chair, Department of Anesthesiology, Karolinska Hospital and Institute, Stockholm, Sweden. David D. Fuller, Ph.D. Assistant Professor, Department of Physical Therapy University of Florida, Gainesville, Florida. David Gozal, M.D. Professor and Children’s Foundation Chair for Pediatric Research, Vice Chair for Research, Director, Kosair Children’s Hospital Research Institute, Department of Pediatrics University of Louisville Louisville, Kentucky.

xv

xvi

Contributors

Jeffrey B. Gross, M.D. Professor, Departments of Anesthesiology, University of Connecticut School of Medicine Farmington, Connecticut. Shiroh Isono, M.D. Department of Anesthesiology, Chiba University Graduate School of Medicine, Chiba, Japan Shahrokh Javaheri, M.D. Professor, Department of Medicine, University of Cincinnati College of Medicine Cincinnati, Ohio and SleepCare Diagnostics Mason, Ohio. Suzanne Karan, M.D. Assistant Professor, Department of Anesthesiology University of Rochester School of Medicine and Dentistry, Rochester, New York. Gordon S. Mitchell, Ph.D. Department of Comparative Biosciences, University of Wisconsin, School of Veterinary Medicine, Madison, Wisconsin. Shakeeb H. Moosavi, Ph.D. Harvard School of Public Health, Boston, Massachusetts, and Imperial College School of Medicine, London, England. Takashi Nishino, M.D. Professor and Chair, Department of Anesthesiology, Chiba University Graduate School of Medicine, Chiba, Japan. Colin A. Nurse, Ph.D. Department of Biology, McMaster University, Hamilton, Ontario, Canada. Erik Olofsen, M.S. Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands. Susheel P. Patil, Ph.D. Instructor, Division of Pulmonary and Critical Care Medicine Department of Medicine, Johns Hopkins University Baltimore, Maryland. David Paydarfar, M.D. Associate Professor of Neurology and Physiology, Department of Neurology, University of Massachusetts, Medical School Worcester, Massachusetts. Frank L. Powell, Ph.D. Professor, Department of Medicine, Director, White Mountain Research Station, University of California, San Diego, La Jolla, California. Raymonda Romberg, M.D., Ph.D. Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands. Jacob Rosenberg, M.D.

University of Copenhagen, Hellerup, Denmark.

Contributors

xvii

Elise Sarton, M.D. Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands. Hartmut Schneider, M.D., Ph.D. Department of Medicine and Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland. Alan R. Schwartz, M.D. Department of Medicine and Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland. Steven A. Shea, Ph.D. Associate Professor, Department of Medicine, Harvard Medical School, and Director, Medical Chronobiology Program, Brigham and Women’s Hospital, Boston, Massachusetts. Philip L. Smith Sleep Disorders Center, Johns Hopkins University Baltimore, Maryland. Kingman P. Strohl, M.D. Professor, Departments of Medicine, Human Genetics, and Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio. Astrid G. Stucke, M.D. Research Fellow, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin. Eckehard A.E. Stuth, M.D. Associate Professor, Department of Anesthesiology, Medical College of Wisconsin, and Director, Cardiac Anesthesia, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin. Luc J. Teppema, Ph.D. Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands. Rajbala Thakur, M.D. Associate Professor, Department of Anesthesiology, University of Rochester School of Medicine and Dentistry, Rochester, New York. Denham S. Ward, M.D., Ph.D. Professor, Departments of Anesthesiology and Biomedical Engineering, University of Rochester Medical Center, Rochester, New York. David O. Warner, M.D. Professor, Department of Anesthesiology, Mayo Medical School and Mayo Clinic Rochester, Rochester, Minnesota. Edward J. Zuperku, Ph.D. Research Professor, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin.

ABBREVIATIONS

ABBREVIATION

DEFINITION

5-HT 5-HTP ACE ACh ACLS AG II AHI AHR AMPA

serotonin 5-hydroxytryptophan angiotensin converting enzyme acetylcholine advanced cardiac life support angiotensin II apnea-hypopnea index acute hypoxic response a-amino-3-hydroxy-5-methylisoxazole4-propionate acute mountain sickness atrial naturetic peptide area postrema action potential 2-amino-5phosphonovalerat artificial brainstem perfusion apolipoprotein E adenosine triphosphate arginine vasopressin blood-brain barrier brain blood flow bilevel positive airway pressure bispectral index large conductance Kþ body mass index blood pressure

AMS ANP AP AP AP5 APB APOE ATP AVP BBB BBF BiPAP BIS BK BMI BP

xix

xx BPM BSA CA CA CAD CB CBD CCHS CCK CDR CH CIH CMS CNS COPD CPAP CPG CPR CRP CSF CSN CVLM CVMS CVO CVRG D2-R DA DEF DI DLCO DM DRG DZ E EBSN EC50 ECE-1 ECF EDIA EMG EPI EPSP ERV FEF

Abbreviations breaths per minute body surface area carbonic anhydrase catecholamine coronary artery disease carotid body carotid body denervation congenital central hypoventilation syndrome cholecystokinin chronic dorsal rhizotomy chronic hypoxia chronic intermittent hypoxia chronic mountain sickness central nervous system chronic obstructive pulmonary disease continuous positive airway pressure central pattern generator cardio-pulmonary resuscitation C-reactive protein cerebrospinal fluid carotid sinus nerve caudal ventrolateral medulla caudal ventral medullary surface circumventricular organs caudal ventral respiratory group D2 receptor dopamine dynamic end-tidal forcing diaphragm single breath diffusion capacity dorsomedial hypothalamus dorsal respiratory group dizygotic expiratory expiratory bulbospinal neuron concentration yielding 50% effect endothelin-converting enzyme-1 extracellular fluid EMG activity in the diaphragm electromyelogram epinephrine excitatory postsynaptic potential expiratory reserve volume forced expiratory flow

Abbreviations fMRI fR FRC FVC GABA GER GG GiA GPN HAPE HCVR HD HEPES HERG HIF HM HPVR HR HTN HVA HVD HVR I IBSN IBW IC IH IM ION IPPB IPSP IT IV Kca Kir KO LC LHB LPB LPG LRN LTF LV LVA

xxi functional magnetic resonance imaging respiratory frequency functional residual capacity forced vital capacity g-aminobutyric acid gastroesophageal reflux genioglossus gigantocellular nucleus pars a glossopharyngeal nerve high-altitude pulmonary edema hypercapnic ventilatory response hypoxic desensitization 2-hydroxyethyl-l-piperazineethane sulfonic acid human ether-a-gogo-related gene hypoxic-inducible factor hypoglossal motoneuron hypoxic pulmonary vasoconstrictor response heart rate hypertension high-voltage activated hypoxic ventilatory depression hypoxic ventilatory response inspiratory inspiratory bulbospinal neuron ideal body weight intercostal intermittent hypoxia intramuscular infraorbital nerve intermittent positive-pressure breathing inhibitory postsynaptic potential intrathecal intravenous Ca2þ-activated Kþ channel inward rectifying Kþ channel knock-out locus coeruleus lateral habenular nucleus lateral parabrachial nucleus lateral paragigantocellular nucleus lateral reticular nucleus long-term facilitation left ventricle low-voltage activated

xxii LVH MAC MAP MFBS MI MP MRI MS MVV MZ NA nACh receptor nAChR NADP NBQX NE NEB NEP NEPI NF NHE NIMV NMBA NMDA NO NOS NOS-1 NPY NREM NTS ODI OHS OSA OSAH OSAS OT P PACU PAG PB PBW PCA PCEA

Abbreviations left ventricular hypertrophy minimum alveolar concentration mean arterial pressure multi-frequency binary sequence myocardial infarction membrane potential magnetic resonance imaging morphine sulfate maximum voluntary ventilation monozygotic nucleus ambiguus nicotinic acetylcholine receptor nicotinic acetylcholine receptor nicotinamide adenine dinucleotide phosphate 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f )quinoxaline norepinephrine neuroepithelial body neutral endopeptidase norepinephrine neurofilament Naþ/Hþ exchanger non-invasive mechanical ventilation neuromuscular blocking agent N-methyl D-aspartate nitric oxide nitric oxide synthase nitric oxide synthase-1 neuropeptide Y non-rapid eye movement nucleus tractus solitarius oxygen desaturation index obesity hypoventilation syndrome obstructive sleep apnea obstructive sleep apnea-hypopnea syndrome obstructive sleep apnea syndrome optic tract pyramidal tract postanesthesia care unit periaqueductal gray matter periodic breathing predicted body weight patient-controlled analgesia patient-controlled epidural analgesia

Abbreviations p-CPA Pcrit PDGF-b PEEP PEFR PEMAX PET PG PGCI pHe PHFD pHi PIIA PIIMAX PKC PK-PD PN PNDS PNG PNN POMC PPL pre-Bo¨tc PSR RAR rCBF REM RM RN ROS ROS RP RTN RV RVLM RVMM rVMS SAR SCI SD SDB SDH SIDS SK

xxiii para-chlorophenylalanine critical pressure platelet-derived growth factor b positive end-expiratory pressure peak expiratory flow rate maximum expiratory pressure positron emission tomography petrosal ganglion paragigantocellular nucleus extracellular pH post-hypoxia frequency decline intracellular pH post-inspiratory inspiratory activity maximum inspiratory pressure protein kinase C pharmacokinetic-pharmacodynamic nasal pressure post-nasal drip syndrome phrenic neurogram posterior nasal nerve proopiomelanocortin pleural pressure pre-Bo¨tziger complex pulmonary stretch receptor rapidly adapting receptor regional cerebral blood flow rapid eye movement raphe magnus raphe nuclei radical oxygen species reactive oxygen species raphe pallidus retrotrapezoid nucleus residual volume rostral ventral lateral medulla rostral ventral medial medulla rostral ventral medullary surface slowly adapting receptor spinal cord injury Sprague-Dawley strain of rats sleep-disordered breathing succinate dehydrogenase sudden infant death syndrome small-conductance Kþ channel

xxiv SLN SMA SNO SON SP STD sTEA STP SWS TASK TE TH TI TIA TLC TMP TMS TRH TTX TV UA UARS VAH VC VCO2 VDH VEGF vIPAG VMH VO2 VRG VT

Abbreviations superior laryngeal nerve supplementary motor area S-nitrosothiol supraoptic nucleus substance P short-term depression segmental high thoracic epidural anesthesia short-term potentiation slow wave sleep tandem pore acid sensitive Kþ channel expiratory phase duration tyrosine hydroxylase inspiratory phase duration transient ischemic attack total lung capacity transmural pressure transcranial magnetic stimulation thyrotropin-releasing hormone tetrodotoxin tidal volume upper airway upper airway resistance syndrome ventilatory acclimatization to hypoxia vital capacity CO2 production ventilatory deacclimatization from hypoxia vascular endothelial growth factor parabrachial nucleus ventromedial hypothalamus oxygen consumption ventral respiratory group tidal volume

CONTENTS

Introduction Preface Foreword Contributors Abbreviations

Claude Lenfant Thomas L. Hornbein

vii ix xi xv xix

I.

NEUROPHARMACOLOGY AND PHYSIOLOGY

1

1.

Peripheral Chemoreceptors: Sensors of Metabolic Status Colin A. Nurse

3

I. II.

3

Introduction Cellular Organization and Innervation of the Carotid Body III. Carotid Body Chemoreceptors: O2-Sensitive Kþ Channels IV. What is the O2 Sensor and How is it Linked to Kþ Channel(s)? V. Role of Fast-Acting Neurotransmitters in Chemosensory Processing VI. Glucose Sensing in the Carotid Body VII. Neuromodulation in the Carotid Body VIII. Future Directions Acknowledgments References 2.

4 6 8 8 12 12 15 15 16

Central Chemoreceptors Luc J. Teppema and Albert Dahan

21

I.

21

Introduction

xxv

xxvi

Contents II. III. IV.

3.

Location of Central Chemoreceptors Mechanism of Central Chemoreception Central Chemoreceptors and Breathing References

Suprapontine Control of Breathing Shakeeb H. Moosavi, David Paydarfar, and Steven A. Shea

71

I. II. III. IV. V. VI.

71 72 72 75 84

Introduction Definitions and Terminology Volitional Control Involuntary Emotional Influences Tonic Excitatory and Inhibitory Drives Interaction between ‘Behavioral’ and ‘Automatic’ Control VII. Learned Respiratory Behaviors VIII. Summary Acknowledgments References 4.

5.

24 38 47 55

86 88 90 91 91

Measurement of Drug Effects on Ventilatory Control Denham S. Ward

103

I. II. III. IV. V. VI. VII. VIII.

103 106 108 109 110 117 120 122 124

Introduction Measurement Techniques Quantification of Drug Pharmacodynamics Resting Measurements Hypercapnic Ventilatory Response Hypoxic Ventilatory Response Changes in Airway Pressure Other Stimuli References

Response Surface Modeling of Drug Interactions: Model Selection and Multimodel Inference Using the Bootstrap Erik Olofsen and Albert Dahan I. II. III. IV. V. VI.

Introduction Pharmacodynamic Interaction Models Model Selection and Multimodel Inference The Bootstrap Applications Conclusions References

133 133 134 137 140 143 151 152

Contents 6.

Respiratory Neuroplasticity: Respiratory Gases, Development, and Spinal Injury David D. Fuller, Gordon S. Mitchell, and Ryan W. Bavis I. II. III. IV. V. VI.

7.

xxvii

Introduction Plasticity Induced by Respiratory Gases in Adult Mammals Developmental Plasticity and the Control of Breathing Sex Hormones Spinal Cord Injury (SCI) and Respiratory Plasticity Conclusion Acknowledgments References

155 158 174 187 187 198 199 199

Airway Reflexes in Humans Takashi Nishino

225

I. II. III.

225 226

Introduction Central Nervous System Afferent Innervation of the Upper Airway and Receptors IV. Reflex Responses from the Upper Airway V. Afferent Innervation of the Lower Airway and Receptors VI. Integrative Aspects of the Airway Reflexes Elicited from the Lower Airways VII. Clinical Problems Associated with Airway Reflexes VIII. Conclusions Acknowledgments References 8.

155

226 229 234 238 244 250 251 251

Inheritance and Ventilatory Behavior in Animal Models Kingman P. Strohl

261

I. II.

262

III. IV. V. VI. VII.

Introduction Evidence and Implications for Inheritance of Ventilatory Traits in Humans Targeting Ventilatory Traits in Small Animals Evidence for the Inheritance of Ventilatory Traits in Rodents Estimates of the Strength of Inheritance Studies of Gene Effects in Rodent Models A Physiogenetic Map of Ventilatory Behavior

262 266 271 274 276 279

xxviii

Contents VIII. Overview and Future Directions Acknowledgments References

282 283 283

II.

PATHOPHYSIOLOGY

293

9.

Congenital Central Hypoventilation Syndrome: Should We Rename it Congenital Autonomopathy? David Gozal

295

I. II. III. IV. V.

10.

11.

Introduction Definition and Diagnosis Pathophysiology Animal Models Structural Central Nervous System Abnormalities VI. Physiologic Abnormalities of Ventilatory Control VII. Autonomic Nervous System Dysfunction VIII. Summary and Conclusions Acknowledgment References

295 296 298 300

Upper Airway Obstruction in Sleep Apnea Susheel P. Patil, Hartmut Schneider, Philip L. Smith, and Alan R. Schwartz

313

I. II. III. IV. V.

313 314 317 333 335 336

Introduction Epidemiologic and Clinical Risk Factors Pathogenesis of Upper Airway Obstruction Therapeutic Implications Summary and Conclusions References

300 301 304 304 304 304

High Altitude Frank L. Powell and Philip E. Bickler

357

I. II. III. IV.

357 358 360

V.

Introduction Ventilatory Response to High Altitude Time Domains of the HVR Increases in the Hypercapnic Ventilatory Response (HCVR) with Acclimatization High Altitude Diseases and Ventilatory Control References

372 372 376

Contents 12.

xxix

Obesity and the Control of Breathing Khalid F. Almoosa and Shahrokh Javaheri

383

I. II. III. IV.

383 384 388

V. VI.

Introduction Overview of Obesity Effects of Obesity on the Respiratory System Disorders of Ventilatory Control Associated with Obesity Effects of Treatment of Obesity and OSAH on Ventilatory Control and PaCO2 Conclusion References

399 408 412 412

III.

CLINICAL PHARMACOLOGY

423

13.

Pain Management and Regional Anesthesia Peter L. Bailey and Rajbala Thakur

425

I. II. III. IV. V.

425 426 433 438

Introduction Postoperative Respiratory Dysfunction Other Effects of Pain and Surgical Trauma Pain Control and Chronic Pain Effects of Pain and Pain Management on Respiratory Function VI. Regional Analgesia VII. Other Analgesic Agents VIII. Pre-Emptive Analgesia IX. Perioperative Analgesia and Pulmonary Outcome X. Cardiac Surgery XI. Summary References 14.

Ventilatory Effects of Medications Used for Moderate and Deep Sedation Jeffrey B. Gross and Dante A. Cerza I. II. III. IV. V. VI.

Introduction Sedatives Opioids Drug Combinations Strategies for Minimizing Respiratory Risks of Sedation Conclusion References

438 454 473 483 483 487 489 490

513 513 516 540 553 557 559 559

xxx

Contents

15.

Central Effects of General Anesthesia 571 Eckehard A.E. Stuth, Edward J. Zuperku, and Astrid G. Stucke I. II. III. IV. V. VI.

16.

The Influence of Inhalational Anesthetics on Carotid Body Mediated Ventilatory Responses Albert Dahan, Raymonda Romberg, Elise Sarton, and Luc J. Teppema I. II. III. IV. V.

17.

18.

Introduction General Effects of Anesthetics on Respiration Anesthetic Effects on Fast Synaptic Neurotransmission Overview of the Brainstem Respiratory Network Paradigms of Anesthetic Effects on Respiratory Neurotransmission Summary and Outlook Acknowledgment References

Introduction Influence of Inhalational Anesthetics on the Ventilatory Response to Hypoxia and Hypercapnia Pain and Behavioral Responses Short-Term Potentiation of Breathing (STP) Conclusions References

571 572 583 597 613 630 630 630

653

653 655 671 674 677 677

General Anesthesia and Respiratory Mechanics David O. Warner

687


687 688 694 709 714 722 722

Introduction Normal Function of the Respiratory Pump Effects of Anesthesia on Chest Wall Mechanics Effects of Anesthesia on Upper Airway Mechanics Effects of Anesthesia on Lung Mechanics Summary References

Recovery from Anesthesia Shiroh Isono and Jacob Rosenberg

737

I. II.

737

III.

Introduction Impairment and Recovery of Upper Airway Function after Anesthesia Impairment and Recovery of Chemical Control of Breathing after Anesthesia

738 750

Contents IV. V.

19.

20.

xxxi Impairment and Recovery of Lung Function after Surgery Late Postoperative Nocturnal Hypoxemia References

752 755 764

Neuromuscular Blocking Agents and Ventilation Lars I. Eriksson

779

I. II.

780

Regulation of Breathing Respiratory Pump Function and the Control of the Upper Airways References

785 789

Cardiovascular Drugs and the Control of Breathing Denham S. Ward and Suzanne Karan

793


793 794 800 801 802 804 805

Introduction Catecholamine Agonists and Antagonists Renin–Angiotensin System Calcium Channel Blockers Purinoceptor Agonists and Antagonists Other Agents References

Author Index Subject Index

815 823

Part I Neuropharmacology and Physiology

1 Peripheral Chemoreceptors: Sensors of Metabolic Status

COLIN A. NURSE McMaster University Hamilton, Ontario, Canada

I.

Introduction

The mammalian carotid body (CB) is a major peripheral chemoreceptor organ that helps in maintaining the chemical composition of arterial blood via the control of respiration. Consistent with its ability to sense bloodborne chemical stimuli, as originally suggested by the earlier studies of DeCastro, the organ is richly innervated and is supplied by an elaborate vascular network that has earned it the reputation as the tissue with the highest blood flow per unit weight [1,2]. Despite their small size, ranging from a few hundred microns to a few millimeters depending on species, these bilaterally paired organs occupy a strategic location near the carotid bifurcation, at the junction where the common carotid artery divides into its internal and external branches, supplying blood to the brain. Since the pioneering work of Heymans and collaborators, it is now well established that the CB is excited by a variety of chemical signals in arterial blood including low PO2, elevated PCO2 and acidic pH [2–5]. These chemoexcitants cause an increased action potential discharge in the carotid sinus nerve (CSN) whose projections to the central pattern generator in the 3

4

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brainstem ultimately lead to a compensatory increase in ventilation. There is strong evidence that the receptor cells for these stimuli are the catecholamine-producing glomus or type I cells [2,6–8], which occur in clusters and receive sensory innervation from the petrosal ganglia via the CSN [1–3]. More recent studies suggest an expanded role of the CB as a polymodal arterial chemosensor. For example, the recent demonstration that CB type I cells can respond to low glucose by secreting catecholamines [9] raises the idea that the organ might be broadly categorized as a ‘metabolic sensor’, that is, it is able to detect a range of circulating products and precursors of cellular metabolism. Moreover, these cells can also sense temperature and osmolarity [10–12] that is dependent on the concentration of circulating water and ions in plasma and extracellular fluid. These advances impinge on a wealth of information available about the transductive events in type I cells and on more recent information about the neurotransmitter mechanisms that translate the receptor potential into an increased afferent central nervous system (CNS) discharge. This review will highlight some of these recent advances. The interesting effects of chronic and intermittent stimuli (e.g., hypoxia) on CB plasticity are areas of physiological and pathophysiological significance, but will not be discussed here due to space constraints. The reader may wish to consult other recent reviews on this topic [13,14]. Also, though pulmonary neuroepithelial bodies (NEBs) represent another well-studied class of peripheral chemoreceptors that sense airway hypoxia [15,16], they have been the subject of recent reviews [17,18] and will not be considered in any detail here. II.

Cellular Organization and Innervation of the Carotid Body

The major cell types of the mammalian CB are the chemoreceptor type I cells which are organized in clusters of 20 or more cells, in intimate association with glial-like sustentacular or type II cells [1]. These chemoreceptors contain biogenic amines, especially dopamine [2,3], and accordingly express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis (Figure 1.1A,B). Other biogenic amines present in type I cells include norepinephrine and the indoleamine 5-HT or serotonin [2,19,20]. There is also substantive evidence that type I cells can synthesize and release acetylcholine in several species [2–4,21–23], though surprisingly, attempts to localize cholinergic gene expression in these cells have been unsuccessful in the rat [24]. A variety of other neurotransmitter candidates has been linked to CB chemoreceptors including substance P [5], GABA [19], and ATP [23]. The chemoreceptor clusters are surrounded by a rich network of fenestrated capillaries that permit ready access of blood-borne chemicals,

Peripheral Chemoreceptors 5

Figure 1.1 Immunofluorescence staining of a tissue section from 13-day-old rat carotid body. In A, the scattered type I cell clusters are immunopositive for tyrosine hydroxylase (TH); the TH antibody was raised in rabbit and visualized with an FITC-conjugated goat antirabbit IgG. The area enclosed by box in A, is shown in B,C after dual immunofluorescence staining for TH and neurofilament (NF), respectively. In C, a mouse anti-NF antibody was used and visualized with Texas red-conjugated goat anti-mouse IgG. The nerve processes and terminals in C (arrows) are in close apposition with the TH-positive type I cells shown in B. Calibration bars represent 40 mm in A, and 20 mm in B,C.

6

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including intravenously administered large dye molecules, e.g., Evans blue dye [25]. Gap junctions link chemoreceptor cells to each other and possibly to type II cells and sensory nerve endings [1,26], and this is likely the basis for electrical coupling observed between adjacent receptor cells [27]. Additionally, ultrastructural studies suggest chemical reciprocal synapses are present between neighboring chemoreceptor cells and between chemoreceptor cells and sensory nerve endings [1], suggesting a role for local circuits in the processing of chemosensory information in the CB. While neurofilament (NF)-positive, afferent fibers (Figure 1.1C), arising mainly from TH-positive neurons in the petrosal ganglion [28], provide the major sensory innervation to chemoreceptor cells, both autonomic and sensory fibers supply innervation to the whole organ [29]. Among these are a plexus of NO-synthesizing nerve fibers, originating from neurons within the glossopharyngeal (GPN) nerve, which are thought to provide efferent inhibition of the CB [30]. Recent evidence suggests that, in addition to causing excitation of type I cells, hypoxia can also directly excite these GPN neurons [31], thereby providing a pathway for negative feedback control of CB activity during natural stimulation.

III.

Carotid Body Chemoreceptors: O2-Sensitive Kþ Channels

Carotid body chemoreceptors or type I cells respond to hypoxia with membrane depolarization and/or increased excitability (e.g., Figure 1.2D), which facilitates entry of extracellular Ca2þ and neurosecretion [2,5–7,32,33]. Since the original description in the rabbit by Lopez-Barneo and colleagues [34], the main hypoxia-sensing mechanism in type I cells appears to involve inhibition of Kþ channels, though the particular subtype(s) of Kþ channel may differ among species and more than one O2-sensitive Kþ channel may occur in the same cell [5,8,35]. For example, inhibition of two distinct O2-sensitive Kþ channels are thought to play key roles in hypoxic chemotransduction in rat type I cells [8,36,37]. One type is the voltageand Ca2þ-dependent large conductance Kþ (BK) channel [7,8]. This is illustrated in Figure 1.2A–C, where inhibition of the BK current by removal of extracellular Ca2þ abolishes hypoxic inhibition of the voltage-dependent outward Kþ current. The other type is a voltage-insensitive background Kþ channel which shares biophysical and pharmacological properties of the acid-sensitive, 2P-domain Kþ channel, TWIK-related acid sensitive Kþ channel (TASK) [36]. While the latter alone satisfactorily accounts for membrane depolarization or receptor potential in single-isolated type I cells during hypoxia [37], both channels most likely contribute to the response of type I cells in their native clustered arrangement (Figure 1.1).

Peripheral Chemoreceptors

7

A

3000

B

2000 Control Wash Hypoxia 0 Ca2+ Hypoxia + 0 Ca2+

1 nA

1000

10 ms −120

C Hypoxia

2000

0 Ca2+ + Hypoxia

0 Ca2+

−80

−40

40

80

D

I (pA)

1600 10 mv 4s

1200 Time (s) 800

Hypoxia 0

10

20

30

Figure 1.2 Effects of hypoxia on whole-cell currents and membrane potential in rat type I cells in culture. A. Voltage step from a holding potential of 60 mV to þ 50 mV elicits an outward Kþ current which is reversibly suppressed (20%) on exposure to a hypoxic solution (PO2 ¼ 20 mmHg). In nominally Ca2þ-free solution, the outward current is suppressed by almost the same amount as hypoxia, and hypoxia had no additional effect on the residual current in Ca2þ-free solution. B. I–V relation for the cell in A shows the effects of hypoxia, Ca2þ-free solution, and both together on the whole-cell currents. C. Time-series plot of the effects of hypoxia and Ca2þ-free solution on the same cell as in A and B. These results confirm that hypoxia suppresses a Ca2þ-dependent Kþ current in rat type I cells [7,8]. D. Hypoxia evoked membrane depolarization that was sufficient to elicit action potentials in a different type I cell (Data from Ref. 33).

Evidence for this view is supported by the observations that blockade of BK channels with iberiotoxin leads to catecholamine secretion from clustered type I clusters in culture [38] and in CB tissue slices [39], suggesting BK channels are normally open under resting ‘‘normoxic’’ conditions in these clusters. Interestingly, Naþ channel density is low or absent in rat type I cells [7,40] though the hypoxia-induced depolarization or receptor potential seen in these usually quiescent cells may in some cases lead to action potentials (Figure 1.2D), presumably mediated largely by Ca2þ influx through voltage-dependent L-type Ca2þ channels [32]. However, there is evidence that spontaneous spike activity may occur in the larger clusters of rat type I cells [20], and in these cases hypoxia can cause an increase in spike

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frequency and broadening of the action potential, where the latter is thought to arise from inhibition of BK channels. In rabbit type I cells the O2-sensitive Kþ channels are different from rat, and include both a voltage-dependent, 4-AP-sensitive, transient Kþ channel [2,6,35] and an inward rectifier, HERG (human ether-a-gogorelated gene)-like Kþ channel [5]. Since rabbit type I cells show spontaneous activity, the voltage-dependent, 4-AP-sensitive Kþ channels are frequently open and their closure by hypoxia increases firing frequency and neurotransmitter release [41]. IV.

What is the O2 Sensor and How is it Linked to K1 Channel(s)?

A major unsolved issue in CB function is the molecular identity and location of the O2 sensor and the signal transduction pathway that couples the sensor to O2-sensitive Kþ channels [35]. One candidate heme protein, the neutrophil-like NADPH oxidase, which satisfies the main criteria for the O2 sensor in pulmonary neuroepithelial bodies [16], does not play this role in type I chemoreceptor cells [42,43, our unpublished observations]. Other candidates include heme proteins in the mitochondrial electron transport chain and heme proteins that are closely associated with the O2-sensitive plasma membrane Kþ channels [35,41,44]. In cell-attached patches of rat type I cells, hypoxia closes TASK-1like Kþ channels, but regulation is lost in excised inside-out patches, suggesting a cytoplasmic messenger or cofactor is required for conferring O2-sensitivity [36]. A suggestion that mitochondria might be involved in the link between the O2 sensor and plasma membrane TASK-1-like Kþ channels in these cells was indicated by the observation that inhibitors of the electron transport chain mimic the effects of hypoxia on background Kþ channels [45]. On the other hand, in rabbit type I cells, hypoxic modulation of single Kþ channels has been observed in excised membrane patches [46], suggesting that these channels may either possess intrinsic O2-sensitivity, or may be closely associated with a separate O2-sensor, e.g., hemoprotein. Thus, there remains the question whether there is a single O2-sensor in type I cells regardless of the Kþ channel type being modulated, or whether there may be more than one sensor, even in the same cell, for different O2-sensitive Kþ channels. V.

Role of Fast-Acting Neurotransmitters in Chemosensory Processing

While strong evidence that CB type I cells act as chemosensors for hypoxic, hypercapnic, and acidic stimuli has accumulated over the last roughly


9

15 years [2,7,8], there has been slower progress on the neurotransmitter mechanisms that translate the receptor potential in type I clusters to an increased afferent discharge in the CSN. Dopamine (DA), one of the best studied CB neurotransmitters, had received much attention as a leading transmitter candidate in chemoreception [2], but fell into disfavor with the demonstration that depletion of CB dopamine had little or no effect on the hypoxia-induced increase in CSN discharge [47,48]. The more popular view to date is that DA acting on presynaptic and possibly postsynaptic D2 dopamine receptors likely plays an inhibitory role in modulating CB function [24]. On the other hand, acetylcholine (ACh) has for many years remained an attractive candidate for mediating CB chemoexcitation [3,4]. However, the skeptics were not satisfied because blockers of both nicotinic and muscarinic ACh receptors could not inhibit completely the CB response to natural stimulation [4]. A satisfactory solution to this impasse was recently provided with the demonstration that co-release of ATP and ACh was likely the main mechanism mediating hypoxic chemotransmission in the rat CB [23]. These studies were greatly facilitated by the development of a co-culture model in which rat type I cell clusters formed de novo functional synaptic connections with dissociated petrosal (afferent) neurons in vitro [21,49]. The main advantage of this co-culture preparation, compared with the commonly used isolated CB-sinus nerve preparation, was that subthreshold postsynaptic responses could be recorded from neuronal cell bodies that were fortuitously juxtaposed to type I clusters [21–23,49,50]. As illustrated in Figure 1.3A,B, transmission of two natural CB stimuli, i.e., hypoxia and isohydric hypercapnia, can be demonstrated in these co-cultures and in both cases the postsynaptic response recorded in the neuron was reversibly inhibited by reduction of the extracellular Ca2þ: Mg2þ ratio. These results are consistent with the idea that chemical synaptic transmission is indeed required and that the responses are not due to a direct action of the chemostimuli on the neurons. In Figure 1.3A, the neuronal soma was close enough to the type I cluster to allow recording of the postsynaptic depolarization, which in this case was near the spike threshold and caused a burst of action potentials. In this configuration, soma recordings provide a replica of synaptic events occurring at the nerve terminals. The equivalent experiment in situ would require electrophysiological recordings from intact nerve terminals apposed to CB type I cells. This experimental approach has only met with limited success due to technical difficulties in obtaining stable recordings from these nerve endings in situ [51]. The ability to record the postsynaptic depolarization following application of a chemosensory stimulus in co-culture permitted pharmacological identification of the neurotransmitters involved. As illustrated

10

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A 20 mV 2s

Hypoxia

Hypoxia

Hypoxia

0.1 mM Ca, 6 mM Mg

20 mV 4s

B

10% CO2

10% CO2

10% CO2

Figure 1.3 Role of chemical transmission in mediating the effects of CB chemostimuli on petrosal neurons that functionally innervated type I clusters in co-culture. Perforated patch recordings of membrane potential were obtained from petrosal neurons that were adjacent to a type I cluster. In A, hypoxia (PO2 5 mmHg; duration indicated by horizontal bar) caused depolarization and a burst of action potentials in a functional petrosal neuron and the effect was reversibly abolished when the extracellular Ca:Mg ratio was decreased from 2 mM Ca2þ:1 mM Mg2þ (left and right traces) to 0.1 mM Ca2þ:6 mM Mg2þ (middle trace). A similar result was obtained when isohydric hypercapnia (10% CO2; pH ¼ 7.4) was used as the chemosensory stimulus in a different co-culture (B). Note spontaneous activity in the co-cultured neuron in B, under control (normocapnic) conditions (5% CO2; pH ¼ 7.4), i.e., to the left and right of the horizontal bars. These data suggest that chemical transmission is involved in the transfer of chemosensory information from the type I cluster to the neurons. The neuronal resting potential was 55 mV in A and B.

in Figure 1.4A, the hypoxia-induced postsynaptic response could be completely inhibited by the combined presence of a nicotinic ACh receptor blocker (100 mM hexamethonium) and a purinergic receptor blocker (50 mM suramin). Similar results were obtained when isohydric and acidic hypercapnia were used as chemostimuli in functional co-cultures (50, our unpublished observations). Other nicotinic and purinergic blockers successfully used in these co-cultures include mecamylamine (1–2 mM) and reactive blue 2 (10–50 mM), respectively [22,49]. In almost all cases, even high concentrations of a single blocker were insufficient to abolish


11

A 20 mV 50 µM hex + 25 µM sur

Hypoxia

B

100 µM hex + 50 µM sur

Hypoxia

Hypoxia

2s

Hypoxia

50 µM hex + 25 µM sur

Hypoxia

Hypoxia

20 s

Hypoxia

Figure 1.4 Evidence that co-release of ACh and ATP mediates hypoxic chemotransmission in co-cultured petrosal neurons, and in the isolated carotid body-sinus nerve preparation in the rat. In A, combined application of hexamethonium (hex) and suramin (sur), blockers of nicotinic ACh and purinergic receptors respectively, caused a dose-dependent inhibition of postsynaptic excitatory response recorded in a petrosal neuron, juxtaposed to a type I cluster (Data from Ref. 23). Similarly, in the intact CB–sinus nerve preparation in vitro (B), the hypoxiainduced sensory discharge recorded extracellularly in the sinus nerve was reversibly abolished in the combined presence of hexamethonium and suramin. The effect of either drug alone produced only partial inhibition of the sensory discharge (not shown). These data support the idea that co-release of ACh and ATP is the major mechanism that mediates hypoxic signaling in the rat carotid body.

the hypoxic response though partial inhibition was consistently obtained [23,49]. The conclusion that co-release of ACh and ATP from type I cells was the principal mechanism underlying hypoxic chemotransmission was not biased by the artificial culture conditions, since in the intact rat CB– sinus nerve preparation the same combination of nicotinic and purinergic blockers was required to abolish the chemosensory discharge, recorded extracellularly in the sinus nerve (Figure 1.4B; [23]). While the molecular composition of the functional postsynaptic AChR must yet be determined, there is strong evidence based on electrophysiological, molecular (RT-PCR), and immunocytochemical techniques that the functional purinergic receptor is a heteromultimer of P2X2–P2X3 subunits [23,50]. Both nicotinic and purinergic receptors are co-expressed in chemosensory neurons where they function as fast-acting, ligand-gated ion channels [23]. Importantly, immunoreactive P2X2 and P2X3 subunits co-localize in sensory nerve terminals apposed to type I receptor cells in the rat CB in situ [23].

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Nurse VI.

Glucose Sensing in the Carotid Body

It was recently demonstrated that low glucose stimulates catecholamine (CA) release from type I cells in tissue slices of the rat CB [9]. Similar to other CB chemostimuli, the effect of low glucose on CA secretion was dosedependent and was abolished by blockers of voltage-gated Ca2þ channels, suggesting that the response depended on membrane depolarization [9]. While low glucose selectively inhibited voltage-dependent outward Kþ current in rat type I cells, the fact that the effect persisted after blockade of the O2-sensitive Ca2þ-dependent maxi-Kþ (BK) current with iberiotoxin indicated a transduction pathway distinct from the one used to signal hypoxia [9]. These data suggest that type I cells are also physiological glucose sensors and, as such, are capable of integrating sensory information arising from a variety of chemical signals that contribute to general metabolic status. Integration of these signals would be expected to influence the frequency of afferent sinus nerve discharge and subsequent activation of the cardiorespiratory and cardiovascular system. Since in our co-culture experiments discussed above, recordings from functional petrosal neurons, co-cultured with type I receptor clusters, were routinely carried out in the presence of high (10 mM) glucose [23], we decided to test whether or not glucose withdrawal (substituted with equimolar sucrose) could stimulate neuronal activity. Indeed, as illustrated in Figure 1.5, the same chemosensory unit that responded to hypoxia with a reversible increase in neuronal spike discharge was similarly excited following glucose withdrawal. Moreover, this postsynaptic excitatory response induced by low glucose was inhibited by combined application of mecamylamine and suramin (our unpublished observations), suggesting that, as was the case for hypoxia (see above), co-release of ACh and ATP from type I cells was required for signaling low glucose. Though other organs are involved in the sensing of blood glucose [52], the strategic location of the CB may be important for brain function since neurons are especially sensitive to simultaneous glucose and O2 deficiency [9]. VII.

Neuromodulation in the Carotid Body

A contributing factor to the slow progress made in identifying the key neurotransmitters that mediate CB chemoexcitation is the wealth of transmitter candidates localized to type I cells [2]. There is evidence that several of them play modulatory roles, where the effect may be either presynaptic, i.e., at the level of the type I cell, or postsynaptic, i.e., at the sensory nerve endings. For example, one presynaptic effect of dopamine acting via D2 receptors on type I cells is to inhibit L-type Ca2þ channels [53]. Such an effect could lead to a negative feedback regulation of

Peripheral Chemoreceptors Control

13 Hypoxia

0 glucose

Wash

20 mV 2s

Figure 1.5 Chemosensory units in co-culture are excited by both hypoxia and low extracellular glucose. Recordings were obtained from a petrosal neuron that was near a type I cluster in co-culture. Note that the neuron, which was quiescent under control normoxic conditions (upper left and right traces), increased firing when exposed to hypoxia (upper middle trace). In this same neuron, removal of extracellular glucose (10 mM glucose that is normally present in the recording medium was replaced by 10 mM sucrose) also caused excitation in the neuron (lower middle trace) and the effect was reversible. Thus, the same chemosensory unit can be excited by hypoxia and low glucose.

neurotransmitter release during hypoxia by an autocrine–paracrine mechanism. Our recent studies suggest that 5-HT (serotonin) and GABA, which are both expressed in rodent type I cells [19,20,54,55], may act as positive and negative modulators of type I chemoreceptor function, respectively. We found that the 5-HT receptor blocker, ketanserin, inhibited the spontaneous spike discharge that is occasionally seen in large type I clusters in culture, and exogenous 5-HT can induce membrane depolarization or rhythmic-like spiking in type I clusters [20]. The effect of 5-HT appears to be mediated via G-protein-coupled 5-HT2a autoreceptors and protein kinase C-dependent inhibition of Kþ channels [55]. Whereas 5-HT can potentiate transmitter output from type I clusters by autocrine–paracrine mechanisms, GABA has the opposite effect. In particular, GABA appears to inhibit transmitter output via G-protein coupled GABAB autoreceptors and PKAdependent augmentation of a TASK-1-like background Kþ current [54]. Other neuromodulators are likely to play important roles in the regulation of type I cell function. For example, ACh activates both nicotinic and muscarinic autoreceptors which are known to be expressed in type I cells of several species [4]. There is also the possibility that ATP, which as described above is an important fast-acting transmitter that is co-released with ACh during CB chemoexcitation, may act presynaptically as well. So far, we have been unable to detect ATP sensitivity in type I cells in

14

Nurse Type II

Type I

100 pA 20 ms

5 mV 4s

50 µM ATP

50 µM ATP

Figure 1.6 Comparison of ATP sensitivity in cultured rat CB type I and type II cells. Top traces show voltage clamp recordings of ionic currents in a type II cell (left) and a type I cell (right) after a few days in culture. Note the absence of voltagedependent outward Kþ currents in the type II cell, but their presence in the type I cell. Currents were evoked during 10 mV incremental voltage steps from 180 mV to þ 60 mV; holding potential ¼ 60 mV. Lower traces show current clamp recordings of membrane potential in the same type II and type I cells (as in upper traces) after rapid perfusion of ATP over the soma. Note that ATP causes membrane depolarization in the type II, but not type I cell. The resting potential was 40 mV in the type II cell and 47 mV in the type I cell.

voltage or current clamp studies (e.g., Figure 1.6). However, the possibility that ATP may influence intracellular Ca2þ homeostasis in type I cells via regulation of intracellular Ca2þ stores is not excluded. Interestingly, we have preliminary data indicating that sustentacular or type II cells, which are intimately associated with type I cell clusters in situ [1] and in culture [56], may express ATP receptors (Figure 1.6). Further studies are required to determine the molecular identity of these purinergic receptors, though the possibility is raised that type II cells may contribute to the overall autocrine regulation of CB function. For example, type II cells may help spread electrical activity within type I clusters via their long, slender processes which tend to surround and penetrate the clusters [56]. ATP released during chemosensory transmission


15

may also contribute indirectly to autocrine–paracrine signaling in the CB after its enzymatic breakdown to adenosine by ectonucleotidases. Adenosine, derived in this way or as a circulating metabolite whose levels in plasma increase during hypoxia, may itself act on adenosine A2A autoreceptors on type I cells and contribute to CB excitation [57], perhaps via inhibition of a 4-AP-sensitive, voltage-dependent outward Kþ current [58]. Thus, the purine ATP may be a central player in CB function, acting directly as a fast co-transmitter in mediating the postsynaptic response and as a presynaptic neuromodulator via type II cells, or indirectly, as a modulator of type I cell function via its breakdown product, adenosine. VIII.

Future Directions

One of the key issues that needs to be clarified is the molecular identity and location of the O2 sensor in CB chemoreceptors and the signaling pathway leading to Kþ current inhibition in low oxygen. Current methodologies using patch clamp techniques at the whole-cell and single-channel level, carbon fiber amperometry to assay for amine secretion in tissue slices, and the application of mitochondrial inhibitors will need to be complemented by functional genomic approaches where the sensor function can be directly manipulated. It will also be of interest to identify the glucose sensor in these cells and the transductive process leading to increased secretion from type I cells in low extracellular glucose. The complexity surrounding the need for such a broad range of neurotransmitters/neuromodulators in type I cells is still puzzling; however, the possibility that different chemostimuli might release a different array of these chemical signals needs further exploration. Finally, since these neurotransmitters and their receptors appear to endow the organ with a remarkable ability to fine tune its responses to any particular stimulus, their likely contribution to chemoreceptor plasticity following chronic stimulation needs to be explored in the future. Acknowledgments The work attributed to my laboratory was supported by grants from the Canadian Institutes of Health Research. I wish to thank Cathy Vollmer for expert technical assistance, Min Zhang for carrying out many of the experiments on co-cultures and providing most of the figures illustrated in the text, Huijun Zhong for providing the data for Figure 1.2, and Mike Jonz for assistance with Figure 1.1. I am indebted to several colleagues for contributing to the ideas expressed in this article, particularly Min Zhang, Veronica Campanucci, and Ian Fearon.

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noradrenaline, serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body, Cell Tiss. Res. 278, 249–254, 1994. Zhang, H. and Nurse, C.A., Does endogenous 5-HT mediate spontaneous rhythmic activity in chemoreceptor clusters of rat carotid body? Brain Res. 872, 199–203, 2000. Zhong, H., Zhang, M. and Nurse, C.A., Synapse formation and hypoxic signalling in co-cultures of rat petrosal neurones and carotid body type 1 cells, J. Physiol. 503, 599–612, 1997. Nurse, C.A. and Zhang, M., Acetylcholine contributes to hypoxic chemotransmission in co-cultures of rat type 1 cells and petrosal neurones, Respir. Physiol. 115, 189–199, 1999. Zhang, M., Zhong, H., Vollmer, C. and Nurse, C.A., Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors, J. Physiol. 525, 143–158, 2000. Gauda, E.B., Gene expression in peripheral arterial chemoreceptors, Micros. Res. Tech. 59, 153–167, 2002. McDonald, D.M. and Blewett, R.W., Location and size of carotid-body like organs (paraganglia) revealed in rats by permeability of blood vessels to Evans blue dye, J. Neurocytol. 10, 607–643, 1981. Kondo, H., Are there gap junctions between chief (glomus, type I) cells in the carotid body chemoreceptors? A review, Micros. Res. Tech. 59, 227–233, 2002. Abudara, V. and Eyzaguirre, C., Electrical coupling between cultured glomus cells of the rat carotid body: observations with current and voltage clamping, Micros. Res. Tech. 59, 249–255, 2002. Katz, D.M. and Black, I.B., Expression and regulation of catecholaminergic traits in primary sensory neurones: relationship to target innervation in vivo, J. Neurosci. 6, 983–989, 1986. Ichikawa, H., Innervation of the carotid body: immunohistochemical, denervation, and retrograde tracing studies, Micros. Res. Tech. 59, 188–195, 2002. Wang, Z.Z., Stensaas, L.J., Dinger, B.G. and Fidone, S.J., Nitric oxide mediates chemoreceptor inhibition in the cat carotid body, Neuroscience 65, 217–229, 1995. Campanucci, V.A., Fearon, I.M. and Nurse, C.A., A novel O2-sensing mechanism in glossopharyngeal neurones mediated by a halothane-inhibitable background Kþ conductance, J. Physiol. 548, 731–743, 2003. Buckler, K.J. and Vaughan-Jones, R.D., Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells, J. Physiol. 476, 423–428, 1994. Zhong, H., Electrophysiology and Transmitter Sensitivities of Isolated Rat Petrosal Neurons: Synapse Formation and Hypoxic Signaling in Co-culture with Carotid Body Chemoreceptors, Ph.D. Dissertation, McMaster University, Hamilton, Ontario, 1997. Lopez-Barneo, J., Lopez-Lopez, J.R., Urena, J. and Gonzalez, C., Chemotransduction in the carotid body: Kþ current modulated by PO2 in type I chemoreceptor cells, Science 241, 580–582, 1988.

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Lopez-Barneo, J., Pardal, R. and Ortega-Saenz, P., Cellular mechanisms of oxygen sensing, Ann. Rev. Physiol. 63, 259–287, 2001. Buckler, K.J., Williams, B.A. and Honore, E., An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium current in rat arterial chemoreceptor cells, J. Physiol. 525, 135–142, 2000. Buckler, K.J., A novel oxygen-sensitive potassium current in rat carotid body type I cells, J. Physiol. 498, 649–662, 1997. Jackson, A. and Nurse, C., Dopaminergic properties of cultured rat carotid body chemoreceptors grown in normoxic and hypoxic environments, J. Neurochem. 69, 645–654, 1997. Pardal, R., Ludewig, U., Garcia-Hirschfeld, J. and Lopez-Barneo, J., Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium, Proc. Natl. Acad. Sci. USA, 97, 2361–2366, 2000. Stea, A. and Nurse, C.A., Whole-cell and perforated-patch recordings from O2-sensitive rat carotid body cells grown in short- and long-term culture, Pflu¨gers Archiv. 418, 93–101, 1991. Lopez-Barneo, J., Ortega-Saenz, P., Molina, A., Franco-Obregon, A., Urena, J. and Castellano, A., Oxygen sensing by ion channels, Kidney Int. 51, 454–461, 1997. Roy, A., Rozanov, C., Mokashi, A., Daudu, P., Al-mehdi, A.B., Shams, H. and Lahiri, S., Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca2þ]i and respiratory responses to hypoxia, Brain Res. 872, 188–193, 2000. He, L., Chen, J., Dinger, B., Sanders, K., Sundar, K., Hoidal, J. and Fidone, S., Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice, Am. J. Physiol. Cell Physiol. 282, C27–C33, 2002. Kummer, W. and Yamamoto, Y., Cellular distribution of oxygen sensor candidates—oxidases, cytochromes, Kþ-channels—in the carotid body, Micros. Res. Tech. 59, 234–242, 2002. Buckler, K.J. and Vaughan-Jones, R.D., Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells, J. Physiol. 513, 819–833, 1998. Ganfornina, M.D. and Lopez-Barneo, J., Single Kþ channels in membrane patches of arterial chemoreceptor cells are modulated by O2 tension, Proc. Natl. Acad. Sci. USA 88, 2927–2930, 1991. Donnelly, D.F., Chemoreceptor nerve excitation may not be proportional to catecholamine secretion, J. Appl. Physiol. 81, 657–664, 1996. Iturriaga, R., Alcayaga, J. and Zapata, P., Dissociation of hypoxia-induced chemosensory responses and catecholamine efflux in cat carotid body superfused in vitro, J. Physiol. 497, 551–564, 1996. Nurse, C.A. and Zhang, M., Synaptic mechanisms during re-innervation of rat arterial chemoreceptors in co-culture, Comp. Biochem. Physiol. Part A, 130, 241–251, 2001. Prasad, M., Fearon, I.M., Zhang, M., Laing, M., Vollmer, C. and Nurse, C.A., Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemosensory signalling, J. Physiol. 537, 667–677, 2001.

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Hayashida, Y. and Hirakawa, H., Electrical properties of chemoreceptor elements in the carotid body, Micros. Res. Tech. 59, 243–248, 2002. Thorens, B., A gene knockout approach in mice to identify glucose sensors controlling glucose homeostasis, Pflu¨gers Archiv. 445, 482–490, 2003. Benot, A. and Lopez-Barneo, J., Feedback inhibition of Ca2þ currents by dopamine in glomus cells of the carotid body, Eur. J. Neurosci. 2, 809–812, 1990. Fearon, I.M., Zhang, M., Vollmer, C. and Nurse, C.A., GABA mediates autoreceptor feedback inhibition in the rat carotid body via presynaptic GABAB receptors and TASK-1, J. Physiol. 553, 83–94, 2003. Zhang, M., Fearon, I.M., Zhong, H. and Nurse, C.A., Presynaptic modulation of rat arterial chemoreceptor function by 5-HT: role of Kþ channel inhibition via protein kinase C, J. Physiol. 551, 825–842, 2003. Nurse, C.A. and Fearon, I.M., Carotid body chemoreceptors in dissociated cell culture, Micros. Res. Tech. 59, 249–255, 2002. Sebastiao, A.M. and Ribeiro, J.A., Adenosine A2 receptor-mediated excitatory actions in the nervous system, Prog. Neurobiol. 48, 167–189, 1996. Vandier, C., Conway, A.F., Landauer, R.C. and Kumar, P., Presynaptic action of adenosine on a 4-aminopyridine-sensitive current in the rat carotid body, J. Physiol. 515, 419–429, 1999.

2 Central Chemoreceptors

LUC J. TEPPEMA and ALBERT DAHAN Leiden University Medical Center Leiden, The Netherlands

I.

Introduction

Some 50 years ago, Leusen, in his ventriculocisternal perfusion experiments in dogs, showed that variations in hydrogen ion concentration in the cerebrospinal fluid (CSF) had a pronounced influence on pulmonary ventilation [1]. These pioneer experiments were followed by the studies of Pappenheimer and co-workers in goats in which they showed that ventilation could be expressed as a unique function of a calculated extracellular pH existing at a location about three-fourths along the artificial steady-state concentration gradient of bicarbonate between the CSF and brain capillary plasma [2,3]. Following these classical studies, this view of central CO2 chemoreceptors that are uniquely sensitive to the pH in their microenvironment was also used by other groups in their attempts to define the ‘functional’ or ‘anatomical’ location of the central chemoreceptors (i.e., their relative distance between CSF and blood and their distance from the irregularly folded ventral surface of the medulla oblongata, respectively, e.g., [4–6]).

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22

Teppema and Dahan

V VI

VII VIII M IX X XI

S

L

XII

C1

Figure 2.1 Ventral medullary surface in the cat with the three ‘‘classical’’ chemosensitive areas: M (Mitchell’s area), S (Schlaefke’s area) and L (Loeschcke’s area).

A major breakthrough in the localization of the chemosensitive regions was achieved by Mitchell, Loeschcke and Schlaefke and co-workers who described three separate superficial (at a depth between 0 and 400 mm) areas on the ventral medullary surface of the cat not only containing pHsensitive cells but also neurons that were crucial in the integration of the afferent central chemosensory input to the respiratory centers (Figure 2.1, [7,8]). For many investigators these studies were the basis to further explore the ventral medullary surface of several species both in vitro and in vivo using local lesion, cooling, electrophysiological means and pharmacological agents administered by way of perfusion, superfusion, or topical application. Meanwhile, from several studies in intact animals it became clear that the assumed role of an extracellular pH as the sole stimulus was no longer tenable [9–11]. Despite this, the existence of chemosensitive regions in several species (e.g., rat, goat) analogous to those described in the cat was never seriously disputed. However, even within these chemosensitive areas, it was very difficult to identify individual respiratory CO2 chemoreceptors defined as (non-respiratory?) sensory neurons that are activated in vivo during hypercapnia (or inhibited during hypocapnia), respond directly

Central Chemoreceptors

23

(i.e., not transsynaptically) to CO2/Hþ and convey their afferent input to the respiratory centers. To date, a universal picture of respiratory CO2/Hþ chemoreceptors with characteristic morphological, neurochemical and electrophysiological properties has not yet been composed. This may have several reasons. One or more of the above criteria may be false or too loose and not specific enough. For example, in vivo and in vitro data indicate that respiratory neurons themselves may behave as central chemoreceptors [12,13], suggesting that not all chemoreceptors are purely sensory neurons. In addition, some chemoreceptors may be inhibited rather than stimulated by a decrease in pH as has been demonstrated for neurons in the ventromedial medulla [14,15]. It is also unknown if central chemoreceptors display a respiratory rhythm: some may and some may not, and they may be a heterogeneous population of cells consisting of both respiratory and nonrespiratory neurons. Another complicating factor is that there are many ways to depolarize or stimulate central chemoreceptors, but a unique intracellular factor on which all these stimuli converge and that we could use as the most direct tool to localize them, has not yet been isolated. Furthermore, the central chemoreceptors are intermingled with other brainstem neurons, complicating their identification. Finally, most of the techniques that were used may have too low a resolution to identify chemoreceptors at the singlecell level. Over the last decade, new techniques have been designed and, due to the enormous progress of molecular biology, it is now possible to use alternative approaches and to study brain function with much higher resolution. This has led to a much broader understanding of the central chemoreceptors and has shed a new light on their distribution: widespread within the brain stem and even more rostral vs. the classical view of chemosensitive regions limited to three areas on the ventral medullary surface. It also becomes progressively more evident that not only the intracellular pH may play a key role in the chemosensitive mechanism, but also ion-specific membrane channels, particularly potassium and possibly also calcium channels. By using the appropriate techniques and experimental paradigms, it is now within our reach to achieve a better understanding of the physiological role of the central chemoreceptors in health and disease. In this chapter it is our intention to focus on some major tools used over the last decade to locate or identify the central chemoreceptors and to summarize the state of the art in this regard. Secondly, we will discuss some recent data that may shed new light on the mechanism of CO2 chemoreception. Finally, we will briefly discuss the physiological role of the central chemoreceptors in the control of breathing.

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Teppema and Dahan II.

Location of Central Chemoreceptors

A. Functional and Anatomical Location

Initially, in the search for the central chemoreceptors the terms functional and anatomical location were used to define their relative distance between CSF and blood and their distance from the irregularly folded ventral surface of the medulla oblongata, respectively (see above). Both definitions are vague and may give rise to confusion. Replacing functional location by relative position or distance to blood vessels is also not very practical. Because some central chemoreceptors are more closely apposed to vessels than others, it is impossible to determine a location of chemoreceptors relative to vessels that is representative for all members. Assuming that one common qualitative feature of all chemoreceptors refers to their stimulus environment, it might be more useful to discuss whether the chemoreceptors measure a parameter that represents the PCO2 of arterial blood or rather one that reflects the tissue PCO2. In the next section we will focus on this aspect. A more practical means to estimate the distance from the medullary surface would be to describe the exact topographical anatomical location of the chemoreceptors without overlooking the sometimes considerable distance between their somata and chemosensitive (dendritic) sites. We will end this section by giving a brief overview of some major techniques used over the last decade to find the anatomical regions that contain chemoreceptors and to localize individual chemosensitive neurons. B. Central Chemoreceptors Measure Tissue PCO2

Studies in rat have shown that putative chemoreceptor cells, particularly in superficial ventral medullary tissue, are very closely associated with blood vessels. Okada et al. [16] described an intimate anatomic relationship between cells that were activated by hypercapnia (positive staining for Fos, even after synaptic blockade; see below) and surface vessels. Confirming and extending previous observations of Gorcs et al. [17], Bradley and co-workers [18], using confocal imaging and electron microscopy, showed a close association between processes of medullary serotonergic raphe neurons and large arteries in areas almost devoid of veins. Because this close association between serotonergic neurons and arteries occurs both in medullary and pontine raphe nuclei and in mesencephalon, and because many of these neurons are very sensitive to changes in pH, the authors suggested that they function as central chemoreceptors involved in ventilatory control to maintain pH/PCO2 homeostasis. The close proximity of these serotonergic cells and their


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processes to brainstem arteries in regions nearly devoid of veins would provide an optimal means to monitor a local CO2 concentration that is relatively unaffected by metabolism and would thus be an ideal location for monitoring the effectiveness of lung ventilation [18]. It is quite possible that the chemosensitive cells described by Bradley et al. function as central respiratory chemoreceptors, but not because they measure arterial PCO2 (this is already provided by the arterial chemoreceptors with their strategic location at the port of the brain circulation). Because the arterial PCO2 has no unique relationship with alveolar ventilation (this relationship alters with changes in metabolism and inspired CO2), it cannot be considered a unique parameter that represents the level or effectiveness of alveolar ventilation. Monitoring a variable that reflects the tissue PCO2 would provide more useful information. According to the mass balance for CO 2 , the P CO 2 in brainstem tissue (where the chemoreceptors are located) is determined by local metabolism and blood flow; CO2 reactivity of brainstem vessels; the arterial PCO2; the slopes of the CO2 dissociation curves for blood and tissue, and a parameter

that locates the chemoreceptor PCO2 somewhere between the tissue and arterial PCO2 [19–21]. For this very reason, by measuring the tissue PCO2, the central chemoreceptors provide an optimal feedback signal for metabolic ventilatory control. Thus, chemoreceptors that measure a parameter that closely reflect arterial CO2 concentration before it has been influenced by local tissue metabolism [18] will not provide the most relevant feedback signal for the metabolic control of breathing. The remaining question now is: do the putative respiratory chemoreceptors described by Bradley et al. [18] measure an arterial or tissue PCO2? Assuming that the processes of these chemosensitive cells measure a PCO2/[Hþ] in their immediate environment, i.e., the perivascular tissue space (with a relatively low value of parameter ), then these cells will measure a variable that must also reflect local metabolism simply because (in the case of hypercapnia) the amount of CO2 that diffuses out from local blood vessels will depend on local blood flow and on the amount of metabolically produced CO2 already present. That indeed the tissue PCO2 is the measured variable rather than a parameter closely representing the arterial PCO2 has been shown in numerous studies in men and whole animals. We give a small selection of numerous examples: 1.

In an early study in anesthetized dogs, Dutton et al. [22] found that following perfusion of the vertebral arteries with hypercapnic blood for 2 minutes, the rate of recovery of ventilation was much faster than after steady-state inhalation of CO2, a finding indicating that the central chemoreceptor PCO2 is dominated by the tissue and CSF PCO2 rather than by the PCO2 in capillary blood. Studies using the technique of dynamic end-

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Teppema and Dahan tidal forcing showed that the time course of the central chemoreflex loop to step-changes in end-tidal PCO2 or arterial PCO2 of blood selectively perfusing the brainstem does not reflect that of the (near) step in arterial PCO2 but rather is determined by the speed of CO2 loading and unloading of the brainstem [23–25]. 2. In cats undergoing artificial perfusion of their brainstems, a selective increase in medullary blood flow induced by adding the vasodilator papaverine to the central blood at constant arterial peripheral and central blood gas tensions, resulted in a decrease in ventilation due to increased washout of CO2 from the pontomedullary region [26]. In this preparation at constant peripheral blood gas tensions, when the PO2 of the blood perfusing the brainstem was lowered from a hyperoxic to a normoxic level without changing its PCO2, ventilation decreased with a time constant determined by the increased washout of CO2 [27]. 3. In the cat, the carbonic anhydrase inhibitors benzolamide and acetazolamide, administered systemically in a dose sufficiently large to completely inhibit erythrocytic carbonic anhydrase, a decrease in the end-tidal and arterial PCO2 in vivo is followed (not preceded) by a large rise in ventilation that is accompanied by a considerable brain stem tissue acidosis (rise in PCO2 and fall in pH) in the face of a lower arterial PCO2 of the blood perfusing the central chemoreceptors [28–30].

If the measured parameter is a reflection of the tissue PCO2, does it mean that all chemoreceptors measure an equal P CO 2 or pH? The answer will be no, because one of the determinants of local PCO2 is local blood flow which will not be equal in all chemosensitive regions. This is illustrated by the existence of appreciable pH gradients in the medulla oblongata as demonstrated in the cat [31]. Chemosensitive neurons in close apposition to large arteries could monitor rapid changes in perivascular CO2/Hþ due to fast changes in medullary arterial PCO2 and/or blood flow, provided they have the appropriate machinery for fast monitoring. Apart from a possible chemosensitive role in respiration, however, both the described cholinergic neurons in ventrolateral medulla [16] and the serotonergic cells in raphe [18] may have an excellent strategic position to play a modulating role in the adaptation of vessel caliber to the PCO2 and PO2 of the inflowing arterial blood. Note that also, hypercapnia-induced cerebral vasodilatation is based on a chemosensitive process governed by changes in local PCO2/Hþ.


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C. Searching the Anatomical Location of Central Chemoreceptors

Over the last decade, several new approaches were applied to localize the central chemoreceptors. This has contributed to a growing consensus to a widespread location of CO2 chemoreceptors in the brain stem. Currently, the following brain stem regions are assumed to contain CO2 chemoreceptors [32–39]: Dorsally, the Nucleus of the Solitary Tract (NTS) in the same areas where afferents from the carotid bodies project; the Locus Coeruleus (LC) in the pons; Ventrally (in rostrocaudal order), the RostroVentroLateral Medulla (RVLM) particularly the RetroTrapezoid Nucleus (RTN), Raphe Nuclei (RN) in the Rostral Ventral Medial Medulla (RVMM), the pre-Bo¨tzinger complex and a region within the Nucleus Ambiguous (NA) belonging to the group of respiratory neurons in the caudal ventrolateral lateral medulla. Some of these areas were explored with different techniques yielding corresponding results. Many other areas, however, may also contain chemoreceptors but are not as extensively explored (e.g., parabrachial and Ko¨lliker Fuse nuclei in pons and regions within the caudal mesencephalon—see below). In the following paragraphs we will discuss the most important tools that were (and still are) utilized to localize these many chemosensitive areas. Electrophysiological Studies In Vitro

In the last decade, the first evidence that several brain regions outside the ventral medullary surface contain CO2/Hþ chemoreceptors came from in vitro studies. In the neonatal rat spinal cord-brainstem preparation at depths between 50 and 700 mm from the ventral surface, respiratory neurons possessing extensive dendrites to within 50 mm from the surface were discovered, showing inherent CO2 sensitivity in the presence of synaptic blockade [13]. This preparation also contains CO2 sensitive neurons in the locus coeruleus [32,33]. In coronal brain slices, CO2/Hþ sensitive cells activated independently from synaptic events were described in the NTS [34,35], hypothalamus [36], locus coeruleus [37] and in the ventral medial medulla (raphe, 14). Intracellular recordings in ventral medullary raphe showed the existence of two chemosensitive cell types, one stimulated and one inhibited by CO2 [14]. This was confirmed by perforated patch recordings in neurons in cell cultures from isolated ventromedial medullary cells [38]. In addition, all acidosis-stimulated cells from these cultures appeared to be serotonergic and had a morphological appearance that clearly differed from that of the acidosis-inhibited cells that were all non-serotonergic [38,39]. Both types of acid-sensitive neurons showed a high sensitivity to pH changes in the physiological range [38].

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Teppema and Dahan Focal Injections of Acetazolamide

An unresolved issue is whether central chemoreceptors contain carbonic anhydrase, shown to be the case for the peripheral chemoreceptors in the carotid bodies [40–43]. Obviously, the presence of this enzyme in cells that should rapidly respond to changes in intracellular pCO2 would be useful. Unfortunately, however, this would not make it easier to localize and identify neurons that specifically function as CO2 chemoreceptors: like in other organs, carbonic anhydrase is a ubiquitous enzyme in the brain and has also been demonstrated in regions (e.g., hippocampal and cerebellar) that do not contain chemoreceptors (see below). Despite these limitations, local application of acetazolamide has proven to be a useful tool to localize chemosensitive regions in the brain stem, mostly in anesthetized animals. In several regions, namely NTS, RTN, midline raphe, rostral part of the ventral respiratory group, pre-Bo¨tzinger complex, and even fastigial nucleus in the cerebellum, topical application of acetazolamide is followed by increases in phrenic activity as large as 49–69% of the increase observed during overall hypercapnia following a change in end-tidal CO2 from 4 to 9% [44–49]. The injections cause acidotic responses in different focal chemoreceptor regions that, at the center of the injection sites, are equivalent to those following a change in end-tidal CO2 from 28 to 64–75 Torr [44–47]. In evaluating the effects of focal acetazolamide injections, the following points need attention: a. Local acidosis in putative chemoreceptor areas caused by acetazolamide injection does not always result in an increase in ventilation (e.g., 47). The reverse, a pronounced effect in chemosensitive regions (e.g., in midline raphe) known to contain relatively few carbonic anhydrase-containing chemosensitive neurons [50], has also been reported [46]. b. The mechanism by which acetazolamide injection causes focal acidosis is obscure and will remain so until more detailed information becomes available on the resulting effect on intracellular pH and on the identity, distribution and subcellular location of the (blocked) isoenzymes involved. One possible mechanism by which acetazolamide might cause extracellular acidosis could be by inhibiting a membrane-associated isoenzyme attached to neuronal processes which modulates the pH of extracellular fluid [51]. Inhibition of (intracellular) glial CA could also cause extracellular acidosis and this, together with a close association of glial cells with neurons, might explain why, as mentioned above, microinjection of acetazolamide in raphe increases respiratory output although this region possesses relatively few CA-containing chemosensitive neurons.


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c. The effects of rapidly permeating carbonic anhydrase inhibitors such as methazolamide were not systematically compared with those of an impermeable inhibitor such as benzolamide (note that acetazolamide is a slowly permeating inhibitor). The only study in which the effects of acetazolamide and methazolamide were compared showed a faster effect of the latter on phrenic activity suggesting involvement of an intracellular isoenzyme [48]. Another interesting feature of the latter study is that these inhibitors were injected into the pre-Botzinger complex, a region known to contain important respiratory neurons in rhythm generation [52]. Are these respiratory neurons chemosensitive? d. The time courses of changes in phrenic activity and local pH do not match: fast acidosis and gradually developing, long-lasting increases in ventilation [44,47]. This may be related to slow diffusion and permeability characteristics of acetazolamide, although a contribution of slow neuronal dynamics cannot be excluded. It is possible that the time course of the change in phrenic activity would match more closely to the (unknown) time course of the (acid?) change in intracellular pH. In this context it is interesting to draw a parallel with the peripheral chemoreceptors. In type I carotid body cells, acetazolamide causes alkalosis [53] and reduces the output and sensitivity of the carotid bodies [54–56]. This implies that on type I cells, carbonic anhydrase has an acidifying effect, which could be mediated by an extracellular membrane-bound isoenzyme that operates in concert with an HCO pump (bicarbonate out, chloride in; 53). This is an 3 /Cl unlikely scenario for the central chemoreceptors because acetazolamide applied locally [47] or via the cerebrospinal fluid [57] and methazolamide, administered systemically after prior inhibition of carbonic anhydrase in all peripheral tissues [30], all cause an increase in respiratory output and CO2 sensitivity. Full understanding of the role of carbonic anhydrase in central chemoreceptors will not be possible without detailed knowledge of the entire machinery that these cells possess to control intracellular pH: ion pumps (including the direction into which they operate), identity and subcellular distribution of carbonic anhydrase isoforms, possible interaction with glial cells, etc. e. Non-specific and/or other pharmacological actions of acetazolamide other than inhibition of CA alone cannot be excluded. For example, in guinea pig mesenteric arteries and human forearm vessels, acetazolamide seems to stimulate KCa (Maxi-K) channels, possibly by a specific action [58,59]. A similar effect may also be responsible for the reduction in O2 and CO2 sensitivity (and their interaction) of the peripheral chemoreflex loop by

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Teppema and Dahan acetazolamide [60]. It may be difficult to envisage how stimulation by acetazolamide of potassium channels in the membrane of central chemoreceptors could result in their depolarization.

