THE USE OF LOCAL ANAESTHETIC AEROSOLS IN THE STUDY OF THE CONTROL OF BREATHING IN MAN AND DOG$

A Thesis submitted for the degree of Doctor of Philosophy in the Faculty of Science to the University of London

by

Russell Douglas Hamilton Department of Medicine Charing Cross and Westminster Medical School London March 1990

1 ABSTRACT In man, information from vagus nerve endings in the airways and lungs is thought to play a small role in the control of breathing at rest but has the potential to become important when ventilation increases and, more significantly, may cause the tachypnoea and breathlessness of some disease states. Unfortunately, few techniques exist which can be readily used to interrupt such information from the lungs in man.

The main aim of this thesis was to investigate the role of these receptors in the control of breathing in man using inhaled local anaesthetic aerosols to block their discharge in a safe, reversible and selective manner. To achieve this, two aerosol systems were developed using 5% bupivacaine as the local anaesthetic. One aerosol (mass median diameter = 4.9pm) was shown by radioisotope scans to deposit predominantly in the larger airways while the other (mass median diameter = 1.0pm) deposited mainly in the lung periphery. In the studies performed in this thesis, the 1.0pm aerosol had no significant effects on breathing in dog or man, possibly because drug levels at the alveolar surface were too low. In contrast, in dogs the 4.9pm bupivacaine aerosol abolished reflexes believed to arise from receptors in the airways and, after a longer inhalation period, a reflex believed to be mediated by C-fibres in the lung periphery.

In normal man, studies using the 4.9pm bupivacaine aerosol indicated that airway receptors act to minimize fluctuations in tidal volume at rest and are involved in limiting inspiration during exercise. The increased ventilatory response and breathlessness during C02 rebreathing after inhalation of this aerosol in normal and 1aryngectomized man demonstrates that the disruption of discharge from receptors in the airways and/or lungs can, under some circumstances, have a considerable effect on the control of breathing. Central intravenous injection of capsaicin produced a burning sensation in the chest (and cough) believed to be mediated by C-fibre endings in the lung; the burning could be abolished by the 4.9pm aerosol. Despite these encouraging results in normal subjects, inhalation of this aerosol had no effect on the tachypnoea or breathlessness of patients with interstitial lung disease during exercise. Further experiments with this useful technique are indicated to determine whether lung receptors produce the tachypnoea in active inflammatory lung disease.

2 For Ron and June Hamilton my parents

3 CONTENTS

List of Tables 7 List of Illustrations 8 Abbreviations 10 Statement 11 Acknowledgements 12

SECTION I - INTRODUCTION AND BACKGROUND

CHAPTER 1: INTRODUCTION 1.1 Overview 13 1.2 Afferent vagal information from the lung 14 1.3 Relative contribution of different 20 afferent vagal inputs 1.4 Rationale for the present studies 22

CHAPTER 2: AEROSOLS 2.1 THEORY AND BACKGROUND 2.1.1 Basic considerations 23 2.1.2 Mechanisms of deposition 23 2.1.3 Factors affecting deposition 24 2.1.4 Previous studies on aerosol deposition 27 2.1.5 Methods of aerosol deposition 29 2.1.6 Choice of generators 31 for the present studies 2.2 EXPERIMENTAL 2.2.1 Large particles 32 2.2.2 Small particles 42

CHAPTER 3: LOCAL ANAESTHETICS 3.1 Background 54 3.2 Structure 54 3.3 Mechanism of action 55 3.4 Differential sensitivities of nerve fibres 57 3.5 Structure-activity relationships 57 3.6 Metabolism 58 3.7 Systemic toxicity 58

4 3.8 Choice of local anaesthetic 59 for the present studies 3.9 Determination of bupivacaine concentration 63 in blood

SECTION II - PHYSIOLOGICAL STUDIES

DOGS - RESPIRATORY REFLEXES 4.1 Introduction 64 4.2 Methods 65 4.3 Results 68 4.4 Discussion 75

NORMAL MAN - RESTING STUDIES 5.1 Introduction 81 5.2 Methods 83 5.3 Results 87 5.4 Discussion 95

NORMAL MAN - SEARCH FOR PULMONARY CHEMOREFLEX 6.1 Introduction 99 6.2 Methods 104 6.3 Results 107 6.4 Discussion 114

NORMAL MAN - EXERCISE 7.1 Introduction 119 7.2 Methods 120 7.3 Results 123 7.4 Discussion 136

NORMAL AND LARYNGECTOMIZED MAN - C02 REBREATHING 8.1 Introduction 141 8.2 Methods 142 8.3 Results 145 8.4 Discussion 156

PATIENTS WITH INTERSTITIAL LUNG DISEASE - EXERCISE 9.1 Introduction 166

5 9.2 Methods 168 9.3 Results 171 9.4 Discussion 178

SECTION I I I - CONCLUSIONS

CHAPTER 10 - CONCLUSIONS AND PLANS FOR FUTURE WORK 10.1 Conclusions 183 10.2 Proposals for future studies 187

APPENDICES 190

REFERENCES 191

6 LIST OF TABLES

Table Description Page

2.1 Particle size distribution of large particle bupivacaine 34 aerosol (DeVilbiss 35B ultrasonic nebulizer). 2.2 Particle size distribution of small particle bupivacaine 47 aerosol (fluidized bed generator with Optimist). 2.3 Particle size distribution of small particle bupivacaine 50 aerosol (Unicorn nebulizer with Optimist). 2.4 Particle size distribution of small particle bupivacaine 51 aerosol (Turret nebulizer with Optimist). 3.1 Chemical and biological properties reported for four 56 typical local anaesthetics. 3.2 Duration of blockade of the cough reflex by three local 62 anaesthetic agents as large particle aerosols. 5.1 Scores from visual analogue scales used to assess central 90 nervous system effects of large particle aerosol inhalation. 5.2 Vigilance and reaction time before and after 91 large particle aerosol inhalation. 5.3 Effect of large particle aerosol inhalation on the 92 pattern of resting breathing in normal subjects. 6.1 Time of onset of the chest sensation after intravenous 113 injection of capsaicin and circulation time to the ear. 8.1 Details of 1aryngectomized subjects. 143 8.2 Cough reflex to mechanical probing in laryngectomized 149 subjects before and after large particle aerosol. 9.1 Details of patients with interstitial lung disease. 168

7 LIST OF ILLUSTRATIONS

Figure Description Page

2.1 Example of the particle size distribution of an ideal 26 aerosol showing the log-normal nature of the distribution. 2.2 Fraction of aerosol particles deposited in the different 28 regions of the lung as a function of particle size. 2.3 Large particle aerosol generator: Devilbiss 35B ultrasonic 33 nebulizer with inspiratory and expiratory one-way valves. 2.4 Lung scan after inhalation of large and small particle Tc- 39 labelled bupivacaine aerosols together with perfusion scan. 2.5 Gamma counts in the right lung following inhalation of Tc- 40 labelled bupivacaine aerosols as a function of time. 2.6 Bupivacaine concentration and Tc activity in plasma 41 following inhalation of Tc-labelled bupivacaine aerosols. 2.7 Small particle aerosol generator: fluidized bed with 46 particle filter (Optimist). 2.8 Small particle aerosol generator: modified jet nebulizer 49 (Turret) and particle filter (Optimist). 4.1 Cardiovascular and respiratory effects of aerosol 69 inhalation and intravenous infusion of bupivacaine in dogs. 4.2 Pulmonary chemoreflex evoked by capsaicin injected into 71 the right ventricle in on dog. 4.3 Pulmonary chemoreflex to capsaicin before and after aerosol 72 inhalation and intravenous infusion of bupivacaine in dogs. 4.4 Hering-Breuer inflation reflex before and after aerosol 73 inhalation and intravenous infusion of bupivacaine in dogs. 4.5 Differential block of the pulmonary chemoreflex and the 74 inflation reflex during recovery from aerosol anaesthesia. 5.1 Copy of the visual analogue scales used to assess central 84 nervous system effects of saline and bupivacaine aerosol. 5.2 Concentration of bupivacaine in plasma during and after a 89 10 min inhalation of large particle bupivacaine aerosol. 5.3 Concentration of bupivacaine in plasma during and after a 94 20 min inhalation of small particle bupivacaine aerosol. 6.1 Effect of central and peripheral intravenous injection of 109 capsaicin in one normal human subject.

8 Figure Description Page

6.2 A&B: Effect of central injections of saline and capsaicin 110

on VT, TI? Te and fc for 5 breaths before and after injection. 7.1 Parts 1&2: Effect of large particle saline and bupivacaine 124 aerosols on the ventilatory response to exercise in normals. 7.2 Mean data over the last 5 min of exercise for normals after 126 large and small particle aerosols, and on repeat exercise. 7.3 Mean data for Tj and TE over the last 5 min of exercise in 127 normals after large particle saline and bupivacaine aerosol. 7.4 Effect of intravenous saline and bupivacaine infusion on 129 the ventilatory response to exercise in normal subjects. 7.5 Parts 1&2: Effect of performing two exercise tests 3 hours 131 apart without aerosols on the ventilatory response to exercise. 7.6 Parts 1&2: Effect of small particle saline and bupivacaine 134 aerosols on the ventilatory response to exercise in normals. 8.1 Effect of large particle saline and bupivacaine aerosol on 146 the ventilatory response to C02 in normal man. 8.2 Effect of large particle saline and bupivacaine aerosol on 147 breathlessness during C02 rebreathing in normal man. 8.3 Effect of large particle saline and bupivacaine aerosol on 150 the ventilatory response to C02 in laryngectomized man. 8.4 Effect of large particle saline and bupivacaine aerosol on 151 breathlessness during C02 rebreathing in laryngectomized man. 8.5 Effect of small particle saline and bupivacaine aerosol on 154 the ventilatory response to C02 in normal man. 8.6 Effect of small particle saline and bupivacaine aerosol on 155 breathlessness during C02 rebreathing in normal man. 9.1 Parts 1-3: Effect of large particle saline and bupivacaine 173 aerosol on the ventilatory response and breathless during exercise in patients with interstitial lung disease. 9.2 Mean data over the last 5.5 min of exercise in patients 177 with interstitial lung disease.

