Hyperbaric Oxygenation: Mitochondrial Activity and Brain Physiological Functions 3031496809, 9783031496806

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Avraham Mayevsky

Hyperbaric Oxygenation Mitochondrial Activity and Brain Physiological Functions

Hyperbaric Oxygenation

Avraham Mayevsky

Hyperbaric Oxygenation Mitochondrial Activity and Brain Physiological Functions

Avraham Mayevsky Faculty of Life Sciences Bar-Ilan University Ramat-Gan, Israel

ISBN 978-3-031-49680-6    ISBN 978-3-031-49681-3 (eBook) https://doi.org/10.1007/978-3-031-49681-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Preface

This new book is the third one, in the set of books, summarizing my research in the field of hyperbaric oxygenation affecting brain functions and mitochondrial NADH redox state monitored in real time. At the end of 1940, I was born in Tel Aviv, Israel, about the same time that Britton Chance earned his Ph.D. and started his interest and research on mitochondrial biochemistry. Shortly after my arrival to Philadelphia in 1972 I start my postdoctorate training with Prof. Britton Chance at the University of Pennsylvania, Philadelphia, studying brain mitochondrial function in vivo using NADH fluorescence in real time. A few months later I studied the mitochondrial NADH in vivo and tried to test the effect of hyperbaric oxygenation on brain energy metabolism evaluated by monitoring the redox state of the mitochondria. I continued the studies, performed by Dana Jamieson and Britton Chance in the mid-1960s in Philadelphia. During the early period of my stay in Philadelphia, I developed the technology enabling the monitoring of mitochondrial NADH redox state in the un-anesthetized rats. I started the studies by adapting the available hyperbaric chambers, and by using the newly developed fiber-optic probes, I opened up a new era of studying the process of oxygen toxicity in vivo while monitoring the mitochondrial NADH redox state. In the next step in 1980–1981, I adopted the large available hyperbaric chamber and introduce to it the multiparametric monitoring system that I developed. This system enabled the monitoring up to 10 biochemical and physiological parameters from the brain of un-anesthetized rats exposed to hyperbaric oxygenation. Using the two hyperbaric chambers we were able to collect a large volume of results and published around 20 full-length publications. The first chapter is an introduction and an historical review of the subject of hyperbaric oxygenation. The second chapter provides the various technologies used in all our studies. The scientific background is presented in the third chapter. Chapters 4, 5, 6, 7, 8, 9, and 10 contained the results of all the experimental protocols performed in my laboratory during the years. Chapter 11 reviews our publications regarding the use of hyperbaric oxygenation in patients. The last chapter contains the discussion and conclusions. Ramat Gan, Israel

Avraham Mayevsky v

Acknowledgments

This book is dedicated to my dearest wife, Zipora, for her vision, patience, and support for more than six decades. Without her encouragement, my academic achievements and writing of this book were impossible to be accomplished. I dedicate this book to my four children, Amotz, Sarit, Hagit, and Shalev, who understood my devotion to the unusual career and enabled me to fulfill all of my obligations and academic activities. My deep appreciation is given to my parents, Shabtai and Rachel Mayevsky, for their devotion and support throughout my life span. My appreciation is given to the late Prof. Britton Chance, an outstanding scientist, teacher, and colleague for almost four decades of fruitful collaboration. Many thanks are given to Prof. Gennady Rogatsky who immigrated to Israel in the early 1990s and was active in my laboratory until his retirement. His contribution to the studies of hyperbaric oxygenation was critical, and without his involvement, the studies were less successful. Especially his contributions to Chap. 11 were critical. The contribution of Dr. Elhanan Meirovitch to the design and performance of the studies described in Chap. 9 was most significant. A special appreciation is given to Judith Sonn, Ph.D., who joined my laboratory as a Ph.D. student and contributed significantly to the studies. Special thanks are given to Mrs. Avrille Goldreich for her dedication to help and bring this book to press in its excellent form. During almost 60 years of research activities at Bar Ilan University, I trained many M.Sc. and Ph.D. students. I want to thank all of them for their confidence and the efforts that they did during the experimental work as well as in preparation and publication of the manuscripts. Special appreciation is given to Nili Zarchin, M.Sc., and Efrat Barbiro-Michaeli, Ph.D., who joined my research team after their graduation. Their assistance in training new undergraduate and graduate students is appreciated very much.

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Contents

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 Introduction and Historical Background����������������������������������������������    1 1.1 The Discovery of Oxygen ����������������������������������������������������������������    1 1.1.1  Ibn al-Nafis (1213–1288)����������������������������������������������������    2 1.1.2 Leonardo da Vinci (1452–1519) ������������������������������������������    3 1.1.3 Michael Servetus (1511–1553) ��������������������������������������������    3 1.1.4 Michael Sendivogius (1566–1636) ��������������������������������������    3 1.1.5 John Mayow (1641–1679)����������������������������������������������������    4 1.1.6 Carl Wilhelm Scheele (1742–1786)��������������������������������������    5 1.1.7 Joseph Priestley (1733–1804) ����������������������������������������������    6 1.1.8 Antoine Laurent Lavoisier (1743–1794)������������������������������    6 1.1.9 Henry Cavendish (1731–1810) ��������������������������������������������    8 1.2 The Discovery of “Oxygen Toxicity” ����������������������������������������������   10 1.2.1 Paul Bert – The Pioneer��������������������������������������������������������   11 1.3 The Discovery of Mitochondria��������������������������������������������������������   21 1.4 Energy Metabolism in Animal Tissues and Organs��������������������������   25 1.5 Mitochondrial Function and Metabolic States����������������������������������   29 References��������������������������������������������������������������������������������������������������   32

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 Basic Concepts of Brain Monitoring Systems ��������������������������������������   37 2.1 Mitochondrial NADH Fluorometry��������������������������������������������������   37 2.1.1 Fiber Optic Fluorometer/Reflectometer��������������������������������   39 2.1.2 Factors Affecting the Monitored Signals������������������������������   40 2.1.3 Preparation of the Brain for NADH Monitoring������������������   42 2.2 Multiparametric Monitoring Systems ����������������������������������������������   44 2.2.1 Introduction��������������������������������������������������������������������������   44 2.2.2 Methods��������������������������������������������������������������������������������   45 2.2.3 Results and Interpretation ����������������������������������������������������   48 2.3 Monitoring of NADH and Brain Functions Inside Hyperbaric Chambers������������������������������������������������������������������������������������������   54 2.3.1 Monitoring of NADH Fluorescence ������������������������������������   54 2.3.2 Monitoring of NADH Fluorescence Together with EEG ����   56 ix

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2.3.3 Simultaneous Measurement of NADH, EEG, pO2, K+e, and DC Potential ������������������������������������������������������������������   58 2.3.4 NADH, Reflectance, Microcirculatory Blood Flow, and Hb Oxygenation ������������������������������������������������������������   62 2.3.5 NADH, Reflectance, ECoG, K+, Ca2+, and DC Potentials����   64 2.3.6 CBF, NADH, Reflectance, ECoG, K+, Ca2+, H+, and DC Potentials ����������������������������������������������������������������   66 2.3.7 CBF, NADH, Reflectance, ECoG, K+, Ca2+, H+, and DC Potentials + ICP Probe��������������������������������������������   71 References��������������������������������������������������������������������������������������������������   72 3

 Scientific Background to Hyperbaric Oxygenation (HBO) ����������������   77 3.1 Effects of Normobaric Hyperoxia on Mitochondrial and Brain Functions��������������������������������������������������������������������������   78 3.1.1 Introduction��������������������������������������������������������������������������   78 3.1.2 The Effects of Normobaric Hyperoxia on Mitochondrial Activities ������������������������������������������������������������������������������   79 3.1.3 Brain Multiparametric Responses to Normobaric Hyperoxia������������������������������������������������������������������������������   81 3.2 Hyperbaric Hyperoxia Affecting Brain and Other Body Organs������   86 3.2.1 Studies by J.W. Bean������������������������������������������������������������   86 3.2.2 Effects of Hyperbaric Hyperoxia on Brain Physiology��������   89 3.3 Hyperbaric Oxygen Therapy������������������������������������������������������������   95 3.4 Hyperbaric Oxygen Therapy – Clinical Data ����������������������������������   98 3.5 Hyperbaric Oxygenation and Mitochondrial Function ��������������������   98 References��������������������������������������������������������������������������������������������������   99

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Typical Brain Mitochondrial Responses to Hyperbaric Oxygenation����������������������������������������������������������������������������������������������  105 4.1 In Vivo Real Time Typical Responses����������������������������������������������  105 4.1.1 Introduction��������������������������������������������������������������������������  105 4.1.2 Methods��������������������������������������������������������������������������������  106 4.1.3 Results����������������������������������������������������������������������������������  107 4.1.4 Discussion ����������������������������������������������������������������������������  112 4.2 Mitochondrial Respiration In Vitro After In Vivo HBO Exposure����������������������������������������������������������������������������������  114 4.2.1 Introduction��������������������������������������������������������������������������  114 4.2.2 Methods��������������������������������������������������������������������������������  114 4.2.3 Results����������������������������������������������������������������������������������  116 4.2.4 Discussion ����������������������������������������������������������������������������  118 References��������������������������������������������������������������������������������������������������  119

5

 Effect of the Pressure Level on the Oxygen Toxicity Process��������������  123 5.1 Introduction��������������������������������������������������������������������������������������  123 5.2 Methods��������������������������������������������������������������������������������������������  124

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5.3 Results����������������������������������������������������������������������������������������������  124 5.4 Discussion ����������������������������������������������������������������������������������������  130 References��������������������������������������������������������������������������������������������������  131 6

Responses to Oxygen Toxicity After Various Treatments��������������������  133 6.1 First Study – Effects of Seizure Protectant – Trimethadione (TMO) ����������������������������������������������������������������������������������������������  134 6.1.1 Introduction��������������������������������������������������������������������������  134 6.1.2 Methods��������������������������������������������������������������������������������  134 6.1.3 Results����������������������������������������������������������������������������������  135 6.1.4 Discussion ����������������������������������������������������������������������������  139 6.2 Second Study – Effects of Hypercapnia ������������������������������������������  141 6.2.1 Introduction��������������������������������������������������������������������������  141 6.2.2 Methods��������������������������������������������������������������������������������  142 6.2.3 Results and Discussion ��������������������������������������������������������  142 6.3 Third Study – The Interaction Between HBO and the Ischemic Brain��������������������������������������������������������������������������������������������������  145 6.3.1 Physiological Responses to HBO of the Partial Ischemic Brain����������������������������������������������������������������������  145 6.3.2 Fourth Study – HBO Therapy After Global Brain Ischemia����������������������������������������������������������������������  150 6.4 Fifth Study – Effects of Pentobarbital and Diazepam����������������������  155 6.4.1 Introduction��������������������������������������������������������������������������  155 6.4.2 Methods��������������������������������������������������������������������������������  155 6.4.3 Results����������������������������������������������������������������������������������  156 6.4.4 Discussion ����������������������������������������������������������������������������  159 References��������������������������������������������������������������������������������������������������  160

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 Interaction Between Carbon Monoxide(CO) and Hyperbaric Oxygenation����������������������������������������������������������������������������������������������  163 7.1 First Study – Multiparametric Recording of the CO Intoxicated Brain After Treatment by Hyperbaric Oxygen��������������  164 7.1.1 Introduction��������������������������������������������������������������������������  164 7.1.2 Methods��������������������������������������������������������������������������������  165 7.1.3 Results and Discussion ��������������������������������������������������������  166 7.2 Second Study – Responses to Cortical Spreading Depression of the CO Intoxicated Brain Treated by Hyperbaric Oxygenation��������������������������������������������������������������������������������������  170 7.2.1 Introduction��������������������������������������������������������������������������  170 7.2.2 Methods��������������������������������������������������������������������������������  171 7.2.3 Results����������������������������������������������������������������������������������  172 7.2.4 Discussion ����������������������������������������������������������������������������  175 References��������������������������������������������������������������������������������������������������  177

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 Effects of Age on the Responses to Hyperbaric Hyperoxia������������������  181 8.1 Study in the Newborn Dog���������������������������������������������������������������  181 8.1.1 Introduction��������������������������������������������������������������������������  181 8.1.2 Methods��������������������������������������������������������������������������������  185 8.1.3 Results����������������������������������������������������������������������������������  188 8.1.4 Discussion ����������������������������������������������������������������������������  192 8.2 Study in Rats of Various Ages����������������������������������������������������������  193 8.2.1 Introduction��������������������������������������������������������������������������  193 8.2.2 Methods��������������������������������������������������������������������������������  194 8.2.3 Results����������������������������������������������������������������������������������  195 8.2.4 Discussion ����������������������������������������������������������������������������  197 References��������������������������������������������������������������������������������������������������  199

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 Brain Multiparametric Responses to Hyperbaric Hyperoxia�������������  201 9.1 HBO Affecting Brain NADH, pO2, K+, and EEG Activities������������  202 9.1.1 Introduction��������������������������������������������������������������������������  202 9.1.2 Methods��������������������������������������������������������������������������������  203 9.1.3 Results����������������������������������������������������������������������������������  205 9.1.4 Discussion ����������������������������������������������������������������������������  210 9.2 HBO Effects on Brain Hemodynamics, Hb Oxygenation, and Mitochondrial NADH����������������������������������������������������������������  212 9.2.1 Introduction��������������������������������������������������������������������������  212 9.2.2 Methods��������������������������������������������������������������������������������  214 9.2.3 Results����������������������������������������������������������������������������������  220 9.2.4 Discussion ����������������������������������������������������������������������������  241 9.3 Hyperbaric Hyperoxia and the Brain In Vivo: The Balance Between Therapy and Toxicity ��������������������������������������������������������  250 9.3.1 Introduction��������������������������������������������������������������������������  250 9.3.2 Methods��������������������������������������������������������������������������������  251 9.3.3 Results����������������������������������������������������������������������������������  253 9.3.4 Discussion ����������������������������������������������������������������������������  255 References��������������������������������������������������������������������������������������������������  258

10 Hyperbaric  Oxygenation Affecting Traumatic Brain Injury��������������  263 10.1 First Study: Brain Injury, Intracranial Pressure, and Cortical Spreading Depression-CSD����������������������������������������  264 10.1.1 Introduction ����������������������������������������������������������������������  264 10.1.2 Methods����������������������������������������������������������������������������  265 10.1.3 Results ������������������������������������������������������������������������������  266 10.1.4 Discussion ������������������������������������������������������������������������  273 10.2 Second Study: Effect of HBO on ICP Elevation Rate During Severe Traumatic Brain Injury��������������������������������������������  276 10.2.1 Introduction ����������������������������������������������������������������������  276 10.2.2 Methods����������������������������������������������������������������������������  277 10.2.3 Results ������������������������������������������������������������������������������  278 10.2.4 Discussion ������������������������������������������������������������������������  281 References��������������������������������������������������������������������������������������������������  283

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11 Hyperbaric  Hyperoxia in Patients After Chest Injury or Ischemic Stroke��������������������������������������������������������������������������������������������������������  289 11.1 First Study – Monitoring of Patients During Hyperbaric Oxygenation������������������������������������������������������������������������������������  289 11.1.1 Introduction ����������������������������������������������������������������������  289 11.1.2 Monitoring of Hemodynamic Parameters������������������������  290 11.2 Second Study – Effect of Hyperbaric Oxygenation During Severe Blunt Chest Injuries������������������������������������������������  301 11.2.1 Introduction ����������������������������������������������������������������������  301 11.2.2 Methods����������������������������������������������������������������������������  301 11.2.3 Results ������������������������������������������������������������������������������  302 11.2.4 Discussion ������������������������������������������������������������������������  303 11.3 Third Study – ARDS in Patients After Blunt Thoracic Trauma: The Influence of Hyperbaric Oxygen Therapy����������������  306 11.3.1 Introduction ����������������������������������������������������������������������  306 11.3.2 Methods����������������������������������������������������������������������������  307 11.3.3 Results ������������������������������������������������������������������������������  309 11.3.4 Discussion ������������������������������������������������������������������������  311 11.4 Forth Study – Optimal Dosing of Hyperbaric Oxygen Therapy in Acute Ischemic Stroke��������������������������������������������������  313 11.4.1 Introduction ����������������������������������������������������������������������  313 11.4.2 Conclusion������������������������������������������������������������������������  317 11.5 Fifth Study – Hyperbaric Oxygenation as Treatment of Acute Ischemic Stroke: Future Perspectives������������������������������  317 11.5.1 Introduction ����������������������������������������������������������������������  317 11.5.2 Pathophysiological Basis for Hyperbaric Oxygen Therapy in Stroke��������������������������������������������������������������  318 11.5.3 Hyperbaric Oxygen and Ischemic Stroke: Experimental Data������������������������������������������������������������  319 11.5.4 Hyperbaric Oxygen Therapy: Application in Stroke Patients������������������������������������������������������������������������������  324 References��������������������������������������������������������������������������������������������������  327 12 Discussion and Conclusions��������������������������������������������������������������������  343 12.1 Responses of Brain Energy Metabolism to HBO ��������������������������  344 12.2 The Functioning Awake Brain Under HBO������������������������������������  345 12.2.1 Surface Fluorometry/Reflectometry����������������������������������  347 12.2.2 Multiparametric Monitoring���������������������������������������������  348 12.2.3 Discussion of Events Recorded During the Phases of Oxygen Toxicity������������������������������������������������������������  349 References��������������������������������������������������������������������������������������������������  353 Index������������������������������������������������������������������������������������������������������������������  357