Microdialysis of CO2

Local application of CO2 in vivo with the aid of a CO2 microdiffusion pipette is a technique developed by Nattie and co-workers to examine the effect of local acidosis by a more physiologic stimulus than acetazolamide [61]. The design of the pipette tip is such that the tissue fluid injection can be avoided; the local acidosis is caused by diffusion of CO2 from the CO2-enriched artificial CSF flowing through the pipette. In contrast to the effects of local acetazolamide injections, the resulting acidotic stimuli rise and reverse quickly and have relatively short-lasting effects on ventilation. Thus, within one animal repeated ‘injections’ of CO2 can be made and dose-response relations studied. Similar to acetazolamide and dependent on the arousal state (anesthetized, asleep or awake), local application of CO2 causes large increases in ventilation when applied in caudal NTS, raphe and RTN, indicating the presence of CO 2 chemoreceptors in these regions. Focal dialysis in RTN with CSF equilibrated with 25% CO2, for example, caused a ventilatory response equivalent to that induced by a rise in arterial PCO2 by 1 kPa (about a 25% increase in ventilation; 61). In particular, application of this technique in awake and sleeping animals (rats) has revealed some other interesting properties of different chemosensitive regions. Microdialysis of CO2 in medullary raphe causes an increase in breathing (frequency) during sleep but not wakefulness [62], but the reverse was found for microdialysis in RTN, where an increase in tidal volume was found in the awake state only [63,64]. Acidosis in caudal NTS causes an increase in tidal volume and frequency while both awake and asleep [65]. Although it cannot be excluded that the same CO2 concentration in the pipette may cause different acidotic stimuli or stimulus spread in the awake state and during sleep, these results indicate that different chemoreceptor areas may have distinct roles depending on the arousal state. Carbonic Anhydrase Immunohistochemistry

In the brain, carbonic anhydrase is widely distributed. The most ubiquitous isoenzyme is CA II, which is found in the cytoplasm of oligodendroglia, microglia, choroid epithelium, in astrocytes and some neurons [66]. Because carbonic anhydrase-containing oligodendrocytes occur throughout the brain, it has been suggested that the enzyme plays an important role in


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controlling the excitability of neurons by rapidly converting metabolic CO2 to Hþ [67]. Membrane-bound CA II has also been demonstrated and constitutes about 50% of the total CA activity in rodent brains [68]. Another isoenzyme, CA IV, a membrane-associated isoform, has been localized both in capillary endothelium, glia and myelin and to a lesser degree in neurons [69–72]. Generally, with the exception of the rostromedial and rostroventrolateral medulla, a detailed topographic description of CA isoenzymes in chemosensitive areas is not available. In rostroventrolateral medullary areas associated with central chemoreceptors and in chemosensitive neurons cultured in vitro, cytosolic and membrane-bound isoenzymes have been demonstrated [73,74]. Wang and co-workers [38] showed that within the medullary raphe many serotonergic neurons do not show immunoreactivity to CA II and/or CA IV. Furthermore, they showed that many other neurons, e.g., hippocampal or cerebellar, do stain for CA, although these regions contain only small percentages of chemosensitive neurons. They also showed examples of serotonergic raphe neurons cultured in vitro (not stained for CA) which were highly sensitive to CO2 but insensitive to acetazolamide. The above data indicate that CA cannot be considered a unique marker for chemoreceptors. Why do some chemoreceptors contain some isoform of the enzyme while others do not? We suggest that this may be related to their location relative to blood vessels. If the arterial PCO2 rises upon exposure to CO2, chemoreceptors that are closely associated with larger, superficial arteries will be more rapidly subjected to a rise in perivascular PCO2 than those supplied with blood from smaller vessels further downstream. Local presence of CA could enable these cells to translate these alterations rapidly into changes in intracellular pH. In this context we refer to an older study in carotid body denervated cats in which we found that changing the extracellular pH in the medulla by way of exposing the animal to squarewave changes in end-tidal CO2 led to a change in phrenic activity that could be well described with a model (with the extracellular pH as input) containing both phasic and tonic components [29]. In this study, the extracellular fluid (ECF) pH changed with a time constant of about 40 s. Identification of a fast and slow component, possibly representing fast and slow chemoreceptors, would be easier if it were possible to induce squarewave changes in medullary ECF pH. Due to (perfusion-limited) slow washin or wash-out of CO2, this is very difficult. It would be interesting, however, to investigate if the distribution of carbonic anhydrase within chemosensitive neurons may be related to their proximity to blood vessels. For example, double staining for Fos and carbonic anhydrase in animals exposed to CO2 breathing could tell us if a preferential staining for carbonic anhydrase may occur in activated (Fos-positive) neurons with cell bodies or processes closely associated with vessels.

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Teppema and Dahan c-fos Immunohistochemistry–A Tool to Localize Central CO2 Chemoreceptors?

The nuclear proto-oncogene c-fos is an immediate early gene that may link short-term cytoplasmic and membrane responses with adaptive alterations in gene activity resulting in both short- and long-term changes in neuronal function [75]. When a cell is exposed to a stimulus, a second messenger causes the induction of immediate-early genes, which then is followed by synthesis of protein (complexes) that function in signal transduction [76,77]. Thus, proto-oncogenes can encode ligands, receptors, GTP-binding proteins, tyrosine-kinases, nuclear receptors and DNA-binding proteins. Expression of c-fos is considered as a marker of activation of individual neurons after synaptic activation and influx of Ca2þ through voltagesensitive Ca2þ channels [76–79]. The protein product of c-fos, the nuclear protein Fos, is rapidly and transiently induced and remains in the nucleus for several hours, where it can be identified with the aid of specific antibodies. Initially, in respiratory physiology, Fos immunohistochemistry was presented as a tool to identify central chemoreceptors [80], and many investigators have used the technique to map neuronal pathways in the brain that are activated during hypercapnia (and also hypoxia). Here we give a brief overview of the most important sites that appear to contain increased Fos levels after exposure of intact animals to inhalation of CO2 (nomenclature after [81]; data from [80,82–91]): a. in dorsal medulla: the commissural, dorsomedial and medial subnuclei of the Nucleus Tractus Solitarius (NTS) at caudal level and at the obex, with little staining in the ventrolateral subnuclei; b. in the Caudal Ventrolateral Medulla (CVLM): A1 noradrenergic cells, but also non-catecholaminergic neurons within and ventral to the Lateral Reticular Nucleus (LRN) and in the vicinity of the nucleus (retro)ambiguous; c. in the Rostral Ventral Medial Medulla (RVMM): neurons in raphe pallidus (RP) and magnus (RM) and gigantocellular nucleus pars a (GiA); d. in Rostral VentroLateral Medulla (RVLM): many neurons within the area overlapping the A1 (noradrenergic) and C1 (adrenergic) regions, within the C1 region, also called the subretrofacial area, retrofacial lateral ParaGigantoCellular nucleus (PGCl) and rostral ventrolateral nucleus [84]. The ventral border of this area is analogous to the intermediate chemosensitive area in the cat [7]. At the rostral border of the RVLM, just caudally from (and in the cat ventrally to) the facial nucleus many c-fos expressing cells are present in a region called the RetroTrapezoid Nucleus (RTN) (Figure 2.2, taken from the cat medulla);


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Figure 2.2 Activation of RTN (retrotrapezoid nucleus) cells in the rostroventrolateral medulla of an anesthetized cat that was exposed to 10% CO2 for 1 hr. Black spots are nuclei that stained for the protein Fos which is the product of the immediate early gene c-fos (Data from Ref. 82).

e. at pontine level: within the Locus Coeruleus (LC), Kolliker-Fuse nucleus and A5 noradrenergic cell group, and more rostrally the external subnucleus of the parabrachial nucleus (vlPAG); f. in caudal mesencephalon: the ventrolateral periaqueductal gray and dorsal raphe structures; g. in more rostral brain areas: the medial supramammillary nucleus, the central nucleus of the amygdala, the paraventricular and supraoptic nucleus in the hypothalamus, the medial preoptic nucleus, the bed nucleus of the striae terminalis, and the lateral septum nucleus; h. in one study, expression of c-fos was reported to occur in rostral regions of the fastigial nucleus in the cerebellum [92]. The above data come from studies showing considerable differences in outcomes, which is not surprising for several reasons. First, the time pattern of Fos expression strongly depends on the strength, duration and nature of the stimulus. Second, the precise experimental paradigm may be of utmost importance because the Fos protein acts as a negative regulator of its own expression: after an initial stimulation (for example, a short period of hypercapnia preceding the test period), there may be a refractory period for a re-induction of c-fos expression that may last for several hours [75]. Thus

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studies with different experimental paradigms may be expected to yield different results. Third, variations in perfusion, fixation, and preparation techniques as well as in antibody specificity or dilution may significantly influence the staining results. In addition, some studies were performed in awake animals, while in others an anesthetic preparation was used. Various anesthetics influence the expression of c-fos differently [93–95]. Anesthesia may also alter hypercapnia-induced expression of Fos in some regions. For example, hypercapnia induces c-fos expression in LC and medial preoptic nucleus in awake rats but not in those anesthetized with a-chloralose urethane [96]. An additional source of confusion may be born from the fact that many Fos-expressing neurons are located in the reticular formation of the brainstem where clearly defined functional regions with wellcircumscribed structures and boundaries are absent (some authors use coarse rather than precise anatomical descriptions). Apart from the above factors, a few other factors should be taken into account. First, hypercapnia is not a specific stimulus to the ventilatory control system because it also elicits cardiovascular, sympathetic and neuroendocrine responses. Second, expression of c-fos is not limited to specific cells but may play a role in stimulus-response coupling that is common to most cell types. Multiple second-messenger pathways appear to converge on c-fos, which is compatible with the finding that many cell types express the gene [75]. In this context, it is of interest to note that there is considerable overlap of brain(stem) regions showing increased expression of c-fos during both hypercapnia and hypoxia [84,88–90,97]. Brain stem regions of the rat showing increased expression during hypercapnia but not hypoxia are RTN, juxtafacial PGCl, GiA, LC and vlPAG [84]. Finally, expression of c-fos may be dissociated from neuronal firing; in some cases increased expression may mirror an increased level of second messengers rather than neuronal activity [75,98]. On the other hand, despite the ability of many cells to express c-fos, the absence of increased Fos levels does not prove absence of activation, so that negative outcomes cannot be excluded. The above limitations and uncertainties may give the impression that using Fos immunohistochemistry raises more questions than it answers. It does not mean, however, that it could not be a useful tool in the identification of central chemoreceptors. First, there is fair agreement between the location of neurons in the brain stem with increased Fos levels after a hypercapnic challenge and those chemosensitive cells that are localized with microinjection of CO2 and acetazolamide and with electrophysiological means. This is particularly true for neurons in the NTS, CVLM, raphe structures in the RVMM, subretrofacial area and RTN in the RVLM and locus coeruleus in the pons. Figure 2.3 shows similar locations of chemosensitive neurons in ventral raphe of the rat medulla identified with electrophysiological means (left panel), and those of


1 mm

Acidosis-stimulated Acidosis-inhibited

LPG

B

Pyr

0.25 mm

Figure 2.3 Chemosensitive cells in rat medullary raphe localized with electrophysiological means in vitro (left panel, data from Ref. 15) and with Fos immunohistochemistry after exposing the whole animal to 15% CO2 for 1 hr (right panel, data from Ref. 84). B: basilar artery; Pyr: pyramidal tract; LPG: lateral paragigantocellular nucleus.

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Figure 2.4 Expression of Fos in the external lateral subnucleus of the parabrachial nucleus of a rat exposed to 10% CO2 for 1 hr. Whether the stained nuclei are from chemosensitive cells is unknown (Data from Ref. 84).

Fos-positive cells after exposure to CO2 in vivo (right panel). Other regions containing immunostained and possibly chemosensitive cells, cells such as the parabrachial nucleus in the pons (Figure 2.4) and more rostral regions in the hypothalamus are relatively unexplored with these alternative techniques, with the exception of electrophysiological studies showing CO2 responsive neurons in the caudal hypothalamus [36]. Note also that Fos immunohistochemistry is a method to localize somata (nuclei) of activated neurons and does not provide any information about the location of the possibly distant chemosensitive sites. Fos immunohistochemistry is a technique with single-cell resolution, a clear advantage. Thus it has an enormous potential to characterize individual activated neurons neurochemically, provided Fos staining is combined with the use of specific antibodies directed against neuromediators, transmitters, second messengers and enzymes. In this way, for example, we know that the majority of neurons in the RVLM that are activated during hypercapnia do not contain noradrenaline or dopamine [84]. By using antero- and retrograde tracers, functional properties of neuronal circuitries can be studied in different physiological circumstances. Fos immunohistochemistry can also be used in combination with pharmacological tools, for example to reveal the second messenger associated with hypercapniainduced Fos expression or to investigate the causal relationship between the increase in ventilation and the increased expression (e.g., by using anti-sense Fos mRNA).


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LHB LPB VMH

SON OT

LC

DM

AP NTS

Pons

7

PreBotc cVRG PGCL VRG 10

rVMS RTN

BotC

P

cVMS

Figure 2.5 Fos expression in the rat brain in response to stimulation of the rostral chemosensitive area at the brain stem surface of the rat with acidic CSF (pH 7.2). CVRG: caudal ventral respiratory group; DM: dorsomedial hypothalamus; 10: inferior olive; LC: locus coeruleus; LHB: lateral habenular nucleus; LPB: lateral parabrachial nucleus; NTS: nucleus tractus solitarius; PGCL: lateral paragigantocellular nucleus; preBotc: pre-Bo¨tziger complex; RTN: retrotrapezoid nucleus; 7: facial nerve; VMH: ventromedial hypothalamus; SON: supraoptic nucleus; OT: optic tract; rVMS: rostral ventral medullary surface; cVMS: caudal ventral medullary surface; P: pyramidal tract. AP: area postrema (Data from Ref. 99).

In summary, more insight is needed into the mechanism(s) by which so many neurons in the brain stem (and more rostral brain regions) show increased expression of c-fos during hypercapnia: are all these neurons directly activated by CO2 or are synaptic events involved? In this context, two recent studies are of particular interest. Douglas et al. [99] showed increased expression of c-fos in many neurons in brainstem, hypothalamus and more rostral regions after application to the caudal and rostral ventral chemosensitive areas of pledgets soaked in artificial CSF with an acidic pH of 7.2. This shows that exposing the ventral medullary surface areas to acid initiates c-fos induction in many distant areas (Figure 2.5), several of which are also activated during exposure to CO2 of the whole animal. Some of these neurons may be activated because they possess (long) processes projecting to the ventral surface. Other neurons, however, may have been activated transsynaptically, as the caudal and rostral ventrolateral medulla have many connections with other brain(stem) areas [84]. Okada et al. [16] studied the expression of c-fos in the ventral medulla after in situ transarterial perfusion of the animal with mock CSF equilibrated with

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8% CO2 and containing low calcium, high magnesium or TTX to block synaptic transmission. The ventral medulla of animals perfused with normal CSF (also equilibrated with 8% CO2) without the synaptic blockers contained many stained large- and small-size nuclei in cells located predominantly in perivascular superficial tissue. In animals that were perfused with CSF containing the synaptic blockers, Fos-containing cells (mostly with small nuclei) were restricted to the marginal glial layer close to the medullary surface and closely associated with blood vessels, suggesting that they may represent true CO2 chemoreceptors. Indeed, in a perforated patch-clamp configuration, the same superficial region appeared to contain cells that were activated by hypercapnia. These well-designed studies show that Fos immunohistochemistry can be a very valuable tool in the identification of central CO2 chemoreceptors.

III.

Mechanism of Central Chemoreception

A. Extracellular pH, Intracellular pH, CO2, and Central Chemoreception

Several in vivo data indicate that the intracellular (pHi) rather than the extracellular pH (pHe) plays a crucial role in central chemoreception. In the anesthetized cat, isocapnic medullary pHe changes have a much smaller effect on minute ventilation or phrenic output than equal changes induced by hypercapnia [9–11]. This can be explained by a major role of pHi in chemoreception or, alternatively, by a direct effect of CO2. After systemic administration of acetazolamide in carotid body denervated cats, the time course of the resulting changes in medullary pHe and ventilation do not match [29], indicating that another factor, possibly pHi, acts as stimulus. The same applies to local application of acetazolamide into chemosensitive areas of rat and cat medulla [44,47]. Intracellular acidosis in the ventral medulla, however, does not always increase ventilation. After peripheral chemodenervation, a hypoxia-induced acidification of the ventral medullary surface fails to increase ventilation in anesthetized rabbit and cat [100,101]. The same was found in awake goats undergoing CO-hypoxia [102]. Assuming intracellular acidosis as the source of this extracellular acidosis, it was suggested that the absence of an increase in ventilation could be explained by an increase in the intra- to extracellular Hþ gradient [100–102]. Hypercapnia or a metabolic acidosis originating in the extracellular environment would decrease this gradient and hence stimulate the chemoreceptors. Organotypic medullary cultures in which many features of central chemoreceptors are preserved are successfully used to study chemoreceptor pHi and pHe responses to manipulations such as hypercapnia (causing both


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intra- and extracellular acidosis), replacement of bicarbonate by HEPES in the bath solution (causing a fall in pHi but no change in pHe), ammonia wash-in (extracellular acidosis, intracellular alkalosis) and ammonia washout (extracellular alkalosis, intracellular acidosis; [103]). In all cases, neuronal responses correlate with changes in pHi rather than pHe, with a fall in pHi consistently coupled to an increase in activity [103,104]. Another argument for pHi as the proximate signal for central chemoreception has been the observed steeper pHi–pHe relationship during hypercapnia in neonatal chemosensitive neurons compared with nonchemosensitive cells [105,106]. Most nonchemosensitive neurons in the inferior olive and hypoglossal nucleus show pHi recovery upon exposure to a hypercapnic acidotic solution; chemosensitive neurons from the ventrolateral medulla and NTS, however, fail to show this recovery, except when the intracellular acidosis is caused by an isohydric (i.e., constant pHe) hypercapnia [105,106]. Carotid body type I cells [53] and taste-bud receptor cells [107] show a similar behavior, suggesting that it might be a common feature of CO2-sensitive cells. It has been suggested that this failure of chemosensitive neurons to recover may be caused by inhibition of a Naþ/Hþ exchanger (NHE, acting as an acid extruser and located in the chemoreceptor membrane) by the low extracellular pH. Naþ/Hþ exchangers in the membrane of chemoreceptors would be much more sensitive to changes in pHe than those located in the membrane of nonchemosensitive cells, explaining a much steeper pHi–pHe relationship in the former. Similarly, a high sensitivity of the anion (Cl/HCO 3 ) exchanger of ventral medullary chemoreceptor cells might explain the failure of these cells to show recovery upon an alkaline challenge [105,106,108]. The cause of this difference in pHe sensitivity of chemoreceptor vs. nonchemoreceptor NHE is unknown, but it has been related to the presence of a different isoform of the transporter in the chemoreceptor membrane. From the six NHE subtypes known to occur in the brain [103], the isoform NHE3 seems to be preferably located in neurons of the superficial ventrolateral region of the medulla [103,109]. Also, selective NHE3 inhibitors are reported to mimic CO2 responses of chemosensitive neurons in vitro, to reduce pHi in VLM neurons and to inhibit pHi regulation upon exposure to ammonia [103]. In the anesthetized cat and rabbit, systemically administered selective NHE3 inhibitors reduce the apneic threshold (own unpublished observations; [110]). The idea that central chemoreceptors may differ from other neurons in their pHi regulation machinery for which a specific membrane transporter might be responsible could be useful in future attempts to isolate unique features of these chemoreceptors. It should be noted, however, that the above data showing pHi regulation in nonchemosensitive cells come from neonatal and therefore immature animals. Nottingham et al. [111]

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compared pHi responses in RTN and hypoglossal neurons from neonatal and mature animals. While neonatal hypoglossal but not RTN neurons showed pHi recovery upon a hypercapnic exposure, in cells from mature animals this difference disappeared: both mature cell types showed no pHi recovery, unless amiloride (to inhibit NHE) was added to the bath. Thus the question remains, whether in mature chemosensitive cells a lack of pHi recovery from intra- and extracellular acidosis indeed represents a feature of central chemosensitivity rather than a general mechanism to protect neurons against an NHE-induced influx of Naþ ions which eventually may lead to cell death [111]. Because hypercapnic exposure with a constant pHe results in intracellular acidosis and increased neuronal activity, it is reasonable to ascribe an important role to the intracellular pH in central chemoreception. A steep pHi –pHe relationship in hypercapnia will imply an appreciable sensitivity to changes in pHe under this condition. Because the relation between pHi and pHe (and vice versa) during a given change in acid-base status depends on the origin of the primary disturbance, it is difficult to describe the relation between ventilation (or chemoreceptor activity) and pHe in terms that are generally valid. A further reason for the complex ventilation-pHe relation is that the extracellular pH may influence chemoreceptor activity in a number of ways independently from its effect on pHi. Various types of membrane ion channels, for example, may change their conductance upon pHe changes and initiate an intracellular chemosensitive stimulus-transduction cascade (see below). Changes in pHe may also modulate ongoing synaptic transmission at the chemoreceptor membrane. A notable and possibly important example is the known pH sensitivity of several members of the purinergic receptor family [112]. In the anesthetized rat, microinjection of the selective P2X antagonist suramin in the retrofacial area increased the apneic threshold and reduced the respiratory response to CO2 [112]. An even more interesting observation was that suramin and PPADS (pyridoxal-phosphate-6-azophenyl-20 ,40 disulfonic acid, another P2X receptor antagonist) reduced the ongoing activity of inspiratory and pre-inspiratory neurons in the pre-Bo¨tzinger complex and blocked their activation by CO2 and ATP [113]. If indeed the excitation of these respiratory neurons by ATP and the P2X agonist ab-methyleneATP would involve a pH-sensitive purinergic receptor, then this would be another clear example of respiratory neurons showing direct pH sensitivity [also 12,13]. Cholinergic neurotransmission is a second example of a pHe-modulated transmission. From numerous studies it is clear that, at least in the ventral medulla, the central chemosensitive mechanism involves a muscarinic cholinergic mechanism [114]. In the in vitro brainstem-spinal cord preparation, blockage of M1 and M3 muscarinic receptors resulted in a depression (cessation) of respiratory output, an effect that was easily


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reversed by facilitation of acetylcholine release [115]. In the cat, topical application of atropine to the ventral medullary surface blunted the ventilatory response to CO2 [116]; this effect could be reproduced by using M1 and M3 receptor blockers [117,118]. In the unanesthetized rat and newborn piglet, microdialysis of muscimol in the RTN inhibited the response to systemic hypercapnia by 24% and 40%, respectively [119,120]. In slices from ventral medulla of rat, Fukuda and Loeschcke [121] showed that an intact cholinergic transmission was a prerequisite for activation by acidic solutions. A similar observation was made in spontaneously breathing dogs undergoing ventriculocisternal perfusion: an increase in ventilation in these animals by CSF acidification was abolished in the presence of atropine [122]. What is the link between chemosensitivity, cholinergic transmission and pHe? The acid sensitivity of cholinergic neurotransmission could reside in a pH effect on imidazole groups of the hydrolytic enzyme acetylcholinesterase [114]. From imidazole groups, on the other hand, we know that they are involved in central chemoreception because when applied to the ventral surface of cats, imidazole-binding agents inhibit the ventilatory response to CO2 [123; for a further explanation of the a-stat hypothesis of central chemoreception see 124]. The varying pHi –pHe relation on one hand, and the additional independent effects of extracellular pH on the other, make the relation between chemoreceptor activity and pHi extremely complex. For example, during brain stem hypoxia the chemoreceptor NHE may be activated [125,126], tending to recover pHi from intracellular acidosis. The resulting (actually measured) fall in pHe may now be unable to inhibit the (activated) membrane transporter. If we assume a sustained intracellular acidosis in the case of steady-state hypoxia (to which increased uptake of lactate via a monocarboxylate transporter may also contribute [127]), then the intriguing question remains about what happens to central chemoreceptor output in this situation: is the failure of minute ventilation to increase (see above) due to independent effects of pHi and pHe on the chemoreceptor neuron, or rather to effects unrelated to the mechanism of chemoreception such as inhibitory modulators acting elsewhere on the respiratory network? It may prove to be elusive to isolate a unique stimulus for the central chemoreceptors, because there are many ways to depolarize a neuron (apart from the question whether depolarization is a conditio sine qua non for activation; see below). A given change in pHe or pHi may have entirely different effects on individual chemoreceptor cells depending on their membrane properties and location (see also below). Should neurons that depolarize upon a fall in pHe, despite the fact that they may not possess the specific machinery of chemosensitive cells in VLM and NTS to regulate pHi, also be called central chemoreceptors? (cf. hypoglossal motoneurons that have TASK-1 channels in their membranes and

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respond with depolarization upon acidosis; see below). Finally, although direct effects of CO2 cannot be excluded, it also cannot be considered as the adequate stimulus because there are many data showing that isocapnic changes in pHe and pHi alter the activity of chemosensitive cells. B. Intracellular pH, Gap Junctions, and Central Chemoreception

One possible explanation for the surprisingly large effects of topical application of CO2 and acetazolamide in chemosensitive brainstem areas on ventilation (see above) is that focal stimulation is spread out over an entire network consisting of neurons that can read an electrical (or metabolic) signal generated by the membrane of a neighboring cell via gap junctions. Gap junctions form a low-resistance communication pathway between adjacent cells and are made from two hemi-channels (connexons), each of which consists of a hexamer of connexins (Cx) [128]. Immunohistochemical studies have shown that putative chemosensitive areas in the NTS, retrofacial area and RTN, but also in the pre-Bo¨tzinger complex, contain gap junction proteins: Cx26 in neurons and astrocytes and Cx32 in neurons [129,130]. This suggests the presence of gap junctions in these areas via which central chemoreceptors could be electrotonically coupled. Indeed, a large percentage of CO2-sensitive neurons in NTS appears to be anatomically and chemically coupled, in contrast to most cells that do not respond to CO2 [131,132]. Many CO2-sensitive neurons in LC also show cell-cell coupling [33,131,132]. Gap junctions appear to close in response to a rise in intracellular [Ca2þ] or [Hþ] [133–135]. Because the central chemoreceptors most likely respond to changes in intracellular pH, closing of gap junctions in response to a rise in intracellular [Hþ] could play a role in the chemosensitive mechanism. Interestingly, Cx32, one of the gap junction proteins in the presumptive chemosensitive areas in the ventral medulla, contains a cytoplasmic domain that is critical to CO2/pH gating sensitivity [136,137]. However, spontaneous electrotonic postsynaptic potentials of LC neurons do not seem to be affected by hypercapnic acidosis, and synchronization in pairs of LC neurons does not disappear with acidosis in the physiological pH range [131,138–140]. This may be related to the fact that the coupling coefficient between coupled neurons also depends on non-junctional conductance [128]. Thus, a decrease in nonjunctional (membrane) conductance in chemosensitive cells, for example during an acidosis-induced depolarization, may tend to enhance junctional cell coupling and offset the channel-closing effect of the lower intracellular pH. As mentioned above, gap junctions are sensitive to intracellular pH. After uncoupling, however, chemosensitive neurons in the solitary complex


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and locus coeruleus retain their CO 2 sensitivity, indicating that cell-cell coupling is not a requirement for CO2 sensitivity per se [33,131]. Coupling between cells resulting in synchronization may have considerable effect on the gain of a system [128]. This may be one of the factors that determine the CO2 sensitivity of the entire network of central chemoreceptors or the form in which CO2 sensitivity is expressed, e.g., DC (low frequency) changes in membrane potential vs. changes in spike frequency [33]. Another function of gap junctions in chemoreceptor cells may be related to the exchange of second messengers such as Ca2þ, cAMP and IP3, allowing coregulation of these messengers in pre- and postsynaptic compartments and the modulation of presynaptic release by postsynaptic second messengers [128,141]. Future studies should address several important questions for more insight into the role of electrotonic and/or biochemical coupling in central CO2 chemoreception. First, it is of interest to document the particular (homocellular or heterocelllular) types of potential gap junctions within the ventrolateral medullary chemosensitive areas and their electrophysiological behavior. Second, what is the effect of changes in intracellular pH on cell coupling between chemoreceptor cells? Is the apparent lack of uncoupling by CO2 (low pH) unique to coupled cells that are excited by CO2? Third, what would be the influence of variations in the level of coupling between chemoreceptors on the CO2 sensitivity of individual neurons and on the gain of the entire network? Could various neurotransmitters (e.g., dopamine and serotonin, transmitters known to influence cell-cell coupling [128]), alter the coupling coefficient between chemoreceptor cells via an effect on membrane resistance and, by this, the sensitivity of the entire network? Finally, could the uncoupling effect of a volatile anesthetic such as halothane [128] be responsible for its depressant effect on central chemosensitivity? C. Glia, Extracellular pH and Central Chemoreception

It is well established that glial cells play a key role in the regulation of the ionic ([Hþ] and [Kþ] in particular) extracellular microenvironment of neurons, which means that the glial cells are a crucial element in determining the neurons’ stimulus level and excitability [142]. What does this mean for the central chemoreceptors? Glial cells could influence the central chemoreceptors in several ways; first, by regulating the extracellular pH [143] which at many locations occurs with the aid of carbonic anhydrase, providing a machinery for fast pHe regulation [66–68]. Local injection of the glial toxin flurocitrate into the RTN results in local extracellular acidosis and an increase in phrenic activity and spontaneous ventilation in anesthetized and conscious rats, respectively [144,145]. CO2 sensitivity is retained in these circumstances [145], indicating

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that the integrity of glial cells is not crucial for an intact chemosensitive mechanism per se. The ventilatory stimulation observed in the above studies may indeed be due to the extracellular acidosis, but a possible influence of a disturbed extracellular potassium concentration cannot be excluded. By regulating the extracellular [Kþ] [142,146], glial cells will directly influence the membrane potential and excitability of neurons. Synthesis, release and uptake of neurotransmitters is another way by which glial cells could influence the activity of central chemoreceptors or even the chemosensitive mechanism. For example, glial cells release lactate that is formed from glucose; this process is stimulated by glutamate that is released from neurons. The lactate then serves as a nutrient for the neuron [127]. Finally, the functional role of possible neuron-to-glial coupling or glial-glial coupling in the central chemosensitive network remains to be determined (see above). D. Intracellular pH, Extracellular pH, Membrane Channels, and Central Chemoreception

Even after synaptic blockade, chemosensitive neurons in NTS, raphe and RVLM respond to CO2/Hþ, suggesting that ion channels may be the primary responsive elements initiating an increase in spike frequency [13,14,147,148]. Dean et al. [148] found that CO2/Hþ depolarized NTS neurons via a reduction in potassium conductance of the membrane, without specifying particular candidates. The large family of potassium channels has many members that possess pH sensitivity. Notable examples are TASK (TWIK-related acid sensitive) channels. These two-pore-domain background potassium channels show fast activation and inactivation kinetics, and are extremely sensitive to changes in external pH within a narrow range (midpoint inhibition of TASK-2 ¼ pH 8.3, TASK-1 ¼ pH 7.3 and TASK-3 ¼ pH 6.3; [149,150]). TASK currents have been recorded in chemosensitive locus coeruleus and serotonergic raphe neurons, but also in hypoglossal motoneurons [151–153]. Thus, because inhibition of these currents in (chemosensitive) cells by extracellular acidosis in the physiological pH range results in their depolarization and increase in excitability, it is attractive to consider TASK channels (and particularly TASK-1) as potential primary targets for CO2/Hþ to initiate the chemosensory stimulus-transduction cascade. In type-I carotid body cells from neonatal rats, inhibition of TASK-1 channels by hypoxia and acidosis has also been shown to result in membrane depolarization [154]. It is of interest to note that in hypoglossal pH-sensitive motoneurons and cerebellar granule cells, TASK channels are modulated (inhibited) by various G-protein-coupled neurotransmitters that are also able to stimulate central chemoreceptors or respiratory neurons, e.g., acetylcholine, TRH, glutamate and serotonin [151,153,155].


45 H+

Transmitter Ligand Receptor 5-HT 5-HT2 5P NK1 TRH TRH-R1 α1 NE glutamate mGluR (group 1)

− Gαq-coupled receptors

TASK-1

+

K

−

+

Anesthetic

Figure 2.6 Modulation of Task-1 channel conductivity by pH, anesthetics and transmitters (Data from Ref. 153).

Also, opening of TASK channels is one of the known concentrationdependent effects of volatile anesthetics such as halothane and isoflurane [153,154,156–158]. This could provide a molecular basis not only for the anesthetic-induced decrease in excitability of motoneurons [153,158], but also for the known depressant effects of these agents on the central (but also peripheral) chemoreflex loop [153,154,159–162]. The above modulating influences on TASK-channel conductivity are summarized in Figure 2.6. Inhibition by extracellular acidosis of TASK channels cannot provide a complete scenario for central chemoreception simply because central chemoreceptors can be activated independently from changes in pHe. It also appears possible to increase spike frequency without membrane depolarization in locus coeruleus and medullary raphe neurons by applying an isohydric (i.e., constant pHe) hypercapnic stimulus [72,163]. Also, recent patch-clamp studies have shown that (hypercapnic) acidosis inhibits Ca2þactivated Kþ (KCa) channels in chemosensitive medullary raphe neurons and in fetal medullary neurons either by a direct effect on these channels or by inhibition of voltage-sensitive Ca2þ channels [14,164]. Note that KCa channels are inhibited by intracellular acidosis [165,166]. In locus coeruleus neurons from rat, isohydric and hypercapnic acidosis have been shown to stimulate L-type Ca2þ and multiple Kþ channels that are expressed in these neurons, e.g., KCa, TASK and inward rectifying Kþ channels (Kir) that is, similar to KCa, also show sensitivity to the intracellular pH [37,167]. The Kir channels deserve special mention because some members or heteromeric combinations are extremely sensitive to the intracellular pH, so that these channels could be a potential tool in the localization of central chemoreceptors. Jiang et al. [168] have presented a very elegant approach. Because it was already known that Kirs are involved in the control of membrane excitability and in cellular responses to hypercapnia and acidosis, these investigators used mammalian cell lines to express various homomeric Kirs and identified several subtypes that are sensitive to changes in

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intracellular (Kir1.1 and Kir4.1) and extracellular (Kir2.3) pH. Subsequently, they identified the critical pH-sensitive residues and motifs of these channels, and, after searching the GenBank, found an amino acid sequence in Kir5.1 identical to the critical motif of Kir2.3. Kir5.1 owes its biological significance to the fact that it forms heterodimeres with the Kir4 subfamily [168]. Because the heterodimer Kir4.1–5.1 showed a higher pH sensitivity than other members of the family and a pKa of 7.45, they suggested that it may well function at physiologic pH and PCO2 levels. When coexpressed in Xenopus oocytes, the current through these Kir4.1–5.1 complexes was reduced at high (8%) CO2 levels (leading to depolarization) but enhanced at low CO2 (3%) (leading to hyperpolarization), compared with the baseline current at 5% CO2. This led the authors to suggest that this Kir4.1–5.1 complex could enable cells to increase or decrease their membrane excitability in response to a rise or fall in PCO2, respectively. As a great surprise, using in situ hybridization, the authors then showed coexpression of Kir4.1–5.1 in cells belonging to regions proposed to contain central chemoreceptors: locus coeruleus, nucleus tractus solitarius, and within the caudal and rostral ventrolateral medulla (e.g., subretrofacial area). This well-designed strategy shows that in order to identify the central chemoreceptors, beginning with molecular mechanisms of CO2/Hþ chemoreception may be a very promising approach. E.

Conclusion

To date, there is no general model for the entire stimulus-transduction cascade of CO2 chemoreception. A general picture emerging from the available data is that individual chemoreceptors may display considerable differences in their pHi regulation machineries, receptors and ion-specific membrane channels that are sensitive to changes in either pHi, pHe or both. The fact that central chemoreceptors are so widely distributed over regions with different afferent connections containing specific transmitters and modulators adds to this complexity. Receptors that influence the conductance of ion channels and/or ion channels themselves (e.g., TASK) are molecular targets for these modulators; this may explain the effects on chemosensitivity of agents such as acetylcholine, ATP and other transmitters (e.g., glutamate) and their antagonists. Variations in tonic or phasic influences of these neuromodulators may thus lead to variations in the stimulus-transduction cascade in chemosensitive cells once they are stimulated by CO2/Hþ. All these factors make it an extremely difficult challenge to isolate a neurochemical or electrophysiological property that is unique for central CO2 chemoreceptors. There is growing consensus of a prominent role for the intracellular pH and potassium (but also other) channels during hypercapnia, but also of an


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important influence of the extracellular pH. Involvement of (acid-sensitive) potassium channels seems to fit in a general framework of CO2 chemoreception in which closure of these channels leads to depolarization and activation. Particularly, a significant role of TASK channels may emerge because they are sensitive not only for changes in extracellular pH but also for neuromodulators and anesthetics that are known to alter chemosensitivity.

IV.

Central Chemoreceptors and Breathing

The central chemoreceptors are an essential part of the control system regulating pulmonary ventilation. By providing a feedback signal representing local blood flow, metabolism and acid-base status of the body to the respiratory controller, they are a crucial factor in pH homeostasis of the body. Central chemoreceptors are the major structures responsible for the ventilatory response to CO2. They also provide a tonic drive to the respiratory motoneurons; especially during sleep, an intact central chemosensitive network seems crucial for the maintenance of an adequate level of ventilation that is important particularly during NREM sleep. The widespread distribution of central CO2 chemoreceptors throughout the brainstem suggests that they may participate in various specific functions associated with the control of breathing but also in functions associated with the control of the arousal state. All these aspect will be briefly discussed in the following section. A. Central Chemoreceptors and Ventilatory Response to CO2

The exact quantitative contribution of the central chemoreceptors to the ventilatory response to CO2 is somewhat controversial, although there is general consensus about a central contribution of at least 50–60%. The issue has been addressed with various approaches, discussed briefly below. Ventriculocisternal Perfusion

In their classic ventriculocisternal perfusion experiments, Fencl et al. [3] attributed the ventilatory adaptations to metabolic and respiratory acidbase disturbances entirely to central chemoreceptors functionally located at two-thirds of the distance between cerebrospinal fluid and arterial blood. In their calculations, the authors treated the extracellular pH as the adequate chemoreceptor stimulus but later findings by other groups were in disagreement with this assumption (e.g., [9–11]). Furthermore, by neglecting the influence of the carotid bodies in hyperoxia, the role of the central chemoreceptors in this preparation was overestimated.

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Teppema and Dahan Hyperoxia to Separate Peripheral and Central Inputs

From dynamic CO2 studies in man under hypoxic, normoxic and hyperoxic conditions, it was initially concluded that hyperoxia virtually eliminates the contribution of the peripheral chemoreceptors to the CO2 response, and thus would provide a means to study central CO2 sensitivity independently of the peripheral chemoreceptors [169,170]. However, now there is ample experimental evidence to show that this assumption overestimated the role of the central chemoreceptors, because even during hyperoxia the carotid bodies may possess considerable CO2 sensitivity. In the anesthetized cat, for example, hyperoxia does not eliminate the CO2 sensitivity of afferent carotid sinus nerve afferents [171–173]. This may be a species-dependent phenomenon, however, because in the anesthetized rabbit, the carotid bodies are virtually silent at PO2 values 430 kPa [174]. Similar to the cat, humans show a considerable peripheral CO2 sensitivity during hyperoxia (references below). Lesion or Cooling of Ventral Medullary Surface Areas

Both in the awake and anesthetized cat, lesioning of specific subretrofacial areas on the ventral medullary surface designated as area S and RetroTrapezoid Nucleus (RTN), respectively, virtually abolishes the ventilatory or phrenic nerve response to CO2 [175–179]. Studies using focal cooling of caudal, intermediate and more rostral ventral medullary surface areas showed profound effects on the pattern of breathing and CO2 sensitivity in cat (e.g., [180–182]), dog [183], and rabbit [184]. These studies were interpreted to mean that the central chemoreceptors were the main responsive elements in the CO2 response and also that at least one of the chemosensitive areas (area S) would serve as an integrator for all afferent chemosensory input [175,181]. More recent data obtained from awake goats after cooling the rostral M and the caudal M-rostral S areas could not confirm this hypothesis because the cooling did not eliminate CO2 sensitivity, even after carotid body denervation [185]. Also, in the same awake animal preparation, bilateral injection of an unselective excitatory amino acid receptor antagonist did not abolish the CO2 response but only reduced it by about 40% [186]. The latter findings are consistent with the view that if focal cooling and treatment of the classical chemosensitive areas with neurotoxins are not able to reduce the CO2 response by more than 60%, the remaining sensitivity can be explained by the presence of central chemoreceptors elsewhere. Thus local lesions or cooling are inappropriate instruments to estimate the overall contribution of central chemoreceptors to the CO2 response in normal conditions. In addition, following lesions (but also following carotid body denervation; see below), any possible peripheral-central interaction could disappear. However, the effects of focal cooling (and stimulation) have revealed some other


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interesting features about the role of ventral medullary chemosensitive areas, because these effects strongly depend on the arousal state of the animals (see below). Carotid Body Denervation

Shortly after Carotid Body Denervation (CBD) in awake adult rats [187], anesthetized and awake cats [188,189], awake dogs [190,191], and goats [192], but not in anesthetized rabbits [193], the ventilatory response to CO2 can be attenuated as much as 20–60%. The effects of CBD, however, are time- and age-dependent. In the goat, for example, CO2 sensitivity returns gradually (i.e., over about 15 days) to normal [192]. Over about three weeks, a similar recovery could not be observed in awake dogs showing a depression of the CO2 response by about 40%, two days after CBD [191]. This estimation, however, was made by using a modified Read rebreathing technique with different step sizes in CO2 and possibly different cerebral blood flow before and after CBD. This may lead to considerable differences between steady-state and rebreathing slopes [21]. After CBD in neonatal goats, piglets and rats, a normal CO2 sensitivity appears to have developed between three weeks and three months after the denervation [194]. These time- and age-dependent effects of CBD on the CO2 response make it questionable whether denervated animals (or carotid-body-resected humans) can provide a realistic picture of the normal contribution of the central chemoreceptors to total CO2 sensitivity. In addition, if the peripheral chemoreceptors provide a tonic facilitatory input to the central chemoreceptors, then an acute resetting by CBD of the central chemoreceptor gain cannot be ruled out and central CO2 sensitivity could be underestimated [195,196]. A tonic input of the carotid bodies into the RostroVentroLateral Medulla (RVLM) could be mediated by setting the local balance between glutamate and GABA turnover [197]. Note the many connections that have been found to exist between the caudal, commissural and medial subnuclei of the NTS on one hand and the RVLM, where many central chemoreceptors are located, on the other [84]. Note also that compared with normoxia, the number of noncatecholaminergic neurons activated by hypercapnia (inhalation of 15% CO2) in the RVLM is substantially reduced in hyperoxia, suggesting a reduced facilitatory input from the peripheral chemoreceptors in this condition [84]. If it can be ruled out that the recovery of the CO2 response after CBD is due to recovery of a peripheral chemoreflex originating in the aortic or carotid bodies, then the denervation experiments are an excellent means to show a remarkable CNS plasticity that may reside in the central chemosensitive network, the mechanism of which remains to be determined [192,194].

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Teppema and Dahan Selective Perfusion of Carotid Bodies and Brainstem

Selective perfusion of carotid bodies and brainstem is an elegant means to study separate central and peripheral contributions to total CO2 sensitivity. Studies with artificial brainstem perfusion in the anesthetized cat have revealed a contribution of the peripheral chemoreceptors as large as 20–58% (mean 48%) during hyperoxia [198]. In dogs undergoing selective perfusion of their carotid bodies, similar results were obtained [199]. Theoretically, a low stimulus level at the carotid bodies in these preparations could also diminish or remove a tonic facilitatory input to the central chemoreceptors and reduce central CO2 sensitivity. The pontomedullary perfusion experiments in the cat, however, have shown a constant central CO2 sensitivity over a wide range of peripheral CO2 tensions [198], suggesting the absence of a peripheral-central CO2 interaction. Dynamic End-tidal Forcing

This approach is based on the different speeds of response of the fast peripheral and slow central chemoreflex loops so that the responses of both reflex loops upon a step change in end-tidal PCO2 would be separable [23,24]. In both the anesthetized cat and humans (awake and sedated), the dynamic ventilatory response to square-wave changes in end-tidal CO2 fits a twocompartment model comprising a fast and slow compartment quite well [23,24]. In the anesthetized cat, the technique was validated by showing that the magnitudes of both components closely corresponded to those obtained during artificial brainstem perfusion [24]. The dynamic end-tidal forcing technique was refined by Pedersen et al. [200] by varying the end-tidal PCO2 with a multi-frequency binary sequence (MFBS), by which the identification and quantitative estimation of the fast peripheral component is improved. Generally all these dynamic studies yielded at least a 50–80% contribution of the central chemoreflex loop while that from the peripheral chemoreflex loop varied between 15 and 50%, with greater contribution at lower PO2 levels. In humans, the peripheral contribution to the total CO2 response during hyperoxia (estimated with MFBS and square-wave changes in endtidal PCO2, respectively) can be as large as 27% at a PO2 background of 200 Torr and 13% at a PO2 greater than 500 Torr [200,201]. B. Central Chemoreceptors in Metabolic Acid-Base Disturbances

The mechanisms by which the body defends against metabolic acid-base disturbances is a complex interplay between intra- and extracellular buffering, adjustment of acid excretion by the kidneys, and adjustment of CO2 removal by the lungs. Ventilatory adaptation to metabolic acid-base alterations is mediated via the peripheral and central chemoreceptors [202].


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The fact that the central chemoreceptors have an impressively high pH sensitivity does not mean that they are responsible for the ventilatory response to a metabolic acidosis or alkalosis. Using the arterial pH as an independent variable shows that the arterial chemoreceptors have a pH sensitivity twice as high as the central chemoreceptors [203]. The crucial factor here is the extent to which and how fast a primary metabolic alteration in arterial pH is reflected in the medullary extracellular fluid. Upon a bolus infusion of NaHCO3 or HCl performed in isocapnic conditions, this appears to be about 30% in the anesthetized cat [204,205], with a time course rapid enough to initiate fast ventilatory changes [205]. In a closed-loop situation, however, when the arterial PCO2 is not clamped as in the above cat experiments, metabolic arterial pH changes develop more slowly. In this situation, the peripheral chemoreceptors sense the change in pH, initiate a change in ventilation and induce a change in arterial PCO2 that will be rapidly followed by a change in medullary ECF PCO2. Thus in the acute phase of a metabolic acid-base disturbance, the medullary pHe (and pHi, but to a lesser degree) will undergo a paradoxical change and the central chemoreceptors will dampen down the ventilatory response initiated by the peripheral chemoreceptors that sense the original disturbance [203,206]. In the chronic phase, this dampening effect may gradually extinguish if compensatory changes in medullary extracellular bicarbonate concentration occur which would tend to normalize local extracellular pH. If a primary acidosis would exist in the brain, the situation would be more complex (cf., a condition of brain hypoxia where ventilatory output is reduced; see above). After CBD, the mechanism of ventilatory adaptation to a metabolic pH disturbance will be quite different, because in this case the central chemoreceptors will initiate a response depending on the extent to which the original disturbance penetrates into the CNS. C. Central Chemoreceptors and Tonic Drive to Breath

The classical areas L, S and M on the ventral medullary surface were originally described as regions with chemosensitive (M and L) and integrative (S) properties [7]. The most complex area of these is area S, which forms the ventral border of the RVLM with its known crucial role in the mediation of pain and the control of cardiovascular and arousal functions [84]. As discussed above, lesions or cooling of the classical areas (particularly S) have profound respiratory depressant effects in anesthetized cats, dogs and rabbits. In the anesthetized goat, focal bilateral cooling of area S and M for 30 s causes an apneic episode that outlasts the period of cooling [207]. In the anesthetized rat and cat, local lesions in the RTN and retrofacial area (which belong to the RVLM and partly overlap area S) cause a decrease in phrenic activity and CO2 sensitivity [177–179,208]. Cooling rostroventral (presumably M and S) chemosensitive areas in the cat

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releases a tonic activity in expiratory muscles [182]. Apart from chemosensitive cells, the RTN also contains neurons with respiratory-modulated activity [209,210]. These studies all indicate that the RVLM, particularly the superficial area extending from the rostral part of area S to the caudal region of area M (containing the RTN) are not only chemosensitive but also provide a tonic drive to the respiratory controller and even may influence the pattern of breathing. The total tonic input to the respiratory controller is composed of inputs from cortical and limbic structures, the reticular activating system, mechanoafferents from nonrespiratory muscles, and from the peripheral and central chemoreceptors. When during anesthesia and, to a lesser degree, during sleep the cortical, limbic and mechanoafferent inputs are reduced, the relative importance of chemical feedback from the chemoreceptors will increase, which is consistent with the current idea that during (NREM) sleep and anesthesia ventilation is predominantly under metabolic control. The dependency of ventilation on central chemoreceptor drive in sleep and anesthesia appears from many animal data. For example, while focal cooling of the rostral chemosensitive areas of goats resulted in complete apnea during anesthesia, during wakefulness a ventilatory depression of only 30% was induced. This effect tended to be somewhat greater during NREM sleep [185,207,211,212]. The crucial importance of central chemoreceptor drive during sleep is also illustrated in patients suffering from the congenital central hypoventilation syndrome (CCHS). Generally, these patients do not show appreciable sensitivity to CO2 during wakefulness and sleep. However, when they are aroused, exercise or execute cognitive tasks, most of them breathe adequately and maintain relatively normal blood gases [213]. When they are asleep, however, they may severely hypoventilate, resulting in high PCO2 levels [213,214; see also Chapter 9 by Gozal, this volume] (note that some CCHS patients also hypoventilate when awake; [214]). Hypercapniainduced arousal is another interesting phenomenon in CCHS patients lacking ventilatory CO2 sensitivity [215]. Could this be mediated by a subset of the many neurons in the brainstem activated during hypercapnia? Note that the picture of these neurons in the brain activated by CO2 is incomplete. Recent fMRI data indicate that hypercapnic challenges elicit discrete changes in activity in multiple brain sites including diencephalic and (sub)cortical structures [216]. Many of these regions are also activated in CCHS patients, but some pontomedullary, cerebellar and (sub)cortical regions showed a reduced hypercapnia-induced activation [217]. More illustrative evidence for the importance of central chemical drive during sleep (or the importance of wakefulness drive when awake) comes from patients with unilateral focal lesions in the rostrolateral medulla. During wakefulness, most of the nine patients studied by Morell et al. [218] maintained a normal PCO2 during rest and exercise, but had a lower than


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normal CO2 sensitivity. All patients, however, displayed a disturbed and fragmented sleep pattern, often accompanied by obstructive apneas [218,219]. D. Central Chemosensitive Areas Throughout the Brainstem: Different Functions for Distinct Areas?

An intriguing property of the central chemoreceptors is their widespread distribution throughout the brainstem. It was suggested that this distribution may have evolutionary grounds based on gradual development of functions associated with the transition from water- to air-breathing, temperature regulation and sleep [124]. That so many neurons are activated by hypercapnia (data from Fos studies) is not very surprising, because apart from respiratory responses, CO2 also elicits cardiovascular, endocrine and thermoregulatory reactions. Consequently, not all brainstem neurons that stain for Fos upon hypercapnic exposure will be respiratory CO 2 chemoreceptors. On the other hand, it is relevant to consider that at least in the medulla, all chemosensitive areas described with the aid of focal stimulations lie well within the regions containing Fos-positive neurons. Suppose then that all these distinct areas serve to influence respiratory output during hypercapnia (which, given the invariable respiratory effects of focal lesions and stimulations seems a reasonable assumption, at least in anesthetized animals), what would be the advantage of their distant locations? It is evident that by this organization various specific respiratory adaptations can be effectuated simultaneously, because with their own efferent connections, all areas involved have distinct specific functions in controlling the extremely complex breathing apparatus. Of utmost importance is also the fact that all the chemosensitive areas have different afferent connections, some of which are clearly under the influence of the arousal state [84]. This then could have the consequence that some areas behave differently in different arousal states, and indeed this appears to be the case. A first example is the RVLM. The different effects of superficial RVLM cooling in the goat during anesthesia and wakefulness may be caused by a decreased tonic input of chemosensitive neurons to the respiratory controller during wakefulness. This could be due to inhibitory influences from the cortex on RTN neurons, which of course does not preclude a simultaneous powerful facilitatory influence of the cortex on respiratory neurons. It is also interesting to speculate about a possible contribution to the observed phenomena of the locus coeruleus with its extensive noradrenergic projections throughout the brainstem [220]. Locus Coeruleus (LC) neurons are activated during hypercapnia in awake but not anesthetized rats [84,96]. Do noradrenergic projections from the (chemosensitive) LC influence the excitability of RVLM neurons, and if yes, in

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NTS −11.00 VII

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Figure 2.7 CO2 microdialysis in the retrotrapezoid nucleus (RTN, left panel) and ventral medullary raphe (right panel) in awake and sleeping rats. CO2 has stimulatory effects (increase in tidal volume) in the RTN during wakefulness but not sleep. In raphe the opposite occurs: stimulation (increase in breathing frequency) during sleep but not while awake. NTS: nucleus tractus solitarius; VII: motor nucleus of the facial nerve (Data from Ref. 227).

what sense? (For a review of central noradrenergic effects on breathing, see [221].) An alternative explanation for the above findings in goats could be a smaller relative contribution of area S-M to total central chemical drive during wakefulness, so that removal of a given amount of their output would have a smaller effect on ventilation than during anesthesia or sleep. A surprising finding in rats (in the light of the above results of cooling in goats) was that microdialysis of CO2 in the RTN increased breathing in awake but not sleeping animals (Figure 2.7, left panel; [63,64]). In anesthetized animals, however, focal CO2 in the RTN increased phrenic activity [61]. At first sight these results in rats and goats seem hard to reconcile, but they may not necessarily prove to be conflicting. Some crucial questions to be answered here are: 1) What is the effect of sleep vs. anesthesia on the CO2 sensitivity of RTN neurons? 2) Do sleep and anesthesia alter the sensitivity of respiratory neurons to input from the RTN


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and other chemosensitive regions? 3) What are the effects of sleep and anesthesia further downstream in the ventilatory control system? The stimulatory effect of focal CO2 in the RTN in awake but not sleeping animals could offer one of the possible explanations for a generally observed higher CO2 sensitivity during wakefulness when compared with sleep. Another example of a chemosensitive area with a possibly specific role in respiratory regulation is the ventral medullary raphe region with a high concentration of serotonergic chemosensitive neurons. In the rat, microdialysis of CO2 in this region increases breathing frequency in sleep but not wakefulness (Figure 2.7, right panel; [62]). During NREM sleep, an increased GABA-ergic input into serotonergic raphe neurons may be responsible for their reduced activity in this state [222,223]. Assuming that this GABA-ergic input leads to hyperpolarization, this may lead to a situation in which the conductance of ion channels in the membrane of these neurons is changed (hyperpolarization, apart from probably direct effect of GABA on specific ionic conductances). If CO2/Hþ would act on raphe neurons by a direct effect on voltage-dependent ion channels that open when the membrane hyperpolarizes, this could lead to a situation in which a CO2/ Hþ-induced effect either could get lost or rather arise following a membrane hyperpolarization. Kir channels could be a candidate for such channels because they open with hyperpolarization [37], may be very pH sensitive [168], and may close upon a decrease in pH which would restore the membrane potential to the level existing without the GABA-ergic input. Various facilitatory inputs that impinge on raphe neurons during wakefulness could be filtered away during sleep, thus allowing CO2 to increase their output. Theoretically, for raphe neurons it seems to make sense to increase their output upon a rise in PCO2 during sleep. By providing a facilitatory input to hypoglossal motoneurons, serotonergic raphe neurons play a role in regulating upper airway patency [224]. During sleep, a decrease in activity of these cells may eventualy result in reduced genioglossal activity [225]. In man, upper airway mechanoreceptors are much less active in NREM sleep than when awake, with a low genioglossal response to large inspiratory resistive loads [226]. By increasing their CO2 sensitivity during sleep, raphe neurons could thus help to prevent, reduce or reverse obstruction of the upper airways. By increasing respiratory frequency in response to CO2 during sleep [62,227], raphe neurons could also reduce the negative airway pressure per breath that is needed to achieve a given level of alveolar ventilation.

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3 Suprapontine Control of Breathing

SHAKEEB H. MOOSAVI

DAVID PAYDARFAR

Imperial College of London Faculty of Medicine, NHLI London, UK

University of Massachusetts Medical School Worcester, Massachusetts

STEVEN A. SHEA Harvard Medical School Division of Sleep Medicine Medical Chronobiology Program Brigham and Women’s Hospital Boston, Massachusetts

I.

Introduction

The current chapter focuses on the functional importance and neuroanatomical basis of suprapontine control of breathing in humans, which complements the pharmacological and pathological perspectives covered in other sections of this book. For this purpose, suprapontine influences are divided into three broad categories: (1) volitional control; (2) involuntary influences associated with emotions and psychological disturbances, and (3) tonic drives including excitatory drives associated with wakefulness. Recent reviews in suprapontine respiratory control focus primarily on functional aspects [1–3], or primarily on the underlying neural substrate [4–6]. This chapter updates these reviews with particular regard to recent studies in humans. We focus more on the second category (involuntary influences), as this has never been reviewed extensively. We have also highlighted two key issues that continue to guide research in the field: (1) interaction between suprapontine- and brainstem-based mechanisms that influence respiratory pump activity, and (2) capacity for learning and adaptation in respiratory control. 71

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Definitions and Terminology

Suprapontine control of breathing refers to any influence on the amount or pattern of breathing resulting from neural drives to the respiratory pump muscles that originate in areas of the brain rostral to the pons. Suprapontine control of respiratory muscle activity does not primarily serve metabolic needs and is anatomically distinct from the automatic respiratory controller in the bulbopontine region of the brainstem (selfsustaining respiratory pattern generating neural circuits that automatically adapt to changes in arterial blood gases, thereby subserving metabolic homeostasis). It is convenient to use the functional term behavioral control as a synonym for the anatomical term suprapontine control (and this is distinct from the functional term automatic control which is used as a synonym for the anatomically defined brainstem respiratory controller). In reality, the functional/anatomical distinction is not absolutely precise [7] as some forms of rudimentary behaviors can be elicited by both suprapontine and brainstem sites [8]. These include protective maneuvers and adjustments to breathing necessary to coordinate gustatory brainstem reflexes, which can also be initiated volitionally (e.g., coughing in the absence of irritation) or modulated through reticular mechanisms of arousal [9]. Other synonyms used for the term ‘suprapontine’ include: forebrain, higher-center, non-metabolic, non-automatic and volitional. However, in this chapter we only use the term ‘volitional control’ in regard to ‘willful’ control. III.

Volitional Control

Adult humans can willfully breathe considerably more than is necessary to meet resting metabolic demands. When breathing as fast and deep as possible, ventilation can increase to as much as 40 times resting ventilation. Full recruitment of all inspiratory motor units is possible through willful control [10]. Breathing also can be completely interrupted by willful suppression. When breath-holding from Functional Residual Capacity (FRC), PaO2 will drop by as much as 50 torr and PaCO2 will rise by about 10 torr within 35 sec of breathing cessation [11]. Increased chemoreceptor afferent activity, reflex brainstem respiratory drive and a sensation of an urge to breathe will irresistibly override the voluntary suppression of respiratory muscle activity beyond this time. Starting the breath-hold at a higher lung volume [12], breathing elevated FiO2, hyperventilating beforehand, or practice will all increase breathhold durations by 2–3 min at best. The behavioral advantage conferred by the ability to completely override metabolic demands (even for only a short time) is obvious. Swallowing can occur, breathing muscles can

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be used to alter abdominal pressure necessary for defecation and parturition, noxious fumes can be avoided while moving to a safer environment, sprinters can achieve faster running speeds, and swimming underwater is possible. Why we are able to breathe willfully so much more than we need is less obvious. The maximum voluntary ventilation achievable is approximately 200 l/min, which provides a generous degree of metabolic freedom to accomplish most behaviors requiring willful control. Willful increases in breathing usually have little to do with blood gas and pH homeostasis and may instead incur additional metabolic cost [13]. However, in healthy subjects, ventilation would have to be as high as 140 l/min before any further increase results in a metabolic cost that exceeds the energy potential of the added oxygen transfer [14]. Maximum Voluntary Ventilation (MVV) can only be sustained for a short time (530 sec) before respiratory muscle fatigue sets in [15]. If isocapnia is maintained, as much as 70% of the MVV can be sustained for 4 min [16] and about 50%–60% of the initial MVV can be sustained as long as motivation continues [16,17]. As well as being able to increase and suppress breathing at will, we can also produce precisely guided breathing motion that simultaneously fulfills behavioral as well as metabolic needs. The ability to sing, speak or play musical instruments demonstrates a high degree of precision with which voluntary changes in the amount and pattern of breathing can be manifested. The respiratory pump is essentially a skeletal muscle system, as such willful contractions can be made with response times within 100 msec [18,19]. Volitional tracking of ventilation is as accurate as a control motor task (hand control in operating a joystick) when the rate of tracking is below 20 perturbations per minute [20,21]. In healthy subjects, this degree of control permits regulation of airway pressures to within 2 cm H2O, about 1% of that generated by maximal voluntary effort [22]. Willful control of breathing can become automated as with other rhythmic motor acts (e.g., walking) [23]. Control of breathing for the purpose of speech production exemplifies this form of subconscious suprapontine control. Both metabolic and behavioral needs are usually met without the need to consciously attend to breathing. In a healthy individual, the majority of breaths during wakefulness occur without awareness. However, awareness of breathing increases in the presence of an obstruction to breathing (e.g., nasal congestion); when metabolic demands increase (e.g., heavy exercise); if gas exchange is compromised (e.g., cardiopulmonary disease), or if attempts are made to measure breathing in the laboratory environment. This awareness can itself influence breathing, presenting a substantial confounding factor in experimental procedures. Minimizing the influence requires careful control of measurement procedures, for example avoiding the use of breathing apparatus that can direct attention towards breathing [3].

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There are conflicting messages in the literature about the role of voluntary drives to breathe in patients with severe lung disease. Early studies led to the proposal that patients with increased inspiratory load suffer respiratory failure when the energy consumption of respiratory muscles reaches a critical value, triggering muscle fatigue [24]. Other evidence suggested respiratory failure was due to blunting of voluntary or automatic drives to breathe, perhaps to compensate for or delay the onset of respiratory muscle fatigue [25]. More recent studies paint a somewhat different picture. The ability to track perturbations in ventilation, as compared with the ability to track perturbations in a hand control task, is not compromised in patients with COPD [26]. Healthy subjects fail to breathe to target mouth pressures against inspiratory resistive loads because of dyspnea associated with carbon dioxide retention [10,27,28]. Task failure was not associated with reduction in twitch amplitude of the diaphragm in response to bilateral phrenic nerve stimulation [10,27], whereas earlier studies had assumed that voluntary cessation of the breathing task (task failure) was indicative of respiratory muscle fatigue. Changes in diaphragmatic activity during transient maximal inspiratory pressure maneuvers indicate that the ability to drive breathing volitionally is actually increased in COPD patients with chronic hypercapnia [29,30]. A shift towards volitional drive during loaded breathing may confer some advantage, since volitional control can fully activate all motoneurons to inspiratory muscles whereas automatic control may not be capable of optimal recruitment during maximal reflex stimulation of the bulbopontine controller by chemoreceptor afferents [10]. Voluntary alterations in airflow profile (e.g., slowed expiration) or in end-expiratory lung volume may also increase gas exchange capability. Life without the ability to exercise volitional control over the respiratory apparatus is dramatically illustrated by a rare clinical condition referred to as the locked-in syndrome. A discrete lesion of the corticospinal pathway in the ventral pons [31] characterizes the condition. The usual level of irregularity of the respiratory rhythm during wakefulness is lost (the breathing pattern when awake becomes more like that normally seen in deep non-rapid eye movement sleep) and a reduction in PCO2 of as little as 1 mmHg from normocapnia results in apnea [32], demonstrating regulation of breathing solely for metabolic demands. The decision to take a breath or expire volitionally has its neural origin in the motor cortex. The earliest recognition of this was during neurosurgery in patients under local anesthesia; stimulation of a focal site in the primary motor cortex close to the vertex resulted in contraction of the diaphragm producing a hiccough [33]. The introduction of transcranial electrical stimulation [34] made it possible to demonstrate pathways from the cortex to the diaphragm in normal man [35]. Later, the use of Transcranial Magnetic Stimulation (TMS) [36] confirmed


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that the optimal site of stimulation was a few centimeters anterior and lateral to the vertex [37] at a site consistent with the observations of Foerster [33]. Studies employing Positron Emission Tomography (PET) to detect changes in regional Cerebral Blood Flow (rCBF) (as an index of neuronal activity), have identified specific foci of increased rCBF in the primary motor cortex, the premotor area, the supplementary motor area and the cerebellum during volitional breathing [38–40]. More recent studies employing functional Magnetic Resonance Imaging (fMRI) have verified these activation sites and identified additional cortical and sub-cortical regions that may be involved in mediating a conscious decision to breathe (inferolateral sensorimotor cortex, striatum, thalamus, globus pallidum, lentiform and caudate nuclei, and medulla) [41–43]. Consistency in cerebral activation sites between studies from independent labs and between studies using different imaging techniques strengthens the findings. However, there is uncertainty about the precise role played by individual foci of activations in the programming and execution of voluntary breathing. What is becoming clear is that a distributed neural network is necessary, the extent of which will depend on the degree of learning, planning, attention and concentration involved in the task. The pattern of cortical/subcortical neural activations associated with voluntary breathing resembles that which would be seen with volitional movements in general. The sensitivity and temporal resolution of fMRI now appears to be capable of distinguishing between clusters of activations within the superior motor cortex that are associated with diaphragmatic contraction (anterior) and clusters associated with thoracic muscle contractions (posterior) [43]. Clinical evidence suggests that volitional control involves corticospinal and/or corticobulbar pathways [44]. Studies using TMS to investigate volitional activation of breathing muscles in patients with cerebral ischemia have shown that the cortico-respiratory projections are located in the pyramidal tracts [45] (see also Section VI). IV.