9 ABBREVIATIONS MMAD mass median aerodynamic diameter GSD geometric standard deviation Tc technetium fR respiratory frequency VT tidal volume VE expired ventilation Tj inspiratory time

Te expiratory time V02 oxygen consumption VC02 carbon dioxide production PETC02 end-expiratory partial pressure of carbon dioxide

Pet02 end-expiratory partial pressure of oxygen PaC02 partial pressure of carbon dioxide in arterial blood Sa02 percent saturation of haemoglobin with oxygen in arterial blood FEV1 forced expired volume in 1 second sGaw specific conductance of the airways FVC forced vital capacity FRC functional residual capacity TLC total lung capacity VTG volume of thoracic gas Kco carbon monoxide transfer coefficient PD35 provocative dose of methacholine required to produce a 35% fall in sGaw fc cardiac frequency ECG electrocardiogram BP blood pressure VAS visual analogue scale PDG phenyldiguanide

10 STATEMENT

I have been the principal investigator in all the work presented in this thesis, being involved in the planning, execution and analysis of all studies. However, the nature of many of the studies, involving the administration of local anaesthetic agents to man, required that a medically qualified colleague be involved in these procedures. This assistance is gratefully recognized in the Acknowledgements section along with credit for the drug analysis which was not performed by me.

Some of the results of the studies described in this thesis have been previously presented elsewhere; a list of the publications in which they appear is given at the end of the References section.

Russell Douglas Hamilton

11 ACKNOWLEDGEMENTS

The first person traditionally thanked in the Acknowledgements section of a thesis is the student’s supervisor; although my supervisor has described me as a great iconoclast, this is one icon which I cannot break. Professor Abraham Guz is a man whose character does not easily submit to description but the most essential thing about him to me is his passionate enthusiasm, both for intellectual rigor and for physical achievement. He has led me through the years of work contained in this book by example and by expectation; for this and much more, I thank him.

Throughout the majority of the time spent on the work towards this thesis, I have had the pleasure of working with Dr. Andrew Winning; his help, friendship and devotion to making the studies happen have been greatly appreciated. My thanks go also to Dr. Ken MacRae for melting huge sections of the statistical iceberg which waits to sink all modern research and to Dr. Kevin Murphy for making physics behave the way the textbooks say it does.

My grateful recognition is also expressed to the following: Dr. Marc Ashun, Department of Pharmacy for help with the preparation of bupivacaine, citric acid and capsaicin solutions; Dr. Felicity Reynolds and her colleagues in the Anaesthetic Unit, St. Thomas’ Hospital for measurement of plasma bupivacaine; the staff of the Department of Forensic Medicine, Charing Cross Hospital for analysis of blood alcohol concentration; Mr. K. Jeyasingh, for assistance with the techniques of Nuclear Medicine; Pam Pate, Department of Comparative Biology for help with studies in her department; Alison Perry, Department of Speech Therapy for help with recruiting laryngectomy patients and to the laryngectomy and interstitial lung disease patients themselves. The many people who steered me through the mists of aerosol science are thanked in the text of the thesis.

The Department of Medicine at Charing Cross Hospital as an ideal, as a group and as individuals has helped to sustain me throughout this work and for this I am extremely grateful. I would like to thank specifically Dr. Lewis Adams and Dr. Alastair Innes for invaluable discussions and Dr. Bernard Fox for the push.

12 CHAPTER 1: INTRODUCTION

1.1 Overview

The way in which we breathe - the timing and magnitude of inspiration and expiration - is the result of a need to fulfil both metabolic and behavioural requirements. Superimposed on this are changes in breathing which form responses to defend the airways and lungs from foreign agents. The basic rate and depth of breathing is determined by the discharge of groups of neurons located in the brain stem, and especially the medulla oblongata, which act as a "central pattern generator" for respiration (Von Euler, 1986). Descending axons from this region project to phrenic motoneurons innervating the diaphragm (inspiration) and to intercostal motoneurons supplying the external (inspiration) and internal (expiration) intercostal muscles (Mitchell and Berger, 1981). The characteristics of this rhythm are modified by the arrival in the medulla of afferent information from receptors positioned such that they can monitor the physical changes produced by breathing (receptors in the respiratory muscles, chest wall, airways and lungs; Shannon, 1986; Widdicombe, 1986; Sant’Ambrogio, 1987b) and changes in metabolic substrate (02) and byproducts (C02, H+) (central and peripheral chemoreceptors; Fitzgerald and Lahiri, 1986). The sensory cortex also receives afferent information from some of these receptors, especially those involved in afferent feedback from the respiratory apparatus; evidence for projections on the cortex of afferent nerves from the airways and lungs comes from electrophysiological studies (Kazakov, 1966; O ’Brien et al., 1971) and from studies involving the perception of afferent information (Banzett et al., 1987; Eckenhoff and Comroe, 1951). The cortex appears to be capable of exerting motor control of breathing via connections to neurons in the medulla and also by cortico-spinal fibres bypassing the brain stem to act directly on motoneurons in the spinal cord (Mitchell and Berger, 1975; Plum, 1970; Hugelin, 1986). Although the site of integration of behavioural and automatic control of breathing is the subject of current discussion (Orem and Netick; 1986), the result of this integration is that the respiratory pattern necessary to achieve a given metabolic need, such as exercise, or behavioural desire, such as speech, can be adopted.

13 The studies described in this thesis investigate the role of afferent information from receptors in the airways and lungs in the control of breathing concentrating on man. In this introductory chapter, the background to, and rationale for, this work will be discussed.

1.2 Afferent vagal information from the lungs

The importance of afferent neural information from the lungs in determining the pattern of breathing in animals was first described by Breuer (Hering, 1868; Breuer, 1868) who showed that inflation of the lungs in anaesthetized rabbits and dogs inhibited inspiration by a mechanism dependent on the integrity of the pulmonary vagus nerves and proposed that this comprised a reflex acting via the respiratory centre in the medulla. This proposal was significant, not only for respiratory physiology, since it provided the first description of a feedback control mechanism; in the words of Hering (1868) "every inspiration, therefore, in that it distends the lung brings about its own end by means of this distension, and thus initiates expiration".

The receptors in the airways and lungs involved in the control of breathing are regarded as being of three types: slowly adapting stretch receptors, rapidly adapting stretch receptors and C-fibre endings; C-fibres are further divided into bronchial and pulmonary endings. Afferent nerve fibres from these receptors run in the vagus (Xth cranial) nerves and terminate in the medulla oblongata, especially in the nucleus of the tractus solitarius (Mitchell and Berger, 1981). Using light microscopy, Agostoni et al. (1957) determined that there were approximately 6000 fibres in the bronchial branches of the vagus nerves to one lung in the cat: of these, 1000 were efferent (30% myelinated; 70% unmyelinated) and 5000 were afferent (30% myelinated; 70% unmyelinated). A more recent study in the same species using light and electron microscopy (Jammes et al., 1982) found a greater number of fibres in the vagal branches to one lung (3415 efferent; 5558 afferent) and a higher proportion of these were unmyelinated (92%).

The enormous body of work examining the location, structure, discharge properties and reflex effects on breathing of vagal receptors in the airways and lungs has been comprehensively examined in a number of

14 excellent reviews (Sant’Ambrogio, 1982, 1987b; Paintal, 1973; Fillenz and Widdicombe, 1972; Widdicombe 1981, 1982; Coleridge and Coleridge, 1984, 1986) and it would be inappropriate for me to repeat this process here. This introductory chapter will, therefore, provide a summary of current thought concerning the characteristics of these receptors and their involvement in the control of breathing; this will necessarily focus on studies in animals but will encompass what little is known of them in man. The involvement of these receptors in specific aspects of the control of breathing will be considered in detail in Chapters 4 to 9 of this thesis. It should be said that, although a great deal is known about these receptors, considerable gaps exist in our understanding, particularly in our ability to assign a given physiological response to discharge in a nerve fibre and to relate that discharge to a receptor of known structure and location; in other words the relationship between physiology and anatomy.

Slowly adapting pulmonary stretch receptors

It now seems generally agreed that slowly adapting pulmonary stretch receptors are located in the intrathoracic and, to a lesser extent, the extrathoracic airways (Sant’Ambrogio, 1987b; Coleridge and Coleridge 1986). The concentration of slowly adapting receptors appears to be highest in the larger airways and progressively decreases towards the terminal bronchioles (Miserocchi et al., 1973; Miserocchi and Sant’Ambrogio, 1974a; Kohl et al., 1986) although a few studies have reported relatively high concentrations of these receptors in the lung periphery (Paintal and Ravi, 1980; Keller et al., 1989). Good physiological evidence exists that the receptors are closely associated with smooth muscle in the airway wall (Bartlett et al., 1976). This has been supported by histological studies in the rat bronchus (von During, 1974) and dog trachea (Krauhs, 1984) describing myelinated fibres which often branch to form unmyelinated endings associated with the connective tissue of the lamina propria and smooth muscle cell layer.

The primary stimulus for slowly adapting pulmonary stretch receptors seems to be a change in tension in the airway wall (Bartlett et al., 1976); in the intact lung this would allow the receptors to perform what is considered to be their main role of sensing changes in lung

15 volume (Knowlton and Larrabee, 1946) or transmural pressure (Miserocchi and Sant’Ambrogio, 1974). The receptors increase their frequency of discharge in response to lung inflation (Adrian, 1933); this frequency slowly decreases if the inflation is maintained. The exceptions to this are slowly adapting receptors in the extrathoracic trachea which, because of the reversal of transmural pressure during the breathing cycle at this site, increase their discharge during expiration (Sant’Ambrogio and Mortola, 1977). Some slowly adapting receptors are tonically active at functional residual capacity (Adrian, 1933).

Slowly adapting stretch receptors are believed to be responsible for the inhibition of inspiratory activity resulting from lung inflation (Hering-Breuer inflation reflex) (Adrian, 1933; Coleridge and Coleridge, 1986). In some species, their static discharge at constant lung volume (Bartoli et a/., 1973) and their continuing discharge during expiration (Knox, 1973; Trenchard, 1977) lengthens expiratory time. Afferent impulses from slowly adapting stretch receptors are carried by nerve fibres with conduction velocities characteristic of myelinated fibres (Paintal, 1953).

In man, nerve endings believed to be slowly adapting pulmonary stretch receptors have been found in histological studies (Larsell and Dow, 1933; Spencer and Leof, 1964). Direct recordings of afferent activity in the vagus nerves indicate that volume-related activity from slowly adapting receptors exists in man (Langrehr, 1964; Guz and Trenchard, 1971a); furthermore, this activity is present within the tidal volume range. Although this information does not seem to be important in determining the pattern of breathing in man at rest (Guz et a/., 1964, 1966), studies on the Hering-Breuer inflation reflex in man (Widdicombe, 1961; Guz et a 7., 1964; Hamilton et a/., 1988) indicate that it may be involved in the control of breathing at higher tidal volumes.