Chapter 1

Introduction and Historical Background

Abstract  This chapter contains the introduction to the five main subjects that will be discussed in the present book summarizing the knowledge on the process of hyperbaric oxygenation as follows: 1 . The discovery of oxygen 2. The discovery of oxygen toxicity 3. The discovery of the mitochondrion 4. Energy metabolism in animal tissues and organs 5. Mitochondrial function and its metabolic states Keywords  Brain metabolism · Tissue energy balance · Oxygen supply · Oxygen gradient · Tissue monitoring

1.1 The Discovery of Oxygen In 2022, J.B. West reviewed the history of atmospheric oxygen as follows: Many of us think a lot about oxygen. This includes how the normal body handles oxygen in health, but particularly how this is complicated by lung disease. Few of us are aware that as human inhabitants of the earth, we have a unique privilege. This is that as air breathers, we and most other animals on Earth, are the only living creatures in the known universe that have an unlimited supply of oxygen. This situation came about through one of the greatest miracles of nature, that is photosynthesis, the ability to release oxygen from water using the energy of sunlight. One consequence of this was that the first atmospheric oxygen came from the metabolism of microorganisms, the cyanobacteria, that used photosynthesis, but for which oxygen was an unwanted by-product. In fact, the oxygen had to be discarded for the organisms to thrive. When a major increase of oxygen concentration in the atmosphere occurred some 2 billion years ago, and the partial pressure of oxygen in the air rose to perhaps 200 mmHg, this Great Oxidation Event as it was called, was a death sentence for the large population of anaerobic animals for whom oxygen was toxic. Today much of the oxygen in the atmosphere comes from photosynthesis in microorganisms, including the

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Mayevsky, Hyperbaric Oxygenation, https://doi.org/10.1007/978-3-031-49681-3_1

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1  Introduction and Historical Background Ibn al Nafis

1

Leonardo da Vinci

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Michael Servetus

3 John Mayow

Michael Sendivogius

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Henry Cavendish

Carl Wilhelm Scheele

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Joseph Priestley

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Antoine Laurent Lavoisier

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Fig. 1.1  The list and pictures of the nine scientists that were involved and contributed to the discovery of oxygen (see also reference by Severinghaus 2016a) cyanobacteria, and the recently discovered Prochlorococcus, that discard this unwanted by-­ product. The result is that the pO2 in our atmosphere at sea level remains nearly constant at about 150 mmHg, although the factors responsible for this are not understood.

The discovery of oxygen is a process that started more than 750 years ago (about 1250) and finished after around 500  years later in 1780. There are at least nine names that are mentioned as related to this discovery and are presented in Fig. 1.1. The first five names contributed by playing an indirect and minor part to the discovery process, while the last four scientists added the main information to the process of oxygen discovery within a 10-year period.

1.1.1 Ibn al-Nafis (1213–1288) An Arab physician that was active in the thirteenth century (1213–1288). He was the first scientist that described the pulmonary circulation (Haddad and Khairallah 1936; Loukas et al. 2008; West 2008). West (2008) summarized the discovery as follows: Ibn al-Nafis (1213–1288) was an Arab physician who made several important contributions to the early knowledge of the pulmonary circulation. He was the first person to challenge the long-held contention of the Galen School that blood could pass through the cardiac interventricular septum, and in keeping with this he believed that all the blood that reached the left ventricle passed through the lung. He also stated that there must be small communications or pores (manafidh in Arabic) between the pulmonary artery and vein, a prediction that preceded by 400 years the discovery of the pulmonary capillaries by Marcello Malpighi. Ibn al-Nafis and another eminent physiologist of the period, Avicenna (ca. 980–1037), belong to the long period between the enormously influential school of Galen in the 2nd Century, and the European Scientific Renaissance in the 16th Century. This is an epoch often given little attention by physiologists but is known to some historians as the Islamic Golden Age. Its importance is briefly discussed here.

1.1  The Discovery of Oxygen

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1.1.2 Leonardo da Vinci (1452–1519) The second scientist that was cited as a discoverer of oxygen in 1515 is Leonardo da Vinci (Reti 1952; Mitzner and Wagner 1992; West 2017). As mentioned by West in 2017 regarding Leonardo (West 2017): Leonardo da Vinci (1452–1519) enjoys a reputation as one of the most talented people of all time in the history of science and the arts. However, little attention has been given to his contributions to physiology. One of his main interests was engineering, and he was fascinated by structural problems and the flow patterns of liquids. He also produced a large number of ingenious designs for warfare and a variety of highly original flying machines. But of particular interest to us are his contributions to bioengineering and how he used his knowledge of basic physical principles to throw light on physiological function. For example, he produced new insights into the mechanics of breathing including the action of the ribs and diaphragm. He was the first person to understand the different roles of the internal and external intercostal muscles. He had novel ideas about the airways including the mode of airflow in them. He also worked on the cardiovascular system and had a special interest in the pulmonary circulation. But, interestingly, he was not able to completely divorce his views from those of Galen, in that although he could not see pores in the interventricular septum of the heart, one of his drawings included them. Leonardo was a talented anatomist who made many striking drawings of the human body. Finally, his reputation for many people is based on his paintings including the Mona Lisa that apparently attracts more viewers than any other painting in the world.

1.1.3 Michael Servetus (1511–1553) Michael Servetus was the third person involved in the discovery of oxygen via description of the pulmonary circulation in 1552. Stefanadis et al. (2009) summarized his discovery as follows: Michael Servetus was the first doctor ever to challenge and scientifically argue against the theories of Galen, which predominated for 14 centuries in medical schools worldwide. Even though he was relatively correct in scientific terms, Servetus was punished because of his boldness in challenging Galen’s theories and was condemned to death by the Holy Inquisition. Yet, by publicly challenging Galen’s and Hippocrates’ predominant and unquestionable lessons on medicine for the first time, Servetus opened the door for other doctors to challenge and correct those theories and subsequently to bring about a new view of human anatomy and physiology. This article underlines the contribution of Servetus to the description of the pulmonary circulation.

1.1.4 Michael Sendivogius (1566–1636) The fourth sage was a famous sixteenth-century Polish noble physician and alchemist, Michael Sendivogius. In 1604, he published that air contains what he named the “secret food of life.” He had realized that this “food” was the same gas emitted by saltpeter (potassium nitrate) when heated. He called it part of the “saltnitre of the

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1  Introduction and Historical Background

earth.” Sendivogius’s writings were frequently copied and read for over a century. However, no one grasped the chemical importance of his discovery, not even physicists Boyle and Newton at Oxford, who read his work 60 years later. Sendivogius’ “food of life” story was only recently rediscovered. He discovered that air is not a single substance and contains a life-giving substance  – later called oxygen  – 170 years before Scheele’s discovery of the element.

1.1.5 John Mayow (1641–1679) John Mayow was the fifth scientist that was involved in the discovery of oxygen. He was a chemist, physician, and physiologist who is remembered today for conducting early research into respiration and the nature of air. Mayow worked in a field that is sometimes called pneumatic chemistry. As a chemist and physiologist, he discovered, about a hundred years before Joseph Priestley and Antoine-Laurent Lavoisier, and identified spiritus nitroaereus (oxygen) as a distinct atmospheric entity. He also found out that animals take up the same gas in their blood during respiration and that the air exhaled by animals had a less amount of spiritus nitroaereus gas than the one present in the open air. Mayow’s first experiment (as seen in Fig. 1.2) involved placing a candle in an inverted jar and placing the jar in a tub of water. The purpose of the water was to seal the vessel. To equalize the initial air pressure on both sides of the jar, Mayow used a straw, but quickly withdrew the straw once the pressures were equalized. As the candle burned, the water in the jar was drawn slightly upward until the candle was extinguished (because it had consumed the oxygen, not because the water had

Fig. 1.2  From John Mayow experiments related to the discovery of oxygen. (By John Mayow, 1674. Public domain ... www.researchgate.net)

1.1  The Discovery of Oxygen

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extinguished it). Mayow correctly explained that the air inside the jar had lost some of its “elasticity” and was no longer able to supply a force to the water equal to the atmospheric pressure. He suggested that this occurred because the air had been “deprived of nitro aerial and elastic particles” by the burning of the flame “so that the air there is not able as before to resist the pressure of the atmosphere.” Despite the critical need of oxygen and its significant use in body energy metabolism, the final steps of oxygen discovery in the atmosphere happened about 250 years ago (1771–1775). Until about 250 years ago, the story of oxygen discovery was described and discussed in terms of oxygen as a chemical element and the most important discovery of chemistry and science (Anon. 1970; Cassebaum and Schufle 1975; Smeaton 1992; Marshall and Marshall 2005; Palmer 2006; West 2014a).

1.1.6 Carl Wilhelm Scheele (1742–1786) The sixth discoverer of oxygen, Carl Wilhelm Scheele, was born in German-­ speaking Pomerania. Trained in Sweden, he became a very clever apothecary and chemist. In 1768, he published new studies of metal chemistry. In 1770, he became director of Locke Pharmacy in Uppsala. He worked at the university there with the famous chemist Torbern Bergman, who helped him publish his work in Latin in the journal Nova Acta. In 1774, Lavoisier sent his new chemistry textbook to Bergman, including a copy for Scheele, whose published work he much admired. Scheele promptly wrote to Lavoisier to thank him for the book. He explained how, in 1771, he had generated a strange new gas by heating certain metallic earths. He had named it fire air because it greatly brightened a candle flame and supported life in mice. Because neither he nor Bergman could relate it to the phlogiston theory, Scheele had delayed publishing it. He asked Lavoisier to repeat the experiment and then help him explain it. The activity of Scheele was summarized by West (2014a) as follows: Carl Wilhelm Scheele (1742–1786) has an important place in the history of the discovery of respiratory gases because he was undoubtedly the first person to prepare oxygen and describe some of its properties. Despite this, his contributions have often been overshadowed by those of Joseph Priestley and Antoine Lavoisier, who also played critical roles in preparing the gas and understanding its nature. Sadly, Scheele was slow to publish his discovery and therefore Priestley is rightly recognized as the first person to report the preparation of oxygen. This being said, the thinking of both Scheele and Priestley was dominated by the phlogiston theory, and it was left to Lavoisier to elucidate the true nature of oxygen. In addition to his work on oxygen, Scheele was enormously productive in other areas of chemistry. Arguably he discovered seven new elements and many other compounds. However, he kept a low profile during his life as a pharmacist, and he did not have strong links with contemporary prestigious institutions such as the Royal Society in England or the French Académie des Sciences. He was elected to the Royal Swedish Academy of Science but only attended one meeting. Partly as a result, he remains a somewhat nebulous figure despite the critical contribution he made to the history of respiratory gases and his extensive researches in other areas of chemistry. His death at the age of 43 may have been hastened by his habit of tasting the chemicals that he worked on.

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1.1.7 Joseph Priestley (1733–1804) Oxygen’s seventh discoverer was Unitarian minister Joseph Priestley. Two churches fired him as being too radical, but he soon became famous while teaching grammar and science at Warrington Academy near Manchester, helping to make it a leading school for dissenters – in fact, the “cradle of Unitarianism,” as one scholar called it. On August 1, 1774, Priestley, by heating red mercury calc, made a gas that caused a glowing splinter to burst into flame and supported life in a mouse in a bottle. He named it dephlogisticated air. In October 1774 in Paris, he described his method of making it to Lavoisier, who never acknowledged his help. West (2014c) summarized his activities as follows: Joseph Priestley (1733–1804) was the first person to report the discovery of oxygen and describe some of its extraordinary properties. As such he merits a special place in the history of respiratory physiology. In addition, his descriptions in elegant 18th-century English were particularly arresting, and rereading them never fails to give a special pleasure. The gas was actually first prepared by Scheele (1742–1786) but his report was delayed. Lavoisier (1743–1794) repeated Priestley’s initial experiment and went on to describe the true nature of oxygen that had eluded Priestley, who never abandoned the erroneous phlogiston theory. In addition to oxygen, Priestley isolated and characterized seven other gases. However, most of his writings were in theology because he was a conscientious clergyman all his life. Priestley was a product of the Enlightenment and argued that all beliefs should be able to stand the scientific scrutiny of experimental investigations. As a result, his extreme liberal views were severely criticized by the established Church of England. In addition, he was a supporter of both the French and American Revolutions. Ultimately his political and religious attitudes provoked a riot during which his home and his scientific equipment were destroyed. He therefore emigrated to America in 1794 where his friends included Thomas Jefferson and Benjamin Franklin. He settled in Northumberland, Pennsylvania although his scientific work never recovered from his forced departure. But the descriptions of his experiments with oxygen will always remain a high point in the history of respiratory physiology.

1.1.8 Antoine Laurent Lavoisier (1743–1794) Lavoisier had become the world’s most brilliant chemist by the age of 30. In the spring of 1775, he began his studies of Priestley’s newly published gas. He named it principe oxigene but continued to refer to it as “vital air.” However, after 8 years of extensively examining its chemistry, he was still unable to prove whether vital air was a new element or a compound. And he doubted but still couldn’t disprove the phlogiston theory. By 1783, he was stalled by these dilemmas. In his Elements of Chemistry, Lavoisier falsely wrote that “This species of air was discovered almost at the same time by Mr. Priestley, Mr. Scheele, and myself,” both a bold lie and proof that he had read Scheele’s 1774 letter (Fig. 1.3b), using his method of making fire air while failing to acknowledge Scheele. West (2013) discussed and summarized the collaboration between Antoine and his wife Marie-Anne Lavoisier in the discovery of oxygen as well as the first measurements of human oxygen consumption as follows:

1.1  The Discovery of Oxygen

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Fig. 1.3  Antoine Laurent Lavoisier (1747–1794) with his wife Marie-Anne (1759–1836). On the far left is the drawing board used by Marie-Anne. On the far right is a glass jar containing water and resting on a porcelain dish. This jar was presumably used for collecting gas. Next to it is a narrow tube partly filled with mercury. This is a eudiometer for measuring the oxygen concentration of gases. To the left of that is a mercury receiver for collecting gas. On the floor there is a large glass vessel that was used for experiments to make water from hydrogen and oxygen. Next to it is a hydrometer for measuring the specific gravity of fluids. This magnificent portrait by Jacques Louis David (1788) is in the Metropolitan Museum of Art, New York. Reproduced by permission. The Metropolitan Museum of Art, Purchase, Mr. and Mrs. Charles Wrightsman Gift, in honor of Everett Fahy, 1977 (1977.10). (Image © The Metropolitan Museum of Art) “ANTOINE-LAURENT LAVOISIER (1743–1794) enjoys a reputation as one of the most eminent scientists of the late 18th Century (Fig.  1.3a). His wife, Marie-Anne Pierrette Paulze Lavoisier (1758–1836), collaborated in many of his studies, but her contributions have received relatively little attention. Antoine Lavoisier’s interests ranged over a wide area of chemistry. One of his major advances was to clarify the concept of an element as a substance that could not be further broken down by chemical analysis. His extensive classification of known elements was described in his book Traité élémentaire de chimie (Elementary Treatise on Chemistry) published in 1789.” “In the life sciences Lavoisier was the first person to recognize the true nature of oxygen. This had been isolated by Joseph Priestley (1733–1804) and Carl Wilhelm Scheele (1742–1786), but its role in respiration was not understood because of confusion caused by the erroneous phlogiston theory. Antoine with the help of his wife demolished this. Lavoisier also clarified the similarities between respiration and combustion, and he was responsible for the first studies of human oxygen consumption under various conditions”. “Lavoisier was the first person to clearly state the role of oxygen, carbon dioxide, and nitrogen in respiration. Here he built on the previous work of other investigators particularly Priestley. This English Nonconformist minister carried out an experiment in August 1774 when, on heating some red mercuric oxide, he found that a remarkable gas was produced. He stated “but what surprised me more than I can yet well express, was that a candle burned in this air with a remarkably vigorous flame and a piece of red-hot wood sparkled in it” (Priestley 1775). Furthermore, he showed that a mouse was able to survive longer in this gas than in ordinary air, and he famously surmised that it might be useful for people with disease. Thus there was no doubt that