Involuntary Emotional Influences

The study of breathing in relation to emotions spans more than a century and has traditionally been the preserve of psychologists and psychoanalysts (for a thorough and critical review of the extensive body of work see [46]). Our aim in this section is to highlight key observations underlying current notions regarding the functional role of emotion-related breathing modifications. For convenience, we have separated breathing behaviors associated with centrally generated emotions (i.e., psychological factors including depression and anxiety, and higher mental functions such as

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thoughts and memory), from those resulting from the emotional consequences on breathing of ascending sensory afferent information (respiratory and non-respiratory). We recognize that this distinction is somewhat arbitrary since perception of sensory afferent information is likely to influence breathing through psychological disturbance, and conversely, psychological disturbance may modulate perceptual sensitivity [47]. Chemoreceptor and mechanoreceptor afferent activity reaches the suprapontine brain with information about respiration, either directly or via the automatic respiratory controller in the brainstem. These signals provide information about motion, position, local irritation of the respiratory apparatus, blood gas tensions, and sensations of respiratory discomfort (dyspnea) [48]. In addition, a constant barrage of nonrespiratory information is available including sound, sight, smell, taste and pain. Automatic reflex responses can be triggered by direct projection of respiratory (and non-respiratory) sensory afferent activity to structures within the brainstem. For instance, local irritation of the upper airways can trigger a cough to clear the airways. A non-respiratory example is a sudden loud noise, which may elicit a sharp intake of breath. Such reflex behaviors fall outside our definition of suprapontine control; reviews of this form of behavioral control can be found elsewhere (e.g., [8]). We limit our discussion in this section to volitional or emotional influences on respiratory muscle activity that are consequent to psychological state or perceptual processing of sensory information in higher centers. Sensory afferent information may also be involved in maintaining the wakefulness drive to breathe (Section V). A. Psychological Influences

Many emotions are expressed by changes in breathing; for example, laughter normally expresses happiness and sobbing expresses sadness. Less obvious changes can occur; for example, deception is associated with significantly shorter breaths preceding a dishonest response when compared with those preceding an honest response [49]. Various methods have been used to evoke specific emotions in healthy subjects including: self-induction by recall of previously experienced emotions [50], suggestion or hypnosis [51,52], cued presentations of affect-laden words or pictures [53,54] and introduction of artificially created threatening situations [55,56]. In addition, psychiatric assessments of subjective responses to interview have been used to correlate breathing patterns with differing thoughts or moods in the clinical setting [57,58]. Reported breathing modifications include changes in amount or pattern of breathing (see [46] for the various patterns of change reported), changes in the ratio of inspiratory to expiratory durations [50], changes in within-breath parameters [59], changes


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in coordination between ribcage and abdominal motion [60], and irregularities of breathing [61]. Are specific breathing patterns associated with specific emotions? This is a central question raised in the earliest studies, and remains a key question. Two early studies generated interest in a specificity model of emotion-breathing association. Changes in breathing pattern tended to be characteristic of the specific self-induced emotion when subjects were asked to revive thoughts of pleasure, pain, anger, disgust, wonder, fear, laughter or hatred [50], or when subjects were asked to revive joy-laughter, sadnesscrying, anger, fear, erotic arousal and tenderness [62]. While a specificity model cannot be discounted [63], other models of association that relate breathing behaviors to groups of emotions based on different affective dimensions (e.g., pleasant-unpleasant, calm-excited, active-passive) or different response requirements (e.g., fight-flight or passive-active coping) may be more relevant but hard to distinguish [46]. In a study that fits with a dimension model, healthy female subjects asked to generate internal thoughts in response to cued presentations of words, responded with increased respiratory frequency when the affective content of the words was stressful, and responded with decreased respiratory frequency when the affective content was relaxing (relative to neutral words) [53]. Other observations fit better with a response requirement model. In one such study, psychotherapeutic interviews of asthmatic subjects were divided into segments that were judged to be indicative of different emotions based on independent psychiatric assessments. The emotions identified were subsequently divided into four different clusters: neutral, distress, giving-up and arousal. Different patterns of breathing modifications emerged for different emotion clusters as well as with personality differences among individuals [57]. Specific breathing behaviors may also distinguish different psychological derangements. For instance, schizophrenic patients breathe more rapidly and shallowly than either control subjects or melancholic patients [65]. More recently, there is a great deal of interest in the nature of breathing associated with anxiety disorders. If patients with hyperventilation syndrome are asked to recall stressful situations specific to their experience, pronounced hyperventilation can be elicited—this think test has been shown to be more effective in identifying hyperventilators than forced hyperventilation provocation tests alone [65]. Hyperventilation is also a feature of a panic attack that characterizes panic disorder. Breathing is more irregular with greater numbers of sighs and an inappropriate tonic hypocapnia in patients with panic disorder when compared with controls [66–68]. Breathing abnormalities in panic disorder patients are now believed to be due, not to an inherently abnormal respiratory controller, but rather to a hypersensitive fear network comprising the amygdala, its projections to the brainstem, the hippocampus and the medial prefrontal

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cortex [69]. It will not be easy to determine the exact pathways involved in panic disorder in the claustrophobic environment of an MRI or PET scanner. It is also notable that the areas activated by anxiety are close or overlapping with areas activated by the perception of a number of different sensations (pain, nausea, air hunger); anxiety may be an element of unpleasant perceptions that is experimentally inextricable. Whether emotional influences on breathing are characteristic of specific emotions, or of clusters of emotions, is only half the story. In terms of functional significance, we must also consider what role such associations might serve. The functional role of psychological influences on breathing is not known. The changes in breathing associated with psychological factors usually occur without awareness and are unrelated to metabolic requirements. Expression of emotions can greatly reduce the ability of the respiratory apparatus to participate in its metabolic role. For instance, fits of laughter produce substantial reductions in lung volume and increase in dynamic airway compression [70]. However, breathing adjustments are not always counterproductive. It is possible that the effects are not simply an epiphenomenon of psychological state but serve some physiological, behavioral or adaptive purpose. Sighs and yawns counter atelectasis of the lungs; such recruitment maneuvers may be especially beneficial in the presence of pulmonary disease [71]. In certain stressful situations, slowing breathing can alleviate anxiety [72]. Subjects consciously performing physical relaxation techniques produce more coordinated ribcage and abdomen motion [60]. Attention and motivation are likely to be necessary for behavioral adaptation of volitional or reflex breathing responses. For instance, attention or motivation can substantially prolong breathholds [73]—being able to breath-hold for longer is a distinct advantage in some circumstances. There is much speculation about the broader impact of emotional influences on breathing, which remains largely unexplored and unsupported by experimental evidence. One possibility is that the changes in breathing brought about by psychological factors are inherently involved in the establishment and self-regulation of emotions. This idea stems from an early theory that physical changes play an important role in formation and experience of emotions [74]. Accordingly, breathing, among other physiological activities, may play a vital role in forming negative emotions that ensure avoidance of harmful situations, thereby contributing to evolutionary survival. Breathing adjustments by psychological factors may also represent a subconscious primeval form of communication; yawns and sighs may communicate boredom and melancholy, but may also indicate contentment [75], and are highly contagious [76]. Interestingly, yawn contagion appears to be found exclusively in humans [77]. What are the cerebral correlates of emotion perception in humans? Within the last decade, a vast literature has built up on this subject


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based on functional neuroimaging, electrophysiological and lesion studies (see [78] for a critical review). In general, there appears to be two neural networks. The emotional impact of a stimulus and its behavioral consequences are processed within a ventral neural network (comprising the amygdala, insula, ventral striatum, and ventral regions of the anterior cingulate and prefrontal cortex). Regulation of the behavioral responses is thought to be the predominant function of the dorsal neural network (comprising dorsal regions of the anterior cingulate gyrus and the prefrontal cortex). What do we know about the descending pathways with respect to emotion-related breathing behavior? Emotion-related breathing behavior can be expressed through involuntary control with no awareness of breathing changes. There is some evidence that this form of influence on the respiratory pump muscles can occur through descending suprapontine pathways that are separate from volitional suprapontine control pathways. For example, there are changes in breathing associated with a display of emotion in patients who have lost volitional control of breathing due to a lesion of the corticospinal tract [31]. Several involuntary motor patterns involving respiratory muscles have been observed in these patients including whining, moaning, sighing and yawning [79], but there is still much to be learned from these unfortunate patients. For instance, it is not yet known if they can learn to use the emotional pathway to enable voluntary activation of the respiratory muscles despite obstruction of corticospinal/corticobulbar pathways. B. Perception of Respiratory Discomfort

Respiratory sensations are always available to the forebrain to direct motor activity; these sensations and the consequent conscious or subconscious suprapontine influences upon breathing may be important in shaping much of our breathing behavior while we are awake. A broader discussion of the influence of respiratory sensations on breathing can be found in earlier volumes in this series [80,81]. Here we have focused specifically on perceptions of respiratory discomfort since much progress has been made in recent years with regard to identifying separable qualities of respiratory discomfort and their most likely afferent sources. Clearly, the framework of perceptual mechanisms for unpleasant breathing sensations has grown in complexity. Physicians refer to sensations of respiratory discomfort as dyspnea (though some use the term in its literal sense, meaning difficult and labored breathing). Patients commonly report dyspnea as shortness of breath or breathlessness. At least three separate qualities of respiratory discomfort can be perceived by patients and healthy subjects: air hunger, effort or work and tightness. This classification is based on studies that analyzed

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the quality of unpleasant sensations evoked by different respiratory stimuli in normal subjects [82,83] or by different clinical conditions in patients [84,85]. Air hunger is readily recognizable as the unpleasant, uncomfortable, imperative urge to breathe at the culmination of a long breath-hold. The sensation of breathing effort or work arises when ventilation requires excessive respiratory muscle activity, e.g., due to fatigue or increased impedance to inspiration [86–89]. The sense of chest tightness is usually associated with episodes of bronchoconstriction [84,90,91]. The various forms of respiratory discomfort coexist in the clinical arena. The relative contribution of each to the overall perception will depend on the underlying pathology. Laboratory evidence shows that these distinguishable sensations arise from different afferent sources [86,87,90,91]. Intense dyspnea can elicit emotional disturbance leading to behavioral adaptations. Dyspnea is a powerful symptom of disease that motivates patients to seek medical attention and to adopt other non-specific changes in lifestyle. Modification of breathing behavior can result in a number of ways. Psychological factors can subconsciously influence breathing behavior directly. As discussed in the previous section, the purpose of such influences is uncertain in some situations, changes may alleviate the perception or the underlying breathing difficulty while in others they may be counterproductive. Dyspnea perception may also directly influence volitional drives to breathe or influence drives to other muscle systems that indirectly influence breathing behavior. These forms of modulation may serve to minimize unpleasant breathing sensations [92]. The impact of dyspnea on breathing behavior will depend on the degree to which the perception is affect-laden. Since air hunger is a particularly unpleasant form of dyspnea, one might expect that this form of dyspnea would more readily influence breathing. Normal subjects and patients with COPD are able to distinguish between the intensity of dyspnea and the distress or anxiety associated with it [93,94]. However, it is yet to be demonstrated that the relationship between unpleasantness and intensity differs among the various qualities of dyspnea, as has been shown for different qualities of pain perception [95]. It is very likely that dyspnea perception will likewise be found to be multi-dimensional in nature [96]. What evidence is there that perception of respiratory discomfort directly influences volitional drives to breathe? It makes intuitive sense that conscious awareness of unpleasant breathing sensations leads individuals to access volitional control pathways in an attempt to minimize the sensation. Based on a growing body of evidence, air hunger depends on excitatory ascending projections of efferent respiratory drives from the brainstem (that report prevailing ventilatory demand) modulated by inhibitory afferent information from pulmonary mechanoreceptors (that report the present level of ventilation). Thus, substituting part of the reflex ventilatory demand from the brainstem with volitional drive may serve to alleviate the urge


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to breathe. It has been proposed that the ventilatory response to hypercapnia may involve a cortical facilitatory component aimed at reducing the urge to breathe that accompanies hypercapnia [97]. This was later challenged by the observation that the minimum ventilation required to alleviate the air hunger of hypercapnia was less than the spontaneous breathing during hypercapnia [98]. Brain imaging studies aimed at elucidating the cerebral activations associated with CO2-stimulated breathing in awake man provide conflicting evidence with regard to the contention that hypercapnia induces cortical facilitation of breathing. Those based on PET studies have found no evidence of primary motor cortex activation [99] in areas previously shown to activate with volitional breathing [39,40]. Other studies based on fMRI techniques suggest that motor cortical activation does accompany the ventilatory response to hypercapnia [100]. Although in some circumstances it would make sense to voluntarily activate breathing muscles for the purpose of minimizing unpleasant respiratory sensations (e.g., terminating a prolonged breath-hold if it is safe to do so), it would be counterproductive in other situations. For instance, a diver must continue to hold his/her breath, despite tremendous metabolic drive, until the swim back to the surface is complete. The decision to begin the swim back to the surface must be made well in advance of intolerable air hunger. Voluntary activation of breathing muscles would also be counterproductive when the ventilatory apparatus is already compromised by disease. This could generate more unpleasant sensations, thereby setting up a positive feedback situation. Thus, it is possible that the perception of air hunger evolved not to directly influence breathing but to generate more general adaptive behaviors serving to distance the individual from situations that threaten respiratory function. A number of behavioral responses exhibited by patients and healthy subjects that may represent subconsciously learned strategies to avoid or minimize unpleasant breathing sensations are discussed in Section VII. Several patient groups have been identified in whom inappropriate perceptions of respiratory discomfort are apparent. An inappropriately high sensitivity of air hunger is a very common symptom in hyperventilation syndrome [101,102], and it is possible that this sensation is itself the trigger for psychogenic hyperventilation. Hyperventilation syndrome may develop as a vicious circle between breathing response and the negative emotional content of symptoms associated with it [103,104]. Increased dyspnea sensitivity to hypercapnia is also apparent in panic disorder; it is uncertain whether this is integral to an inappropriate fear response [69]. At the other end of the spectrum, patients with blunted perception of dyspnea (so-called poor perceivers) may account for many asthma fatalities [105]. A recent study has also reported blunted perception of dyspnea in stroke patients [106]. Depression in chronic fatigue syndrome can increase the effect of work or effort of breathing associated with resistive loading [47] and may

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impair voluntary activation of the diaphragm in asthmatics, making them more vulnerable to ventilatory failure [107]. What are the cerebral correlates of dyspnea perception? Three PET studies [108–110] and one fMRI study [111] have recently mapped brain activations during dyspnea induced in normal subjects. Principal activation was in the insula (extending to the operculum), cerebellum, and the anterior cingulate (other activations included thalamus and SMA). Activation of the anterior insular cortex was common in all of these studies, raising the possibility that the insula is essential for dyspnea perception. The insular cortex is a para-limbic structure. One of its many roles is thought to be formulating behaviors and learning visceral homeostasis [112]. Electrophysiological and tracer studies in animals indicate that it receives afferents from respiratory chemoreceptors, pulmonary receptors and medullary respiratory neurons [113–115]. The insula is activated in imaging studies of other unpleasant visceral sensations [116,117], including pain [118]. What are the different afferent sources of dyspnea perception? The sensation of air hunger (e.g., during blood gas derangements) most likely arises from perception of respiratory motor drive from the brainstem [86,119], a corollary copy of which is transmitted to the cerebral cortex [120,121]. In addition, a direct projection of chemoreceptor afferents cannot be discounted. Respiratory muscle activity is not involved in generating air hunger, since complete neuromuscular blockade does not diminish this sensation [122,123]. Nonetheless, afferent information from respiratory muscle activity and other mechanoreceptor afferent information reporting the level of pulmonary ventilation are known to relieve dyspnea [124,125]. Evidence points to vagal afferent information from pulmonary receptors, activated by bronchospasm or inflammation of the airways, as the most likely source of tightness [90]. The sense of breathing effort or work is thought to arise from a combination of respiratory muscle afferent activity and from corollary discharge of central neural motor drives to the respiratory muscles [86,89]. In contrast to air hunger, pulmonary afferent information is not thought to have a role in relieving sensations of breathing effort or work. C. Perception of Music

Anticipation of experimental procedures [126,127] and states of anxiety [128] can give rise to increased ventilation and/or irregularity of breathing patterns. Respiratory studies on human subjects therefore often employ music or other auditory stimuli (such as white noise or talking stories) in order to distract subjects from the test stimuli or more generally to relax naive subjects or patients who may be anxious. However, a number of studies have shown that auditory stimulation may itself introduce a source of ‘‘behavioral noise’’ that can affect the amplitude and cycle period of


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respiration. Poole et al. [129] demonstrated systematic effects of different tone bursts on the breathing cycle and suggested that such effects could form the basis of objective hearing tests in children or patients with psychological pathologies. Early studies by Skaggs [130] employed novel forms of auditory stimulation (a car horn and an empty can dropped on the floor) to demonstrate, not surprisingly, sudden reactive inspiratory movements. Other studies [131,132] investigated these effects using transient presentations of white noise and showed that the respiratory cycle during which they were presented, as well as subsequent cycles, were affected. Since the effects decayed with repeat presentations, this supports the view that they represent orienting or defensive reflexes to which the subjects habituate [131]. Nonetheless, continued white noise over many respiratory cycles at a comfortable auditory level systematically increases breathing frequency, perhaps via an arousing influence [133]. Indeed, direct stimulation of the reticular activating system may represent a general arousal mechanism through a change in the contribution of a tonic wakefulness drive on respiratory control (see Section V). The above forms of direct stimulation may bear a teleological significance in eliciting fight/flight reflex responses. The effects of musical or rhythmical auditory stimulation over many respiratory cycles have also been reported [134,135]. The close anatomical proximity of auditory nuclei to respiratory neural networks in the brainstem and the abundance of collaterals and interneurons in the auditory pathway make it likely that some form of direct stimulation of the automatic respiratory controller is possible when subjects attend to musical auditory inputs. Haas et al. proposed that music rhythm acts as an external pacemaker (‘zeitgeber’) that ‘modulates’ respiratory timing [135] by impinging on the respiratory central pattern generator (CPG) and entraining breathing. In support of this hypothesis, these authors were able to demonstrate a reduced coefficient of variation in breath period when subjects listened to music as well as to demonstrate integer ratios between beat period (of a metronome or of various musical segments) and breath period; they also demonstrated a significant respiratory phase coupling in 12 out of 20 subjects (half of whom were musically trained). The study by Harrer [134] is often cited as a particularly dramatic example of behavioral modulation of breathing (e.g., cited in [2]). This study demonstrated that, in a human subject listening to a melodic excerpt of music by Chopin, respiratory volume became markedly irregular when the music was suddenly replaced by the dissonant atonal electronic music of Stockhausen. However, we are not aware of any other experimental verification of this single observation. Music (especially classical music) is often touted as a ‘relaxant’ or ‘mood enhancer.’ Music has been shown to reduce anxiety and respiratory rates in women awaiting surgery [136] and reduces spontaneous breathing efforts in ventilator-dependent patients [137]. Functional neuroimaging

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studies suggest that the perceptual processing of the pleasantness or unpleasantness of music involves the paralimbic and neocortical regions [138]. V.

Tonic Excitatory and Inhibitory Drives

Sections III and IV indicate that during the awake state there are at any instance a great many potential influences on breathing. A recurring notion that would explain the subconsciously increased breathing in many different behavioral situations in awake subjects is that the increased breathing is a by-product of arousal. Fink [139] coined the term the wakefulness drive to breathe. In his study, thirteen subjects underwent passive (mechanically ventilated) and active hyperventilation to reduce PaCO2 and increase PaO2. Hence, any ‘chemical’ drive to breathe was absent at the end of either of these periods. But, it was found that rhythmic breathing continued at the normal frequency in all subjects who remained awake. What was keeping the respiratory rhythm going? Fink observed that one of the subjects stopped breathing when he became drowsy. This momentary response in the drowsy subject is the same as occurs in humans under general anesthesia or sleep wherein apnea is elicited consistently by hypocapnia (e.g., [140– 143]). Fink concluded that cerebral activity associated with wakefulness is a component of the normal respiratory drive. However, it has never been proven that it is cerebral activity per se which enhances breathing, and it could represent activity in many of the brainstem and basal forebrain areas that are associated with arousal. For instance, the loosely defined reticular activating system, which embeds the brainstem respiratory complex, can provide a stimulatory effect upon certain brainstem respiratory-related neurons [144,145]. There are other more specific arousal related neurons throughout the brainstem and basal forebrain [146] and it is possible that these stimulate breathing directly. Absence of this tonic drive to breathe at sleep onset can cause hypoventilation and even central apnea (this appears more likely when sleep transitions are rapid). For a comprehensive review of this area, see Shea [3]. In slow-wave sleep, there is a consistent reduction in ventilation, associated with an increase of several mmHg in the arterial PCO2 [147]. Medullary and pontine inspiratory related neurons show a significant decrease in activity [148]. This phenomenon is essentially independent of influences from peripheral sensory input; extensive peripheral denervation does not abolish these sleep-related decrements in the firing of respiratoryrelated neurons. It has been suggested that this decrease in respiratory activity probably represents loss of tonic excitatory activity associated with wakefulness [149]. Support for this idea in humans comes from a study of breathing during ventilator-induced hypocapnia in normal subjects [141].


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During wakefulness, only brief periods of apnea were obtained. However, during sleep, at comparable levels of hypocapnia, apnea lasted longer than in awake subjects. Furthermore, the duration of apnea was longer in deeper stages of non-REM sleep. Hypoxia could shorten the duration of apnea. These and other studies conclusively demonstrate that in slow-wave sleep, breathing is critically dependent upon arterial PCO2 and PO2. Breathing during REM sleep is much more variable and less influenced by chemical drives, and is thought to be essentially controled by the behavioral control system (reviewed in [3]). Therefore, derangements in the automatic or homeostatic control of respiration are manifested mainly during non-REM sleep and may not be obvious during wakefulness or REM sleep. A dramatic example of this is Congenital Central Hypoventilation Syndrome whereby patients breathe normally during wakefulness and REM sleep but seriously hypoventilate during non-REM sleep (see Chapter 9, this volume, and [150]). Animal studies in which different parts of the brain were electrically stimulated, showed that there were more regions providing an inhibitory influence on the automatic respiratory controller than excitatory influences [151]. Seizures are known to elicit prolonged apnea, especially when their focus is in the limbic areas [152]. Conversely, bilateral damage to the hemispheres can ‘release’ involuntary behavioral control of respiratory muscles that are normally inhibited. For example, pathological involuntary laughter or crying can occur if limbic projections to subcortical structures are impaired [7]. Such ‘disinhibition’ is commonly seen as a psychiatric consequence of traumatic brain injury. Cerebral infarcts are also known to result in increased ventilatory sensitivity to hypercapnia [153,154]. Existence of cortical inhibition of diencephalic structures is strongly supported by lesion experiments in animals. Thus the ventilatory response to hypoxia is unchanged by midcollicular section but dramatically enhanced by decortication [155]. The activity of the automatic controller in the brainstem is therefore influenced not only by tonic excitatory influences associated with wakefulness, but also by tonic inhibitory drives that provide cortical braking of ventilatory reflexes. Neuroanatomically, sources of tonic excitatory influences are located in subcortical structures such as the hypothalamus and reticular formation, whereas tonic inhibitory influences descend from more rostral limbic areas serving to modulate the excitatory influences or directly braking activity of the automatic controller in the brainstem. It has been proposed that tonic excitatory and tonic inhibitory influences on the metabolic controller exist in a state of balance that can be shifted in certain circumstances such as hyperthermia, chronic exposure to high altitude and when different doses of certain general anesthetics are given [4]. It has been suggested that cortical inhibition may be a remnant of a diving reflex [156]. Cerebral damping of metabolic reflexes may also play an important role

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in protecting against instability of respiratory control as in Cheyne Stokes breathing [153]. VI.

Interaction between ‘Behavioral’ and ‘Automatic’ Control

The many reasons for which we breathe can be broadly divided into those exclusively serving ‘metabolic’ needs and those primarily serving ‘behavioral’ needs. This functional hierarchy is partly reflected in an anatomical hierarchy. The existence of a separate automatic controller in the brainstem is supported by evidence from a large number of studies using different methodological approaches [157], including tissue ablation [155]. The behavioral system of control is less well defined and more widely distributed, extending from the brainstem to the highest areas of the brain [3,6,8]. The majority of sensory inputs and the final neural output to pump muscles are common to both brainstem and suprapontine control mechanisms. The respiratory pump apparatus is often described as unique among motor systems since two separate controllers drive it concomitantly. However, the uniqueness of the respiratory motor system may simply be that its automatic central pattern generator resides at a lower neural threshold. This viewpoint is indirectly supported by evidence from animal work [158] that implicates gating mechanisms capable of triggering automatic rhythmic muscle contractions for locomotion without cortical input. The neurological basis and extent to which the behavioral and metabolic controls of breathing interact were realized as key issues several decades ago [7,159]. The signal that underlies automatic/metabolic control of breathing is transmitted to spinal motoneurons via ventrolateral tracts in the spinal cord. There is clinical and experimental evidence that suprapontine drives are transmitted to the same group of motoneurons involved in automatic respiration, but via dorsolateral spinal cord pathways [160,161]. However, the pathways within and rostral to the brainstem are less certain. One possibility is that the voluntary signal originates in the contralateral cortex and bypasses the automatic control system in the brainstem. In this model, the integration of the voluntary and automatic signals occurs exclusively at the level of respiratory motoneurons in the spinal cord. However, it has been demonstrated in conscious animals that a learned voluntary respiratory task (abrupt termination of inspiration and prolongation of expiration) was associated with concomitant termination in brainstem inspiratory neurons and activation of some brainstem expiratory neurons [162]. These findings suggest that the voluntary signal may be integrated at least in part at the level of the automatic system. A recent fMRI study has reported the presence of medullary activity associated with voluntary breathing in humans [43], but could not exclude the possibility


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that this activity was related to sensory afferent input from the lungs or chest wall. There are many situations in which the behavioral and metabolic control systems have conflicting influences on breathing and must therefore compete for control over the respiratory pump. It has been suggested that during much of the awake state, breathing is under the exclusive control of suprapontine mechanisms [3,163]. The observation that the precision with which ventilation can be volitionally tracked is the same irrespective of whether a background of hypocapnia or one of eucapnia is maintained, is consistent with the notion that chemical control plays little or no part during resting breathing [21]. Thus, in the awake state, the relative effectiveness of regulating influences will vary depending on the imperativeness of behavioral needs as much as upon metabolic needs [139]. To what extent can volition dictate breathing before metabolic needs become imperative? Duration of maximum voluntary breathing efforts is less with inspiratory resistive loading [24,164] or elevations in lung volume [165]. If isocapnia is not artificially maintained, syncope could occur due to cerebral ischemia—a natural limitation to willful hyperventilation. The duration of breath holds also depends on lung volumes as well as upon blood gas levels. When metabolic demands are increased, as during exercise, the ability to exert willful control on breathing is further curtailed as exemplified by significant reduction in breath hold durations [166] and by a reduction in speech intelligibility [167]. Motivation, which in turn will depend on the level of attention or arousal, emotional state and other psychological factors, plays an important role in determining the degree to which the metabolic controller can be challenged. Thus, the duration of a breath hold will increase substantially in highly motivated situations such as competition. Elite synchronized swimmers can hold their breath for significantly longer durations than control subjects despite the fact that their hypercapnic ventilatory sensitivity is not different [168]. Presumably they can tolerate higher CO2 loads. Dejours [169] pointed out that during 100 m sprints, elite athletes might not breathe at all despite a tremendous metabolic burden, implying that all proposed automatic drives to breathe can be overridden by the cortex at least for a short time. Speech provides a good example of the competition between the behavioral and automatic respiratory control systems. The flow requirements for normal speech exceed resting ventilation, and so subjects hyperventilate when they speak at rest (e.g., [159,170]). However, as the automatic drive is increased, say by exercise, the subject will have some control of whether to maintain their speech quality at the expense of reduced breathing, or to increase the airflow during speech (thereby altering speech quality) to maintain their gas exchange. The first approach appears to predominate—for example, there were 73% reductions in the ventilatory responses to hypercapnia during speech [159,170], and a 55% reduction

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in the ventilatory response to exercise during speech [171]. A more recent study reports similar findings [172]. Nevertheless, at the extremes of automatic ventilatory drive, subjects may not be able to speak because of the high flows required for gas exchange. For example, during very heavy exercise only gasped phrases are possible [167,171]. It should be pointed out that in these studies, the air used for speech has already been used for gas exchange, so the behavioral and automatic respiratory drives are not completely mutually exclusive. However, in ventilator-dependent tracheostomized patients, there is complete antagonism between the airflow requirements for speech and gas exchange; in this situation, air is stolen from alveolar ventilation during the ventilator’s inflation phase to produce sound [173]; thus there is hypoventilation and relative hypercapnia during speech. During induced hypercapnia (15 mmHg increase in PETCO2 ) that caused shortness of breath, all subjects could still speak adequately. Two of five subjects adapted by reducing the air used for speech during inflation. In contrast, one subject reacted, as normal subjects do during hypercapnic speech, by increasing the airflow per syllable (a maladaptive strategy in ventilated subjects, because this causes relatively greater hypoventilation). These adaptive or maladaptive changes were modest despite the strong hypercapnic stimulus. No subject adapted fully, by consistently not speaking on the ventilator’s inflation phase. It was concluded that the behavioral drive to speak was modified but never fully suppressed by increased automatic respiratory drive. VII.

Learned Respiratory Behaviors

Behavioral strategies to avoid respiratory discomfort include stopping exercise, altering eating habits and speech patterns (and possibly language), some of which involve modulation of muscle activity not normally part of the breathing act. Compared with healthy subjects, patients with sarcoidosis, asthma, or emphysema adopt different breathing patterns during conversational speech, thereby avoiding shortness of breath [174]. Nasal congestion is associated with uncomfortable perceptions of resistance to airflow; even in healthy subjects, this alerts the individual to increase respiratory muscle force or to switch to mouth breathing. Patients with severe cardiopulmonary disease often adopt pursed-lips breathing, brace their arms, or elevate functional residual capacity with tonic inspiratory muscle contraction (e.g., [175,176]). Pursed-lips breathing improves some mechanical indices of lung function by promoting slower and deeper breathing [177–179]. Alleviation of dyspnea and improvements in arterial blood gases have been observed in COPD [179], but pursed-lips breathing may not relieve certain forms of dyspnea [177]. Arm bracing allows normal subjects to increase by a small amount the maximal ventilation they can


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sustain [180]; this may explain why athletes brace their arms on their knees at the end of a sprint to repay the oxygen debt. A more recent study has shown that the increased capacity to sustain hyperpnea cannot be explained by improved inspiratory muscle function that is in fact impaired by arm bracing [181]. Learned behavioral respiratory adaptations may be based upon the improvement in performance of a task. The precisely guided voluntary respiratory movements involved in singing or playing wind instruments are an example of learned respiratory adaptation motivated by a desire to improve performance outcome. Feedback of internal respiratory sensations (proprioceptive cues from the lungs and chest wall) as well as external sources of feedback (e.g., auditory) help to shape the complex pattern of efferent motor drives needed. Repetition and practice will reduce the need for mental concentration and attention to feedback, indicating a reduced dependence on cortical motor planning and greater involvement of subcortical structures such as basal ganglia and cerebellum. Experimental verification exists of the idea that a voluntarily altered breathing pattern can be improved with training, especially when there is feedback (in this case electromyographic) [182]. Breathing retraining techniques based on these principles have had some success in rehabilitation of patients with lung disease (e.g., [183,184]) and hyperventilation syndrome [185,186]. The longterm clinical effectiveness of these breathing retraining techniques is as yet inconclusive. It has yet to be determined how long one can retain a voluntarily modified breathing behavior, and whether the learned pattern ever becomes completely automatic. In this respect, sleep may be a useful tool to distinguish automatic from subconsciously controlled breathing patterns. Exercise hyperpnea in humans may be in part a learned response that has been forged by adaptive adjustments [187,188]. This scheme of control would be independent of any explicit exercise input and thereby differs from the idea that the descending motor command to exercising muscles simply irradiates to respiratory neurons in the brainstem [127]. Feedforward control of this type presumably depends on a memory of the prior experience of exercise and might therefore require plasticity in respiratoryrelated neurons within suprapontine neural networks. The possibility that such plasticity can occur at the level of the brainstem cannot be discounted. The first experimental evidence for this hypothesis came from a study in awake goats [189] in which the goats hyperventilated during exercise after they had been subjected to training trials in which the same exercise was performed repeatedly but with added respiratory deadspace in the breathing circuit. Several recent reports have generated conflicting claims about whether such long-term modulation of exercise hyperpnea is possible in humans. One study found greater ventilatory responses at exercise onset following several trials of arm exercise with added respiratory deadspace [190].

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However, a clear demonstration of long-term modulation of the steady-state phase of exercise in humans is lacking [191,192]. It is possible that greater numbers of conditioning trials or more elaborate associative conditioning paradigms may be required to alter a response that has been forged over many years. There have been many attempts to demonstrate learned respiratory behaviors through classical conditioning of respiratory responses to changes in blood gases (see [193] for review of the early work). In a well-controlled study in humans, it has been demonstrated that following eight pairings of a sound and a brief hypoxic challenge, the sound alone produced an increase in breath duration [194]. Cats have been conditioned to hold their breath when presented with a tone by initially presenting the tone in combination with an inhalation of ammonia [162]. The marked changes in breathing which can occur during the anticipation of performing exercise are thought to be due to a learned feedforward response to avoid blood-gas disturbances [126]. Such adaptability of the ventilatory responses to, for example, exercise and hypoxia may be important in maintaining normal blood gases in the face of changes in physiological, mechanical or environmental conditions. VIII.

Summary

Without the various forms of volitional control of breathing we would not be able to sing, speak, whistle, play wind instruments, sniff, swim or perform yoga—activities that define humans both as individuals as well as social beings. Emotional influences on breathing may occur as a result of internally generated psychological disturbances that are pathological (depression, anxiety, panic disorder) or non-pathological (mental stress, thoughts and memory). Emotional influences may also occur through perceptual processing of respiratory or non-respiratory sensory afferent information. Such influences may modulate respiratory as well as nonrespiratory muscle systems. The functional significance of such influences is less certain; possible roles include rudimentary forms of social communication; protection of the respiratory apparatus from threatening situations that cannot be dealt with by simply changing breathing; improving mechanical efficiency of the respiratory apparatus, and in the development of emotions themselves. The operation of the automatic controller in the brainstem is modulated by tonic excitatory influences (from the subcortical structures) as well as tonic inhibitory influences (from more rostral limbic areas). Many of the emotional influences may be by-products of a shift in the balance of these opposing influences. This balance may also serve to stabilize respiratory control. How and to what extent this suprapontine system of control interacts with the automatic system of control and adapts, remain


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key issues in this field. Direct study of these issues in human subjects has benefitted in recent years from rapid advances in functional neuroimaging techniques and non-invasive transcranial stimulation techniques, guided by pathological case reports. Acknowledgments SHM is a Parker B. Francis Fellow in Pulmonary Research. DP is supported by grants from the National Institutes of Health (NIH R01 HL49848, HL071884) and an Established Investigator Award from the American Heart Association (AHA). SAS received support from the National Institutes of Health (NIH R01 HL64815 and NIH K24076446). The contents of this chapter are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the AHA.

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183. Johnston, R. and Lee, K., Myofeedback, a new method of teaching breathing exercises in emphysematous patients, Phys. Ther. 56, 826–831, 1976. 184. Donner, C.F. and Howard, P., Pulmonary rehabilitation in chronic obstructive pulmonary disease (COPD) with recommendations for its use, Report of the European Respiratory Society Rehabilitation and Chronic Care Scientific Group (S.E.P.C.R. Rehabilitation Working Group) Eur. Respir. J. 5, 266–275, 1992. 185. van Doorn, P., Folgering, H. and Colla, P., Control of the end-tidal PCO2 in the hyperventilation syndrome, effects of biofeedback and breathing instructions compared, Bull. Eur. Physiopathol. Respir. 18, 829–836, 1982. 186. Cowley, D.S. and Roy-Byrne, P.P., Hyperventilation and panic disorder, Am. J. Med. 83, 929–937, 1987. 187. Somjen, G., The missing error signal—regulation beyond negative feedback, News in Physiological Sciences 7, 184–185, 1992. 188. Shik, L.L., [Regulation of respiration during muscular exertion] Nauchnye Doki. Vyss. Shkoly. Biol. Nauki. 18–29, 1985. 189. Martin, P.A. and Mitchell, G.S., Long-term modulation of the exercise ventilatory response in goats, J. Physiol. 470, 601–617, 1993. 190. Helbling, D., Boutellier, U. and Spengler, C.M., Modulation of the ventilatory increase at the onset of exercise in humans, Respir. Physiol. 109, 219–229, 1997. 191. Turner, D.L. and Sumners, D.P., Associative conditioning of the exercise ventilatory response in humans, Respir. Physiol. Neurobiol. 132 159–168, 2002. 192. Moosavi, S.H., Guz, A. and Adams, L., Repeated exercise paired with imperceptible dead space loading does not alter VE of subsequent exercise in humans, J. Appl. Physiol. 92, 1159–1168, 2002. 193. Bykov, K., The Cerebral Cortex and the Internal Organs, New York, Chemical, 1957. 194. Gallego, J. and Perruchet, P., Classical conditioning of ventilatory responses in humans, J. Appl. Physiol. 70, 676–682, 1991.

4 Measurement of Drug Effects on Ventilatory Control

DENHAM S. WARD University of Rochester School of Medicine and Dentistry Rochester, New York

I.

Introduction

Drug effect studies are generally performed either to determine the effects (or side effects) of a clinically used drug, or to use the drug as a pharmacological probe to learn more about the functioning of ventilatory control mechanisms. An example of the former might be the comparison of the ventilatory depressing effects of different opioids [1] and of the latter, the use of a receptor antagonist to determine if an endogenous neurotransmitter is involved in ventilatory control [2]. Many techniques have been used to study and quantify drug effects on the control of breathing, but few standard methods for clinical testing have evolved. This chapter will primarily review methodology used to characterize drug effects on ventilatory control in humans in order to obtain useful clinical or physiological information. The other chapters in this book provide a wide range of the many different techniques that have been used, both in human and in invasive animal studies. Few drugs are studied for their positive effects on ventilatory control. Examples might be the use of almitrine [3,4] or doxapram [5] as ventilatory 103

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stimulants or the effects of naloxone [6] or flumazenil [7] in reversing the respiratory depressing effects of opioids or benzodiazepines. More often drug effect studies on the control of breathing are investigations of the ventilatory side effects of drugs used, for example, to provide pain relief. When studying the effects of drugs on the ventilatory control system, both the drug characteristics (pharmacokinetic and pharmacodynamic) and the difficulties present in making measurements on the ventilatory controller must be taken into account. This can present experimental problems quite different from those seen when experiments are designed to test a physiological hypothesis. However, most tests for drug effects use techniques originally designed as physiological experiments, often in animal models. There is no agreement if a particular test best predicts clinically important outcomes (e.g., post-operative respiratory complications and use in obstructive sleep apneic patients) or is best used to gain physiological information. Recurring themes in drug effect studies include how to handle the closed-loop nature of the respiratory controller and whether or not stimulated (e.g., with inhalation of CO2) or unstimulated (resting) measurements best reflect the drug effect [8,9]. If the clinical situation is studied, the feedback loops are left intact and no stimulation is applied. However, the well-known insensitivity of the controlled variables (outputs) to system parameter variation in a negative feedback control system often limits the observed effect of the drug, resulting in small changes in the measured variables [10]. This is illustrated in Figure 4.1 with a hypothetical drug causing general anesthesia and depression of the hypercapnic ventilatory response. If the drug effect is measured by the change in resting ventilation, then only a small effect is observed. Since the metabolic hyperbola is relatively flat around the normal resting values, there is a moderate increase in the PaCO2, but if the decrease in ventilation at a fixed hypercapnic level (e.g., PaCO2 ¼ 55 mm Hg) is measured, then there is a very large drug effect. When various maneuvers are used to open the feedback loops, the effect of the drug may be more apparent but the clinical interpretation may be called into question [9]. The classic investigation of the respiratory control system is to characterize the input–output relationship (static or dynamic) of all or part of the system and then determine how this relationship is modified by the administration of a drug. Invasive animal studies can focus on a single component of the system such as the carotid sinus nerve firing rate. However, the complete intact system has many inputs and outputs, as well as many internal feedback loops besides the dominant CO2-ventilation closed loop. These feedback loops present considerable difficulty in reliably estimating system performance when characteristics or parameters of the individual components are measured. In humans or in intact animal preparations, inputs to the control system can be divided into closed-loop

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Figure 4.1 Effect of a hypothetical anesthetic on measurements of the ventilatory control. This drug causes a large reduction in the hypercapnic response slope, a relatively small increase (shift to the right) of the zero ventilation intercept (2 mm Hg), and an elimination of the wakefulness drive to breath. This results in a relatively small decrease in the alveolar ventilation at rest (2 l/min); but because of the flatness of the metabolic hyperbola a larger increase in the resting CO2 (15 mm Hg). However, if the drug effect was quantified by the reduction of ventilation at a fixed CO2 (55 mm Hg has been commonly used—V55), a large decrease in ventilation is measured (30 l/min).

(chemoreflex as labeled by Dejours [11]) and open-loop (non-chemoreflex) [12]. The sensitivity of a chemoreflex (e.g., hypercapnic chemoreflex) is often characterized by a gain with units related to the change in output divided by the change in input (e.g., l min1 mm Hg1), but the fact that this gain is determined by all factors from input to output is important to remember. It is also tempting to consider the non-chemoreflex inputs as those setting the baseline or resting ventilation, and the chemoreflexes as those setting the gain of the closed loops; however, it is important to note that the non-chemoreflex inputs can modulate the chemoreflexes (see Ward and Karan [13] and Chapter 3, this volume, for recent reviews). Many of the non-chemoreflexes, including volitional inputs [14], have projections into or through the brainstem respiratory centers [15] and thus have the potential to alter the chemoreflex loop gain. For example, in nonrapid-eye movement (non-REM) sleep the hypoxic response is markedly reduced with only a small decrease in baseline ventilation [16,17]. Also, it has long been known that sleep and opioids have a strong interaction in

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depressing ventilation [18,19]. These interactions underscore the need to carefully design and perform experiments assessing drug effects on ventilatory control. An essential part of assessing drug effects is the parameterization of the respiratory response. This requires some type of model, statistical or parametric, that can be fitted to the respiratory data. Today, breathby-breath measurements are the norm and are used as the raw data for the models. Modeling in the control of breathing has had a long history [20] (see Khoo and Yamashiro [21] for a recent review); these models have played an integral part in the development and validation of methods to assess the ventilatory control system [22]. However, these models often require many parameters determined from several experiments. If the model parameters are to be estimated from the measured data of a single brief experiment, as is usually required when testing for a drug effect, then a greatly simplified model is needed. For the clinical assessment of drugs (and frequently used drug combinations), there might be expected some degree of standardization on the appropriate approach. However, this is not the case and there is a very wide range of published techniques. In the design of a study to ascertain a drug’s clinical effects, many factors need to be considered (Table 4.1) and the principles of good clinical trial design applied, whether the study is an outcome clinical trial or a laboratory study [23]. For example, the particular study design will have a major impact on the required size of the study. Thus, a laboratory investigating the effect of a drug on a particular chemoreflex, with a carefully selected, homogeneous subject population and with the drug levels tightly controlled, may require only a few subjects to demonstrate an effect. However, a clinical trial attempting to show a change in the post-operative respiratory morbidity with one analgesic regimen versus another may require a large number of subjects. II.

Measurement Techniques

Standard measurement techniques include airway gas concentrations (partial pressures) and flows, usually on a continuous basis and using measurement devices with very fast response times. These continuous signals are then digitized with an analog-to-digital converter and stored in a digital computer. For most breath-to-breath data calculations a sample rate of 50 Hz is sufficient. This digital data can then be processed to calculate a set of commonly utilized respiratory parameters, e.g., inspired and end-tidal (PET) O2 and CO2, tidal volume, inspiratory time, expiratory time, minute ventilation, etc. Both commercial and laboratory-designed hardware and software are commonly used for these purposes [24]. Although there are many technical difficulties in making these measurements


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Table 4.1 Example Characteristics of Studies to Determine the Ventilatory Effects of Drugs in Human Subjects (1)

Clinical study (a) Clinical setting (e.g., recovery room, post-operative, out-patient) (b) Patient population (e.g., age, sex, weight, co-existing diseases, concurrent medications) (c) Drug administration method (e.g., patient-controlled analgesia, spinal, oral) (d) Stimulation (e.g., none, hypoxia, hypercapnia, negative airway pressure) (e) Outcome measurement (e.g., hypoxia, ventilation, complications)

(2)

Laboratory study (a) Subject population (e.g., normal, age, sex) (b) Drug administration (e.g., target controlled infusion, measurement of plasma levels) (c) Stimulation (i) Closed loop (chemoreflex) (e.g., hypoxia, hypercapnia, acidosis; ramps vs. steps) (ii) Non-closed loop (e.g., airway load, exercise, pain, audiovisual) (iii) Combined chemoreflex and non-chemoreflex stimulation (d) Measurements (e.g., ventilation, tidal volume, frequency, abdominal/chest wall excursions, esophageal/airway pressure, genioglossal EMG) (e) Response model (e.g., steady-state vs. transient, mixed effect, population PK-PD)

and ensuring proper instrument calibration, the problems have been well worked out and there should be no issues in making accurate measurements in the laboratory [25]. Confirmation of the measured end-tidal values with arterial blood gas measurements is a good practice when a new protocol or new equipment is used. An accurate, motor-driven syringe pump providing a consistent sinusoidal volume waveform is also invaluable in the set-up and maintenance of a flow/volume measurement system. Several reviews are available on the technical aspects of these measurement techniques (including a still useful special section in Chest, 70, 109–195, 1976) [26–29]. Measurement of arterial oxygenation is often made with pulse oximetry rather than with measurement of end-tidal O2. Although the PETO2 can often differ from the arterial PO2 by several mm Hg, the measurement of saturation with pulse oximetry can also be inaccurate and has a time delay that can differ between rapidly increasing versus decreasing oxygen tensions [30]. Since oxygenation is critically important, monitoring of patients who are at risk for respiratory depression with a pulse oximeter has become standard, both in routine clinical practice and as an outcome in clinical trials studying respiratory depression [31–35].

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Wheatley et al. used timed-epoch (1 h) histograms of the continuous saturation to show differences in the patterns of desaturation between pre- and post-surgery [34]. Obviously, while severe prolonged hypoxemia is dangerous, less severe episodic hypoxemia (e.g., in the post-operative period) has not been clearly linked to adverse outcomes [36]. However, chronic episodic desaturations (e.g., in sleep apnea patients) are more clearly associated with changes in health status [37,38]. While much work has focused on the pharmacologic alteration of the chemoreflexes, upper airway obstruction is clinically a predominant drug effect in many patients, especially those with obstructive sleep apnea. Inductance plethysmography has been used for many years to measure the relative motions of the ribcage and diaphragm [39] and to prevent some of the alterations in respiratory pattern caused by airway instrumentation [40,41]. Determination of upper airway obstruction can be done using inductance plethysmography and analyzing the signals for asynchrony [42]. However, other drug effects may cause asynchrony between the rib cage and abdomen without any overt airway obstruction and caution must be taken in interpreting these signals [43–45]. In fact, alterations in the relative amount of respiratory drive to the diaphragm versus the intercostal muscles, as assessed by inductance plethysmography, is an important drug effect, particularly in infants [46]. Inductance plethysmography may also be useful in clinical studies where long-term respiratory monitoring is needed and thus direct measurement of ventilation via airway instrumentation is not practical [35]. For many studies, the subject can breath through either a facemask or mouthpiece. A facemask is preferred since it causes less disruption of the normal respiratory pattern and permits normal nasal breathing. However, when testing sedatives, it is important that a good mask seal is maintained even when the mouth muscles become slack.

III.

Quantification of Drug Pharmacodynamics

To fully characterize the effects of a drug on ventilatory control, it is necessary to construct a full dose (or even better, a plasma concentration) response curve. This curve can then be parameterized with standardized pharmacodynamic parameters (e.g., plasma concentration for a 50% maximum effect, EC50). Selecting the particular respiratory responses to parameterize as well as the particular equation to use are important decisions. Since respiratory depression is often being evaluated, it is common for a study to select a dose in the therapeutic range (e.g., for an opioid it could be a dose providing adequate analgesia) and then investigate the respiratory depression for that single dose. However, much more


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information about the relative effects of respiratory depressants is obtained if full drug concentration–response curves are investigated. For example, Mildh et al. [47] used computer-controlled infusions of fentanyl and alfentanil to set plasma levels. They then used measurements of resting ventilation, PaCO2 and arterial plasma drug levels to fit either a Hill (ventilation) or a linear (PaCO2) equation. This approach permitted them to compare potencies of the drugs for respiratory depression. Detailed modeling of both the pharmacokinetic and the pharmacodynamic effects of drugs can be done; this may allow a better understanding of the mechanisms of the differences between drugs. Bragg et al. used a population-based approach to model the pharmacodynamics and kinetics of morphine and fentanyl to determine how developmental age changes the respiratory depression of the drugs [48]. The next chapter discusses in more detail the methods that can be applied to determine, from respiratory data, the pharmacodynamic models for single drugs and combinations of drugs. IV.

Resting Measurements

The simplest and arguably the most clinically relevant [9] data measurement is the effect of a drug on resting ventilation and/or CO2 (either arterial or end-tidal). However, interpretation of this measurement becomes more complex due to many factors (in addition to the chemoreflexes) determining resting ventilation. The functioning of the hypercapnic chemoreflex feedback loop when CO2 is increased will tend to minimize the measured effect of the drug. The resting ventilation is determined by the intersection of the ventilatory response to CO2 with the metabolic hyperbola (Figures 4.1 and 4.2). The ventilatory response to CO2 is often characterized as linear above a threshold and thus has three, relatively independent factors: wakefulness drive (i.e., ventilation below the CO2 threshold), slope, and position (i.e., the CO2 intercept at zero ventilation) of the hypercapniainduced ventilation. Other parameterizations of the response have also been used [10,49]. Many factors (e.g., hypoxia, acid–base status, pain, arousal, etc. [13,50,51]) influence the specific value of each of these parameters and it is important that the control and drug conditions are identical except for the drug under study. When studying the respiratory effects of a drug, the changes in the resting values need to be supplemented with further information about specific drug effects on the elements of the controller. Most frequently these are measurements of the effects on the hypercapnic or hypoxic chemoreflexes. In clinical studies, it is difficult to control the many factors involved; particularly in patients, it is often difficult to make reliable measurements. In many clinical studies, then, the resting control of breathing, particularly

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Figure 4.2 Illustration of how an increase in the ‘wakefulness’ drive, caused by an arousal, can affect the resting ventilation. The control response has a resting ventilation at the intersection of the wakefulness drive and the chemoreflex response. The drug caused a decrease in the slope with a small right shift of the chemoreflex and a reduction (but not elimination) of the wakefulness drive such that the resting ventilation is now determined by the intersection of the metabolic hyperbola and the chemoreflex line. The arousal caused an increase in the wakefulness drive and an elimination of the small right shift. Note that even though there was no change in the reduction of the slope, the resting ventilation and PaCO2 returned almost back to normal.

in post-operative patients, is often summarized by simply measuring the oxygen saturation with pulse oximetry over time. These studies can be of great clinical interest since post-operative desaturation has been associated with myocardial ischemia and arrhythmias. However, these studies are greatly aided by, at the least, a qualitative measurement of ventilation [31] and sleep state [52] as done during polysomnography in the sleep laboratory. V.

Hypercapnic Ventilatory Response

The hypercapnic ventilatory response has been used for 100 years to assess the ventilatory response to drugs [53,54]. While originally only different levels of inspired CO2 were used, it soon became apparent that measurement or estimation of the arterial CO2 is also required. The development of the rebreathing technique by Read provided a relatively simple technique to measure the hypercapnic response quickly, and thus it rapidly


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became popular for many drug studies [22]. The response is usually parameterized with a slope and intercept obtained by linear regression of the breath-by-breath ventilation on the PETCO2 [55]. The steady-state technique (constant inspired CO2 concentration for a long enough period of time for the ventilation to reach steady-state) has evolved with control over the endtidal CO2 via manipulation of the inspired gas tension (dynamic end-tidal forcing—DEF) using automatic computer feedback control [56–58]. It is used to obtain step responses [57] or pseudo-rebreathing inputs [59] and leads to more sophisticated input function designs, thereby obtaining more accurate estimates of the chemoreflex loop parameters [60]. These DEF techniques have often been aimed at estimating both a peripheral and a central CO2 gain based on separation of the response times. While both techniques (steady-state and rebreathing) yield several characteristic parameters of the respiratory system that are related to underlying mathematical models, the primary parameter of interest for drug studies is often the overall CO2 gain—the increase in ventilation resulting from an increase in CO2. While Read’s original work indicated that the rebreathing and steady-state gains were identical [22], subsequent analysis [61] and work by others indicate that gain measured by rebreathing is larger than when measured by the steady-state technique [22,61–65] and more importantly, may also result in differences in the estimation of the effects of drugs on the hypercapnic response [66]. The essential features of the Read rebreathing test are that the subject begins the process with a vital capacity breath and then starts rebreathing from a small bag (4–6 l) filled with 7% CO2 in oxygen. The purpose of these maneuvers is to start the rebreathing at a value close to the mixed venous level and thus quickly obtain an equilibrium in mixed venous, lung, rebreathing bag, arterial, and brain tissue PCO2 [22,61]. Since external gas exchange no longer occurs, the rise in PETCO2 is independent of the level of ventilation, effectively opening the CO2 control feedback loop. The top panel in Figure 4.3 shows the continuous waveform from a single subject undergoing a Read rebreathing test. Note the vital capacity breath at the start of the rebreathing as well as the initial step in PETCO2 . There is a short equilibrium period and then the PETCO2 slowly increases at a rate determined by the subject’s metabolic rate and body CO2 buffering capacity. Thus, the CO2 stimulus can be characterized as an initial step of A (mm Hg) and a rate of rise of R (mm Hg min1) [59]. The differences in ventilation between the steady-state data and the rebreathing data at the same PETCO2 , and the resulting difference in the slope of the response, are illustrated in the bottom panel of Figure 4.3. The steady-state data (four periods of 8 min of elevated PETCO2 ) are shown by the open circles. The initial rebreathing data (filled squares) show an almost vertical increase in ventilation without a change in PETCO2 , representing the initial step and equilibrium period for the rebreathing. These points cannot be used in

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Figure 4.3 Continuous tidal volume and CO2 waveforms from a subject performing a rebreathing experiment (upper panel) and the regression fits to rebreathing data (squares) and steady-state data (open circles) (Data from Ref. 63).

the calculation of the response slope. The open squares then show the breath-to-breath rebreathing data. These points are used to calculate the rebreathing ventilatory response. Note that the rebreathing data show the expected right shift, but also that the slope is steeper. This increase in slope can be predicted from theoretical analysis [59,61,63,67] and is due to the increase in cerebral blood flow with hypercapnia reducing the arterialto-tissue CO2 gradient. If the gradient is reduced to zero and it remains


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constant throughout the rebreathing period, then the measured slope should approximate the slope of the tissue PCO2-to-ventilation relationship. However, it is difficult to verify that these conditions are, in fact, achieved and maintained [67]. This analysis also shows that the measured slope will depend on the size of the initial step, A, which can present difficulties when, in a pharmacological study, the starting PETCO2 is increased due to the ventilatory depression of the drug. Bourke and Warley directly compared the hypercapnic responses measured with the steady-state and rebreathing methods following two doses of morphine [66]. They found that the control slopes were higher when measured with the rebreathing method than with the steady-state method, and that after morphine the steady-state method showed a rightward shift and no change in slope while when measured with the rebreathing method, there was a decrease in the slope. Applying the theoretical analysis of Berkenbosch et al. [59], they determined that most of this change in slope was due to the smaller A after morphine due to the increase in PETCO2 . Their conclusion was that the specific opioid effect was a rightward shift of the hypercapnic ventilatory response without a change in sensitivity, and that this was best measured with a steady-state technique [66]. Similarly, Linton et al. found that while the slopes of the CO2 response determined by steadystate and rebreathing were identical under control conditions, acidosis and alkalosis had quite different effects on the response as measured by the two methods [64]. The rebreathing technique has been further refined by Duffin and colleagues [49,62,68,69], who have advocated preceding the rebreathing not with a single vital capacity breath, but rather with a period of hyperventilation that reduces the PETCO2 into the mid-20s mm Hg. Rebreathing is started and ventilation is maintained by the resting (wakefulness) ventilatory drive (see above) as the PETCO2 gradually rises. Once a threshold is reached, the ventilation then increases linearly with PETCO2 until a second breakpoint is reached, when the ventilation then increases faster due to a rapid increase in ventilatory frequency (Figure 4.4). This technique still provides an estimate of the hypercapnic response slope that is larger than the steady-state response [62]. While this method may provide further information about drug effects on different parts of the response (e.g., basal ventilation, threshold T1 and slope S 2), it has not been applied to many drug studies [70,71]. For most drug studies, the use of a steady-state technique is the most appropriate for determining central chemosensitivity. Since these tests are often performed in hyperoxia, the peripheral chemoreceptors contribute little to the overall ventilatory drive. Special test or data analysis needs to be applied if separation of the peripheral and central chemosensitivities is desired.

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Figure 4.4 Duffin’s modified rebreathing test in which the commencement of rebreathing is after a period of hyperventilation in which the PETCO2 is lowered to below 30 mm Hg (data segment not shown). Several parameters can be estimated from these experiments: The basal ventilation prior to the increase with hypercapnia; the PETCO2 threshold T1 at which ventilation first increases; the initial segment slope, S1; the second threshold when respiratory frequency starts to greatly increase, T 2; and the final augmented slope, S2 (Data from Ref. 49).

A single breath test of CO2 has been used as a clinical test to determine the sensitivity of the peripheral chemoreceptors [72]; this test has been applied in determining the effects of spinal anesthesia [73]. While this test is quick to perform, the signal-to-noise ratio is poor and the estimation of the peripheral gain can be contaminated with other


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factors [74]. Interestingly, the sensitivities of the single-breath CO2 test and the acute hypoxic response were not found to be correlated [75]. This test for peripheral sensitivity should not be used in place of an hypoxic test if the effect of a drug on the ventilatory response to hypoxia is desired. The main advantage of the rebreathing technique, aside from the relatively simple equipment required, is that a hypercapnic response can be obtained in several minutes, a fact which may be important if the drug effect is rapidly changing. The steady-state technique, if a constant inspired CO2 is used, may require over 10 min for ventilation to reach a steady state [64]. However, the technique of controlling the inspired CO2 such that the endtidal CO2 reaches a constant value immediately (e.g., step in PETCO2 [57]) or follows some other pre-described waveform (e.g., sinewave [76] or a timevarying binary sequence [60,77]) can permit a much shorter time period in which to make the ventilatory measurements. These shorter DEF steps can be combined with a two-compartment mathematical model of the ventilatory controller and the response separated into a fast (peripheral) and a slow (central) component [78,79]. However, from human experiments it cannot be certain that the dynamic component separation with statistical parameter estimation actually results in a central and peripheral component. Using an artificially perfused brainstem (ABP), spontaneously breathing cat preparation, DeGoede et al. directly compared the results of the DEF step response parameter estimation method with the determination of the central and peripheral gains from the ABP method [80]. Good agreement was found between the gains and the CO2 (extrapolated) threshold determined by these two methods over a range of peripheral gains. In carotid-body-resected human subjects, the DEF technique also found no peripheral component [81]. More recently, the DEF technique was used with a multifrequency binary sequence of PETCO2 in patients with unilateral and bilateral resections of the carotid bodies [82]. Interestingly, when using this technique, the bilaterally resected patients had lower sensitivity for the single slow component than the control subjects. Although the step input seems to satisfactorily separate the gains, its use has been criticized on the grounds that the input is not persistently exciting enough to obtain good estimates of the peripheral gain [60]. In essence, this is because the fast-component gain is only estimated during the initial rapid increase in ventilation, and the total gain is determined by the estimated final ventilation. A proposed solution to this problem is to use a waveform that consists of multiple up and down steps of different duration, resulting in a multi-frequency binary sequence (Figure 4.5, top panel). The number of transitions and the duration of the steps can be determined theoretically to provide an input that is appropriate for the assumed central and peripheral time constants [60]. A drawback of this technique is that an experimental period of over 20 min is required; however,

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Figure 4.5 Use of a specialized dynamic end-tidal CO2 forcing function to obtain the estimates of the effects of a drug on the parameters of the chemoreflex loops. The top panel shows a multifrequency binary sequence (theoretical) in end-tidal CO2. This input function is used to excite the ventilatory controller (bottom panel) before and after propofol administration. This waveform allows for the parameters of both fast (peripheral) and slow (central) chemoreflex loops to be estimated from the ventilatory response. This divides the response into central, Vc, and peripheral, Vp, components (Data from Ref. 83).

it provides a more reliable and accurate estimate of the peripheral gain. This technique has been applied to drug studies in which the drug level was constant for the time period, allowing estimation of changes in the central and peripheral CO2 gains (Figure 4.5, bottom panel) [83]. Besides understanding the effects of a drug on the hypercapnic response when the drug concentration is constant, it is also of interest to study the time course of the drug effect. For many drugs that have slowly changing respiratory effects, repeated hypercapnic studies can be made, but with drugs that produce rapid alterations in ventilation (e.g., anesthetic induction drugs), other techniques must be employed. An obvious method


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is to hold the end-tidal CO2 constant by adjusting the inspired CO2 as ventilation changes with the drug effect. Since respiratory depressants are usually studied, it is necessary to start at a hypercapnic level so that the inspired CO2 can be lowered as the ventilation declines. By repeating the experiment twice, the so-called dual iso-hypercapnic technique, the time course of the change in the hypercapnic sensitivity can be constructed [84]. The major drawback to this technique is the need to give two drug doses, most likely on different days, unless the drug has very fast pharmacokinetics (e.g., remifentanil [85]). The day-to-day variability in the hypercapnic response will greatly diminish the ability to accurately measure changes in the hypercapnic sensitivity. Bouillon and coworkers have applied an indirect response model to the ventilatory depression caused by alfentanil [10] and remifentanil [86] in the non-steady state. This modeling technique permits the prediction of the time course of respiratory depression after a bolus dose of the drug, considering the indirect effect of the slow rise in CO2-stimulating ventilation [87]. While this technique is attractive and provides a clinically relevant model of druginduced respiratory depression, it requires estimation of the hypercapnic response for each subject prior to the drug experiment. The method also requires assumptions on the shape of the hypercapnic response, particularly in the transient, when CO2 is less than its final steady-state value, e.g., below the metabolic hyperbola (Figure 4.2). This model has not been fully experimentally validated and has the additional drawback of not being able to predict the apnea that is observed clinically at high drug doses [88]. VI.

Hypoxic Ventilatory Response

Although the ventilatory response to hypoxia has been described since the early part of the 20th century, the effect of morphine on the hypoxic response was not studied until 1975 [89]. Although hypoxia is a common and dangerous clinical condition, the effects of drugs on the hypoxic response have been difficult to study. The hypoxic response is a nonlinear function of the arterial O2 [90], is strongly affected by the CO2 level [91,92], and has a time-dependent effect [93]. The time dependence of the ventilatory response can be seen as a pronounced decrease in ventilation (hypoxic ventilatory decline—HVD) after 5–15 min of isocapnic hypoxia [90,94,95]. In studying the hypoxic response, the negative feedback from the increased ventilation lowering the CO2 and raising the O2 has to be eliminated. Thus isocapnic conditions have to be maintained not only throughout the time period of the hypoxic stimulus, but also during the different drug conditions. It is important that the CO2 chosen be high enough to ensure that the hypercapnic chemoreflex is active. Since many of the tested drugs will cause hypercapnia at rest, the control hypoxic

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responses need to be made at similar hypercapnic levels. While a rebreathing type of hypoxic test has been advocated [96], the biphasic nature of the hypoxic response can readily affect ventilation before the end of the rebreathing period. Thus, hypoxic responses should be tested with a DEF-type system so that PETCO2 can be controlled on a breath-to-breath basis and precise control over PETO2 (and saturation) can be maintained. However, since in the clinical situations PETCO2 will obviously not be controlled, poikilocapnic hypoxic tests have been advocated as a better measurement of the clinical effects of drugs [97–99]. The results for a drug effect may be different depending on whether an isocapnic or a poikilocapnic hypoxic test is used. For example, Sjogen et al. found that the isocapnic acute hypoxic response was 4.7 2.3 l min1 but only 1.4 1.0 l min1 for the poikilocapnic response; 0.6 MAC of isoflurane did not significantly reduce the poikilocapnic response (1.3 0.8 l min1) but did reduce the isocapnic response (2.3 1.4 l min1, p 5 0.02) [98]. The first issue in quantifying the hypoxic response is to determine the parameters of the nonlinear relationship between ventilation and arterial PO2 (Figure 4.6). There is no underlying physiological basis for any particular parameterization and the cellular mechanisms underlying the non-linear response are poorly understood (see Nurse, Chapter 1, this volume). Either a hyperbola [100–102] or linear-in-saturation [96] model has been the most commonly used. The availability of pulse oximetry has made the use of the linear-in-saturation model popular. Several studies have compared the fit of the various models to the initial hypoxic response (note that it is not appropriate to characterize this as the steady-state hypoxic response since the development of the acute response and HVD overlap, and no real steady state is seen in the first 20–30 min of hypoxia [93]). The studies have not found any significant difference in the fit between the hyperbolic equation and the linear-insaturation equations [103,104]. Figure 4.6 shows a comparison of seven models to the hypoxic response of a single subject. By visual inspection, it is readily apparent that the variability in the data makes all the models, except for a direct linear fit to PETCO2 , seem to be suitable. Because the linear slope with respect to saturation is the most readily interpretable, it is the most common model used to quantify a drug effect (expressed in units of l min1 % sat1, while noting that since the ventilation increases with decreasing saturation, this gain is negative, but is commonly expressed as the change in ventilation for a decrease in saturation giving a positive value). There are no studies of whether the shape of the acute hypoxic response is altered by a drug, but given the variability of the data it is doubtful that minor changes could be detected.


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Figure 4.6 Examples of different model fits to the hypoxic ventilatory response in one subject. Top and center panels show the fit to PETO2 and the bottom panel shows the fit to the measured saturation (Data from Ref. 103).

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The ventilatory response to sustained hypoxia has several time domains, and therefore it is not possible to define a true steady-state response [93]. The response over the first 20 min of hypoxia is markedly biphasic, with ventilation declining by approximately 50% of the initial rise over this period of time [95]. This response has been modeled in a way that is similar to the dynamic hypercapnic response model, with two exponential terms but with one stimulatory and the other inhibitory [105–107]. However, few drug studies have examined specific changes in these model parameters, but rather used pseudo-steady-state methods of parameterizing the hypoxic response. These methods make the assumption that the ventilation at 3–5 min after the onset of hypoxia is not affected by HVD and that an average ventilation during this time period will be a measure of the peripheral (carotid body) chemoreflex. The ventilation occurring between 15 and 20 min of hypoxia will reflect the development of HVD and can be used to calculate the sustained ventilatory response. An interesting characteristic of HVD is that a brief period of normoxia (e.g., 5 min) will not reverse the depression [108]. The magnitude of this repeated hypoxic response, when compared with the initial hypoxic response, can be used as a measure of HVD. A drug’s effect can be assessed both on the acute hypoxic response and HVD [109]. Figure 4.7 shows how the characteristics of the hypoxic response can be calculated. A drug may have independent effects on the acute response or HVD. However, it is important to understand how HVD is assessed. The amount of HVD is not fixed, but rather depends on the magnitude of the acute response [92,110]. Thus, a drug that reduced the acute response would also seem to increase the amount of HVD if measured only by the sustained hypoxic sensitivity. In drug effect studies, it is better to express the magnitude of HVD by the ratio of the increase in ventilation from the baseline to the acute peak to the amount of ventilatory decline from the acute peak ventilation to the ventilation after 15–20 min of hypoxia. These changes in ventilation can be normalized by the saturation at these time points if the saturation is not held adequately constant throughout the whole hypoxic period. While ventilation is most commonly used as the measured variable, it is important to note that HVD seems to occur primarily in tidal volume, and separating the response into tidal volume and ventilatory frequency may reveal an effect on one or the other [111]. VII.

Changes in Airway Pressure

Since airway obstruction is a common occurrence after many respirationdepressing drugs [112], there has been development of techniques to


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Figure 4.7 Response to 20 min of isocapnic hypoxic and a repeat hypoxia exposure after 5 min of normoxia before (top panel) and after alfentanil (bottom panel). The acute hypoxic response for the first step (intervals 1 and 2) and the second step (intervals 4 and 5) can be calculated. The HVD can be calculated as the ratio of these acute steps or as the absolute difference between the ventilation during intervals 2 and 3. Normalizing the change in ventilation by the change in saturation results in calculation of hypoxic sensitivities (Data from Ref. 109).

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quantify this effect. This can be done in several ways, either through an added resistance or through the application of negative airway pressure. The former studies have been developed to simulate the effects of airway disease while the latter, to simulate obstructive sleep apnea (see Chapter 10 by Schwartz, this volume). In an awake subject, the application of negative airway pressure activates compensating activity in the pharyngeal dilator muscles; complete airway obstruction seldom occurs with negative pressures even as low as 30 cm H2O. However, during sleep, a negative nasal pressure of 10 cm H2O can induce a syndrome-like obstructive sleep apnea in normal subjects [113], and a positive pressure is required to maintain a patent airway in patients with obstructive sleep apnea. Application of different airway pressures (from positive to negative) has been used to test for changes caused by diazepam [114], midazolam [115], isoflurane [116] and vecuronium [117]. Typically, the outcome measurement in these studies is the pressure that causes airway collapse (critical pressure, Pcrit), measured either directly [115] or by extrapolation from the flow versus airway pressure on flow-limited breaths [116]. The measurement of flow limitation requires the measurement of esophageal pressure; the additional measurement of genioglossial EMG may also be useful (Figure 4.8). While a lower Pcrit in anesthetized patients correlated with the severity of sleep-disordered breathing [118], it is unknown how the drug-induced propensity for airway obstruction as measured by Pcrit correlates with clinically important outcomes. VIII.

Other Stimuli

Pain and exercise are other common stimuli that have pronounced ventilatory effects. The ventilatory response to exercise has been extensively studied throughout the 20th century without complete understanding of all the mechanisms involved. This makes it difficult to interpret any drug effects on the exercised induced ventilatory responses. Two studies have found that opioids do little to blunt the link between exercise and ventilation even at doses that caused considerable resting hypercapnia [119,120]. Since painful stimuli can only be quantified subjectively and introducing pain is an additional variable in drug effect studies, the interpretation of the results of these studies can be quite complex. Surgical skin incision has been studied for its effects on ventilation and ventilatory pattern [121,122]. The interaction of painful stimuli and drug-induced depression of the chemoreflexes has also been studied for both the hypercapnic and hypoxic responses. Pain seems to have little effect on the control response [123] and does not counter the effects of inhalational anesthetics on the hypoxic response [124]. When studies involve the use of painful stimuli it is important to consider anxiety and arousal induced by


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Figure 4.8 Top panel show experimental setup to change the airway pressure (both positive and negative) to test the effects of isoflurane on the collapsibility of the upper airway. Bottom panel shows the physiological response during isoflurane anesthesia when the airway pressure is reduced from þ20 cm H2O (A) to levels that cause flow limitation (B) or complete obstruction (C). Note that even a brief pulse of 30 cm H2O (D) did not activate any EMGgg even though there is a robust signal when the subject is awake (E) and is asked to protrude the tongue (F). Pm—mask pressure; Pnp—nasopharyngeal pressure; Pop—oropharyngeal pressure; Php—hypopharyngeal pressure; Pes—esophageal pressure; Piso—isoflurane concentration; EMGgg—genioglossus electromyogram; MTA—moving time average (Data from Ref. 116).

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the stimuli as well as the specific type of stimuli employed (e.g., electrical [123], pressure [125] or thermal [126]). References 1.