Rapidly adapting pulmonary stretch receptors

Rapidly adapting pulmonary stretch receptors, like slowly adapting receptors, are located in the extrathoracic and intrathoracic airways down to terminal bronchioles; their concentration is highest in the

16 larger airways especially at airway bifurcations (Widdicombe, 1954a; Mortola et al., 1975; Sampson and Vidruk, 1975; Sant’Ambrogio et al., 1978). The sensitivity of rapidly adapting receptors to inhaled irritants (Widdicombe, 1954a) led to the proposal that these receptors were located in the airway epithelium. The epithelial nerve endings observed by light (Larsell, 1921; Elftman, 1943) and electron microscopy (Fillenz and Woods, 1970; Das et al., 1979) are, therefore, generally considered to represent rapidly adapting receptors (Fillenz and Widdicombe, 1972; Widdicombe, 1982).

Rapidly adapting receptors show a brief, irregular, burst of activity in response to lung inflation with very rapid adaptation if the inflation is maintained; they are also strongly stimulated by lung deflation (Knowlton and Larrabee, 1946). During normal breathing, they have an infrequent and irregular discharge often unrelated to the respiratory cycle (Hills et al., 1969; Armstrong and Luck, 1974; Sampson and Vidruk, 1975; Bergren and Sampson, 1982) which can increase during hyperpnoea (Sellick and Widdicombe, 1969; Pack and Delaney, 1983). As well as being stimulated by the generalized airway deformation associated with large lung inflations or deflations, rapidly adapting receptors respond to local probing (Knowlton and Larrabee, 1946). When the mucosa of receptive field is removed, this response is abolished while the response to inflation and deflation remains (Sant’Ambrogio et al., 1978) indicating that nerve fibres innervate both superficial and deeper layers of the airway wall.

The increase in inspiratory activity which results from large, rapid inflation of the lungs in animals (gasp reflex) is believed to be mediated by an increase in activity in rapidly adapting pulmonary stretch receptors (Larrabee and Knowlton, 1946; Widdicombe, 1954b). Although rapidly adapting receptors throughout the airways are stimulated by a wide variety of inhaled dusts and chemicals (Mills et al., 1969; Mills et al., 1970; Sellick and Widdicombe, 1971), stimulation of receptors in the extrathoracic airways is believed to produce cough, while stimulation of those in the intrapulmonary airways may produce hyperpnoea but is not believed to cause cough (Fillenz and Widdicombe, 1972; Widdicombe, 1981). Afferent impulses from rapidly adapting stretch receptors are carried by nerve fibres with conduction velocities slower than those of slowly adapting

17 stretch receptors but still characteristic of myelinated fibres (Paintal, 1953).

In man, histological studies demonstrate bronchial intra-epithelial nerve endings which are more numerous in the larger bronchi and at points of airway branching (Larsell and Dow, 1933; Spencer and Leof, 1964). No direct recordings of vagal activity believed to originate from rapidly adapting receptors have been made in man to my knowledge.

C-fibre endings

C-fibre endings in the lungs have been divided into two groups, with presumed anatomical locations, on the basis of their accessibility to chemicals injected into the pulmonary or systemic circulation (Coleridge and Coleridge, 1977,1984,1986). According to this schema, pulmonary C-fibres would lie in or near the alveoli while bronchial C- fibres would lie in intrapulmonary and extrapulmonary airway walls. However, it has been argued that the criterion of vascular accessibility, although able to separate C-fibres into two groups, cannot be relied on to assign anatomical locations to fibres in these groups (Sant’Ambrogio and Sant’Ambrogio, 1982; Sant’Ambrogio, 1987b); this is considered in more detail in Chapter 4 (page 75). Unmyelinated fibres, thought from their structure to be afferent in nature, have been described between epithelial cells in the trachea of man (Rhodin, 1966) and in the alveolar walls of animals (Meyrick and Reid, 1971; Hung et a/., 1973) and man (Fox et al., 1980).

In general, pulmonary C-fibre endings are sensitive to mechanical changes while bronchial C-fibre endings are relatively insensitive to such changes (Kaufman et al., 1982; Coleridge and Coleridge, 1986). The activity in all bronchial and many pulmonary C-fibres is sparse and irregular during normal breathing although some pulmonary C-fibres show activity which is related to respiratory rhythm (Coleridge and Coleridge, 1977a, 1977b). Similarly some pulmonary C-fibres are sensitive to lung inflation while bronchial C-fibres are not (Coleridge and Coleridge, 1977b; Kaufman et al., 1982). Deflation does not appear to stimulate either type of ending (Armstrong and Luck, 1974; Coleridge et al., 1965; Coleridge and Coleridge, 1977b). Both pulmonary and bronchial C-fibre endings are strongly stimulated

18 by chemicals although the nature of the substances involved is generally different for the two types of ending. Bronchial C-fibre endings are especially responsive to mediators of inflammation which can be made and released by cells in the airway walls including histamine, prostaglandins, 5-hydroxytryptamine and bradykinin (see Coleridge and Coleridge, 1986) while pulmonary C-fibres are especially sensitive to exogenous substances such as phenyl diguanide (Paintal, 1955) and capsaicin (Coleridge et a/., 1965).

The reflex effects on breathing of pulmonary and bronchial C-fibre stimulation are essentially the same and consist of rapid, shallow breathing often preceded by apnoea (Coleridge and Coleridge, 1986); they may also be involved in the production of cough (Coleridge and Coleridge, 1986; Sant’Ambrogio et al., 1984; Sant’Ambrogio, 1987a; Karlsson et al., 1988). Recently, interest has been directed at proposals that activation of pulmonary C-fibre endings may be responsible for the tachypnoea of certain disease states (Frankstein and Sergeeva, 1966). Experimental support for this has come from studies which demonstrate that C-fibres mediate the increase in respiratory frequency produced by pneumonia (Trenchard et al., 1972), pulmonary embolism (Guz and Trenchard, 1971b) and pulmonary venous hypertension (Coleridge and Coleridge, 1977a). Afferent impulses from C-fibre endings are carried by vagal fibres with conduction velocities characteristic of unmyelinated fibres (Coleridge and Coleridge, 1977b).

As mentioned above, unmyelinated fibres believed to correspond to pulmonary and bronchial C-fibres have been found in man (Rhodin, 1966; Fox et al., 1980). To my knowledge, no direct recordings of activity believed to originate from C-fibre endings have been made in vagal nerve fibres in man. Attention has recently focused on the possibility that pulmonary C-fibre stimulation may mediate the tachypnoea of certain disease in man (Guz et al., 1970) and also on the suggestion by Paintal (1969) that these receptors may be may be involved in the genesis of the sensation of breathlessness.

19 1.3 Relative contribution of different afferent vagal inputs

A major difficulty in determining the role played by different lung receptors in the control of breathing is that, under a given set of circumstances, all or none of them may be discharging and all or none of the resulting afferent information may be processed by the central nervous system to influence the pattern of breathing. This has led to the use of techniques aimed at interrupting the passage of afferent neural information in a selective fashion. Three main techniques - cooling, anodal polarization and compression - have been used to block conduction in myelinated fibres while permitting that in unmyelinated fibres to continue; this appears, at least for the first two techniques, to be due to selective blockade of saltatory conduction (Franz and Iggo, 1968).

Although vagal cooling has been widely used, some controversy exists regarding its limitations (see Coleridge and Coleridge, 1986; Pisarri et al., 1986; Jonzon et al., 1988). Two points seem particularly important: firstly, myelinated fibres of all diameters are blocked completely at approximately the same temperature (Paintal, 1965; Franz and Iggo, 1968) and secondly, cooling affects conduction, especially with respect to discharge frequency, in both myelinated and unmyelinated fibres (Franz and Iggo, 1968; Jonzon et al., 1988). Anodal polarization involves the application of direct current to the vagus nerve to block the ’A’ and ’B’ wave of the electroneurogram while leaving the ’C’ wave intact (Guz and Trenchard, 1971; Trenchard et al., 1972). Although this technique does not seem to interfere with discharge frequency in C-fibres to the same extent as does vagal cooling, it has the problems of extraneous stimulation at the cathode and may result in progressive nerve damage (Thoren et al., 1977). Nerve compression is a rather coarse technique and does not seem to be in general use for vagal blockade.

The suggestion that the susceptibility of a nerve fibre to local anaesthesia was inversely related to its diameter (Gasser and Erlanger, 1929), led to the proposal that local anaesthetics could be applied to a nerve trunk in a concentration which would block conduction in unmyelinated fibres while preserving that in myelinated fibres. This proposal is now known to be incorrect and reliable

20 differential blockade on this basis is not possible (Nathan and Sears, 1961); the reasons for this are discussed in Chapter 3 (page 57).

The techniques described above are only suitable for use in animals. In man, complete vagal blockade has been achieved by direct application of local anaesthetic to the exposed vagus nerves in the neck (Guz et a l., 1964, 1966b, 1970) and by injection of local anaesthetic at the base of the skull, which also blocks the glossopharyngeal nerves (Guz et a l., 1966a, 1966b; Guz and Widdicombe 1970). However, this technique is difficult to perform and not widely applicable on moral grounds; furthermore it does not allow differential blockade of nerve fibre types. In contrast, it is theoretically possible to use a local anaesthetic to block receptors at different anatomical sites in the lung if the agent is inhaled in the form of an aerosol. This can be done by controlling the physical characteristics of the aerosol particles and the pattern of breathing by which they are inhaled, such that the aerosol particles will preferentially deposit either in the central airways or in the lung periphery. The state of knowledge and technology concerning aerosols has for many years permitted the administration of aerosols which deposit in the large airways; one of the first medical nebulizers was introduced more than fifty years ago (Collison, 1935) and the Wright nebulizer (Wright, 1958) is still used as a standard for aerosol studies. However, although our understanding of the behaviour of aerosols has improved considerably in recent years, it is still technically difficult to deliver an aerosolized drug to the lung periphery in appreciable quantities.