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1  Introduction and Historical Background Priestley had produced oxygen. However, unfortunately he did not understand its nature. Priestley was a follower of the phlogiston theory that had been promoted by Georg Ernst Stahl (1659–1734) and that stated that all combustible materials are composed of ash (calx) and phlogiston (Greek for inflammable), and that during burning, phlogiston escaped, leaving the dephlogisticated ash behind. This is in fact the reverse of what happens during combustion when oxygen combines with a combustible material, but the theory was enormously influential in the mid-18th Century. Priestley is often credited with “discovering” oxygen just as Columbus “discovered” America but neither Priestley nor Columbus correctly identified what they found. Carl Wilhelm Scheele (1742–1786) in Sweden had independently produced oxygen even before Priestley although the publication of his findings was delayed (Scheele 1777). Scheele called the gas “fire-air” but again he was influenced by the phlogiston theory and did not recognize its true nature. Priestley visited Paris in October 1774 with his patron Lord Shelburne and they had dinner with Lavoisier and some other chemists. At that time Priestley described the experiments that he had carried out with mercuric oxide; naturally, these evoked great interest. As a result, Lavoisier repeated the experiments and was eventually able to understand the chemical processes. For example, in 1775  in what became known as the Easter Memoir, Lavoisier stated that “the substance which combines with metals during calcination, thereby increasing their weight, is nothing else than the pure portion of the air which surrounds us and which we breathe.” This was the coup de grâce to the phlogiston theory. As we shall see later, Marie-Anne Lavoisier played an important part in this advance because it was she who translated the English texts of Priestley and another proponent of phlogiston, Richard Kirwan (1733–1812), so that Antoine could read them.

1.1.9 Henry Cavendish (1731–1810) In 1766, Cavendish discovered and published how to make an inflammable gas (H2) by putting iron filings in strong acid. In the late 1770s, he and Priestley noted that burning inflammable air caused dew to form on glass walls. In 2014, J.B. West published a paper summarizing the activities of Cavendish as follows: Henry Cavendish (1731–1810) was an outstanding chemist and physicist. Although he was not a major figure in the history of respiratory physiology he made important discoveries concerning hydrogen, carbon dioxide, atmospheric air, and water. Hydrogen had been prepared earlier by Boyle but its properties had not been recognized; Cavendish described these in detail, including the density of the gas. Carbon dioxide had also previously been studied by Black, but Cavendish clarified its properties and measured its density. He was the first person to accurately analyze atmospheric air and reported an oxygen concentration very close to the currently accepted value. When he removed all the oxygen and nitrogen from an air sample, he found that there was a residual portion of about 0.8% that he could not characterize. Later this was shown to be argon. He produced large amounts of water by burning hydrogen in oxygen and recognized that these were its only constituents. Cavendish also worked on electricity and heat. However, his main contribution outside chemistry was an audacious experiment to measure the density of the earth, which he referred to as “weighing the world.” This involved determining the gravitational attraction between lead spheres in a specially constructed building. Although this was a simple experiment in principle, there were numerous complexities that he overcame with meticulous attention to experimental details. His result was very close to the modern accepted value. The Cavendish Experiment, as it is called, assures his place in the history of science. (West 2014b)

1.1  The Discovery of Oxygen

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In many published papers and Internet sites, the credit of oxygen discovery was given either to the American Joseph Priestley (Partington 1962; Miller 1987; Williams 2003) or to the German–Swedish Carl Wilhelm Scheele (Partington 1962; Smeaton 1963; Taylor 2019). The available information indicates that Scheele was the first one to discover oxygen in 1771–1772, but he published it officially a few years later in 1777 (Scheele 1777). He claimed that in 1774, he sent a letter to the famous French chemist Antoine de Lavoisier, regarding his discovery, but Lavoisier claimed that he never received the letter. Priestley discovered oxygen and in 1774 he notified his findings to Antoine de Lavoisier who studied oxygen chemistry for a number of years. Priestley actually published his discovery in 1775. In 2004, Wilkinson published a paper (Wilkinson 2004) describing the significant contribution of Lavoisier, Scheele, and Priestley to the understanding of respiratory physiology in the eighteenth century. Here are his conclusions: There appear to be a great desire in history to define who was the first to do something. In reality it makes no great difference to today’s work who was first to define oxygen in the eighteenth century. What can be seen from the above is the ability of these three great men to set new paradigms and work to prove their theories. Some were more accurate than others but it is the ability to take that lateral step that sets these characters apart from their peers. It could be considered that if Scheele first isolated oxygen then Priestley defined its properties and Lavoisier showed it was an element but in fact they all added a significant set of pieces to the physiological jigsaw puzzle of which oxygen was just a part.

In 1996, John Severinghaus (Fig. 1.4) became interested in the story of the discovery of oxygen and published his first paper on this subject (Severinghaus 2002). In the introduction to the cited paper, Severinghaus wrote the next paragraph: Americans are taught that the English Unitarian minister Joseph Priestley discovered oxygen in 1774. Scandinavians are taught that the Swedish apothecary Carl Wilhelm Scheele generated oxygen in Uppsala in 1771–2, several years before Priestley. Scheele claimed that he wrote Lavoisier, describing the experiments in September, 1774. However, Lavoisier denied seeing or receiving his letter. Among Nordic historians, Lavoisier has never been forgiven for this rebuff. Some historians have doubted whether such a letter was ever sent or received, because Scheele first published in 1777. Priestley brought the news of his discovery to Lavoisier in October and in publications overturning the conventional Phlogiston Theory, causing a revolution in chemistry.

One year later, Severinghaus published an extended paper on this topic (Severinghaus 2003) and came to the conclusion that Marie-Anne Lavoisier received the original letter of Scheele and hid it until 219  years later the letter was found (Fig. 1.3b). Later, Severinghaus combined the discovery of Oxygen as an element, in the eighteenth century to the description of the pulmonary circulation in the thirteenth century (Severinghaus 2016a, b). This subject was discussed in other papers describing the Islamic Golden age in the thirteenth century (Haddad and Khairallah 1936; West 2008).

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1  Introduction and Historical Background

Fig. 1.4 (a) The “Master Tinker” and the blood  – Gas analyzer. (b) John Severinghaus near a patient that was monitored in the hospital of the University of California San Francisco California, USA. (From: Severinghaus, 1922 to 2021, Anesthesiology. 2021; 135(4): 555–557)

1.2 The Discovery of “Oxygen Toxicity” The connection between the time to discover the element “Oxygen” and its possible toxic effects (developed by breathing high levels of oxygen) is amazing. In a review published by Bean (1945), he opened his paper as follows: Shortly after having isolated O2 Priestley (1775) wrote, “From the greater strength and vivacity of the flame of a candle, in this pure air, it may be conjectured, that it might be peculiarly salutary to the lungs in certain morbid cases … But, perhaps, we may also infer from these experiments, that though pure dephlogisticated air might be very useful as a medicine, it might not be so proper for us in the usual healthy state of the body: for, as a candle burns out much faster in dephlogisticated than in common air, so we might, as may be said, live out too fast, and the animal powers be too soon exhausted in this pure kind of air. A moralist, at least, may say, that the air which nature has provided for us is as good as we deserve…. The feeling of it to my lungs was not sensibly different from that of common air; but I fancied that my breast felt peculiarly light and easy for some time afterwards. Who can tell but that, in time, this pure air, may become a fashionable article in luxury. Hitherto only two mice and myself have had the privilege of breathing it.

1.2  The Discovery of “Oxygen Toxicity”

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Fig. 1.5  The unique published book written in France originally (a) by Paul Bert (b) and the translated book to English by Hitchcock MA and Hitchcock FA (c) in 1943

1.2.1 Paul Bert – The Pioneer It took about 100 years between the discovery of oxygen by Scheele, Priestley, and Lavoisier in the 1770s and the famous publication of Paul Bert (Fig. 1.5b) on oxygen toxicity in 1878 (Bert 1943). His book was translated from French (Fig. 1.5a) into English (Fig. 1.5c) in 1943 by Hitchcock MA and Hitchcock FA and published as Barometric pressure: Researches In Experimental Physiology, College Book Company, Columbus, Ohio (Bert 1943). Few papers described the research activities of Paul Bert related to the effects of barometric pressure (Fulton 1843; Kellogg 1978; Dejours and Dejours 1992; Acott 1999). Actually, Bert started his research activities on animal tissue grafting or skin grafting and, in 1865, was awarded a prize in experimental physiology in this field. A few years later, in 1870, he became interested in respiration physiology and disorders such as asthma. According to J.F. Fulton (that wrote the Foreword to the book of Bert seen in the next paragraph), “after Bert spent time on asthma research, a friend of Bert, Dr. Denis Jourdanet, returned from a research period in Mexico and suffered from mountain sickness, they decided that Bert should dedicate his time performing experiments on the effect of pressure on plants, animals and humans. Jourdanet was a wealthy man and he offered to provide any funds that Bert needed.”

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1  Introduction and Historical Background

1.2  The Discovery of “Oxygen Toxicity”

13

This research activity funded by Jourdanet led to the publication of Bert’s book. In one of the first pages of the book, Bert acknowledged and dedicated the book to Dr. Jourdanet, as seen in Fig. 1.6. Figure 1.7 shows the chambers that were used in studying the effects of hypobaric and hyperbaric studies reported by Bert. Figure 1.8 shows the setup of the equipment and chamber that were located in the Sorbonne. Most of Bert’s book described the effects of decrease in oxygen supply due to exposure to high altitude or low oxygen partial pressure in the respiration mixture. This subject was investigated originally by Denis Jourdanet (Fig.  1.9a) and was published (1875b) 3 years before Bert’s book, as seen in Fig. 1.9b. The history of Jourdanet’s research activities was described and summarized, in 2013, by West and Richalet (2013) in a paper entitled “Denis Jourdanet (1815–1892) and the early recognition of the role of hypoxia at high altitude.” The abstract of this paper was as follows:

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Fig. 1.6  The dedication of the book written by Paul Bert made to his friend Dr. Jourdanet

Fig. 1.7 (a) Apparatus of M. Jourdanet for the therapeutic use of compressed or expanded air. (b) This is a compression chamber that allowed Bert to measure the effects of barometric pressure on himself and others. Inside was an Indian rubber bag so he could inhale oxygen. Access points allowed him to take blood samples to determine changes in blood oxygen tensions. He exposed himself to decreased pressures. Once he started to feel symptoms, he inhaled oxygen. He wrote: “But all these symptoms disappeared as by enchantment so soon as I respired some of the oxygen in the bag; returning, however, when I again breathed the air in the cylinder”

Fig. 1.8  A unique setup system to measure the effects of hypobaric and hyperbaric air or oxygen located in the laboratory of physiology in the Sorbonne, Paris

Fig. 1.9  Dr. Denis Jourdanet (a) and his book published in 1875 (b)

16

1  Introduction and Historical Background Denis Jourdanet (1815–1892) was a French physician who spent many years in Mexico studying the effects of high altitude. He was a major benefactor of Paul Bert (1833–1886), who is often called the father of high-altitude physiology because his book La Pression Barométrique was the first clear statement that the harmful effects of high altitude are caused by the low partial pressure of oxygen. However, Bert’s writings make it clear that the first recognition of the critical role of hypoxia at high altitude should be credited to Jourdanet. Jourdanet noted that some of his patients at high altitude had features that are typical of anemia at sea level, including rapid pulse, dizziness, and occasional fainting spells. These symptoms were correctly attributed to the low oxygen level in the blood and he coined the terms “anoxyhémie” and “anémie barométrique” to draw a parallel between the effects of high altitude on the one hand and anemia at sea level on the other. He also studied the relations between barometric pressure and altitude, and the characteristics of the native populations in Mexico at different altitudes. Jourdanet believed that patients with various diseases including pulmonary tuberculosis were improved if they went to altitudes above 2,000 m. This led him to recommend “aérothérapie” in which these patients were treated in low-pressure chambers. Little has been written about Jourdanet, and his work deserves to be better known.

The book by Jourdanet was published in French and only the abstract was translated into English, as seen in the following citation (Jourdanet 1875a): After having practiced medicine for six years on the borders of the Gulf of Mexico, and rendered himself familiar with the diseases and conditions of life of the inhabitants of low levels, M. Jourdanet removed to the elevated plateau of Anahuac – more than 2,000 meters above the sea level. Here, as might have been anticipated, he found the pathological conditions different, but to his surprise he discovered that the differences were not simply such as result from temperature, or are paralleled in places of lower level and higher latitude, but presented peculiarities which he conceived to be dependent on the elevation of the situation alone. A residence of twenty years in the locality enabled him to confirm this idea and to prove that, while the blood of the inhabitants presented no poverty of corpuscles, the corpuscles themselves were deficient in oxygen, on account, as he believed, of the too feeble pressure of the atmosphere in these high regions. This led him to undertake the study of the whole question of the influence of the atmospheric pressure on health, and to call to his aid M. Paul Bert, Professor of Physiology at the Sorbonne, by means of whose experiments he believes himself to have arrived at some definite results. These, with every other possible point of interest connected with the subject, he now presents us with, in two large and beautifully illustrated volumes; leaving, however, the details of the physiological experiments to be published in a forthcoming work by P. Bert himself.

The results of Bert’s experiments in which hyperbaric oxygenation was used are summarized in the next paragraphs published in pages 578–580 (Bert 1943).

1.2  The Discovery of “Oxygen Toxicity”

Experiments Subchapter III SUMMARY AND CONCLUSIONS Summarizing. If we clear the principal results from the incidental questions which we have brought up and settled to the course of our research, the study of death in confined air under different pressures brings us to the following formulae. In ordinary air: A. – At pressures lower than one atmosphere, the death of animals occurs when the oxygen tension of the air is reduced to a certain constant value (which for sparrows equals on the average O2  P = 3.6). B. – For pressures included between 2 and 9 atmospheres, death occurs when the carbonic acid tension rises to a certain constant value (which for sparrows equals on the average CO2  P = 26).