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Ward steady-state method are not equal in man, J. Physiol. (Lond.) 411, 367–377, 1989. Linton, R.A,, Poole-Wilson, P.A., Davies, R.J. and Cameron, I.R., A comparison of the ventilatory response to carbon dioxide by steady-state and rebreathing methods during metabolic acidosis and alkalosis, Clin. Sci. Mol. Med. Suppl. 42, 239–249, 1973. Jacobi, M.S., Patil, C.P. and Saunders, K.B., Transient, steady-state and rebreathing responses to carbon dioxide in man, at rest and during light exercise, J. Physiol. (Lond.) 411, 85–96, 1989. Bourke, D.L. and Warley, A., The steady-state and rebreathing methods compared during morphine administration in humans, J. Physiol. (Lond.) 419, 509–517, 1989. Dahan, A., Berkenbosch, A., DeGoede, J., Olievier, I.C.W. and Bovill, J.G., On a pseudo-rebreathing technique to assess the ventilatory sensitivity to carbon dioxide in man, J. Physiol. (Lond.) 423, 615–629, 1990. Duffin, J. and McAvoy, G.V., The peripheral-chemoreceptor threshold to carbon dioxide in man, J. Physiol. (Lond.) 406, 15–26, 1988. Mohan, R.M., Amara, C.E., Vasiliou, P., Corriveau, E.P., Cunningham, D.A. and Duffin, J., Chemoreflex model parameters measurement, Adv. Exp. Med. Biol. 450, 185–193, 1998. Katzman, M.A., Duffin, J., Shlik, J. and Bradwejn, J., The ventilatory response to cholecystokinin tetrapeptide in healthy volunteers, Neuropsychopharmacology 26, 824–831, 2002. Vovk, A., Duffin, J., Kowalchuk, J.M., Paterson, D.H. and Cunningham, D.A., Changes in chemoreflex characteristics following acute carbonic anhydrase inhibition in humans at rest, Exp. Physiol. 85, 847–856, 2000. McClean, P.A., Phillipson, E.A., Martinez, D. and Zamel, N., Single breath of CO2 as a clinical test of the peripheral chemoreflex, J. Appl. Physiol. 64, 84–89, 1988. Steinbrook, R.A. and Concepcion, M., Respiratory effects of spinal anesthesia: Resting ventilation and single-breath CO2 response, Anesth. Analg. 72, 182–186, 1991. Khoo, M.C.K., A model-based evaluation of the single-breath CO2 ventilatory response test, J. Appl. Physiol. 68, 393–399, 1990. Chua, T.P. and Coats, A.J., The reproducibility and comparability of tests of the peripheral chemoreflex: Comparing the transient hypoxic ventilatory drive test and the single-breath carbon dioxide response test in healthy subjects, Eur. J. Clin. Investig. 25, 887–892, 1995. Daubenspeck, J.A., Frequency analysis of CO2 regulation: Afferent influences on tidal volume control, J. Appl. Physiol. 35, 662–672, 1973. Yang, F. and Khoo, M.C., Ventilatory response to randomly modulated hypercapnia and hypoxia in humans, J. Appl. Physiol. 76, 2216–2223, 1994. Ward, D.S. and Bellville, J.W., Effect of intravenous dopamine on hypercapnic ventilatory response in human, J. Appl. Physiol. 55, 1418–1425, 1983. Dahan, A., van den Elsen, M.J.L.J., Berkenbosch, A., DeGoede, J., Olievier, I.C.W., van Kleef, J. and Bovill, J.G., Effects of subanesthetic halothane on the


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ventilatory responses to hypercapnia and acute hypoxia in healthy volunteers, Anesthesiology 80, 727–738, 1994. DeGoede, J., Berkenbosch, A., Ward, D.S., Bellville, J.W. and Olievier, C.N., Comparison of chemoreflex gains obtained with two different methods in cats, J. Appl. Physiol. 59, 170–179, 1985. Bellville, J.W., Whipp, B.J., Kaufman, R.D., Swanson, G.D., Aqleh, K.A. and Wiberg, D.M., Central and peripheral chemoreflex loop gain in normal and carotid body-resected subjects, J. Appl. Physiol. 46, 843–853, 1979. Fatemian, M., Nieuwenhuijs, D.J.F., Teppema, L.J., Meinesz, S., van der Mey, A.G.L., Dahan, A. and Robbins, P.A., The respiratory response to carbon dioxide in humans with unilateral and bilateral resections of the carotid bodies, J. Physiol. (Lond.) 549, 965–973, 2003. Nieuwenhuijs, D., Sarton, E., Teppema, L.J., Kruyt, E., Olievier, I., van Kleef, J. and Dahan, A., Respiratory sites of action of propofol: Absence of depression of peripheral chemoreflex loop by low-dose propofol, Anesthesiology 95, 889–895, 2001. Blouin, R.T., Conrad, P.F. and Gross, J.B., Time course of ventilatory depression following induction doses of propofol and thiopentathal, Anesthesiology 75, 940–944, 1991. Babenco, H.D., Conard, P.F. and Gross, J.B., The pharmacodynamic effect of a remifentanil bolus on ventilatory control, Anesthesiology 92, 393–398, 2000. Bouillon, T., Bruhn, J., Radu-Radulescu, L., Andresen, C., Cohane, C. and Shafer, S.L., A model of the ventilatory depressant potency of remifentanil in the non-steady state, Anesthesiology 99, 779–787, 2003. Jusko, W.J. and Ko, H.C., Physiologic indirect response models characterize diverse types of pharmacodynamic effects, Clin. Pharmacol. Ther. 56, 406–419, 1994. Gross, J.B., When you breathe IN you inspire, when you DON’T breathe, you . . . expire: New insights regarding opioid-induced ventilatory depression, Anesthesiology 99, 767–770, 2003. Weil, J.V., McCullough, R.E., Kline, J.S. and Sodal, I.E., Diminished ventilatory response to hypoxia and hypercapnia after morphine in normal man, N. Engl. J. Med. 292, 1103–1106, 1975. Bisgard, G.E. and Neubauer, J.A., Peripheral and central effects of hypoxia, in Regulation of Breathing, Dempsey, J.A. and Pack, A.I., eds., New York, Marcel Dekker, Inc., pp. 617–668, 1995. Rebuck, A.S. and Woodley, W.E., Ventilatory effects of hypoxia and their dependence on PCO2, J. Appl. Physiol. 38, 16–19, 1975. Easton, P.A. and Anthonisen, N.R., Carbon dioxide effects on the ventilatory response to sustained hypoxia, J. Appl. Physiol. 64, 1451–1456, 1988. Powell, F.L., Milsom, W.K. and Mitchell, G.S., Time domains of the hypoxic ventilatory response, Respir. Physiol. 112, 123–134, 1998. Weiskopf, R.B. and Gabel, R.A., Depression of ventilation during hypoxia in man, J. Appl. Physiol. 39, 911–915, 1975. Easton, P.A., Slykerman, L.J. and Anthonisen, N.R., Ventilatory response to sustained hypoxia in normal adults, J. Appl. Physiol. 61, 906–911, 1986.

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Rebuck, A.S. and Campbell, J.M., A clinical method for assessing the ventilatory response to hypoxia, Am. Rev. Respir. Dis. 109, 345–350, 1974. Sjogren, D., Sollevi, A., Ebberyd, A. and Lindahl, S.G.E., Poikilocapnic hypoxic ventilatory response in humans during 0.85 MAC isoflurane anesthesia, Acta Anaesthesiol. Scand. 38, 149–155, 1994. Sjogren, D., Sollevi, A., Ebberyd, A. and Lindahl, S.G., Isoflurane anaesthesia (0.6 MAC), hypoxic ventilatory responses in humans, Acta. Anaesthesiol. Scand. 39, 17–22, 1995. Sjogren, D., Lindahl, S.G., Gottlieb, C. and Sollevi, A., Ventilatory responses to acute and sustained hypoxia during sevoflurane anesthesia in women, Anesth. Analg. 89, 209–214, 1999. Lloyd, B.B., Jukes, M.G.M. and Cunningham, D.J.C., The relation between alveolar oxygen pressure and the respiratory response to carbon dioxide in man, Q. J. Exp. Physiol. 43, 214–227, 1958. Weil, J.V., Bryne-Quinn, E., Sodal, I.E., Friesen, W.O., Underhill, B., Filley, G.F. and Grover, R.F., Hypoxic ventilatory drive in normal man, J. Clin. Investig. 49, 1061–1072, 1970. Weil, J.V. and Zwillich, C.W., Assessment of ventilatory response to hypoxia: Methods and interpretation, Chest 70, 124–128, 1976. van Klaveren, R.J. and Demedts, M., A mathematical and physiological evaluation of the different hypoxic response models in normal man, Respir. Physiol. 113, 123–133, 1998. Kronenberg, R., Hamilton, F.N., Gabel, R., Hickey, R., Read, D.J.C. and Severinghaus, J.W., Comparison of three methods for quantitating respiratory response to hypoxia in man, Respir. Physiol. 16, 109–125, 1972. Smith, W.D., Poulin, M.J., Paterson, D.H. and Cunningham, D.A., Dynamic ventilatory response to acute isocapnic hypoxia in septuagenarians, Exp. Physiol. 86, 117–126, 2001. Ward, D.S., Dahan, A. and Mann, C.B., Modelling the dynamic ventilatory response to hypoxia in humans, Ann. Biomed. Eng. 20, 181–194, 1992. Painter, R., Khamnei, S. and Robbins, P., A mathematical model of the human ventilatory response to isocapnic hypoxia, J. Appl. Physiol. 74, 2007–2015, 1993. Easton, P.A., Slykerman, L.J. and Anthonisen, N.R., Recovery of the ventilatory response to hypoxia in normal adults, J. Appl. Physiol. 64, 521–528, 1988. Cartwright, C.R., Henson, L.C. and Ward, D.S., Effects of alfentanil on the ventilatory response to sustained hypoxia, Anesthesiology 89, 612–619, 1998. Dahan, A., Ward, D.S, van den Elsen, M., Temp, J. and Berkenbosch, A., Influence of reduced carotid body drive during sustained hypoxia on hypoxic depression of ventilation in humans, J. Appl. Physiol. 81, 565–572, 1996. Easton, P.A. and Anthonisen, N.R., Ventilatory response to sustained hypoxia after pretreatment with aminophylline, J. Appl. Physiol. 64, 1445–1450, 1988. Hillman, D.R., Platt, P.R. and Eastwood, P.R., The upper airway during anaesthesia, Br. J. Anaesth. 91, 31–39, 2003.

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5 Response Surface Modeling of Drug Interactions: Model Selection and Multimodel Inference Using the Bootstrap

ERIK OLOFSEN and ALBERT DAHAN Leiden University Medical Center The Netherlands

I.

Introduction

In clinical practice, drugs are often administered in combination to optimize the balance between their therapeutic and adverse effects. Ideally their interaction is synergistic (supra-additive) with respect to the desired effect and only additive or even antagonistic (infra-additive) with respect to their side effects. We studied depression of ventilation and the ventilatory response to hypoxia during combined sevoflurane and alfentanil administration [1] and depression of ventilation and the ventilatory response to hypercapnia during combined propofol and alfentanil administration [2]. To that end, we developed response surface models that capture the relationships between drug concentrations and resulting effects. In the following, we will focus on possible formulations of response surface models and on how to choose between competing models.

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Pharmacodynamic Interaction Models

Pharmacodynamic interaction models attempt to describe the relationship between the concentrations Ci of n simultaneously administered drugs and a resulting measurable effect parameter E under study, e.g., ventilation. If the drugs have the effect of depression from a baseline E0, one can write E ¼ E0 f ðC1 , C2 , . . . , Cn Þ þ 2

ð5:1Þ

where the function f represents the knowledge postulated about the relationship and 2 denotes unexplained variability in measurements of the effect. The function f, visualized in n þ 1-dimensional space, will be referred to as a response surface. A. The Richards Model

The function discussed by Richards [3] attempts to model reaction rates rather than the equilibrium situation, but it may be of value for empirical models. It can be written as

C EðCÞ ¼ E0 1 C50

!!1= 1 1 2

ð5:2Þ

where C50 is the concentration and E ¼ ð1=2ÞE0, and and are shape parameters. When ¼ 1, the equation reduces to the familiar inhibitory sigmoid Emax model EðCÞ ¼ E0

1 1 þ ðC=C50 Þ

ð5:3Þ

while when ¼ 1, it reduces to

C EðCÞ ¼ E0 1 C50

1 2

ð5:4Þ

In the following, the latter model will be referred to as the power model. Parameter C50 may be poorly estimable if it lies outside the concentration range used in the study. It may therefore be better to write 1= C EðCÞ ¼ E0 1 1 l Ch

ð5:5Þ

Response Surface Modeling of Drug Interactions

135

where is the fraction of E0 attained when the concentration equals Ch, the midpoint of the concentration range in the study design. An extension for two drugs that respects Loewe additivity (i.e., additivity should hold, irrespective of the values of and , when C1 and C2 both denote concentrations of the same drug [4]) reads as 1= 1= 1= C1 C2 EðC1 , C2 Þ ¼ E0 1 þ 1 1 1 2 Ch,1 Ch,2 ð5:6Þ Its limit when ! 0 equals 1= 1= ! ! C1 1 C2 1 þ log log : 1 2 Ch,1 Ch,2

EðC1 ,C2 Þ ¼ E0 exp

ð5:7Þ B. A Mechanism-based Approach

Suppose that for simultaneous binding of drug molecules to a receptor d½RD ¼ kon ½D ½R koff ½RD dt

ð5:8Þ

where [D], [R] and [RD] are the drug, receptor, and bound receptor concentrations, respectively. For an inhibitory effect model, where effect measure E ranges from E0 to zero, one usually writes ½RT ½RD ½RD ¼ E0 1 E ¼ E0 ½RT ½RT

ð5:9Þ

where [RT ] is the total receptor concentration. Let us instead assume ½RD E ¼ E0 1 ½RX

ð5:10Þ

where [RX ] may possibly be much smaller than [RT ] so that E may be reduced to zero when only a fraction of the receptors is occupied, similar to the concept of receptor reserve. So dE E0 d½RD E0 ¼ ¼ ðkon ½D ½R koff ½RDÞ dt dt ½RX ½RX

ð5:11Þ

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with ½R ¼ ½RT ½RD

and

E ½RD ¼ ½RX 1 E0

ð5:12Þ

which gives dE ½RT ¼ kon ½D E0 ðE0 E Þ þ koff ðE0 E Þ dt ½RX

ð5:13Þ

When [RX ] ¼ [RT ], this reduces to dE ¼ kon ½D E þ koff ðE0 E Þ: dt

ð5:14Þ

In steady-state this yields E ¼ E0

1 , 1 þ ðkon =koff Þ ½D

ð5:15Þ

which is equivalent to Eq. (5.3). However, when ½RX ½RT , ½RT = ½RX E0 ðE0 E Þ, and, by discarding the latter term and incorporation [RT ]/[RX ] in kon, Eq. (5.13) reduces to dE ¼ kon ½D E0 þ koff ðE0 E Þ dt

ð5:16Þ

Note that the interaction between E and D in Eq. (5.13) has disappeared so that the dynamics are closer to those of a classical effect-site model [5], although the presence of in the differential equation still makes them distinct. Furthermore, kon will be dominated by wash-in effects rather than receptor kinetics. In the steady-state we have kon E ¼ E0 1 ½D ð5:17Þ koff which is equivalent to Eq. (5.4). Steady-state relationships between those extremes can be found by introducing a parameter ¼ ½RX =½RT , so that EðCÞ ¼ E0

1 ð1 Þ K ðC=Ch Þ 1 þ K ðC=Ch Þ

with

K¼

1 1 ð1 Þ

ð5:18Þ

where C and denote, like in the previous section, concentration and the fraction of E0 attained when the concentration equals Ch, respectively. C. Modeling Interaction

The current state-of-the-art modeling of interaction is based on two fundamental ideas [6]. First, the combination of two drugs is considered


137

to act like a single drug with a certain concentration–effect relationship. Second, the properties of this virtual drug, and therefore the parameters of its concentration–effect relationship, are only dependent on the ratio of the concentrations of the two drugs, given by Q ¼ U1/(U1 þ U2), where U1 ¼ C1/Ch,1 and U2 ¼ C2/Ch,2. In the studies described below, interaction was incorporated by writing 1= 1= 1= C1 C2 EðC1 ,C2 Þ ¼ E0 1 þ IðQÞ 1 1 1 2 Ch,1 Ch,2 ð5:19Þ where I(Q) is a spline function with parameters Imax and Qmax, which denote the maximum value of the interaction and the value of Q at which I(Qmax) ¼ Imax, respectively. For further details, see Ref. [1]. III.

Model Selection and Multimodel Inference

In the previous section, we discussed two competing model formulations that differently link the same two extremes: the power model (Eq. (5.19) with ¼ 1) and the inhibitory sigmoid Emax model (Eq. (5.19) with ¼ 1). In order to determine which of those two models is best suited for our experimental data, we need an appropriate criterion of what is best. We will now briefly introduce the likelihood ratio test and Akaike’s information-theoretic criterion (AIC); for a complete discussion, see Ref. [7]. The likelihood ratio test is based on the statistic C2 ¼ 2 ðLLr LLf Þ

ð5:20Þ

where LLr and LLf are the maximized log-likelihoods of the reduced and full models, respectively, C2 is approximately 2 distributed with q degrees of freedom and q is the number of fixed parameters in the reduced model. It can be used to test the hypothesis that a parameter has a certain fixed value. However, the -level of the test is rather arbitrary, especially for a chain of nested models, and this criterion cannot be used for nonnested models. Alternatives are Akaike’s information-theoretic criterion (AIC) and its variant (AICc) with a correction for small sample sizes, which are given by AIC ¼ 2LL þ 2k AICc ¼ 2LL þ 2k

N Nk1

ð5:21Þ ð5:22Þ

where LL is the maximized log-likelihood of the model under consideration, k is the number of adjustable parameters and N is the number of samples.

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AIC is an estimate of (2 times) the relative information loss when truth is approximated by a model; therefore the model with the lowest AIC can be considered to be best. As truth is unknown, only relative values can be evaluated. The bias (equal to k) originates from the fact that the parameters of the model have to be estimated. It may well be that alternative models are almost equally suited to capture the information in the data and discarding them may result in biased and incorrectly precise parameter estimates and inferences. Models can be assigned [7] so-called Akaike weights which are given by expðð1=2Þi Þ w i ¼ PR r¼1 expðð1=2Þr Þ

ð5:23Þ

where R is the number of models, and i the Akaike differences i ¼ AICi AICmin

ð5:24Þ

and AICmin is the value of AIC for the best model (note that the wi do not depend on it). When parameters have equivalent interpretations across models, the weights can be used to obtain model-averaged estimates of parameters and their variances; otherwise the predicted expected response variables from each model may be averaged. When more than one data set is available, model weights can also be obtained by model selection frequencies which are given by the number of times a particular model is chosen for each data set. In the following example, multiple data sets are generated by means of Monte Carlo simulation. A. An Example

To illustrate the use of AIC, we now focus on the following simple model: yi ¼ a þ 2 i

ð5:25Þ

where yi ði ¼ 1, . . . , NÞ are samples, a is a constant and 2i are independent Gaussian variates with variance 2 : The maximum likelihood estimators of the parameters are well known, and given by a^ ¼

N 1X yi N i¼1

and

^ 2 ¼

N 1X ðyi a^ Þ2 N i¼1

ð5:26Þ

The question now arises whether the data support estimation of parameter a; it could be better to fix it at some value,pfor ffiffiffiffi example zero, which simplifies the model. From a^ and VARða^ Þ ¼ ^ 2 = N, a confidence interval can be constructed to test the hypothesis H0: a ¼ 0. However, we will proceed in


139

A

B 0.6 Selection frequency

Selection frequency

0.3

0.2

0.1

0

0.5 0.4 0.3 0.2 0.1 0

-2

-1

0

1 a

2

3

4

-2

0

1 a

2

3

4

C

0.8 Selection frequency

-1

0.6 0.4 0.2 0 -2

-1

0

1 a

2

3

4

Figure 5.1 Normalized selection frequencies for the illustrative model given by Eq. (5.25) using data from Monte Carlo simulations. A. Model set consisted of seven alternatives with fixed values of a. B. Model set consisted of M0 with fixed value of a ¼ 0 and M1 where a was adjustable. C. Model set consisted M0 with fixed value of a ¼ 1 and M1 where a was adjustable.

a different way, since in practice we can be sure that H0 is false, and our aim is to minimize the loss of information that occurs when the data are approximated by a model. Monte Carlo simulation is a general method for assessing the properties of estimators [8]. From the model, B ¼ 1,000 realizations were generated, with a ¼ 1, 2 ¼ 25, and N ¼ 25. Seven models were fitted to the data using NONMEM [9]. Parameter a was fixed to each of the values 2, 1, . . . , 4; 2 remained adjustable. The Akaike criterion was used to select the best model for each of the B realizations. Figure 5.1A shows the fraction of times each model was selected. The probability of selecting the model with a ¼ 0 was 0.241. This value corresponds nicely to the probability of 0.242 that a^ is between (0.5, 0.5) obtained from its theoretical distribution function (although this relationship is not necessarily exact).

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The model with a fixed value of a ¼ 1 is best. However, in practice, the value of a is unknown and the models with alternative values of a also have considerable weight. A more often used model set would consist of M0 where a is fixed to zero, together with M1 where a is adjustable. Figure 5.1B shows the probability density function of model selection and the corresponding value of a, estimated from 10,000 realizations. The best model was M0 with selection probability 0.6955. Again, alternative values of a have considerable weight. A possibility [7] here is to calculate a weighted value, namely a^ ¼ 0:6955 0 þ 0:3045 mean of alternative estimates of að2:044Þ 0:6225: So this procedure removes part of the bias caused by considering only the best model (a ¼ 0, while true a ¼ 1). It is also possible [7] to calculate a model-averaged value of the variance of a^ , since the variance of the fixed a ¼ 0, being zero, is surely underestimated. In this example, VARfa^ g 0:9783: Notice that the hypothesis H0: a ¼ 0 would not have been rejected, while the single model with fixed a ¼ 0 is not optimal. We could, by accident, have chosen M0 with fixed a ¼ 1. By using the procedure produced above, we find that the probability that AIC selects this model is approximately 0.8780, a^ 1:008 and VARfa^ g 0:070 (Figure 5.1C). Of course, the model set must be carefully defined a priori; it is statistically incorrect to locate a fixed value of a of M0 that minimizes its variance after the data have been collected. When it has to be located, it should be a free parameter or else many fixed values could be tried as the distribution of the model weights is approximately equivalent to the one of a^ if a were free (Figure 5.1A). There should be no variability in the way a fixed value of a is chosen; eliminating a parameter by fixing it to zero is a general way to simplify a model. It is clear that it is not appropriate to consider only the best model (with a ¼ 1) as this gives a false sense of precision. The larger the standard error of a when it is free, the more likely it can be fixed, as the data do not provide information about its value. When there is an estimate available from another study, this estimate could be incorporated using the Bayesian approach or again fixed if the uncertainty is negligible, such as for fundamental constants from physics. In practice, there is usually only one data set and model selection frequencies are obviously not available. Model selection or averaging could be done using the Akaike weights as defined above, but we will now consider estimating model selection frequencies by a method called the bootstrap. IV.

The Bootstrap

A. Introduction

The bootstrap is an alternative method for assessing the properties of estimators [10]. To explain the principle underlying the bootstrap, consider


141

the definition of the mean of a discrete probability distribution pk with K possible events: ¼

K X

k pk

ð5:27Þ

k¼1

In practice, the pk are unknown, but they can be estimated by the number of times k is observed divided by the total number of observations N, by assigning pi ¼ 1/N to each observation xi. Therefore can be estimated by the well-known formula ¼

N 1X xi N i¼1

ð5:28Þ

So the true distribution F (discrete or continuous) is approximated by an empirical distribution F^ consisting of weights 1/N at the observations, which can be plugged-in to obtain estimates of its parameters. As the observations xi are samples from F^, one realizes that repeating an experiment (for example to construct confidence intervals) can be simulated by resampling, with replacement, from F^: The estimator under study is applied to each replicate data set and hence its bias and variance can be assessed. This is important in particular when other means of estimating them are lacking, such as the properties of the median. The fact that this is possible may be surprising, but actually it is equally surprising that standard errors can be obtained from a maximum likelihood estimator which is also based on a single data set. Maximum likelihood estimators do have welldefined properties, but in general only asymptotically (so when the number of data points is infinite); the bootstrap can then be used to study them when the amount of data is small. But in the following, we will study bootstrapaided model selection in particular. B. An Example

We go back to the simple example of the previous section and study how the bootstrap can be employed to guide model selection. One data set was generated from the model with parameters given earlier. From this one data set, B ¼ 1,000 replicates were generated by sampling with replacement such that the number of samples remained N ¼ 25. To each replicate, the seven models were fitted and the fraction of times each model was selected according to the Akaike criterion is shown in Figure 5.2A. This distribution resembles the Normal distribution of a^ with a^ 0:468 and ^ 2 1:16 (these estimates are obtained from the single data set used for the bootstrap). Figure 5.2B and Figure 5.2C show the probability density functions of model selection and the corresponding value of a, estimated

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Selection frequency

Selection frequency

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from 10,000 realizations with the previously defined model sets. It is important here to forget that the true value of a ¼ 1 because in most life science experiments, the true values are unknown, and reality is always more complicated than the models under consideration. The best we can do is find a good approximation of the information contained in the data, and ensure that parameter estimates are associated with a proper measure of their uncertainties. In this example, we could select model M0 with confidence factor 0.8371, which differs from 0.6955 as obtained from the Monte Carlo simulations. However, this factor itself is a random variable and varies between experiments (just as a confidence interval does). The point is that the bootstrap enables us to obtain a measure of model selection uncertainty. Model selection frequencies are similar, but in general not equal to, the Akaike weights. In this example, the Akaike weight of model M0 ¼ 0.7480


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and model M1 has associated a^ ¼ 0:468: Further research is needed to assess their respective properties. However, a practical advantage of using the Akaike weights is that no computer-intensive bootstrap runs are needed. V.

Applications

A. Interaction between Sevoflurane and Alfentanil

Nine healthy male volunteers participated, after approval of the protocol by the local Human Ethics Committee, in a study designed to assess the effects of sevoflurane and alfentanil, given separately and in combination, on resting normoxic ventilation and on ventilation due to acute isocapnic hypoxia (duration of hypoxia ¼ 3 min, PETO2 ¼ 48 mmHg). The end-tidal concentrations of sevoflurane (Csev) used were 0, 0.1, 0.2 and 0.3%; the target alfentanil plasma concentrations (Calf) used were 0, 10, 20, 30, 40, and 50 ng/ml. The nature of the interaction was assessed for, among other things, the steady-state ventilation ðV_ i Þ and its dependence of the arterial blood saturation ðV_ i =Sp O2 Þ: For further details, see Ref. [1]. A population analysis using NONMEM [9] yielded a linear relationship between both sevoflurance and alfentanil concentration and (hyperisocapnic, normoxic) ventilation with synergistic interaction between sevoflurane and alfentanil. Parameter estimates were associated with a relatively large standard error and since C50 cannot be negative, the distributions were most probably skewed. We employed the bootstrap to assess these parameter distributions and confidence intervals (using the bias-corrected and accelerated (BCa) method [10]) from B ¼ 1,000 replicate data sets. Histograms of the parameter estimates are shown in Figure 5.3. The peaks in the bins at the maximum x-value correspond to quite a number of large parameter estimates and are another sign of their skewed distributions. The 50% and 95% confidence intervals were: C50, alf 62.8–92.2 and 45.9–360 ng/ml; C50, sev 1.14–2.81 and 0.567–308%; Imax 1.71–2.24 and 1.38–6.66. In particular, the C50 of sevoflurane was poorly determined form the data as the concentration range used in the study (0–0.4%) was well below the estimated C50. In Ref. [2], we found that transforming the model parameters from C50 to as described in Section II considerably improved parameter estimation. With this model, parameter Qmax could be fixed to 0.5, which means that interaction is maximal at the diagonal that crosses the concentration plane designed for the experiment, and is therefore not a coincidental value. Histograms of parameter estimates using the transformed model with fixed ¼ 1, fixed Q max ¼ 0.5 and free Imax are shown in Figure 5.4. The 50% and 95% confidence intervals

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were: alf 0.738–0.812 and 0.657–0.890; sev 0.849–0.922 and 0.757–0.971; Imax 1.27–1.52 and 0.969–1.97. The value of maximum interaction Imax was determined more precisely, but was also closer to 1. Furthermore, we wondered which model would best fit the data: the inhibitory sigmoid Emax model or the power model described in Section II. The bootstrap model selection frequencies in Table 5.1 provide answers to both questions: the information in the data was best captured by the sigmoid Emax model with interaction. Rather than discarding the alternative models, we keep all eight of them, but with weight factors determined by the model selection frequencies. Averaging model parameters across the different models is not meaningful here as some have different typical values. However, it is possible to obtain mode-averaged predictions, using the selected models and their parameters from each replicate data set, and to determine their median prediction, and 50%, 80%, and 90% prediction intervals as shown in Figure 5.5. It shows prediction intervals for the single drugs and for their combination when interaction is maximal (at Q ¼ Qmax). Note that the concentrations extend those used in the experiments by 50% so the intervals


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plotted in that range are extrapolations. Furthermore, the drug effects were normalized to baseline. Sevoflurane was less potent than alfentanil in depressing ventilation, also with respect to their interaction, since adding sevoflurane (going from the top left to the top right panel) has almost no

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effect, while adding alfentanil (going from the bottom to the top right panel) has a large effect. Parameter V_ i =Sp O2 , the hypoxic ventilatory response, was found to be linearly dependent on alfentanil and sevoflurane concentration, without interaction. In Figure 5.6 histograms of parameter estimates are shown from a bootstrap analysis with fixed ¼ 1, fixed Qmax ¼ 0.5 and free Imax. Parameter Imax was estimated to be very close to 1. Model selection frequencies are given in Table 5.2. In this case the linear, additive interaction model was best. The multimodel median response surface is shown in Figure 5.7; the prediction intervals are shown in Figure 5.8. The median curves show remarkable linearity, but the top curves (with smallest depression) show some sigmoidicity. The curvature at low concentrations

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Figure 5.6 Modeling depression of the hypoxic ventilatory response Vi =Sp O2 by sevolflurane and alfentanil: histograms for the estimates of parameters alf ðAÞ, sev ðBÞ and Imax (C).

Table 5.2 Depression of the hypoxic ventilatory response (Vi/SpO2) by sevoflurane and alfentanil: power and sigmoid Emax model selection frequencies obtained from 1,000 bootstrap data sets. The linear model is the power model with ¼ 1; additive interaction is present when Imax ¼ 1

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cannot be captured by the linear model. However, the sigmoid Emax model is a symmetric curve so that a curvature at low concentrations is always mirrored at high concentrations. The question now is what would happen at concentrations higher than those used in the experiments. It is most probably not true physiologically that parameter V_ i =Sp O2 will reach zero only at infinite drug concentrations. Furthermore, predictions with negative values should be admissible since the depressant effects of central hypoxia may more than counteract the effects of hypoxia at the peripheral chemoreceptors [1,11]. Multimodel inference allows us to deal with these possibilities and avoids falsely precise, and therefore less accurate, predictions.

B. Interaction between Propofol and Remifentanil

In another study, 22 healthy male volunteers participated after approval of the protocol by the local Human Ethics Committee. They received targetcontrolled infusions of propofol and remifentanil such that at least four concentration combinations were achieved. The target concentrations were chosen at evenly spaced design points between 0 and 2 ng/ml remifentanil

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and 0 and 2 mg=ml propofol. At these drug levels, ventilation and the ventilatory response to hypercapnia were measured. Response surfaces _ i and ventilation at a fixed end-tidal PCO of for resting ventilation V 2 55 mmHg V_ i,55 were constructed using a population analysis with NONMEM [9]. For further details, see Ref. [2]. In that study, we found that both V_ i and V_ i,55 were best described by the power model with free , fixed Qmax ¼ 0.5 and free Imax. Model selection frequencies (with free and fixed Qmax ¼ 0.5; number of replicate data sets B ¼ 1,000) are given in Table 5.3 for both V_ i and V_ i, 55 : Clearly, the power model with interaction is best, especially for V_ i,55 : As parameter distributions were approximately normal, showing their histrograms would not provide much information

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Table 5.3 Depression of ventilation (Vi) and ventilation at 55 mmHg (Vi,55) by propofol and remifentanil: power and sigmoid Emax model selection frequencies obtained from 1,000 bootstrap data sets. Additive interaction is present at Imax ¼ 1 Power Model

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beyond the standard errors. Baseline V_ i and V_ i,55 of the best models were 9.4 0.3 (SE) and 31.4 1.5 (SE), respectively. The multimodel median response surface for V_ i,55 obtained by the bootstrap model selections is shown in Figure 5.9; prediction intervals are shown in Figure 5.10 for V_ i and Figure 5.11 for V_ i,55 . The curves for V_ i,55 show a rapid decrease of the respiratory controller’s sensitivity to CO2 and subsequently follow the depression of baseline ventilation. Parameter Imax was 1.9 0.2 (SE) for V_ i and 1.2 0.1 (SE) for V_ i,55 of the best models. Propofol was less potent in depressing V_ i than V_ i,55, but the larger value of Imax partly compensates for this. Their interaction is such that adding propofol (going from the top left

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to the top right panels) has a small effect, while adding remifentanil (going from the bottom to the top right panels) has a large effect. VI.

Conclusions

Respiratory depression by combined administration of sevoflurane and alfentanil, or propofol and remifentanil, was described by response surface modeling. The adequacy of competing model formulations was assessed by a bootstrap-aided model selection strategy based on AIC. The bootstrap allowed us both to assess model selection stability and to obtain multimodel inferences such as predicting the balance between therapeutic and adverse effects. Multimodel inference

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avoids biased and incorrectly precise predictions based on a single, possibly marginally better, model. However, from our analyses we do conclude that the power model is more suitable to describe respiratory depression that the classical sigmoid Emax model, especially if we want to take loss of ventilatory stability and apnea into account. References 1.

Dahan, A., Nieuwenhuijs, D.J.F., Olofsen, E., Sarton, E.Y., Romberg, R.R. and Teppema, L.J., Response surface modeling of alfentanil–sevoflurane interaction on cardiorespiratory control and bispectral index, Anesthesiology 94, 982–991, 2001.

Response Surface Modeling of Drug Interactions 2.

3. 4. 5.

6.

7. 8. 9. 10. 11.

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Nieuwenhuijs, D.J.F., Olofsen, E., Romberg, R.R., Sarton, E.Y., Ward, D.S., Engbers, F.H.M., Vuyk, J., Mooren, R.A.G., Teppema, L.J. and Dahan, A., Response surface modeling of remifentanil–propofol interaction on cardiorespiratory control and bispectral index, Anesthesiology, 98, 312–322, 2003. Richards, F.J., A flexible growth function for empirical use, J. Exp. Botany 10, 290–300, 1959. Berenbaum, M.C., What is synergy? Pharm. Rev. 41, 93–141, 1989. Sheiner, L.B., Stanski, D.R., Vozeh, S., Miller, R.D. and Ham, J., Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine, Clin. Pharm. Ther. 24, 358–371, 1979. Minto, C.F., Schnider, T.W., Short, T.G., Gregg, K.M., Gentilini, A. and Shafer, S.L., Response surface model for anesthetic drug interactions, Anesthesiology 92, 1603–1616, 2000. Burnham, K.P. and Anderson, D.R., Model Selection and Multimodel Inference, 2nd edn., New York, Springer, 2002. Kotz, S. and Johnson, N.L., Encyclopedia of Statistical Science, Vol. 5., New York, Wiley, 1985. Beal, S.L. and Sheiner, L.B., NONMEM User’s Guides, Nonmen Project Group, University of California at San Francisco, San Francisco, 1999. Efron, B. and Tibshirani, R.J., An Introduction to the Bootstrap, New York, Chapman and Hall, 1993. Sarton, E.Y. and Dahan, A., Sites of respiratory action of opioids, in On the Study and Practice of Intravenous Anaesthetics, Vuyk, J., Engbers, F.H.M. and Groen-Mulder, S.M., eds., Dordrecht, The Netherlands, Kluwer Academic Publishers, 2000.

6 Respiratory Neuroplasticity: Respiratory Gases, Development, and Spinal Injury

DAVID D. FULLER and GORDON S. MITCHELL University of Wisconsin Madison, Wisconsin

RYAN W. BAVIS Assistant Professor of Biology Bates College Lewiston, Maine

I.

Introduction

Since prolonged failure of the respiratory control sysem is not compatible with life, the mechanisms underlying respiratory control must be robust under a wide range of conditions. However, a robust neural system need not be a rigid circuit. Neuroplasticity enables appropriate adaptations to frequent or chronic disturbances that initiate active modifications in respiratory control such as weight gain or loss, altitude exposure or injury [1]. Studies of respiratory plasticity are intended to reveal how and why experience or changing conditions influence the control of breathing. Further, the respiratory system provides an ideal model to explore fundamental mechanisms of neuroplasticity for at least two reasons. First, the respiratory neural control networks produce a spontaneous rhythmic and quantifiable motor output under a wide range of in vivo and in vitro conditions. Second, clear functional significance can be ascribed to respiratory motor output: it represents breathing. Many influential models of neuroplasticity (e.g., hippocampal long-term potentiation; [2,3]) lack these advantages. 155

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We have begun to make progress toward understanding the mechanisms and manifestations of respiratory neuroplasticity [1,4,5]. In this chapter, we will review the literature concerning three forms of respiratory plasticity including plasticity induced by altered respiratory gases in adults, plasticity specific to development, and plasticity following injuries to the spinal cord. These topics illustrate diverse mechanisms and functional significance of plasticity in respiratory control. Other interesting and important examples of respiratory plasticity are omitted to allow a more comprehensive review of these topics, which represent the most thoroughly studied aspects of respiratory neuroplasticity to date. The reader is also referred to a recent series of review articles on respiratory neuroplasticity [1,6–10] . These articles provide an in-depth overview, and illustrate the emerging interest and excitement regarding this topic. A. Plasticity vs. Modulation

Plasticity and modulation are related but distinct properties of neural systems [1]. These properties are not mutually exclusive, which may lead to confusion. For example, modulation of a neural process may induce or maintain altered system characteristics, thereby eliciting plasticity. To promote effective communication among respiratory physiologists, Mitchell and Johnson [1] proposed the following working definitions of plasticity and modulation pertaining to respiratory motor control: Plasticity: ‘‘a persistent change in the neural control system (morphology and/or function) based on prior experience.’’ Modulation: ‘a neurochemically induced alteration in synaptic strength or cellular properties, adjusting or even transforming neural network function.’ In other words, respiratory plasticity describes an effect that outlasts the stimulus for a period of seconds to years, whereas neuromodulation generally occurs on a relatively short time scale (e.g., seconds to minutes) and is rapidly reversed when the neuromodulator is no longer present and functionally active. Plasticity and modulation are not static processes; they are susceptible to modification by prevailing conditions or experience [11–13]. Metaplasticity (i.e., plastic plasticity) describes a change in the expression of plasticity due to experience [11,12,14]. Similarly, metamodulation refers to modulation of a modulatory process [1,13]. B. Where does Respiratory Neuroplasticity Occur?

The neurons that control breathing represent a complex, integrative network (Figure 6.1). Redundancy in respiratory control mechanisms is evident in the mammalian central nervous system (CNS) [7,15,16], and

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Figure 6.1 A. Potential sites of plasticity in respiratory motor control. Alterations in respiratory motor output could reflect neuroplasticity at multiple sites including: (1) peripheral chemoreceptors; (2) the nucleus of the solitary tract; (3) the ventral respiratory group, including the pre-Bo¨tzinger complex; (4) supramedullary structures including the cerebral cortex, thalamus and cerebellum; (5) afferent inputs to the spinal cord; (6) respiratory motoneurons or local interneurons (see Figure 6.1B); or (7) the neuromuscular junction. Each of these possibilities is discussed in the text. B. Potential cellular and synaptic mechanisms underlying respiratory motor plasticity. Respiratory neuroplasticity at any site may result from the cellular/synaptic mechanisms exemplified here for a respiratory neuron located in the spinal cord. Plasticity may involve multiple mechanisms including: (1) changes in neuron properties such as somal size and membrane properties (e.g., resistance, rheobase current, etc.); (2) changes in synaptic efficacy induced by neuromodulators such as serotonin (see Figure 6.2); (3) alterations in synaptic efficacy due to prior activity within that synapse (i.e., activity-dependent synaptic plasticity); (4) revelation of pre-existing synaptic connections that were previously ineffective (i.e., silent synapses); (5) alterations in neuromodulator function due to changes in the neuromodulator (i.e., metaplasticity) or in release, reuptake and/or receptor activation; (6) growth of new synapses (excitatory or inhibitory) or changes in arborization of existing connections (i.e., sprouting).

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this redundancy has a physiological advantage: failure in a single component will not necessarily cripple the system. Similarly, plasticity in respiratory control need not be restricted to a discrete location. Sites suspected to contribute to different models of respiratory plasticity include peripheral chemoreceptors or chemosensory neurons [6,17–19], the nucleus of the solitary tract [20–22], pre-motoneurons, modulatory neurons (e.g., serotonergic or noradrenergic; [1,10,23]), pontine respiratory neurons [24,25], brainstem rhythm-generating neurons [26,27], spinal neurons ([28–30]; Figure 6.1), and the neuromuscular junction [9,31]. Plasticity rostral to the brainstem may also impact respiratory control as shown by classical and operant conditioning studies [32–36]. Figure 6.1 presents a simplified scheme for respiratory control and identifies potential sites of respiratory neuroplasticity; presumably plasticity can occur at any level in the network, and possibly many levels at once. II.

Plasticity Induced by Respiratory Gases in Adult Mammals

Changes in respiratory gases and respiratory plasticity are of particular interest for several reasons. First, it is common for humans to experience alterations in blood gases, particularly during pulmonary disease, sleepdisordered breathing or ascent to altitude. In healthy individuals, plasticity may optimize or smooth respiratory motor output during or following alterations in respiratory gases. A failure of plasticity may contribute to disorders such as obstructive sleep apnea (OSA) or sudden infant death syndrome (SIDS). Here we review the ventilatory response during and following acute and chronic exposure to both sustained and intermittent alterations in inspired oxygen and carbon dioxide. Acute has been defined as one hour or less [37]. The distinction between sustained and intermittent exposures is important since it has become clear that the specific pattern of respiratory stimulation can be an important determinant of the forms of plasticity that will be induced [4,38]. A. Hypoxia

Reductions in arterial oxygen tension activate a complex feedback system that typically increases alveolar ventilation in adults. The primary source for reflex ventilatory stimulation during hypoxia is the carotid body [37]. Hypoxia also stimulates chemosensitive cells in the aortic bodies [39,40] and some neurons in the brainstem [41–43], although their contributions to breathing under normal circumstances are either minimal or not understood. However, their minimal role in normal conditions does not rule out increased contributions to the control of breathing due to


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plasticity. For example, the aortic chemoreceptors become increasingly important following carotid denervation, particularly in young animals [7,44,45]. The nucleus of the solitary tract in the medulla provides the initial central synaptic relay for peripheral chemoafferent neurons. Subsequent projections include the ventrolateral medulla and midline raphe neurons [46], ultimately reaching respiratory motoneurons or other CNS regions including the cerebellum [47,48]. Acute Hypoxia (51 h)

The acute hypoxic ventilatory response is characterized by a hyperbolic increase in minute ventilation as the partial pressure of arterial oxygen (PaO2) declines [37,49]. However, even during isocapnic hypoxia, the temporal pattern of breathing (i.e., changes in inspiratory tidal volume vs. frequency) is variable within and among species [37,49]. Certain neurochemicals that contribute to the acute hypoxic response have been reviewed in detail [21,50]. Following the acute increase in ventilation, hypoxia evokes several time-dependent changes in respiratory motor output including short-term potentiation (STP), short-term depression (STD), and hypoxic ventilatory depression (HVD). These responses represent discrete, time-dependent mechanisms that depend on the severity, duration and pattern of hypoxia [37,49,51]. One consideration is whether the underlying mechanisms represent modulation or plasticity. For example, STP (see below) occurs within seconds to minutes of the initial acute response. Increases in ventilation during STP could represent plasticity initiated during the early stages of chemoafferent input. Alternatively, ongoing modulation of chemoafferent stimulation could also produce STP. Resolution will require a more thorough understanding of the mechanisms underlying these respiratory responses. Short-term potentiation (STP)

Slow (second to minute) increases in ventilation following the initial, abrupt increase in ventilation during hypoxia are known as STP [49]. Following hypoxia, the slow decline of respiratory motor output towards pre-hypoxia baseline values may represent decay of the STP mechanism [49,51]. However, the onset of STP is more rapid than the decay in ventilation following hypoxia [52], which may indicate that the decay in ventilation occurs via a distinct mechanism. STP has been demonstrated in awake humans [53,54], goats [55], ducks [56], and mice [57–59], and anesthetized cats [52], dogs [28,60], and rats [61,62]. It is usually expressed as an increase in tidal volume or respiratory neurogram amplitude (vs. frequency; [49]) after the acute hypoxic response. The physiological significance of STP is unknown, but this mechanism may ‘smooth’ ventilation and promote

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stability of respiratory motor output during and following hypoxia [51,63]. Failure of the STP mechanism is associated with pathologies including OSA and congestive heart failure since neither patient population exhibits STP during or following hypoxia [64,65]. STP can be evoked by electrical stimulation of carotid chemoafferent neurons, indicating that it results from a central neural mechanism [20,51,61]. The manifestation of STP is modified by vagal inputs [60], the magnitude and duration of the hypoxic response [66,66a], and prevailing CO2 levels [55,67]. STP may be genetically determined as it is absent in certain mouse strains [57]. Several laboratories have attempted to identify molecules necessary for the expression of STP. Millhorn et al. [68] showed that serotonergic, dopaminergic, and noradrenergic drugs do not affect STP in anesthetized cats. Since nitric oxide (NO) is important for other forms of plasticity, several investigators have hypothesized that it plays a role in STP [21,58,59]. STP is not seen in mice treated with a nitric oxide synthase-1 (NOS-1) inhibitor [69] or in mutant mice deficient in NOS-1 [69]. Kline et al. [69] suggest that NO alters ionic conductances in central respiratory neurons during hypoxia, resulting in STP. Further, NO may be involved in STP through the formation of nitrosothiols [70]. An N-methyl-D-aspartate (NMDA) receptor involvement in STP is suggested by observations that STP is attenuated by NMDA receptor antagonists in anesthetized rats, although the location of the relevant receptors is uncertain [62,63,71]. Other postulated but unproven mechanisms of respiratory STP include calcium accumulation in premotoneurons [52] and substance P release in the nucleus tractus solitarius (NTS) [20]. Phrenic STP may reflect a spinal mechanism, possibly within bulbospinal synaptic connections to spinal phrenic motoneurons [28]. At least three experiments have produced data consistent with this hypothesis [28,29,72]. McCrimmon and colleagues [28] showed that descending synaptic inputs to phrenic motoneurons can be potentiated via NMDA receptor-dependent mechanisms. A short latency (1–2 ms) phrenic compound action potential was uniquely revealed following paired pulse spinal cord stimulation. The new potential was eliminated after systemic administration of the NMDA antagonist MK-801, consistent with the NMDA dependence of STP [62]. A form of STP is induced by high frequency (100 Hz) stimulation of the C1–C2 lateral funiculus in anesthetized rats [29]. Intracellular recordings indicate that short-lasting phrenic motoneuron depolarization and enhanced excitatory post-synaptic potentials (EPSP) occur coincident with STP in this model, thereby providing strong evidence for a spinal site of action [29]. Potentiation of spinal inputs to respiratory motoneurons has also been demonstrated in an in vitro turtle preparation [72]. High-frequency electrical stimulation of spinal synaptic inputs to respiratory motoneurons caused a persistent potentiation (minutes) of


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evoked potential amplitude [72]; the short latency of the evoked potentials (5 1 ms) indicates that spinal synaptic transmission was enhanced. The studies discussed above demonstrate a spinal effect similar to STP, but currently it is unknown if similar mechanisms contribute to STP during and following hypoxia. Nevertheless, these data strongly suggest that plasticity in or around respiratory motoneurons may contribute to STP. For example, potentiation following spinal stimulation [28,29,72,73] could result from plasticity within motoneurons, interneurons, or pre-synaptic inputs (all of which could reflect a spinal mechanism). However, STP of supraspinal motor output (e.g., hypoglossal) could occur by a similar mechanism. Thus, one possibility is that STP arises within discrete motor nuclei (e.g., phrenic, hypoglossal), and does not reflect an overall increase in descending respiratory drive. This hypothesis is supported by studies showing that phrenic and hypoglossal STP can be dissociated in anesthetized cats [74]. Specifically, stimulation of the carotid sinus nerve (CSN) elicits STP in both phrenic and hypoglossal motor output, whereas superior laryngeal nerve stimulation evoked STP of hypoglossal, but not phrenic, motor output. Since the hypoglossal motor output was uniquely affected following superior laryngeal nerve stimulation, STP must be induced independently in different respiratory motor outputs, although the cellular/synaptic mechanisms may be the same. Hypoxic ventilatory depression (HVD) or ‘roll-off’

When hypoxia is sustained for minutes to hours, HVD develops (also known as hypoxic ventilatory roll-off; [37,75]). In adult humans, steadystate isocapnic hypoxia induces an initial acute increase in ventilation that peaks after approximately 3–5 min. This peak is followed by a ‘roll-off’ to a new, lower ventilation that is subsequently maintained [37,75]. As reviewed by Bisgard and Forster [75], potential mechanisms underlying HVD include (1) altered pulmonary mechanics; (2) reduced peripheral (carotid body) chemosensitivity; (3) a direct effect of carotid chemoafferent neurons on medullary excitability; (4) cerebral hypocapnia and/or alkalosis; (5) direct effects of hypoxia on the CNS (i.e., hypoxic brain depression), or (6) reduction in metabolic rate during hypoxia. The mechanism(s) of HVD have not yet been resolved, but there is general agreement that changes in pulmonary mechanics, altered metabolic rate or hypocapnia cannot explain HVD in most circumstances [37,49]. Furthermore, most experimental evidence suggests that a reduction in peripheral chemosensitivity cannot explain HVD, at least in adult animals [37]. The preponderance of evidence indicates that HVD results from a central neural mechanism activated by input from peripheral chemoreceptors. The neurochemicals required for HVD remain under investigation. Serotonin and/or opioids are not necessary for HVD [37]. However adenosine, dopamine, GABA, protein kinase C (PKC), and platelet-derived

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growth factor b (PDGF-b) are strong candidate molecules. An adenosine receptor antagonist (aminophylline) attenuates HVD in humans [76]. However, another adenosine receptor antagonist, theophylline, does not prevent HVD in goats [77]. Dopamine may be involved in HVD because the dopamine antagonist haloperidol blocks HVD in cats [78]. A build-up of the inhibitory neurotransmitter GABA has also been implicated in HVD [37]. The decline in phrenic motor output during 20 min of hypoxia (10% O2) corresponds to a gradual increase in GABA in the ventrolateral medulla [79]. Gozal and Gozal [80] showed that a ventilatory decline occurs in parallel to reductions in NTS PKC activity in hypoxic rats. Because PKC may facilitate respiratory motor output during hypoxia, it was suggested that reductions in PKC may contribute to HVD. Based on known links among PKC, NMDA receptors, and PDGF-b, Gozal and colleagues hypothesized that hypoxia triggers PDGF-b release in the brainstem, thereby contributing to HVD. They showed that pharmacological inhibition of PDGF-b receptors abolishes HVD in mice [80a]. The overall picture that emerges is that HVD may involve a complex interaction between multiple neurochemicals, and may reflect several distinct mechanisms. Short-term depression (STD) and post-hypoxia frequency decline (PHFD)

Another time-dependent respiratory response during acute hypoxia is STD [61]. Although similar to HVD, STD was tentatively classified as a distinct mechanism by Powell et al. [49]. The primary difference is that STD is expressed as a decline in burst frequency (vs. tidal volume in HVD) and is seen within seconds to minutes of the acute hypoxic response [49,61]. STD has been reported only in rats and little is known about the underlying mechanism. However, a variant of STD is observed following a single hypoxic episode. A short-term reduction in respiratory burst frequency is often observed in anesthetized adult and geriatric rats, regardless of sex [61,81–84]. This decline in respiratory burst frequency is known as PHFD [81] and occurs due to an increase in the interval between inspiratory bursts (i.e., increased expiratory time) with little change in inspiratory burst duration. As such, the underlying mechanism may reflect potentiation of expiratory duration rather than inhibition of inspiratory drive per se [24]. Post-hypoxia frequency decline may not require carotid chemoafferent input since it is evoked by hypoxia in carotid denervated, awake rats [85]. However, persistent hypocapnia following hypoxia was not ruled out as a cause of PHFD in these experiments [85]. PHFD is abolished following lesions in the ventrolateral pons (A5 region; [81]). Moreover, A5 pontine electrical stimulation decreases inspiratory burst frequency in anesthetized rats [24]. Within the ventrolateral pons, noradrenergic neurons with respiratory related activity have been identified [86]. Taken together, the works of Guyenet et al. [86] and Dick and Coles [24] suggest that pontine adrenergic mechanisms are necessary for PHFD. Systemic a-2 adrenergic


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receptor antagonism attenuates PHFD in anesthetized rats [82], and PHFD is mimicked by systemic application of a-2 adrenergic agonists [87]. In contrast, intracerebroventricular injection of a different a-2 antagonist does not alter PHFD in rats [88]. Discrepancies between these reports may reflect different populations of laboratory rats (i.e., genetic differences; [89,90]), differences in experimental protocol, or differences in drug delivery, efficacy or specificity. Serotonergic and glutamatergic mechanisms may also influence PHFD. Pre-treatment with a 5HT2 receptor antagonist (ketanserin) accentuates PHFD in anesthetized rats [91]). The 5HT1 receptor agonist 5-carboxamidotryptamine also increased PHFD, an effect most likely attributable to diminished raphe neuron activity following activation of autoinhibitory serotonin type I receptors [91]. Excitatory glutamatergic transmission also influences PHFD because it is prevented by brainstem application of NMDA receptor antagonists [92]. Finally, PHFD in rats is subject to a degree of metaplasticity since prior hypoxic episodes diminish its expression following subsequent hypoxic exposures [82,93]. In summary, PHFD may result from complex interactions between different neuromodulatory systems (e.g., serotonergic, adrenergic, glutamatergic). The ventrolateral pons may be a critical neuroanatomical location for PHFD [24]. Brief Intermittent Hypoxia

When hypoxia is experienced in an intermittent or episodic pattern, unique forms of plasticity are revealed [4,19,94]. Following brief but continuous hypoxic exposures, respiratory motor output (electroneurograms or electromyograms) or ventilation return to pre-hypoxic baseline within minutes [37,94]. In contrast, brief periods of episodic hypoxia elicit a longlasting (minutes to hours) enhancement of inspiratory motor output known as respiratory long-term facilitation (LTF, Figure 6.2; [49,61]). LTF is usually expressed as increased inspiratory motor output or tidal volume in anesthetized animals [4,95], and as an increase in breathing frequency in awake animals [58,59,96,97]. Long-term facilitation is an excellent example of pattern sensitivity in respiratory plasticity since comparable periods of continuous hypoxic stimulation do not produce LTF [38,98,99]. The magnitude and pattern (augmenting vs. decrementing; tidal volume vs. frequency) of LTF are variable between preparations and laboratories. However, the fundamental observation is consistent: respiratory motor output (e.g., electroneurograms, electromyograms, tidal volume or frequency) remains elevated for a sustained period following episodic chemoafferent stimulation [4,95]. LTF has been demonstrated in sleeping humans [100–102], awake ducks [56], goats [96], dogs [103], rats [97,104] and mice [58], and anesthetized cats [52]

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A

Long term facilitation

Baseline 50% O2 0 min 11% O2

60 min

5min

B Serotonin release

Respiratory drive

Figure 6.2 Phrenic long-term facilitation (LTF). A. Representative tracing of phrenic LTF in an anesthetized, paralyzed, vagotomized, and ventilated rat. A typical experiment consists of three, 5-min bouts of isocapnic hypoxia followed by 1 h of isocapnic hyperoxia (lower tracing). Amplitude of the integrated phrenic activity (upper trace) increases during each hypoxic exposure, but returns to near baseline (dotted line) upon return to hyperoxia. Following the third hypoxic exposure, phrenic activity gradually increases; this increase in phrenic motor output is LTF. B. Working model for phrenic LTF. We propose that phrenic LTF results (at least in part) from potentiation of excitatory glutamatergic inputs to phrenic motoneurons and therefore represents a spinal mechanism (Data from Refs. 1, 4, and 95). In this model, intermittent hypoxia causes intermittent release of serotonin, thereby activating serotonin type 2 (5-HT2) receptors on phrenic motoneurons. Activation of metabotropic 5-HT2 receptors initiates intracellular signaling cascades that lead to increased translation of new proteins, such as BDNF. These proteins may act on the phrenic motoneuron, or on excitatory glutamatergic bulbospinal neurons, thus enhancing glutamate-dependent currents during inspiration. Similar mechanisms likely contribute to LTF in other respiratory motor outputs (e.g., hypoglossal, intercostal).


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and rats [4,95]. It should be noted that certain preparations or protocols have failed to yield LTF [104–106]. The reasons underlying this apparent contradiction are not clear in all cases, but LTF is susceptible to modulation and metaplasticity. Accordingly, the presence (or absence) of certain factors could eliminate (or enhance) LTF expression. For example, the magnitude of LTF is altered by the severity, duration, and number of hypoxic episodes [104,107] as well as the magnitude of the acute hypoxic response [95,108]. LTF is also influenced by age, sex, sex hormones, the estrus cycle [109,110], and genetics [90,111]. The probability of LTF also decreases as baseline respiratory motor output increases [95]. That is, if the difference in motor activity between baseline and maximum (e.g., maximal hypercapnic ventilatory response) conditions is (relatively) small, the capacity to exhibit plasticity may also be small (i.e., minimal LTF). Therefore, conditions which increase respiratory drive at baseline (e.g., mild hypercapnia; respiratory tract infections) may limit or prevent LTF. Our laboratory has observed on several occasions that respiratory viral or bacterial infections in our rat colony substantially reduce the probability of evoking phrenic LTF (Mitchell, G.S. and colleagues, unpublished observations). However, the latter observation remains anecdotal and awaits systematic investigation. Long-term facilitation is more robust in anesthetized preparations in which many confounding variables (e.g., PaCO2) can be rigorously controlled [95]. Vagal influences, which are often absent in reduced preparations, are inhibitory to or obscure LTF [112]. Nevertheless, LTF can be evoked in awake, vagally intact animals [59,96,97,104]. LTF has not been shown in awake humans [106], but can be activated during sleep [100,102]. In particular, sleeping humans who demonstrate significant inspiratory flow limitation develop substantial LTF following hypoxic episodes. LTF of upper airway muscle activity (e.g., genioglossus) has been postulated to stabilize breathing in patients with sleep-disordered breathing [49,102], consistent with recent reports of upper airway muscle LTF in humans [101,102,113]. Pharmacologic studies indicate that LTF is serotonin (5-HT) dependent [91,114–116]. Further, serotonin 5-HT2 receptor activation is necessary during (but not following) bouts of hypoxia [117]. Thus, 5-HT2 receptor activation during hypoxia initiates events that lead to, and maintain LTF. This observation reconciles potential inhibitory effects of serotonin [118] with serotonin-dependent facilitation. Inhibitory effects (possibly via pre-synaptic 5-HT1 receptors [119], may be restricted to the period of serotonin exposure, whereas the excitatory effects initiated by 5-HT2 receptor activation persist following receptor activation [117]. Brain-derived neurotrophic factor (BDNF) plays a critical role in LTF in anesthetized rats [4]. BDNF is necessary and sufficient for some forms of synaptic plasticity, and BDNF increases serotonin levels and turnover (reviewed in Ref. [4]). Episodic hypoxia increases BDNF in the

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ventral cervical spinal cord through a serotonin-dependent, protein synthesis-dependent mechanism [120]. Further, BDNF seems to be necessary and sufficient for phrenic LTF because pharmacological blockade of its receptor (TrkB) prevents LTF and spinal application of BDNF mimics LTF [120a]. Long-term facilitation may require NO [19,59], a molecule that also contributes to breathing stability during hypoxia [69,121]. Awake mice show LTF following 3 episodes of hypoxia (7% O2), however, LTF is not present following pharmacological block of NOS-1 or in NOS-1 knockout mice [59]. Kline et al. [59] suggest that nitric oxide may permit LTF by modulating 5-HT release from raphe neurons or by prolonging the actions of 5-HT on respiratory neurons. Although NO may increase with 5-HT2 receptor activation [122], nitric oxide may also inhibit 5-HT2 receptor function [123]. Thus, the role of nitric oxide in LTF will require further investigation. Where does the mechanism of LTF reside? The seminal experiments by Millhorn et al. [114,124] suggest that LTF can be accounted for by a central neural mechanism because it was evoked by CSN electrical stimulation. However, LTF may not require carotid chemoafferent inputs since a form of LTF can be evoked by intermittent hypoxia in carotiddenervated rats, although the amplitude is significantly reduced [108]. Morris, Lindsey and colleagues [10,45,46,125,126] suggest that LTF may be a consequence of pre- and/or post-synaptic changes in effective connectivity between inspiratory driver and premotoneurons in the medulla. Their data show that pontomedullary neural networks generate spatiotemporal patterns of synchrony, and enhanced synchrony may contribute to LTF [10]. However, responses of multiple respiratory neurons have been recorded only during the initial minutes of LTF [10]. The putative contribution of altered functional connectivity in the medulla to sustained LTF is unknown. While LTF can be evoked in different respiratory motor outputs (e.g., hypoglossal, phrenic, intercostals), a growing body of evidence suggests that in the case of phrenic activity, serotonin may act via spinal mechanisms to initiate phrenic LTF. First, hypoxia elicits serotonin release in the phrenic motor nucleus (detected by carbon fiber electrodes; [23]) and serotonergic terminals are present in the region of the phrenic motor nucleus (for review see Ref. [118]). Second, spinal application of methysergide (a serotonin receptor antagonist) or protein synthesis inhibitors blocks phrenic but not hypoglossal LTF [120], indicating a spinal site of action. Third, short latency phrenic potentials evoked by spinal stimulation at C2 are augmented following LTF [127]. Finally, a pre-conditioning lesion (cervical dorsal rhizotomy—CDR) that increases serotonin terminal density near identified phrenic motoneurons also enhances phrenic LTF in anesthetized rats [128]. Collectively, these observations support the hypothesis that phrenic LTF results (in large part) from spinal cord mechanisms. However, this


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hypothesis does not preclude additional effects at supra-spinal or peripheral locations [4,10,19,114,124]. We recently proposed working network and cellular/synaptic models of phrenic LTF (Figure 6.2; [4,5,116,118]. First, caudal raphe neurons projecting to the brainstem and spinal cord are activated during hypoxia [129,130]. Serotonin release from raphe neurons [23] during (but not following) hypoxia [117] activates post-synaptic 5-HT2A receptors on phrenic motoneurons [131], leading to kinase activation and new protein synthesis (translation from existing mRNA). These new proteins enable LTF by strengthening descending excitatory bulbospinal inputs to phrenic motoneurons [4,120]. Since bulbospinal respiratory drive in adult rats involves both NMDA and non-NMDA receptors [132,133], we postulate that newly synthesized BDNF phosphorylates glutamate receptors in the spinal cord [134]. By phosphorylating glutamate receptors, increased currents or insertion of additional glutamate receptors into the postsynaptic membrane may result [135]. Potentiated glutamate receptor currents would amplify descending respiratory drive, increasing phrenic output for the same pre-synaptic glutamate release (i.e., LTF). BDNF could also increase pre-synaptic glutamate release [136]. Sustained Hypoxia (41 h)

Detailed reviews of this topic can be found in Dempsey and Forster [137], Bisgard and Neubauer [37], Bisgard and Forster [75], Bisgard [18] and Powell et al. [49,138]. Hypoxic exposures greater than 1 h lead to at least two time-dependent ventilatory responses. The first response is a progressive increase in ventilation that reaches a plateau within hours to days, depending on the species (ventilatory acclimatization to hypoxia, VAH; [49]). The new, steady-state ventilation often exceeds the acute hypoxic ventilatory response prior to VAH [37]. The second mechanism is revealed as a decrease in hypoxic chemosensitivity in long-term high altitude residents, and is not considered further in this section (for review see Ref. [139] Chapter 11 by Powell and Bickler in this volume). Ventilatory acclimatization to hypoxia is characterized by a progressive decrease in PaCO2 due to the increase in minute ventilation during hypoxia. The length of time required for complete VAH is variable among species, ranging from hours to days [37,75], potentially reflecting distinct mechanisms or different time courses of the same general mechanism. Bisgard [18] provides an overview of peripheral chemoreceptor function during and following sustained hypoxia. Goats experiencing carotid body hypoxia with systemic (including CNS) normoxia exhibit VAH [140]. VAH is unique to hypoxia in goats, as perfusion of isolated carotid bodies with hypercapnic blood did not evoke this response [141]. Further, progressive increases in carotid chemoafferent activity are observed

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during VAH in anesthetized goats [142] and cats [143]. Thus, carotid body mechanisms contribute to (or are solely responsible for) VAH in certain preparations [18]. What mechanisms are active within the carotid body? Carotid body neuropeptides, such as endothelin, change in a manner consistent with increased carotid body sensitivity during sustained hypoxia [144–146]. In contrast, carotid body dopaminergic and noradrenergic mechanisms may be upregulated during sustained hypoxia, changes that would seem to favor a reduction in carotid chemosensitivity [146a]. Thus, the increase in carotid body hypoxic sensitivity may reflect a complex balance between excitatory and inhibitory neuromodulators [18]. Powell et al. [138] review the evidence for CNS mechanisms of VAH. They propose that a given chemoafferent input to the CNS produces a greater hypoxic ventilatory response following sustained hypoxia (e.g., days). This suggestion is supported by data from rats showing that integrated hypoxic phrenic responses to chemoafferent nerve activation are enhanced following days to weeks of hypoxia [138,147]. Neurochemical mechanisms of this plasticity are unknown. Serotonin, which enables respiratory LTF following intermittent hypoxia, probably does not play a critical role in VAH [118]. Huey and Powell [148,149] investigated the role of dopamine in VAH. After an extensive series of experiments on rats, they concluded that alterations in dopaminergic function do not explain increased CNS gain of the hypoxic ventilatory response after sustained hypoxia. In summary, sustained hypoxia induces plasticity, manifest as an increase in the acute hypoxic ventilatory response. This process may be initially dominated by peripheral chemoreceptor sensitization [37], followed by progressively increasing contributions from the central neural integration of carotid chemoafferent neurons [147,150,151]. Chronic Intermittent Hypoxia (CIH)

Chronic intermittent hypoxia is a more common experience than sustained hypoxia [19]. The physiological effects of CIH have been studied since the 1930s [152]. Scientists in the former Soviet Union initiated these studies in an attempt to better prepare Soviet pilots for flights in open cockpits up to 6000 m. Serebrovskaya [152] provides an excellent summary of this early Soviet research. CIH reportedly has been used as a treatment for disorders ranging from depression to radiation sickness, although the mechanistic bases for these reported effects are unclear [152]. CIH has also been reported to enhance athletic performance [153]. However, there may be shortcomings that limit or constrain the potential of CIH as a therapeutic tool. For example, certain CIH protocols elicit pathophysiology such as systemic hypertension [154], altered sympathetic chemoreflexes [155] and hippocampal apoptosis [156,157].


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Plasticity induced by CIH may be determined by the pattern and duration of hypoxic exposures [4,19]. Three patterns of CIH will be discussed: 1.

Repeated hypoxic episodes lasting 5 1 min separated by periods of normoxia ranging from seconds to minutes. This pattern is similar to what is experienced by patients with central or OSAs. As a result, several laboratories have developed CIH protocols within these general guidelines [19,154]. 2. Hypoxic episodes lasting minutes separated by similar durations of normoxia. This type of CIH protocol has been used extensively in studies of respiratory neuroplasticity [4,158]. 3. Long-lasting (hours to days) periods of hypoxia separated by similar periods of normoxia (e.g., repeated, prolonged ascent to altitude).