Presumably for this reason, the majority of studies in the literature reporting the use of a local anaesthetic aerosol to study the control of breathing, have used an aerosol which would be expected to deposit in the larger airways. The first such study was that of Petit and Delhez (1970) who reported that inhalation of an aerosol of 20% lignocaine with 1% adrenaline could abolish the tachypnoea, hyperventilation and sensation of breathlessness produced by the inhalation of histamine aerosol in three out of ten asthmatic subjects. Although the presence of adrenaline in the aerosol made the interpretation of that study difficult, the reported success of the local anaesthetic delivered in this way to inhibit the ability to

21 cough and swallow prompted Jain et ah (1973) and Dain et ah (1975) to examine the ability of a 5% bupivacaine aerosol to block respiratory reflexes in animals. In turn, the general success (see Chapter 4 of this thesis for full discussion) of these studies led Cross et ah (1976) to repeat the reflex studies in dogs and perform an extensive study on the safety and reflex effects of inhalation of 5% bupivacaine aerosol in man. The results of this and other work using the inhalation of a local anaesthetic aerosol to study the control of breathing in man are discussed in the appropriate sections of this thesis.

1.4 Rationale for the present studies

The objective of the studies described in this thesis was to produce blockade, in a reversible and selective fashion, of afferent information from C-fibres in the lung periphery while preserving information from slowly adapting and rapidly adapting receptors in the airways, and vice versa. In order to achieve this goal, two aerosol systems were developed: one producing deposition of local anaesthetic primarily in the lung periphery and the other depositing local anaesthetic predominantly in the airways. The ability of these aerosols to block respiratory reflexes was then assessed in the dog and the effect of inhalation of the aerosols on the control of breathing in a number of conditions was determined in man.

The reflexes examined in dogs were the Hering-Breuer inflation reflex, the cough reflex to mechanical stimulation of the airway and the pulmonary chemoreflex to capsaicin injection. The conditions examined in man were those of resting breathing and stimulated breathing (exercise and carbon dioxide rebreathing) in normal subjects, carbon dioxide rebreathing in laryngectomized subjects, and exercise in patients with interstitial lung disease. A study was also performed to investigate whether the pulmonary chemoreflex, believed to be mediated by pulmonary C-fibres in animals, could be elicited by central intravenous injection of capsaicin in normal subjects and determine whether the inhalation of local anaesthetic aerosol could block any effects resulting from such injection.

22 CHAPTER 2: AEROSOLS

2.1 THEORY AND BACKGROUND

2.1.1 Basic considerations

An aerosol may be defined as a suspension of solid or liquid particles in air (after Stuart, 1973). It is usually accepted that particles with a diameter between 0.01 to 100 pm can maintain sufficient stability as a suspension to form an aerosol (Stuart 1973). When inhaled, aerosol particles with a diameter greater than 10 pm generally deposit in the oropharynx; those with a smaller diameter can pass into the respiratory tree where they may deposit in the tracheobronchial or alveolar region (Lippmann et a 7., 1980). In order to determine the likely site of deposition of an aerosol particle in the lungs, it is necessary to consider the mechanisms of aerosol deposition and the factors which affect these mechanisms.

2.1.2 Mechanisms of deposition

The two main mechanisms by which aerosol particles deposit in the lung are inertial impaction and gravitational settlement (Hatch and Gross, 1964). A third mechanism, diffusion by Brownian motion, is important only for very small particles (<0.5pm diameter) which contain little mass (Hatch and Gross, 1964). Inertial impaction is the most important mechanism of deposition for larger particles and may be of importance for all particles greater than 1pm diameter (Hatch and Gross, 1964; Stuart, 1973). It occurs when a particle travelling at a high velocity is unable to follow the changes in direction of the airstream in which it is being carried. The probability of inertial impaction, I, of a particle when the airstream changes direction by an angle of 9 is proportional to Ud2sin9 R where U is the velocity of the airstream (and particle), d is the diameter of the particle and R is the airway radius (Stuart, 1973). The main sites of deposition are, therefore, in the oropharynx and at or near bifurcations in the larger airways where total cross-sectional area is small and the air velocity high. Aerosol deposition by this

23 mechanism is increased by turbulent airflow caused, for example, by airway obstruction (Goldberg and Lourenco, 1973).

Gravitational settlement is the major mechanism of deposition for particles in the smaller airways and alveoli where the total cross section area is high and the air velocity low. A particle settles under gravity, accelerating until it reaches a terminal settling velocity, Ut, equal to (p-cQgd2 181 where p and o are the density of the particle and air respectively, g the acceleration due to gravity, d the diameter of the particle and the viscosity of air (Stuart, 1973). Gravitational settlement becomes less efficient when the terminal settling velocity falls below about 0.001 cm/sec which, for a sphere of unit density, corresponds to a diameter of 0.5pm (Lippmann et a/., 1980).

2.1.3 Factors affecting deposition

Aerosol characteristics

Although there are several ways in which the size of an aerosol particle can be described, the most useful and the most widely accepted is in terms of aerodynamic diameter. The aerodynamic diameter (D) of a particle may be defined as the diameter of a unit density sphere with the same terminal settling velocity as the particle in question (Newman et a 7., 1982). This takes into account both particle shape and density and is, therefore, the most appropriate unit for assessing deposition by inertial impaction and gravitational settlement (Lippmann, 1980). All particle sizes quoted in this thesis are expressed in terms of this parameter. However, since an aerosol consists of a collection of particles, the distribution of particle size must be considered. If the particles which make up the aerosol are all of the same size the distribution is said to be monodisperse. If, on the other hand, the particles cover a range of sizes, the distribution is described as heterodisperse. Most methods of aerosol generation produce heterodisperse aerosols which are not normally distributed but which can often be fitted to a log-normal distribution (Morrow, 1974).

24 Since the effect of an aerosol when deposited in the lung depends not simply on the number of particles of a certain size, but on the mass contained in those particles, an aerosol is best described by the distribution of mass with respect to particle size. A ideal example of such a distribution is shown in Fig 2.1 A. Replotting this using a logarithmic scale on the abscissa (Fig 2.1 B) demonstrates the log­ normal nature of the distribution. In the same way as a normal distribution is conventionally described by a mean (or median) and standard deviation, a log-normal distribution is described by a geometric mean (or median) and geometric standard deviation. The derivation of these parameters is shown in Fig 2.1 C. On the ordinate, the cumulative mass contained in particles up to a given size is plotted on a probability scale; on the abscissa, particle size is plotted on a logarithmic scale. The geometric mean particle size is that corresponding to 50% on the cumulative frequency plot; this value is generally referred to as the mass median aerodynamic diameter (MMAD) since half the mass of the aerosol is contained in particles larger and half in particles smaller than it (Agnew, 1984).

The geometric standard deviation (GSD) is calculated by dividing the particle size corresponding to 84.1% by the particle size corresponding to 50% on the cumulative frequency plot (Fig 2.1 C). This is by analogy to the normal distribution where 34.1% of the distribution lies one standard deviation above the mean. The GSD describes the degree of heterodispersity of an aerosol particle distribution (Morrow, 1981). Theoretically a GSD of 1.0 indicates monodispersity but for practical purposes a GSD of 1.0 - 1.2 is considered to be monodisperse (Fuchs and Sutugin, 1966). Although the deposition of all particles in an aerosol at the same site should require the aerosol to be monodisperse, it has been suggested that a heterodisperse aerosol with a GSD of less than 2.5 will deposit in a similar fashion to a monodisperse aerosol of the same MMAD (Morrow, 1981; Clarke et ah, 1981).

The significance of particle size in the potential effectiveness of an inhaled drug is enormous since the mass of drug contained in a particle is proportional to the cube of its radius. For example, a single 10 pm diameter particle has the same mass as a thousand 1 pm diameter particles.

25 Fig 2.1 Example of the particle size distribution of an ideal aerosol showing the log-normal nature of the distribution. The figures show mass as a function of particle size. A, relative frequency of mass in particles of a given size (linear scale on abscissa); B, as in A with logarithmic scale on abscissa; C, cumulative mass, expressed as a percentage, in particles up to a given size (probability scale on ordinate; logarithmic scale on abscissa). The MMAD is the particle size corresponding to 50% on the cumulative frequency plot. The GSD is ratio of the particle size at 84.1% to the particle size at 50% on the cumulative frequency plot.

Particle Size (Aerodynamic Diameter) (/i,m)

Particle Size (Aerodynamic Diameter) (jum)

26 Pattern of breathing

The amount of aerosol deposited and its distribution within the lung will depend on the pattern of breathing during aerosol inhalation. Aerosol inhalation via the mouth reduces deposition in the extrathoracic region as compared with inhalation via the nose (Agnew, 1984). Increased inspired airflow increases deposition, by inertial impaction, in the oropharynx and larger airways (Lippmann and Albert, 1969; Goldberg and Lourenco, 1973). As the volume inhaled is increased the aerosol particles penetrate deeper into the lung and increase deposition in peripheral airways and alveoli (Pavia, 1977). Breath-holding after inspiration or a slower respiratory rate enhances deposition, by gravitational settlement, in the lung periphery (Palmes, 1973).

2.1.4 Previous studies on aerosol deposition

Although a theoretical consideration of particle size and pattern of breathing allows general predictions to be made about the site of aerosol deposition, the exact site of deposition can only be determined by direct observation. The most useful technique to achieve this in man involves the inhalation of an aerosol labelled with a gamma radiation-emitting isotope and the measurement, with external detectors, of radioactivity deposited in different parts of the respiratory tract (Lippmann and Albert, 1969). An example of the results of such a study is shown in Fig 2.2 where the data of Stahlhofen et a 7. (1980) have been redrawn to show the regional deposition of monodisperse, unit-density particles inhaled by normal subjects to a fixed pattern (VT = 1 1; fR = 7.5 breaths/min; inspiratory flowrate = 250 ml/min). Although a detailed review of this topic is outside the scope of this thesis, the important conclusions from such work are as follows. Alveolar deposition occurs with a wide range of particles and, although maximum alveolar deposition occurs with particles of 3 - 4 pm diameter (Lippmann and Albert, 1980; Heyder, 1982), exclusively alveolar deposition requires particles with a diameter below 2 pm (Stahlhofen et al., 1980; Heyder, 1982). The situation for exclusively tracheobronchial deposition is less simple since particles of a size which deposit in this region (2 - 10 pm diameter) can also deposit in the oropharyngeal

27 Fig 2.2 Fraction of aerosol particles deposited in the extrathoracic (Ext), tracheobronchial (Tb), and alveolar (Alv) regions of the lung as a function of particle size. Data obtained in normal subjects inhaling monodisperse, unit-density aerosol particles with VT = 1 1; fR = 7.5 breaths/min; inspiratory flowrate = 250 ml/min. (Redrawn from the data of Stahlhofen et a h , 1980).