17

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1  Introduction and Historical Background

C. – For very high pressures, death is due exclusively to the too great tension of the ambient oxygen. It comes quickly when the tension of the gas reaches 300 or 400. D. – For pressures of 1 to 2 atmospheres, death seems to be due especially to the lowering of the oxygen tension but in part also to the rise of the CO2 tension. E. – Starting with 3 or 4 atmospheres, the fatal effect of the oxygen begins to be felt and becomes very evident at about 9 or 10 atmospheres. Experiments made either with gaseous mixtures mor or less rich in oygen, or in the presence of alkalis capable of absorbing the carbonic acid as it is formed, cause us to give to these laws an even greater character of generality and we can formulate them in the following manner (applying them for greater clearness, to sparrows). The tension of a gas being representede by the product of its percentage multiplied by the barometric pressures, we see that death occurs. A. – When the oxygen tension drops bewlow 3.6, whether the barometric pressure is above or below the normal pressure: of course, in the first case, the carbonic acid must be removed by an alkali. B. – When the carbonic acid tension rises above 26, whether the pressure is above or below the normal pressure; of course, in the latter case superoxygenated mixtures must be used. Death in Closed Vessels What we say of carbonic acid is general for all poisonous gases (CO, HS, etc): only the numeric value of the lethal

1.2  The Discovery of “Oxygen Toxicity”

tension will change. We shall return to this point when we speak of the hygiene of workmen in compression tubes. C. – Where the oxygen tension reaches about 300, whatever the percentage and the pressure are (the latter evidently cannot be lower than 3 atmospheres, with pure oxygen). D. – These kinds of death can be combined by twos, A with B and B with C, according to the pressures and gaseous compositions used. Death A is a real asphyxia for lack of oxygen; death B is a plisoning by carbonic acid; death C can be called, for convenience and in spite of the strangeness of the expression, a poisoning by oxygen. We see – and this is the most general result reached – that in all cases the barometric pressure in its variations is never directly, of itself, the cause of the phenomena. It is only one of the conditions which alter the tension of the gases, and the other factor, the percentage, can completely offset its effects. If its progress is in the other direction, just as it will increase them rapidly, if its progress is in the same direction. If now we leave out the carbonic acid produced, to place ourselves in conditions nearer those in which our present problem appears in nature or industry, setting aside certain phenomena which are quite secondary and to which we shall return at the appropriate time, we reach these conclusions: 1. That three animals, the first of which exhausts by its respiration a closed space full of air, the second of which is compelled to breathe in a current of air of diminishing oxygen content, the third of which is subjected to a gradual decrease

19

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of pressure, are all three, by these different procedures threatened by the same symptoms and the same death, a death from lack of oxygen, a real asphyxia; 2. That two animals, one of which breathes in a current of air of increasing oxygen content while the other is subjected to a barometric pressure increasing from 1 to 5 atmospheres, are in identical conditions. That, besides, the animal which breathes pure oxygen at 2, 3, 4 atmsopheres, etc., is in the same conditions as the one which breathes pure air at 10, 15, 20 atmospheres; both are, by these different procedures, threatened by the same symptoms and the same death, a death from excess of oxygen, a poisoning of a sort hitherto unknown. All the influence which barometric modifications exercise on animals is summed up in these terms: too low an oxygen tension or too high an oxygen tension. Such is the very simple explanation given us by experiments in which we considered the ambient medium much more than the animal. But this too low or too high tension of the oxygen must be studied now, not only its measure, but in its immediate consequences; the animal itself must also be examined with more care. The first question which I shall now consider is that of the composition of the gases contained in the blood of animals subjected to different pressures.

1.3  The Discovery of Mitochondria

21

1.3 The Discovery of Mitochondria The understanding of mitochondrial function has been a challenge for various investigators, since its discovery more than 150 years ago. In (1981), Ernster and Schatz reviewed the history of mitochondrial structure and function studies. There is no real single answer regarding who discovered mitochondria. The process of discovery and identification was a gradual one and four scientists that were involved in the discovery of the mitochondria are presented in the next few figures. The Swiss anatomist and histologist Albert von Kölliker (1856), shown in Fig. 1.10, described in 1856 what he called “granules” in the sarcoplasm of striated muscle (Fig. 1.11). According to A. L. Lehninger, “Kölliker was among the first to notice the arrangement of granules in the sarcoplasm of striated muscle over a period of years beginning around 1850”. These granules were later called sarcosomes by Retzius (Fig. 1.12) in 1890. These sarcosomes have come to be known as the mitochondria – the power houses of the cell. In the words of Lehninger, “Kölliker should also be credited with the first separation of mitochondria from cell structure. In 1888 he teased these granules from insect muscle, in which they are very profuse, found them to swell in water, and showed them to possess a membrane.” As mentioned, Retzius, in 1890, called the granules, Sarcosomes which later on were identified as the mitochondria (Retzius 1890). Retzius published more than 300 scientific works in anatomy, embryology (Fig.  1.13), eugenics, craniometry, zoology, and

Fig. 1.10  Albert von Kölliker (a) that contributed initially to the discovery of the mitochondrion (b)

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1  Introduction and Historical Background

Fig. 1.11  The study of Albert von Kölliker that contributed to the identification of “Grannules” in the sarcoplasm of striated muscle

Fig. 1.12  Gustav Retzius and his publication that called the granules “sarcosomes”

1.3  The Discovery of Mitochondria

23

Fig. 1.13  One of the main publication of G.Retzius on the Sperm cells

botany. He gave his name to the 60 micrometer-diameter Retzius cells in the central nervous system of the leech (Hirudo medicinalis). During his time at the Karolinska Institute, he made important contributions to anatomical descriptions of the muscles of the eardrum, the bones of the middle ear, and the Eustachian tube. The discovery of mitochondria in general came in 1890 when Richard Altmann (Fig. 1.14a), a cytologist, identified the organelles and dubbed them “bioblasts,” as presented in his publication seen in Fig. 1.14b (Altmann 1890). The fourth scientist involved in the discovery of the mitochondria was Carl Benda (Fig. 1.15), that in 1898, coined the term mitochondria, Greek for thread, mitos, and granule, chondros (Benda 1898).

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Fig. 1.14 (a) Richard Altman that gave the name “bioblasts” that later on was identified as the mitochondria. (b) The book published by Altmann (1890)

A

B

Carl Benda (30 December 1857 Berlin – 24 May 1932 Turin) was one of the first microbiologists to use a microscope in studying the internal structure of cells. In an 1898 experiment using crystal violet as a specific stain, Benda first became aware of the existence of hundreds of these tiny bodies in the cytoplasm of eukaryotic cells and assumed that they reinforced the cell structure. Because of their tendency to form long chains, he coined the name mitochondria ("thread granules"). These bodies had first been noted in 1857 by the physiologist and pioneer of the light microscope, Albert von Kölliker, and were later termed "bioblasts" by Richard Altmann in 1886.

Fig. 1.15  Prof. Carl Benda (a) that coined the term mitochondria in 1898 (b)

1.4  Energy Metabolism in Animal Tissues and Organs

25

1.4 Energy Metabolism in Animal Tissues and Organs The functional capacity of any tissue is related to its ability to perform its work. It is possible to assess this ability through the knowledge of changes in the oxygen balance, i.e., the ratio of oxygen supply to oxygen demand in the tissue. As seen in Fig. 1.16a, this concept was suggested by Barcroft 110 years ago (Barcroft 1914). He described the relationship between tissue activity, oxygen consumption, and increase in blood supply as a compensation mechanism, as presented also in Fig. 1.17. This observation that was published in 1914 was and is supported widely by many studies published in the last 110 years. Schematic presentation of the balance between oxygen supply and demand in a typical tissue is shown in Fig. 1.16b. The supply of oxygen is dependent upon microcirculatory blood flow (TBF), blood volume (TBV), and the level of oxygen bound to the hemoglobin (HbO2) in the small blood vessels, namely, in the microcirculation. The level of oxygenated hemoglobin in the microcirculation is affected by two factors, namely, oxygen consumption by the mitochondria and the microcirculatory blood flow. The demand for oxygen is affected by the specific activities taking place in each organ. The intracellular level of mitochondrial NADH (the reduced form) is a parameter related to the oxygen balance in the tissue.

A

B

Oxygen Homeostasis in Tissues Brain

Microcirculatory

Liver

Oxygen Molecule

Microcirculatory Blood Flow &Volume

Tissue pO2

O2

Kidney

1.Ionic Homeostasis 2. Signal Conduction 3. Muscle contraction 4. Glandular Secretion 5. G-I tract Activity

ATP

Fig. 1.16 (a) The title page of the book published by J. Barcroft in 1914. (b) Schematic presentation of tissue energy balance evaluated by energy supply and demand. Energy supply is measured by tissue blood flow (TBF), tissue blood volume (TBV), and oxygenated hemoglobin (HbO2), which is similar in all tissues. Energy demand varies between the different tissues and may include: Ionic Homeostasis, Signal Conduction, Muscle contraction, Glandular Secretion, and G-I tract and kidney function. Mitochondrial NADH redox state serves as an indicator for tissue energy or oxygen balance. (Mayevsky and Barbiro-Michaely 2013a)

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Fig. 1.17  The hypothesis formulated by Barcroft in 1914 regarding the connection between organ activity, oxygen consumption, and blood flow. (Barcroft 1914)

Oxygen Gradient from Air to Mitochondria O2

150

160

N2

End Tidal CO2

100

Heart Rate Rhythm &ECG

95

A

Systemic HbO2

3

Macro circulation

Systemic Blood Pressure

Lung Output HbO2

100

Cardiac Output

Systemic Saturation (Pulse Oximetry)

Skeletal Muscle

50 20-40

Microcirculation blood flow and Hb oxygenation NADH redox state in mitochondria

Micro-circulation

Brain

1-3

0 Air

Alveoli

Arterial Blood

Tissue

Intracellular

Intramitochondrial

Fig. 1.18  Oxygen gradient from air to the mitochondria. Clinical monitoring of patients includes the various parameters along the oxygen gradient. (Mayevsky and Barbiro-Michaely 2013b)

Figure 1.18 shows the gradient of oxygen between room air through the lungs, the large arteries and small arterioles to the intracellular space and finally the mitochondrion. Since 90–95% of the total body consumed oxygen is utilized in the mitochondria, it is important to understand its gradient from the atmospheric air (the source) to the site of utilization in the mitochondria. In Fig. 1.18, the decreased level of oxygen is illustrated in the main sites between air and the mitochondria. In insert A, the association/dissociation curve of hemoglobin (Hb/HbO2) is presented. Under normal conditions, the blood collected from the various organs of the body is re-­ oxygenated in the alveoli of the lungs that are surrounded by blood capillaries. The

1.4  Energy Metabolism in Animal Tissues and Organs

27

oxygenated arterial blood (HbO2 = 97–98% saturation) reached the left ventricle of the heart and distributed to all organs of the body via the arterial macro circulation part of the blood system. The systemic oxygenated blood is shown also in insert A part 1. It’s important to note that the release or download of oxygen from of oxygenated hemoglobin is possible only by passive diffusion process dependent on the oxygen gradient across the blood vessels and the extracellular and intracellular spaces. Therefore, most of the oxygen will be provided to the cellular compartment of the various organs via the microcirculation and not the macro-circulation as seen in the lower right part of Fig. 1.19. The level of HbO2 saturation in the microcirculation of the various organs is dependent on the blood flow to the organ and the rate of oxygen consumption by the specific tissue. For example, in skeletal muscle at the resting state, the saturation (around 80%) is much higher (Fig. 1.18A3) as compared to the resting brain (40–50%), seen in point 2 in the insert A. The reason is that the “resting” brain consumes much more oxygen as compared to the muscle and the download of oxygen from the HbO2 is much higher. The main approaches to monitor oxygen metabolism under in vivo conditions and in real time are to measure oxygen levels in the tissue, and the second way is to measure hemoglobin saturation. In this scheme, the various points of patients’ monitoring are presented. As can be seen, the last usual parameter, in the oxygen gradient, is the pulse oximeter that measures the saturation of hemoglobin in the large arteries. As of today, monitoring of the microcirculation and especially mitochondrial function is not a standard approach and clinical tool.

D

A

Mitochondrial NADH (Fluorometry)

Large Artery

O2

Typical cell

H2O

ADP+Pi

Pyruvate Lactate

E

Tissue Reflectance At 366nm (Blood Volume)

Glycolysis

Glucose

ATP

Capillary

B

Systemic Hemoglobin Oxygenation ( Pulse Oximeter)

Microcirculatory Hemoglobin Oxygenation (Tissue Oximeter)

Microvascular Arterioles & capillaries

C Tissue Blood Flow (Laser Doppler Flowmetry)

Fig. 1.19  The available techniques for real-time monitoring of energy metabolism at the tissue level. Part a shows the coupling between the macro-circulation monitored by the Pulse oximeter and the microcirculation. (b–e) monitoring of vascular and intracellular compartments (see text for details). (Mayevsky and Barbiro-Michaely 2013a)

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Figure 1.19d presents the involvement of the mitochondria in cellular and tissue energy metabolism. Substrates and O2 are supplied and regulated by the blood in the microcirculation, namely, from the very small arterioles and the capillary bed. The main function of the mitochondria is to convert the potential energy stored in various substrates and its metabolites (e.g., glucose) into ATP. The inner membrane of the mitochondria contains five complexes of integral membrane proteins, including NADH dehydrogenase (Complex 1). Three of those proteins are involved in the respiratory chain activity. The main function of the respiratory chain is to gradually transfer electrons from NADH and FADH2 (originating from the Krebs cycle) to oxygen (O2). With the addition of protons (H+), water molecules (H2O) are generated in Complex 4. NADH is a substrate or a coenzyme for the enzymatic activity of dehydrogenases that form part of the respiratory chain and reside in the inner membrane of the mitochondria. Further details on the biochemical properties of NADH can be found in publications by Chance and his collaborators. The formation of ATP depends on the sufficiency of substrate (i.e., glucose) and oxygen supply to the tissue by the blood flow and hemoglobin oxygenation in the microcirculation (Fig.  1.19) as well as on the integrity of mitochondrial activity. Without sufficient supply of ATP, cells cannot function properly and can, ultimately, die. Since most of the energy consumed by tissues is dependent upon the availability of oxygen, under normal mitochondrial function, there is high coupling between oxygen supply and energy production. When mitochondrial activity is damaged, due to various reasons, this coupling is disrupted. The electron transfer (oxidation/ reduction) down the respiratory chain results in the production of adenosine tri-­ phosphate (ATP). Concomitantly with the electron transport, the respiratory chain components switch between reduced and oxidized states, each of which has different spectroscopic properties. Hydrolysis of the pyrophosphate bonds provides the energy necessary for the cell’s work. In order to assess the energy demand, it is necessary to measure different organ-specific parameters. For example, in the brain, the energy demand can be evaluated by measuring the extracellular levels of K+ that reflect the activity of the major ATP consumer – Na+/K+ATP’ase (Mayevsky and Chance 1982; Mayevsky 1984). In the heart, most of the energy is consumed by muscle contraction activity. On the other hand, the energy supply mechanism is the same for all tissues: oxygenated blood reaching the capillary bed releases O2 that diffuses into the cells. Therefore, it is possible to evaluate tissue energy supply by monitoring the following four parameters in all tissues: tissue blood flow (TBF), tissue blood volume (TBV), tissue oxyhemoglobin (HbO2) and mitochondrial NADH redox state (See Fig. 1.19). The production of available energy (ATP) depends on pO2 (partial oxygen pressure) in the various compartments of the tissue. Information regarding pO2 in the tissue, therefore, is helpful for the evaluation of tissue metabolic activity. Oxygen levels can be measured by oxygen electrodes; however, the information collected is an average of the compartments around the electrode. In the past, the sensitivity and accuracy of oxygen electrodes in the range of 1 mmHg (intracellular level) was not sufficient for the evaluation of mitochondrial function. As of today, the newly developed techniques could provide more accurate values of oxygen. Other recent

1.5  Mitochondrial Function and Metabolic States

29

published studies have suggested that intracellular oxygen levels are in a higher range (Springett and Swartz 2007; Wilson 2008; Pittman 2011; Harms et al. 2012; Schober and Schwarte 2012). The need for an intracellular pO2 indicator, as a physiological and biochemical parameter of living tissue, has emerged more than 60 years ago. Mitochondria are the intracellular organelles that consume most of the oxygen. Therefore, the redox state of electron carriers in isolated mitochondria in vitro as well as in vivo as a function of oxygen concentration has been extensively studied. Chance et al. concluded that “For a system at equilibrium, NADH is at the extreme low potential end of the chain, and this may be the oxygen indicator of choice in mitochondria and tissue as well” (Chance et al. 1973). Lubbers, in 1995, concluded that “the most important intrinsic luminescence indicator is NADH, an enzyme of which the reaction is connected with tissue respiration and energy metabolism” (Lubbers 1995). As of today, the ability to measure tissue energy metabolism at the microcirculation and cellular levels is not available for clinical applications although a device was developed.