CIH with short-duration hypoxia (51 min)

This pattern of CIH exposures is produced in the laboratory in an attempt to mimic the clinical condition of sleep apnea. However, apneic episodes in OSA patients are accompanied by mild hypercapnia as well, in effect producing an intermittent asphyxia (i.e., hypoxia and hypercapnia). Accordingly, it is important to ascertain differences between poikilocapnic vs. isocapnic or hypercapnic hypoxia, similar to OSA patients. Interestingly, the magnitude of post-apnea ventilation is highly correlated with the CO2 load during the preceding apneic episode [159]. This observation led to the suggestion that a CO2-dependent facilitation of respiratory motor output may occur transiently after apneic episodes [159]. Currently, the impact of OSA on respiratory control is controversial [19]. Some investigators report that ventilatory chemoresponses are enhanced in OSA patients, possibly contributing to ventilatory instability [160], whereas others report either no change [161,162] or a reduction in ventilatory chemosensitivity in OSA patients [163]. Peng et al. [107] measured hypoxic ventilatory responses following 10 days of CIH in rats (5% O2 for 15 sec, alternating with 21% O2 for 5 min, 8 h/day, 10 days). However, in this protocol, the 15-sec period of 5% O2 is followed by 4150 seconds of transient hypoxia. Rats were subsequently anesthetized and phrenic nerve activity was monitored during hypoxia. Rats treated with CIH had an almost 50% greater phrenic motor output during hypoxia than controls. This increase in the hypoxic phrenic response was mirrored by increases in carotid body chemosensitivity. Thus, the augmented hypoxic response could be accounted for, at least in part, by peripheral (carotid body) mechanisms. These data do not rule out central neural mechanisms [93]. Peng et al. [107] reported additional plasticity evoked by CIH. A robust (4200%) LTF of carotid chemoafferent activity

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could be evoked by episodic hypoxia (15 sec 5% O2, 5-min normoxia, 5-min intervals) in CIH-treated, but not control rats [107]. No obvious changes in carotid body morphology were found. Similar CIH protocols cause systemic hypertension in rats, although respiratory responses were not evaluated in these studies (reviewed in Ref. [154]). CIH with moderate-duration hypoxia (1 to 20 min)

Our laboratory developed a model of CIH in which rats experience one week of nocturnal intermittent hypoxia (11–12% O2/air at 5-min intervals, 12 h/night; [93]). The short-term and long-term phrenic responses to additional hypoxic episodes were examined in anesthetized, paralyzed, vagotomized, and artificially ventilated rats. CIH augments the short-term hypoxic phrenic response (during hypoxia), abolishes PHFD and enhances phrenic LTF by more than 100% [93]. Since these effects are reversed by methysergide, serotonin receptor activation is necessary to maintain these forms of plasticity [93]. However, ketanersin, a more selective 5-HT2 receptor antagonist, was less effective, suggesting an involvement of non5-HT2 serotonin receptors [93]. Since integrated phrenic responses to CSN stimulation are amplified following CIH [93], CIH-induced plasticity results, at least in part, from a central neural mechanism. This pattern of CIH may have therapeutic value, as it can restore blunted hypoxic phrenic responses following neonatal hyperoxia [164] and enhances existing, but ineffective, synaptic pathways below spinal cord injury [30]. Most studies of human responses to CIH have employed relatively short bouts of CIH (e.g., 1 h) repeated daily. The following CIH protocols have all been reported to enhance subsequent hypoxic ventilatory responses: 3 6 min hypoxia (end-tidal PO2 ¼ 35–50 mm Hg) separated by 4 min normoxia, 14 consecutive days [165]; 3–4 7 min progressive hypoxia (end-tidal PO2 reaching 35–40 mm Hg), 14 consecutive days [166]; 20 min isocapnic hypoxia per day, 14 consecutive days [167]. While these studies did not address the underlying mechanisms, they document that the hypoxic ventilatory response is subject to plasticity following CIH in humans. CIH with long-duration hypoxia (hours – days)

Several investigations have exposed humans to hypoxia for 1–2 h per day for approximately one to two weeks [152,168–170]. While these studies have protocol differences, they all report that the hypoxic ventilatory response was significantly enhanced following CIH. Another study followed Chilean miners commuting to work from sea level to 4500 m on a weekly basis [171]. A type of ventilatory acclimatization, characterized by an increased ventilatory response to hypoxia, began to emerge after 12 months of this CIH pattern. Elite athletes using the live high, train low approach [153] are another population exposed to CIH. There is some evidence that spending nights in simulated altitude environments and days at sea level (where the


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acute effects of altitude will not impair training) can improve athletic performance, presumably through adaptations in oxygen transport (e.g., increased red blood cell number). This pattern of CIH may also induce plasticity in respiratory control. For example, this pattern of CIH enhances acute hypoxic responses in humans [172]. To our knowledge, LTF has not been assessed following CIH in humans. In summary, neuroplasticity following CIH of various durations and patterns is manifest as an increase in the acute hypoxic response; most investigations suggest that the hypercapnic ventilatory response is not affected. CIH also induces a form of metaplasticity by enhancing LTF. The mechanisms underlying these changes may not be consistent across CIH paradigms. For example, repeated, short, intense hypoxic exposures may preferentially evoke peripheral (carotid body) plasticity while chronic exposure to longer, more moderate hypoxic bouts may induce central neuroplasticity. Confirmation of this hypothesis and elucidation of the detailed mechanisms underlying the effects of CIH will require further investigation. B. Hyperoxia

In mammals, hyperoxia (i.e., FiO2 4 0.21) is experienced only in experimental and clinical therapeutic conditions. There are no naturally occurring causes of hyperoxia in humans. Brief hyperoxia has been shown to induce plasticity in respiratory control [173,174]. A brief bout of hyperoxia (10 min, 100% O2) can enhance hypoxic ventilatory responses assessed shortly thereafter [173]. These authors speculated that the increased hypoxic ventilatory response reflected enhanced glutamate release in the nucleus of the solitary tract following hyperoxia. Gozal [174] extended these observations by demonstrating that a 10-min pre-exposure to hyperoxia (100% O2) caused a nitric oxide-dependent augmentation of the hypoxic ventilatory response in rats. Gozal speculated that activation of NMDA receptors led to NOS activation and potentiation of the hypoxic response. Sustained hyperoxia (100% O2; 12–60 h) blunts subsequent hypoxic responses in rats and cats [175–177] by reducing carotid but not aortic chemosensitivity [177,178]. The carotid body undergoes striking morphological changes during prolonged hyperoxia including type I cell necrosis [179,180]. Ren et al. [181] reported that 8 h of hyperoxia (end-tidal O2 ¼ 300 mm Hg) blunts the hypoxic ventilatory response in humans. In contrast, prolonged hyperoxia, either sustained or intermittent, does not blunt the hypoxic ventilatory response in humans [182]. Many studies of respiratory LTF have used anesthetized rats or cats with hyperoxic baseline conditions [83,84,90,94,116,117]. In these studies, the animals were ventilated with hyperoxic gas mixtures (FiO2 0.5) to prolong viability of the experimental preparation. One consequence of using

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a background of hyperoxia in episodic hypoxia studies is that, in addition to hypoxic episodes, animals are exposed to rapid rises in oxygen to hyperoxic levels at the end of each hypoxic bout. This led to an investigation of the effects of repeated, brief exposures to hyperoxia on resting respiratory motor output [108]. Although there was no evidence for time-dependent changes in phrenic activity following episodic hyperoxia (FiO2 ¼ 1.0), this finding does not preclude the possibility that episodic hyperoxia and/or exposure to reactive oxygen species (ROS) initiates other forms of respiratory plasticity. C. Hypercapnia

Hypercapnia results when alveolar ventilation is low relative to metabolic rate, such as following CNS injury (e.g., spinal cord injury), during central or OSAs, or chronic pulmonary diseases that exhibit CO2 retention (e.g., chronic obstructive pulmonary disease). Increases in inspired CO2 represent a potential cause of hypercapnia in closed environments (e.g., submarines, spacecraft) and are experienced by many burrowing species [183,184]. Although CO2 is a powerful respiratory stimulus and a primary determinant of respiratory motor output [185], it may also elicit inhibitory mechanisms of respiratory neuroplasticity as described below. Acute Sustained Hypercapnia

The initial ventilatory response to hypercapnia is an increase in tidal volume or its neural equivalent (phrenic neurogram or diaphragm EMG) with a lesser increase in burst frequency. Tidal volume generally reaches a plateau within a few minutes, and does not show much, if any, roll-off within this time frame. However, following a 25-min bout of hypercapnia, phrenic motor output was depressed for up to an hour in anesthetized rats [38]. This long-term depression (LTD) was evident as depressed phrenic burst amplitude for up to 60 min following hypercapnia. Phrenic burst frequency exhibited a transient (15-min) depression following hypercapnia, but then returned to baseline values. The mechanisms underlying LTD following sustained hypercapnia are pattern-sensitive since intermittent hypercapnia (of comparable cumulative duration) did not produce significant LTD in the same rat substrain [38]. Acute Intermittent Hypercapnia

Dong et al. [186] exposed anesthetized cats to intermittent 5% CO2 challenges. Facilitation of inspiratory output was seen during successive bouts of hypercapnia as inspiratory tidal volume and diaphragm electromyogram (EMG) activity increased despite concurrent decreases in frequency. Optical reflectance of the ventrolateral medullary surface was


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measured to provide an index of respiratory neural activity. Successive hypercapnic challenges increased the optical reflectance, suggesting that neural activity was enhanced by successive hypercapnic episodes. Unfortunately, long-term changes in respiratory motor output following episodic hypercapnia were not assessed [186]. Morris et al. [46] recorded phrenic motor output following episodic intracarotid injection of CO2-saturated saline. This intermittent hypercapnia protocol induced persistent facilitation of respiratory motor output similar to hypoxia-induced LTF (see above). However, intracarotid injections directly activate only carotid chemoreceptors with little, if any, direct effects on the central nervous system. Accordingly, these data do not reflect the effects of systemic or CNS hypercapnia on respiratory motor output. Episodic systemic hypercapnia induces LTD of respiratory motor output in anesthetized rats [187]. In this study, LTD was prevented or attenuated if rats were pretreated with a2-adrenergic receptor antagonists. Subsequently, Baker et al. [38] found that a similar pattern of hypercapnia caused a (non-significant) trend for LTD in anesthetized rats of a different substrain of Sprague–Dawley rats, suggesting possible genetic influences on LTD. Gozal and colleagues examined the ventilatory response to intermittent hypercapnia in awake humans [188,189]. Over the course of six, 2-min hypercapnic episodes (5% CO2) a significant shift in the pattern of breathing (increased tidal volume, decreased breathing frequency) with no change in the overall minute ventilation occurred. This effect was abolished if the interval between hypercapnic episodes was extended from 5 to 15 min, indicating that the underlying mechanism was sensitive to interval time. A similar change in breathing pattern during episodic hypercapnia occurs in healthy children, but not in pediatric OSA patients [189]. Gozal et al. speculate that the altered ventilatory response in pediatric OSA patients reflects plasticity induced by repeated asphyxic events during OSA. Thus, repeated episodes of hypercapnia (i.e., during apneic episodes) may change the subsequent pattern of ventilatory response to hypercapnia. Chronic Sustained Hypercapnia

The ventilatory response to chronic hypercapnia was investigated by exposing humans to 1.5% CO2 for 42 days [190]. Minute ventilation and alveolar CO2 were increased throughout the entire CO2 exposure although there was little change in these variables after the first few days. Alveolar CO2 and minute ventilation remained significantly elevated for 10 days upon return to normoxia. Dempsey and Forster [137] reviewed the existing literature on sustained hypercapnia in mammals. They concluded that the ventilatory response to chronic hypercapnia is biphasic [137,191]. After the acute onset of hypercapnia, ventilation remains elevated, but declines

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somewhat during subsequent days to weeks of hypercapnia. Recent studies in rats confirmed this conclusion [192] and indicate that ventilation during chronic hypercapnia follows the normal circadian rhythm [193]. Although arterial CO2 remains elevated for the duration of hypercapnia, ventilation, plasma and CSF pH can all return toward normal [137]. Accordingly, the mechanism for respiratory stimulation during chronic CO2 exposure remains controversial. During chronic hypercapnia, ventilatory sensitivity to CO2 decreases in mammals and birds [137,194], although exceptions have been reported [195]. Following chronic hypercapnia, ventilation and arterial CO2 can remain elevated for several weeks in humans upon return to normoxia [190]. However, CO2 chemoresponses return to control values within three to four weeks after return to normoxia [190]. Chronic Intermittent Hypercapnia

To our knowledge, there are no studies directly addressing the impact of chronic intermittent hypercapnia on respiratory plasticity. III.

Developmental Plasticity and the Control of Breathing

The expression of plasticity may depend on the age and/or developmental stage during which the environmental or experimental stimulus is presented. Developmental plasticity refers to plasticity that is specific to periods of development [1,6]. Although plasticity is not unique to development, immature animals may exhibit greater capacity for plasticity, including forms of plasticity that cannot be elicited in the mature animal or that persist for longer periods (e.g., weeks to years). This developmental specificity suggests the existence of critical periods (also known as developmental windows, Figure 6.3), when the phenotype is particularly sensitive to prevailing external or internal conditions. Critical periods may vary in their timing, duration and underlying mechanisms [196]. Understanding developmental plasticity is critical to appreciate normal development of respiratory control mechanisms, various respiratory disorders (e.g., sudden infant death), as well as phenotypes associated with altitude or burrowing life-styles. In the following sections, we briefly review developmental plasticity in mammalian respiratory control induced by altered respiratory gases, altered metabolic rates, neural injury (i.e., chemoafferent denervation), and stress. Further, we consider how this plasticity may relate to pathophysiology. Developmental plasticity in respiratory control is not limited to mammals and has been demonstrated in a variety of vertebrate and invertebrate species, emphasizing that this topic has evolutionary as well as biomedical significance.


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A. Time 250 200 (∆bl, %bl)

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l % 0% % % % tro 0% 60 60 6 60 60 ts) 6 2 4 3 1 4 a 4 – r ks ek ek ek 1– s 1 atric ee We We We k eri ks e W e e g e W ( W n Co

Figure 6.3 A. During development, there are critical periods (also known as developmental windows) during which respiratory phenotypes are sensitive to environmental conditions. B. The critical period for hyperoxia-induced developmental plasticity in rats extends into the second postnatal week. The figure shows phrenic nerve responses to isocapnic hypoxia (PaO2 40 mm Hg) in anesthetized rats exposed to 1–4 weeks of developmental hyperoxia (60% O2); control animals were raised in normoxia. Adult (3–5 month) and geriatric rats (14–15 month) exposed to 4 weeks of hyperoxia from birth have substantially blunted hypoxic phrenic responses. Adult rats exposed to developmental hyperoxia for either the first or second postnatal week (but not the third or fourth week) also have blunted hypoxic responses. Accordingly, the critical period for this plasticity includes the first two post-natal weeks. It is not known whether the critical period extends prenatally as well (figure compiled from previously published data). (Data from Refs. 251, 252.)

A. Prenatal Hypoxia

Exposing pregnant dams to the equivalent of 12% O2 throughout gestation causes persistent hyperventilation in the newborn rat [197]. These effects are similar to VAH in adult rats [198], although somewhat greater effects on metabolism are observed. Resting ventilation and the ventilation to metabolism ratio remains elevated at 1 and 3 (but not 9) weeks of age following prenatal hypoxia (10% O2; [199]). Thus prenatal hypoxia induces persistent hyperventilation of longer duration than VAH in adults [137].

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Prenatal hypoxia also has lasting effects on hypoxic ventilatory and metabolic responses [199], generally enhancing ventilation and abolishing metabolic changes during hypoxia at 1 and 3 weeks of age. These effects are transient, however, and the hypoxic ventilatory response is attenuated relative to normal by 9 weeks of age [199]. It is not known whether altered normoxic and hypoxic ventilation following prenatal hypoxia reflect plasticity in the control of breathing per se since blood gases were not measured in these studies; hypoxia-treated rats could have experienced more severe hypoxia and/or hypercapnia due to impairments in gas exchange. Effects of prenatal hypoxia could also result from changes in respiratory mechanics [200]. However, prenatal hypoxia alters the postnatal expression of dopamine, noradrenaline, and related enzymes in the carotid body and brainstem respiratory centers [199,201], suggesting neuroplasticity. Further, carotid body glomus cells isolated from neonatal rats following prenatal hypoxia (second day of gestation through postnatal days 5–8) exhibit membrane current properties consistent with increased excitability [202,203]. While it is unknown if these carotid body effects persist beyond the first few days post-hypoxia, these data suggest a mechanism for enhanced hypoxic responses following prenatal hypoxia. Most studies of prenatal hypoxia produce fetal hypoxemia by exposing the mother to environmental hypoxia (i.e., low inspired PO2), as may occur at altitude. However, plasticity in the control of breathing may result from other sources of prenatal hypoxia. Intrauterine growth restriction, such as that caused by chronic placental insufficiency or experimental manipulation (e.g., ligation of the uterine artery, maternal anemia), may result in fetal hypoxemia. Intrauterine growth restriction attenuates the postnatal hypoxic ventilatory response in young sheep during wakefulness [204,205], but not during sleep [206,207], and may increase the ventilatory response to progressive asphyxia (i.e., hypercapnic hypoxia) in newborn guinea pigs [208]; none of these studies found a change in steady-state hypercapnic ventilatory responses. Prenatal exposure to carbon monoxide (CO) produces tissue hypoxemia by reducing O2 content of fetal blood. In guinea pigs, repeated exposure to prenatal CO (10 h/day) for the last 6 weeks of gestation increases hypercapnic ventilatory responses in 4–5 day old pups, despite normal resting ventilation [209]. Additional studies are needed to verify that fetal hypoxemia is the common stimulus in these examples, but collectively, these data suggest that prenatal hypoxia can have lasting effects on control of breathing. B. Sustained Neonatal Hypoxia

Neonatal hypoxia induces short- and long-term respiratory plasticity distinct from adult plasticity. For example, rats raised in hypoxia (10% O2) for the first postnatal week hyperventilate when transferred to room


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air ([210]; see also [211]). Hyperventilation is persistent, lasting 6–7 weeks after return to normoxia despite normal PaO2 [212,213]. A similar hypoxic exposure in post-puberty rats has no enduring effects, indicating that the critical period for this plasticity does not extend into adulthood [212,213]. Whereas prenatal and adult exposures to chronic hypoxia generally enhance acute hypoxic ventilatory responses, hypoxia during early postnatal development appears to blunt hypoxic responses. Indeed, chronic neonatal hypoxemia may explain blunted hypoxic responses in humans born with cyanotic heart disease [214–216]. Humans born in hypobaric hypoxia also exhibit reduced hypoxic ventilatory responses as adults, even after moving to sea level [139,217]. This effect can only be partially attributed to genetic mechanisms [139]. For example, high-altitude residents may be born with normal hypoxic responses and gradually acquire blunted responses during postnatal development [139,218–221]; similar changes are not observed, or occur more slowly and to a lesser extent, in sea level natives after moving to altitude as adults [218,219,221]. How long hypoxic ventilatory responses remain blunted once normoxia is restored is unclear [139,214–218,222], though any recovery appears to be gradual. Hypercapnic ventilatory responses are generally reported to be unaltered by neonatal hypoxia in humans [214,218,219,223], although other studies have found both reduced [224] and enhanced [225] ventilatory responses to CO2 in high-altitude natives. Similar effects of neonatal hypoxia on hypoxic ventilatory responses have been observed for non-human mammals, confirming long-lasting developmental plasticity. In young rats, cats and sheep born and raised in hypoxia, ventilatory responses to acute hypoxia are abolished or greatly reduced 0–24 h after return to normoxia [226–231]. In rats and sheep, hypoxic ventilatory responses remain blunted for weeks to months following 1–2 weeks of neonatal hypoxia despite normal blood gases and/or metabolic rates [213,229,232]. In rats, this long-lasting plasticity cannot be elicited after puberty [213] and is expressed only by males [232]. Hypercapnic ventilatory responses are generally normal following neonatal hypoxia [213,234,235], indicating that the effects of neonatal hypoxia are specific to the hypoxic response. The mechanism(s) for long-lasting attenuation of the hypoxic ventilatory response following neonatal hypoxia are unclear. Neonatal hypoxia (from birth) delays maturation of carotid body responses either to hypoxia [228,230,235,236] or to combined hypoxia and hypercapnia [237]. However, even if rats are born and raised in hypoxia, carotid body O2 chemosensitivity appears to recover spontaneously despite continued hypoxia [226] or following return to normoxia [235,236]. Moreover, phrenic nerve responses to isocapnic hypoxia are unaltered in adult male rats raised in hypoxia for postnatal week 1, despite having blunted hypoxic ventilatory

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responses [232]. Thus, changes in peripheral chemosensitivity, chemoafferent pathways or central neural integration of chemoafferent inputs cannot explain the long-lasting attenuation of hypoxic ventilatory responses following neonatal hypoxia; the impairment appears to be downstream of phrenic activity (Figure 6.1). Consistent with this conclusion, neonatal hypoxia alters respiratory system mechanics and lung morphology [212,238], and diaphragm function [31,239]. Thus, neonatal hypoxia may have short-term effects on carotid bodies whereas anatomical and/or neural changes in respiratory muscles or respiratory mechanics may be responsible for long-lasting plasticity. C. Intermittent Neonatal Hypoxia

Intermittent hypoxia is observed clinically, often in combination with mild hypercapnia due to periodic apneas or impaired gas exchange (e.g., OSA, apnea of prematurity, asphyxia during birth, bronchopulmonary dysplasia). In neonatal rats, pre-treatment with short periods of intermittent hypoxia reduces HVD during subsequent exposures [121,158]. Thus, intermittent hypoxia enhances hypoxic ventilatory responses in both neonatal and adult rats [93,158]. This facilitatory plasticity may diminish with longer periods of intermittent hypoxia in both neonates and adults [19,158]. In experimental studies with rats, both prenatal and neonatal CIH impairs anoxic gasping [240]. Moreover, 30 min of severe hypoxia (6% O2) twice daily for the first four postnatal days reduces the hypoxic ventilatory response one day later in rats, but these effects are no longer evident at two weeks of age [241]. Consistent with rapid recovery following neonatal intermittent hypoxia, CIH during development does not appear to alter the adult hypoxic ventilatory response [242]. However, while daily bouts of anoxia (100% inspired N2) had no detectable effect on the hypoxic ventilatory response at 7 days of age [243], a single 20-min anoxic episode in 3–4 day old neonatal rats enhanced the hypoxic ventilatory response measured at 25 days of age [244]; no effects on the hypoxic response were detected at 9 days of age. Thus, even a solitary severe hypoxic episode during the neonatal period can have lasting effects on the control of breathing that are only revealed later in life, emphasizing that multiple post-exposure time points may be necessary to adequately assess the expression of developmental plasticity. Several studies have considered the effects of intermittent hypoxia during development in piglets. Piglets (3–5 week old) exhibit blunted hypoxic ventilatory responses following five daily exposures (30 min/d) to poikilocapnic hypoxia [245], consistent with data in rats [241]. However, there is some evidence that rapid cycles of hypoxia within a single day (7 cycles, 3 min hypoxia/3 min air) also attenuate the hypoxic ventilatory response in this species at 2–3 week of age [246]. Since adults of other species


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exhibit enhanced hypoxic sensitivity following intermittent hypoxia [19,93,158], this manifestation of plasticity may be unique to development in piglets. However, until similar studies are completed in adult pigs, it remains possible that blunted hypoxic responses following intermittent hypoxia is a species-specific trait. A very different picture emerges when neonatal piglets (1–2 weeks old) are exposed repeatedly to hypercapnic hypoxia [247]. Acute hypoxic ventilatory responses, measured 24 h following the last of seven daily exposures to 10% O2 and 6% CO2, are enhanced in piglets despite normal blood gases; acute responses to hypercapnia or hypercapnic hypoxia are generally reduced. Moreover, the pattern of hypercapnic hypoxia during each daily episode (intermittent vs. continuous) appears to influence the resulting plasticity [247]. Thus, the effects of CIH during development on ventilatory control are complex, depending on the exposure pattern and duration, background PaCO2 and species studied. D. Hyperoxia

Exposure to 30–60% O2 for a week or more during early (primarily postnatal) life reduces hypoxic ventilatory responses in rats and cats [228,248,249,249a]. In kittens born and raised in 30% O2 for the first 12–13 days of life, acute hypoxic ventilatory responses are absent immediately following the hyperoxic exposure [229]; it is not known if these effects are persistent. In contrast, rats exposed to 30% O2 for the first 5 days of life exhibit sustained increases in ventilation during acute hypoxia (vs. the biphasic hypoxic response typical of neonatal rats), suggesting accelerated maturation of the hypoxic ventilatory response [250]. However, the carotid body response to hypoxia was abolished in rats after 5–10 weeks of hyperoxia from birth in the same study [250]. It is possible that 5 days of 30% O2 is not long enough to blunt the hypoxic ventilatory response, or that the exposure period did not encompass the critical period for this plasticity (see below). Our laboratory has demonstrated that 1–4 weeks of developmental hyperoxia (30–60% O2) causes long-lasting attenuation of the hypoxic ventilatory and/or phrenic nerve responses in rats [17,164,248,249,249a,251,252]. After controlling for blood gases and metabolic rate, hypoxic ventilatory responses are significantly reduced despite normal normoxic ventilation in awake, adult rats (43 months of age) exposed to 60% O2 for the first week [249a] or month [248] of life, primarily due to a smaller increase in respiratory frequency; hypercapnic ventilatory responses are unaffected by developmental hyperoxia [248]. Phrenic responses to isocapnic hypoxia are also reduced in anesthetized rats exposed to hyperoxia during development [17,164,249,249a,251,252]. Although the effects of one month of 60% O2 on hypoxic phrenic responses appear permanent [251], rats experiencing shorter (i.e., one week) hyperoxic

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exposures exhibit a degree of spontaneous recovery [249a]. However, hypoxic responsiveness can be restored (at least temporarily) to rats exposed to one month of 60% O2 during development by treatment with chronic sustained or intermittent hypoxia as adults [164]. It is not known whether this functional recovery reverses the effects of developmental hyperoxia or enhances phrenic responses by inducing normal, adult-type plasticity of the residual hypoxic response (see section above on Chronic intermittent hypoxia). Adult rats exposed to 60% O2 for one month exhibit no lasting changes in ventilatory [248] or phrenic [249] responses to hypoxia, indicating that the plasticity following developmental hyperoxia is specific to development. A recent study revealed that the critical period for hyperoxia-induced respiratory plasticity extends into the second postnatal week ([252]; Figure 6.3); one week of 60% O2 has no lasting effect on the hypoxic phrenic response of rats treated during the third or fourth postnatal week. However, since hyperoxic exposures begin a few days prior to birth in most of these studies [17,164,248,249,249a,251,252], there is some possibility of pre-natal effects of hyperoxia as well. The effects of prenatal hyperoxia have not been studied explicitly. Nevertheless, prenatal hyperoxia is not necessary for this form of developmental plasticity since hypoxic responses are reduced in adult rats that have been exposed to 60% O2 during the second postnatal week only [252]. Since phrenic responses to isocapnic hypoxia are blunted in rats following developmental hyperoxia, the effect of hyperoxia on the hypoxic ventilatory response cannot be (wholly) explained by changes in respiratory muscles or respiratory mechanics. Moreover, electrical stimulation of the CSN (i.e., bypassing hypoxic chemotransduction in the carotid body) produces equivalent increases in phrenic motor output in hyperoxia-treated rats and untreated controls [251,253], indicating that central integration of afferent information from peripheral chemoreceptors is not impaired. Several lines of evidence indicate that developmental hyperoxia induces plasticity at the carotid body chemoreceptors. For example, carotid body morphology is altered by 1–4 weeks of 30–60% O2 during development, with reduced carotid body volume (total volume and volume occupied by glomus cells), fewer carotid chemoafferent neurons and altered dopamine content [164,251,254,255]. Attempts to measure hypoxic sensitivity of carotid body chemoreceptors through single fiber recordings have been unsuccessful in adult rats exposed to developmental hyperoxia [17], possibly reflecting the reduced number or fragile condition of chemoafferent neurons. In the absence of single unit recordings, carotid body responses have been assessed from whole CSN preparations. These studies consistently demonstrate reduced carotid body responses to hypoxia, asphyxia and/or intravenous cyanide injection [17,250,251,255,256]. It is not clear from these whole nerve studies whether individual glomus cells are less sensitive to


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hypoxia or if hypoxic responses are reduced because there are fewer glomus cells and/or chemoafferent neurons. However, it was recently reported that carotid body dopamine release is reduced during hypoxia, but not in response to extracellular Kþ, in adult rats several months after developmental hyperoxia [255], suggesting decreased hypoxic sensitivity. Moreover, immediately after 1–3 weeks of hyperoxia from birth, single unit carotid body responses to hypoxia are depressed in rats [257] and kittens [228], as are the depolarization and intracellular calcium responses to hypoxia in carotid body glomus cells isolated from hyperoxic rats [258]. Thus, developmental hyperoxia may attenuate hypoxic responses through lasting effects on both the absolute numbers and O2 sensitivity of carotid chemoreceptor cells. Conversely, since phrenic responses to CSN stimulation in rats exposed to developmental hyperoxia are normal [251,253], the reduced number of axons from chemoafferent neurons [254] may not be a major contributor to impaired hypoxic ventilatory responses. The pathways by which developmental hyperoxia alters carotid body function are currently unknown. By raising PaO2 above the threshold for carotid body activity, hyperoxia may reduce or prevent depolarization of the carotid body chemosensory cells. This may in turn diminish the activitydependent synthesis and/or release of neurotransmitters and trophic factors in the carotid body chemosensory system during critical periods of development [259,260]. For example, carotid chemoafferent neurons (and perhaps glomus cells themselves) require the release of trophic factors from intact carotid bodies during the early postnatal period [261]. The synthesis and release of trophic factors may be activity dependent [260] and there is suggestive evidence that trophic factors such as BDNF are reduced in the carotid body following chronic hyperoxia (unpublished data cited in Ref. [254]). Thus, carotid chemoreceptor inactivity could result in inadequate trophic factor availability during developmental hyperoxia, thereby causing carotid body hypoplasia and degeneration or withdrawal of chemoafferent neurons [254]. Carotid chemoafferent neurons no longer require trophic support by the third postnatal week, perhaps earlier, [261], consistent with the critical period for hyperoxia-induced changes in carotid body morphology [254] and hypoxic phrenic responses [252]. In addition to reducing neural activity, hyperoxia could alter the expression of genes regulated by O2. Many genes directly related to carotid body function are regulated by hypoxia, including genes related to O2 sensing (e.g., ion channels), neurotransmitters and neurotransmitter release [262], and these genes may also be susceptible to hyperoxia [263]. Other O2-sensitive genes and gene products could have indirect effects on carotid body function, such as vascular endothelial growth factor (VEGF), which has been identified in the carotid body (Wang, Z.-Y. and Bisgard, G.E., personal communication). If hyperoxia downregulates VEGF production in the carotid body,

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as it does elsewhere in the body [264], developmental hyperoxia could lead to poor carotid body vascularization. Loss of vasculature could alter tissue O2 profiles, or could cause a loss of glomus tissue (and thus trophic support for developing chemoafferent neurons). Finally, the effects of hyperoxia on carotid body function could be mediated by ROS, either through altered gene expression or cellular toxicity [263,265–269]. The neural effects of developmental hyperoxia (60% O2) seem to be specific to the carotid body and related chemoafferent pathways, with no impairment of neurons in the nodose ganglion [254], the hypercapnic ventilatory response [248] or the central neural integration of chemoafferent inputs [251,253]. Thus, the effects of developmental hyperoxia on respiratory control are not due to widespread, non-specific cellular toxicity. However, given its high blood flow relative to metabolic rate, the carotid body may be more susceptible to hyperoxia than other tissues [175,178]. Accordingly, local ROS-mediated toxicity could directly or indirectly lead to the loss of glomus cells and/or chemoafferent neurons. Developmental hyperoxia is rare in non-experimental settings, and is probably limited to mechanical ventilation and/or supplemental oxygen therapies in infants. Attempts are made to maintain SaO2 (and therefore PaO2) within limits to minimize or prevent hyperoxic toxicity and conditions such as retinopathy of prematurity. However, periods of hyperoxia are likely to occur during oxygen therapy [270], and the lower limits of hyperoxic exposure required for hyperoxia-induced respiratory plasticity are poorly understood. In human infants, there is a relationship between oxygen therapy and blunted hypoxic ventilatory responses [271]. It is not known whether this relationship is causal, or if hypoxic chemosensitivity was reduced in these infants due to prior exposure to intermittent hypoxia associated with bronchopulmonary dysplasia or septicemia [271–274], but this relationship highlights the need to understand the effects of oxygen therapy on ventilatory control from a biomedical perspective. Relative hyperoxia at (premature) birth (i.e., the abrupt rise from fetal PaO2) may also induce hyperoxia-related plasticity in respiratory control, either as part of normal development or pathologically, but this has not been studied [6]. E.

Hypercapnia

Although many burrowing mammals and birds regularly experience high levels of inspired CO2 during development [183,184], environmental hypercapnia is rare in humans, particularly during development. However, hypercapnia may occur during some diseases, often in conjunction with hypoxia (e.g., chronic lung disease). Moreover, current strategies for neonatal respiratory therapy may include permissive hypercapnia, a strategy


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in which low tidal volumes are used during mechanical ventilation to minimize lung trauma while allowing PaCO2 to rise to 45–55 mm Hg or higher [275]; the effects of permissive hypercapnia on the development of respiratory control have not been studied. The few studies examining developmental hypercapnia and the control of breathing in mammals have reached opposite conclusions. Birchard and colleagues [276] studied male rats exposed to 6% CO2 from fertilization through the third postnatal week (i.e., throughout prenatal and postnatal development). When studied after 6 weeks in room air, blood gases, metabolic rates and ventilation were similar to control rats at three levels of inspired CO2 (0–5%), indicating no long-lasting effects on the control of breathing. In a similar set of experiments, Rezzonico and Mortola [277,278] studied male and female rats exposed to 7% CO2 for six days (beginning 24 h after birth). Two days after the return to room air, CO2-treated rats exhibited greater resting ventilation and reduced ventilatory responses to 10% inspired CO2 (expressed as percentage increase from baseline) [278]. When studied at 45–50 days of age (i.e., 6 weeks post-exposure), CO2-treated rats had reduced resting ventilation, normal metabolic rates, normal hypoxic ventilatory responses (10% O2), and reduced hypercapnic ventilatory responses (10% CO2) [277]. Thus, in contrast to the earlier study by Birchard and colleagues, this study found evidence for long-lasting plasticity in respiratory control following developmental hypercapnia. Several methodological differences could contribute to these divergent conclusions, including the timing, duration and severity of developmental hypercapnia and the levels of CO2 used to assess the hypercapnic ventilatory response. Alternatively, the differences may be explainable by the sex of the rats studied. In female Japanese quail (Coturnix japonica) treated with 2% CO2 throughout embryonic development, adult hypercapnic ventilatory responses are reduced despite similar blood gases, metabolic rates, and resting ventilation [279]; however, the hypercapnic ventilatory responses of male quail were unaffected by developmental CO2. Re-analysis of an earlier study [280] revealed a similar pattern for zebra finches (Taeniopygia guttata): only female finches exhibit blunted hypercapnic ventilatory responses following embryonic CO2 exposure [279]. Thus, sex may have profound effects on the expression of developmental plasticity and may contribute to the different conclusions reached in studies with rats. Indeed, Birchard and colleagues [276] studied only male rats, whereas Rezzonico and Mortola [277,278] report data for a combination of males and females (Mortola, J.P., personal communication). Acclimation to CO2 as adults produces only transient effects on the hypercapnic ventilatory response (reviewed above), suggesting that the longlasting effects (weeks to months) of developmental CO2 exposure in rats and birds are specific to development. No studies have attempted to define the critical period for this CO2-induced developmental plasticity, although data

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from birds suggest that both embryonic (i.e., prenatal) and nestling (i.e., postnatal) CO2 exposures produce qualitatively similar effects on the hypercapnic ventilatory response [279,280]. Likewise, the mechanisms underlying this plasticity have not yet been investigated. Although changes in metabolism, gas exchange and blood buffering do not appear to be involved in the altered ventilatory responses in adult quail [279], early postnatal CO2 exposure in rats causes changes in respiratory mechanics that persist into adulthood [281]. Therefore, it is currently not possible to determine whether changes in the hypercapnic response reflect changes in CO2 chemosensitivity, CNS integration or respiratory mechanics. However, since the hypoxic response was not affected by neonatal hypercapnia in rats [277], the role of the observed changes in respiratory mechanics in the blunted hypercapnic response following developmental hypercapnia is questionable. F.

Metabolism

Changes in metabolic rate are typically matched by corresponding increases or decreases in ventilation [282]. Sant’Anna and Mortola [283,284] investigated whether chronic alterations in metabolic rate induce developmental plasticity in respiratory control. In the first set of experiments, rats were raised for the first three postnatal weeks in small (6 pups/litter) or large (16 pups/litter) litter sizes [283]. Large-litter rats had reduced growth and lower metabolic rates prior to weaning (presumably reflecting reduced caloric intake due to increased food competition). Ten days later, resting ventilation was greater in large-litter vs. small-litter rats. However, the increased ventilation was matched by a greater mass-specific metabolic rate (i.e., the ventilation to metabolism ratio was unchanged). Hypoxic and hypercapnic ventilatory responses were unaffected by developmental litter size. In a second study [284], rats were exposed to cold (14 C) for the first three postnatal weeks, resulting in a chronic elevation of metabolic rate during this period. Aside from a small change toward a slower and deeper breathing pattern, no changes in resting ventilation or metabolism or in the hypoxic and hypercapnic ventilatory responses were detected in cold-reared rats when studied approximately 10 days later. These studies suggest that chronic alterations in metabolic rate during development have minimal consequences for the respiratory control system. However, it is likely that the experimental treatments affected more than metabolic rate (e.g., stress hormones) and the data should be interpreted with caution. As reviewed in the preceding sections, developmental perturbations of respiratory gases may cause long-lasting changes in respiratory control. However, the effects of prolonged changes in ventilation induced by altered respiratory gases (i.e., increased respiratory drive) vs. effects of altered


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respiratory gases themselves (i.e., tissue PO2 and PCO2) are unknown. Since, chronic alterations in metabolic rate modify ventilation with minimal effects on blood gases (i.e., ventilation to metabolism ratio unchanged; [282], studies of developmental metabolic derangements may shed light on this issue [284]. Specifically, the studies by Sant’Anna and Mortola [283,284] suggest that the change in blood gases may be the stimulus for long-lasting plasticity following developmental hypoxia, hyperoxia, and hypercapnia. G. Sensory Denervation

Sensory denervation during development may induce plasticity in respiratory control. Carotid body denervation typically results in hypoventilation during normoxia and eliminates, or greatly reduces, ventilatory responses to hypoxia in neonatal and adult mammals [7]. In many mammalian species, both young and adult animals exhibit substantial plasticity following carotid denervation, spontaneously recovering resting blood gases and hypoxic ventilatory responsiveness over a period of weeks to years, although this recovery is somewhat less in humans (reviewed in Ref. [7]). Studies in rats, pigs and goats suggest that this recovery may occur sooner or more completely if the carotid body denervation occurs in early postnatal life [7,44,45,285]. In adults, recovery following carotid denervation may involve an upregulation of aortic body chemoreceptor function, abdominal and/or central hypoxia-sensitive tissues, or chemosensory pathways [286–291]. In contrast, functional, O2-sensitive aortic chemoreceptors may already be present during the neonatal period. Aortic chemosensitivity (assessed by ventilatory responses to local NaCN injection) is normally lost at around 8 days of age in piglets, but remains prominent in piglets denervated neonatally [45,285]; similar observations have been reported for rats [44]. Moreover, there appears to be a critical period during development (around the second week) when piglets are more likely to exhibit irregular breathing and apneas following carotid denervation [292]; denervation before or after this critical period has no effect on respiratory stability. The relatively normal breathing observed following denervation prior to this critical period may reflect the immediate availability of functional aortic chemoreceptors [285]. Together, these data suggest that neonatal animals are able to capitalize on redundancy in the respiratory control system that is normally lost during maturation, and this capacity may contribute to age-dependent differences in recovery from carotid denervation. Given the dramatic recovery of hypoxic ventilatory responses following carotid denervation, it is interesting that hypoxic responses are still reduced at 3 months of age in rats exposed to developmental hyperoxia, and may remain so permanently [251]. Indeed, residual hypoxic sensitivity is

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unaffected by vagotomy in these rats and is abolished following bilateral section of the CSN [249], suggesting that aortic chemoreceptors do not upregulate in this model. It is possible that this difference between carotid denervation vs. developmental hyperoxia reflects the impairment of both carotid and aortic chemoreceptors by hyperoxia in the latter case. Alternatively, aortic chemoreceptor upregulation may be inhibited in hyperoxia-treated rats. Aortic chemosensitivity following carotid body denervation is serotonin-dependent [45], and serotonin and serotonin receptors are upregulated in the chemosensitive portion of the aorta following bilateral carotid denervation in neonatal rats and piglets [293]. Since unilateral carotid denervation elicits no change in serotonin or serotonin receptors (unpublished data cited in Ref. [293]) the residual carotid body function in hyperoxia-treated rats may be sufficient to prevent compensatory aortic chemosensitivity. H. Neonatal Maternal Separation

Stressful conditions, such as restraint or immobilization, alter ventilatory control in adult mammals [294–296]. These effects may persist at least 24 h after immobilization has ended, indicating plasticity in ventilatory control mechanisms [296]. Recent evidence indicates that stress during early postnatal development caused by maternal separation has lasting effects on the control of breathing as well. In these studies, rat pups were isolated from their mothers for 3 h/day on 10 consecutive days, beginning on postnatal day 3 [297,298]. As adults, rats that experienced neonatal maternal separation exhibited reduced hypercapnic ventilatory responses, possibly related to changes in the paraventricular nucleus of the hypothalamus [297]. Although a decreased hypercapnic response is also observed following immobilization stress in adult rats [296], the enduring nature of this plasticity suggests that these effects could be unique to development. Moreover, neonatal maternal separation enhances adult hypoxic ventilatory and phrenic responses in male (but not female) rats ([298]; Kinkead, R., personal communication). In contrast, hypoxic responses were unaffected by immobilization stress in adult male rats [296]. It is unknown whether neonatal maternal separation and adult immobilization produce comparable stress responses, or to what extent the duration of exposures (10 days in neonates, 1–2 days in adults) alters this plasticity. However, these studies suggest the existence of critical periods for stress-related respiratory plasticity. It is important to note that altered respiratory gases can induce stress responses (e.g., stress hormone secretion), although this responsiveness may be relatively low during development [299–301]. Thus, activation of stress responses during development may be one mechanism by which altered respiratory gases induce developmental plasticity. However, there is no


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direct evidence to support a causal role for a generalized stress response in the examples of developmental plasticity (i.e., hypoxia, hyperoxia, and hypercapnia) described in the preceding sections. Indeed, the variability in plasticity (magnitude, direction, and altered reflex) and underlying mechanisms (e.g., chemosensitivity vs. respiratory mechanics) argue against a single mechanism for developmental plasticity in respiratory control. IV.

Sex Hormones

Sex is emerging as an important consideration in the study of developmental plasticity. Several examples of developmental plasticity are specific to either males or females. Examples include plasticity induced by neonatal hypoxia [233,302] and maternal separation [298] in rats, and embryonic hypercapnia in birds [279]. Other forms of developmental plasticity are sex insensitive (e.g., developmental hyperoxia; [251]), but most models have not been studied with respect to sex. This is an important consideration as sex may impact the assessment of plasticity, and the value of therapeutic strategies intended to induce plasticity may be sex-specific. Future studies will benefit by explicitly considering sex in their experimental designs. The mechanisms by which sex interacts with plasticity are unknown, although hormones are likely to be involved in both developmental and adult neuroplasticity [303–305]. Sex hormones are believed to influence the control of breathing [306] and have recently been implicated in adult respiratory plasticity following intermittent hypoxia (i.e., LTF, [109,110]). Moreover, manipulation of sex hormones during development can in itself influence adult respiratory control. Male and female adult rats exhibit distinct ventilatory responses to injection of aspartic acid, an NMDA receptor agonist [307,308]. Neonatal treatment with sex hormones (testosterone in females or estradiol in males) can switch the female ventilatory response into the male-type response, and vice versa, possibly by altering NMDA receptor expression [308–310]; these effects of neonatal hormone treatment do not appear until after sexual maturity [308]. In addition, male rats treated with estradiol at 5 days of age had reduced hypercapnic ventilatory responses as adults despite similar resting ventilation [310]. Consequently, environmental factors that alter perinatal hormone levels (e.g., maternal stress in rats; [311]), could have long-term effects on the control of breathing. V.

Spinal Cord Injury (SCI) and Respiratory Plasticity

One strategy for promoting functional motor recovery following incomplete SCI is to strengthen (and better utilize) existing, non-injured pathways [312].

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The inherent plasticity of the respiratory neural control system makes this approach a viable therapeutic option for improving respiratory muscle control following SCI. Respiratory deficits following cervical SCI are determined by the severity and segmental level of the injury. High cervical SCI (lesions rostral to C4) are immediately life threatening because inspiratory premotor drive to spinal respiratory motoneurons is interrupted. Thus, high cervical lesions generally require immediate and sustained ventilatory support, whereas spinal lesions caudal to C4 produce comparatively less respiratory dysfunction. However, even low cervical and thoracic SCI patients are at risk for respiratory-related disorders (e.g., pneumonia; [313]) and have impaired airway protective reflexes (e.g., cough; [314]). Early spontaneous recovery of motor function following SCI is often attributed to resolution of spinal shock, inflammation and edema in the spinal cord [315]. Subsequent improvements may reflect increased respiratory muscle strength [316], altered pulmonary mechanics [317], and recruitment of accessory inspiratory muscles not normally used to generate airflow [316,318]. An additional possibility is that time-dependent neuroplasticity in surviving neurons/networks improves respiratory motor function in the months to years following SCI. Much evidence suggests that neuroplasticity can improve respiratory motor function in animal models of SCI (reviewed below). A recent case report provides hope that the same is true for human cervical SCI patients [319]. A C2 injured, ventilator-dependent patient with complete loss of motor and sensory function (grade A; American Spinal Injury Association) experienced no significant motor recovery in the first five years post-injury, although an MRI indicated that up to 20% of C2 spinal tissue was intact. The patient then underwent a rigorous activity-based recovery program. Over years five to eight post-injury, the patient demonstrated substantial motor recovery (two ASIA grades) that was attributed to the training. EMG studies suggest that the patient may have regained some voluntary control of the diaphragm. The significance of this respiratory motor recovery is evident in the following quotation from the patient, A ventilator failure . . . would have been a terrifying experience because I couldn’t really breathe. Now, I can breathe quite well . . . I am able to move my diaphragm, an ability that was achieved by exercise and training [319]. This delayed motor recovery probably reflects plasticity in the small amount of spared respiratory pathways. Several laboratories are currently investigating if and how neuroplasticity can enhance respiratory function following SCI in animal models. In this section, we will review respiratory neural control and plasticity following injuries to the spinal cord including hemisection, contusion, and chronic deafferentation via dorsal rhizotomy.


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A. Spinal Hemisection

Animal models of respiratory dysfunction following SCI represent a compromise between what is seen clinically, what is survivable by laboratory animals without extensive respiratory support, and a clearly definable lesion. The most common experimental lesion used to study respiratory neural control following SCI is cervical hemisection (from midline to the lateral edge of the cord) rostral to the phrenic motor nucleus (for review see Refs. [8,320]). Cervical hemisection is reproducible, quantifiable and has a low mortality rate in rats. The major advantage of the hemisection model is that it permits study of existing, but normally ineffective, spinal pathways that cross the spinal midline caudal to the injury and project to phrenic motoneurons (reviewed in detail below). However, a limitation of spinal hemisection as an SCI model is that it is not representative of the most common clinical injuries (i.e., spinal contusion; [312]). Minute ventilation is sufficient to maintain arterial blood gases during normoxia within a few days following C2 hemisection [321], but hypoventilation may occur in the first 1–2 h post-injury (Fuller, D.D., Golder, F.J. and Mitchell, G.S., unpublished observations). Respiratory insufficiency following C2 hemisection may also be revealed during anesthesia [322,323] or hypoxic or hypercapnic challenge. Rats with chronic C2 hemisection breathe with increased frequency and decreased tidal volume at 1 and 2 months post-injury when anesthetized [323] or awake (Fuller, D.D. and Mitchell, G.S., unpublished observations). Tidal volume continues to diminish over a period of weeks post-C2 hemisection, but breathing frequency progressively increases resulting in a constant minute ventilation (Fuller, D.D. and Mitchell, G.S., unpublished observations). The mechanisms producing the alterations in breathing pattern following C2 hemisection are not fully known, but partly result from pulmonary vagal feedback [323]. In addition, the altered breathing pattern may reflect supraspinal plasticity [324] or changes in pulmonary mechanics (e.g., reduced chest wall compliance; [317]). During breathing at rest, the phrenic nerve or hemidiaphragm ipsilateral to C2 hemisection is silent (i.e., no inspiratory bursting) in the days to weeks following the injury [30,321,323–325]. However, chemical or pharmacological stimulation of respiratory drive produces rhythmic inspiratory phrenic nerve activity below the hemisection [8]. Inspiratory bursts occur at the same frequency as those in the contralateral phrenic nerve, but amplitude is considerably less [30,322–324]. How does respiratory activity recur despite elimination of all ipsilateral bulbospinal inputs? In rats, bulbospinal respiratory neurons project axons bilaterally to phrenic motoneurons [326–328]. Although most crossed pathways decussate in the brainstem, an apparently ineffective synaptic pathway

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crosses the spinal midline in rats caudal to C2 [326]. Phrenic motor output caudal and ipsilateral to cervical hemisection results from activation of these existing but previously ineffective synaptic pathways to phrenic motoneurons [8]. This effect has been termed the crossed phrenic phenomenon [8,329]. Many published studies of the crossed phrenic phenomenon do not use histological techniques to document that all descending ipsilateral tracts have been severed. Accordingly, the existence of a crossed-spinal pathway is not required to explain the crossed phrenic phenomenon in all published studies. One alternative neural pathway to phrenic motoneurons could be dendrites from contralateral motoneurons that cross the spinal midline as originally suggested by Ref. [330]. Phrenic motoneuron dendrites that cross the spinal midline are more prevalent in neonatal rats [331,332], but still may be present in adult animals [332]. A crosseddendritic pathway could transmit inspiratory impulses to motoneurons below the hemisection. Regardless of the anatomical substrate, the crossed phrenic phenomenon is one of the best examples of motor recovery following SCI and it provides an opportunity to examine the mechanisms underlying recruitment and plasticity of functionally latent synapses [333]. The crossed phrenic phenomenon occurs in a wide range of species including rats [30,321,323,324], rabbits [334,329], dogs [329,335], cats [335,336], guinea pigs [333] and woodchucks [329]. However, Rosenbluth and Ortiz [329] did not observe the crossed phrenic phenomenon in 3 of 4 acutely hemisected monkeys. In the fourth monkey, cervical spinal hemisection did not paralyze the ipsilateral diaphragm, but ispilateral hemidiaphragm contractions were abolished by cutting the contralateral phrenic nerve [329]. Rosenbluth and Oritz concluded that diaphragm contractions were produced by impulses traveling in axons of the phrenic nerve contralateral to hemisection, and therefore did not represent crossed phrenic activity. However, the post-hemisection time interval before crossed phrenic pathways can be recruited is variable between species (an observation that may account for the failure of Rosenbluth and Ortiz [329] to find crossed phrenic activity in primates). The crossed phrenic phenomenon can be induced immediately post-hemisection in cats, dogs, rabbits and woodchucks [329]. In contrast, young rats and guinea pigs require a delay ranging from 3 to 24 h before chemical stimulation will reveal crossed phrenic pathways [337,338]. However, crossed phrenic pathways can be activated immediately following hemisection if rats are treated with the serotonin precursor 5-hydroxytryptophan (5-HTP) [30,339]. Thus, following acute hemisection in rats, crossed phrenic pathways are present, but functionally latent [8]. One possibility is that activation of crossed phrenic pathways is prevented by inhibitory influences associated with acute SCI (e.g., hemorrhage, swelling, etc.; [312]). A more likely


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scenario is that activation of crossed phrenic pathways requires necessary preconditions (e.g., removal of active inhibition, [321]; morphological plasticity, [8]; insertion of glutamate receptors into post-synaptic membranes, [135]) not present immediately post-hemisection [30]. Consistent with this interpretation, robust crossed phrenic responses occur in acutely C2 hemisected rats given a pre-conditioning lesion CDR but not in control or sham-operated rats [340]. CDR may create necessary preconditions by enhancing serotonergic terminal density [128] or neurotrophin concentration in the cervical spinal cord [341]. Both of these putative changes could increase efficacy in existing spinal synapses (see section above on Brief intermittent hypoxia). The crossed phrenic phenomenon is influenced by age of the animal at the time of injury [338,342,342a]. Older rats require no delay between hemisection injury and recruitment of crossed phrenic pathways during asphyxia [338]. Goshgarian [338] speculates that crossed phrenic pathways mature during the normal aging process, resulting in more effective synaptic transmission. However, this hypothesis may not be true of all synaptic inputs to phrenic motoneurons. For example, long-latency (41.0 ms) crossed phrenic potentials evoked by spinal cord stimulation are revealed following systemic treatment with 5-HTP [339]. These long-latency potentials are present in young (3–5 month) but not old (1.5–2 years) rats [342]. Thus, age-related developmental plasticity of crossed phrenic pathways is a complex process that requires further investigation. Sex-related influences on crossed phrenic pathways have not been specifically investigated. Separate laboratories studying exclusively male or female rats following C2 hemisection have reported qualitatively similar data during chemical or pharmacological induction of crossed phrenic activity [8,30,323,324]. However, the rate of spontaneous recovery of inspiratory motor activity in rats may be more rapid in males [30]. Male rats also appear to recover locomotor function more rapidly than female rats following C2 hemisection (Golder, F.J., personal communication). Further, sex has a critical influence on spinal respiratory plasticity in the form of phrenic LTF [83,84,109,110]. Genetic differences in crossed phrenic expression are also unknown. Genetics are suspected to influence spinal respiratory plasticity [90] and motor recovery following SCI [343]. Moreover, deficits in inspiratory tidal volume following C2 hemisection are different between rat strains (Fuller, D.D. and Mitchell, G.S., unpublished observations). Collectively, these observations suggest that sex and genetic influences on crossed phrenic activity merit further investigation. Although the crossed phrenic phenomenon has been an important model of respiratory motor recovery following SCI for over 100 years, it was not known whether crossed phrenic activity made a functionally

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meaningful contribution to inspiratory tidal volume. By comparing ventilation between rats with either C2 hemisection alone or C2 hemisection þ ipsilateral phrenicotomy (i.e., preventing crossed phrenic activity from reaching the diaphragm), Golder and colleagues [322] assessed the contribution of these crossed pathways to breathing in rats. Ipsilateral phrenicotomy did not alter the rapid shallow breathing pattern in C2 injured rats. However, the ability to generate large inspiratory volumes (e.g., during augmented breaths) was significantly impaired if crossed phrenic activity was prevented. Thus, crossed phrenic activity is physiologically significant in terms of (large) tidal volume generation [322]. B. Serotonin and Crossed Phrenic Pathways

Serotonin may be a key element in the spontaneous improvement of locomotor and respiratory function following SCI [131,324,344–349]. Following pre-treatment with the serotonin synthesis inhibitor parachlorophenylalanine (p-CPA), the crossed phrenic phenomenon is observed in fewer rats, and the magnitude of phrenic activation on the hemisected side is reduced [350]. Similarly, treatment with the serotonergic neurotoxin 5,7 dihydroxytryptamine prior to injury significantly reduces crossed phrenic activity 2 months following hemisection [324]. Conversely, the serotonin precursor 5-HTP activates crossed phrenic pathways in spinally hemisected rats [30,339]. Recruitment of crossed phrenic pathways following 5-HTP is reversed following serotonin receptor antagonism with methysergide [339,342,351,352]. The specific serotonin receptor subtype necessary to recruit crossed phrenic pathways appears to be the 5-HT2A receptor [348,131]. 5-HT2A receptors located on phrenic motoneurons [131] may contribute to recruitment of crossed phrenic pathways. Further evidence for a spinal mechanism underlying crossed phrenic pathway activation comes from studies utilizing spinal cord electrical stimulation [30,339,342]. Shortly after i.v. 5-HTP, a short-latency (0.7 ms) crossed-phrenic-compound action potential is evoked by stimulating the ventrolateral spinal cord at C2 [339]. The latency of this response indicates that spinal cord neurons must be responsible, since this is insufficient time for a relay through supraspinal structures. C. Spontaneous Motor Recovery in Crossed Phrenic Pathways

Time-dependent plasticity occurs following cervical hemisection, strengthening crossed phrenic pathways [8]. For example, hours to days posthemisection in rats, inspiratory motor output ipsilateral to the injury can be induced only through increased ventilatory drive or by pharmacological


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means [8]. However, within two weeks, small crossed phrenic inspiratory bursts are observed at baseline conditions (Pa CO 2 40 mm Hg, PaO2 4 100 mm Hg) in approximately 50% of anesthetized rats ([30], Golder, F.J., personal communication; but see Ref. [325]). Spontaneous recovery grows stronger with time; one to two months post-C2 hemisection, spontaneous inspiratory bursts during quiet breathing (eupnea) are observed in the phrenic nerve or hemidiaphragm ipsilateral to injury in most C2 hemisected rats [324,325], although their amplitude remains small. The mechanisms underlying the spontaneous appearance of crossed phrenic activity are unknown, but may involve serotonin (see above). Initially, cervical hemisection decreases serotonin terminal density in the ipsilateral phrenic nucleus; however, over a period of days to weeks, serotonin terminal density increases to levels above that in normal rats [353]. Thus, descending serotonergic inputs project bilaterally to the phrenic nucleus in rats, which may provide a neuroanatomical basis for the timedependent recruitment of crossed phrenic pathways following chronic spinal hemisection. Spontaneous motor recovery in crossed phrenic pathways may develop in part secondary to morphological plasticity in the spinal cord following C2 hemisection [8,9]. For example, an increase in the number of dendro-dendritic appositions and synaptically active zones is observed in the ipsilateral phrenic motor nucleus a few hours post-C2 hemisection [354]. These rapid morphological effects are blunted when animals are given a serotonin synthesis inhibitor (para-chlorophenylalanine) prior to hemisection [355], consistent with effects of para-chlorophenylalanine on the crossed phrenic phenomenon [350]. The surface area of ipsilateral phrenic motoneuron somata are significantly decreased two weeks post-C2 hemisection, suggesting that motoneuron excitability increases following injury, a change that would enhance crossed phrenic activity [356]. D. Strengthening Crossed Phrenic Motor Output

One strategy for promoting functional recovery of respiratory motor output following cervical SCI is to strengthen (and better utilize) existing, non-injured pathways. This is a viable clinical strategy because the majority of SCIs are incomplete, and residual neural pathways are intact at the injury level [357]. Crossed phrenic pathway recruitment during heightened respiratory drive reflects ongoing recruitment or modulation (vs. plasticity) of an existing motor pathway since this effect is not persistent. However, chronic treatments may induce plasticity, strengthening crossed spinal synaptic inputs to phrenic motoneurons [30,340,358,359].

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Chronic pharmacological stimulation of respiratory drive with the adenosine receptor agonist theophylline appears to strengthen crossed phrenic pathways [358]. Following chronic (28 days) SCI theophylline treatment, 29 of 32 rats showed enhancement of crossed phrenic motor output. However, animals were studied shortly after theophylline treatment was terminated, raising the possibility that the motor recovery could reflect continued theophylline effects. The effects of theophylline were mediated by both adenosine A1 and serotonin 5HT2 receptors, possibly located on spinal motoneurons [348]. Our laboratory recently found that exposing C2 hemisected rats to CIH increased the efficacy of crossed phrenic pathways (Figure 6.4; [30]). C2 hemisected rats were exposed to CIH (5 min 11% O2, 5-min normoxia; 12 h/night) on day 7–14 post-injury, and were then studied in acute neurophysiological experiments. CIH-treated rats had substantially greater A

Spontaneous

B

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Chronic Intermittent Hypoxia

Phrenic Recording

Figure 6.4 Chronic intermittent hypoxia strengthens normally ineffective pathways to phrenic motoneurons. The left panel (A) depicts crossed phrenic pathways to phrenic motoneurons (Data from Refs. 326, 376, and 377). Bulbospinal respiratory neurons have cell bodies in the medulla (ventral respiratory group) and project bilaterally to phrenic motoneurons. Bulbospinal projections which cross the spinal midline in the cervical spinal cord are known as crossed phrenic pathways. The terms ipsilateral and contralateral are generally used relative to the hemisection. Panel B depicts spontaneous inspiratory activity and compound action potentials (evoked via ventrolateral funiculus electrical stimulation) recorded in the phrenic nerve ipsilateral to C2 hemisection. Following chronic (2 week) C2 hemisection, ipsilateral phrenic inspiratory burst amplitude was significantly greater at baseline and during hypoxia in CIH-treated vs. normoxia-treated rats. Phrenic burst frequency was not different between groups at any time point. The panel on the far right shows crossed phrenic potentials evoked by 1000 mA stimulation current. Whereas the normoxia-treated rat has only a small evoked potential, the rat conditioned with CIH following chronic hemisection displays a clear evoked potential in the ipsilateral phrenic nerve suggesting that crossed phrenic pathways are enhanced by a spinal mechanism following CIH (arrow ¼ stimulus artifact) (figure modified from Ref. 30).


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inspiratory phrenic activity below hemisection at baseline and during chemoreceptor activation. In addition, short-latency (0.5–0.7 ms), spinally-evoked potentials recorded in the phrenic nerve ipsilateral to hemisection were of greater amplitude following CIH (Figure 6.4). These data strongly suggest increased efficacy of a monosynaptic spinal synapse [30]. Interestingly, pre-treatment (i.e., before C2 hemisection) with CIH had no discernable effect on crossed phrenic motor activity. Therefore, CIH-induced plasticity of crossed phrenic pathways requires necessary preconditions created by SCI. How might existing crossed phrenic activity be enhanced by CIH or other treatments? Our working hypothesis is that augmented crossed phrenic inspiratory burst amplitude following CIH represents increased synaptic strength of pre-existing crossed spinal pathways to phrenic motoneurons. Transformation of ineffective or silent synapses to functionally effective synaptic connections has considerable precedent [352]. For example, silent glutamatergic synapses in the rat dorsal horn are transformed into functional synapses by serotonin [360]. Serotonin may be of particular importance as CIH enhances phrenic motor output via serotonin-dependent mechanisms in spinal-intact rats [93] and can modulate crossed phrenic pathways [339,342]. Neurotrophins such as BDNF have the potential to strengthen existing synapses to phrenic motoneurons and may be involved in enhancing crossed phrenic activity. BDNF is critical for some forms of neuroplasticity (e.g., hippocampal long-term potentiation, [136]), can enhance neurotransmission at glutamatergic synapses [361] and, importantly, is upregulated in the cervical spinal cord following brief episodic hypoxia [362]. Correlative evidence provides further support for a role of BDNF in enhancing crossed phrenic pathways: a pre-conditioning lesion (CDR) increases BDNF protein levels in the ventral spinal cord [341] and also enhances crossed phrenic pathways [340]. However, preliminary data from our laboratory indicate that neither BDNF nor serotonin are significantly altered in the ventral cervical spinal gray matter two weeks following C2 hemisection, either with or without CIH-treatment [363]. This observation does not rule out a role for these molecules in CIH-induced spinal plasticity, but limits potential involvement to initiation vs. maintenance of increased synaptic strength. For example, in a number of other models of serotonin-dependent neuroplasticity, serotonin receptor activation is necessary to initiate, but not maintain the plasticity [117,364,365]; neurotrophins such as BDNF may play a similar role. Consistent with this idea, BDNF and receptor tyrosine kinase B mRNA expression transiently increase following C2 hemisection, but return to control levels within two weeks [356].

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Contralateral and Supraspinal Effects of Cervical Hemisection

Unilateral SCI often induces plasticity in contralateral motor pools. For example, after thoracic spinal hemisection, perinatal chicks regain posture and locomotor skills via increased reliance on contralateral motor activity [366]. Similarly, rats with cervical or thoracic hemisection injuries have altered locomotor patterns with increased reliance on contralateral motoneurons [367]. In contrast, contralateral respiratory motor output may be inhibited under certain conditions following unilateral spinal cord injury [324]. Two months post-hemisection, anesthetized, paralyzed, and ventilated rats have a decreased inspiratory burst frequency during quiet breathing and reduced contralateral phrenic burst amplitude during hypercapnia [324]. These adaptations may reflect supraspinal (e.g., medullary) plasticity because hypoglossal motor output is also attenuated following cervical spinal hemisection [324]. This diminution of contralateral respiratory motor output following a unilateral lesion is difficult to explain, particularly because chronic C2 hemisected rats maintain adequate ventilation ([321]; Fuller, D.D. and Mitchell, G.S., unpublished observations). However, rats that do not spontaneously express the crossed phrenic phenomenon do not exhibit reductions in contralateral phrenic output [320]. Further, rats receiving a dual injury (C2 hemisection and ipsilateral phrenicotomy) that prevents crossed phrenic motor output from reaching the diaphragm also do not show reductions in contralateral phrenic output [322]. Indeed, the contralateral phrenic nerve actually shows enhanced inspiratory burst amplitude during chemoreceptor stimulation in dual injury rats [322]. Thus, contralateral phrenic motor plasticity is critically influenced by the presence (or absence) of crossed phrenic activity. Compensatory plasticity in contralateral motor pools (i.e., greater motor output) may require necessary preconditions that are established only when crossed phrenic activity is prevented. F.

Cervical Contusion Injuries

Respiratory motor plasticity following cervical spinal contusion injuries is largely unexplored. The clinical literature documents recovery of respiratory motor function following cervical contusion (particularly with low cervical injury), but it is unknown if this recovery reflects neuroplasticity, a change in pulmonary mechanics, or other factors. El-Bohy et al. [368] demonstrated the feasibility of cervical contusion in rats as a model of respiratory dysfunction following SCI. Rats were able to survive a lateralized contusion at C2 created with the New York University contusion system (impact height ¼ 12.5 mm). Phrenic nerve recordings five weeks post-injury suggested a reduced dynamic range of phrenic motor output ipsilateral to the contusion. In specific, baseline motor output averaged almost 80% of


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that observed during asphyxia [368]. Normalization concerns make these data difficult to interpret and additional studies are needed. Following T8 contusion injury in rats (weight drop device, [369]), inspiratory tidal volume is reduced, breathing frequency is elevated, and minute ventilation is unchanged [370]. However, these changes in the pattern of breathing do not persist beyond the first week post-injury [370]. Interestingly, a single spinal injection of basic fibroblast growth factor at the time of injury prevented the initial changes in breathing pattern [370]. This report highlights the potential use of breathing as a functional outcome measure to assess experimental therapeutic approaches following SCI [371]. G. Dorsal Rhizotomy

Acute CDR reveals the crossed phrenic phenomenon in female rats, suggesting a contralateral afferent pathway that may inhibit crossed pathways to phrenic motoneurons [321]. Thus, crossed phrenic motor output following acute CDR may reflect removal of inhibitory afferent projections to phrenic motoneurons via polysynaptic pathways (reviewed in Ref. [372]). This effect may be species specific, as Rosenbluth and Ortiz [329] reported no effect of acute CDR on crossed phrenic activity in cats and rabbits. Chronic dorsal rhizotomy elicits morphological and neurochemical plasticity within the spinal cord. Chronic CDR increases serotonin terminal density in the immediate vicinity of labeled phrenic motoneurons and also increases phrenic motoneuron size [128]. Chronic dorsal rhizotomy also increases serotonin terminal density in the spinal dorsal horn [349,373,374]. Chronic CDR is associated with increased ventral cervical spinal concentrations of BDNF and a related neurotrophic factor, neurotrophin-3 [341]. In association, CDR has little impact on the shortterm hypoxic phrenic response and PHFD [128]. However, serotonindependent phrenic LTF is enhanced following chronic CDR [128]. The enhanced LTF is blocked by the 5-HT2 receptor antagonist ketanserin, indicating that this form of metaplasticity (i.e., enhanced LTF) exhibited normal serotonin receptor dependence [128]. Chronic CDR also reveals normally ineffective crossed phrenic pathways [340]. In acutely C2 hemisected rats, crossed phrenic potentials were evoked at a lower current in rats with chronic CDR [340]. Importantly, the phrenic nerves were sectioned prior to recording in these experiments in both control and CDR rats; thus inhibitory phrenic afferent inputs were absent in both experimental groups. In contrast to the LTF data (see above), serotonin receptor antagonism had minimal effect on crossed phrenic activity following chronic CDR [340]. Thus, serotonin receptor activation is not necessary to maintain enhanced crossed phrenic pathways following CDR,

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although this does not rule out a potential role for serotonin in initiating spinal plasticity. It is tempting to speculate that changes in spinal neurotrophins following CDR [341] contribute to enhancement of crossed phrenic pathways, but this hypothesis requires rigorous evaluation. Alternatively, respiratory plasticity following spinal sensory denervation does not necessarily result from a spinal mechanism. CDR can induce plasticity distant to the surgically affected area. For example, CDR (C3–C5) enhances LTF of hypoglossal motor output [375], even though the hypoglossal motor nucleus is rostral to the site of denervation. Therefore, changes in respiratory motor output following chronic dorsal rhizotomy may reflect plasticity at distant sites (see below). Bilateral thoracic (T2–T12) dorsal rhizotomy in goats induces functional and morphological plasticity [349]. After recovery from surgery, an intriguing pattern of severe ventilatory failure during even modest exercise, followed by progressive functional recovery, was observed [349]. In association with these functional deficits and/or recovery, increased serotonin concentrations and serotonin-immunoreactive terminal density were found in the thoracic and cervical spinal cord, suggesting that serotonin may be involved in the mechanism underlying functional recovery [349]. However, there was also evidence for increased spinal dopamine and norepinephrine concentrations [349]. One intriguing aspect of these changes in spinal neuromodulators is an observation that serotonin and dopamine increased at spinal segments C6–C7, the segments associated with the phrenic motor nucleus in goats [349]. One possible interpretation of these findings is that neurochemical changes outside the segments directly affected by surgery (and denervation) compensate for the loss of respiratory function and thereby contribute to functional recovery. These hypotheses remain to be tested. V.

Conclusion

The neurons and networks controlling ventilation express plasticity and metaplasticity in response to a myriad of stimuli including alterations in environmental gases and neural injury. Several unifying principles emerge from our review of respiratory plasticity. First, the pattern, duration, and severity of experimental or environmental stimulation is an important determinant of respiratory plasticity. Second, both sex and genetics must be considered when evaluating phenotypes associated with, or mechanisms responsible for, respiratory plasticity. Finally, development and aging exert considerable influence on the expression of respiratory neuroplasticity. Some forms of respiratory plasticity are unique to development; others may be unique to later periods (e.g., adult or geriatric animals).