Particle Size (Aerodynamic Diameter) (jllm)

28 region (especially if inspiratory flow rates are high), and in the alveolar region (especially if inspiratory flow rates are low) (Heyder, 1982). The best compromise to achieve exclusively tracheobronchial deposition appears to be to use an aerosol with a particle size around 5 pm diameter (to minimize alveolar deposition) with a low inspiratory flow rate (to minimize oropharyngeal deposition).

2.1.5 Methods of aerosol generation

All aerosol generation processes fall into two fundamental categories: condensation of a gaseous substance and comminution (break-up) of a liquid or solid substance. However, most pharmacologically active compounds have such low vapour pressures that they undergo thermal degradation before sufficient vapour for condensation can be produced. Condensation processes are, therefore, unsuitable for generating aerosols from such substances.

Aerosol generation from a liquid

The most common method of comminution of a liquid to give an aerosol with a particle size appropriate for lung deposition, is by pneumatic atomization. An example of this type of nebulizer is shown in Fig 2.8. In this process, a high-velocity airstream is forced over one end of a narrow tube, the other end of which is immersed in the liquid to be atomized. The liquid is drawn up the tube by the Bernoulli effect and sheared into droplets by the high-velocity airstream. The atomization process generally produces an aerosol with a significant mass in particles greater than 20 pm diameter and a very heterodisperse distribution (Swift, 1980). For this reason, the larger particles are usually removed by one or more baffles placed in front of the jet as shown in Fig 2.8. A generator modified in this way is known as a jet x nebulizer rather than an atomizer because the aerosol produced is a more stable 'cloud’ of droplets. Several factors are important in determining the characteristics of an aerosol produced by a jet nebulizer. Increasing the number of baffles, or other impaction devices used in the nebulizer, will reduce the particle size and make the distribution more monodisperse but at the cost of a decreased mass output. Increasing the airflow rate through a jet nebulizer will

29 decrease the particle size and increase the mass output. Over the last 10 years a considerable number of jet nebulizers have become commercially available and are widely used to generate therapeutic aerosols. However it is only recently that these have been properly characterized (Newman et ah, 1986).

A different principle of droplet formation is used in the ultrasonic nebulizer; a piezoelectric transducer creates high frequency pressure waves in the liquid to be nebulized, resulting in a fountain of droplets being released from the surface of the liquid. An example of this type of nebulizer is shown in Fig 2.3. Because liquid comminution in this device does not require airflow, the dispersion of particles is achieved by passing air over the surface of the liquid. The mass concentration of the resulting aerosol is, therefore, generally considerably higher than that from a jet nebulizer for the same MMAD (Sterk et ah, 1984). Aerosols produced by ultrasonic nebulizers are heterodisperse, the MMAD being determined mainly by the oscillating frequency of the transducer, higher frequencies producing smaller particles. This method is unsuitable for the production of very small particles since the thermal energy associated with oscillation of the piezoelectric transducer would cause the liquid to boil at oscillation frequencies significantly above the 5MHz required for their generation (Swift, 1985).

Although several other methods exist for producing aerosols from liquids, including some for producing monodisperse aerosols (e.g. spinning disc and vibrating orifice generators), the aerosols produced by them have very low mass concentrations and are therefore not useful for the purposes of the present work.

Aerosol generation from a solid

The production of an aerosol from a solid usually requires that the comminution and dispersion processes are separate. The difficulties are therefore twofold: firstly to produce a powder consisting of appropriately sized particles and then to disperse it as an aerosol such that the particles do not reaggregate. The two common methods for micronizing powders are by means of a grinding mill in which the powder is crushed by hard grinding elements or by a fluid energy mill

30 in which high velocity air is used to bring powder particles into rapid collision with each other. Although small particles can be readily produced by these methods, neither creates particles which are all exactly the same size.

Successful dispersion of the powder to form an aerosol requires that the particles be completely separated and that aerosol is produced in a steady fashion over a period of time. Most such devices employ either a compressed cake of powdered material from which particles are slowly removed by a knife edge (e.g. Wright dust feed) or a loose powder which is fed into an aerosolizing region (e.g. NBS dust generator) (Swift, 1985). Although these devices can produce aerosols with very high mass concentrations they are not generally useful for producing small particle aerosols with a narrow range of particle size. The reason for this lies in the attractive forces between particles which become relatively more important as particle size decreases.

Perhaps the best generator to produce a nearly monodisperse aerosol of solid, small particles is the fluidized bed generator. An example of this type of generator is shown in Fig 2.7. In this device, the powder to be aerosolized is mixed with a bed of larger, solid particles (often bronze beads) which are 'fluidized’ by passing a stream of air through them; this action deagglomerates the particles and allows them to be carried away by the air stream as an aerosol. The details of the construction and operation of such a device is described later (page 45).

2.1.6 Choice of generators for the present studies

For the studies described in this thesis I wished to employ two different aerosols: a 'large particle’ aerosol which, ideally, would deposit exclusively in the airways and a 'small particle’ aerosol which, ideally, would deposit exclusively in the lung periphery. In order to produce this selective deposition, the aerosols needed to be as monodisperse as possible, and inhaled using a controlled pattern of breathing. Furthermore, in order that the local anaesthetic delivered as an aerosol should have the greatest pharmacological action

31 possible, a generator which produced aerosol with the highest mass concentration was required.

Several setups using different nebulizers and configurations of breathing tubing were developed to determine the best system for large particle aerosol generation and inhalation. These included a system involving two jet nebulizers run in parallel used by Jain (1975) and an ultrasonic nebulizer used in its standard clinical configuration as described by Palva et a l. (1975). After a detailed examination of such methods, an ultrasonic nebulizer, with a modified system of breathing tubing to achieve maximum aerosol delivery to the subject, was chosen to produce the large particle aerosol. The choice of the small particle generator was less clearcut. Several potential small particle generators were tested and rejected (page 42-44) before deciding to use a modified jet nebulizer. However, because of concern that the lack of physiological effect of this aerosol (described in detail in later chapters) was due to a low mass concentration of local anaesthetic particles, a fluidized bed generator was built and used in some of the later studies in the hope that it would produce an aerosol with a higher mass concentration.

2 .2 EXPERIMENTAL

2.2.1 Large particles

Aerosol generation

Large particle aerosol was produced by a DeVilbiss 35B ultrasonic nebulizer (DeVilbiss Health Care, Feltham, UK) from a 5ml aqueous solution of either 5% (w/v) bupivacaine hydrochloride (local anaesthetic) or 0.9% (w/v) sodium chloride (control solution). The apparatus is shown in Fig. 2.3. The output dial on the nebulizer was set to maximum. In order to maximize the amount of aerosol inhaled by a subject, a breathing system incorporating inspiratory and expiratory one-way valves was attached to the nebulizer. Aerosol was continuously generated in the nebulizer, but flow of aerosol to the subject only occurred on inspiration. This system ensured that all the aerosol produced was available to the subject and that no rebreathing occurred. To reduce the amount of aerosol lost

32 Fig 2.3 Large particle aerosol generator: DeVilbiss 35B ultrasonic nebulizer with tubing incorporating inspiratory and expiratory one-way valves.

^ aerosol

, solution to be nebulized

one-way valve

33 Table 2.1 Particle size distribution of large particle bupivacaine aerosol (DeVilbiss 35B ultrasonic nebulizer).

Lower Size Upper Size Mass Of Particles Cumulative Mass Of Band Of Band In Band Of Particles (pm) (pm) (%) (%)

23.7 33.7 0.1 100.0

17.7 23.7 0.9 99.9

13.6 17.7 2.5 99.0

10.5 13.6 3.4 96.5

8.2 10.5 9.5 93.1

6.4 8.2 21.2 83.6

5.0 6.4 11.7 62.4

3.9 5.0 15.5 50.7

3.0 3.9 26.2 35.2

2.4 3.0 2.4 8.9

1.9 2.4 0.1 6.6

1.5 1.9 0.0 6.5

1.2 1.5 0.4 6.5

0.0 1.2 6.1 6.1

Particle size distribution determined by Malvern 2300 Pulse Particle Sizer. Nebulizer filled with 5 ml of 5% bupivacaine hydrochloride solution and air passed through at 10 1/min. Particle sizes are given as aerodynamic diameter in pm. The mass of particles in each size band is given as a percentage of the total mass of aerosol. The cumulative mass is the mass of particles, as a percentage of the total, whose aerodynamic diameter lies below the upper size limit of the band.

34 by deposition in the apparatus, the length of tubing from the nebulizer to the subject was kept to a minimum. The tubing and valves were those used in anaesthetic circuits (Intersurgical, Twickenham, UK) and had an internal diameter of 22 mm.

Aerosol administration

Normal subjects breathed the aerosols through a mouthpiece to a pattern designed to produce maximum deposition of this aerosol in the tracheobronchial region while minimizing the initially unpleasant effects from bupivacaine depositing in the oropharyngeal region. The pattern of breathing during aerosol inhalation therefore consisted of a 5 sec inspiration, a 10 sec breath-hold and a 5 sec expiration for the first 5 min followed by a 5 min period of resting tidal breathing. Patients with interstitial lung disease also breathed the aerosols through a mouthpiece for 10 min. However, due to their breathlessness, they were unable to follow the initial 5 min pattern adopted by the normal subjects; they were therefore instructed to take slow, deep breaths for the entire 10 min period. To allow aerosols to be given to patients with tracheal stomas, the area around the stoma was cleaned with 70% isopropyl alcohol and a plastic tracheostoma ring (V Mueller, Chicago, USA) attached with silicone skin adhesive (V Mueller, Chicago, USA). This permitted the connection of standard 22 mm diameter tubing. These patients also breathed the aerosols for 10 min and were instructed to take slow, deep breaths.

In all groups of subjects it was occasionally necessary, due to the initial oropharyngeal irritation produced by bupivacaine aerosol, to remove the subject from the aerosol generator. After a period of 1 to 2 min the irritation abated as the bupivacaine already inhaled produced anaesthesia. The subject could then resume aerosol inhalation, usually with no further discomfort.

Dogs inhaled the aerosols through an endotracheal tube by spontaneous breathing for 10 or 20 min.