1.5 Mitochondrial Function and Metabolic States As seen in Fig.  1.16, the mitochondrial NADH redox state represents the tissue oxygen balance (Mayevsky 1992; Rampil et al. 1992). At this stage, we were able to monitor simultaneously microcirculatory blood flow, blood volume, hemoglobin oxygenation, and mitochondrial NADH redox state. As we know today, the best monitored parameter that represents mitochondrial function in  vivo is the redox state of NADH; therefore, I will describe in this section the historical background of its monitoring under in vivo conditions. Chance et  al. (1973) reviewed the historical studies that made the connection between the activity of mitochondria and oxygen utilization as follows: “The accumulation of evidence since the pioneer work of Otto Warburg an ‘Atmungsferment’ (Warburg 1949) and David Keilin on cytochromes (Keilin 1925) as the keystones of cellular oxygen utilization led us to the study of the redox states of electron carriers in isolated mitochondria as a function of oxygen concentration and to develop techniques to measure the states of anoxia and normoxia in living tissues.” During the early 1950s, the study of mitochondrial activity in  vitro, and after 10  years in  vivo, was accelerated by the development of new optical monitoring devices. The UV-A light (Fig.  1.20c) was applied to measure NADH absorption (Theorell and Bonnichsen 1951; Theorell and Chance 1951; Chance 1952, 1953), and later on, the measurement of NADH fluorescence (Fig. 1.20a, b) was started (Duysens and Kroneberg 1957; Duysens and Amesz 1957; Chance and Baltscheffsky 1958). During those years, a large number of papers on the monitoring of NADH were published from the laboratory of Prof. B. Chance and he became the leader of in  vitro and in  vivo monitoring of NADH fluorescence. Figure  1.21 presents the pictures of the five cited scientists that affected significantly the development of the

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1  Introduction and Historical Background

Fig. 1.20  The fluorescence spectra (b) of the in vitro mitochondria (a) exposed to UV light. (c) the absorption spectra of NAD and NADH. (d) The definition of mitochondrial metabolic state, in vitro, in 1955, by Chance and Williams, opened up a new era in spectroscopic measurements of respiratory chain enzyme’s redox state in vitro as well as in vivo. (Chance and Williams 1956)

Fig. 1.21  The five scientists that enabled our understanding of mitochondrial function and NADH monitoring in vitro and in vivo situations

1.5  Mitochondrial Function and Metabolic States

31

theoretical and experimental technology for the monitoring of mitochondrial NADH function in vitro and in vivo. The detailed descriptions of the respiratory chain and oxidative phosphorylation in the mitochondria – published in 1955 by Chance and Williams – established our basic knowledge of the mitochondrial function (Chance and Williams 1955b, c, d, e; Chance et al. 1955). In those five papers, Chance and Williams defined, for the first time, the metabolic states of isolated mitochondria in vitro, depending on the substrate, oxygen, and ADP levels. In addition, they correlated those metabolic states to the oxidation-reduction levels of the respiratory enzymes, as seen in Fig. 1.20d. The physiological significance of those metabolic states was discussed in 1956 by Chance and Williams (1956). Detailed explanation of the table is given here as cited from the original paper by Chance and Williams (1956). In the case of the phosphorylating chain, the variety of steady states is much greater (see Fig. 5 and Table V in Chance and Williams 1956), and it is essential to define both the substrate and ADP levels in order to identify the steady-state condition of the mitochondria during the experiment (Chance and Williams 1955a, d). States 2, 3, 4, and 5 are the ones relevant to the present discussion and will be described very briefly. State 2 is a “starved” state in which ADP has been added in order to exhaust the endogenous substrate. Alternatively, the mitochondria are freed of endogenous substrate by any process that will increase the endogenous ADP level sufficiently to promote rapid respiration and exhaust the substrate. In addition, uncoupling agents soon lead to state 2. The mitochondria are rather fragile in this condition in agreement with the data on the rapid deterioration of mitochondria caused by lack of ATP. Spectroscopically, state 2 is important because the components of the respiratory chain become nearly completely oxidized, and this gives one of the three levels necessary for a measurement of percentage reduction in the steady state. State 5, like State 2, is characterized by zero respiration, but in this case oxygen is lacking, and instead of the components being oxidized, all those associated with the respiratory pathway are reduced, since reducing substrate was added in order to produce State 5. The ADP level will be high in State 5. State 4 is an aerobic state characterized by a low respiration rate – a “resting” state – even though substrate is present. Several components show large percentage reductions in this state, especially DPN. State 4 requires definite conditions for its establishment; mitochondria must be carefully prepared and be supplied with substrate and without phosphate acceptor. State 3 is the “active” state of rapid respiration and phosphorylation, with adequate supplies of substrate and phosphate acceptor. One aspect of State 3 that is of great significance in relation to thermo-dynamic aspects of phosphorylation (see below) is the steady-state level of the cytochrome is maintained constant while the ADP level changes over a wide range. Experimentally, these states are of great significance because they involve different oxidation-reduction levels for all the components of the respiratory chain and allow us to determine the nature of components of the respiratory chain involved in oxidative phosphorylation.

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An intensive use of the real time in vivo NADH monitoring approach started in 1962. The “classical” paper on in vivo monitoring of NADH was published in 1962 by Chance et al. (1962a). They were able to simultaneously monitor the brain and kidney of anesthetized rats using two microfluorometers. Chance and collaborators elaborated on this kind of in vivo monitoring and used it in other rat organs (Chance et al. 1962b, c; Chance and Schoener 1962).

References Acott, C. 1999. Oxygen toxicity: A brief history of oxygen in diving. SPUMS Journal 29 (3): 150–155. Altmann, R. 1890. Die Elementarorganismen Und Ihre Beziehungen Zu Den Zellen. Leipzig: Veit & comp. Anon. 1970. Carl Wilhelm Scheele (1742-1786) Swedish apothecary. JAMA 212 (13): 2258–2259. Barcroft, J. 1914. The respiratory function of blood. Cambridge: Cambridge University Press. Bean, J.W. 1945. Effects of oxygen at increased pressure. Physiological Reviews 25: 1–147. Benda, C. 1898. Ueber die Spermatogenese der Vertebraten und höherer Evertebraten, II. Theil: Die Histiogenese der Spermien. Archives of Analytical Physiology 73: 393–398. Bert, P. 1943. Barometric pressure: Researches in experimental physiology. [First published in French in 1878]. [Translated by: Hitchcock, M.A., Hitchcock, F.A. Columbus, OH, College Book Company]. Cassebaum, H., and J.A. Schufle. 1975. Scheele’s priority for the discovery of oxygen. Journal of Chemical Education 52: 442. Chance, B. 1952. Spectra and reaction kinetics of respiratory pigments of homogenized and intact cells. Nature 169: 215–221. ———. 1953. Dynamics of respiratory pigments of ascites tumor cells. Transactions. New York Academy of Sciences 16 (2): 74–75. Chance, B., and H.  Baltscheffsky. 1958. Respiratory enzymes in oxidative phosphorylation (VII – Binding of intramitochondrial reduced pyridine nucleotide). The Journal of Biological Chemistry 233 (3): 736–739. Chance, B., and B.  Schoener. 1962. Correlation of oxidation-reduction changes of intracellular reduced pyridine nucleotide and changes in electro-encephalogram of the rat in anoxia. Nature 195: 956–958. Chance, B., and G.R. Williams. 1955a. A method for the localization of sites for oxidative phosphorylation. Nature 176 (4475): 250–254. ———. 1955b. Respiratory enzymes in oxidative phosphorylation (I- Kinetics of oxygen utilization). The Journal of Biological Chemistry 217: 383–393. ———. 1955c. Respiratory enzymes in oxidative phosphorylation (II- Difference spectra). The Journal of Biological Chemistry 217: 395–407. ———. 1955d. Respiratory enzymes in oxidative phosphorylation (III- The steady state). The Journal of Biological Chemistry 217: 409–427. ———. 1955e. Respiratory enzymes in oxidative phosphorylation (IV- The respiratory chain). The Journal of Biological Chemistry 217: 429–438. ———. 1956. The respiratory chain and oxidative phosphorylation. In Advances in enzymology, ed. F.F. Nord, 65–134. New York: Interscience Publisher, Inc. Chance, B., G.R. Williams, W.F. Holmes, and J. Higgins. 1955. Respiratory enzymes in oxidative phosphorylation (V- A mechanism for oxidative phosphorylation). The Journal of Biological Chemistry 217: 439–451. Chance, B., P. Cohen, F. Jobsis, and B. Schoener. 1962a. Intracellular oxidation-reduction states in vivo. Science 137: 499–508.

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Chance, B., V. Legallias, and B. Schoener. 1962b. Metabolically linked changes in fluorescence emission spectra of cortex of rat brain, kidney and adrenal gland. Nature 195: 1073–1075. Chance, B., B. Schoener, and J.J. Fergusson. 1962c. In vivo induced oxidation by adrenocorticotrophic hormone of reduced pyridine nucleotide in the adrenal cortex of hypophysectomized rats. Nature 195: 776–778. Chance, B., N.  Oshino, T.  Sugano, and A.  Mayevsky. 1973. Basic principles of tissue oxygen determination from mitochondrial signals. In Oxygen transport to tissue, Advances in experimental medicine and biology, vol. 37A, 277–292. Dejours, P., and S. Dejours. 1992. The effects of barometric pressure according to Paul Bert: The question today. International Journal of Sports Medicine 13 (Suppl 1): S1–S5. Duysens, L.N.M., and J.  Amesz. 1957. Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region. Biochimica et Biophysica Acta 24: 19–26. Duysens, L.N., and G.H. Kroneberg. 1957. The fluorescence spectrum of the complex of reduced phosphopyridine nucleotide and alcohol dehydrogenase from yeast. Biochimica et Biophysica Acta 26 (2): 437–438. Ernster, L., and G. Schatz. 1981. Mitochondria: A historical review. The Journal of Cell Biology 91: 227s–255s. Fulton, J.F. 1843. Forward to Paul Bert’s book “Barometric Pressure”. Columbus: The F. J. Heer Printing Company; Bert. Haddad, S.I., and A.A. Khairallah. 1936. A forgotten chapter in the history of the circulation of the blood. Annals of Surgery 104 (1): 1–8. Harms, F.A., S.I.A. Bodmer, N.J.H. Raat, R.J. Stolker, and E.G. Mik. 2012. Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin. Optics Letters 37: 2625–2627. Jourdanet, D. 1875a. Influence de la pression de l’air sur la vie de l’homme. Nature 12 (309): 472–474. ———. 1875b. Influence de la pression de l’air sur la vie de l’homme. Paris: Mason. Keilin, D. 1925. On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants. Proceedings of the Royal Society of London – Series B: Biological Sciences 98: 312–339. Kellogg, R.H. 1978. “La Pression barometrique”: Paul Bert’s hypoxia theory and its critics. Respiration Physiology 34 (1): 1–28. Kolliker, A. 1856. Einige Bemerkungen uber die endigungen der Hautnerven un deu bau der Muskelzellen. Zeitschrift für Wissenschaftliche Zoologie 8: 311–325. Loukas, M., R. Lam, R.S. Tubbs, M.M. Shoja, and N. Apaydin. 2008. Ibn al-Nafis (1210-1288): The first description of the pulmonary circulation. The American Surgeon 74 (5): 440–442. Lubbers, D.W. 1995. Optical sensors for clinical monitoring. Acta Anaesthesiologica Scandinavica Supply 39 (104): 37–54. Marshall, J.L., and V.R.  Marshall. 2005. Rediscovery of the elements: Carl Wilhelm Scheele. University of North Texas Libraries, UNT Digital Library. Spring 2005; Indianapolis, Indiana. Crediting UNT College of Arts and Sciences. Mayevsky, A. 1984. Brain NADH redox state monitored in vivo by fiber optic surface fluorometry. Brain Research Reviews 7: 49–68. ———. 1992. Cerebral blood flow and brain mitochondrial redox state responses to various perturbations in gerbils. Advances in Experimental Medicine and Biology 317: 707–716. Mayevsky, A., and E. Barbiro-Michaely. 2013a. Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: I. Basic methodology and animal studies. The Journal of Clinical Monitoring and Computing 27: 1–34. ———. 2013b. Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: II: Human studies. The Journal of Clinical Monitoring and Computing 27: 125–145. Mayevsky, A., and B. Chance. 1982. Intracellular oxidation-reduction state measured in situ by a multicannel fiber-optic surface fluorometer. Science 217: 537–540. Miller, F.A. 1987. Joseph Priestley, preeminent amateur chemist. Journal of Chemical Education 64: 745.

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Mitzner, W., and E.  Wagner. 1992. On the purported discovery of the bronchial circulation by Leonardo da Vinci. Journal of Applied Physiology (Bethesda, MD: 1985) 73 (3): 1196–1201. Palmer, W. 2006. Carl Wilhelm Scheele (1742–1786): The life of a great chemist. In IPS-USA-2006 Internet conference on advances in the Internet, Processing, Systems and Interdisciplinary Research. Palo Alto, California, USA. Partington, J.R. 1962. The discovery of oxygen. Journal of Chemical Education 39: 123–125. Pittman, R.N. 2011. Oxygen gradients in the microcirculation. Acta Physiologica (Oxford, England) 202: 311–322. Priestley, J. 1775. Experiments and observations on different kinds of air. London: Johnson. Rampil, I.J., L. Litt, and A. Mayevsky. 1992. Correlated, simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation. Journal of Clinical Monitoring 8 (3): 216–225. Reti, I. 1952. Leonardo da Vinci’s experiments on combustion. Journal of Chemical Education 29 (12): 590–596. Retzius, G. 1890. Muskelfibrflle und Sarcoplasman. Biol Untersuch Stockholm NF 1: 51–88. Scheele, C.W. 1777. Chemische Abhandlung von der Luft und dem Feuer (Chemical studies on air and fire). Uppsala/Leipzig: Verlegt von Magn. Swederus Buchhandler, zu finden bey. Schober, P., and L.A. Schwarte. 2012. From system to organ to cell: Oxygenation and perfusion measurement in anesthesia and critical care. Journal of Clinical Monitoring and Computing 26: 255–265. Severinghaus, J.W. 2002. Priestley, the furious free thinker of the enlightenment, and Scheele, the taciturn apothecary of Uppsala. Acta Anaesthesiologica Scandinavica 46 (1): 2–9. ———. 2003. Fire-air and dephlogistication. Revisionisms of oxygen’s discovery. Adv. Exp. Med. Biol. 543: 7–19. ———. 2016a. Eight sages over five centuries share oxygen’s discovery. Advances in Physiology Education 40 (3): 370–376. ———. 2016b. The Most important discovery of science. In Oxygen transport to tissue XXXVII, Advances in experimental medicine and biology, ed. C.E.  Elwell, T.S.  Leung, and D.K. Harrison, vol. 876, 1–16. Smeaton, W.A. 1963. New light on Lavoisier: The research of the last ten years. History of Science 2: 51–69. ———. 1992. Carl Wilhelm Scheele (1742-1786): Provincial Swedish pharmacist and world-­ famous chemist. Endeavour 16: 128–131. Springett, R., and H.M. Swartz. 2007. Measurements of oxygen in vivo: Overview and perspectives on methods to measure oxygen within cells and tissues. Antioxidants & Redox Signaling 9: 1295–1301. Stefanadis, C., M. Karamanou, and G. Androutsos. 2009. Michael Servetus (1511–1553) and the discovery of pulmonary circulation. Hellenic Journal of Cardiology 50 (5): 373–378. Taylor, G. 2019. The chemical works of Carl Wilhelm Scheele. Ambix 66: 90–92. Theorell, H., and R. Bonnichsen. 1951. Studies on liver alcohol dehydrogenase I. Equilibria and initial reaction velocities. Acta Chemica Scandinavica 5: 1105–1126. Theorell, H., and B. Chance. 1951. Studies on liver alcohol dehydrogenase II. The kinetics of the compound of horse liver alcohol dehydrogenase and reduced diphosphopyridine nucleotide. Acta Chemica Scandinavica 5: 1127–1144. Warburg, O. 1949. Heavy metal prosthetic groups and enzyme action. T.  A. Lawson. Oxford: Clarendon Press. West, J.B. 2008. Ibn al-Nafis, the pulmonary circulation, and the Islamic Golden Age. Journal of Applied Physiology 105 (6): 1877–1880. ———. 2013. The collaboration of Antoine and Marie-Anne Lavoisier and the first measurements of human oxygen consumption. American Journal of Physiology. Lung Cellular and Molecular Physiology 305 (11): L775–L785. ———. 2014a. Carl Wilhelm Scheele, the discoverer of oxygen, and a very productive chemist. American Journal of Physiology. Lung Cellular and Molecular Physiology 307 (11): L811–L816.