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The physiological significance of respiratory plasticity is clear in some examples (e.g., recovery of ventilation following spinal injury) and perhaps less so in other cases. Through continued study we may come to appreciate the physiological significance of these and other forms of respiratory plasticity. For example, LTF of upper airway muscle activity may stabilize the pharyngeal airways during sleep, and a loss of LTF with age may contribute to OSA. Respiratory neurobiologists are beginning to unravel the mechanisms underlying certain forms of plasticity (e.g., LTF, VAH, etc.) and these efforts are leading to a greater understanding of respiratory neural control. Equally important, such studies may lead to insights concerning mechanisms of plasticity throughout the CNS. It is our hope that careful exploration of respiratory plasticity, using molecular through behavioral approaches in comparative and clinical studies, will lead to an understanding of the role played by plasticity (or lack of plasticity) in respiratory disorders. Further, an understanding of respiratory neuroplasticity mechanisms may provide the rationale to harness this plasticity for rehabilitative efforts (e.g., following SCI) or for the treatment of respiratory-related pathologies such as OSA. Acknowledgments D.D. Fuller was supported by a Parker B. Francis Fellowship in Pulmonary Research. R.W. Bavis was supported by NIH NRSA post-doctoral fellowship award HL70506. Work conducted in G.S. Mitchell’s laboratory was funded by the National Institutes of Health (HL/NS 69064, HL53319, and HL 65383). We thank Brad Hodgeman for preparation of the figures and Drs. T. Baker-Herman, F.J. Golder and S.M. Johnson for their helpful critique of the manuscript. References 1.

Mitchell, G.S. and Johnson, S.M., Neuroplasticity in respiratory motor control, J. Appl. Physiol. 94, 358–374, 2003. 2. Bliss, T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of anaesthetized rabbit following stimulation of the perforant path, J. Physiol. 232, 331–356, 1973. 3. McGaugh, J.L., Memory—A century of consolidation, Science 287, 248–251, 2000. 4. Mitchell, G.S., Baker, T.L., Nanda, S.A., Fuller, D.D., Zabka, A.G., Hodgeman, B.A., Bavis, R.W., Mack, K.J. and Olson, E.B., Jr., Intermittent hypoxia and respiratory plasticity, J. Appl. Physiol. 90, 2466–2475, 2001. 5. Feldman, J.L., Mitchell, G.S. and Nattie, E.E., Breathing: Rhythmicity, plasticity, chemosensitivity, Annu. Rev. Neurosci. 26, 239–266, 2003.

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7 Airway Reflexes in Humans

TAKASHI NISHINO Chiba University Chiba, Japan

I.

Introduction

In general, a reflex consists of a neural receptor, afferent pathway, central synapses, efferent motor pathway, and effector organ; this general rule can be applied to airway reflexes (Figure 7.1). The effects of airway reflexes are very diverse and include changes in breathing pattern, maintenance of airway patency, defensive or protective reactions, alterations in bronchomotor tone, mucus-secreting response, and cardiovascular changes. These reflex responses are influenced by the nature and strength of the stimulant. In addition, some of these reflex responses are highly specific for the particular respiratory site. This chapter will deal chiefly with airway reflexes in humans, describing general characteristics and the function of airway reflexes, and some clinical problems related to anesthesia and respiratory care.

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Nishino

Central nervous system (airway reflex program)

Afferent pathway

Afferent pathway

Nervous receptors

Effector organs

Stimulation

Airway reflexes

Figure 7.1 Simplified model of airway reflex system.

II.

Central Nervous System

It is obvious that the central nervous system (CNS) plays a crucial role in initiation of airway reflexes. However, studies on the neurophysiology of airway reflexes have mainly defined the afferent pathways, leaving the CNS as a black box, and little is known about the central neural organization of airway reflexes. Since primary afferents from the receptors in the airways travel in the vagus (X), glossopharyngeal (IX), and trigeminal nerves (V), and converge in the solitary tract destined for synaptic contact with secondorder neurons in the nucleus tractus solitarius (NTS), the NTS must be viewed as the single most important site of early integration of afferent input relevant to the control of airway reflex systems. The NTS also is richly endowed with neuropeptides and other neuroactive substances [1–3] and recent studies have demonstrated the presence of various receptors in the central pathways such as opioid, serotonin, dopamine and N-methyl-D-aspartate (NMDA) receptors [4–6]. Although the study of CNS pharmacology modulating airway reflexes provides some important information, the brainstem neuronal network mechanisms that mediate airway reflexes are still poorly understood.

III.

Afferent Innervation of the Upper Airway and Receptors

The upper airway is composed of the nose, the pharynx, the larynx, and the extrathoracic portion of the trachea. The anatomy of the upper airway is very complex and its structural complexity reflects diverse functions such as

Airway Reflexes in Humans

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phonation, olfaction, air conditioning, digestion, preservation of airway patency, and protection of the airways. Sensory nerve endings in and under the epithelium of the upper airway that respond to various stimuli are called upper airway receptors. Upper airway receptors can be classified into nasal, pharyngeal, laryngeal, and tracheal receptors, based on their location. We know little about the structure of these receptors although numerous afferent terminals have been described in the upper airways: free nerve endings, distributed among the epithelial cells, having either myelinated or non-myelinated fibers, as well as more organized structures like corpuscles and taste buds [7,8]. There are free nerve endings with myelinated afferent nerve fibers under the epithelium of the upper airways, which not only respond to a wide variety of irritant gases and aerosols but also show a rapidly adapting response to maintained mechanical deformation [9]. These nerve endings are especially sensitive to chemical and mechanical stimulation and are classified as irritant receptors [10] (Figure 7.2). With single-unit action potential recordings, Sant’Ambrogio et al. [11,12] showed in the larynx of the dog that in addition to irritant receptors, there are three groups of receptors classified on the basis of their responses to airflow and mechanical changes (Figure 7.3). These are: (1) cold (flow) receptors which are affected by changes in laryngeal temperature; (2) pressure receptors which are sensitive to changes in laryngeal transmural pressure, and (3) drive receptors which are affected by laryngeal motion. Although the extrapolation of such classification to other sites of the upper airways may not be entirely valid,

Cigarette smoke AP % CO2

5 0

Pes (kPa) −0.5 −1.0 Water AP Pes (kPa) −0.5 −1.0

Figure 7.2 Responses of a laryngeal irritant receptor to cigarette smoke and distilled water. A.P.: action potentials. Exposure of the upper airway to smoke is indicated by the CO2 signal (Data from Ref. 10).

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(a)

0 −1.5

. 0.5 V 0 (1/s) 0.5 Lar. Temp. 34 36 (C) 3s A

A A.P.

A.P.

0 Pes [k Pa] –1

+0.8 Plr [k Pa] 0.0 −0.8 B

B

(b)

A.P.

(c)

0 Pes [k Pa] –1 –2

A.P. +0.8 Plr [k Pa] 0.0 −0.8 −1.6 C

C A.P.

A.P. 0 Pes [k Pa] –1

Plr [k Pa]

–2 1s

+0.8 0.0 −0.8 −1.6 −2.4 2s

Figure 7.3 Different types of laryngeal receptors responding to airflow and mechanical changes. (a) Behavior of laryngeal cold (flow) receptor during inhalation of warm air. Dog is spontaneously breathing through upper airway. Note that inspiratory modulation of receptor disappears when laryngeal temperature during inspiration is raised to expiratory level by inhalation of warm air (between arrows). A.P.: action potentials; Pes: esophageal pressure; V; airflow; Lar. Temp: laryngeal temperature. (b) Laryngeal pressure receptor responding to negative transmural pressure. Note that maximum activity is seen during upper airway occlusion, in which larynx is subjected to increased negative pressure. Recording from left SLN. A: upper airway breathing changed to tracheostomy breathing at arrow. B: upper airway breathing and upper airway occlusion. C: tracheostomy breathing and tracheal occlusion. (c) Laryngeal drive receptor responding to distortion caused by action of upper airway muscles. Abbreviations are the same as Figure 7.3(b). Note that an inspiratory activity is present and is not influenced by flow. Inspiratory activity increases when inspiratory drive increases (occluded efforts) (Data from Refs. 11 and 12).


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receptors in different sites of the upper airways have many properties in common. For example, an activity related to cooling of the nasal cavity has been demonstrated in the ethmoidal nerve of cats [13,14], rats [15], and guinea pigs [16]. Also, the presence of pressure-responsive receptors within the nasal passages has been demonstrated in the ethmoidal nerve of rats [17] and cats [14]. Although non-myelinated afferent fibers from the larynx have been described in the larynx of cats [18,19] and guinea pigs [20], they are thought to be scanty and information about their properties is apparently lacking. However, it has been reported that intralaryngeal capsaicin, given to stimulate C-fiber receptors, causes no respiratory response in dogs, but induces apnea and hypertension in rats [21]. Therefore, in some species, the functional role of C-fiber receptors may not be ignored.

IV.

Reflex Responses from the Upper Airway

Reflex activity in the upper airway is associated with two important respiratory functions subserved by the upper airway: (1) defense and protection of airway and (2) maintenance of airway patency.

A. Defense and Protection of Airway Nose

Mechanical and chemical irritation of the nasal mucosa can elicit various reflex responses. Although the sneeze is one of the representative respiratory reflexes from the nose, it has been studied far less than other nasal reflex responses. This is probably because the sneeze is easily blocked by anesthesia. Nevertheless, there is evidence to show that, in anesthetized cats, electrical stimulation of the anterior ethmoidal nerve, the posterior nasal nerve (PNN) or the infraorbital nerve (ION), all of which are branches of the trigeminal nerve, can elicit a sneeze identical to that induced by mechanical stimulation [22]. The trigeminal nerves carry most of the afferent fibers, which respond to chemical and mechanical irritation on the nasal mucosa. Therefore, the trigeminal nerve is considered the main afferent pathway for the sneezing reflex. However, strangely enough, the sneeze is inhibited when the ION is stimulated together with either the anterior ethmoidal nerve or the PNN [22], suggesting the presence of inhibitory pathways in the ION. Sneezing can also be elicited by local application of capsaicin, nicotine or formalin to the nasal mucosa, but higher doses of capsaicin that deplete substance P-containing nerves of the neuropeptide can prevent the sneeze caused by inhaled irritants [23].

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During sneezing, the respiratory changes are quite similar to those of coughing. Thus, the expulsive effort is preceded by a deep inspiration. Unlike the cough, however, the pharynx seems to be constricted and the forced expiration is via both the nose and the mouth. Bronchoconstriction is characteristically present with coughing, but occurs only infrequently with sneezing. While it is possible to perform voluntary coughing, it is virtually impossible to reproduce sneezing voluntarily. Stimulation of the nasal mucosa can also produce depression of breathing or even apnea (Figure 7.4). However, it is not known why the same stimulus may cause different reflex responses and what determines which reflex will occur. Depression of breathing and apnea are more commonly observed than sneezing, at least in experimental conditions, presumably because these reflexes are more resistant to general anesthesia than the sneeze reflex [24]. The apneic reflex, which is possibly related in mechanism to the diving reflex of aquatic animals, would prevent or limit penetration of water or irritant gases in the respiratory tract. Despite the fact that the nose is an important reflexogenic site in humans, depression

60 0

HR

100 BP (mmHg) 0 Ptr (cmH2O)

. V (I/sec)

10 0

10 0

VT

4

(I)

0

PET

CO2

40

(mmHg)

0

Fiso

5

(%)

0

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Figure 7.4 Respiratory response to nasal insufflation of a pungent anesthetic (isoflurane) in a lightly anesthetized human. HR: heart rate; BP: arterial blood pressure; Ptr: intratracheal pressure; V_ : airflow; VT: tidal volume; PETCO2 : end-tidal PCO2: Fiso: isoflurane concentration.


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of breathing or apnea may be relatively insignificant in clinical situations. For instance, mechanical stimulation in the nose in the newborn rabbit can cause death [25], but this never happens in human babies. Larynx

The larynx is one of the most important reflexogenic sites. Defensive and protective reflexes originating from the larynx include cough, apnea, the expiration reflex, laryngeal closure, swallowing, bronchoconstriction, and mucous secretion. The richness of the reflex response is properly represented by the abundance of sensory endings in various laryngeal structures. Among various reflex responses, cough, the expiration reflex, and swallowing can be identified as defense reflexes that aim at removal of the irritant agent, while apnea, laryngeal closure and bronchoconstriction are described as protective reactions that aim at stopping harmful penetration of the noxious agent from the outside environment [26]. The reflex responses elicited from the larynx vary with the nature and strength of the stimulant. For example, weak mechanical and chemical stimulations may introduce only the laryngeal closure reflex [27,28]. With stronger stimuli, forceful expiratory efforts such as the expiration reflex and coughing may be induced. The expiration reflex consists of a brief expiratory effort not preceded by an inspiration [29]. In contrast, the cough reflex usually starts with a brief rapid inspiration, followed immediately by a compressive phase in which the glottis is closed and the expiratory muscles contract forcefully together with the diaphragm [30]. An expulsive phase follows immediately, when the glottis is suddenly reopened, allowing the expulsion of a blast of air. During that time, oscillation of tissue and gas causes a characteristic explosive sound and may play a role in suspending secretions in the moving gas stream. Coughing elicited from the larynx appears to be similar in its fundamental characteristics to that evoked from the trachea. However, it has been shown in animal studies that the pattern of coughing from the larynx is slightly different from that induced from the trachea (laryngeal coughing has a higher frequency, a paroxysmal nature and stronger inspiratory efforts) [30]. Although there is no doubt that the larynx is an important tussigenic area, several studies raised the question about the importance of laryngeal afferent input in the cough reflex. Stockwell et al. [31] showed that anesthetic block of the superior laryngeal nerve (SLN) in man did not affect the cough threshold to inhaled nebulized citric acid in awake humans, suggesting that SLN afferents do not play a necessary role in initiation of citric acid-induced cough. A similar conclusion may be derived from the study of Higenbottam et al. [32] who showed that patients with cardiopulmonary transplants showed a poor or absence cough response to distilled water aerosols. It seems that coughing due to inhaled irritants is not

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much changed by laryngeal denervation; however, this does not necessarily mean that the larynx cannot be an important site for the initiation of cough. Pharynx

Mechanical stimulation of the mucosa of the nasopharynx in cats can elicit a sniff-like aspiration reflex that produces repeated powerful contraction of the diaphragm [33]. The purpose of this reflex response may be removal of foreign materials from the back of the nose into the pharynx to be swallowed or expelled by coughing. Korpas and Tomori [30] have shown that this reflex is exceptionally resistant to anesthesia, hypercapnia, asphyxia, hypothermia, and antitussive drugs. Although this reflex has been demonstrated in many other mammalian species, it seems that it is very weak in humans, including in neonates. The most obvious reflex response elicited from the pharynx is reflex swallowing. Although the main function of reflex swallowing is the propulsion of food from the oral cavity into the stomach, it also can serve as a protective reflex for the respiratory tract [34]. In general, swallowing can be divided into three states: (1) the initial oral preparatory state, (2) the subsequent pharyngeal state, and (3) the esophageal state. Of the three states, the involuntary control of the pharyngeal state of swallowing is the most important state from the standpoint of airway protection. Swallowing results in reflex closure of the glottis, the single most vital function of the larynx. Strong adduction of the true vocal cords is supplemented by the closure of the false cords and approximation of the aryepiglottic folds, although adduction of the true cords alone suffices to prevent entrance into the trachea of swallowed material. Swallowing must interact with respiration so that a swallow causes minimal or no disturbance of continual respiration [35]. In awake human adults, approximately 80% of swallows occur during the expiratory phase, and respiratory movement resumes after a swallow in the same expiratory phase as has been interrupted [36]. The preponderant coupling of swallows with the expiratory phase may be a useful mechanism for clearing the airway of foreign materials before the subsequent inspiration and thus may exert a physiologic role in preventing low-grade recurrent aspiration. This coupling of swallows with the expiratory phase is lost in unconscious adults, suggesting that the mechanism responsible for the preferred timing of swallows during the expiratory phase may have both centrally programmed and learned behavioral components [37]. The preponderant coupling of swallows with the expiratory phase is also lost during hypercapnia [38] and with the addition of respiratory elastic loading in conscious humans [39]. In these conditions, the timing of swallows shifts from the expiratory phase towards the inspiratory phases of the next breath. It may be possible that with this shift, incompletely eliminated pharyngeal content is aspirated into the


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larynx during the subsequent inspiration. In fact, it has been suggested that swallows coinciding with the expiratory–inspiratory transition phase are the most liable to produce aspiration [40].

B. Maintenance of Airway Patency

Until recently, only the defensive role of upper airway reflexes had been studied in detail, although it has been known for many years that pressure and airflow in the upper respiratory tract can change breathing [41]. The results of several studies [42–48] that analyzed the mechanisms of sleep apnea and sudden infant death syndrome indicate that upper airway pressure reflexes have important actions on the musculature of the pharynx and therefore on its patency, on breathing, and on arousal. For example, negative pressure in the upper airway produces an excitatory effect on upper airway dilating muscles while exerting an inhibitory effect on breathing patterns by prolonging inspiratory and expiratory durations and by decreasing the rate of rise of diaphragmatic activity [42–44,47,48]. The main reflexogenic site responding to pressure changes is thought to be the larynx that is supplied by the internal branch of the SLN, whereas the nose plays a subsidiary role and the oropharynx does not seem to be important. Thus, the negative upper airway reflex can be abolished by mucosal application of topical anesthetics or by cutting the SLN [43,49]. Both the excitatory influences on the upper airway controlling muscles and the inhibitory influences on chest wall muscles can be interpreted as contributing to airway stability in that they increase the dilating forces and reduce the collapsing forces, respectively. In fact, the elimination of sensory feedback from the upper airway impairs the ability of upper airway muscles to respond to adverse conditions such as airway obstruction [50]. Other studies [51,52] also showed that in awake humans, a strong activation of the genioglossus muscle occurs in response to negative pressure applied in the upper airway, which can be abolished by upper airway anesthesia. Although application of negative pressure causes genioglossus muscle activation in both wakefulness and sleep, the increase in genioglossus activity is attenuated during non-Rapid Eye Movement (non-REM) sleep in adult subjects as well as in neonates [53,54]. Regarding maintenance of airway patency, the ability to switch the breathing route appears to be another important function. Acute obstruction of nasal passages leads to opening the mouth and oral breathing. The ability to change from the nasal route of airflow to the oral route during nasal obstruction is crucial for maintenance of adequate ventilation. Two major muscles responsible for the switching of breathing route are the palatoglossus muscle, which directs the soft palate caudally and ventrally, and the levator veli palatini, which pulls the soft palate cephalad and in

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a dorsal direction. Activation of these muscles results in nasal breathing and oral breathing, respectively. Consciousness is an important factor in the control of the route of breathing [55], but the switching of breathing route could also be triggered by some reflex mechanisms. It has been shown that in awake subjects, application of topical lidocaine in the nasal passages delays the onset of oral breathing in response to nasal occlusion (Figure 7.5), suggesting that in humans, sensory information from receptors in the nasal passage has an important role in controlling the shift of breathing route [56]. V.

Afferent Innervation of the Lower Airway and Receptors

The vagus nerve and its bronchial branches innervate the tracheobronchial tree and lung parenchyma. In contrast to the dense parasympathetic nerve supply to the lower airways, adrenergic innervation is sparse in humans [57]. Furthermore, there is no evidence for functional sympathetic innervation in isolated human smooth muscle [58]. It is generally agreed that in the lower airway, there are at least four different types of receptors: (1) slowly adapting receptors (SARs); (2) rapidly adapting receptors (RARs); (3) C-fiber endings, and (4) sensory receptors in neuroepithelial bodies (NEBs). Of these, the structure of only two types of receptor, i.e., SARs and NEBs, can be stated with confidence despite numerous studies on the morphology of the sensory innervation of the airways. The reflex function of the latter is unclear [59].

Nasal chamber pressure (cm H2O) Mouth chamber pressure (cm H2O) SaO2 (%)

5 0 1 0

BEFORE ANAESTHESIA

AFTER ANAESTHESIA

100 80

End-tidal CO2 nose (mm Hg)

40 0

End-tidal CO2 mouth (mm Hg)

40 0

10 S

Figure 7.5 Respiratory responses to nasal occlusion in one nasal breather. The subject was breathing through a face mask, which separates nasal and oral passages. Horizontal bars indicate nasal obstruction and arrows indicate the beginning and end of oral breathing. Note that oral breathing starts immediately after nasal obstruction before topical nasal anesthesia whereas the start of oral breathing delays considerably after topical nasal anesthesia (Data from Ref. 56).


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A. Slowly Adapting Receptors (SARs)

Slowly adapting receptors are found in the smooth muscle of the larger airways and correspond to the myelinated afferent nerve fibers in the vagus. The SARs are the more easily identifiable of the endings, having a respiratory modulation because of the regular characteristics of their discharge both during transient changes in lung volume and during maintained inflations. Some SARs have a continuous discharge during the respiratory cycle with an increasing rate during inspiration, indicating the activation threshold of these receptors is below functional residual capacity (FRC), whereas others can fire only above FRC [60] (Figure 7.6). In general, they are not chemosensitive, but they are stimulated by drugs such as histamine and acetylcholine, which contract smooth muscle [61,62]. Inhalation of CO2 and volatile anesthetics is also known to affect the

(a)

(b)

A.P.

Ptr

cmH20 5 0 1s

(c)

(d)

A.P.

cmH20 Ptr

5 0

Figure 7.6 Subtypes of pulmonary stretch receptors (SARs). (a) Low-threshold SARs with tonic expiratory discharge; (b) low-threshold SAR with cardiac modulation; both (c) and (d) are high-threshold SARs, but tracheal threshold pressure of (c) is much higher than that of (d) (Data from Ref. 60).

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activity of SARs [63]. Inhalation of CO2 inhibits their activity by a direct action on the receptor with action on 4-aminopyridine-sensitive Kþ channels [64], whereas volatile anesthetics may inhibit or stimulate the receptors, depending on the concentration and the type of SAR [60]. B. Rapidly Adapting Receptors (RARs)

Although the structure of RARs has not been fully delineated, RARs are known to have non-myelinated terminals connected to thin myelinated vagal afferents (A) [59,63]. They are found throughout the lower airways but are scanty in the smaller bronchi, and none has been identified in the bronchioles and alveoli. As the name implies, these receptors adapt rapidly to a maintained inflation or deflation of the lungs. The respiratory modulation of the RARs is irregular in both its timing with the breathing cycle and its pattern of discharge. RARs are activated by a large number of mechanical and chemical irritant stimuli (ammonia, ether vapor, cigarette smoke, etc.), by inflammatory and immunological mediators, and by airway and lung pathological changes. All the stimuli that can induce coughing can also stimulate RARs, and therefore, it is likely that RARs are directly involved in elicitation of the cough reflex [65]. Also, most of the mechanical and chemical irritants that stimulate RARs are effective bronchoconstrictors, although cough and bronchoconstriction are two separate reflexes [32]. C. C-fiber Endings

Two groups of C-fiber receptors have been distinguished on the basis of their circulatory accessibility through either the pulmonary or the bronchial circulation [66,67]. Pulmonary C-fiber receptors are those arising from the endings located in the lung parenchyma, and are directly accessible to a challenging drug injected into the pulmonary artery, whereas bronchial C-fiber receptors located further down stream, innervating the airway mucosa, are accessible to the challenging drug injected into the left atrium or directly into the bronchial artery (Figure 7.7). Although the distinction between pulmonary and bronchial C-fiber receptors was based initially on the location and circulatory accessibility, the two have been found to differ somewhat in their afferent properties. For example, pulmonary C-fiber receptors show a relatively greater mechanosensitivity than do bronchial C-fiber receptors in dogs [67,68]. Pulmonary C-fiber endings are relatively insensitive to autacoids such as bradykinin, histamine, serotonin, and prostaglandins, whereas bronchial C-fiber endings are sensitive to a wide range of intrinsic chemicals including histamine, bradykinin, and prostaglandins, either injected into the bronchial artery or administered as aerosol


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Pulmonary C-fiber and rapidly adapting receptor

5 sec.

ENG Pes 0 (cmH20) −10 0 Ptr −10 (cmH20) −150 100 BP (mmHg) 50

PTO2

21 16 5 0

(%)

PTCO2 (%)

Marker

Inj.

Bronchial C-fiber ENG Pes

0

(cmH20) −10 Ptr 0 (cmH20) −10 −150

BP 100 (mmHg) 50 PTO2 (%)

PTCO2 (%)

21 16 5 0

Marker Inj.

Figure 7.7 Responses of pulmonary C-fiber (small spikes) and rapidly adapting receptor (larger spikes) (upper panel) and a bronchial C-fiber (lower panel) to the right atrial injection of capsaicin (10 mg/kg) in an anesthetized, spontaneously breathing dog. Note that the pulmonary C-fiber was activated immediately after the capsaicin injection, but the bronchial C-fiber had a longer latency, whereas in both cases apnea was followed by hypotension and bradycardia. The pulmonary C-fiber was located in the central part of the left lower lobe and the bronchial C-fiber ending was located at the hilum of the left lower lobe. ENG: electroneurogram; Pes: esophageal pressure; Ptr: intratracheal pressure, BP: arterial blood pressure; PTO2 : PO2 in tidal air; PTCO2 : PCO2 in tidal air. The injection time is marked on the bottom trace (Data from Ref. 66).

[67–69]. In contrast, the two groups of C-fiber receptors respond similarly to inhalation of volatile anesthetics [66]. D. Neuroepithelial Bodies (NEBs)

The airway and alveolar epithelia contain pulmonary neuroendocrine cells that contain a large range of neuroendocrine markers and bioactive substances such as serotonin, calcitonin gene-related peptide, and the mitogen bombesin. These cells are sometimes collected into clusters called neuroepithelial bodies (NEBs). The NEBs are innervated and the recent evidence shows that there are two separate populations of sensory nerve fibers that selectively contact the NEBs, i.e., the vagal sensory component

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and the spinal sensory component [70]. The NEBs are thought to be sensors of hypoxia, and hypoxia-sensitive Kþ channel have been identified in their membranes [71]. Hypoxia is presumed to release mediators from the NEBs, which in turn stimulate the sensory nerves and exert local actions on airway smooth muscle and the bronchial and pulmonary vascular beds [59]. However, there is no evidence to suggest that sensory nerves associated with NEBs are involved in elicitation of airway defensive reflexes. VI.

Integrative Aspects of the Airway Reflexes Elicited from the Lower Airways

A. Lung Inflation and Activation of SARs

It is generally believed that inflation of the lungs activates SARs and causes the classical Breuer-Hering inflation reflex that provides the predominant mechanism for regulating the depth and rate of breathing in anesthetized animals. Although there are some reports to suggest that the Breuer-Hering inflation reflex is operative at normal tidal volume in human subjects [72,73], this reflex is considered very weak in healthy eupneic man, particularly in conscious conditions. Hamilton et al. [74] studied the effect of passive lung inflation during wakefulness and during stable non-REM sleep in laryngectomized patients. During sleep, apnea, as the evidence of the Breuer-Hering inflation reflex, was produced by lung inflations only when inflation volumes exceeding 1 liter were applied at end-inspiration, whereas no change in respiratory timing was apparent during wakefulness even with greater inflation volume, suggesting that the Breuer-Hering inflation reflex can be demonstrated above the resting tidal volume range in adult man only in the absence of the behavioral control of breathing. In contrast to the responses observed during lung inflation, deflation of the lungs causes reflex tachypnea and a reflex increase in inspiratory drive in most species including man [75]. Considering the experimental results that deflation reduces the input from SARs and stimulates RARs [76,77], the reflex excitatory effect on breathing appears to result from stimulation of RARs. Inflation of lungs causes not only Breuer-Hering inflation reflex but also reflex relaxation of smooth muscle [78]. This reflex is predominantly attributable to inhibition of parasympathetic cholinergic motor tone since atropine can prevent this reflex action. Although clinical significance of this reflex is not entirely clear, it has been proposed this reflex could adjust airway geometry in different patterns of breathing to optimize the relationship between airways resistance and deadspace [79]. The significant role of SARs on coughing was implicated first by Bucher [80] who suggested that during the inspiratory effort, which is an


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integral part of the cough reflex, SARs are more intensively stimulated, thereby increasing the inhibitory influence on central inspiratory activity and thus strengthening the subsequent expiration. The studies of Hanacek et al. [81] and Sant’Ambrogio et al. [82] showed that in anesthetized rabbits, stimulation of tracheal mucosa failed to elicit cough when high concentrations of SO2 had abolished the Breuer-Hering inflation reflex (Figure 7.8). In anesthetized humans, lung inflation with CPAP of 10 cmH2O did not exert any influence on the reflex responses to tracheal irritation in terms of the types, latencies, and duration of reflex responses, suggesting the role of SARs in elicitation of cough is speculative in anesthetized humans [83]. However, information obtained in anesthetized subjects may not be entirely applicable to awake subjects. Since it has been reported that the cough response to inhalation of nebulized distilled water is remarkably diminished in awake, heart-lung transplantation patients whose lungs are denervated below the level of the tracheal anastomosis, compared with normal subjects [32], it is possible that loss of the facilitatory influence of SARs on

Control BP

SO2

100

(mmHg) 0 Dia. EMG Abd. EMG 10 Pes 0 (cmH2O) –10 PLC 10 0 (cmH2O)

BP

100

(mmHg) 0 Dia. EMG Abd. EMG Pes 10 (cmH2O) 0 –10

Figure 7.8 Effects of SO2 administration on the cough reflex in an anesthetized rabbit. In the control situation, lung inflation to 10 cmH2O inhibited breathing (Hering-Breuer inflation reflex). Cough (indicated by increased activity in diaphragm and abdominal muscles) could be elicited by tracheal stimulation. After SO2 administration (200 ppm), the Hering-Breuer inflation reflex has disappeared and cough could not be elicited with tracheal stimulation. Left-hand tracings: before SO2 administration; right-hand tracings: after SO2 exposure. Upper panels: effects of lung inflation. Lower panels: effects of tracheal stimulation (Data from Ref. 82).

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coughing could contribute to the diminished cough response during laryngeal irritation in these patients. B. Reflex Responses Elicited by Mechanical and Chemical Stimulation of the Lower Airways

Considerable differences exist among species with respect to the type and magnitude of reflex responses elicited from the lower airways. For example, mice and ferrets show no cough response to mechanical stimulation of the tracheal mucosa [84,85] whereas the same stimulation elicits vigorous coughing in cats and dogs [86,87]. The reflex responses evoked from the stimulation of the lower airways are linked to arousal. Sullivan et al. [88] demonstrated that a stimulus sufficient to cause coughing during wakefulness failed to do so in either SWS or REM sleep in the absence of arousal. When the stimulus was sufficient to cause arousal, coughing always followed arousal, suggesting that coughing involves supra-medullary neural processes that are normally active only during wakefulness. In anesthetized animals, irritation of the trachea with mechanical stimuli causes coughing, hypertension and laryngeal constriction, whereas irritation of the bronchi with chemical stimuli causes hyperpnea [86,87]. Also, in general the trachea and its vicinity are very sensitive to mechanical stimulation whereas the bronchi are more sensitive to chemical stimulation. These differences in the reflex responses seem to depend on the difference in the characteristics of RARs in different sites, since it is known that RARs in the trachea and larger bronchi are very mechanosensitive whereas RARs in bronchi are more chemosensitive [59]. In anesthetized humans, at least six different types of respiratory responses are observed during stimulation of the tracheal carina induced by injection of a small amount of distilled water [89]: namely (1) the apneic reflex, (2) the expiration reflex, (3) the cough reflex, (4) spasmodic panting, (5) slowing of breathing, and (6) rapid, shallow breathing. However, the level of anesthesia has a marked influence on these reflex responses. Thus, among these reflex responses, the cough reflex is the most sensitive and the apneic reflex is the most resistant to deepening anesthesia, whereas the other types of reflex responses were in between (Figure 7.9). Airway receptors responsible for eliciting airway reflexes do not appear to be uniformly distributed in the airways [62], and therefore it is not surprising to observe the differences in the reflex responses from different sites in the airways. In contrast with the reflex responses elicited from the tracheal carina, the same stimulation given to the bronchus causes little or no reflex response [90] (Figure 7.10). This observation is in agreement with the observation made by Jackson [91] that a small mechanical irritant in the trachea causes vigorous coughing whereas mechanical stimulation of the


% RESPONSES

100

241 Expiration reflex

Apnea

Spasmodic, panting breathing

80 60 40 20 1.3

% RESPONSES

100

1.0

0.7 MAC

Slowing of breathing

Cough reflex

80

Rapid, shallow breathing

60 40 20

1.3

1.0

0.7 MAC

Figure 7.9 Occurrence of various respiratory responses to injection of distilled water into the trachea at three different depths of enflurane anesthesia in humans. Ordinate, percent of positive responses. MAC: minimum alveolar concentration (Data from Ref. 89).

finer subdivisions of the tracheobronchial tree causes less cough production during bronchoscopic procedures in awake patients, suggesting the peripheral bronchial branches are less important as reflexogenic areas in humans. Cigarette smoke is one of the most common inhaled irritants in human airways and is known to evoke coughing and reflex bronchoconstriction. Although it has been reported that cigarette smoke activates RARs in the larynx and tracheobronchial trees and thereby elicits coughing and bronchoconstriction [26], the study of Lee et al. [92] demonstrated that in dogs, inhaled cigarette smoke triggers a consistent and vigorous stimulation of pulmonary C-fiber afferents. Nicotine is considered to be primarily responsible for triggering this response, since the response is completely abolished by pretreatment with hexamethonium, a nicotinic acetylcholinereceptor antagonist. In agreement with this idea, in healthy human nonsmokers, the intensity of cigarette smoke-induced airway irritation and cough responses following cigarette smoke inhalation are greatly attenuated by premedication with aerosolized hexamethonium [93] (Figure 7.11).

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(bpm) HR 120 80 (mmHg) BP 120 80 (cmH2O) AP 5 0 (1/min) AF 0.5 0

VT

l 0.3

0 (mmHg) 40 PET CO2 0

(bpm) HR 120 80 (mmHg) BP 120 80 (cmH2O) AP 5 0 (1/min) AF 0.5 0 VT

l 0.3

0 (mmHg) 40 PET CO2 0

Figure 7.10 Respiratory responses to stimulation by distilled water (given at arrows) of trachea and bronchus. Upper panel: tracheal stimulation; Lower panel: bronchial stimulation. Note that stimulation of trachea causes vigorous reflex responses of respiration and circulation whereas bronchial stimulation caused no reflex response. HR: heart rate; BP: arterial blood pressure; AP: airway pressure; AF: air flow; VT: tidal volume; PETCO2 : end-tidal PCO2 (Data from Ref. 90).

Ozone is another potentially harmful chemical stimulus with irritant effects on the lower airways. In dogs, inhalation of ozone into the lower trachea causes tachypnea and bronchoconstriction [94]. In human subjects, breathing ozone for even relatively short periods causes rapid shallow


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Airway irritation (total number of push-button signals)

After placebo 10

After hexamethonium 10

10

8

8

8

6

6

6

4

4

4

2

2

2 0

0

0 0-5

5-15

15-30

Time after smoke inhalation (sec)

Figure 7.11 Comparison of responses to inhalation of high-nicotine cigarette smoke after premedication with aerosolized saline placebo and hexamethonium (10%) (Data from Ref. 93).

breathing, cough, inspiratory chest pain, and bronchoconstriction [95,96]. These reflex responses are thought to be mediated by the increase in vagal C-fiber receptors in the lower airways. In fact, there is evidence to show that the sensitivity of bronchial C-fiber receptors can be increased by ozone in dogs [97] and rats [98]. However, ozone can also activate RARs [97]. Therefore, it is unlikely that all the ozone-induced reflex responses are mediated by bronchial C-fiber receptors. Although a predominant role of C-fiber receptors in the changes of breathing pattern, such as apnea and rapid shallow breathing in response to extraneous chemicals, is well recognized, the role of airway C-fibers in initiating cough remains uncertain and has been the subject of considerable debate. Inhalation of capsaicin aerosol reproducibly causes cough in humans [99] and conscious guinea pigs [100]. In addition, tachykinins, especially substance P, can induce coughing in guinea pigs [101]. In normal and asthmatic subjects, substance P aerosols cause no cough [102], but this agent induces cough in patients with upper respiratory tract infections [103]. By contrast, systemic administration of capsaicin does not usually elicit cough [26] with an exception in one study with conscious humans [104]. Furthermore, there is experimental evidence to show that C-fiber activation can inhibit cough in anesthetized animals [105] (Figure 7.12). In addition, capsaicin is not sufficiently specific as a C-fiber stimulant, since capsaicin can stimulate not only C-fiber endings but also the endings of RARs [106].

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Nishino (a)

(b)

EMG

0.4 V′ (l.s-1)

0.2 0 0.2 0.4

BP (kPa)

20 10 0

30 s

Figure 7.12 Effect of pulmonary C-fiber reflex on coughing induced from the tracheobronchial tree of unanesthetized cat. EMG: electromyographic activity of genioglossus muscle; V 0 : airflow from tracheal cannula; BP: arterial blood pressure. (a) The tracheobronchial mucosa was stimulated mechanically during the signal marks, causing increased EMG activity and airflow, corresponding to cough efforts. The cough efforts continued long after the stimulus had stopped. (b) Phenylbiguanide (25 g/kg) was injected intravenously at the arrow, causing hypotension, bradycardia and apnea due to stimulation of pulmonary C-fiber receptors. During the apnea, the tracheobronchial stimulus was repeated at the signal marks, causing no change in airflow but some increase in EMG activity. Later, during the phase of rapid shallow breathing, the tracheobronchial stimulus was repeated and caused cough efforts, with no coughing after the end of stimulus (Data from Ref. 105).

Thus, it has been argued that inhaled capsaicin could cause cough by stimulating RARs, either directly or indirectly by releasing tachykinins from C-fiber endings, which sensitizes the endings of RARs, or by initiating reflex bronchoconstriction that in turn stimulates the RARs [59,65] (Figure 7.13). VII.

Clinical Problems Associated with Airway Reflexes

A. Pulmonary Aspiration

The pulmonary aspiration of oropharyngeal or gastric contents into the lower respiratory tract is a major complication observed during perioperative periods and a life-threatening danger to every comatose and debilitated


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C-fiber receptor

RAR Capsaicin Irritants, etc.

Mucus Mechanical Epithelium

Tachykinins

Mucosa

Peptidases Tachykinin Antagonists Antipeptidases Inhibit cough

CNS

Excite cough

Cough

Figure 7.13 Diagram of hypothetical role for tachykinins in cough. Tachykinins may be released from C-fiber receptors, and diffuse to RARs, which they stimulate causing cough. They can be broken down by peptidases, which in turn can be inhibited by antipeptidases. Tachykinin antagonists can prevent the action of tachykinins on the RARs. If there is sufficient stimulation of C-fiber receptors, these can cause a central inhibition of cough. CNS: central nervous system; RAR: rapidly adapting receptor (Data from Ref. 65).

patient [34,107]. Several pulmonary syndromes including aspiration pneumonitis, aspiration pneumonia, airway obstruction, lung abscess and chronic interstitial fibrosis may occur after aspiration, depending on the amount and nature of the aspirated material, the frequency of aspiration, and the host’s response to the aspirated material [107]. Among these pulmonary syndromes, aspiration pneumonitis and aspiration pneumonia are the two main syndromes, and although there is some overlap between these syndromes, they are distinct clinical entities. Aspiration pneumonitis, known as acid aspiration syndrome or Mendelson’s syndrome, is a chemical lung injury that occurs in patients who have a marked disturbance of consciousness such as resulting from stroke, drug overdose, or general anesthesia. On the other hand, aspiration pneumonia is an infectious process caused by the inhalation of oropharyngeal secretions that are colonized by pathogenic bacteria. Reliable incidences of clinical aspiration pneumonitis and aspiration pneumonia are difficult to determine since the epidemiologic study of aspiration syndromes is not sufficient. It has been reported that aspiration

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pneumonitis occurs in approximately 10% of patients who are hospitalized after drug overdose [108]. A multicenter, prospective study of nearly 200,000 operations in France from 1978 to 1982 found the overall incidence of aspiration to be 1.4 per 10,000 anesthetics [109]. A review of computer-based records of approximately 185,000 anesthetics administered to adults from 1967 to 1983 in Sweden noted an incidence of 4.7 aspirations per 10,000 anesthetics [110]. The incidence of aspiration pneumonia is more difficult to determine. Several studies indicate that 5–15% of cases of communityacquired pneumonia are aspiration pneumonia [111–113]. There is no doubt that defensive airway reflexes, including the swallowing reflex, play a crucial role in prevention of pulmonary aspiration [34,107]. On the other hand, there are certain clinical situations where a suppression of defensive airway reflexes is desirable. For example, the SLN block may be the choice of technique to facilitate diagnostic laryngoscopy and bronchoscopy as well as to allow the comfortable placement of a tracheal tube in those patients in whom a difficult intubation is anticipated, since this technique can minimize the adverse effects of upper airway reflexes during airway maneuvers [114]. Impairment of defensive airway reflexes may result from a defect or disorder in any part of the reflex arc of the defensive airway reflexes shown in Figure 7.1. Since afferent nerve endings are the natural starting of all reflex activity, it is natural that impairment of triggering defensive airway reflexes occurs after application of local anesthetics into the upper airway. As mentioned above, nerve blocks of the afferent pathway with local anesthetics are useful in elimination of various defensive airway reflexes. The impairment of defensive airway reflexes may also occur in the CNS. It is a common observation that depression of defensive airway reflexes can occur not only during general anesthesia but also during sedation in surgical patients [115,116]. Dysfunctions of efferent neural pathways and effector organs may also seriously impair the defensive airway reflexes. Muscle disorders, disorders of the neuromuscular junction, and disorders that affect peripheral nerves all can lead to impairment of reflex responses and may result in pulmonary aspiration [117]. B. Cough

Coughing is very rare in complete health. On the other hand, chronic cough is one of the most common symptoms seen in ambulatory practice [118]. It is also well recognized that cough is associated with many kinds of diseases such as upper airway infections, asthma, bronchitis, and pneumonia as well as allergy, various occupational exposures, and cigarette smoking [119]. Thus cough is an index of disease and can be used as a tool in differential diagnosis. Although cough is a normal protective reflex, in disease it may be


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persistent, impairing bodily functions and becoming an embarrassment [120]. For example, distal bronchial secretions tend to be spread peripherally during cough and may spread disease within the lung. Trauma to the tracheobronchial wall or larynx may lead to bleeding or may play a role in the development of infection. The muscular effort involved in coughing is relatively great and may aggravate heart failure in patients who have passive congestion of the lungs. Ribs may be fractured as the result of cough [121], and this is a not uncommon cause of severe chest pain. Muscle soreness likewise develops in chronic prolonged cough. During a prolonged paroxysm of cough, the persistent high intra-thoracic pressure may so impede venous return that cardiac output falls and cerebral ischemia occurs [122]. This form of cough syncope may be associated with fainting and convulsions, and is most common in the elderly with cerebral arteriosclerosis. Chronic cough is associated with the risk of myocardial infarction [123]. Cough may also be harmful in a public health sense because of its potential for spreading infections. When the cough is troublesome and serves no useful purpose, the cough has to be treated effectively. The rational clinical approach to the management of chronic cough is to search for an underlying cause and to treat the cause. This approach is usually easy and successful in the majority of patients with chronic cough. Indeed, Irwin et al. [124] showed that utilizing the anatomic, diagnostic protocol, the causes of cough were determined in 99% of their patients, leading to specific therapy that was successful in 98%. It has been reported that the cough reflex in patients with chronic cough is sensitized in several associated conditions, including angiotensin-converting enzyme (ACE) inhibitor cough, gastroesophageal reflux (GER), and cough-variant asthma [125]. However, the mechanism responsible for sensitization of the cough reflex remains obscure. Angiotensin-converting enzyme inhibitor cough occurs in 0.2–33% of patients treated with ACE inhibitors [126], recurring with reintroduction of the same or another ACE inhibitor. It is more common in women. The mechanism may involve accumulation of endogenous tachykinins or bradykinin in the airway due to prevention of breakdown of kinins by ACE, which may stimulate RARs and induce coughing [65,127] (Figure 7.13). Gastroesophageal reflux is a common cause of chronic cough [124,128]. Although some patients may have reflux up to the pharynx and may even aspirate, it is unlikely that aspiration of acid reflux may be a prerequisite. There is evidence to indicate that the cough reflex can be caused by activation of sensory receptors in the distal esophagus [129]. Cough-variant asthma is an occult form of asthma of which the only sign or symptom is chronic cough [130,131]. The diagnosis of cough-variant asthma is made only by demonstration of airway hyperresponsiveness to methacholine or histamine challenge and an excellent response to treatment

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with bronchodilators and corticosteroids [132]. Why some patients with asthma have cough as the principal feature of their disease is totally unclear. Postnasal drip syndrome (PNDS), either singly or in combination with other conditions, is the single most common cause of chronic cough in nonsmoking patients [124]. Although the mechanisms causing cough in patients with PNDS are unclear, it is likely that secretions including inflammatory mediators penetrate to the larynx, and possibly to the trachea, and stimulate the afferent limb of the cough reflex in the upper respiratory tract [133]. C. Laryngospasm

Exaggeration of upper airway defensive reflexes is also a clinical problem. An example of exaggerated reflex responses is laryngospasm. Laryngospasm usually does not occur during wakefulness but it frequently occurs during a light depth of anesthesia, suggesting that light anesthesia may potentiate or consciousness may attenuate the upper airway reflexes. Laryngospasm consists of prolonged intense laryngeal closure in response to direct laryngeal stimulation from inhaled irritant agents, secretions or foreign bodies, leading to severe hypoxemia. In both anesthetized animals and anesthetized humans, there is some evidence to suggest that the laryngeal responses to airway irritation interact with background chemical ventilatory drive. For example, in anesthetized cats hypercapnia alone and hypoxia alone decreases the degree and duration of laryngospasm due to SLN stimulation, whereas hypocapnia augments and prolongs the duration of this laryngospasm [134]. In addition, in humans the addition of 5% CO2 to inspired isoflurane significantly reduces the frequency and severity of airway complications such as breathholding, coughing, and laryngospasm [135]. D. Airway Obstruction

Recognition of clinical problems such as obstructive sleep apnea (OSA) has generated an immense interest in the patency of the upper airway in recent years. Upper airway collapse is often seen in anesthetized patients and in patients with OSA syndrome. Upper airway obstruction in unconscious subjects has been attributed primarily to a result of the tongue falling back [136], but more recent studies indicate that the most common site at which obstruction occurs is the pharynx [137,138]. Upper airway obstruction occurs easily in older, male, and overweight patients who frequently have reduced pharyngeal size or pharyngeal structural abnormalities, suggesting that anatomical factors play an important role in initiating the upper airway obstruction [139]. Upper airway patency during wakefulness is in large part attributable to continual control by the higher nervous system, which regulates inspiratory motor output to the muscles of the pharynx and related structures. In addition to


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the effects of upper airway dilating muscles, the thoracic muscles may also influence the upper airway. During inspiration with the diaphragm, a negative pressure is generated in the pharynx and this negative pressure during inspiration is considered an important factor in promoting occlusion of the upper airway. However, the negative-pressure reflex would be initiated during upper airway obstruction and would tend to compensate for airway obstruction while increasing the pharyngeal size or stiffness. In healthy subjects, nasal occlusion with large negative pressures does not collapse the upper airway, presumably because the collapsing effect of negative pressure is opposed by the action of airway dilating muscles. Moreover, in normal human subjects, topical anesthesia of the upper airway increases the incidence of episodes of airway obstruction during sleep, particularly if the pharynx is anesthetized [140,141]. Thus, at least in some circumstances, upper airway reflex responses may contribute to upper airway patency. Patients with OSA have small pharyngeal size and thus may have greater changes in upper airway pressure with ventilation, and hence reflex activation of the muscles of the airway. However, any pressure reflex may not be adequate to keep the upper airways patent in these patients during sleep. Patients with OSA may be at risk for hypoxemia not only during anesthesia but also after anesthesia and surgery. Although the effects of anesthesia and surgery on postoperative hypoxemia have not been fully examined in patients with OSA, extreme episodic hypoxemia has been reported in these patients [142,143]. The study of Isono et al. [144] showed that postoperative episodic hypoxemia is associated with the presence of preoperative sleep-disordered breathing. Although anesthesia and sedation often reduce both respiratory drive and airway reflex activity, the reduction in activity of upper airway muscles seems to be greater than that of the diaphragm [145]. Thus, it is possible that the imbalance between dilating and collapsing forces may be a precipitating factor of upper airway obstruction. The majority of awake patients are nasal breathers during quiet breathing. Thus, even in the presence of a patent upper airway, airway obstruction may occur if the ability to change from the nasal route of airflow to the oral route in response to nasal obstruction is impaired [146]. E.

Cardiovascular Responses

Mechanical or chemical stimulation of most parts of the respiratory tract causes hypertension as a primary reflex effect, suggesting that release of catecholamines from the adrenal medulla and/or sympathetic stimulation of the heart are involved in this primary reflex response [24]. However, quantitative evaluation of the circulatory response to airway irritation

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during spontaneous breathing is by no means easy, because the primary reflex changes in blood pressure may be masked by the secondary homodynamic effects of respiratory responses such as coughing, expiration reflex, and breath-holding, which cause considerable changes in intrathoracic pressures and thereby venous return. In lightly anesthetized, paralyzed humans, sudden increases in blood pressure and heart rate are frequently observed during laryngoscopy and tracheal intubation and these responses may be potential problems to patients with coronary artery disease [147]. The responses of hypertension and tachycardia may be diminished by pretreatment with topical anesthesia, intravenous lidocaine, narcotics, or b-adrenergic receptor blocking drugs; and ensuring an adequate depth of anesthesia at the time of airway stimulation [148]. VIII.

Conclusions

The majority of afferent nerves arising from the airways are conducted in the trigeminal nerve, vagal nerve, and their branches. The importance of these nerves’ afferent innervation of the airways and their roles in elicitation of airway reflexes can be demonstrated by selective blockade of these afferents. Although the types of airway reflexes vary with the site of stimulation, airway reflexes fall into two categories: (1) defensive or protective reflexes that protect the respiratory tract from potentially harmful influences, and (2) regulatory reflexes that determine the pattern of breathing, control smooth muscle tone, and maintain airway patency. In experimental studies, different types of receptors have been identified in the upper and lower airways and their properties have been clarified, based on single-fiber recordings. There are at least five different types of receptors in the upper airway and four different types of receptors in the lower airways. Although some of these receptors have been implicated in playing important roles in particular airway reflexes, it is rather difficult to say exactly which receptors are responsible for which reflexes. In other words, the link between a given receptor and a particular reflex is unclear. Concerning airway receptors and airway reflexes, most information has been obtained in anesthetized animals and relatively few studies have evaluated the role of airway reflexes in humans. The application of experimental studies to pathophysiology in human subjects and patients would be significant, although it remains to be determined whether the results obtained from animal studies may be extended to humans. The better understanding of airway reflexes is essential to the better management of patients who need respiratory care.


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8 Inheritance and Ventilatory Behavior in Animal Models

KINGMAN P. STROHL Case Western Reserve University Cleveland, Ohio

This review concerns the genetic, non-environmental factors that influence normal variations in ventilation and/or its components, frequency and tidal volume. The question of ‘is there a genetic effect?’ is now answered a number of times in both human studies and rodent models, and there is moderate evidence to suspect that specific gene regions operate in determining the apnea–hypopnea index, a defining value for human sleep apnea. There is the best evidence in animal models to the address questions of ‘how strong is the genetic component?’ and ‘what genes might be involved?’ There is consensus regarding the collection of phenotype values for frequency and tidal volume and the response to chemosensory challenges, and qualitative and quantitative differences exist among rodent strains in both steady-state and transient changes in these traits to chemosensory challenge. Some gene regions could be interesting in regard to explaining the risk of progression or of severity of diseases in which disorders of ventilatory control operate to produce hypoxic complications. Computational analyses of datasets and breeding of animals offer 261

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opportunity to create a functional map of the connections between genes and ventilatory behavior. I.

Introduction

Ventilatory rate and depth (ventilatory behavior) alters its characteristics during growth and development as well as in response to both short- and long-term perturbations to preserve homeostasis in cellular respiration, acid–base balance, and heat exchange. In mammals, ventilation is achieved through combinations of tidal volume and frequency [1], yet there exists a wide range of respiratory frequencies that are mechanically efficient [2,3]. This flexibility allows ample scope for genetic factors to modify ventilatory behavior in a homeostatic manner without an increase in energy cost. Diseases of the chest wall and lungs restrict this range, produce a mismatch between respiratory control and ventilatory effects, and are associated with feelings of breathlessness or dyspnea [4,5]. In sleep-disordered breathing, the problem in ventilatory control is state-related; disturbances in ventilation fragment sleep and produce hypoxic stress resulting in daytime symptoms of sleepiness and cardiovascular sequelae [6]. This chapter will review the evidence that reproducible differences in ventilatory behavior occur between otherwise healthy animals. While individual values are modified over time by factors such as chronic exposure to altitude, respiratory loading, physical fitness, age, hormones, sex hormones, and sleep [7,8], the focus of this review will be the inheritance of ventilatory traits in animal models (mice and rats). Such models can yield insight into clinical disorders of respiratory control, such as sleep apnea. II.

Evidence and Implications for Inheritance of Ventilatory Traits in Humans

One can identify an individuality in respiratory patterning of tidal volume and frequency in humans during both wakefulness and sleep [9]. The shape and timing of tidal breaths are more similar in monozygotic twins than in unrelated individuals, not only during breathing at rest but also during behavioral tasks as well [10]. These and other observations suggest that variations in respiratory patterning occur in healthy humans and may have an inherited basis. Furthermore, such variation is not merely the result of differences in mechanical properties or in respiratory gas exchange. Thus, the causes for this phenomenon reside in central patterning of respiratory rate and the size and pattern of tidal volume [11]. Additional evidence indicates that humans exhibit heritable variations in the response to standard ventilatory challenges. There are absent or weak hypercapnic responses in natives of Papua New Guinea in contrast to the

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two- to four-fold increases in ventilatory responsiveness to carbon dioxide present in residents of Australia [12]. Sibling similarity occurs in the blunted ventilatory responses to hypercapnia first reported in athletes [13]. Others suggest familial clustering of hypoxic sensitivity is a distinguishing characteristic in this same population [14]. There are greater similarities in hypoxic ventilatory responsiveness between monozygotic (MZ) twins than between dyzygotic (DZ) twins at sea level [15–19]. Some but not all studies reported concordance of patterns of ventilatory responsiveness to hypercapnia between MZ twins [20,21]. Reports indicate abnormal ventilatory responsiveness to hypercapnia or hypoxia is present in first-degree relatives of patients with excessive hypercapnia, hypoxia, or unexplained respiratory failure [20,22–25] or those with sleep apnea [26]. This literature forms one basis for concluding that there occurs a rather wide range of variation in chemoresponsiveness in the healthy human population, and that such variation is to some significant degree likely to result from inheritable factors. The literature on familial factors that operate in the presentation of sleep apnea is rather good [27]. While direct studies of genetic factors in the cause of clinical disorders are limited in number, there is more direct evidence in assignment of genetic strength. There is an increased prevalence (two-fold) in a polymorphism for apolipoprotein E in patients with sleep apnea [28], a polymorphism also associated with cardiovascular disease and Alzheimer’s disease. This is not observed in other studies [29,30], possibly because of ascertainment bias. In the Cleveland cohort, there is statistical evidence for an oligogenic transmission explaining some 27% of the variation in apnea–hypopnea index (AHI) expression in the community. Having two or more family members with elevated values for AHI is associated with increased likelihood of finding another (Figure 8.1) [31]. In addition, families in Cleveland with a history consistent with sudden infant death more often have two or more members with obstructive sleep apnea (OSA), when compared with families without such history. Other associations in this dataset include a tendency for a more blunted hypoxic response, and cephalometric measures of brachycephaly, a smaller mean posterior nasal spine–basion distance (smaller posterior airway), and smaller mandibular/maxillary ratio (shorter jaw) [32]. Thus there are interesting components in the respiratory control system that appear to infer inheritable risk for complex process like sleep apnea. Even more direct is the evidence from a 10 cM genome scan on some members of the Cleveland Family Study [33]. Individuals were selected to be maximally informative for AHI, a defining metric for obstructive sleep apnea. Subjects were genotyped for 375 autosomal microsatellite markers at a sex-averaged spacing of 9.1 cM (Marshfield Center for Human Genetics, set 10). The sample included 66 extended pedigrees of European–American origin comprising 344 subjects and 59 African–American pedigrees (n ¼ 257

264

Strohl 4 3.5 3 2.5 1 2

2

1.5

>3

1 0.5 0 1

2

>3

Number of family members with sleep apnea

Figure 8.1 Shown are the odds ratios for the probability of finding another person with an elevated apnea–hypopnea index (AHI) based upon the number of people in a family currently identified with an elevated AHI. There are instances where it appears that sleep apnea has a strong familial component (Data from Ref. 27).

individuals). A multipoint model-free linkage analysis of the logtransformed AHI and body mass index (BMI) was performed (modelbased analyses also were performed). In order to utilize all of the available phenotypic and genotypic information, a variance components approach appropriate for extended pedigrees was used [33]. A summary of these findings is presented in Table 8.1. The linkage scores are modest; however, the sensitivity of the scan is also only modest, being model-free with an 10 cM average length. As well, there is the possibility of heterotypes, with pedigrees in which different gene regions contributing to the expression of AHI and/or BMI phenotype. One area of linkage was to a marker on chromosome 19 that resides in the region of the apolipoprotein E (APOE) locus, a region already of interest in sleep apnea [28]. A second biologically plausible and clinically interesting candidate gene, proopiomelanocortin (POMC), resides in the region of maximal linkage on chromosome 2. The sleep apnea phenotype and the POMC linkage are consistent with the proposed relationship of sleep-disordered breathing to the metabolic syndrome [34]. Finding such linkage allows one to consider studies on the separate and combined effects of obesity and diet in rodent models. There is an interplay between melanocortin and neuropeptide Y (NPY) in pathological weight loss and weight gain in murine models [35]. POMC is of central importance to energy metabolism,


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Table 8.1 Gene Regions in the Human Associated with Sleep Apnea Trait

Human chromosome

Distance (cM)

LOD scorea

AHI

2 19 8c 19 8c 2 12

74b

1.4–1.6

65 137 54 72

1.2 1.5 1.8 1.7

BMI a

Adjusted for age and gender. Region-of-interest includes proopiomelanocortin (POMC). c A–A only. Source: Data from Ref. 182. b

leptin levels, and other features of the metabolic syndrome [36]. The human POMC gene is homologous to mouse Ch. 14 (4 cM), where polymorphisms among mouse strains are linked with the metabolic syndrome. Indeed, leptin resistance differs between strains of mice without genetically determined obesity at this linkage site, the A/J mouse being resistant and the C57BL/6J being sensitive to diet-induced obesity [37], and there exist differences between strains at a level of POMC expression [38]. Yet leptin supplementation in the C57BL/6J animals that show low plasma leptin levels in response to fat feeding slows but does not prevent the subsequent development of diet-induced obesity [39], suggesting a polygenic influence on trait expression. There may be gene polymorphisms that are disease-modifying rather than disease-causing. One gene of interest is the angiotensin-converting enzyme gene (ACE, human Chromosome 27). There are now two reports that link ACE I/D polymorphisms to apnea length and sleep hypoxemia in OSA patients [40,41]. In one European study, the frequency distribution of the I/D polymorphism was similar in the control population as in OSA patients; also, the levels of ACE were higher in OSA patients and fell with treatment [42], providing some support for ACE as a disease-modifying allele. Another set of candidate genes relates to the nitric oxide synthase (NOS) enzymes that produce NO (nitric oxide). There is some data that NO production is reduced in OSA patients [43,44], and that levels rise with treatment [45]. Yet one study suggests that NO levels increase from before sleep to after sleep in patients with sleep apnea [46]. This is an interesting result since at sea level acute and sub-acute exposures to hypoxia reduce NOS activity and NO production; moreover, high altitude natives at 4000 m often have higher levels of exhaled NO than sea level natives [47].

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Presumably a downregulation in the NO system in sleep apnea could be a factor in the altered vascular reactivity seen in this disorder [48], and be modeled by intermittent hypoxia [49]. A theme in all these studies may be that gene polymorphisms affect the quality, quantity, or consequences of abnormal breathing events during sleep. Therefore, there is reason to believe that efforts to quantify and identify the chromosomal mechanisms of sleep apnea may unravel the physiology of normal breathing and of hyper- or hypo-ventilation states. Studies of genetic factors complement studies of cellular and organ system models, and precedent exists for identifying homologues in the rat, mouse, and human genome [50,51]. Genetic homologies among species also provide an extraordinary opportunity for understanding the evolution of ventilation.

III.

Targeting Ventilatory Traits in Small Animals

Ventilatory behavior is one part of a respiratory control system that operates to provide sufficient oxygen to meet cellular metabolic requirements and to remove enough carbon dioxide so that cell function is not impaired by excessive change in hydrogen ion concentration. What is remarkable is that in the healthy state, the respiratory control system will maintain arterial PO2 and PCO2 within a fairly narrow range despite changes in metabolism and environmental conditions with growth, development, and extremes of daily living. Respiratory control operates using principles of feedback and feedforward control [7]. Figure 8.2 illustrates essential components of this engineered system. The controller regulates ventilatory behavior (the controlled system) through efferent neural pathways coordinating contraction and relaxation of the controlled variable (respiratory muscles). A multiplicity of inputs to the respiratory neurons ensures that ventilation will be maintained when disease affects one or more afferent pathways or when the perception of some sensory cue is blunted by anesthesia, sleep, or neural injury. Conflicting demands, signals, or both, from different receptors may be responsible for dyspnea, a common symptom in respiratory disease, or for hiccups, cough, and postural events that also utilize respiratory muscles. The system is also driven by feed-forward mechanisms. One example is the anticipation of ventilation that may precede exercise even in the absence of CO2 sensitivity [52]. Feed-forward mechanisms play a critical role in smoothly integrating breathing with swallowing, speaking, singing, and defecating. Pontine and medullary respiratory groups initiate ventilatory behavior [53]; these areas regulate the firing and pattern of discharge in the motor


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Cortical State O2 Consumption Hypothalamic Regulation

CO2 Production

Pontomedullary Outflow Neural Transmission

Weight Medulla (VMS) Carotid Body

Frequency

Muscle Controlled Variables: O2 content, pH

Minute Ventilation

Tidal Volume

Measured Variables

Figure 8.2 This diagram identifies some of the anatomic and functional elements in the creation of ventilatory behavior. It is very simplistic. The controller elements are the cortex, pons and medulla; the controlled elements are the neuromuscular and anatomic elements of the upper airway and chest wall; the controlled variables are identified as arterial levels of CO2 and O2. Variables that are measured in studies of unanesthetized and unrestrained animals are noted. Weight is a loading factor to the controlled elements, but also is a symbol of metabolic influences on ventilatory behavior through hypothalamic function.

neurons of chest wall and upper airway muscles in a sequential and nonrandom fashion. Discharge patterns in some pontine cells appear to be dependent on afferent vagal feedback but become more clearly phasic after vagotomy. Thus, in the adult mammal neither the medulla nor the pons alone generates the respiratory pattern. In addition, there is an interaction between states of alertness and the activity of higher brain centers and the brain stem bulbopontine respiratory neurons that determines respiratory rhythm and depth. A detailed discussion of the theory and the experimental data on the generation of respiratory rate and depth [53] is beyond the scope of the current review; however, the notion that there are several current models is important if one is to think of linkage between genes and ventilatory behavior. Electrical (neural) and mechanical outputs of the central inspiratory activity involve the anatomic structures of the chest wall (to inflate the lungs) and the upper airway (to stabilize the air channel to the lungs) [7]. In the diaphragm there is a progressive rate of rise in inspiratory activity

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that allows the musculature to overcome the progressive increase in the elastic recoil of the lung during inflation. The rate of rise of central inspiratory activity, and hence the rate of lung inflation, is controlled by mechanisms different from those that terminate inspiration. Hypoxia and hypercapnia increase the steepness of the ramp of inspiratory activity and hence increase the rate of inspiratory airflow and tidal volume, but they have little effect on the duration of inspiration and, therefore, on the breathing frequency. This means that ventilatory responses to hypercapnia and hypoxia will depend on the sensitivity of both chemoreceptors and stretch receptors. Chemoreceptor sensitivity, since it influences the rate of increase in central inspiratory activity, is related more closely to the level of average airflow during inspiration than to minute ventilation. The change in the tidal volume/inspiratory time ratio, rather than the change in ventilation itself, more closely reflects chemical drive. On the other hand, the change in inspiratory time as a fraction of total breath duration may relate to afferent feedback such as that from the activity of stretch receptors. Additional factors alter the effect of respiratory central drive on tidal volume and frequency. With diseases of the lungs and chest wall, with obstructive apnea during sleep, or during certain stages of sleep when nonrespiratory drive is altered in muscles of the chest wall, there can be a mismatch between neural activity and mechanical result [7]. Hence, features of the controlled elements of the chest wall and upper airway can obscure central drive, especially if the intervention substantially alters the controlled system. The choice of using a single or double chamber for the barometric methods is in part determined by whether one suspects lung mechanics to substantially change during testing [54], since tidal volume and inspiratory flow are not always a precise copy of central drive. Ideally, one would pick a collection of phenotype traits that are sensitive to single features of ventilatory behavior; however, it is not feasible to examine all parts of respiratory control at once. The need to study large numbers of small animals and at times permit these test subjects to breed has necessitated the use of non-invasive measures of ventilation, namely tidal volume and frequency, using the barometric method (Figure 8.3) [55]. There is anguish over this approach [54]. Most agree that frequency is a very precise value, but tidal volume is semi-quantitative [60]. The latter is estimated from temperature changes which in turn can be affected by the mammal’s temperature and the temperature and humidity of the chamber [55,61]. A major strength of this approach is the ability to measure an animal more than once [62]. Variability is induced by day-to-day alterations in such environmental factors as light, smell, noise, etc., but can be reduced by careful attention to the animal protocol [54]; time of day also influences trait values [63,64]. These variables enter into estimates

Inheritance and Ventilatory Behavior in Animal Models Br. Norway

269

Zucker fatty F2 F2BN 1 2 3 4 5 6 7* 8 9

F1

Key: fa−

fa+

950 bp 700 bp

3 = fa/fa 4 = fa/fa 6 = fa/− 7*=-/-

F2 1/4

1/2

1/4

Fatty

Figure 8.3 An intercross strategy is shown for a cross between the Zucker and the Brown Norway rat. The allele illustrates the transmission of the mutant fatty (noted as faþ) gene. One estimates the strength of inheritance by the difference in the variance in trait values among the F1 generation (which is genetically identical and whose variance is environmental) and the variance in the F2 generation (where the genes are mixed and the environmental component is assumed to be the same as with the F1 animals). Such estimates can be made for animals in the F2 generation that carry the faþ and/or fa null (fa) allele.

of the environmental component of the protocol, and if too large or uncontrolled may obscure a genetic effect. Hence, studies of unanesthetized animals require larger numbers or statistical adjustments that reduce such influences. Reduced preparations or anesthetized models require more considerable attention to reproducibility of trait values. In anesthetized mice there is good correspondence between estimates of pulmonary mechanical problems identified by the barometric method when compared with more direct measures of mechanics like force oscillatory techniques [65]. Therefore, one can conceive of approaches that compare animal strains in regard to dose– response relationships in chemical drive, or the anatomic or physiologic characterization of brain regions and/or the individual influences of resistance and compliance of upper airway and chest wall structures on total respiratory mechanics. However, the exclusive use of reduced preparations and more technologically advanced approaches is appropriate for times in which the genes or proteins are better characterized. The use of a reduced preparation is most convincing in regard to evolutionary fitness when used in tandem with other methods to disclose mechanisms for genetic effects [66]. Another source of worry is the reproducibility of results across laboratories. There is some empiric evidence for this general concern, although not directly related to ventilatory behavior. After standardizing strain source and testing systems, there were systematic differences in

270

Strohl

behavior across laboratories in a battery of six tests for neurobehavior. For some tests, the magnitude of difference ascribed to strain depended upon the specific testing lab [67]. Thus, care must be taken in characterizing the behavior in both common strains and mutants, as results could be idiosyncratic to a particular laboratory. At present there is no such comparable study for ventilatory behavior. There is some data to suggest that absolute values for such traits as frequency and tidal volume vary between laboratories that use slightly different methods of testing. Despite such differences, the ranking of animals to each other, in particular the pattern of breathing for the C57BL/6J, is similar between laboratories [68–71]. Animal models of human problems like sleep apnea exist in larger animals (dogs, elephant seals, and pigs) but the rodent models (mice and rats) are amenable to genetic dissection. Studies in these models can address environmental exposures, consequences, and intermediate phenotypes found in humans, which may relate to OSA expression or progression [72–74]. Utilizing inbred strains reduces genetic variability and permits more control over age effects and environmental factors that confound human studies. Simultaneous use of proteomics and expression arrays in such models may efficiently screen many biochemical processes that could contribute to trait variance, and can be useful in locating candidate genes within the regions-of-interest identified through the study of in-bred lines. This approach is used to identify genomic elements operating in strain differences in sleep in the mouse [75]. Complementary and parallel studies in animals and humans can be more efficient in understanding the biology of sleep-disordered breathing and sleep hypoxemia when compared with a sequential approach. Compared with the mouse, the rat’s larger size and betterdefined brain nuclei are amenable to follow-up studies of cardiovascular and respiratory control in the central nervous system. Respiratory mechanics of the upper and lower airways are in relative proportion more similar between the rat and the human than between the human and the mouse [2]. If one considers a need for arterial blood gas measurements as a phenotype trait, one would have to consider not only the technical accuracy and reproducibility of the measurement but also the variability in the state of the animal. Both effects limit the power of intermittent sampling of arterial blood gases to detect differences in gas exchange or acid–base between strains or in their progeny. Technology for on-time sampling of gas exchange, particularly if such methods were non-destructive, might be useful. Finally, the laboratory must be directed toward a goal of having reproducible testing conditions over the life of the rodent study or cross [76]. Changes in testing methods or personnel, and in animal quarters, may all obscure detection of small (53%) but important gene effects. In short,


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the more uniform the testing conditions, the less influence there is for environmental variance and the more likely one will detect genetic effects and interactions. Identification of gene loci that link to intermediate traits has given insight into risk factors for cardiovascular and metabolic disease [77,78]. The start of this field in regard to respiratory control was the empirical observation that ventilatory phenotype was a factor in the variations among mouse strains in the model of environmental ozone exposure [73]. What is needed is the examination of other models, including those gene knock-outs for cardiovascular or neurologic functions that might operate in the risk for or recognition of sleep apnea [79]. A survey of respiratory frequency at rest and with chemoresponsiveness could provide insight into markers for risk of unexpected death in infancy or for screening adults with hypoventilation complications with illnesses like sleep apnea, COPD, and asthma [78,80,81], or lethal human illnesses such as idiopathic or congenital hypoventilation syndromes [32,82]. In summary, the key to any approach to the genetics of respiratory control is an intimate knowledge of the trait value and the animal. Initial results are likely to be confounded by sources of variance and by major confounding variables. The assignment of gene action linked to ventilatory behavior to a particular candidate physiologic process should be a statistical or empirical rather than an intuitive process. Bias introduced at every level of collection of the phenotype data may enter into analysis and produce or obscure linkage. The physiologist will need to prove suspected biologic linkage in any number of ways [6]. Nevertheless, in this postgenome era there is enormous opportunity to measure ventilatory traits in any number of mammalian models and to begin to describe disorders of respiratory control in terms of molecules relevant to whole system needs, like ventilation, metabolism, and responses to sleep and chemosensory stress. IV.