35 Aerosol particle size distribution

To determine the particle size distribution of the aerosol produced by the ultrasonic nebulizer, 5 ml of 5% bupivacaine hydrochloride solution was placed in the nebulizer and air passed through it at 10 1/min. To ensure that the size distribution was the same as when the aerosol was administered to subjects, the tubing and one-way valves were connected as in Fig 2.3. The resulting aerosol was passed through a Malvern 2300 Pulse Particle Sizer (Malvern Instruments, Malvern, UK). In this device the size of an aerosol particle is measured by the degree to which it diffracts a beam of laser light through which it passes thereby allowing the size of particles to be determined 'in flight’. The particle size distribution for the large particle aerosol is shown in Table 2.1. The aerosol had a MMAD of 4.9 pm with a GSD of 1.7.

Mass of aerosol generated

To determine the amount of aerosol generated per unit time a constant flow of air at 10 1/min was passed through the nebulizer. The resulting particles were trapped in a Pall BB50 high efficiency filter (Pall, Portsmouth, UK) which has been reported to remove 99.999% of particles greater than 0.02 pm diameter (Ball and Saunders, 1987) while offering minimum resistance to flow. 10 ml of 5% bupivacaine solution was placed in the ultrasonic nebulizer and, after the nebulizer had run for 10 min, an additional 10 ml of bupivacaine added and the nebulizer run for a further 10 min. The collection filter was changed at 5, 10, 15 and 20 min. Filters were dried in an oven at 70°C before use. They were weighed immediately after aerosol collection and again after drying in the oven as before. The mass output of wet aerosol over 20 min was 600 mg/min. The mass output of dry bupivacaine hydrochloride was 54.6 mg/min; this corresponds to a concentration of drug per volume of aerosol of 5.46 mg/1 (5,460 mg/m3).

Deposition in the lung

To determine the pattern of aerosol deposition and the amount of bupivacaine deposited in the lung ideally required a gamma-emitting

36 radioisotope (for example " raTc or 123I) to be chemically bound to bupivacaine hydrochloride such that, when the drug was nebulized and inhaled, the activity seen on a lung scan would correspond to a known quantity of bupivacaine. It was important that the isotope chosen should have a half-life in the order of hours so that it could be used safely in human subjects, and that its association with bupivacaine should not disrupt the characteristics of the local anaesthetic molecule. Expert advice on this was sought from a commercial manufacturer of radioisotopes (Amersham Ltd, Amersham, UK), the company which supplies bupivacaine (Glaxo Ltd, Greenford, UK) and Mr. K. Jeyasing of the Department of Nuclear Medicine at Charing Cross Hospital. Their advice was that, although it may be possible to covalently bind 123I to bupivacaine (and this was by no means certain), the process would almost certainly change the physical and chemical properties of the drug. It was recommended that Technetium, in the form 99mTc04~, be mixed with the bupivacaine hydrochloride solution allowing simple ionic bonding to occur between the Technetium and bupivacaine. This technique has been used successfully by other workers to examine the deposition of aerosols in the lung (Dashe et ah, 1974; Ruffin et a h , 1978).

Studies using this radioisotope were performed in two normal subjects. The maximum possible radiation dose the lungs was calculated to be 315 mrad (Roedler et ah, 1978); this is approximately 1/5th of the radiation dose to the lungs delivered by an X-ray computerized tomography scan. The studies had the approval of the Radiation Protection Advisor of Charing Cross Hospital. 11 mCi (4.1 X 1011 Bq) of 99mTc04“ in 0.5ml of normal saline were added to 5 ml of 5% bupivacaine hydrochloride solution and nebulized as previously described (page 32-35). The subjects inhaled the aerosol for 2 min rather than the usual 10 min to minimize any clearance from the lung of deposited aerosol before lung scans could be made. Immediately after aerosol inhalation, posterior gamma scans were performed on seated subjects using a Nuclear Enterprises 8960 LF scintillation camera (Nuclear Enterprises, Edinburgh, UK). Scans of 2 min duration were made at 1, 5, 10, 20 and 30 min after the end of aerosol inhalation. The number of gamma emissions detected by each 6mm X 6mm area of the gamma camera was stored in a 64 X 64 matrix by a Nodecrest computer (Nodecrest, Byfleet, UK) for subsequent analysis.

37 The outline of the lungs was determined from a lung perfusion scan made in each subject after an intravenous injection of 1.5 mCi (0.56 x 1011 Bq) 99mTc04"-labelled macroaggregated albumin. The lung outline was fitted, by computer analysis, to the perfusion scan at the gamma count level corresponding to 18% of the maximum number of counts (Burton et al., 1984) and subsequently superimposed on the aerosol scans. Fig 2.4 A shows an aerosol scan taken 1 min after aerosol inhalation in subject 1. The central pattern of aerosol deposition can clearly be seen. The lung perfusion scan, in which the technetium is lodged diffusely throughout the lung, is shown for comparison (Fig 2.4 C). The decrease in gamma counts in the right lung as technetium is cleared from the lung is shown for both subjects in Fig 2.5 A (the left lung cannot be used for such analysis since radioactivity in the stomach due to swallowed aerosol overlies it). Note that approximately 50% of the initial activity remains in the lung 10 min after the end of aerosol inhalation.

To relate the counts obtained from the lung scans following aerosol inhalation to the amount of radioactivity, and therefore mass of drug deposited in the lung, required a scan to be made of a calibrating 'phantom*. The technique used for this was that of Chung et al. (1988) in which a subject’s perfusion scan provides a calibrating phantom specific to their lungs. Since virtually all the labelled albumin injected for a perfusion scan becomes lodged as microemboli in the lung, and since the amount of radioactivity injected is known, the number of counts obtained from the perfusion scan correspond to a known technetium activity. Once this calibration factor has been calculated, the number of gamma counts in the lung on the aerosol scans can be converted into technetium activity and then into mass of bupivacaine. Since this technique has not been described in detail in the literature, an example of the calculations involved is given in Appendix 1. By this method, the estimated mass of bupivacaine deposited in the lungs during a 10 min aerosol inhalation was 29.3 and 26.7 mg for the subjects 1 and 2 respectively.

In an attempt to determine whether bupivacaine and technetium were cleared from the lungs at the same rate, venous blood samples were taken at 1, 10 and 20 min after the end of aerosol inhalation in

38 Fig 2.4 A, Posterior gamma lung scan 1 min after inhalation of large particle technetium-labelled bupivacaine aerosol in normal subject 1; B, as A after small particle aerosol using Unicorn/Optimist; C, perfusion scan following injection of technetium-labelled macroaggregated albumin in the same subject. The central pattern of deposition of the large particle and the peripheral pattern of deposition of the small particle aerosol can be seen.

A : Large Particle

B : Small Particle

39 Fig 2.5 Gamma counts in the right lung following inhalation of technetium-labelled bupivacaine aerosol as a function of time after inhalation in two normal subjects. A, large particle aerosol; B, small particle aerosol.

cn c □ CO c D o O) o hd o c c co c u o o o

0 5 10 15 20 25 30 Time from end of aerosol inhalation (min)

40 Fig 2.6 Bupivacaine concentration and technetium activity in plasma following inhalation of technetium-labelled bupivacaine aerosol in normal subjects. A, large particle aerosol; B, small particle aerosol In both sets of data the ratio of technetium to bupivacaine in the first sample after the end of aerosol inhalation (A=0.031 pCi/pg;B=0.225 pCi/pg) is similar to that in the original solution nebulized (A=0.036 pCi/pg;B=0.204 pCi/pg), indicating that technetium and bupivacaine are being deposited in and, at least initially, cleared from the lung together.

E

CJ> 0.500 r B: Small Particle Aerosol Bupivacaine • 1 0.100 5 Subject 2 TechnetiumA c o 0.400 0.080

fflc cO 0.300 0.060 uo ’o. 0.100 - 0.020 _Q a E .000 O) 0.000 Plasma Technetium Activity (/zCi/ml) Plasma Technetium Activity (/zCi/m 5 10 15 20 25 30 CL Time from end of aerosol inhalation (min)

41 subject 1. The radioactivity of the blood was measured using a Nuclear Enterprises scintillation counter and plasma bupivacaine concentration determined by gas chromatography (modified from Reynolds and Beckett, 1968). Fig 2.6 A shows this data. It can be seen that the ratio of technetium to bupivacaine in the first sample after the end of aerosol inhalation (0.031 pCi/pg) is virtually the same as that in the original solution nebulized (0.036 pCi/pg) indicating that bupivacaine and technetium are being deposited in and, at least initially, cleared from the lung together. However after this the amount of technetium in the blood continues to rise while that of bupivacaine falls. Interpretation of this later data is difficult since bupivacaine and technetium are being cleared into the blood from the lungs at the same time as they are being cleared from the blood by catabolic mechanisms.

2.2.2 Small Particles

Because of the anticipated difficulty in being able to achieve sufficient peripheral lung deposition of a local anaesthetic aerosol to block conduction in any nerve fibres which may be there, a considerable time was spent, before any physiological studies were performed, in the selection and evaluation of nebulizers which were likely to produce a monodisperse, small particle aerosol with a very high mass concentration. Although the inclusion of details on all the generators considered is beyond the scope of this thesis, some mention of two generators which were tested and rejected is essential since these have been used by other workers to deliver local anaesthetic aerosols.

Preliminary studies

Heated Tube Generator

Following the suggestion by Professor A.S. Paintal (personal communication) that it may be able to block 'J’ receptors located in the alveoli with a suitably sized aerosol of a local anaesthetic, Sister Joseph’s group at St Bartholomew’s Hospital (London, UK) constructed a generator designed to produce an aerosol with 100% of particles below 4pm in diameter (Lunt et a h , 1981). The device

42 consists of a Bird Micronebulizer attached to a steel tube which is electrically heated by insulated wire wrapped in a spiral around it. Its principle of operation is that, although the Bird nebulizer produces an aerosol with a MMAD considerably greater than 4 pm, the subsequent passage of the aerosol through the heated tube would cause water to evaporate from the particles thereby reducing their size.

The device which I tested was kindly loaned to me by Sister Joseph’s group and used in accordance with her instructions. The nebulizer contained 5 ml of 5% bupivacaine hydrochloride solution, it was operated at an airflow rate of 7.5 1/min and the temperature of the tube was adjusted to give an air temperature at the exit of 37°C. The particle size distribution was determined by laser light diffraction as described above (page 36). The MMAD was 1.9 pm with a GSD of 3.7. The mass output was determined over a 5 min period using the technique described above (page 36). The mass concentration of dry bupivacaine in the aerosol was 0.46 mg/1 (460 mg/m3).