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———. 2014b. Henry Cavendish (1731-1810): Hydrogen, carbon dioxide, water, and weighing the world. American Journal of Physiology. Lung Cellular and Molecular Physiology 307 (1): L1–L6. ———. 2014c. Joseph Priestley, oxygen, and the enlightenment. American Journal of Physiology. Lung Cellular and Molecular Physiology 306 (2): L111–L119. ———. 2017. Leonardo da Vinci: Engineer, bioengineer, anatomist, and artist. American Journal of Physiology. Lung Cellular and Molecular Physiology 312 (3): L392–L397. West, J.B., and J.P. Richalet. 2013. Denis Jourdanet (1815–1892) and the early recognition of the role of hypoxia at high altitude. American Journal of Physiology. Lung Cellular and Molecular Physiology 305 (5): L333–L340. Wilkinson, D.J. 2004. Resuscitation great. The contributions of Lavoisier, Scheele and Priestley to the early understanding of respiratory physiology in the eighteenth century. Resuscitation 61 (3): 249–255. Williams, K.R. 2003. The discovery of oxygen and other priestley matters. Journal of Chemical Education 80: 1129–1131. Wilson, D.F. 2008. Quantifying the role of oxygen pressure in tissue function. The American Journal of Physiology-Heart and Circulatory Physiology 294: H11–H13.

Chapter 2

Basic Concepts of Brain Monitoring Systems

Abstract  The basic principles and technology of NADH monitoring was discussed in more details in the book published by Mayevsky in (2015). Nevertheless, in this chapter, the more specific details of NADH monitoring during exposure to hyperbaric oxygenation are presented. In addition to the monitoring of mitochondrial NADH, this chapter will present the development of the multiparametric monitoring system that provides real-time information on the cerebral hemodynamic, ionic, and electrical activities during CSD. The various combinations of multiparametric assemblies will be discussed in details. Keywords  NADH fluorometry · Ionic homeostasis monitoring · Brain oxygen level monitoring · Hyperbaric brain oxygenation · Oxygen toxicity In studying the effects of hyperbaric oxygenation, we used a few monitoring systems developed in our laboratory since 1973. In the early stages, we monitored in the hyperbaric chamber the EEG and the mitochondrial NADH redox state using surface fluorometry/reflectometry. Later on, we added a few more parameters to the monitoring system and developed the multiparametric assembly that enables to correlate the hemodynamic, metabolic, ionic, and electrical parameters while monitoring the brain in real time inside the hyperbaric chamber.

2.1 Mitochondrial NADH Fluorometry As described in Chap. 1, NADH could be measured by utilizing its absorption spectrum in the UV range, as well as by the blue fluorescence spectrum emitted under UV illumination. In the early stages, NADH monitoring was based on the difference in the absorption of NADH and NAD+. At the range of 320 nm to 380 nm, only the reduced form – NADH absorbs light, while NAD+ does not (Fig. 1.20c). Therefore, when a mixture of NADH and NAD+ is illuminated in a cuvette by 320–380 nm,

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Mayevsky, Hyperbaric Oxygenation, https://doi.org/10.1007/978-3-031-49681-3_2

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only NADH will affect the absorption spectrum peak at 340 nm. This property of NADH was used in the early 1950s by a number of investigators, as reviewed in Sect. 1.4. Chance and collaborators utilized this technique to measure NADH in muscle homogenates or intact cells (Chance 1952) and published a large number of papers concerning the unique absorption spectrum of NADH.  The absorption approach is not practical for measuring NADH in a thick tissue; hence another property of NADH was used. Since the early 1950s, fluorescence spectrophotometry of NADH has been employed in various in vitro and in vivo models. The emission of NADH fluorescence, under illumination at 320–380 nm, has a very wide spectrum (420–480) with a peak at 450–460 nm (Fig. 1.20b). NADH fluorescence has been identified by Chance and his collaborators as a good indicator of the intramitochondrial oxidation-reduction state (Chance et al. 1973). Since the early 1950s, different groups have developed a number of fluorometers adapted to their specific experimental protocols. As of today, most of the groups using NADH fluorometers construct the devices in-house, since no suitable commercial products are, in fact, available. During the past four decades, the workshop of the Johnson Research Foundation at the University of Pennsylvania Medical School (Philadelphia, PA, USA), headed by Prof. Britton Chance, has manufactured a few types of laboratory instruments purchased and used by various investigators (Chance et al. 1975). The basic features of NADH fluorometers consist of the following components: 1 . A light source (including appropriate filters). 2. An optical path to the preparation and back to the detection unit. 3. Detection and signal processing units. 4. Signal recording and storage units. In our earlier review published in 1984, we extensively specified the light-guide-­ based fluorometry used in our studies (Mayevsky 1984a). The effect of blood on NADH fluorescence was discussed early by Chance et al. (1962). In order to monitor NADH in vivo, Chance’s group had to avoid areas containing large blood vessels which interfere with the emission and excitation light. The monitoring of a second channel in tissue fluorometry in vivo was reported by Chance et al. (1963). They showed that “changes due to the deoxygenation of oxyhemoglobin do not interfere with measurement of the time course of fluorescence changes in the tissue studies.” The addition of a second monitoring signal, namely, tissue reflectance at the excitation wavelength was reported by Jobsis and Stainsby (1968). It was based on a previous model described by Jobsis et al. (1966). In another two papers by Jobsis and collaborators (1971a, b), the measurement of 366 nm reflectance was used for the correction of the NADH fluorescence signal from the brain. The reflectance signal was subtracted from the fluorescence signal.

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2.1.1 Fiber Optic Fluorometer/Reflectometer In order to enable the monitoring of NADH fluorescence in unanesthetized animals or other in vivo preparations, a flexible means was needed to connect the fluorometer with the tested organ, for example, the brain. This was achieved in 1972 when UV-transmitting quartz fibers became available (Schott Jena Glass, Germany). We have used the light-guide-based fluorometer for in  vivo monitoring of the brain (Chance et al. 1973; Mayevsky and Chance 1973) subjected to anoxia or cortical spreading depression. The historical development of light-guide-based fluorometry–reflectometry is shown in Fig. 2.1. The original device functioned on the time-­ sharing principle (Fig. 2.1a), where four filters were placed in front of a 2 arms light guide. Filters 1 and 3 enabled the measurement of NADH fluorescence, while filters 2 and 4 were used to measure tissue reflectance at the excitation wavelength. The reflectance trace was used to correct the NADH signal for hemodynamic artifacts, and to indicate changes in the blood volume of the sampled tissue. In this original system, only one photomultiplier tube was used for the detection of the two signals. Figure 2.1b presents one of the first in vivo brain monitoring time-sharing setups, connected to the brain of an anesthetized rat (Mayevsky and Chance 1975, 1976). In order to simplify the monitoring system, the time-sharing approach (AC mode) was replaced by splitting the light emitted from the tissue into two unequal fractions for the measurement of fluorescence and reflectance signals. This model, named the DC type fluorometer, had originally a 3-way light guide, which was later replaced by a two arms light guide probe. In all the configurations, the reflectance signal was used for the correction of the fluorescence signal. The model shown in Fig. 2.1 was used to study the brain (Mayevsky and Chance 1973, 1974, 1975, 1976; Mayevsky 1975, 1976; Mayevsky et al. 1993) (Fig. 2.2). Other groups used optical fibers to connect the monitored tissue to the fluorometer differently than the models shown in Figs. 2.1a and 2.3a. Photomultiplier

B Time Sharing Fluorometer/Reflectometer

Water Cooled Hg Arc

Emission and Reflectance Light Guide (450nm and 366nm)

Excitation Light Guide 366nm Air Line (20 psi)

Brain Cannula

Fig. 2.1 (a) Time-sharing fluorometer/reflectometer connected to a rat brain cannula by a flexible y shape light guide (Mayevsky 1984a). (b) Picture of the time-sharing fluorometer reflectometer used in the 1970s. The rat brain is connected to the device by a flexible fiber-optic probe (Mayevsky 1984a)

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Fig. 2.2 (a) The effects of anoxia on NADH fluorescence, 366 nm reflectance, EEG, and blood pressure. SB, the animal stopped breathing; SN, stop nitrogen; AR, short artificial respiration (Mayevsky and Chance 1975). (b) The repetitive response of the brain to spreading depression evoked by application of 0.4 M KCI on the dura. The third trace is on an expanded amplitude scale. The arrow direction shows an increase in the optical signals. Time proceeds from left to right (Mayevsky 1984a)

2.1.2 Factors Affecting the Monitored Signals The excitation and emission spectra of NADH are affected by the redox state of this fluorochrome and by other factors, leading to artifacts in the fluorescence measurements. In this section, various NADH unrelated factors affecting the measured signal will be discussed. Since most fluorometers involve the measurement of the total backscattered light at the excitation wavelength (i.e., 366 nm), the discussion will concern changes in NADH fluorescence as well as in tissue reflectance.

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Fig. 2.3 (a) The structure of the standard DC fluorometer/connected to the brain of a small animal (Mayevsky 1984a). Excitation(right), Emission(left) – optical fibers for the monitoring of NADH redox state. H.V. high voltage, PM photomultiplier. (b) The effect of the monitored tissue volume (the diameter of the fiber-optic probe) was tested under anoxia, as shown in B, upper 3 traces (2 mm diameter) and lower 3 traces (1 mm diameter) (Mayevsky 1984a)

The following factors may affect the two measured signals, 366 nm reflectance (R) and 450 nm fluorescence (F): 1 . Tissue movement due to mechanical or intracranial pressure changes in the brain. 2. Extracellular space events, such as volume changes or ion shifts between intraand extra-cellular spaces. 3. Vascular and intravascular events, for example, oxy-deoxy Hb changes and blood volume changes due to autoregulatory vasoconstriction under pathological conditions. 4. Intracellular space factors, such as O2 level, ATP turnover rate, substrate availability, and mitochondrial redox state. This subject will be discussed later on. In order to compensate for the various unrelated factors affecting the NADH fluorescence signal, various approaches have been developed. In the paper published by Ince et al. (1992), the various correction techniques were listed and discussed in detail. It appears that the correction technique is also dependent on the instrument configuration (Harbig et al. 1976; Balaban et al. 1980). Most of the published materials are based on the 1:1 correction factor or ratio, when subtracting the 366 nm reflectance signal from the fluorescence signal. In order to determine the “correction factor” value, the use of saline flushes toward the brain was the main approach. The results obtained in the cat brain are shown (Harbig et al. 1976). When a bolus of saline is injected into the brain via the carotid artery, a fast response in the reflectance and fluorescence signals is recorded.

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A similar study was performed in the rat model by our group (Mayevsky 1978). The saline flushes were done into the left carotid artery and the response was recorded in the left hemisphere. The correlation between the changes in the R and the F signals measured. As of today, a new approach is still lacking to compensate for non-NADH factors affecting the NADH fluorescence signal. Bradley and Thorniley (2005) published a review article dealing with the various correction techniques for tissue fluorescence. They summarized their review by the following conclusion: “even though research has been conducted into correction techniques for over thirty years, the development of a successful and practical correction technique remains a considerable challenge.”

2.1.3 Preparation of the Brain for NADH Monitoring Using of the optical technique for monitoring of NADH Fluorescence requires a stable contact between the fiber-optic probe and the tissue. Movement artifacts will affect the stability of the monitored signals. In order to achieve this stability, we developed the following technique of cementation that was applied to the brain, as shown in Fig. 2.4.

Fig. 2.4  Stages in the preparation of the rat brain for NADH monitoring. (a) Location of screws enabling the fixation of the light guide holder to the skull by dental acrylic cement. (b) The light guide holder located above the brain surface. (c) The view of the skull after the end of the operation. (d) After the insertion of the fiber-optic probe to its holder, the animal is ready for the monitoring (Mayevsky and Barbiro-Michaely 2013)

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A special table and probe-holding device was constructed in order to perform brain preparation for the monitoring period. The brain is operated while the head is connected to a special head holder for the period of the operation (20–30 min) and then could be released, for the monitoring period, as shown in Fig. 2.4. The brain was the main organ monitored by our group. In order to be acquainted with the NADH monitoring device, it is recommended to start with brain monitoring and later on to move and use the selected organ. The reason for it is that the connection between the fiber-optic probe and the monitored tissue must be constant during the monitoring session, and in the brain, it is easily achieved by connecting a probe holder to the skull using acrylic cement, as shown in Fig. 2.4c, d. A midline incision was made in the skin, exposing the skull. Three holes were drilled in the skull and appropriate small screws were inserted into the skull (less than 1 mm in depth), as shown in Fig. 2.4a. An appropriate hole (3–5 mm in diameter) was drilled in the right or left parietal bone for the fixation of a light guide holder in which the monitoring probe was inserted later on. The light guide holder and the three screws were then fixated to the skull using dental acrylic cement (Fig. 2.4c). Ten minutes later, the head of the animal was released from the head holder and the probe was inserted to a predetermined depth and fixed by a set screw (Fig. 2.4d). The structure of the standard DC fluorometer/reflectometer connected to the brain of a small animal is shown in Fig. 2.3a. Various types of light guide holders are presented (Mayevsky 1984a). The thread outside the bottom of this cannula enabled screwing it into the skull and also gave a better connection between the cannula and the cement. The cannula shown in Fig. 2.4b was used in experiments in which animals were exposed to nitrogen/oxygen-breathing cycles or to hyperbaric pressure of oxygen. The EEG was measured between the two electrodes, 1–2, cannula type B was used in all experiments in which spreading depression was elicited by application of KCl solution above the Dura (Mayevsky and Chance 1975). The KCl electrodes 1, 2–3, 4. The third type, C, has two compartments at the bottom, the small one for KCl application 3–4 and the large compartment, 1–2, where chemicals such as Metrazol were applied and were affecting a larger area. A fifth electrode was located at 180° to electrodes 3–4, so the EEG was measured at the same time (Mayevsky and Chance 1975). Almost all publications on brain NADH were related to the surface of the brain. Monitoring deep structures of the brain is possible, but the insertion of the probe into the brain causes injury to the monitored brain tissue. Therefore, it is recommended to start the monitoring of the brain after appropriate time (at least 30 min) needed for the recovery of the tissue. We have used a short anoxic episode (20–30 sec) in order to test the intactness of the tissue. If the NADH response to anoxia was too small, we stopped the experiment, because it indicated that the brain was damaged and NADH was already elevated.

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2.2 Multiparametric Monitoring Systems 2.2.1 Introduction Evaluation of brain physiological functions, in real time, could be done using various methodological approaches that could be classified according to the invasiveness of the technologies used. The noninvasive group includes most of the techniques that are used in patients on a daily basis. In this approach, the sensors may touch the skin but will not penetrate the skull. This group could be subdivided into the mapping/imaging option or the local measurement approach. In the second group, the sensors may be located epidural (below the bone) or subdural touching the pia mater. In the third approach, the sensor or the probe is introduced into the tissue itself, creating a new microenvironment and some damage. I introduce here the term “physiological mapping” as presented in the minimally invasive group named MPA or the Multi Parametric Approach or Multi Parametric Assembly. The MPA was developed mainly to study the brain, but the same MPA concept and technology was used in other organs such as the heart and kidney. The aim of the MPA is to provide real-time data describing the relationship between hemodynamic, metabolic, ionic, and electrical activities in the cerebral cortex. Normal brain mitochondrial function is a precondition for the performance of all other brain functions; therefore, a short introduction on brain energy metabolism is presented here. The aim of this chapter is to demonstrate the historical development of the technology used in monitoring the brain and other organs’ functions under various pathophysiological conditions. We are presenting the stages of the development and typical results obtained in our laboratory. It’s important to note that in our monitoring system, all the probes were placed on the surface of the brain and never penetrated the tissue itself. Most of the references cited in this chapter were published by our group. We were interested in the microenvironment of the brain containing neurons, glia, synapses, and the microcirculatory elements (small arterioles and capillaries). The various parameters and the technology developed are presented in Fig.  2.5. During the development process, we pursued the goal of being minimally invasive in terms of the penetration to the cortical tissue itself. It was obvious that the various probes could not monitor the same volume of tissue due to the size of each probe used. Therefore, we attempted to minimize the diameter of the various probes located in the MPA that had a 5–6 mm contact area within the cerebral cortex. In most of the perturbations used, such as global ischemia, anoxia, hypoxia, or hemorrhage, most of the areas in the cortex will respond in the same way. The initial step in the development of the MPA was the establishment of the fiber-optic-based NADH monitoring system in 1972 when the first UV-transmitting optical fibers appeared. It was a continuation of the long-term usage of old devices for NADH monitoring in vivo, where the animal was located in an optic-based rigid device. The connection of the brain to the fluorometer via optical fibers enabled us to monitor, for the first time, the brain of unanesthetized animals. The initial data on the use

2.2  Multiparametric Monitoring Systems

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Brain Physiological Mapping NADH Fluorescence

Mitochondrial Function

ElectroCortico Graphy (ECoG)

Cerebral Blood Flow and Volume

Microcirculatory Hemodynamic Hemoglobin Saturation

Electrical Activities

Tissue Oxygenation

DC Steady Potential

Extracellular K+

Tissue pO2

Ionic Homeostasis

Fig. 2.5  Schematic presentation of the concept “Brain Physiological Mapping” (Modified Mayevsky 2015)

of this technology appeared in two papers (Chance et  al. 1973; Mayevsky and Chance 1973). All details of the technological aspects and animal preparation appear in the original relevant publications; therefore, a short description of technology relevant to each parameter will appear in the initial part of the methods section. In the results section, typical responses to various types of perturbations will be presented together with the technology used in the specific study. Our approach was to develop a new upgraded version of the monitoring system and presenting initial preliminary results. The next step was to run a large well-designed study on few groups of animals and the data was quantitated and analyzed for its statistical significance. In order to save space, we are presenting here only typical results collected during the developmental stage.