Evidence for the Inheritance of Ventilatory Traits in Rodents

In 1984, Ou et al. [83] reported differences in the ventilatory behavior between two colonies of Sprague-Dawley rats in response to chronic exposure to hypoxia. Despite these being out-bred strains (no conscious in-breeding by brother–sister pairing for 420 generations), consistent differences in the ventilatory response to hypoxia before the exposure remained between colonies over the course of several years, and correlated to differences in the erythropoietic and pulmonary vascular pressor responses to chronic hypobaric hypoxia [84]. By the vendors maintaining such closed populations, there had occurred genetic drift between the

272

Strohl

populations. The problem with the study of these Sprague-Dawley strains is that the similarities between the genomes are so great that finding the responsible gene is extremely difficult. In 1992, Connelly et al. [85] reported (almost as an afterthought) the observation that the effect of N-methyl-D -aspartate (NMDA) receptor-channel activation in the production of respiratory patterns differed by strain in an anesthetized rat model. The predominant respiratory response to systemic MK-801 administration, a pharmacologic blocker of NMDA activation, was an increase in inspiratory duration and a decrease in amplitude of diaphragm electromyogram and phrenic nerve discharge. Effects on inspiratory timing and amplitude were most pronounced when the rats were vagotomized. Respiratory timing changes in response to systemic MK-801 administration differed between the Wistar and SpragueDawley strains. Breathing patterns resembling apneusis, i.e., irregular inspiratory durations prolonged two- to thirty-fold, occurred in 60% of the vagotomized, spontaneously breathing Sprague-Dawley rats and in none of the Wistar rats. The authors concluded that the breathing pattern in Sprague-Dawley rats is more sensitive to interference with NMDA-mediated mechanisms. This study is important as it illustrates that strain differences may occur in ventilatory behavior even in the anesthetized preparation, so that models of the neurochemistry of breathing based upon findings from one strain need to be tested for face validity in another strain. In 1995, Tankersley et al. [73] reported results from the more traditional approach of surveying many in-bred strains, in this case mice, in order to detect if any strains showed significant divergence in trait values. There were significant inter-strain differences in respiratory frequency, hypoxic responsiveness, and hypercapnic responsiveness between several strains [70]. Other reports from this group indicate success in an intercross strategy between C57BL/6J (B6) and C3H/HeJ (C3) to uncover gene regions involved in ventilatory behavior. This group has subsequently reported, in a similar intercross, that a strain distribution pattern for respiratory rate when exposed to hypoxia is consistent with the hypothesis that two genes regulate parental strain differences [86,87]. Cosegregation analyses suggest that the genetic control of frequency during hypoxia differs from the genes that control differential baseline frequency [88]. Although the genetic control of tidal volume appears more complex, differences in the minute ventilation during hypoxia is determined by tidal volume responses. In this instance, the major gene regions that may relate to lung mechanics appear to be distinct from those correlated to inspiratory drive [89]. Therefore, this study suggests that the phenotypic variation in hypoxic ventilatory response between the two parental strains, especially related to frequency


273

during hypoxia, is possibly regulated by only a few major genetic determinants. In 1997, Strohl et al. [90] reported measurements of ventilation and metabolism in four strains of rats, chosen for a wide variation in traits for body weight and/or blood pressure regulation. The conclusion is that strain, more than the effect of body mass or sex, had a major influence on metabolism, the pattern and level of ventilation during air breathing, and ventilation during loading or unloading of chemoreceptor input in the unanesthetized rat. A more recent study found similar results in four strains of rat, including a confirmation of the low CO2 response in the Brown Norway [91]. These strain differences indicate that the ventilatory response to chemosensory input appears to be genetically regulated. There are indications that inheritance operates in some of the nonsteady-state responses. The Dejours phenomenon is very clearly demonstrated under non-steady-state conditions involving rapid transitions from room air to hypoxia and then to hyperoxia. This transition is quite complex when compared with steady-state responses to hypoxia and/or hypercapnia and, at least intuitively, is thought to be due to an unnatural environmental exposure. To test this null hypothesis, we exposed two rat strains known to differ in ventilatory behavior to oxygen after exposure to hypoxia. We examined the ventilatory behavior (frequency, tidal volume and minute ventilation) in Sprague-Dawley and Brown Norway animals. The phenomenon of ventilatory decline in response to 100% oxygen after 5 min of exposure to hypoxic gas (10% oxygen, 90% N2) was not common in the Sprague-Dawley strain, but was quite evident in the Brown Norway strain (Figure 8.4). This study rejected the null hypothesis and suggests that genetic factors do influence the ventilatory response to hyperoxia following acute hypoxic exposure [92]. Such non-steady-state responses could be important intermediate traits relevant to recurrent cycles of hypoxia-reoxygenation present in sleep-disordered breathing. The presence of post-hypoxic ventilatory decline is found in those patients with heart failure and Cheyne–Stokes respiration, rather than those without Cheyne–Stokes respiration [93]. The above studies in common strains of mice and rats offer the most compelling argument for ventilatory behavior being affected by genetic drift and natural selection and offer the foundation for physiological genomic studies aimed at elucidating the genetics of these ventilatory control mechanisms. As will be discussed below, the effects seen in these models can be quite modest, and the assignment of cause-and-effect relationships can be complex given that adjustments in one gene may influence another gene or gene product and one gene can have an influence on more than one trait or part of the system for respiratory control.

274

Strohl

SUCTION Monitor Temp., humidity , %O2 , %CO2

Reference chamber

GAS MIXTURE

Ventilatory behavior (freq, TV, Ve)

PRESSURE TRANSDUCER

Figure 8.4 This diagram illustrates the elements of a barometric method for measuring ventilatory behavior in rodents (Data from Ref. 90). Not shown in detail is the use of a sealed reference chamber, identical in size as the animal chamber.

V.

Estimates of the Strength of Inheritance

The techniques used to examine experimental crosses are now more powerful and flexible [6]. Current approaches can accommodate environmental influences, make minimal assumptions about the mode of inheritance of the trait, and can accommodate effects of factors such as age, growth, and cohort effects, unknown ascertainment, heterogeneity, and heterozygosity in parental lines. Observational data should include real or potential temporal changes in litter size, food vendor and brand of rat chow. These quantitative and qualitative variables are handled as co-variate shared environmental variables. Animal husbandry reduces heterozygosity and the intercross strategy will provide a reliable means for estimating environmental effects and permit heritability estimates for a variety of important ventilatory phenotypes [94]. For the assessment of the gross effects of genes and environmental factors on phenotypic values, variance components models are used [94]. The total phenotypic variance of each trait is partitioned into a genetic


275

Sprague Dawley

insp

Air

10% O2

100% O2

5thmin

1stmin

Brown Norway

time

1999

Figure 8.5 The barometric tracings from two animals (one Sprague-Dawley and one Brown Norway) are compared with regard to the pattern of breathing at rest, in the fifth minute of hypoxia, and in the first minute or so after re-oxygenation from hypoxia (adapted from Ref. 92).

component and an environmental component. One then estimates the strength of inheritance by the difference in the variance in trait values among the first generation animals (which is genetically identical and whose variance is environmental) and the variance in the second generation (where the genes are mixed and the environmental component is assumed to be the same as with the first generation animals). Experience with cross-breeding of the Sprague-Dawley and Brown Norway and of the Brown Norway and the Zucker strains has permitted us to make such calculations. A schematic diagram of the strategy for this cross is presented in Figure 8.5. A summary of the estimates for some traits for ventilatory behavior is presented in Table 8.2. In this instance, estimates are provided from analyses of the variance [95] in first- and second-generation animals of a cross between a Zucker fat male and Brown Norway females. From this data we suggest that some but not all trait values related to ventilation and metabolism show higher (440%) inheritance. The strength of inheritance for some ventilatory traits is similar to that described for airway responsiveness in mice [61].

276

Strohl

Table 8.2 Heritability Estimates from a Cross between the Zucker and Brown Norway Strains

Trait Weight Lee index (mass) Carbon dioxide production Frequency at rest Minute ventilation Frequency in hypercapnia Ventilation in hypercapnia Frequency in hypoxia Minute ventilation in hypoxia

Total heritability (h2)

Variance attributed to the fa allele

Variance attributed to the rest of the Genome

0.78 0.72 0.55 0.59 0.11 0.28 0.38 0.60 0.54

47% 38% 10% 9% 6% 17% 0% 11% 0%

53% 62% 90% 91% 94% 83% 100% 89% 100%

The Zucker rat strain carries a mutant allele, the fa, that alters the leptin receptor; two such mutant alleles results in an obese phenotype due to leptin resistance (Data from Ref. 129).

VI.

Studies of Gene Effects in Rodent Models

If one were to identify the best evidence for genetic effects, it would be in the literature on genetically engineered animals and studies of drug-by-strain interactions. These studies indicate that genetic background directly influences ventilatory behavior (respiratory frequency, tidal volume, and/ or minute ventilation) either at rest or with hypercapnic challenges. There is evidence demonstrating the action of genes that arises out of studies of nitric oxide synthase (NOS). There are three isoforms of NOS (NOS-1, NOS-2, and NOS-3) arising from three different regions of the genome, and these enzymes are involved in the generation of nitric oxide (NO). The concept of genetic transmission of post-hypoxia ventilatory behavior is directly supported by reports of nearly absent respiratory depression in response to brief hyperoxia in nitric oxide synthase (NOS)-3 mutant mice [96]. In addition, an altered breathing pattern was seen in NOS-1 mice where function was eliminated by knock-out [97,98]. Other studies of knock-out (KO) mouse models report loss of or reduction in hypoxic response attributed to endothelin-converting enzyme-1 (ECE-1) [99], to endothelin-1 [100], to dopamine [101], to the neutral endopepidase (NEP) [102], and to HIF [103]. On the other hand, animals knocked-out for other supposedly critical factors for hypoxic or hypercapnic responses show no effect on ventilation; this is the case for endothelin-3 [104]. Some genes may have functions in developmental programs. The reports on the NK-1 KO suggest that NK1 receptors are important in the response to acute hypoxia in the adult mouse; however, NK1 receptors


277

are not obligatory for the prenatal development of the respiratory network, for the production of the rhythm, or for the regulation of breathing by short-lasting hypoxia in neonates [105]. When comparisons are made between monoamine oxidase A-deficient transgenic (Tg8) mice with control (C3H), the implication is that an elevated serotonin during the perinatal period alters respiratory network maturation and produces a permanent respiratory dysfunction, whereas a high serotonin level present in adults depresses chemosensation; the authors concluded that the metabolism of serotonin plays a crucial role in the maturation of the respiratory network and in ventilatory response to hypoxia [106]. The NADPH system is of interest for its presumed role in oxygen sensing at a mitochondrial level. There is a role for the subunit of NADPH, gp91, in cellular sensing of oxygen in airway neuroepithelial cells [107], but not in adrenal cells [108]. Yet in KO models there is little effect on pulmonary vascular [109] or carotid body [110] cellular responses to hypoxia, or in ventilatory behavior [111]. In contrast to the interest in hypoxia, there is less work in models on chemoresponsiveness to CO2. Development of carbon dioxide responses is impaired in KO models for the mammalian achaete-scute homologous gene (Mash-1, now called Ascl 1) [112]. Adult monoamine oxidase A-deficient transgenic mice, when compared with control (C3H) mice, show blunted CO2 responses which are restored when serotonin levels are pharmacologically suppressed [106]. One interesting study that utilized CO2 loading was able to disprove the null hypothesis that exposure to high levels of carbon dioxide would not result in differences in early-gene (c-fos) expression among strains [113]. This approach probably raises more questions than it answers, but in short it does document the possibility for intra-species variations in the widespread brain networks that could act not only on ventilatory behavior but on acid–base balance as well [114]. A more complex model that is still based on single mutant genes includes the ob/ob mutant mouse [86,115,116]. This model is based upon the C57BL/6J strain and is a good example of a modifier gene effect. The basic structure of resting breathing is unaffected but the function of carbon dioxide responses is modified by the leptin system, even in the absence of obesity. Thus, a candidate gene/protein approach has shown proof-of-principle for providing insight into functions of individual genes relevant to ventilatory behavior and human conditions like obesity and sleep-disordered breathing. Knock-out models, while of significant interest, are often not relevant to understanding risk factors since evolutionary fitness requires actions of both the real and latent variance in several genes and gene products, respiratory system capacity, and learning. Understanding the role of genes

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Strohl

in normal and pathologic control of ventilation will require approaches that will accommodate the probable collective effects of multiple loci. Another way to illuminate genetic influences is to examine drug effects in different strains of rodent. Since studies in NOS KO mice have shown that the nitric oxide system can influence post-hypoxic ventilatory behavior [66,96,117], the question arises whether nitric oxide synthase (NOS) blockade would similarly affect ventilatory behavior in two rat strains. Post-hypoxic decline in frequency and minute ventilation (the Dejours phenomena) is present in some but not all studies of Sprague-Dawley rats, a finding that might relate to colony differences [92]. In Sprague-Dawley animals without this response, global NOS blockage produced the Dejours phenomenon, while NOS blockade resulted in a greater increase in resting ventilation in the Sprague-Dawley than in the Brown Norway strain, and uncovered the Dejours phenomenon in the Sprague-Dawley strain [118]. Based on this observation we proceeded to administer a selective neuronal NOS (NOS-1) blocking agent (7-nitroindazole or 7-NI, 60 mg/kg) and found the same effect as the non-specific NOS inhibitor but without altering metabolism (unpublished findings). These findings are consistent with the effect of NOS inhibition in the control mice studied in relation to their NOS-1 KO cousins [98]. These approaches are intended to determine if a specific system, in this case the nitric oxide system, operates similarly in all rodents. As the results indicate, the various strains of rats and mice do not utilize the NO pathway in the same way with regard to the regulation of ventilatory behavior. The strength of the knock-out approach or drug-by-strain approach is the comfortable connection between a known gene, peptide, or protein and the ability to make a rational connection to ventilatory behavior. Falsepositive findings may occur due to selection bias and/or over-interpretation of the significance of results from knock-out models and/or reduced preparations, especially if one uses only this information to screen human populations. Multiple approaches are preferable, and there are a number of incremental steps that must be taken before one can assign a specific role to a gene. The pioneering work by Tankersley and his group has identified some gene regions that are statistically linked to differences in ventilatory behavior between C57BL/6J and C3H/HeJ mice [74,88,119,120]. Quantitative statistics of animals derived from these strains suggest that the genetic control of hypoxic ventilatory responses exhibits a relatively simple Mendelian inheritance in terms of respiratory timing characteristics. Furthermore, differences in inspiratory time at baseline is linked to a putative genomic region on mouse chromosome 3 [88]; however, a likely candidate gene related to neuronal receptors does not explain this association [121]. Of interest, this region does not correlate with the variations in lung mechanics between these two strains [89]. This literature


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encourages the use of functional genomics in an effort to link ventilatory behavior to molecular structure [120]. VII.

A Physiogenetic Map of Ventilatory Behavior

A literature on the genetics of ventilatory behavior is emerging with animal models and traits that appear relevant to human physiology and pathophysiology. One theme is that a limited number of genes can have a measurable impact on ventilatory behavior. The overview of the general idea of how genes act to produce the ventilatory phenotype is illustrated in Figure 8.6. From this perspective it is remarkable that studies of ventilatory behavior have identified any genetic effects (Table 8.3). It is likely the genes that ultimately influence ventilation at rest may also affect ventilation when breathing is stimulated or may correlate with regulatory systems for the cardiovascular system, and perhaps even metabolism. This makes sense when one considers how well integrated, from a physiological standpoint, the systems and mechanisms that regulate respiration are. It will, therefore, be important to consider the presence of gene interactions in the data analysis. Such interactions are revealed not only as preliminary data for the mapping of gene loci, but also as a way of gaining insight into the interrelationships and nature of the genetic mechanisms influencing ventilatory responsiveness [122]. There are also patterns of interactions among the physiologic traits we use for describing ventilatory behavior, and associations that differ among

Ventilation during sleep and wakefulness

f

TV

SYSTEMS LEVEL

………. ………

CELL LEVEL

Genes or Gene Regions

Figure 8.6 The pathways between genes and the ventilatory traits of respiratory frequency ( f ) and depth (tidal volume or TV) are complex and involve several levels of cellular and system integration.

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Strohl

Table 8.3 Candidate Genes or Regions Identified (February 2003) Gene

Human region

ACE POMC APOE Unknown NOS-1 neuronal NOS-3 (eNOS) Unknownd

17, q23 2, p23 19, q13.2 8, 137cMc 12, q24.2 7, q36 4, 3, and 13

Unknown Endothelin-1 (Edn-1) ECEC-1 HIF-1a Dopamine (1a) receptor Monoamine oxidase A Ascl 1

6 and 15 6, p24.1 6, q36–37 14, q21–24 11, q11 X, p11.4–11.3 12, q22–23

Mouse region 11 (65 cM) 14 (4 cM) 7 (4 cM) Unknown 5 (65 cM) 5 (5 cM) D3mit7 (approximately 26.4 cM) (site of ob mutation in B6 strain) D9mit207 13, 26 cM Unknown 12, 31 cM 9 (drd2) X, 5.2 cM 10 (ascl1)

Referencea Speciesb [40,41] [128] [28] [128] [98] [96] [88]

Human Human Human Human Mouse Mouse Mouse

[87] [100] [99] [103] [101] [106] [130]

Mouse Mouse Mouse Mouse Mouse Mouse Mouse

a

Primary observation(s). Species in which the observation was first made. c No gene mapped to this region as yet. d Contributes to the strain differences to susceptibility to diet (fat) induced obesity. Source for homology information: http://www.informatics.jax.org/. b

strains. Figures 8.7a and b result from our work in the Sprague-Dawley and Brown Norway rat strains and a collaboration with Physgen, the Program in Genomic Application, based at the Medical College of Wisconsin, and headed by Dr. Howard Jacob. Even in a grey scale, the eye can detect pattern differences clustering among traits in the Sprague-Dawley (SD) strain. Also the eye can detect differences in these patterns between SpragueDawley and Brown Norway animals (n ¼ 55 in each group). This approach results in the creation of a systems map for ventilatory function [122]. This map can be used alone or in combination with whole genome mapping to disclose interactions among ventilatory traits. Some effects that may be disclosed are pleiotropy, epistasis, and sexual dimorphism [94]. Pleiotropy, one gene or the mutation of a gene resulting in the production of apparently unrelated (multiple) effects at a phenotypic level, has been observed by us in an association between values for resting breathing and hypoxic ventilatory response in humans [123]. Epistasis is an interaction of two or more loci on any given trait value; more technically, it is a form of gene interaction whereby one gene interferes with the phenotype


281

Bivariate Coefficients of 24 Traits

Metabolism Resting Breathing 5thmin. Hypoxia 5thmin. Hypercapnia 10% O2 Responsiveness Hypercapnic Resp. Isocap. Hypox Resp. Re-oxygenation (Dejours)

CORRELATIONS DIFFER BETWEEN STRAIN

Figure 8.7 (a) In this example we performed Pearson coefficient correlations among the 24 traits comprising eight groups of systems actions in a group of Sprague-Dawley animals (n ¼ 55). The correlation coefficient is presented as a set of colors (shown here in grey scale) from þ1 right hand of the scale to 1. The original graphs are color codes; however the structure is disclosed by grey-scale. (b) Shown for comparison are the correlations for the Sprague-Dawley (Figure 8.7a) and the Brown Norway (n ¼ 55). These figures were constructed as examples of physiogenomic approaches in collaboration with Peter Tonellato and the Program for Genomic Application, the Medical College of Wisconsin (see Ref. 108 for other examples).

282

Strohl

expression of another non-allelic gene [94]. It is a mechanism by which two loci can have more (or less) than an additive effect. Sexual dimorphism occurs in many physiologic variables and can occur at both chromosomal or hormone environment level. Imprinting refers to a differential effect, at either a chromosomal or allelic level, depending upon whether the genetic material came from a male or female parent. A clinical example is transmission of either Prader-Willi or Angelman syndrome, human conditions associated with respiratory disturbances during sleep [124–127]. Only gene mapping proves this effect, but some inference about its action can be gained by pedigree analysis. VIII.

Overview and Future Directions

Results of these studies of inheritance of ventilation, tidal volume, and frequency may permit the design of appropriate experiments including rational decisions to examine the physiologic systems for the loci identified by gene mapping. If results indicate a substantial effect in respiratory mechanics, attention could be directed to specific biochemical, morphometric, and mechanical features. More conclusive investigation would include the more complicated measures of such things as the mechanical system (controlled element), arterial blood gases (controlled as well as controlling element), and upper airway mechanics (controlled element). Identification of trait influencing loci will be relevant to understanding diseases characterized by either hypo- or hyper-ventilation and/or by alterations in respiratory patterning [101]. We would have greater opportunity to understand the heterogeneity in responses to environmental influences and, potentially, to develop a set of markers that helps address breathing patterns in adults with hypoventilation or with illnesses like congestive heart failure, COPD, and asthma. However, it is unrealistic to propose that genetic factors explain all of the variability in the expression of ventilatory behavior or its pathogenic effects on cardiopulmonary disorders of sleep. Rather, the questions currently being addressed relate to the relative strength of the genetic components and the identification of the genes, proteins, and/or systems that produce ventilatory dysrhythmia and the fundamental nature of the periodic hypoxemia found in sleep-disordered breathing. A reasonable expectation related to therapeutic utility would be the identification of disease-modifying genes, including those that amplify a given stimulus, sensitize (desensitize) an effector pathway, or promote a stimulus–response mismatch. Having such markers will permit the design of subsequent studies to identify individuals at greater or lesser risk. Such stratification would complement current clinical practice and begin efforts to identify those at


283

risk, predict morbidity, or otherwise inform in regard to appropriate surveillance or clinical management in cardiopulmonary illnesses in which sleep-disordered breathing occurs. Acknowledgments The author would like to thank the Department of Veterans Affairs and the National Heart, Lung and Blood Institute for support of this line of research. The author was a visiting Scientist to the Program for Genomic Application, the Medical College of Wisconsin (Howard Jacob, Ph.D., Director) as part of a Sleep Academic Award program from the National Institutes of Health. References 1. 2.

3. 4. 5. 6. 7. 8. 9.

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Part II Pathophysiology

9 Congenital Central Hypoventilation Syndrome: Should We Rename It Congenital Autonomopathy?

DAVID GOZAL University of Louisville Louisville, Kentucky

I.

Introduction

There is no doubt that in the context of disorders of respiratory control, congenital central hypoventilation syndrome (CCHS) occupies a unique place in human physiology. This entity, which can be viewed as a true experiment of nature, has allowed for major discoveries about the roles played by chemosensitivity in the maintenance of gas homeostasis as a function of state. However, as always occurs in biology, things turn out to be more complicated than anticipated. As such, the conceptual frameworks that have been proposed to account for the manifestations of CCHS have evolved over recent years to incorporate novel findings derived from physiology, genetics, and medicine. This chapter will review such findings and delineate the evolution of the concept that CCHS, rather than represent a pure phenotype of absent central chemosensitivity, is rather an intrinsic disorder of the autonomic nervous system.

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Definition and diagnosis

CCHS (Ondine’s curse) is traditionally defined as the failure of automatic control of breathing [1–10]. The term ‘‘Ondine’s curse’’ was initially employed by Severinghaus and Mitchell to describe three adult patients who developed central hypoventilation following high cervical/brainstem surgery [11]. When awake and requested to breathe voluntarily, these patients would have no difficulty in performing such task, yet they required mechanical ventilatory support for severe central apnea while asleep [11]. In 1970, an infant with the typical clinical features corresponding to CCHS was reported [1]. In CCHS, ventilation is most profoundly affected during quiet/ non-rapid-eye-movement sleep (NREMS), a state during which automatic neural control is predominant [5]. Ventilatory patterns are also abnormal during active/rapid eye movement sleep (REMS) and during wakefulness, but to a lesser degree [1,3,5,6,12–14]. The severity of ventilatory dysfunction can range from relatively mild alveolar hypoventilation during sleep and almost adequate alveolar ventilation during wakefulness, to complete apnea during sleep with severe alveolar hypoventilation during waking. Other symptoms indicative of brainstem dysfunction may be present, but are not essential to make the diagnosis of CCHS. The clinical presentation of CCHS varies depending on the severity of the phenotype [8,9,15–17]. Most CCHS patients will manifest their disorder in the newborn period; they may be apneic at birth or develop apnea and cyanosis requiring resuscitation in the delivery room or nursery, such that often the concern for preceding perinatal asphyxia usually overrides the suspicion of CCHS. However, while perinatal asphyxia of a magnitude leading to severe respiratory depression is usually associated with significant additional neurological manifestations, CCHS infants will essentially display intact neurological function provided that they receive adequate ventilatory support. The inability to wean these babies from mechanical ventilatory support along with the observation that their respiratory disturbance appears worse while the baby is asleep usually prompts the diagnosis. It should be pointed out that over the first several months or years of life, there is an apparent improvement in the magnitude of the alveolar hypoventilation in CCHS infants. However, rather than reflect a change in the underlying disorder, these dynamic temporal changes most likely represent the normal maturation of the respiratory system. The vast proportion of CCHS infants will not be apneic at birth and will develop cyanosis, tachycardia, and diaphoresis when asleep, and can be mistaken for having a congenital cardiac defect or be confused with apparent lifethreatening events [8]. It is assumed that some SIDS cases may in fact represent unrecognized CCHS. Others have such mild hypoventilation that they can go unrecognized for several years and present with pulmonary hypertension and cor pulmonale. It is estimated that about 30% of CCHS

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patients will require mechanical ventilatory support 24 h/day with the remainder displaying near adequate ventilation and gas exchange during wakefulness. It should be stressed that the diagnosis of CCHS is one of exclusion, i.e., the etiology of hypoventilation is unknown. Therefore, all other known causes of hypoventilation need to be ruled out, and this may be particularly difficult during the newborn period. Obviously, ventilatory muscle weakness, cardiac disease, or any type of respiratory disease all need to be excluded. Magnetic Resonance Imaging (MRI) scans of the brain including the brainstem will usually rule out the presence of anatomic lesions, which are absent in CCHS. Multiple metabolic disorders can present as apnea and cyanosis in the infant, and therefore a metabolic screen should be conducted. Specific disorders such as Leigh’s disease, pyruvate dehydrogenase deficiency [18], and carnitine deficiency should be considered as well as congenital myopathies, diaphragmatic dysfunction, congenital myasthenia gravis, or Mobius syndrome [19]. Because asphyxia, infection, trauma, or tumors may coexist, the decisions about the contribution of these confounding factors to the clinical presentation may be arduous [15]. In general, the initial evaluation should include chest X-rays, fluoroscopic or ultrasound evaluation of the diaphragm, electrocardiogram, echocardiogram, 24 h Holter recording, meticulous neurologic examination in addition to the Central Nervous System (CNS) imaging studies, and urinary and plasma screen for metabolic disturbances. A detailed ophthalmological examination may reveal abnormal pupillary and optical disk features [20–22]. In those cases where mild abdominal distension or delayed defecation is present, a rectal biopsy should be considered to determine whether Hirschprung’s disease (HD) may be present, since it will occur in 15–20% of all patients with CCHS [14,22–30]. A detailed evaluation in a respiratory physiology laboratory is critically important to the diagnosis and management of these patients. Polygraphic recordings of respiratory and cardiac signals during all sleep and wake stages, including chest and abdominal wall motion (respiratory inductance plethysmography), end-tidal CO2, pulse oximetry and waveform, and ECG can be combined to EEG and EMG channels that will define state. Careful observation of the infant’s respiratory (tidal volume and frequency) and cardiac frequency responses to ongoing spontaneous changes in blood gas exchange can provide important information and obviate the need for immediate application of exogenous hypercapnic and hypoxic challenges. Such recordings will also permit determination of the appropriate levels of mechanical respiratory support that the patient needs during the various states. It should be emphasized that these patients can be very unstable, particularly during their early years of life. Indeed, minor respiratory infections can trigger apnea both during wakefulness and during sleep, and the absence of subjective or objective response to hypoxia or

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hypercapnia in these patients, which in normal children would manifest as increased respiratory efforts, retractions, or nasal flaring, will further mask the progression of potentially serious deterioration in clinical status. Thus, unless very subtle symptoms are accounted for, hypoxia for example will be detected only when lethargy, cyanosis, and/or loss of consciousness survene. Thus, continuous monitoring by trained and skilled observers is necessary to prevent significant hypoxemia and its sequelae. Similarly, bradycardia is not uncommon, even if it does not usually require implantation of cardiac pacemakers in infancy [9,31–35]. Syncope, particularly during Valsalva-like maneuvers, is not uncommon as well [36]. Feeding difficulties during infancy and abnormal gastroesophageal motility mostly presenting as gastroesophageal reflux are frequent and can lead to aspiration of intestinal contents, resulting in frequent implementation of gastrostomy and anti-reflux procedures [37–39]. In practical terms, the proposed diagnostic criteria for CCHS have been recently reviewed [15] and essentially consist of the following: 1. persistent evidence of sleep hypoventilation (PaCO2 4 60 mmHg); 2. the onset of symptoms usually occurred during the first year of life, and in most during the first few days of life; 3. absence of primary pulmonary disease or neuromuscular dysfunction, which could explain the hypoventilation; 4. no evidence of cardiac disease; 5. no evidence of metabolic disorders.

III.

Pathophysiology

The exact pathophysiology of CCHS remains unknown. However, most of the evidence now suggests that this is a generalized disorder of the autonomic nervous system which affects many more systems than just the control of respiration. Indeed, in addition to HD, multiple mediastinal and adrenal ganglioneuromas and other tumors of the neural crest have been described in CCHS patients [23,25,40–44]. Decreased heart rate variability is almost universal in CCHS [32], and with increasing age, arrhythmias may occur and may necessitate implantation of cardiac pacemakers [34]. Furthermore, specific testing of cardiac responses to baroreceptor stimulation in children with CCHS, revealed marked alteration in cardiovascular fast-component characteristics of the reflex [45]. The gastrointestinal dismotility that is so frequent in early life further supports the notion of a diffuse autonomic disorder. Ophthalmological abnormalities, especially those mediating the neural control of eye movement and pupillary responses, are frequently seen in CCHS patients [20–22]. In addition, brainstem auditory evoked potentials can be abnormal [46–48].


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Recent and exciting developments have occurred lending support to the genetic hypothesis of CCHS. Indeed, a genetic defect is the probable underlying etiology for many, if not all, CCHS patients, and this assumption is particularly enhanced in the context of CCHS coinciding with the presence of HD. While a single gene hypothesis has been proposed, it is more likely that CCHS is the phenotypic manifestation resulting from the interaction between the environment and a polygenic disease. The rationale for CCHS containing a genetic component is its early manifestation in the newborn period, and its occurrence in families including siblings, female twins, mother with neuroblastoma and child with CCHS, and more recently mothers with CCHS giving birth to infants with CCHS [31,49–53], and of course its association with HD [14,22–30]. In fact, up to 20% of reported cases of CCHS are accompanied by HD, such that the association of these two relatively rare clinical entities suggests a possible common pathogenetic basis. Screening for mutations in CCHS, however, has yielded informative yet somewhat disappointing results. Indeed, since a mutation in the RETprotooncogene is associated with HD [54–56], and since RET may play a critical role in the development of the neural crest and parasympathetic system in both HD [54] and CCHS [40–44], a genetic screening of RET mutations was performed by several research groups and revealed that occasional patients may exhibit RET mutations, even when HD is not present [54–61]. Similarly, mutations in the endothelin 3 gene, brain-derived neurotrophic factor [62–64], and in glial-derived neurotrophic factor [65] have been described in patients with CCHS. However, the significance of such sporadic findings remains to be established. In a major, recent breakthrough discovery in this field, mutations in the Phox2b gene, a gene critically involved in the development of the neural crest during embryogenesis, have been identified in a large proportion of French children with CCHS and their families [66]. These findings open new and exciting venues for the understanding of the pathophysiological mechanisms underlying the phenotypic manifestations of CCHS, and further provide conceptual support for the title of this chapter, especially considering the important role of neural crest-derived neurons in autonomic function. It should be emphasized that the putative genetic etiology of CCHS is at least partially undermined by the fact that family members of CCHS patients do not display any evidence of respiratory control dysfunction [67]. In contrast, a recent case/control study revealed that families and siblings of CCHS patients exhibit higher frequency of symptoms compatible with autonomic alterations, thereby suggesting that subtle phenotypic manifestations may be present and supporting some form of Mendelian inheritance [68]. Creation and implementation of a genomic/phenotype database of all diagnosed patients with CCHS will undoubtedly facilitate future genetic studies in this disorder.

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Animal Models

A true animal model displaying all or most of the phenotypic expression patterns of CCHS has not yet become available. Schla¨fke and colleagues have demonstrated that after either electrocoagulation- or ibotenic acidinduced lesions of the intermediate area within the ventral medullary surface, significant compromise of both hypoxic and hypercapnic ventilatory responses will occur in anesthetized and awake cats [69–71]. This experimental model shares some of the typical respiratory control alterations found in CCHS patients. In recent years, and despite the aforementioned negative genetic findings, several investigators have explored the possibility that genetic manipulation of the RET protooncogene in the mouse may lead to respiratory manifestations reminiscent of CCHS [72]. Indeed, markedly reduced ventilatory responses to hypercapnia were present in RET knockout mice [72]. Using whole-body flow plethysmography, baseline breathing and ventilatory and arousal responses to chemical stimuli were examined in unrestrained heterozygous c-ret þ/ newborn mice and their wild-type c-ret þ/þ litter mates at 10–12 h of postnatal age [73]. The hyperpneic and arousal responses to hypoxia and hypercapnia were not significantly different in these two groups. However, the number and total duration of apnea and periodic breathing episodes were significantly higher in c-ret þ/ than in c-ret þ/þ pups during hypoxia and post-hypoxic normoxia. Thus, different aspects of respiratory control are determined by the activity of neural crest controlling genes. Erickson et al. reported similar results in brain-derived growth factor knock-out mice [74], thereby suggesting that multiple genes may be involved in the regulation and development of respiratory and autonomic control sites during embryogenesis. In this regard, recent work with homeobox genes led to the generation of a knockout mouse for RNX. The genes Tlx1 (Hox11), Enx (Hox11L2, Tlx-2) and Rnx (Hox11L2, Tlx-3) constitute a family of orphan homeobox genes. In situ hybridization has revealed considerable overlap in their expression within the nervous system, but Rnx is singularly expressed in the developing dorsal and ventral region of the medulla oblongata. Transgenic mice in which the RNX gene was knocked-out revealed severe hypoventilation, suggesting that this gene may be implicated in CCHS [75]. However, three recent screens for mutations in the RNX gene failed to reveal any abnormality in CCHS patients [60,76,77]. V.

Structural Central Nervous System Abnormalities

Based on the initial assumption that a centrally located defect would be present in CCHS, many attempts have been made over the years to identify


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structural CNS abnormalities. Earlier reports of hypoplasia of the arcuate nucleus in one patient with CCHS [78], and the presence of abnormal evoked potential responses to auditory stimuli [46–48] further suggested that a brainstem lesion might be present. In addition, central hypoventilation syndrome has been described in occasional patients with cerebrovascular malformations and in patients with CNS infections [24,79–83]. Careful radiological surveys of the brain in several CCHS patients have thus far failed to identify any recognizable lesion accountable for the unique manifestations of this syndrome [84] (Harper, personal communication). Neuronal loss in reticular nuclei and cranial nerve nuclei as well as in the nucleus ambiguus and the hypoglossal and dorsal motor nucleus of the vagus have been reported in an infant with CCHS [81]. Furthermore, Cutz et al. reported that in two CCHS patients, their carotid bodies had volumes less than 50% of those seen in normal individuals with reduced numbers of glomus cells [85]. These investigators also reported substantial hypertrophy in the neuroepithelial bodies of these CCHS patients, possibly as a compensatory mechanism for the carotid body abnormality [85]. More recently, using non-invasive functional MRI approaches which provide functional topographic maps of the brain in response to the application of specific stimulation paradigms [86–90] significantly reduced responses in several brain regions in CCHS patients further suggesting that the ‘‘defect’’ is diffusely represented in regions that are immediately pertinent to the embryogenesis of the neural crest [91–96]. However, such a diffuse pattern of neural recruitment deficits also reinforces the concept that the global defect may involve integration of neural autonomic afferent inputs rather than represent a defect in intrinsic chemosensitivity. VI.

Physiologic Abnormalities of Ventilatory Control

Identification of the putative site(s) underlying the phenotypic manifestations of CCHS could not only lead to better therapeutic approaches in these patients, but also provide extremely important insights into modeling and localization of structures mediating respiratory control. Dissection of each of the components of the various functional elements involved in CCHS is obviously impossible in humans. Within these limitations, the following studies provide important information regarding the behavioral characteristics of respiratory control systems in CCHS. A schematic diagram of such approach is provided in Figure 9.1, and should assist in the rationale and interpretation of the studies conducted below. The initial corollary examined was whether voluntary breathing could be affected. Children with CCHS demonstrate no deficit in generating volitional breathing. The observation that the magnitude of alveolar hypoventilation was principally expressed during NREMS led to

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Gozal (6)

Arousal centers Cortex

CENTRAL CHEMORECEPTORS

(2) (1) ???

Hypothalamus locomotion center

Central respiratory drive (4)

Respiratory muscles (3)

Spinal relays (5) Mechanoreceptors muscle afferents

Pulmonary venous return

· VE Peripheral chemoreceptors PaO , PaCO , pH 2

2

Figure 9.1 Schematic diagram of a simplified model of the respiratory control system with potential chemoreceptive, mechanoreceptive and behavioral inputs to the medullary respiratory controller (pathways 1–5), and to the reticular activating system (pathway 6). In CCHS subjects, cortical pathways (1) are operational. The central chemoreceptive pathway and/or putative integrative chemoreceptive pathway (2) are defective. However, pathway 3 may be partially functional as abrupt peripheral chemoreceptor stimulation may induce some ventilatory changes in CCHS subjects. The hypothalamic/locomotor center (4) and mechanoreceptive pathway (5) have been demonstrated to effectively stimulate ventilation in CCHS. For discussion, see text.

the assumption that central chemoreceptor function was abnormally reduced or absent. Examination of ventilatory responses to endogenous challenges of isolated hypercapnia, hypoxemia, and to combined hypoxia and hypercapnia (asphyxia) revealed negligible responses during sleep [1–5,8,9], as well as during wakefulness [97]. Indeed, Paton and coworkers studied rebreathing hypoxic and hypercapnic ventilatory responses during wakefulness in five children with CCHS aged 6–11 years, and found that ventilatory responses to both hypoxia and hypercapnia were essentially random, with no evidence of progressive ventilatory increases despite increasing stimulus. Interestingly, despite absent rebreathing ventilatory responses to both hypercapnia and hypoxia [97], most CCHS patients are able to sustain adequate ventilation during wakefulness [98], such that redundant mechanisms may be operational. A recent study by Gaultier et al. demonstrated that while alveolar hypoventilation was particularly


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prominent during NREM sleep, hypercapnic chemosensitivity was unaffected by sleep states [99]. These data suggest that the intrinsic defect in CCHS is present at all times, but becomes more prominently expressed during conditions in which other redundant mechanisms are either less active or inoperative. One such mechanism could be related to either intact or residual peripheral chemoreceptor function. In five children with CCHS (ages 9–14 years) who demonstrated adequate ventilation during wakefulness, ventilatory challenges with 100% oxygen breathing, five tidal breaths of 100% N2, and vital capacity breaths of 5% and 15% CO2 in O2 and 5% CO2 in N2 induced overall similar increases in minute ventilation (VE) in CCHS and controls [100], suggesting that during abrupt transients in inspired gas concentrations, the peripheral chemoreceptors can be stimulated and induce intact responses [100]. The greater interindividual variability of responses in CCHS could reflect the lack of modulation of ventilatory control due to defective integration of afferent respiratory neural input [100,101]. In this context, if indeed the deficit in CCHS corresponds to defective integration of autonomic sensory stimuli, then arousal during hypercapnia should occur even if the ventilatory responses to hypercapnia are absent. Indeed, the frequency of arousal from a hypercapnic challenge during sleep was similar in eight CCHS and eight controls [102], suggesting the presence of intact central chemoreceptor sensitivity, and of defective integration of chemoreceptor inputs [102]. Based on the premise that chemoreceptors are important controllers of ventilation during exercise, one would also expect to observe substantial gas exchange alterations during physical activity in CCHS patients. In fact, Silvestri et al. showed severe gas exchange abnormalities in full-time ventilator-dependent CCHS patients during moderate exercise [103]. However, both Paton et al. [104] and Shea et al. [105] showed that exercise-induced hyperpnea can occur in CCHS patients who require ventilatory assistance during sleep, particularly at exercise intensities below the anaerobic threshold [103–105]. Such observations suggest that in the absence of ventilatory response to gradual chemoreceptor stimulation, movement exerts a dominant influence on respiratory rate, and consequently on the increase of VE during exercise [104,105], further strengthening the concept that central integration of chemosensory and metabolic inputs is faulty in CCHS. To further examine this concept, passive lower extremity motion approaches were used, both during wakefulness and sleep. Passive leg motion elicited relative hyperventilation in excess of metabolic requirements, resulting in normalization of end-tidal carbon dioxide tension (PETCO2 ), independent of state [106,107]. Thus, in a setting of deficient integration of respiratory control inputs, either mechanoreceptor afferent input from muscle and joints and/or rhythmic entrainment of respiration takes over, and plays a significant role in the modulation of breathing

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during exercise in children with CCHS [106–108]. Furthermore, normalization of PETCO2 with motion as measured in CCHS would lend support to a basic defect in integration of efferent and afferent neural inputs to respiratory controllers sites. VII.

Autonomic Nervous System Dysfunction

In addition to disturbed moment-to-moment heart rate variability [32–35], significant alterations in dopamine turnover have been found in patients with CCHS [109], and vagally mediated syncope may also occur [36], thereby lending further support to the assumption that significant dysregulation of central autonomic nervous system control is frequently present in CCHS. As mentioned, such alterations are also present in family members, and segregation analysis further indicates that CCHS is the most severe manifestation of a generalized dysfunction of the autonomic nervous system with a family pattern consistent with Mendelian transmission [110]. VIII.

Summary and Conclusions

The clinical and physiological spectrum of CCHS supports major physiological concepts, namely, (a) this disorder represents a congenital and genetically determined disease variant of the autonomic nervous system in general, and more specifically of the embryological development of the neural crest, and (b) the recruitment and activation of redundant mechanisms in these patients allows for the ability to sustain near adequate gas exchange during wakefulness and physical activity. Further efforts to expand on genotype–phenotype relationships and interactions should permit better insights into the normal development and function of the autonomic nervous system in man. Acknowledgment The author is supported by grants from the National Institutes of Health (HL69932, HL63912, and HL66358), and The Commonwealth of Kentucky Research Challenge Trust Fund. References 1.

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10 Upper Airway Obstruction in Sleep Apnea

SUSHEEL P. PATIL, HARTMUT SCHNEIDER, PHILIP L. SMITH, and ALAN R. SCHWARTZ Johns Hopkins University Baltimore, Maryland

I.

Introduction

Obstructive sleep apnea is a common disorder linked to the increasing prevalence of obesity in Western society, leading to recurrent oxyhemoglobin desaturations and arousals from sleep. Obstructive sleep apnea is caused by episodes of upper airway obstruction during sleep, and has been associated with increased morbidity and mortality [1–4] stemming from neurocognitive [5–8], cardiovascular [9–12], and respiratory dysfunction [13–18]. Upper airway obstruction is largely related to an increased propensity of the upper airway to collapse during sleep through a loss of neuromuscular tone. Sedatives and anesthetic agents can mimic the effects of natural sleep, thus increasing the risk for upper airway obstruction by blunting neuromuscular reflex and arousal responses that restore upper airway patency. This chapter will focus on the clinical and epidemiologic risk factors for upper airway obstruction, the pathophysiology of upper airway obstruction, and its implications for monitoring and treatment.

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Epidemiologic and Clinical Risk Factors

Obstructive sleep apnea is a comparatively new disorder with initial medical reports documenting the presence of discrete apneic episodes recurring throughout the night first appearing in the early 1960s [19–21]. While initial clinical reports established a strong association between sleep apnea and morbid obesity in patients with the Pickwickian syndrome [22–24], it was subsequently demonstrated that sleep apnea also occurred quite commonly in mildly to moderately overweight individuals who had no alterations in daytime gas exchange or evidence for cor pulmonale [13,25]. In a landmark publication in 1978, Remmers et al. [26] documented that apneas were due to the development of pharyngeal obstruction and were terminated by arousal from sleep. This report ushered in an era of physiologic investigations that examined the mechanism of upper airway obstruction [27–30], epidemiologic studies that defined the prevalence and risk factors for this disorder [31–42], and numerous investigations that examined the neurocognitive [5–8], behavioral [43–45], neuroendocrine [46–48], metabolic [25,49], and cardiovascular consequences [10,50,51] of sleep apnea. Investigations into the pathogenesis of sleep apnea and its consequences have employed standardized metrics of sleep apnea severity based on counts of apneas (no airflow) and hypopneas (reduced airflow associated with arousals and/or oxyhemoglobin desaturations) per hour of sleep (apnea–hypopnea index, AHI), associated arousals and oxyhemoglobin desaturations. With further refinements in monitoring technology, definitions of disease severity were expanded to include subjects with intermediate degrees of upper airway obstruction during sleep, and variable degrees of microarousals and oxyhemoglobin desaturations [52–59]. As standards for assessing the severity of upper airway obstruction and resulting alterations in gas exchange and sleep architecture have evolved, investigators have recognized a progression in upper airway obstruction that is characterized by snoring, obstructive hypopneas, and obstructive apnea [60]. In recent years, investigators have documented a high prevalence of sleep apnea in the general population. Early studies using a combination of symptom surveys and limited physiologic recordings in normal healthy adult populations suggested a high prevalence of sleep apnea in Western society [38,61–66]. In a subsequent, more comprehensive epidemiologic survey of sleep apnea in the general population, Young and co-workers [31] employed polysomnography to identify those with sleep apnea. The estimated prevalence of sleep apnea syndrome (AHI 5 and daytime hypersomnolence) was 2% in women and 4% in men for the population at large. Even higher point prevalence of sleep apnea (AHI 5) was observed in habitual snorers (9% of women and 24% of men). Furthermore, in an older, multicenter community-based cohort (the Sleep Heart Health Study) enriched with subjects who snored [9], sleep apnea was found in more than half of the

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men and a third of the women, indicating that the prevalence of this disorder may be quite high in older strata of the general population. Several recognized clinical risk factors have been associated with a greatly increased prevalence of obstructive sleep apnea in the general population [48,67]. Among these, obesity is thought to be the major risk factor. Mild to moderate obesity has been associated with a markedly increased prevalence of sleep apnea [25,31–42,68] and extreme obesity has been linked to increases in disease severity [69–73]. Male gender also constitutes a particularly strong risk factor, and confers a two- to three-fold increased risk of sleep apnea in the population at large [67,74]. It has been postulated that this increased risk is related to differences in the distribution of adipose tissue in men [35,48], who exhibit a predominantly central pattern of adiposity around the trunk and abdominal viscera, when compared with peripheral adiposity in women [75,76]. Increases in central adiposity with age may also account for an increase in sleep apnea prevalence in postmenopausal women [77–80]. In addition, recent studies have demonstrated familial aggregation and a racial predisposition to sleep apnea in individuals of African-American and Asian descent, suggesting that heritable factors also contribute to the development of sleep apnea [81–83]. Thus, obesity, and in particular central adiposity, may interact with male gender, age and genetic factors, leading to increases in the prevalence of sleep apnea. Nevertheless, the physiologic mechanisms linking obesity, male gender and sleep apnea have not been fully explained. Although age has also been considered a risk factor for sleep apnea, its effect may not be very pronounced. In a cross-sectional study of moderately obese, healthy male community subjects, no difference in the prevalence of sleep apnea was demonstrated across age groups [84]. Additional longitudinal data suggests that age does not contribute significantly to the prevalence of sleep apnea since there is minimal progression of disease in elderly cohorts over time [85]. Nevertheless, the high prevalence of sleep apnea in older populations may be related to age-related increases in body weight [86–89]. The pivotal role of obesity in the pathogenesis of sleep apnea is further suggested by the observation that modest decreases in body weight have been associated with substantial reductions in AHI [90,91]. Thus, both cross-sectional and longitudinal data suggest that obesity rather than age per se is a major risk factor for this disorder. With progressive increases in weight, patients develop signs of upper airway obstruction, including snoring, snorting, gasping, choking, and witnessed apneic episodes. As these symptoms progress, sleep disruption and daytime hypersomnolence typically ensue. When patients have been systematically queried in sleep disorders centers [92–98], investigators found that symptoms of upper airway obstruction, sleep disruption, and daytime hypersomnolence were highly predictive of the presence of sleep apnea. Of note, signs of upper airway obstruction were particularly predictive of

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this disorder in clinic-based populations. Moreover, when combined with measures of adiposity (i.e., body mass index or neck circumference) and male gender, symptoms of upper airway obstruction, sleep disruption, and excessive daytime somnolence were highly predictive of the presence of sleep apnea [96,97]. In addition to clinical and epidemiologic risk factors, obstructive sleep apnea has been associated with alterations in upper airway anatomy. Structural changes, including tonsillar hypertrophy [99–101], retrognathia [102–104], and variations in craniofacial structures [105–110], have been linked to an increased risk of sleep apnea, presumably by increasing upper airway collapsibility. Similarly, during wakefulness, computed tomographic and magnetic resonance imaging studies have demonstrated increased fatty tissue deposition and submucosal edema in the lateral walls of the pharynx, both of which narrow the pharyngeal lumen and may predispose to obstruction during sleep [19,111–113]. Underlying medical illness may also predispose to alterations in upper airway anatomy that play a role in the pathogenesis of upper airway obstruction. Endocrinopathy including testosterone administration [114–117], hypothyroidism [118–120], and acromegaly [121–123] are recognized risk factors for obstructive sleep apnea, and may produce structural changes in the neck and upper airway muscles that further predispose to upper airway obstruction. Specifically, testosterone replacement has been found to increase neck size, which has been associated with an increase in airway collapsibility [114,115,117,124]. Macroglossia, a recognized feature of hypothyroidism, acromegaly, and amyloidosis, may narrow the pharyngeal lumen and overload dilator muscles that maintain upper airway patency. Finally, hypothyroidism and acromegaly may result in soft tissue swelling and a concomitant myopathy that could narrow the pharynx and further compromise dilator muscle function, respectively. In recent years, clinical and epidemiologic evidence has also suggested a link between sleep apnea and underlying cardiovascular disease. It is now recognized that cardiovascular risk factors including male gender, visceral adiposity, glucose intolerance, and hypertension are highly associated with sleep apnea [32,34,48,125–127]. While some of these risk factors may predispose to sleep apnea (gender, obesity, and central adiposity), it also appears that cardiovascular and metabolic dysfunction are likely a consequence of this disorder. Evidence to support this association include a nearly three-fold increased incidence of hypertension in individuals with sleep apnea over a four-year follow-up period, suggesting that sleep apnea could be an antecedent for daytime hypertension [51,128]. Furthermore, sleep apnea has been associated with an increased risk of hypertension, stroke, angina, myocardial infarction and congestive heart failure in the ongoing multi-centered Sleep Heart Health Study [9,10]. Finally, among men with stable heart failure due to systolic dysfunction, approximately


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two-thirds of patients had sleep apnea [129–131]. Thus, the prevalence of sleep apnea may be particularly high in patients with underlying cardiovascular disease, and likely predisposes to increased cardiovascular morbidity and mortality. The cardiovascular sequelae of obstructive sleep apnea may be related to acute and chronic cardiovascular stress and metabolic dysregulation. Acutely, obstructive apneic episodes in experimental animals and humans are associated with recurrent surges in blood pressure following each apnea cycle [128,132–135]. Acute increases in blood pressure have been related to progressive increases in sympathetic drive throughout the apnea [136,137] and arousals at the termination of apneic episodes [134,137–139]. Investigators have further demonstrated that sleep apnea abolished the usual nocturnal decline in blood pressure [132,140], and that nocturnal blood pressure can rise above daytime levels in sleep apneic patients [140]. Experiments in animals have provided further insight into the mechanism for blood pressure elevations accompanying apneic episodes. The acute hemodynamic sequelae of sleep apnea have been shown to relate to the severity of oxyhemoglobin desaturations during the apneic episodes [141,142]. Blood pressure surges following apneas, however, appear to be caused by arousals and central effects of hypoxemia, which trigger pronounced sympathetic neural discharge and peripheral vasoconstriction, and increase cardiac afterload [137,143–145]. Chronic effects of intermittent hypoxemia and associated sympathetic neural discharges have now been linked to glucose intolerance [25,48,49,146], endothelial dysfunction [147–150], and disturbances in peripheral microvascular control [151]. Thus, evidence from animals and humans indicates that obstructive sleep apnea imposes significant acute and chronic metabolic and cardiovascular dysfunction. III.

Pathogenesis of Upper Airway Obstruction

A. Pathophysiology of Airflow Obstruction

Initial reports investigating the mechanism for upper airway obstruction [26] clearly demonstrated that the pharynx was the site of obstruction. The role of pharyngeal obstruction was further emphasized by the fact that obstructive sleep apnea could be eliminated by treatments that relieved or bypassed the upper airway [152–158]. Concepts concerning the pathogenesis of upper airway obstruction in obstructive sleep apnea were based on early work in normal anesthetized and paralyzed subjects [159,160], demonstrating that mechanical factors related to head, neck, and jaw position predisposed to pharyngeal obstruction. From fluoroscopic studies [161,162], it was shown that the tongue prolapsed during periods of obstruction, emphasizing that bulk soft tissue displacement was required

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for obstruction to occur. In particular, the tongue was noted to play a primary role, since the genioglossus muscle, the primary tongue protrusor, prolapsed into the pharynx whenever neuromuscular tone waned [26]. Further studies examined whether the obstruction occurred primarily at the retroglossal or retropalatal segment. Specifically, upper airway manometric studies during sleep demonstrated that wide pressure gradients developed in either pharyngeal segment during obstructive episodes, suggesting that collapse could occur in either the velo- or oropharynx [163–165]. Additional computed tomographic [166] and endoscopic imaging studies [167] have since documented that collapse can occur simultaneously in both pharyngeal segments, and can vary between non-REM and REM sleep stages within a single individual. These studies have given rise to the concept that while localized anatomic factors, such as adenotonsillar hypertrophy, retrognathia, and alterations in upper airway bony and soft tissue structures may predispose to collapse of certain pharyngeal segments, a more generalized defect in upper airway structural or neuromuscular control may be responsible for collapse along the entire length of the pharyngeal airway during sleep. To elucidate the mechanism of upper airway obstruction in obstructive sleep apnea, several approaches have been adopted to model the factors involved in the pathogenesis of pharyngeal collapse. Initial efforts focused on the interplay between extraluminal upper airway muscles that dilate and negative intraluminal pressures generated by the diaphragm that collapse the pharynx. It was originally postulated that upper airway patency was determined by the balance of pressures between the intraluminal and extraluminal spaces [26]. As intraluminal suction pressures overcame the dilating forces around the pharyngeal lumen, the theory held that the pharynx would progressively collapse and ultimately occlude during sleep. Later studies, however, have minimized the role of intraluminal suction pressures in the pathogenesis of upper airway obstruction by demonstrating that upper airway occlusion could occur spontaneously, even when intraluminal pressures were positive [168,169]. These observations resolved a major question regarding the role of negative intraluminal pressures in the pathogenesis of obstructive sleep apnea, and confirmed that negative pressures were not required for airway occlusion to occur. Rather, the markedly negative intraluminal pressures generated by the diaphragm during periods of upper airway obstruction were the consequence rather than the cause of upper airway occlusion. To further elucidate the mechanism for upper airway obstruction, investigators have examined airflow dynamics during periods of obstruction, and found that pressure-flow relationships were identical to those previously described for other collapsible biologic conduits, i.e., the Starling resistor (Figure 10.1) [170–172]. In a series of studies in sleeping humans


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Figure 10.1 The upper airway can be represented as a mechanical analogue (Starling resistor model) consisting of a rigid tube with a collapsible segment. Upstream (nasal) and downstream (hypopharyngeal) segments have fixed diameters and defined resistances, Rus and Rds, respectively. Pressures in these segments are represented by Pus and Pds, respectively. The collapsible segment has no resistance, but is subject to the surrounding pressure, Pcrit. Collapse occurs only when the surrounding pressure exceeds the intraluminal pressure (adapted from Ref. 60).

both with [29,173] and without sleep apnea [30], investigators demonstrated that upper airway flow dynamics resemble those seen in a Starling resistor in two respects [174]. First, as the downstream (tracheal) pressure falls during inspiration, inspiratory airflow attains a maximal level (VImax) and becomes independent of further decreases in downstream pressure (Figure 10.2A). This phenomenon, known as inspiratory airflow limitation, is characterized by a plateauing of airflow and is often associated with collapse of the pharynx and audible snoring. Second, under conditions of inspiratory airflow limitation, maximal inspiratory airflow varies linearly with changes in upstream nasal pressure, and falls from normal tidal airflow levels (300–500 ml/s) at higher levels of nasal pressure to zero as nasal pressure is lowered below the critical level (critical pressure, Pcrit) (Figure 10.2B). In fact, the critical closing pressure (Pcrit) is a direct measure of the collapsibility of the pharynx, and is operationally defined by lowering the nasal pressure until inspiratory airflow ceases (the airway occludes). In further studies, the upstream nasal pressure has been manipulated systematically, and critical pressures were measured in groups of individuals manifesting varying degrees of upper airway obstruction during sleep (Figure 10.3) [29,59,60]. Critical pressures were markedly negative in normal individuals with evidence of airflow obstruction, whereas critical pressures were positive in apneic patients with complete upper airway occlusion. In patients with partial airflow obstruction during sleep (obstructive hypopnea, upper airway resistance syndrome, and asymptomatic snorers), critical pressures were between these two extremes (minimally to moderately

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negative) [29,30,60]. These observations suggested that varying degrees of upper airway obstruction during sleep were associated with quantitative differences in critical pressures, reflecting differences in upper airway collapsibility across the spectrum from health to disease. Intervention studies have demonstrated that improvements in sleep apnea severity (AHI) are predicted by reductions in critical pressure after specific interventions. For example, in a study of weight loss intervention [69], decreases in critical pressure were directly correlated with decreases in weight, and sleep apnea remitted when critical pressures fell below 4 cmH2O. In a subsequent study examining the effect of uvulopalatopharyngoplasty on critical pressures and sleep apnea severity, it was shown that sleep apnea severity also correlated with reductions in critical pressure below a similar threshold of 4 cmH2O [175]. Finally, postural maneuvers, which lower the critical pressure by only 2–4 cmH2O, were associated with only modest improvements in sleep apnea severity for those in whom the critical pressure fell below atmospheric levels [176–178]. These intervention studies have clearly established that sleep apnea is due to elevations in critical pressure and that specific interventions can lead to predictable falls in

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Figure 10.3 Critical closing pressures of the upper airway (Pcrit) during sleep are plotted for groups of individuals that represent the clinical spectrum of obstructive sleep apnea—non-snoring, snoring, upper airway resistance syndrome (UARS), obstructive hypopnea, and obstructive apnea. Pcrit increases with increasing disease severity over a relatively narrow range of pressures. Note overlap between UARS and obstructive hypopneas, suggesting that the two disorders are indistinguishable in the degree of upper airway function, or in the impact of upper airway obstruction on sleep continuity (adapted from Refs. 30, 59 and 60).

critical pressures and improvements in sleep apnea that are based on the post-treatment value. Experimental studies have demonstrated that the critical pressure also determines the level of maximal inspiratory airflow. When the critical pressure is positive, airflow is zero at atmospheric nasal pressure. Progressive increases in nasal pressure in apneics have been associated with increasing levels of maximal inspiratory airflow and progression from obstructive apneas to hypopneas, to snoring and finally to normal breathing during sleep [29,60,173]. Once the nasal pressure exceeds the critical

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pressure, the airflow response to elevations in nasal pressure is determined by the airway resistance upstream to the site of pharyngeal collapse [173, 179]. Conversely, lowering the nasal pressure in normal individuals induces upper airway obstruction (inspiratory flow limitation) and progressive reductions in maximal inspiratory airflow [30]. As nasal pressure approaches a negative critical pressure in normal individuals, airflow falls and recurrent obstructive hypopneas and apneas ensue, leading to oxyhemoglobin desaturation and arousals from sleep [180]. In fact, a gradient of at least 5–8 cmH2O between the nasal and critical pressure is required to maintain tidal airflow levels, and approximately 10 cmH2O is required to abolish inspiratory airflow limitation completely [30,174,180]. These findings indicate that normal individuals and sleep apnea patients differ solely in their level of critical pressure, suggesting that obstructive sleep apnea is due to differences in upper airway collapsibility during sleep [180].

B. Anatomic Factors

While the critical pressure determines the degree of upper airway obstruction during sleep, the mechanisms leading to alterations in critical pressures are not well understood. In general, elevations in critical pressures have been attributed to either alterations in upper airway anatomical structures or disturbances in upper airway neuromuscular control [29,69]. Several approaches have been exploited to distinguish experimentally between these mechanisms for elevations in critical pressure. By eliminating neuromuscular activity with the administration of neuromuscular blocking agents, Isono and colleagues have recently examined the structural basis for alterations in critical pressure and compared values in normal individuals to those in patients with sleep apnea. In seminal studies [167,181–184], these investigators found elevated critical pressures in sleep apnea patients compared with normal controls, suggesting that alterations in upper airway anatomy contribute to the pathogenesis of upper airway obstruction. These investigators further determined that obesity, jaw position, acromegaly, tonsillar hypertrophy, and a smaller bony enclosure surrounding the pharynx may also elevate critical pressure [106,185–189]. Based on their observations (Figure 10.4), it has been proposed that crowding of soft tissue surrounding the pharynx could elevate the critical pressure by increasing the peri-pharyngeal pressure [106]. In addition to soft tissue crowding the pharyngeal lumen, cervical structures may exert axial forces that also influence upper airway collapsibility. In studies of airflow dynamics in the isolated upper airway of animals [190,191], investigators found that critical pressures fell

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Figure 10.4 Relative differences in the soft tissue and the bony enclosure of the upper airway can influence the critical closing pressure (Pcrit) of the upper airway. As illustrated in this mechanical model, (a) the upper airway can be represented as a rigid bony enclosure with soft tissue contained within the enclosure. Increases in the mass of soft tissue (b) (which can occur with obesity) within a constant bony enclosure can increase the tissue pressure (Ptissue), narrowing and predisposing the upper airway to collapse. Similarly (c), a fixed amount of soft tissue within a smaller bony enclosure, e.g., as seen with retrognathia, can increase the tissue pressure (Ptissue) around the lumen of the upper airway and predispose it to collapse (Data from Ref. 106).

markedly when the trachea was pulled caudally. This decrease was attributed to stretching and unfolding of redundant pharyngeal mucosa. Moreover, it was hypothesized that increases in tension within the pharyngeal wall mucosa would make it better able to withstand the compressive radial forces exerted by surrounding tissues. In further studies examining the interaction between radial and axial forces, Rowley et al. [191] demonstrated that axial traction amplifies the response of radial forces that either dilate or compress the airway (Figure 10.5). Additional evidence in animal and human studies indicate that axial tension increases during inspiration when the diaphragm moves caudally along with mediastinal and tracheal structures [192,193]. As the airway elongates, increases in axial tension have been associated with improvements in upper airway patency during inspiration [194]. Conversely, obesity, which decreases lung volumes and elevates the diaphragm, may predispose to upper airway obstruction through the loss of axial forces on the pharyngeal segment. Thus, structural factors leading to a dynamic interplay between axial and radial forces likely elevate critical pressures in obese individuals and modulate critical pressures throughout the respiratory cycle.

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Figure 10.5 (A) Tracheal and tongue displacement result in axial and radial forces that stabilize the upper airway. (B) Tongue displacement results in dilating forces that prevent upper airway collapse through decreases in critical pressure (Pcrit). (C) Tracheal displacement results in axial tension that can further stabilize the upper airway. (D) Axial traction can potentiate the effect of radial forces, thereby causing further decreases in Pcrit (Data from Ref. 191).

Several lines of evidence suggest obesity is associated with anatomic alterations that may also predispose to upper airway obstruction. In an isolated rabbit upper airway model, Koenig and Thach [195] found that applying lard-filled bags to the cervical area increased the critical pressure, suggesting that local deposition of cervical and peri-pharyngeal fat increases critical pressures through direct pressure on the upper airway. Similarly, obesity has been linked with elevations in neck circumference and increased amounts of peri-pharyngeal fat [196,197], which could narrow and compress the upper airway. In fact, in sleep apnea patients, MRI studies [112,198] have demonstrated greater amounts of peri-pharyngeal fat, correlating with sleep apnea severity. Finally, histologic studies of resected uvular tissue revealed greater amounts of submucosal fatty tissue in patients with obstructive sleep apnea [199]. These findings suggest that the compressive effects of fatty tissue deposited around the pharynx increase upper airway collapsibility, and possibly offset the effects of dilator muscles that maintain airway patency. Anatomic alterations may also account for increases in upper airway collapsibility in obese compared with non-obese individuals [69]. Critical pressures do not decrease with anterior mandibular displacement in obese individuals, a maneuver known to restore upper airway patency in normal


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individuals [187]. The lack of response with anterior mandibular displacement may be attributed to the fact that obesity, and in particular central obesity, has been associated with reductions in lung volume [200], known to increase upper airway collapsibility both in humans and in animals [190–193,201,202]. Thus, obesity imposes mechanical loads on both the upper airway and respiratory system that contribute to upper airway narrowing, obstruction, and collapse during sleep. C. Neuromuscular Factors Neuromuscular Reflexes

In addition to alterations in upper airway anatomy, disturbances in neuromuscular control may also contribute to the pathogenesis of upper airway obstruction during sleep. As neuromuscular activity wanes at sleep onset, its role in the maintenance of upper airway patency may differ between normal individuals and sleep apnea patients. The effect of neuromuscular activity on the critical pressure can be discerned during natural sleep and general anesthesia—with or without complete neuromuscular blockade [167,203,204]. When neuromuscular activity was reduced or eliminated under general anesthesia, critical pressures correlated with the presence and severity of upper airway obstruction during sleep. As seen in Figure 10.6, critical pressures in normal individuals and sleep apnea patients were near atmospheric, at levels known to be associated with severe upper airway obstruction during sleep. Since much lower (more negative) critical pressures are required to maintain upper airway patency during sleep in normal individuals [30], the marked critical pressure elevation in normal individuals during anesthesia and neuromuscular blockade suggests that neuromuscular mechanisms exert a major influence in the normal maintenance of upper airway patency during sleep (Figure 10.7). In contrast, critical pressures in sleep apnea patients do not differ markedly between the intact sleeping and anesthetized/paralyzed states [29,30,60,167]. Whereas neuromuscular mechanisms appear to stabilize the upper airway in normal sleeping individuals, this latter finding in sleep apnea patients suggests that a disturbance in neuromuscular control leads to upper airway obstruction during sleep. This disturbance may be due to a loss of neural drive to the upper airway musculature or to a reduced work efficiency of pharyngeal muscles that maintain upper airway patency. Current evidence suggests that compensatory increases in neuromuscular mechanisms play a critical role in maintaining upper airway patency in both humans and animals. Mezzanotte et al. [205] found that waking genioglossal EMG was elevated in sleep apnea patients when compared with normal controls, and postulated that apneic patients compensate for a narrower pharyngeal airway with higher levels of waking neuromuscular activity. Similarly, Hendricks and colleagues

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Figure 10.6 The apnea–hypopnea index during REM sleep demonstrates positive correlation with measurements of critical closing pressure (Pcrit) during general anesthesia, a state during which neuromuscular responses are minimized (reprinted with permission from Ref. 203).