Although the heated tube generator produced an aerosol with quite a high mass output, a considerable percentage of the particles were too large for the aerosol to be useful in providing purely alveolar deposition. For instance 36% of the mass of the aerosol was contained in particles greater than 4 pm in diameter. The generator was therefore rejected for these studies.

Gage Cyclone

The suggestion to test the Gage Cyclone was made by Dr. R.D. Stark, Pharmaceuticals Division, ICI, Macklesfield, UK. The device was originally described (Gage, 1968) as a method of generating small particle aerosols of pesticides. It consists of a an atomizing jet to produce particles which are forced around a disc-shaped glass chamber (the 'cyclone’) where large particles impact by inertial forces on the walls allowing the remaining small particles to leave the device through a central exit port. The particles are passed through a 15 1 perspex reservoir chamber before being inhaled.

The device which I tested was kindly loaned to me by Dr. Stark’s group and used in accordance with their instructions. 5% bupivacaine

43 hydrochloride solution was fed to the nebulizer jet at 0.5 ml/min; the output airflow rate was 10 1/min. The particle size distribution was determined by a laser light diffraction technique (Particle Measuring Systems, Boulder, USA) similar to that used for the large particle aerosol. The MMAD was 1.5 pm with a GSD of 1.5. The mass output was determined over a 5 min period using the technique described above (page 36). The mass concentration of dry bupivacaine in the aerosol was 0.035 mg/1 (35 mg/m3).

Although the Gage Cyclone produced an aerosol with a reasonable particle size distribution for peripheral lung deposition with only 2.5% of the mass contained in particles greater than 4 pm in diameter, the mass output of drug was much too small to make this nebulizer useful in my studies.

Modified Jet Nebulizer

Experience gained during the search for a suitable small particle aerosol generator led me to the conclusion that best system would consist of a jet nebulizer (to generate small particles) with an integral system of baffles (to further reduce particle size) run at a high flow rate (to decrease particle size and increase mass output) with the addition of some sort of final particle filter (to remove any remaining large particles).

In order for this system to be successful it was important that it utilise the best jet nebulizer available; at the time when this work was started, this was considered to be the Unicorn nebulizer (Medicaid, Pagham, UK). However, during the course of the work, a nebulizer with similar particle size characteristics to the Unicorn but a higher mass output (see page 52) came on the market (Turret nebulizer, Medicaid); this was, consequently, used in the later studies. Thus, the Unicorn was used for the studies described in Chapters 5, 7 and 8, while the Turret was used in those studies presented in Chapters 4 and 6.

The selection of the particle filter was made following discussions with Dr D. Royston, Northwick Park Hospital, London, UK who suggested that a tube containing small spheres would reduce the particle size of

44 an aerosol passed through it by inertial impaction of the larger particles. Although my preliminary experiments were carried out using such a device constructed by me, an improved version became commercially available (Optimist, Medicaid) and this was used for the studies reported in this dissertation.

These systems proved to be the best tested and were therefore used as the small particle generator for the physiological studies described in this dissertation. Their use is described in detail on pages 48 to 53.

Fluidized Bed Generator

Although a modified jet nebulizer was chosen as the small particle generator for the studies described in this thesis, the lack of physiological effect associated with its use (described in later sections) led me to try another aerosol generator in one of the later studies. Since the maximum concentration of drug in an aerosol is obtained if the aerosol consists of solid particles, a system to achieve this was sought. Following discussions with Dr. A. Morgan and colleagues at the Atomic Energy Research Establishment, Harwell, UK, a fluidized bed generator was built similar to that described by Carpenter and Yerkes (1980). Such generators have been used in toxicological studies of inhaled dust particles and in producing calibrating aerosols for particle-measuring equipment but have not previously been used to deliver drugs. The device is shown in Fig 2.7. It consists of a brass tube (5 cm internal diameter, 26 cm high) containing a bed of bronze beads (150 pm diameter) supported by a stainless steel mesh (63 pm aperture) above an air plenum. Before use, 2 gm of bupivacaine powder, which had previously been micronized using a fluid energy mill (Micron Mills, East Peckham, UK) until the particle size was less than 2 pm diameter, was mixed with the bed material. Dry, compressed air was blown into the plenum and flowed up through the bed causing it to fluidize; this allowed the bupivacaine to be released as an aerosol from the device. An airflow rate of 36 1/min was required to fluidize the bed. To ensure that the final aerosol contained no particles greater than 2pm, an inertial impaction device (Optimist, Medic-Aid) was fitted to the top of the fluidized bed. To ensure that a subject’s inspiratory flow rate did not exceed

45 Fig 2.7 Small particle aerosol generator: fluidized bed with particle filter (Optimist).

t

46 Table 2.2 Particle size distribution of small particle bupivacaine aerosol (fluidized bed generator with Optimist).

Lower Size Upper Size Mass Of Particles Cumulative Mass Of Band Of Band In Band Of Particles (pm) (pm) (%) (%)

8.2 10.5 0.1 100.0

6.4 8.2 1.3 99.9

5.0 6.4 1.5 98.6

3.9 5.0 0.9 97.1

3.0 3.9 0.7 96.2

2.4 3.0 0.7 95.5

1.9 2.4 14.5 94.8

1.5 1.9 73.9 80.3

1.2 1.5 3.9 6.4

0.0 1.2 2.5 2.5

Particle size distribution determined by Malvern 2300 Pulse Particle Sizer. The generator was charged with 2 gm of micronized pure bupivacaine hydrochloride powder and driven by air at 36 1/min. Particle sizes are given as aerodynamic diameter in pm. The mass of particles in each size band is given as a percentage of the total mass of aerosol. The cumulative mass is the mass of particles, as a percentage of the total, whose aerodynamic diameter lies below the upper size limit of the band.

47 that provided by the aerosol generator, a 6 1 bag was incorporated in the breathing circuit to act as an aerosol reservoir.

The particle size distribution was determined by laser light diffraction as described above (page 36). The particle size distribution is shown in Table 2.2. The aerosol had a MMAD of 1.7 with a GSD of 1.2. The mass output was determined over a 20 min using the technique described above (page 36). The mass concentration of dry bupivacaine in the aerosol was 0.366 mg/1 (366 mg/m3).

The fluidized bed generator therefore produced an aerosol which consisted of small, relatively monodisperse particles (only 3.8% of the mass was contained in particles greater than 4 pm in diameter) with a reasonably high mass output. The aerosol produced by the fluidized bed was, consequently, ideal for providing purely peripheral deposition and it was used in one of the later studies performed (Chapter 4).

Small particle aerosol used throughout this thesis

Aerosol generation

Small particle aerosol was produced from a 5 ml aqueous solution of either 5% (w/v) bupivacaine hydrochloride (local anaesthetic) or 0.9% (w/v) sodium chloride (control solution) using either a Unicorn or a Turret jet nebulizer connected to an Optimist inertial impaction device. The apparatus using a Turret is shown in Fig 2.8; the system incorporating a Unicorn nebulizer was exactly the same except for the substitution of this nebulizer for the Turret. The nebulizer was operated at an airflow rate of 10 1/min; the nebulization time was 20 min. To maximize the amount of aerosol delivered, the nebulizer was replaced with a fresh one after 10 min of nebulization for the Unicorn and every 2 min throughout the period of aerosol delivery for the Turret. To ensure that the subject’s inspiratory flow rate did not exceed that provided by the nebulizer, a 6 1 bag was incorporated in the circuit to act as an aerosol reservoir.

48 Fig 2.8 Small particle aerosol generator: modified jet nebulizer (Turret) and particle filter (Optimist) with tubing incorporating reservoir bag and inspiratory and expiratory one way valves. The integral inlet and outlet valves of the Turret nebulizer (I and 0) were blocked off for these studies so that the maximum output could be achieved.

one-way mouthpiece valve

compressed air 10 l/min

49 Table 2.3 Particle size distribution of small particle bupivacaine aerosol (Unicorn nebulizer with Optimist).

Lower Size Upper Size Mass Of Particles Cumulative Mass Of Band Of Band In Band Of Particles (pm) (pm) (%) (%)

3.0 3.9 0.0 100.0

2.4 3.0 0.4 100.0

1.9 2.4 4.1 99.6

1.5 1.9 18.8 95.5

1.2 1.5 11.4 76.7

0.0 1.2 65.3 65.3

Particle size distribution determined by Malvern 2300 Pulse Particle Sizer. Nebulizer filled with 5 ml of 5% bupivacaine hydrochloride solution and driven by air at 10 1/min. Particle sizes are given as aerodynamic diameter in pm. The mass of particles in each size band is given as a percentage of the total mass of aerosol. The cumulative mass is the mass of particles, as a percentage of the total, whose aerodynamic diameter lies below the upper size limit of the band.

50 Table 2.4 Particle size distribution of small particle bupivacaine aerosol (Turret nebulizer with Optimist).

Lower Size Upper Size Mass Of Particles Cumulative Mass Of Band Of Band In Band Of Particles (pm) (pm) (%) (%)

3.0 3.9 0.1 100.0

2.4 3.0 0.3 99.9

1.9 2.4 0.3 99.6

1.5 1.9 18.8 99.3

1.2 1.5 10.0 80.5

0.0 1.2 70.5 70.5

Particle size distribution determined by Malvern 2300 Pulse Particle Sizer. Nebulizer filled with 5 ml of 5% bupivacaine hydrochloride solution and driven by air at 10 1/min. Particle sizes are given as aerodynamic diameter in pm. The mass of particles in each size band is given as a percentage of the total mass of aerosol. The cumulative mass is the mass of particles, as a percentage of the total, whose aerodynamic diameter lies below the upper size limit of the band.

51 Aerosol administration

Normal subjects breathed the aerosols through a mouthpiece to a pattern designed to produce maximum deposition in the lung periphery. The pattern of breathing therefore consisted of a 5 sec inspiration, a 10 sec breath-hold and a 5 sec expiration for the 20 min of aerosol inhalation.

Dogs inhaled the aerosols through an endotracheal tube by spontaneous breathing for 20 min.

Aerosol particle size distribution

The particle size distribution was determined by laser light diffraction as for the large particle aerosol (page 36). The particle size distribution is shown for the Unicorn/Optimist in Table 2.3 and for the Turret/Optimist in Table 2.4. The Unicorn/Optimist produced an aerosol with a MMAD of 0.9 pm with a GSD of 1.7; the aerosol produced by the Turret/Optimist had a MMAD of 1.0 pm with a GSD of 1.4. No particles were detected above 4 pm in diameter with either system. The aerosol produced by both generators was therefore ideal for purely peripheral deposition in the lung.