2.2.2 Methods 2.2.2.1 NADH Monitoring NADH can be measured by utilizing its absorption spectrum in the UV range, as well as by the blue fluorescence spectrum under UV illumination. All details regarding the monitoring of NADH appear in Sect. 2.2.1.

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2.2.2.2 Local Cerebral Blood Flow (CBF) To measure in real time the CBF from the same cortical area as the MPA location, we used the LDF technique (Dirnagl et al. 1989; Haberl et al. 1989; Wadhwani et al. 1990). The LDF measures relative flow changes, and readings have been shown to correlate with the relative changes in CBF measured by the two other quantitative approaches (for a review, see Wadhwani et al. 1990). The principle of the LDF is to utilize the Doppler shift, namely, the frequency change that light undergoes when reflected by moving red blood cells. A beam of low-power or diode laser light is transmitted by an optical fiber to the tissue. After the multiple scattering of the light, another optical fiber picks up the reflected light that is recorded by a photo detector. The run signal is analyzed by a complicated algorithm developed by the manufacturers, and the results are presented in percentage of a full scale (0–100%), thereby providing arbitrary relative flow values. The change in the total back-scattered light is an indirect measure of the blood volume in the sampled tissue. To quantify and normalize CBF values, we defined the reading value after death as 0 CBF. The 100% value was defined as percent CBF read on the LDF scale during the control period. 2.2.2.3 Oxygen Electrodes The electrodes were constructed inside 1.6 mm (OD) polyethylene tubing (PE-160). Platinum wire (25 micro meters) was sealed in glass by a flame, and an insulated lead wire was attached to the other end of the platinum wire. Two assemblies, along with Teflon-coated 250-micron silver wire (for use as a reference) were pulled into the PE tubing so that the glass-sealed ends of the platinum were flush with the end of the PE tubing. The Teflon at the end of the silver wire was removed, and this bare section could extend beyond the edge of the PE tubing. The electrode’s zero response was tested in saline bubbled with N2, and its linearity was tested in saline bubbled with different mixtures of O2 and N2. A cellulose diacetate membrane was put on the tip by dipping the electrode in 5% cellulose diacetate solution. The calibration and calculation of pO2 in the brain are only a relative term, since the absolute value of pO2 is very sensitive to the electrode location on the brain. 2.2.2.4 Ions-Selective Electrodes and DC Potential To monitor the extracellular levels of K+, Ca2+ and H+, we used specially designed mini electrodes made by World Precision Instruments (WPI; Sarasota, FL). A flexible tubing made of polyvinyl chloride was sealed at one end with a membrane sensitive to a specific ion. The tube was filled with the appropriate solution and connected to an electrode holder with a salt bridge between the membrane and an Ag– AgCl pellet located inside the holder. The interface between the polyvinyl chloride

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tubing and the holder was glued with epoxy, and such electrodes were usable for a few weeks. The sensitivity of the electrodes to the specific ion was close to the Nernstian value, namely, 50–60  mV/decade for K+ and H+ or 25–30  mV/decade for Ca2+. DC potential was measured concentrically around the ion selective electrodes. Each electrode had a saline bath around its perimeter, and an Ag–AgCl electrode (WPI) was connected to it. 2.2.2.5 Reference Electrode An Ag–AgCl electrode connected via a saline bridge to the neck area of the animal was used as the reference electrode. Polyethylene tubing stuffed with a cotton string that expanded when wet was inserted into the electrode holder (WPI) and glued with 5-min epoxy. 2.2.2.6 Electrocorticography – ECoG Spontaneous electrical activity of the brain surface was measured by two polished stainless steel rods or silver wire inserted into the MPA. 2.2.2.7 Temperature Measurements Brain temperature was measured with a thermistor probe) Yellow Springs Instruments) located inside the MPA and connected to a thermometer. 2.2.2.8 Data Collection and Analysis Relative CBF  The LDF provided arbitrary units (blood flow and volume) that were calibrated in relative terms compared with the normoxic brain that served as the control (0–100% range). NADH Redox State  Measurements of NADH provided a method for evaluating the mitochondrial redox state. The normoxic NADH level is considered to be 100%, and the changes due to the treatment given were calculated in comparison with the normoxic level. Mayevsky (1976) has shown in the past that the NADH redox state correlates well with the brain’s functional activity. The origin of the NADH signal in our system is mainly mitochondrial (for details, see Mayevsky and Rogatsky 2007), whereas the cytoplasmic NADH contribution to the signal is negligible. Nevertheless, because the NADH is not measured in a purified form, we prefer to label the trace in the records as corrected fluorescence (CF).

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Multiparametric Calculation  The MPA provides analog signals of electrical activities and NADH redox states as well as extracellular concentrations of K+, H+, and Ca2+. The mV values measured by the electrodes were transformed to mM values by using the standard calibration technique (Friedli et al. 1982) and the principles of the Nernst equation. All calculations were performed via special software programs. Real-Time Data Acquisition  Because of the large number of parameters measured and the need for the logarithmic transformation of the data from the K+, Ca2+, and H+ electrodes, it was necessary to use a computerized system for data acquisition and storage. Furthermore, to record all the analog signals (at least 16 channels), it was more appropriate to use a computer display instead of the paper capacity for continuously collecting information. 2.2.2.9 Animal Preparation for Monitoring All experimental protocols were approved by the institutional animal care committee under the instruction of the National Institutes of Health. The experimental procedures were detailed previously (Mayevsky and Chance 1982; Mayevsky 1984a). In order to demonstrate the performance of the various monitoring devices, we used male rats and male Mongolian gerbils. The preparation of the animals for monitoring is presented in Sect. 2.1.3.

2.2.3 Results and Interpretation In this section, we will present and discuss the stages of the monitoring system development and data collected by these systems. 2.2.3.1 Fiber Optic-Based Fluorometer and EEG During the initial step, we developed the fiber optic-based fluorometer/reflectometer for monitoring of mitochondrial NADH in anesthetized or unanesthetized animal. Various types of fiber-optic probes were developed during the years. In order to keep a direct and constant contact between the brain and the probe, specially designed holders were developed during the years. The first fiber-optic probe was described in 1973 (Mayevsky and Chance 1973; Mayevsky 1984a), and the other types were presented in our review paper (Mayevsky 1984a). After the end of the operation, the probe was inserted into the holder that was cemented to the skull. Figure 2.2 shows the typical two responses obtained in a rat experiment. As can be seen, we monitored two signals by the Fluorometer, namely, the NADH fluorescence at 450  nm and the total back-scattered light at the excitation wavelength

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(366 nm) called Reflectance. In addition to NADH, we monitored EEG activity by placing two stainless steel electrodes on the brain surface. In Part A, the complete elimination of oxygen led to a large increase in the NADH and the EEG signal disappeared very fast. In this animal, the change in the reflectance was very small as compared to most of the rats. As soon as the rat started to breathe air, the NADH level recovered very fast. In part B, the effect of brain activation induced by Cortical Spreading Depression (CSD) is shown. In this response, the Blood volume changes concomitantly with the mitochondrial NADH.  The NADH redox state was shifted to more oxidized state due to the increase in ATP demand. The CSD developed only in the stimulated hemisphere, as seen in the ECoG, while the contralateral hemisphere served as a control (results are not presented). The same monitoring system was used in other studies (Mayevsky 1984a). 2.2.3.2 The Addition of K+ Monitoring The next step in the development of the MPA was to measure extracellular potassium levels representing the oxygen demand processes. In the initial stage, we used the microelectrode for potassium available in the early 1970s. In 1972, Vyskocil et al. used a potassium selective microelectrode and showed the leakage of K+ from the cells during the wave of CSD (Vyskocil et al. 1972). In order to correlate the responses of NADH and ECoG to changes in extracellular K+, we adopted two approaches. Initially we used a double-barreled microelectrode (Zeuthen 1971; Zeuthen et al. 1974), seen in Fig. 2.6a, inserted 1 mm into the cerebral cortex of a rat (Mayevsky et  al. 1974b). This technology was based on the development of potassium-sensitive ion-exchanger (Walker 1971) filled in one barrel, while the other barrel was filled with 2 M KCl for the measurement of DC steady potential. Typical recording of the response to CSD presented in Fig. 2.6b was made with a double-barreled microelectrode in the cortical tissue. The waves recorded with the microelectrode were more sharply defined, with fast rise times, fast signal-decay times, and a short duration. We combined the measurement of NADH, ECoG, DC potential, and extracellular K+ from the same hemisphere (Mayevsky et  al. 1974b). This electrode was inserted into the cerebral cortex while the NADH was placed on the surface. The results of such an experiment (Mayevsky et al. 1974b) are shown in Fig. 2.7 As can be seen, the leakage of potassium stimulated the energy metabolism recorded as the oxidation of NADH (state 4 to state 3 transitions). In order to minimize the invasiveness of the potassium electrode, we developed a surface electrode for potassium located on the brain in the same configuration as the NADH probe. The technical details of the preparation of the surface potassium electrode, seen in Fig.  2.8a, were provided by Crowe et  al. (1977). The typical response to CSD is shown in Fig. 2.8b. In order to measure the extracellular potassium in the rat brain, it was necessary to remove the dura mater since the diffusion

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Fig. 2.6 (a) Schematic presentation of the microelectrodes used in order to measure brain extracellular K+ and DC steady potential in a rat model. Reprinted with permission by the first author T. Zeuthen 1979 (Zeuthen et al. 1979). (b) Microelectrode recording from brain in CSD using a two-barrel microelectrode and separate ECoG electrodes (Crowe et al. 1977)

of the ion through the dura was very slow and undetectable (Crowe et al. 1977). As can be seen, the responses to CSD were recorded by the two types of electrodes used. 2.2.3.3 NADH and pO2 Measurements Since Mitochondrial NADH is sensitive to intracellular levels of oxygen, we had developed an MPA that contained a surface oxygen electrode in combination with NADH and ECoG (Fig. 2.9a). It is important to note that oxygen electrode readings are averaging the oxygen level in the vascular, extracellular, and intracellular compartments (Mayevsky et al. 1980b). Typical results of this MPA are presented in Fig. 2.9b. The response to anoxic episode, shown in Fig. 2.9b, is typical to the lack of oxygen, namely, a sharp drop in pO2 and a large increase in NADH. At the recovery phase, the pO2 response to CSD has a unique nature. One minute after breathing was started, a biphasic response was recorded inversely related to the reflected light changes, namely, that

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Ref Flu

1 min

NADH EEG Right K+E

K+DC KCl 0.6M

NaCl 0.85%

Fig. 2.7  The effect of cortical spreading depression (elicited by 0.6 M KCI) on the extracellular potassium, DC potential, ECoG, and NADH fluorescence of the exposed rat brain cortex. The potassium and the DC steady potential were measured by microelectrodes (Mayevsky et al. 1974b)

Fig. 2.8 (a) Representation of a combined electrode for extracellular K+, DC steady potential and Electro corticogram – ECoG leads, from the brain surface (Mayevsky et al. 1980a). (b) Recording made using the combined surface electrode during cortical spreading depression (Crowe et al. 1977)

when the reflectance showed an increase, the pO2 trace showed a parallel decrease; and when the reflectance showed a large decrease (increased blood volume), the pO2 showed three- to four-fold increase and then gradually recovered to the normoxic level in parallel to the gradual increase in the reflected light. The biphasic response in the pO2 trace is in correlation to the response of the NADH fluorescence trace and we assume that it is the typical response to CSD, as described previously (Mayevsky et al. 1980b) after anoxic cycle.

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A

B

Ref Flu NADH PO2 EEoG N2 AIR

Fig. 2.9 (a) Schematic presentation of the various probes locations above the brain of the gerbil. The large cannula contained the light guide for NADH measurement, the pO2 and ECoG electrodes. The cannula for KCl application was located 2–3  mm anterior to the large cannula (Mayevsky et al. 1980b). (b) The effects of anoxia on the metabolic and electrical activity of gerbil brain (Mayevsky et al. 1980b)

2.2.3.4 The First Multiparametric Monitoring System Our aim was to provide a new holding system allowing an easy implantation of surface probes in various combinations on the brain of small mammals and to reevaluate the potential of such surface electrode assemblies in tracing physiological and pathological events (Friedli et al. 1982). In this stage, we added to the basic assembly system the measurements of extracellular tissue pH and K+, DC potential, and local temperature, in addition to the other parameters, including NADH fluorescence, reflected light, and electrocorticogram (ECoG). Ideally, the multiprobe assembly designed (Fig. 2.10a) for our project had to be rugged enough to withstand routine use by nonspecialists and yet be miniaturized enough to fit the limited surface available for implantation on rat and gerbil skulls (about 6–7 mm in diameter on each hemisphere). For ischemic studies, our interest was focused on monitoring mean values over large areas, suggesting the use of larger electrodes. In contrast, following propagated events such as spreading depression would have required the tip of the multiprobe assembly to be concentrated in a tiny space, perceiving the same phase of the wave, unless each sensor could be located with respect to the wave front by an auxiliary signal. The second option was retained by recording the wave of the DC potential concentrically to each sensor. In addition, the complete assembly had to provide adequate protection and shielding of the high impedance ion-sensitive electrodes (K+ and pH) and stable enough reference junctions for use in unanesthetized animals. With K+ surface electrodes, concentric DC potential has been proposed as the best approximation for the DC potential component to be subtracted from the sensor signal (Crowe et al. 1977). In this study, we also attempted to check the accuracy of the method by recording DC potential differentially between a central barrel and the peripheral slit and to reevaluate its usefulness in correcting surface K+ and pH measurements under various conditions.