[206,207] have studied the effects of anatomic airway narrowing in a bulldog model of sleep apnea, and found evidence for increased neuromuscular activity during sleep that may lead to muscle fiber injury in the cervical muscles. Series and co-workers [208] biopsied upper airway muscles and noted increased glycolytic capacity and alterations in the length tension characteristics of the muscularis uvulae in apneic patients. These alterations in upper airway muscle properties are consistent with the notion that neuromuscular recruitment during wakefulness may be required to compensate for mechanical loads in snoring and sleep apnea patients. Of note, critical pressures during wakefulness varied directly with physiologic and metabolic disturbances of the muscularis uvulae, suggesting that upper airway muscles still did not fully compensate for mechanical loads (Figure 10.8). Thus, it appears that a complex interaction between airway muscles and anatomic structures is required to maintain upper airway patency during wakefulness, and that a critical failure of compensatory neuromuscular mechanisms occurs during sleep.


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Figure 10.7 Differences in critical closing pressure (Pcrit) during (A) NREM sleep and (B) general anesthesia with neuromuscular blockade. Increases in Pcrit during neuromuscular blockade relative to sleep in normal individuals without sleep apnea suggest the importance of neuromuscular factors in the maintenance of upper airway patency. The apparent lack of a difference in Pcrit during sleep vs. neuromuscular blockade in subjects with sleep apnea suggests that neuromuscular responses are impaired in this group (adapted from Refs. 30, 60 and 167).

Upper airway obstruction is known to trigger various neuromuscular reflexes that activate upper airway dilator muscles (Figure 10.9) [205,209]. In animal studies, pulmonary and upper airway mechanoreceptors and chemoreceptors act individually and in combination to modify upper airway dilator muscle activity [210–217]. While these findings attest to the importance of neural activation, the precise effect of neural activity on upper airway function has not been well characterized. Chemoreceptor and mechanoreceptor reflexes [213,218,219] play a major role in regulating upper airway neuromuscular control and collapsibility, as emphasized by studies in which hypercapnia markedly increased neuromuscular activity and decreased the critical pressure [219]. Thus, studies in animals suggest that neuromuscular mechanisms can play an important role in stabilizing the upper airway, and that the loss of these protective mechanisms may predispose to upper airway obstruction during sleep.

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Figure 10.8 Airflow obstruction stimulates neuromuscular mechanisms that compensate for mechanical loads in individuals with sleep apnea relative to individuals without sleep apnea. As shown, (A) increases in length tension of the muscularis uvulae are associated with increases in critical pressures (Pcrit). In addition, (B) increases in anaerobic enzyme activity are associated with increases in Pcrit measured during wakefulness, providing further evidence of compensation for mechanical loads on the upper airway (Data from Ref. 208).

Studies examining neural EMG activity of various muscles have documented the influence of chemoreceptor and mechanoreceptor reflex responses on upper airway neuromuscular control during wakefulness [220,221]. Nevertheless, the influence of chemoreceptors responses during sleep is unclear, since marked hypercapnia is generally not observed during apneic episodes and the obstruction often persists in the face of significant hypoxemia. Moreover, it is now thought that mechanoreceptors may not respond appropriately to the markedly negative intraluminal pressure generated during periods of upper airway obstruction [211,222–227]. Early studies demonstrated that topical anesthesia of the pharyngeal mucosa increased the number of obstructive apneas and hypopneas during sleep in normal subjects and loud snorers, and/or increased the duration of apneic episodes [228–231]. In addition, it appears that upper airway sensory


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Figure 10.9 Upper airway obstruction can trigger various neuromuscular responses, as illustrated, to prevent upper airway collapse (Data from Ref. 299).

pathways may be impaired, since temperature, two-point discrimination and vibratory thresholds are disrupted in sleep apnea patients compared with normal individuals [232–234]. Sensory receptor dysfunction could also attenuate the response of upper airway dilator muscles to the markedly negative airway pressures generated during periods of upper airway obstruction. Further evidence for sensorimotor dysfunction is provided by graded histopathologic and immunochemical alterations in the palatopharyngeus and muscularis uvulae in sleep apnea patients, relative to asymptomatic snorers and normal individuals [235–239]. Findings of muscle fiber type redistribution and injury (fascicular atrophy and grouped atrophy in muscle fibers), have suggested that myopathic as well as sensory dysfunction may further compromise neuromuscular responses to upper airway obstruction. In recent studies involving tracheostomized patients [169,240], marked decreases in both tonic and phasic genioglossal activity were noted when patients breathed through their tracheostomy compared with breathing through their nares. This finding suggested that cyclic changes in pharyngeal pressures drive phasic firing of the genioglossus muscle during inspiration. More recent work has clearly demonstrated that genioglossal activity correlates with changes in pharyngeal pressure under a variety of experimental conditions, including one in which an iron lung was used to dissociate effects of tidal swings in intraluminal airway pressure from the

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intrinsic respiratory pattern generators. Akahoshi and colleagues [241] demonstrated strong correlations between genioglossal EMG activity and pharyngeal pressure, regardless of whether pressure swings were modulated by hypercapnia or passive breathing in the iron lung (Figure 10.10). These findings confirm that pressure-sensing mechanisms play a prominent role in modulating upper airway neuromuscular activity during wakefulness, although their role during sleep has not been well studied. In addition to changes in neuromuscular activity, a recent study [169] has established marked phasic modulation of upper airway collapsibility throughout the respiratory cycle. In sleeping tracheostomized apneic patients, markedly diminished (more negative) critical pressures were observed when patients breathed through their pharynx as opposed to their tracheostomy, suggesting that neuromuscular responses to negative airway pressure decreased collapsibility (Figure 10.11). In contrast, when patients breathed through their tracheostomies, the critical pressure increased and rose even further during expiration compared with inspiration. These observations in tracheostomized subjects suggest that phasic mechanisms stabilize upper airway patency and that phasic changes in airflow regimen, pressure, and genioglossus EMG activity play a role in dynamically modulating upper airway function during sleep.

Upper Airway Muscles

Which upper airway muscles might be responsible for maintaining upper airway patency? Initially, investigators focused on the role of the genioglossus, a prominent airway dilator muscle. The effect of the genioglossus muscle on upper airway stability has been examined by stimulating the genioglossus electrically in the isolated feline and canine upper airways. In these animal studies [218,242–247], electrical stimulation led to a significant fall in pharyngeal compliance and critical pressures, which stiffened and lowered airway collapsibility. Later studies by Fregosi and co-workers [248] confirmed that electrical stimulation of the genioglossus stabilized the airway, and that co-stimulating the tongue’s protrusor and retrusor muscles augmented this effect. In addition, it appears that both muscle groups play a significant role physiologically in stabilizing and maintaining airway patency, since these muscle groups are recruited simultaneously under hypercapnic and hypoxic conditions [249,250]. The observations in animals have been extended to studies in humans, demonstrating that stimulation of the lingual protrusor (genioglossus) selectively or in combination with retractor muscles reduces airflow obstruction during sleep [251–254]. Studies by Oliven and co-workers confirmed these findings and demonstrated improvements in upper airway flow dynamics in both animals and humans [244,255–259]

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Figure 10.11 Phasic modulation of upper airway collapsibility (Pcrit) is present throughout the respiratory cycle in sleeping patients with tracheostomy (n ¼ 6). Pcrit decreases during inspiration versus peak expiration during tracheostomy (solid bars) breathing, and falls even lower with nasal breathing. Effect of nasal (open bar) vs. tracheostomy breathing and respiratory phase (tracheostomy breathing: peak expiratory, end expiratory, and inspiratory) on Pcrit is illustrated. Values are means SE (from Data Ref. 169).

during electrical stimulation at various lingual sites. Nevertheless, electrical stimulation of the lingual musculature only partially relieved upper airway obstruction, and did not eliminate obstructive apneic episodes completely [253,254], suggesting that other muscles must also be recruited to restore upper airway patency and normalize tidal airflow. It is well known that the intrinsic muscles of the pharyngeal wall and the cervical strap muscles also play a role in the maintenance of airway patency. In the isolated feline upper airway model, studies by Kuna [260,261] and others [262,263] have demonstrated that pharyngeal constrictor muscles are recruited phasically in states of high ventilatory drive, and thus may reduce airway compliance (stiffen the airway). Moreover, responses in upper airway patency to constrictor muscle stimulation


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depend on pharyngeal luminal size. The pharyngeal constrictors were found to narrow the airway when widely patent, yet dilate when narrowed [264,265]. In further studies, specific oro- and velopharyngeal muscles appear to differentially control the patency of specific pharyngeal segments, suggesting that coordinated action of these muscles is required to stabilize the airway along its entire length. In addition to the genioglossus and intrinsic pharyngeal muscles, the cervical strap muscles also play a major role in supporting the airway, primarily by exerting caudal traction on the hyoid bone complex [266]. Thus, coordinated action of muscles that dilate, elongate and stiffen the pharyngeal wall may be required to optimally preserve airway patency.

IV.

Therapeutic Implications

Despite the complexity in delineating the structural and neuromuscular factors modulating upper airway patency, two basic approaches can be taken to relieve pharyngeal obstruction in patients with obstructive sleep apnea (Figure 10.12). Both are predicated on the notion that collapse and airflow obstruction are due to elevations in critical pressure. As previously discussed, continuous positive airway pressure (CPAP) has been designed to overcome the obstruction (positive critical pressure) by elevating upstream pressures at the nose or mouth [152,267]. CPAP has been a mainstay of therapy for obstructive sleep apnea for nearly 20 years, and is effective in relieving obstruction in patients with a broad range of critical pressures. An alternative therapeutic approach to therapy is to lower the critical pressure by augmenting the structural or neuromuscular mechanisms required for the maintenance of airway patency. Current approaches to correcting alterations in upper airway mechanics include weight loss [69,91,268,269], postural maneuvers [176–178], upper airway reconstructive surgery (uvulopalatopharyngoplasty, transpalatal resection, adenotonsillar resection) [101,270–272], and a variety of procedures designed to move the hyoid, mandible and maxillary bones anteriorly [273–276]. Of note, Isono and colleagues have shown that anterior mandibular displacement lowered the critical pressure significantly in lean normal individuals, an effect that is best explained by reductions in tissue pressure surrounding the airway. Nevertheless, anterior mandibular displacement had little effect in obese patients (Figure 10.13). Alternatively, compensatory neuromuscular mechanisms can be improved with maneuvers that increase the recruitment of muscles during sleep, thereby counteracting the sleep-dependent decline in neuromuscular activity leading to upper airway obstruction. Initial efforts to electrically stimulate specific upper airway muscles have offered partial relief of upper

334

Patil et al. THERAPY FOR OBSTRUCTIVE SLEEP APNEA Change nasal or critical pressure PN

0 Pcrit Pcrit

PN

(A)

(B) 0

Figure 10.12 Upper airway obstruction may be relieved through either of two mechanisms. (A) Increases in nasal pressure (PN) beyond the critical pressure (Pcrit) of the upper airway will result in airflow. (B) Alternatively, reductions in tissue pressure through surgical intervention (e.g., UPPP [uvulopalatopharyngoplasty], LAUP [laser-assisted uvulopalatoplasty], mandibular advancement, etc.) or neuromuscular stimulation (e.g., hypoglossal nerve stimulation) will reduce Pcrit, resulting in a patent upper airway (Data from Ref. 300).

airway obstruction [251–254,277], as have pharmacologic strategies that stimulate upper airway motor neuron pools with tricyclic antidepressants [278–282] and serotonergic agents [283–290]. Refinements in these therapeutic strategies will undoubtedly require more precise elucidation of the neurochemical, sensorimotor, and mechanical factors that maintain upper airway patency during sleep. Therapeutic monitoring and intervention is particularly important in the peri-operative setting, where lingering effects of pharmacologic agents can depress upper airway neuromuscular activity and predispose to upper airway obstruction and obstructive sleep apnea. In general, monitoring should be instituted to detect airway obstruction arising from CNS depressants such as alcohol, benzodiazepines, opioids, and general anesthetics, which block compensatory neural responses in several ways. First, alcohol ingestion has been shown to decrease genioglossal muscle activity [291–293], thereby blunting reflex recruitment of the upper airway musculature and predisposing to worsening obstruction. Second, arousal


335 Oropharynx

−5

−5

−10

−10

−15 −20

** Control

Mandibular advancement

es e No n ob es e

Ob

0

Ob

es e No n ob es e

Ob

es e No n ob es e

0

es No e n ob es e

5 Ob

Estimated closing pressure (cmH2O)

Velopharynx 5

**

−15 −20

** Control

Mandibular advancement

Figure 10.13 Mandibular advancement results in minimal decreases in closing pressures in obese individuals relative to non-obese controls in the velopharynx. Modest decreases in closing pressures in the oropharynx are observed in obese individuals, but are of lesser magnitude than seen with non-obese subjects. **p 5 0.01 vs. control. Values are means SE (Data from Ref. 187).

responses to airway occlusion are known to be prolonged in normal sleeping subjects after alcohol ingestion [294]. Finally, the combined effects of CNS depressants on the upper airway musculature and arousal responses could account for observed increases in the frequency and duration of apneic episodes, and worsening of oxyhemoglobin desaturations after alcohol ingestion [295]. Of special note, sedation with midazolam has been associated with upper airway critical pressures equivalent to those during sleep in normal individuals [296,297]. In contrast, general anesthesia (isoflurane) has been associated with markedly elevated critical pressures approximating those found during complete neuromuscular blockade [167,203,298], suggesting that general anesthesia is associated with a nearly complete loss of protective upper airway reflex mechanisms. To avert complications arising from upper airway obstruction postoperatively, oximetry should be monitored, postural maneuvers (semi-recumbent posture) and nasal CPAP should be implemented, and doses of CNS depressants should be minimized.

V.

Summary and Conclusions

In conclusion, obstructive sleep apnea is a common disorder of Western society, particularly in overweight men and postmenopausal women.

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By modeling the upper airway as a simple collapsible conduit, investigators have determined that the severity of upper airway obstruction during sleep is related to quantitative differences in critical pressure, which reflect differences in the degree of upper airway collapsibility. Elevations in critical pressure appear to be due to a complex interaction of mechanical alterations and disturbances in upper airway neuromuscular control. In turn, disturbances in neuromuscular control and responses to upper airway obstruction lead to alterations in radial and axial traction on the pharyngeal airway, as well as variations in the neuromotor tone of the intrinsic muscles of specific pharyngeal segments. Understanding these pathophysiologic mechanisms can guide our approach to the treatment of obstructive sleep apnea. In the perioperative setting, awareness of the effects of anesthetic agents on the upper airway, the acute hemodynamic stresses produced by obstructive apneic episodes, and their associated desaturations and arousals make it particularly important to prevent and relieve airway obstruction when it occurs. References 1.

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11 High Altitude

FRANK L. POWELL

PHILIP E. BICKLER

University of California, San Diego, La Jolla, California

University of California, San Francisco, California

I.

Introduction

Control of breathing at high altitude has been studied intensely for over a century. Although there has been a variety of theories to explain the deleterious effects of high altitude, it is now clear that the primary problem is a decrease in the oxygen partial pressure. The decrease in inspired PO2 during ascent to high altitude is the most common form of environmental hypoxia in humans and terrestrial animals, and the reflex increase in ventilation from arterial hypoxemia is the body’s first line of defense against decreased O2 supply. Hence, understanding the control of breathing at altitude provides the basis for understanding the normal physiological response to hypoxia. We understand the basic elements of the response but many fundamental questions remain, and these are currently under investigation with modern experimental techniques. Although hypoxia is the primary physiological challenge at high altitude, it is important to emphasize that O2 is not the only factor that affects the control of breathing at altitude. Specifically, CO2 and pH are also extremely important because they are both determined by the level 357

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of ventilation, and because they stimulate ventilation. Another important factor in the control of breathing at high altitude is the duration of hypoxic exposure. Ventilatory acclimatization to high altitude provides a useful model to study the changes that can occur in control of breathing during chronic hypoxia, and can provide important insights into the control of breathing under pathological conditions such as heart and lung disease. Finally, there are pathological changes at high altitude and these may involve problems with control of breathing. This chapter reviews recent progress in our understanding of the control of breathing in hypoxia at high altitude, how this changes during prolonged exposure to altitude, and how it may be involved in pathological responses to altitude. II.

Ventilatory Response to High Altitude

Decreasing PiO2 at altitude results in decreased PaO2 , which stimulates arterial chemoreceptors. The ventilatory response to PaO2 is called the hypoxic ventilatory response, or HVR, and is the reflex response to stimulation of arterial chemoreceptors, including the carotid and aortic bodies. Normally, the HVR is not very large until PaO2 falls to a level at which O2-hemoglobin saturation starts decreasing significantly and, in fact, ventilation is a linear function of arterial O2 saturation down to 70–80% [1]. However, this linear relationship is a coincidence and not a mechanistic explanation because arterial chemoreceptors respond only to O2 partial pressure, and not O2 content or saturation [2]. This chapter does not review the mechanisms of chemoreception in the carotid body arterial chemoreceptors, which have been covered elsewhere ([2], and Nurse, Chapter 1, this volume). Arterial PCO2 is generally the most important regulated variable in the control of breathing under resting conditions in normoxia. Even under conditions of hypoxia, the effects of PaCO2 are important and will determine the magnitude of the HVR, as illustrated in Figure 11.1. The lower response curve shows a normal HVR with PaCO2 decreasing as a result of hypoxic ventilatory stimulation. Such a poikilocapnic HVR would occur during exposure to progressively higher altitudes. As PaO2 decreases below 60 mmHg, the arterial chemoreceptors are stimulated and cause a small increase in ventilation. This decreases PaCO2 and, thereby, ventilatory drive from central and arterial chemoreceptor stimulation. The net effect on ventilation is the result of stimulation by hypoxia and inhibition from decreased PaCO2 (i.e., 37 mmHg). As PaO2 decreases more, hypoxic ventilatory stimulation increases, and this causes further reductions in PaCO2 . Ventilation remains a compromise between hypoxic stimulation and hypocapnic inhibition on the non-isocapnic hypoxic ventilatory response.

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Figure 11.1 The hypoxic ventilatory response (HVR) under normal non-isocapnic (or poikilocapnic) conditions, with PaCO2 decreasing as ventilation is stimulated, and under isocapnic conditions at the normal or elevated PaCO2 levels. The synergistic interaction between CO2 and hypoxia as stimulants of the arterial chemoreceptors means that ventilation during hypoxic conditions at altitude is a compromise between hypoxic stimulation and hypocapnic inhibition.

To rigorously quantify the ventilatory response to PO2, one needs to maintain PaCO2 constant, for example by increasing inspired PCO2. This is shown on the isocapnic HVR in Figure 11.1; note that this is steeper than the poikilocapnic HVR. Increasing PaCO2 further above normal reveals a synergistic, or multiplicative, interaction between PaO2 and PaCO2. This multiplicative interaction can be explained by the effects of PaO2 and PaCO2 on the carotid body chemoreceptors, i.e., the afferent activity from the chemoreceptors reflects the multiplicative nature of these stimuli [2]. During longer exposures to hypoxia at high altitude, there are further changes in the ventilatory response to O2 and CO2. This results in ventilatory acclimatization to hypoxia (VAH), which includes both persistent hyperventilation when normoxia is restored, and an increase in the isocapnic HVR (Figure 11.2). The persistent hyperventilation in normoxia, and resulting decrease in PaCO2, is one of the most robust measures of VAH. This apparent increase in CO2 sensitivity can be viewed as a change in the arterial PCO2 set point with chronic hypoxia. The increase in O2 sensitivity has been more controversial, and is often confused by the blunted HVR observed in some high altitude natives, or in residents of high altitude for many years (see Hypoxic Desensitization

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PaO2 (Torr) Figure 11.2 Isocapnic HVR in rats under control, normoxic conditions (squares) and after acclimatization to hypoxia (simulating 6,000 m). Ventilatory acclimatization includes (1) a persistent hyperventilation when normoxia is restored, and (2) an increase in the isocapnic HVR. PaCO2 was maintained at the level measured during 30% O2 breathing, which removed any O2-sensitive stimulation of arterial chemoreceptors, and was lower after acclimatization (32 Torr vs. 37 Torr before), reflecting a change in the arterial PCO2 set-point after chronic hypoxia (Data from Ref. 51).

below). However, it is now clear that the isocapnic HVR is increased by chronic hypoxia in humans and animals. A recent review of the literature found that conflicting reports could be explained by (a) complications from different time domains of the HVR that may arise with different protocols; (b) indices used to quantify the HVR, and (c) the effects of CO2 on the HVR [3]. All studies of the isocapnic HVR in humans after acclimatization to hypoxia have found significant increases in the HVR. The exact choice of PaCO2 for the isocapnic measurements did not appear to be critical. Hence, VAH in normal conscious humans includes increased O2 sensitivity and a change in the arterial PO2 set point. Next, we review the mechanisms of the HVR that are important during different times of exposure to hypoxia at altitude, and then the changes in ventilatory responses to CO2 during acclimatization. III.

Time Domains of the HVR

The HVR is not a single mechanism, but is a complex interplay between several distinct mechanisms operating in different time domains [4]. Among

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the features distinguishing these different mechanisms are: (a) the specific stimuli that elicit them (e.g., pattern and intensity of hypoxic exposure); (b) their time course (seconds to years); (c) the nature of the reflex response (tidal volume vs. frequency); (d) whether the response is excitatory or inhibitory to ventilation, and (e) their neurochemical basis. Some mechanisms are sufficiently long lasting to affect future ventilatory responses to hypoxia, indicating a degree of memory, or functional plasticity in the ventilatory control system. All of these mechanisms can interact to affect the ventilatory response to hypoxia at altitude, and produce the timedependent changes of ventilatory acclimatization described above. Many of the mechanisms have been discussed in more detail in other reviews [4–9], which are cited in place of original references when possible. A. Responses to Acute Hypoxia

Changes in ventilation (in both VT and fR) during or following a brief hypoxic exposure (2–5 min) are shown schematically in Figure 11.3. Although the primary stimulus to these changes is a decrease in PaO2

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HVD min

hrs - days

months - years

HYPOXIA

Figure 11.3 Time domains of the HVR. Top panel—Ventilation, tidal volume and respiratory frequency during acute hypoxia. The acute HVR (AR) is followed by short-term potentiation (STP) of VT and short-term depression (STD) of fR. STP and STD are manifested at the termination of acute hypoxia also. Bottom panel—Ventilation during chronic hypoxia shows hypoxic ventilatory decline (HVD) after the acute HVR, then an increase with ventilatory acclimatization to hypoxia (VAH). Chronic hypoxia for years to a lifetime leads to hypoxic desensitization (HD), although the exact duration necessary for this is not known.

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acting on the arterial (peripheral) chemoreceptors, similar responses with similar time courses can be elicited in anesthetized, paralyzed mammals by electrical stimulation of the carotid sinus nerve. Within this relatively short time domain, at least three distinct mechanisms have been identified: the Acute Response, Short-Term Potentiation and Short-Term Depression. Acute HVR

As discussed above, the acute HVR is the immediate augmentation of ventilatory activity at the onset of hypoxia (within one breath of PaO2 changing at the carotid bodies), and the decrease in ventilatory activity at the termination of hypoxia (Figure 11.3). The acute HVR represents the effects of changes in arterial (peripheral) chemoreceptor afferent input to glutamatergic synapses in the nucleus of the solitary tract, NTS (cf. [7,10]). This synaptic input is gated, such that the immediate response to changes in afferent input depends on the phase of the ongoing respiratory cycle [6]. This is the mechanism responsible for the rapid increase in ventilation during acute exposure to altitude. Short-Term Potentiation (STP)

STP represents a further, progressive increase in ventilatory activity after the acute HVR that proceeds with a time course of many seconds up to one minute [6]. STP is also manifested as a progressive post-stimulation decline in ventilatory activity after carotid sinus nerve stimulation, with a slightly longer time constant of 1–2 minutes (Figure 11.3). Although STP is generally thought to be a reflection of the same mechanism during the on and off responses, this hypothesis has not been rigorously tested. STP was formerly referred to as afterdischarge, based on Sherrington’s concept of a prolonged neural discharge triggered by a brief stimulation [6]. STP has been demonstrated following brief hypoxia in human subjects while awake [11], asleep [12] and during exercise [13]. STP, which increases ventilatory drive and changes the normal level for arterial PCO2, has not been studied after acclimatization to hypoxia (see below). STP does not appear specific to carotid body afferent inputs since other respiratory stimuli elicit a similar phenomenon [6]. Several neurotransmitters and neuromodulators (e.g., serotonin, catecholamines, opiates) have been ruled out as candidates for mediating STP in mammals [6]. Possible explanations for STP include: (a) presynaptic calcium accumulation along premotor pathways, thus mediating enhanced transmitter release when action potentials subsequently reach the terminal [14], or (b) release of modulatory neuropeptides, such as substance P, at key locations in the respiratory neural control system.

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STP has been suggested to play a smoothing role in the control of breathing, preventing reflex activation of the respiratory system from proceeding too rapidly and, thus, imparting system stability [6]. Short-Term Depression (STD)

STD is manifested as the recovery of the transient overshoot in respiratory frequency at the onset of carotid chemoreceptor stimulation, or a transient undershoot in frequency at the termination of stimulation, that lasts many seconds to a few minutes (Figure 11.3). To date, STD has been demonstrated only in the fR response of phrenic nerve activity in anesthetized rats during or following hypoxia or carotid sinus nerve stimulation [15]. Although STD is similar in some respects to hypoxic ventilatory decline (HVD, see below), important differences lead us to classify this time-dependent response as a unique mechanism or class of mechanisms at this time. There are differences in the ventilatory pattern (fR decreases in STD vs. VT in HVD), time course (many seconds to a few minutes with STD vs. many minutes with HVD), and in the neurochemical mechanisms thought to be involved in STD and HVD. It is possible that STD is unique to anesthetized rats, since it is not observed in anesthetized cats following carotid sinus nerve stimulation (cf. [6]) nor in other mammalian species investigated (e.g., goats, [16]). B. Responses to Sustained Hypoxia

Additional mechanisms become apparent when the hypoxic exposure is continuous for a prolonged period of several minutes to months. Among these mechanisms are: Hypoxic Ventilatory Decline, Ventilatory Acclimatization and Deacclimatization to Hypoxia during chronic exposures, and Hypoxic Desensitization with life-long hypoxia (Figure 11.3). Hypoxic Ventilatory Decline (HVD)

HVD is the roll off, or decrease in ventilation relative to the acute HVR, when moderate hypoxemia is sustained for 5 to 30 minutes in adult animals (Figure 11.3) [5]. HVD is distinct from the secondary decrease in ventilation from hypocapnia accompanying the acute response because it occurs during isocapnic hypoxia. As defined here, HVD differs from the biphasic HVR observed in neonates and small rodents, which represents an appropriate decrease in ventilation for the decrease in metabolic rate that occurs with hypoxia in small animals. The onset and resolution of HVD have a similar time course. Once HVD is established, the ventilatory response to subsequent hypoxic challenges is depressed for up to 60 min after normoxia is restored, but this can be shortened by breathing O2 enriched gas mixtures [17].

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HVD has been observed in awake humans, and in awake and anesthetized cats, but it could not be demonstrated in awake dogs [5] and may, or may not, be observed in awake rats [3]. It primarily affects VT, but not rhythm generation, and differs from short-term depression (STD, see above), which decreases fR. The mechanism of HVD is unknown but experiments on different species and preparations suggest specific effects of hypoxia on both (a) the sensitivity of ventilation to O2 and (b) central ventilatory drive independent of any changes in O2 sensitivity. Experiments from several studies in awake humans indicate that O2 sensitivity decreases during HVD, and it has been hypothesized that HVD is a specific effect of hypoxia on arterial chemoreceptors [18]. Some of the strongest support for this theory comes from experiments comparing the ventilatory response with brief stimulation of arterial chemoreceptors by hypoxia vs. hypercapnia during sustained hypoxia [19]. However, it is difficult to quantify the carotid body contribution to the ventilatory response to CO2 in awake humans. Other studies of awake humans have found ventilatory decline during sustained hypoxia without a significant decrease in O2 sensitivity [20,21]. This is consistent with experiments on anesthetized animals which show that the ventilatory response to arterial chemoreceptor or carotid sinus nerve stimulation, (i.e., the gain or slope of the HVR) does not decrease during several minutes of inspired hypoxia or low levels of carbon monoxide inhalation [17]. It is also consistent with neural recordings from carotid body afferents in animals, which, with the possible exception of the rabbit, show no change in chemoreceptor activity over the time course of HVD [5]. These data suggest that HVD is a decline in central ventilatory drive independent of changes in O2 sensitivity of arterial chemoreceptors or ventilation. The neurochemical basis of HVD is not clear. There is experimental evidence implicating ventilatory inhibition by adenosine, GABA, and opioids, but none of these neuromodulators can completely explain HVD [5]. A dopaminergic mechanism may be involved in HVD in some species since haloperidol, a D2 dopamine receptor antagonist, eliminates ventilatory roll-off in awake and anesthetized cats [5], but not in awake humans [22]. D2 receptors in the carotid body are not involved in the decrease in chemoreceptor O2 sensitivity during HVD in humans [5]. One mechanism that is apparently not involved is insufficient energy substrate [17]. Acid-base shifts in the brain at the site of the chemoreceptors have been proposed to contribute to HVD, although evidence for this is not clear. With the acute hyperventilation induced by hypoxia, the fall in tissue PCO2 at the central chemoreceptors should lower the ventilatory stimulus. However, at least in the case when CO2 at the peripheral chemoreceptors remains isocapnic, HVD is still observed [23]. Another test of this concept could be made with the administration of a carbonic anhydrase inhibitor

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such as acetazolamide (DiamoxÕ ), which would eliminate the brain tissue alkalinity resulting from hyperventilation [24], and possibly eliminate the HVD. A role for lactic acid accumulation in the mechanism for HVD has also been suggested. Hypocapnia and hypoxia are synergistic in increasing lactic acid production in the brain [25]. Somewhat surprisingly, lactic acid production in brain cells may depress ventilation, even though acidity generally stimulates it (reviewed by Smith et al. in Ref. [23]). Cells in the ventral medulla chemosensitive zones produce an excess of lactic acid during hypoxia [26,27]. Intracellular acidification may lead to depression of neuronal excitability by hyperpolarizing the membrane potential (by altering the transcellular Hþ gradient, or by activation of acid-sensitive potassium channels) or by reducing neurotransmitter release via reductions in intracellular [Ca2þ]i. This effect decreases respiratory motor output, explaining the decrease in ventilation when compared with that predicted. Testing this hypothesis will require improved identification of chemosensitive respiratory neurons and methods to correlate pHi, [Ca2þ]i and neuronal output. A potential mechanism for the increase in ventilation during altitude acclimatization (described next) is a decrease in HVD with chronic hypoxia. However, HVD has been shown to persist during prolonged exposure to altitude [1]. Ventilatory Acclimatization to Hypoxia (VAH)

VAH is defined as the time-dependent increase in ventilation that occurs with chronic hypoxic exposures of several hours to months (Figure 11.3) [5,8]. Ventilatory acclimatization to high altitude is the classic example of VAH, and its physiological significance, in terms of increasing O2 delivery, is well known [28]. The time course of VAH is species-dependent, and can be complete after only 4 to 6 hrs in goats (in terms of no further changes in ventilation or PaCO2 relative to seven days of hypoxia), or may require more than 10 days in humans [5]. The rat has a time course similar to that in humans, and has been shown to be a good model for human VAH when corrections are made for changes in metabolic rate occurring during the first days of hypoxia [5]. Experiments on VAH usually study the effects of chronic hypoxia and hypocapnia, both of which occur in healthy subjects exposed to hypoxia. The effects of changing CO2 sensitivity are discussed separately below. However, recent experiments show that the isocapnic HVR also increases in humans and animals [3]. Changes in HVR with VAH

Changes in ventilatory and carotid body O2 sensitivity are now generally accepted mechanisms contributing to the increased HVR with VAH. This is

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consistent with the lack of VAH in carotid body-denervated animals, and the increase in the isocapnic HVR during VAH in awake animals and humans [3,5,29]. Animal studies show increased O2 sensitivity of carotid body chemoreceptors, although there are significant species differences. In goats, carotid body chemoreceptor afferent activity increases during 6 hrs of hypoxia, similar to the time course required for complete VAH in this species [5]. In cats, carotid body chemoreceptors increase O2 sensitivity after 48 hrs of hypoxia [30], although the situation after 2 wks of hypoxia is unclear (see Hypoxic Desensitization below). The increase in carotid body chemoreceptor O2 sensitivity can completely explain VAH in goats after 6 hrs and cats after 48 hrs of hypoxia. However, it is important to understand that complete VAH after only hours does not preclude different mechanisms turning off and on during longer time domains. Another mechanism that can increase the HVR with VAH is altered CNS processing of arterial chemoreceptor afferent input. The first evidence for this came from human studies showing an increase in the ventilatory response to arterial chemoreceptor stimulation with doxapram after acclimatization to altitude [31]. If chronic hypoxia does not change the effect of this drug on chemoreceptors, then the change with acclimatization could be explained by an increase in the CNS gain of the HVR that translates chemoreceptor afferent input into ventilatory output. The ventilatory response to arterial chemoreceptor stimulation with doxapram or NaCN also increases significantly after exposure to two or more days of hypoxia in rats and ponies (reviewed by Powell, Huey, and Dwinell [32]). However, the ventilatory response to NaCN is not increased significantly after four hours of hypoxia in goats, so increases in the CNS gain of the HVR may require exposure to hypoxia lasting days or more. The most conclusive evidence for chronic hypoxia increasing the CNS gain of the HVR comes from experiments on anesthetized rats [33]. These experiments produced graded and reproducible changes in arterial chemoreceptor afferent input by electrically stimulating the carotid sinus nerve at different frequencies and measuring ventilatory motor output as integrated phrenic nerve activity. After seven days of hypoxia, the CNS gain of the HVR increased significantly, primarily by increasing the ventilatory frequency response to chemoreceptor stimulation. It was hypothesized that the increased CNS gain of the HVR takes more than two days of hypoxic exposure, which is consistent with it not being observed in the shorter studies (see above). Dopamine (DA) was suggested as a likely candidate for increasing the CNS gain of the HVR based on a significant correlation between increases in ventilation and tyrosine hydroxylase (the rate-limiting enzyme for DA synthesis) in respiratory centers of the brain after chronic hypoxia [34]. Further, DA acting on D2 receptors (D2-R) in the brain increases the CNS gain of the HVR [35]. Pharmacological and transgenic studies in mice

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support an increased excitatory effect of DA acting at D2-receptors (D2-R) in the CNS contributing to ventilatory acclimatization to hypoxia. Acclimatized mice with the D2-R gene ‘knocked out’ [36] or D2-R blocked with a systemic antagonist [37] do not show a normal time-dependent increase in hypoxic ventilation, and the independent effects of D2-R in the carotid body cannot explain these results [35]. However, D2-R in the CNS cannot explain the increased CNS gain of the HVR in acclimatized rats [38,39]. In rats, at least, it appears that changes in dopaminergic modulation of the HVR function to optimize CNS sensitivity to afferent input from arterial chemoreceptors, as well as to O2 sensitivity of the carotid body [35]. Glutamate and GABA are also involved in the HVR but they have not been investigated after chronic hypoxia [32]. Nitric oxide (NO) exerts positive feedback on glutamate release in the respiratory centers where the arterial chemoreceptors synapse, and could be involved in increasing the CNS gain of the HVR also [32]. Serotonin is important for long-term facilitation of ventilation following repeated bouts of hypoxia, but this mechanism cannot explain ventilatory acclimatization to sustained hypoxia [7]. In summary, the neurochemical basis for the increased CNS gain of the HVR with ventilatory acclimatization to hypoxia remains unknown. Changes in ventilatory drive and CO2 sensitivity with VAH

From the early 1960s, acid-base changes associated with the hyperventilation accompanying acclimatization to high altitude have been proposed to contribute to the pattern of ventilatory changes. Mitchell and Loeschke [40] had recently identified pH-sensitive areas on the ventral surface of the medulla that stimulated ventilation. The group of Severinghaus and Mitchell [41] proposed that the acute respiratory alkalosis from the acute HVR restrained the increase in alveolar ventilation because of the alkalinity in the cerebrospinal fluid (CSF) bathing these central chemoreceptors. During acclimatization, compensatory responses were proposed to lower the pH in the CSF (for example, by active transport of HCO 3 out of CSF) and to facilitate a further increase in ventilation. The shifts in CSF acid-base balance could persist for a time after return to low elevation (or during administration of supplemental oxygen at high altitude), explaining the Houston-Riley enigma [42] of continued hyperventilation after return to sea level (see Ventilatory Deacclimatization to Hypoxia below). However, subsequent studies failed to confirm that CSF pH returned to normal with time at altitude [43,44]; in fact, the CSF pH most likely remains increased for a long period. A problem in providing evidence that pH or bicarbonate compensation in the CSF contributes to VAH could be that very small changes in CSF pH have large effects on ventilation. For example, the pH changes in CSF required to produce a 1L/min change in ventilation (sufficient to decrease PaCO2 2–3 mmHg) is only ca. 0.001 pH units, below ready detection.

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Further, decreases in CSF bicarbonate change the relationship between pH and PCO2 making changes in the PCO2/pH relationship even steeper. In their study of CSF pH and respiratory sensitivity to hypercapnia during altitude acclimatization, Crawford and Severinghaus [44] found that only a 0.003 difference of CSF pH (based on the slope of the hypercapnic ventilatory response) could explain the persistent hyperventilation during normoxia after three days at altitude. Another complication for testing this hypothesis is the fact that the chemosensory cells responding to increases in CO2 may be few in number [20] and distributed throughout the brain stem. Nattie [45] reviews data showing that at least five distinct, non-medullary, chemoreceptor areas respond to microdialysis with CO2 or micro injections of acetazolamide by increasing respiratory motor neuron output in awake or anesthetized animals. Also, the pH measured by central chemoreceptors remains controversial [46]. Hence, it has not been possible to demonstrate conclusively that a change in stimulus level of central chemoreceptors contributes to an increase in ventilation with VAH. However, the increased ventilatory drive observed during VAH behaves as if the set point for central CO2 receptors is decreased. Ventilatory Deacclimatization from Hypoxia (VDH)

When normoxia is acutely restored during, or after, chronic hypoxia, ventilation and ventilatory O2 sensitivity do not immediately return to control levels [5]. This persistent hyperventilation in normoxia after chronic hypoxia is termed VDH, and it decays with a time course similar to the time-dependent increase in ventilation with VAH. Given the similar time courses for VAH and VDH, it was generally thought that they were the same mechanism being turned on and off, respectively. However, recent studies have shown that VDH and VAH can be dissociated, and are probably separate mechanisms. For example, VDH does not occur after VAH in goats exposed to isocapnic hypoxia for 4 to 6 hrs, but it does occur when hypoxic exposure is accompanied by hypocapnia [5]. Differences in VDH with CO2 levels support the idea that changes in CO2-sensitive mechanisms explain persistent hyperventilation in normoxia after acclimatization, as discussed above. Increased O2 sensitivity of arterial chemoreceptors, or the CNS gain of the HVR, would not be expected to increase ventilation in normoxia when arterial chemoreceptor afferent activity is minimal. However, a recent study found that hypocapnia is not necessary for VDH in humans [29]. The authors postulated hyperventilation-induced hyperpnea and potential central effects of hypoxia might explain persistent hyperventilation when normoxia is restored after chronic hypoxia.

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Hypoxic Desensitization (HD)

When humans experience chronic hypoxia for years or a lifetime, the HVR becomes blunted. Ventilation in hypoxia is decreased relative to normal subjects acclimatized to altitude for shorter periods of time (Figure 11.3) and ventilatory sensitivity to PaO2 is decreased [8]. This may conserve energy, through reduced work of breathing, when other non-ventilatory modes of acclimatization (e.g., metabolic, vascular, hematological) have had time to occur [8,28]. HD is an acquired characteristic that increases with the level of altitude and time at altitude, but there is disagreement about its reversibility [8]. An interesting exception to the correlation between high altitude exposure and HD is provided by adult Tibetans residing at 3658 m since birth. These high-altitude natives do not have a blunted HVR, in contrast to Chinese born near sea level but who lived at altitude for years. However, Tibetan natives from 4400 m do show HD [47]. This suggests that both genetic and environmental factors contribute to HD. The effects of genetics and potential evolutionary adaptations to hypoxia at altitude in humans have been reviewed in a special issue of the journal High Altitude Medicine and Biology [48–50]. One of the difficulties in studying the physiological mechanisms of HD is the lack of suitable animal models. Cats show HD after two weeks at simulated altitudes of 5500 m, but it reverses quickly with return to normoxia, in contrast to humans [8]. Reports of a blunted HVR in chronically hypoxic rats can be explained by the effects of hypocapnia or anesthesia on an HVR that is actually increased by chronic hypoxia [51]. The profound changes in carotid body structure that occur with chronic hypoxia might be expected to alter chemoreceptor O2 sensitivity, but many of these morphological changes occur before HD [5], so their significance in HD is not clear. Increased dopaminergic inhibition at the carotid body may be involved [5]. Changes in both carotid body chemoreceptor O2 sensitivity and CNS gain of the HVR are reported to contribute to HD in cats [52], but these results are difficult to reconcile with another study which shows increased carotid body chemoreceptor O2 sensitivity in cats after the same period of acclimatization [53]. C. Intermittent Hypoxia

The physiological responses to intermittent hypoxia (IH) are clearly not the same as those to continuous hypoxia, although the effects of the pattern and dose of hypoxic exposure are not yet understood. Much of the basic research in this area has been motivated by the problem of sleep apnea at sea level, although there have been several studies of IH for physical training to improve athletic performance at low altitudes, high-altitude mountaineering, commuting to work at high altitude, and even comparative physiological models in nature (reviewed by Garcia and Powell, Ref. [54]).

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The problem of sleep apnea at altitude is considered separately, because here we review the changes in ventilatory control with IH and high-altitude mountaineering or work. IH has been an integral part of high-altitude mountaineering for years. Climbers ascend from low-altitude base camps to higher altitudes where they establish camps to stage for their summit attempts. Typically they return to base camp for more supplies and sleep. Over the days and weeks that are necessary to climb major peaks, this pattern of up and down results in IH exposures that are typically increasing in the degree of hypoxia. Although this pattern probably originated out of logistical necessity, it has been obvious for many years that climbers should sleep at as low an altitude as possible, while still obtaining adequate acclimatization and meeting the climbing schedule for a successful summit bid. More recently, IH has been viewed as a way to potentially speed up or improve acclimatization. The tragic events on Mount Everest in 1996 focused attention on IH as a means to gain rapid acclimatization. Traditionally, acclimatization in the Himalayas is achieved by slowly ascending to altitude during a long trek. In contrast, modern guided trips taking paying clients to the highest summit on earth have a strong profit motive to minimize the total time for an expedition. Of course, minimizing exposure to hypoxia is a reasonable goal but acclimatization cannot occur without some hypoxia. Several groups have studied the applied physiology of IH as a means of pre-acclimatization to improve climbing performance at high altitude. One of the earliest studies hypothesized that IH could produce acclimatization more efficiently than continuous chronic hypoxia (CH) [55]. To mimic the usual climbing pattern of mountaineers during an expedition, they exposed 12 subjects to simulated altitude in a hypobaric chamber. After three consecutive days simulating 6000 m for 5 hr and 8000 m for the next one hour, they observed an increase in VE and PaO2 in hypoxia, indicating the initiation of ventilatory acclimatization. Subsequent studies exposed resting humans to IH simulating altitudes above 5000 m as a pre-acclimatization training method for mountaineering expeditions [20,45,56–58]. Others used less severe hypoxic stimuli (simulated altitudes of 2500 to 5000 m) but added exercise to the IH and also measured the effects of such IH on the hypoxic ventilatory response (HVR) [59–62]. In general these studies showed increases in the HVR and ventilation and arterial saturation (SaO2) during hypoxia. Hence, the studies show ventilatory acclimatization to IH is qualitatively similar to CH, although they did not specifically compare the two patterns of hypoxia. Also, some of the protocols actually combined stimulus patterns by administering CH before IH [56,57]. The opportunities for work at high altitude are increasing with the extensive development of commercial and scientific activities in high

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mountain ranges. Notable examples include mining in South America and high-altitude observatories for optical and radio telescopes, both involving altitudes of 3500 to 6000 m [63–66]. With these opportunities come large-scale challenges to public health and worker safety issues, as well as problems in maintaining worker productivity and morale. Not only are there physical effects on the employees from working at altitude, but the harsh environment precludes families from residing near the work place. Hence, workers commute between low-altitude homes and high-altitude work sites. In general, a commuting strategy is the same as the acclimatization strategy for high-altitude mountaineering, i.e., descending to low altitude when possible for rest and recovery. One goal in this strategy is to eliminate or minimize the loss of any acclimatization to hypoxia while normoxic (or less hypoxic). The benefits for different commuting schedules remain to be determined but are under active study. For example, a three-year prospective study was begun in February 1998 to characterize miners in North Chile working at 4300 to 4600 m altitude [67]. The literature oriented towards mountaineering leaves many unanswered questions about (a) the time course of acclimatization in IH; (b) the effects of different levels and duration of hypoxic exposure, i.e., the dose of IH; (c) the effects of exercise training responses to IH, and finally (d) differences in mechanisms of acclimatization to IH vs. continuous hypoxia. These issues have been addressed by measuring the time course of ventilatory and hematological changes in humans exposed to a protocol of moderate IH at rest [68,69]. The study was designed to give a lower ‘dose’ of hypoxia than previous IH studies by simulating only 3800 m altitude (PiO2 ¼ 90 Torr) for two hours per day, for 12 consecutive days, and exercise training was not part of the study [68]. The time course of change in the isocapnic HVR appeared quantitatively similar with IH and CH but on a compressed time scale with IH [69]. In IH, the isocapnic HVR (with end-tidal PCO2 held constant as inspired PO2 decreased) significantly increased to a maximum value at five days, although it subsequently decreased towards control levels by 12 days at the end of the protocol. In CH, there was a monotonic increase in isocapnic HVR during the first two weeks, similar to other studies [70]. Another difference between IH and CH was the lack of persistent hyperventilation in normoxia with IH. Despite the increase in the isocapnic HVR, ventilation and SaO2 were not significantly increased in hypoxic or normoxic conditions at any time during the IH protocol. We hypothesize that IH changed O2 sensitivity similarly to the change caused by continuous hypoxia, but it did not change normoxic ventilatory drive and arterial PCO2 set points like acclimatization to continuous hypoxia. More studies are needed comparing IH and CH to determine if fundamentally different mechanisms are involved.

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Increases in the Hypercapnic Ventilatory Response (HCVR) with Acclimatization

Some studies [70], but not all [71,72], have reported increases in the slope of hypercapnic ventilatory response (HCVR) in humans during altitude acclimatization. If one corrects for the log PCO2 relationship, the HCVR slope is not increased during acclimatization [70]. Increased HCVR slopes during acclimatization have been found in cats [30,73] and in goats [74]. As summarized by Weil [8], the mechanisms of changes in slope and intercept of the HCVR during altitude acclimatization are not entirely clear. As is the case with HVR, hyperoxia does not immediately reverse changes in the HCVR seen during acclimatization. Most of the work on the mechanisms of the increase in HCVR has focused on the carotid bodies. Acute hypoxia potentiates the carotid body neural output response to hypercapnia [75], but it is not clear whether chronic hypoxia causes further increases or not [53]. As is the case with the HVR, it remains possible that increases in central chemoreceptor sensitivity to CO2 contribute to augmented HCVR with chronic hypoxia. One possible carotid body mechanism that could increase the HCVR during chronic hypoxia is modulation of dopaminergic neurotransmission. Tatsumi, Pickett and Weil [73] found that in cats, peripheral dopaminergic blockade with domperidone increased responses to both hypoxia and hypercapnia, suggesting that decreased dopaminergic inhibition in the carotid body could explain the increase in chemosensory response in acclimatization. In these same cats, following acclimatization, a substantially diminished response to dopaminergic blockade suggests that lessening of dopaminergic activity is associated with ventilatory acclimatization. V.

High Altitude Diseases and Ventilatory Control

A. Acute Mountain Sickness (AMS)

Although AMS is more correlated with elevated heart rate than with declines in O2-hemoglobin saturation [76], it may be related to incomplete ventilatory acclimatization to altitude, since the symptoms disappear as ventilation increases (reviewed by Smith, Dempsey and Hornbein, Ref. [23]). Accordingly, it is rational to expect that the symptoms of AMS would be improved by drugs that increase ventilation or improve tissue oxygenation. Acetazolamide, a carbonic anhydrase inhibitor, improves AMS symptoms (headache, sleep disturbance, loss of appetite, malaise). Studies reporting an improvement in sleep quality and AMS symptoms with carbonic anhydrase inhibition have recently been reviewed [77]. Although it only minimally increases the HVR and HCVR [78], acetazolamide typically has its most pronounced effects in reducing periodic breathing and episodes

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of desaturation during sleep [79]. However, a recent study [80] found that arterial saturation in subjects was increased after three weeks of treatment with acetazolamide, implying an effect on ventilatory acclimatization over the longer term. The dose of acetazolamide required for benefit is 750 mg/day; 500 mg/day was not found to be effective in a 33-trial metaanalysis of drugs used for treatment of acute mountain sickness [81]. Overall, the effectiveness of acetazolamide in the treatment of the symptoms of acute mountain sickness is probably similar to that of the steroid dexamethasone [80]. The mechanism of acetazolamide’s benefit in the symptoms of acute mountain sickness may relate to improved cerebral blood flow and oxygenation that results from the carbonic acidosis it produces in brain extracellular fluids [24,81]. Almitrine bismesylate, a respiratory stimulant acting at the carotid bodies, has been investigated for its potential in speeding the process of altitude acclimatization. Although it markedly enhances ventilation and the HVR, it does not reduce periodic breathing at altitude [78]. This is very interesting because it has been proposed that an imbalance between peripheral chemoreceptor input and central drive is the basis for periodic breathing (reviewed by Weil and White, Ref. [82]). Furthermore, even though almitrine may have beneficial effects on oxygenation during wakefulness, side effects of weight loss and peripheral neuropathies may explain the lack of recent enthusiasm for the compound and its limitation for use in the treatment of chronic hypoxia [83]. Doxapram is another respiratory stimulant acting on the carotid bodies, but no information is available on the use of this drug in facilitating ventilatory acclimatization to altitude. Theophylline, a respiratory stimulant and bronchodilator, has been proposed to facilitate the respiratory acclimatization to altitude. Theophylline improves the symptoms of acute mountain sickness [76], an effect perhaps related to respiratory stimulation. Oxygenation was improved in the group receiving theophylline, but whether the effect was due to an increase in HVR or to a general increase in respiration was not delineated. Given the importance of respiratory control during sleep to the symptoms of AMS, it would be of interest to know if theophylline decreases periodic breathing or desaturations. Several studies [84,85] suggested that the extract from Ginkgo biloba might be useful for the prevention of acute mountain sickness. The mechanisms remain unknown; specifically, the respiratory effects of Gingko have not been studied. It would be interesting to determine whether the reported benefits of ginkgo correlate with enhanced blood oxygenation, ventilatory acclimatization, or improved cerebral blood flow and oxygenation. While short-acting sedative-hypnotics such as temazepam may have depressant effects on respiration at altitude [86], they may be beneficial overall because they reduce the predominant problem of periodic breathing during sleep at altitude [87]. The non-benzodiazepine sedative-hypnotic

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zolpidem has been shown to have similar benefits on sleep at altitude, without adversely influencing respiration [88]. Paradoxically, sedative drugs may improve overall gas exchange at high altitude, especially during sleep, because of their effect in reducing periodic breathing. This may result from altering the balance between peripheral and central chemoreceptor drives (see below). Several other compounds having sedative effects have been evaluated for their efficacy in treating acute mountain sickness-associated decrements in sleep quality. The mild sedative L-tryptophan does not influence acute ventilatory response at moderate altitudes [89], but its effect on AMS symptoms was not investigated. Nor is it is not known if periodic breathing or oxygen saturation are positively influenced by L-tryptophan. In contrast, alcohol, which in common with benzodiazepines produces its sedative and hypnotic effects via GABA receptors, inhibits the early stages of acute ventilatory adaptation to hypoxia at moderate altitude and is thus not recommended as a way of improving sleep quality [90]. Finally, some evidence suggests that progestational steroids such as medroxyprogesterone may reduce AMS symptoms and improve brain oxygenation [91]. B. Periodic Breathing (PB) During Sleep

Periodic breathing during sleep is a common occurrence during altitude acclimatization. It results from an imbalance in CO2- and O2-sensitive ventilatory drives. Instability arises from a low ventilatory drive during sleep and hypocapnia, with a simultaneously high ventilatory drive from hypoxia. There is a strong correlation between the HVR and PB in normal subjects at altitude [71], and the arterial chemoreceptor stimulant almitrine increases PB at altitude [78]. However, not all studies find an increase in PB with increased HVR (reviewed by Weil and White, Ref. [82]). Given the role of a high HVR in PB at altitude, it is interesting that PB decreases with acclimatization (reviewed by Weil and White, Ref. [82]) while the HVR increases (see above). However, this can be explained by the increased CO2-sensitive ventilatory drive and correction of the respiratory alkalosis during acclimatization. The increased CO2-sensitive ventilatory drive is in harmony with the elevated HVR and is able to maintain more regular breathing during sleep after acclimatization. This is similar to the ventilatory drive from wakefulness preventing PB at altitude. C. Chronic Mountain Sickness (CMS)

CMS is a poorly understood condition found in both high-altitude natives and in lowlanders living for long periods (many months to years) at high

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altitudes. Also called Monge’s disease (reviewed by Monge, Leon-Valarde and Arregui, Ref. [92]), it was first described in high-altitude residents in Bolivia and Peru. The incidence of CMS is greatest in lowlanders, and least in populations of humans genetically adapted to high-altitude life [93,94]. A primary feature of the pathophysiology of CMS appears to be a progressive loss of ventilatory drive in hypoxia, resulting in diminished ventilatory responsiveness to hypoxia, and worsening arterial oxygen saturation. Lahiri, Rozanov and Cherniak [95] reviewed the altered structure and function of the carotid bodies in chronic hypoxia. The other pathologic features of CMS very likely are caused by this progressive hypoxemia, including pulmonary hypertension, heart failure, and excessive erythrocytosis [96]. Viewed from this perspective, the disease represents a failure of respiratory control to deal with long-term environmental hypoxia, i.e., a failure of acclimatization. For unknown reasons, it affects certain individuals much more strongly than others. It would be of interest to know if individuals with pre-existing low hypoxic ventilatory responsiveness are at greater risk of developing CMS. Healthy long-term residents at high altitudes exhibit ventilation and HVR responses that are as great as recently acclimatized newcomers to altitude, indicating that the decline in ventilation in these individuals is indeed pathological, and that certain populations may be genetically advantaged with respect to maintaining ventilation during years of hypoxemia [94]. Further evidence for a fault in ventilatory drive is the correlation between the incidence of CMS and the decrease in ventilation observed with aging [92]. One prominent feature of CMS is dramatically lower arterial oxygen saturation during sleep when compared with healthy highaltitude residents. CMS patients spend a greater time in sleep-disordered breathing, with correspondingly greater periods spent desaturated, without compensatory increases in cerebral blood flow [97], indicating decreased brain oxygen delivery during a considerable portion of the night. The causes of the progressive decline in hypoxic ventilatory responses in CMS patients are not known, but the carotid bodies of patients with CMS are enlarged and structurally different than those of sea-level individuals [95,98]. There is no apparent role for endorphin-induced ventilatory decline in CMS, since naloxone does not alter the depressed ventilation of individuals with the condition [99]. There may be a role for respiratory stimulants such as medroxyprogesterone or almitrine in the hypoventilation associated with chronic mountain sickness. Medroxyprogesterone, which is effective in ameliorating both the excessive polycythemia and impaired oxygenation during sleep, may be of significant benefit. At least one study has suggested similar benefits for almitrine [100]. In general there is very little information available on the effective treatment of CMS.

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HAPE is a pathological response to hypoxia that is exacerbated by a low HVR and brisk hypoxic pulmonary vasoconstrictor response (HPVR) [101]. Susceptibility to HAPE is correlated with a low HVR in several studies [102,103], but it is important to note that not everyone with a low HVR develops HAPE. A low HVR will increase the stimulus for HAPE because alveolar PO2 will be lower for any given PiO2. Further, carotid body stimulation can reduce the HPVR so a weak HVR may lead to a stronger HPVR and increase the possibility of HAPE (101). However, these are only correlations and experiments have not established a mechanistic relationship between low ventilatory sensitivity and HAPE. References 1.

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Bashir, Y., Kann, M. and Stradling, J.R., The effect of acetazolamide on hypercapnic and eucapnic/poikilocapnic hypoxic ventilatory responses in normal subjects, Pulm. Pharmacol. 3, 151–154, 1990. Hackett, P.H., Roach, R.C., Harrison, G.L., Schoene, R.B. and Mills, W.J., Jr., Respiratory stimulants and sleep periodic breathing at high altitude, Almitrine versus acetazolamide, Am. Rev. Resp. Dis. 135, 896–898, 1987. Ha, Z., Zhu, Y., Zhang, X., Cui, J., Zhang, S., Ma, Y., Wang, W. and Jian, X., The effect of rhodiola and acetazolamide on the sleep architecture and blood oxygen saturation in men living at high altitude, Zhonghua Jie He He Hu Xi Za Zhi 25, 527–530 (Chinese), 2002. Dumont, L., Mardirosoff, C. and Tramer, M.R., Efficacy and harm of pharmacological prevention of acute mountain sickness: quantitative systematic review, BMJ 321, 267–272, 2000. Bickler, P.E., Litt, L. and Severinghaus, J.W., Effects of acetazolamide on cerebrocortical NADH and blood volume, J. Appl. Physiol. 65, 428–433, 1988. Weil, J.V. and White, D.P., Sleep, in High Altitude: An Exploration of Human Adaptation, Hornbein, T.F. and Schoene, R.B., eds., New York, Marcel Dekker, pp. 707–730, 2001. Watanabe, S., Kanner, R.E., Cutillo, A.G., Menlove, R.L., Bachand, R.T., Jr., Szalkowski, M.B. and Renzetti, A.D., Long-term effect of almitrine bismesylate in patients with hypoxemic chronic obstructive pulmonary disease, Am. Rev. Respir. Dis. 140, 1269–1273, 1989. Gertsch, J.H., Seto, T.B., Mor, J. and Onopa, J., Ginkgo biloba for the prevention of severe acute mountain sickness (AMS) starting one day before rapid ascent, High Alt. Med. Biol. 3, 29–37, 2002. Roncin, J.P., Schwartz, F. and D’Arbigny, P., EGb761 in control of acute mountain sickness and vascular reactivity to cold exposure, Aviat. Space Environ. Med. 67, 445–452, 1996. Roggla, G., Moser, B. and Roggla, M., Effects of temazepam on ventilatory response at moderate altitude (Letter to editor), BMJ 320, 56, 2000. Dubowitz, G., Effect of temazepam on oxygen saturation and sleep quality at high altitude: randomized placebo controlled crossover trial, BMJ 315, 587–589, 1998. Beaumont, M., Goldenberg, F., Lejeune, D., Marotte, H., Harf, A. and Lofaso, F., Effect of zolpidem on sleep and ventilatory patterns at simulated altitude of 4,000 meters, Am. J. Resp. Crit. Care Med. 153, 1864–1869, 1996. Roggla, G., Moser, B., Wagner, A. and Roggla, M., L-Tryptophan does not influence acute ventilatory response at moderate altitude, Wein Klin. Wochenschr. 112, 634–636, 2000. Roeggla, G., Roeggla, H., Roeggla, M., Binder, M. and Laggner, A.N., Effect of alcohol on acute ventilatory adaptation to mild hypoxia at moderate altitude, Ann. Intern. Med. 122, 925–927, 1995. Imray, C.H., Barnett, N.J., Walsh, S., Clarke, T., Morgan, J., Hale, D., Hoar, H., Mole, D., Chesner, I. and Wrights, A.D., Near-infrared spectroscopy in the assessment of cerebral oxygenation at high altitude, Wilderness Environ. Med. 9, 198–203, 1998.

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Monge, C.C., Leon-Valarde, F. and Arregui, A., Chronic mountain sickness in Andeans, in High Altitude: An Exploration of Human Adaptation, Hornbein, T.F. and Schoene, R.B., eds., New York, Marcel Dekker, pp. 815–838, 2001. Moore, L.G., Comparative human ventilatory adaptation to high altitude, Respir. Physiol. 121, 257–276, 2000. Curran, L.S., Zhuang, J. and Sun, S.F., Ventilation and hypoxic ventilatory responsiveness in Chinese-Tibetan residents at 3,658 m, J. Appl. Physiol. 83, 2098–2104, 1997. Lahiri, S., Rozanov, C. and Cherniak, N.S., Altered structure and function of the carotid body at high altitude and associated chemoreflexes, High Alt. Med. Biol. 1, 63–74, 2000. Ge, R.L. and Helun, G., Current concept of chronic mountain sickness: pulmonary hypertension-related high-altitude heart disease, Wilderness Environ. Med. 12, 190–194, 2001. Sun, S., Oliver-Pickett, C., Ping, Y., Micco, A.J., Droma, T., Zamudio, S., Zhuang, J., Huang, S.Y., McCulbugh, R.G., Cymerman, A. and Moore, L.G., Breathing and brain blood flow during sleep in patients with chronic mountain sickness, J. Appl. Physiol. 81, 611–618, 1996. Heath, D., The morbid anatomy of high altitude, Postgrad. Med. J. 55, 502–511, 1979. Sun, S.F., Huang, S.Y., Zhang, J.G., Droma, T.S., Banden, G., McCullough, R.E., Cymerman, A., Reeves, J.T. and Moore, L.G., Decreased ventilation and hypoxic ventilatory responsiveness are not reversed by naloxone in Lhasa residents with chronic mountain sickness, Am. Rev. Respir. Dis. 141, 1294–1300, 1990. Kryger, M., Glas, R., Jackson, D., McCullough, R.E., Scoggin, C., Grover, R.F. and Weil, J.V., Impaired oxygenation during sleep in excessive polycythemia of high altitude: improvement with respiratory stimulation, Sleep 1, 3–17, 1978. Schoene, R.B., Hultgren, H. and Swensen, E.R., High-altitude pulmonary edema, in High Altitude: An Exploration of Human Adaptation, Hornbein, T.F. and Schoene, R.B., eds., New York, Marcel Dekker, pp. 777–814, 2001. Selland, M.A., Stelzner, T.J., Stevens, T., Mazzeo, R.S., McCullough, R.E. and Reeves, J.T., Pulmonary function and hypoxic ventilatory response in subjects susceptible to high-altitude pulmonary edema, Chest 103, 111–116, 1993. Hackett, P.H., Roach, R.C., Schoene, R.B., Harrison, G.L. and Mills, W.J., Jr., Abnormal control of ventilation in high-altitude pulmonary edema, J. Appl. Physiol. 64, 1268–1272, 1988.

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12 Obesity and the Control of Breathing

KHALID F. ALMOOSA

SHAHROKH JAVAHERI

University of Cincinnati College of Medicine Cincinnati VA Medical Center Cincinnati, Ohio

University of Cincinnati College of Medicine Cincinnati VA Medical Center Cincinnati, Ohio and SleepCare Diagnostics Mason, Ohio

I.

Introduction

Obesity is a common and chronic medical problem in the world today [1,2]. The health significance of obesity lies in its detrimental effects on morbidity and mortality through its direct association with several disorders [3,4]. Obesity-related health care costs in the United States amount to approximately $68 billion per year, with an additional estimated $30 billion per year spent on weight-reduction programs [5]. Obesity affects various body systems in different ways. It has numerous effects on the respiratory system, including chest wall mechanics, gas exchange, cost of breathing, and ventilatory control during both wakefulness and sleep. In this chapter, we discuss the various pathophysiological effects of obesity on pulmonary function and ventilatory control, the associated disorders of ventilatory control such as obstructive sleep apnea and the obesity hypoventilation syndrome (OHS), and the effects of obesity treatment on obstructive sleep apnea and ventilatory control.

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Almoosa and Javaheri II.

Overview of Obesity

A. Definition and Epidemiology

Obesity is defined as the presence of excessive body fat that causes an increase in body mass. This is reflected by an increased body-mass index (weight in kilograms divided by the square of the height in meters, BMI) [6]. A normal BMI is between 18.5 and 24.9 kg/m2, while a BMI between 25 and 29.9 kg/m2 reflects overweight, BMI greater than 30 kg/m2 defines obesity, and a BMI over 40 kg/m2 defines extreme obesity. The incidence and prevalence of obesity are increasing worldwide, particularly in industrialized countries [1,2]. Furthermore, the increase in obesity is occurring in both sexes, in all races and ages, and at all educational levels. According to Mokdad et al. (Table 12.1), the prevalence of obesity (BMI 30 kg/m2) in the United States was about 21% in 2001, while the prevalence of extreme obesity (BMI 40 kg/m2) was 2.3% [2]. Extreme obesity is highest among black women, persons who have not completed high school, and short people [7]. The prevalence of childhood obesity has also increased to 10% among 2- to 5-year olds; 15% among 6- through 11-year olds, and 16% among 12- through 19-year olds [8]. This is particularly evident among minority groups. B. Etiology and Genetics of Obesity

While the cause of obesity is incompletely understood, both genetic and environmental factors play important roles in its development [9,10]. Evidence for genetic contributions to obesity comes from both animal models [11–13] and human studies. Evidence of genetic influences in humans include highly correlated BMIs among first-degree relatives; greater BMI similarity between monozygotic vs. dizygotic twins, and significant

Table 12.1 Definitions and Prevalence of Obesity (Data from Ref. 2). Body size Normal Overweight Class I obesity Class II obesity Class III (extreme) obesity Prevalence Obesity Extreme obesity

BMI (kg/m2) 18.5–24.9 25–29.9 30–34.9 35–39.9 40 21% 2.3%

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correlation of body habitus and BMI between biologic parents and adopted children even when their environments are different [14–16]. Although obesity is a complex polygenic disorder, making it difficult to study its basis, rare genetic mutations have been identified. However, more recently, mutations in melanocortin 4 receptors, which are receptors for the anorexigenic peptide a-melanocyte, have been reported to account for 6% of childhood obesity [17]. Environmental factors have a major impact on the risk of obesity development. Western society’s affluence, easy availability of food, and the rise of fast food culture have undoubtedly all contributed to the increase in obesity over the past several decades. In addition, decreased physical activity and reduced energy expenditure associated with a sedentary life style have contributed to the development of obesity [18,19]. C. Effects of Obesity on Health

Obesity increases the risk of development of many disorders, such as adultonset diabetes mellitus, hypertension, hyperlipidemia, gall bladder disease, some cancers, and heart disease [2,20–22]. Obese subjects have twice the risk of developing heart failure as their non-obese counterparts, with risk proportional to increasing BMI (Figure 12.1) [4]. The risk of death is increased throughout the range of moderate and severe obesity for both men and women in all age groups, with risk being particularly higher for blacks than for whites (Figure 12.2) [3]. This increased risk of all-cause mortality from obesity occurs mainly through its linkage with associated diseases, particularly cardiovascular disorders [23]. Metabolic syndrome, which is linked to obesity, has been recently defined by the National Institutes of Health [24] as a constellation of two clinical and three laboratory findings. These include: (1) abdominal obesity (measured in waist circumference 4102 cm for men and 488 cm for women); (2) blood pressure (130/85 mm Hg); (3) hypertriglyceridemia (150 mg/dl); (4) low HDL (540 mg/dl for men and 550 mg/dl for women), and (5) fasting glucose 110 mg/dl. The syndrome is also associated with insulin resistance and high C-reactive protein concentration (CRP). At presentation, metabolic syndrome is defined by the presence of three or more components noted above. Metabolic syndrome is also associated with endothelial dysfunction and increased risk of cardiovascular disorders. The age-adjusted U.S. prevalence of metabolic syndrome is about 24%. As a precursor of cardiovascular diseases, detection of metabolic syndrome and its treatment may prevent associated cardiovascular pathology. Effects of obesity are not only related to the excess body fat as a percentage of total fat, but also to the distribution of fat, with the

Almoosa and Javaheri A

Cumulative incidence of heart failure (%)

386 Women

25

20

15 Obese 10

Overweight Normal

5

0

0

3

6

9

12

15

18

21

Years No. AT RISK Normal Overweight Obese

1729 955 493

Cumulative incidence of heart failure (%)

1634 880 448

1568 815 409

1477 757 372

1227 634 296

295 248 104

Men

25

B

1688 929 477

Obese

20

Overweight

15 Normal 10

5

0

0

3

6

9

12

15

18

21

Years No. AT RISK Normal Overweight Obese

869 1378 457

822 1322 433

758 1254 403

690 1163 370

637 1071 342

512 871 276

105 171 51

Figure 12.1 The risk of heart failure in obesity in men and women (Data from Ref. 4).

android upper-body distribution carrying a greater risk than the gynecoid lower body allocation [25]. In addition, increased neck size with fat deposition within the upper airway contributes to the presence and degree of obstructive sleep apnea–hypopnea syndrome (OSAH) [26,27].

Obesity and Control of Breathing

387

3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6

Cardiovascular disease Cancer All other causes

< 18 18. .5 5 – 20 20 .5 .4 – 22 21 .0 .9 – 23 23 .5 .4 – 25 24 .0 .9 – 26 26. .5 4 – 28 27. .0 9 – 30 29. .0 9 – 32 31 .0 .9 –3 4. 9 ≥3 5. 0

Relative risk of death

Men

Body-mass index

2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 18

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