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Fig. 2.10 (a) Schematic presentation of the multiprobe assembly used in our study. Left: longitudinal section of cannula and assembly located above cortex. A small push-pull cannula was placed near assembly to initiate spreading depression. Lower right: topical view of gerbil skull together with location of various electrodes. Top right: cross section of wide end of cannula (Friedli et al. 1982). (b) Effects of anoxia (left) and ischemia (center) on metabolic, ionic, and electrical signals measured from a slightly anesthetized gerbil. Spontaneous spreading depression cycle was developed after ischemic episode. In this experiment, we recorded corrected fluorescence alone (CF) without uncorrected fluorescence and reflectance. For abbreviations, see text (Friedli et al. 1982)

Electrode Assembly  The solution adopted for the electrode holder is basically a modification of the Lucite cannula described by Mayevsky et  al. (1980a) for the light guide and the potassium-sensitive electrodes. To offer space for more probes, the new cannula was shaped as a truncated cone instead of a cylinder (Fig. 2.10a). The holes accommodating short electrode probes (K+, pH, and pO2) are made convergent toward the lower surface to occupy less space on the brain and divergent at the top to facilitate handling and sealing of the probes. An additional hole is drilled obliquely from the upper surface to merge with each sensor channel at about mid-­ distance of the lower surface. This hole accommodates an AgCl wire, used to record the local DC potential concentrically to the sensor. The long and rigid steel stem of the light guide (L) used in this study occupies a straight vertical hole in the cannula and serves as an axis to hold the cannula (c) and the cable holder (h) at a convenient distance of each other (4 cm). Steel rods (preferentially threaded) can be used as additional or replacement pillars to fix the cannula to the cables holder. The arrangement leaves optimal access to the electrodes and electrical connections for assembling and replacement. The complete assembly is protected and shielded by a silver-pointed Lucite sleeve sliding over the cable holder. If a stronger construction is desired, the sleeve can also be permanently screwed into the cannula and cable holder, with a half-cylinder piece cut out as a removable cover. When the holder is assembled, the fixed steel pillars are screwed and/or glued in first. To protect the solder point, chloritized silver wires are glued with two-­ component epoxy glue in short pieces of Teflon that fit over the sensor electrode shaft and into the DC channel in the cannula. The Teflon-sleeved wires and glass tubes of the sensors are then fixed in place with hard Elephant wax and

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polyethylene-sleeved probes (Yellow Springs Instrument temperature probe 5  U) with paraffin melted by a thermocautery tip. To avoid tension on the electrodes wires, they are connected to the input cable through a flexible coil of 36-gauge isolated copper wire (Belden). Figure 2.10b shows a typical response of the gerbil brain to anoxia (left) and bilateral carotid occlusion (right). The initial response to anoxia is an increase of the NADH level as shown in the corrected fluorescence (CF) trace. As a result, the extracellular level of K+ increased and returned to the baseline level after restoration of air breathing. The pH increased under anoxia, the DC potential measured in all three electrodes site (DCH+, DCK+, and DCF) did not show any change during the anoxic cycle, and the ECoG showed a typical depression. After occlusion of the right carotid artery (Rocc), the NADH increased by 10% compared with the normoxic level and reached only one-third of the maximal NADH level, measured during the anoxic cycle or bilateral carotid occlusion. The partial ischemia induced by Rocc did not cause any other significant changes during the short period of occlusion. After complete ischemia was induced by occlusion of the left artery (Locc, while the right one was occluded), the NADH showed a 30% increase and returned to the baseline level after both left and right arteries were reopened. During the period of complete ischemia, extracellular K+ level went up and the pH went down (more acidic). A spontaneous CSD developed during the complete ischemia, and the wave propagated to the measuring site after the two carotid arteries were reopened. During the CSD event, typical responses were recorded; namely, K+ was elevated and then pumped back into the cells, and as a result the NADH became more oxidized (oxidation cycle). The pH showed an acidification response to the CSD. As expected, the three DC potential signals showed a transient negative shift during CSD. We found that the resting level of extracellular K+ was 3 ± 1 mM in all undamaged brains. When the initial levels were higher, the animal was not included.

2.3 Monitoring of NADH and Brain Functions Inside Hyperbaric Chambers 2.3.1 Monitoring of NADH Fluorescence In order to expose an organ in vivo to elevated oxygenation–hyperoxia, it is possible to use one of the two options: (a) Normobaric hyperoxia is achieved by making the animal breathe elevated FiO2, namely, between 21% O2 and 100% O2 at atmospheric pressure. (b) Hyperbaric hyperoxia (HBO) is induced by using a hyperbaric chamber in which oxygen pressure is elevated while the animal is located in the chamber. It is well documented that providing animals or man with elevated oxygenation will lead to the development of “oxygen toxicity.” The time needed for the

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Fig. 2.11 (a) Chamber for the study of cell suspensions in mitochondria under hyperbaric conditions. The illustration shows a compensated fluorometer with compensating photomultiplier on the left, excitation source in the center, and measuring photomultiplier on the right. The chamber is also provided for light transmission and measurements with the double-beam spectrophotometer for the purpose of measuring the oxidation-reduction state of cytochromes. The fluorometer for measuring reduced pyridine nucleotide concentration can be replaced by one for measuring flavoprotein only, or flavoprotein and pyridine nucleotide (Chance 1966; Chance et al. 1966). (b) The response of the reduced pyridine nucleotide component of rat live mitochondria to anoxia and to high pressure oxygen (Chance 1966)

development of this toxic event is inversely proportional to the level of oxygenation, namely, the higher the pO2, the shorter the time. On the other hand, providing more O2 may be beneficial in conditions such as carbon monoxide toxicity, body oxygenation pathology (heart or lung problems), and severe trauma. Therefore, it became necessary to understand the relationship between the level of oxygenation and the function of the mitochondria in vivo. In this section, papers that are cited in the reference list include studies where other organs beside the brain were used. In the mid-1960s, Chance and collaborators (Chance et al. 1965, 1966, 1969; Chance 1966, 1967) developed the experimental setup that enabled the exposure of various types of mitochondria as well as of the entire small animal to the hyperbaric chamber. They showed that the NADH of the brain, liver, and kidney became oxidized under hyperbaric oxygenation, and this effect was correlated with a decrease in pyridine nucleotides measured by biochemical analysis of fixed tissue. Figure 2.11 presents the setup (A) and typical results (B) obtained when suspension of rat liver mitochondria was exposed to hyperbaric oxygenation. Initially, the mitochondria were exposed to anoxia as seen in the left side and an increase in NADH fluorescence was recorded. Increase in the pressure of oxygen to 11 atmospheres led to a clear oxidation of NADH in the mitochondria. This pressurization technique was used in later experiments when various organs of the rat were exposed in vivo to HBO, as seen in Fig. 2.12a (Chance et al. 1966). Typical results of an in vivo monitoring are shown in Fig. 2.12b, where the rat liver in vivo was exposed to gradual increase in oxygen pressure (Chance et al. 1966). A clear oxidation of NADH was recorded in the two tested organs.

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Fig. 2.12 (a) Fluorometer attached to small animal chamber. Shown left is the compensating photomultiplier, center is the excitation lamp, and right is the measuring photomultiplier (Chance et al. 1966). (b) The response of rat liver to repetitive pressurization and decompression with oxygen. The values of pressure are included. The sensitivity for measuring fluorescence changes is also indicated (Chance et al. 1966)

Fig. 2.13 (a) Schematic representation of location of cannulas and ECoG electrodes on rat’s skull (Mayevsky and Shaya 1980). (b) Schematic representation of the light pipe holder (cannula) implanted epidurally and cemented to the skull (Mayevsky 1975). (c) Time-sharing fluorometer/ reflectometer, attached to the hyperbaric oxygen chamber enables the measurement of NADH from the cortex of the awake rat exposed to HPO (Mayevsky 1975)

2.3.2 Monitoring of NADH Fluorescence Together with EEG After the introduction of the light-guide-based fluorometry, we were able to expose an awake brain to hyperbaric oxygenation conditions, as seen in Fig. 2.13 (Mayevsky 1975; Mayevsky and Shaya 1980). Figure 2.14 presents the response of the brain to 100% oxygen under hyperbaric conditions of 6ATA O2 (Mayevsky 1975). A clear decrease in NADH (oxidation) was recorded during the shift from 21% to 100% O2

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Fig. 2.14  The responses of awake brain cortex exposed to 75 psi pure oxygen (control animal). (a) 366 nm reflectance; (b) 450 nm fluorescence; (c) 450 nm corrected fluorescence; (d) EEG of right hemisphere; (e) EEG of left hemisphere. Note that the EEG amplitude (traces D and E) was changed during the experiment. Upward deflection of the pen represents an increase in the signal size (Mayevsky and Shaya 1980)

as compared to the increase in NADH that was recorded before death (lower right side). The same type of NADH decrease (oxidation) was recorded during compression of up to 10 atmospheres 100% O2 (Mayevsky et  al. 1974a, 1980c, 1989; Mayevsky and Shaya 1980; Mayevsky 1983a, 1984a, b; Yoles et al. 2000). The reflectance at 366 nm increases during the compression period, and a few minutes later, a large decrease of reflectance occurs. This pattern of reflectance changes was observed in all animals. The third trace from the top – the corrected fluorescence  – represents the difference between the fluorescence emission at 450 nm and the reflectance at 366 nm. By this subtraction, one can eliminate absorbance changes due to hemodynamic effects which produce artifacts in the fluorescence measurements. This correction technique is now used by several groups (Kobayashi et al. 1971a, b; Mayevsky and Chance 1973, 1974; Harbig et al. 1976). During the compression, an oxidation of NADH of 10% of the normoxic fluorescence level is observed, which is maintained for 15  min. A series of oxidation-­ reduction cycles of NADH then appears. Approximately 10 min before the animal stops breathing, a reduction of NADH is observed which increases to 50–60% at the end. The fourth and fifth traces of Fig. 2.14 show the EEG measured from the two hemispheres. In most animals, the two hemispheres of the cortex respond to HBO in the same way. A few minutes after compression, the EEG changes from the typical ‘awake’ pattern to the activated pattern, and then the convulsions appear. The number of bursts of convulsive activity differs between animals. The EEG becomes flat just before the animal stops breathing, and the reduction of NADH is seen. In another study, we tested the effects of unilateral and bilateral carotid artery occlusion on the responses to hyperbaric oxygenation (Mayevsky et al. 1989), as

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KCl

KCl

Fig. 2.15  The experimental setup by which the awake brain was monitored under HBO conditions. The two-channel light guide fluorometer/reflectometer is shown in Part B. The topical view of a rat brain implanted with the light guide holders and the ECoG electrodes is shown in Part A (Mayevsky et al. 1989)

presented in Fig. 2.15. Part A shows the location of the two probes on the two hemispheres. The 2-channel fluorometer is presented in Part B. Figure 2.16 presents typical results measured in the gerbil brain exposed to anoxia (A), left carotid occlusion (B), and right carotid occlusion (C).

2.3.3 Simultaneous Measurement of NADH, EEG, pO2, K+e, and DC Potential To elucidate the mechanism of brain O2 toxicity, it is necessary to measure as many physiological parameters as possible from the same site. Our present study aimed to develop a new multiparameter monitoring system located in a hyperbaric chamber, using an awake animal. The basic features of the multiparameter assembly used under normal conditions were described previously (Friedli et al. 1982; Mayevsky and Sclarsky 1983), and in the present study, we extended and adapted the system for the hyperbaric chamber. The multiparameter assembly can measure up to eight parameters, representing the metabolic, ionic, and electrical activities on the cerebral cortex. Metabolic activity is evaluated by measuring tissue O2 tension and by monitoring the intramitochondrial NADH oxidation-reduction state. The ionic state of the tissue is evaluated by monitoring extracellular potassium (K+) activity. Electrical activity was monitored by bipolar electrocorticography (ECoG) and the DC steady potential. In addition, we monitored the surface temperature with a miniature thermistor. The

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Fig. 2.16  Metabolic and electrical responses to anoxia (a), left carotid (b) and right carotid artery occlusion (c) measured bilaterally from gerbil brain (Mayevsky et al. 1983)

multiprobe assembly has the following main features. (1) All parameters have the same type of noninvasive surface contact with the cortex. (2) The multiprobe assembly can be very easily removed without any damage from the skull at the end of the experiment, so that repetitive experiments can be performed in a short period of time. (3) All signals monitored by the multiprobe assembly show a very small sensitivity to movements of the animal while exposed to hyperbaric pressure. Figure 2.17 presents in a schematic way the connection between the multiprobe assembly and the cerebral cortex of the rat brain (A) and the location of the various probes in relation to the skull (B). The various electrodes were held in the multiprobe assembly as described in detail elsewhere (Friedli et  al. 1982; Mayevsky et al. 1999b). The common part of the light guide was cemented inside the multiprobe assembly in the same way as the other electrodes. A short piece of the excitation and emission bundle of fibers was connected to the common part. Two bundles of fibers were connected inside the wall of the hyperbaric chamber and used to transmit the light into and from the multiprobe assembly. Specially designed connectors were used when the animal was introduced into the chamber for measurements (Fig. 2.17c). The various cables and the light guide were fixed in the chamber wall with screw(s) by epoxy glue, as seen in Fig. 2.18. The following parameters were monitored and recorded on a multichannel recorder (Beckman Dynograph): (1) ECoG, (2) tissue PO2, and (3) corrected K+ (the uncorrected signal was not recorded in our experiments due to a limited number of recording channels). Figure 2.19 shows a typical response to complete anoxia and recovery to the pre anoxic state. The first response to the N2 breathing is the drop in PO2 to its minimal

Fig. 2.17 (a) – Schematic presentation of multiprobe assembly (MPA) used in hyperbaric chamber. (a) longitudinal section of MPA; (b) the relative location of various probes above brain. Ref, reference Ag–AgCI electrode; f, refill tube for Ref or DC electrode; c, Lucite cannula; s. Plexiglas sleeve; L, light guide; h, cable holder; Ek, K+ electrode; DCK, DCL, Ag–AgCI electrode; PO2. Oxygen electrode; ECoG, electrocorticography electrodes (Mayevsky 1983b). (c) Rat connected to multiprobe assembly inside hyperbaric chamber (Mayevsky 1983b)

Fig. 2.18  Interconnection between multiprobe assembly connected to rat inside chamber and monitoring system located outside hyperbaric chamber (Mayevsky 1983b)

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Fig. 2.19  Effects of anoxia (100% N2) on metabolic ionic and electrical activities in the rat brain. ECoG, electrocorticogram; PO2 partial pressure of O2; Ek-, potential measured due to net changes in extracellular K+; DCK+, DC potential measured near K+. electrode or light guide; R, 366-nm reflectance; F, 450-nm fluorescence; CF, 450-nm corrected fluorescence; N2, Nitrogen breathing (Mayevsky 1983b)

level (0), which recovered as soon as air was introduced into the breathing mixture. A large overshoot in the PO2 was recorded followed by a slow recovery to the normoxic level. In parallel to the PO2 changes, the intramitochondrial NADH (CF signal) also responded very quickly to anoxia. We used the 1:1 correction factor to obtain the net change in NADH signal. This factor has also been used by other investigators in the past (Jobsis et  al. 1971a; Harbig et al. 1976). The high steady level of NADH during the anoxia is in parallel to the very low level of the PO2. During the recovery phase, the intramitochondrial NADH (CF) reached the normoxic quickly, while the reflectance and the uncorrected fluorescence recovered more slowly in parallel to the slow recovery in the PO2 trace. During anoxia, a small increase in K+ was recorded. As soon as energy was available again, the K+e returned to the resting level after a small undershoot. The ECoG reached the isoelectric state about 30 s after breathing N2 and was followed by a fast recovery after rebreathing air.

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2.3.4 NADH, Reflectance, Microcirculatory Blood Flow, and Hb Oxygenation In order to assess the hemodynamic and metabolic functions of the cerebral cortex, we used the Tissue Vitality Monitoring System (TVMS) that includes two devices: a time-sharing fluorometer-reflectometer (TSFR) for mitochondrial NADH redox state and microcirculatory hemoglobin oxygen saturation HbO2 measurement (Rampil et  al. 1992), combined with a laser Doppler flowmeter (LDF) for CBF monitoring (Fig. 2.20a). The connection between the brain and the TVMS was done by a flexible light guide inserted through the wall of the hyperbaric chamber. The fiber-optic probe includes fibers that were connected to the two instruments, as shown in the enlargement of the bundle tip. The measurement of HbO2, reflectance at 366 nm and NADH were performed by the same excitation and emission fibers. The CBF was measured by three optical fibers located in the center of the time-­ sharing bundle of fibers. This system includes a rotating wheel with eight specific filters at the appropriate wavelengths (366  nm, 450  nm for NADH and 585  nm, 577 nm for oxyhenoglobin measurement, to four filters for excitation light and four filters for emitted light (Fig. 2.20b)). The wheel rotates at about 2400 rpm, which is a very high speed with respect to the kinetics of physiological changes; thus, NADH and oxyhemoglobin are simultaneously monitored (Rampil et al. 1992; Mayevsky and Rogatsky 2007). Anoxia (100% N2 inhalation for about 20  s) was induced in order to find the maximal reduced NADH and minimal HbO2 levels (Fig. 2.21). The effect of anoxia

Fig. 2.20 (a) Schematic representation of the experimental setup showing the time-sharing fluorometer/reflectometer, laser Doppler flowmeter and electrodes for electrocorticography (ECoG). Ex and Em – excitation and emission fibers for NADH and HbO2 monitoring, respectively; LD in and LD out – optical fibers for blood flow monitoring. The numbers in the spinning disk refer to the wavelength filters. The rat brain is connected to monitoring system via a flexible fiber-optic probe penetrating the wall of the chamber. (b) Absorption spectra of saturated (blue) and unsaturated (pink) hemoglobin (Jourdanet 1875; Frank et al. 1989)

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Fig. 2.21  The effect of anoxia (100% N2) on cerebral blood flow (CBF), reflectance (R366), mitochondrial NADH redox state (NADH), and hemoglobin oxygen saturation (HbO2). Values are shown as mean percent values ± SE. **p