Mass of aerosol generated

The mass output was determined over a 20 min period using the technique described for the large particle aerosol (page 36). The mass concentration of dry bupivacaine in the aerosol from the Unicorn/Optimist was 0.261 mg/1 (261 mg/m3) and from the Turret/Optimist was 0.513 mg/1 (513 mg/m3). The output for the Turret/Optimist was, therefore, the highest of any small particle generator tested while that of the Unicorn/Optimist was somewhat less than that of the fluidized bed generator but higher than the remainder of the useful generators tested.

Deposition in the lung

The technique described for the large particle aerosol (page 36-42) was used to determine the pattern of deposition of aerosol and amount

52 of bupivacaine deposited in the lung using the Unicorn/Optimist system in the same two normal subjects; this procedure was not repeated following the introduction of the Turret/Optimist system. Fig 2.4 B shows an aerosol scan taken 1 min after aerosol inhalation in subject 1. The peripheral pattern of deposition can clearly be seen, especially when this scan is compared to the perfusion scan in the same subject (Fig 2.4 C). The decrease in gamma counts in the right lung as technetium is cleared from the lung is shown for both subjects in Fig 2.5 B. It can be seen that, despite the peripheral nature of aerosol deposition, it is not immediately cleared from the lung; approximately 50% of the activity remains at 10 min after aerosol inhalation.

The estimated total dose to the lungs using the technique described for the large particle (page 38) was 27 and 15 mg for a 10 min aerosol inhalation in subjects 1 and 2 respectively. Data on the activity of technetium and amount of bupivacaine in the blood following aerosol inhalation is given for subject 2 in Fig 2.6 B. The ratio of technetium to bupivacaine for the first sample after the end of aerosol inhalation (0.225 pCi/pg) is very similar to that in the original solution nebulized (0.204 pCi/pg) indicating once again that bupivacaine and technetium are being deposited and, at least initially, cleared together. The data after this time are difficult to interpret for the reasons given previously (page 42).

53 CHAPTER 3: LOCAL ANAESTHETICS

3.1 Background

The selection of a local anaesthetic agent which would provide the greatest degree and longest duration of nerve blockade, without systemic toxicity, required, firstly, a consideration of the structure and mechanism of action of local anaesthetics and, following this, an experimental investigation of the most promising agents to see which provided the best blockade of a lung reflex when given as an aerosol.

3.2 Structure

The general structure of most local anaesthetics is similar; they consist of a hydrophobic aromatic residue separated from a hydrophillic basic group (usually a secondary or tertiary amine) by an ester or amide link. Fig 3.1 shows the structure of four typical local anaesthetics. The nature of the hydrophobic and hydrophillic groups will govern the activity of the local anaesthetic (page 57-58) while the type of linkage group will determine the mechanism of its metabolic degradation (page 58). In the free base form shown in Fig 3.1, local anaesthetics exist as oils or amorphous solids which are generally lipid soluble but insoluble or slightly soluble in water. They are, therefore, usually prepared for pharmaceutical use in the form of a water soluble salt, commonly the hydrochloride. This charged acid form (BH+) of an uncharged free base (B) will dissociate in water as follows: BH+ B + H+

The dissociation constant for this will be:

Ka [B1 [H!1 [BH+]

Since pKa log Ka and pH log [H+] we can write:

pKa = pH + log [BH*1 [B]

54 It can be seen from this equation that the pKa is the pH at which half the local anaesthetic is in the charged form [BH+] and half is in the uncharged free base form [B]. It can also be seen that if the pKa of the local anaesthetic is greater than the prevailing pH, more of the substance will exist in the charged form than in the free base form. Since the pKa for local anaesthetics is between 7.5 and 10.0, this is indeed the case under physiological conditions.

3.3 Mechanism of action

Local anaesthetics may be defined as chemicals that reversibly block action potentials in excitable membranes (Strichartz and Ritchie, 1987). Although the way in which local anaesthetics act is understood in general terms, considerable debate exists with respect to the exact mechanisms involved. The generation and propagation of action potentials in nerves depend on the opening and closing of sodium and potassium channels in the nerve membrane (Hodgkin and Huxley, 1952) and it is agreed that local anaesthetics act by interfering with the function of these channels, in particular preventing the influx of sodium ions via the sodium channel, so as to block the generation of action potentials (Taylor, 1959).

Two fundamentally different theories have been suggested to account for this. The first proposes that local (and volatile) anaesthetics penetrate the cell membrane and the resulting membrane expansion interferes with the function of the sodium channel in a non-specific way (Seeman, 1972; Lee, 1976). The second submits that local anaesthetics block sodium channel function by binding to specific receptors on the sodium channel - the local anaesthetic crosses the nerve cell membrane in the hydrophobic uncharged form but acts on sodium channel receptor sites, accessible only from inner surface of the membrane, in the hydrophillic positively charged form (Narahashi and Frazier, 1971; Strichartz, 1973). The considerable body of work performed since these theories were proposed supports the latter mechanism although general membrane effects may contribute (Rang and Dale, 1987; Ritchie and Greene, 1985; Strichartz and Ritchie, 1987). The current theories of sodium channel blockade by local anaesthetics are based on the modulated receptor hypothesis of Hi lie (1977). In the simplest form of this model, the different voltage-dependent

55 Table 3.1 Chemical and biological properties reported for four typical local anaesthetics.

Name Structure Molecular pKa Lipid Anaesthetic Anaesthetic Weight Solubility Potency Durat ion

Procaine H,N — (// f v N>— C11 — OCH 2C H 2— N / C ’Hs 236 8.92

H 8C 4 CH: , , \ / \ II / Amethocaine n — x>— C — o c h 2 c h ,— N 26^4 8.49 205 3.5 / \ CH:

CDcn

^ C 2H5 Lignocaine 23 A 7.86 I/4 2.0

^ C ,H ,

c 4hs

Bupivacaine // 5 288 8.10 1375 3.5 / ' >— NH — c —*

CH 3

Values for lipid solubility, anaesthetic potency and duration are unitless using procaine as a reference. pKa, negative logarithm of the acid dissociation constant at 25°C; lipid solubility estimated by partitioning into n-heptane/aqueous buffer; potency and duration determined from concentration required to block conduction in frog sciatic nerve and length of resulting block. Data are those of Truant and Takman, 1959; Tucker and Mather, 1980; Conception and Covino, 198A. conformational states of the sodium channel have different binding affinities for local anaesthetic molecules; inactivated channels have the highest affinity, open channels a lower affinity and resting channels the lowest affinity.

3.4 Differential sensitivities of nerve fibres

The classical paper of Gasser and Erlanger (1929) first proposed the principle that nerve fibres are differentially susceptible to local anaesthetic blockade on the basis of fibre diameter: small (slowly conducting) fibres being more sensitive than large (rapidly conducting) fibres. In recent years it has become apparent that, although this may be valid for myelinated fibres (and even this has been challenged ; Gissen et a h , 1980; Wildsmith et ah 1985), it does not hold true for unmyelinated fibres: A6 (myelinated) fibres may be blocked before C (unmyelinated) fibres (Nathan and Sears, 1961; Fink and Cairns, 1984; Raymond and Gissen, 1987). It seems likely that the different mode of conduction in myelinated and unmyelinated fibres (saltatory versus continuous) is partly responsible for these differences (Ritchie and Greene, 1985) although the details remain to be established. Other important factors appear to be the degree of lipid solubility of the local anaesthetic (agents with high lipid solubility can more readily penetrate myelin sheaths; Wildsmith et ah, 1985) and the length of a fibre over which the anaesthetic is applied (smaller fibres require shorter lengths to be exposed due to their shorter internodal distances; Franz and Perry, 1974).

3.5 Structure-activity relationships

The first scientific reports of a local anaesthetic agent appeared during the nineteenth century when the ability to cause local numbing was discovered to be one of the effects of cocaine (see Vandam, 1987 for a detailed historical account). The subsequent search for a synthetic local anaesthetic compound led to the production of procaine in about 1904; some of its chemical and biological properties are given in Table 3.1. This agent has remained the prototype for many of the local anaesthetics synthesized since.

57 The rate of onset of action of a local anaesthetic is related to its pKa; agents with a lower pKa tend to have a faster onset since, at physiological pH, more of the local anaesthetic will exist as the free base form and can more rapidly penetrate both surrounding tissue and nerve cell membranes (Conception and Covino, 1984). In general, the potency of a local anaesthetic increases with its degree of lipid solubility, since this permits it more readily to penetrate the nerve cell membrane (Courtney and Strichartz, 1987). The duration of anaesthesia is related to the degree of lipid solubility since this increases the affinity for neural and extraneural tissues (Tucker and Mather, 1980). Increasing the size of the molecule also tends to increase the duration of anaesthesia probably by increasing the degree of binding to receptor protein (Conception and Covino, 1984).

3.6 Metabolism

Ester-linked local anaesthetics are rapidly hydrolysed by esterases especially those in the plasma and, to a lesser extent, the liver (Arthur, 1987). Because of the non-specific nature of this hydrolysis, it may be expected that, if an ester-linked local anaesthetic were given as an aerosol to the lung, local esterases in the lung would be able to metabolize it. In contrast, amide-linked local anaesthetic agents, appear to be metabolized exclusively by the liver, the initial reaction being N-dealkylation (Arthur, 1987).

3.7 Systemic toxicity

Before any studies involving a drug can be performed safely, the possible toxic effects associated with its use and the dose at which they occur must be known. In the studies described in this thesis local anaesthetics were used at doses very much below those reported to produce any side effects; blood levels were frequently determined to confirm this. Because of the wide usage of local anaesthetics, an enormous amount is known about their toxic effects. The systemic toxicity of local anaesthetics is, in general, related to their ability to stabilize membranes. Local anaesthetics can readily cross the blood-brain barrier and affect the central nervous system (Covino, 1987) initially producing central nervous system excitation (lightheadedness, dizziness, visual and auditory disturbances,

58 drowsiness, disorientation) and at higher doses, shivering, slurred speech, muscle twitching and tremors followed by generalized convulsions. Very high doses can result in central nervous system depression with a cessation of convulsions and respiratory arrest. The cardiovascular side-effects, consisting of hypotension and possible cardiac arrest, require doses several times greater than that for central nervous system toxicity (Covino, 1987).

With respect to bupivacaine, no symptoms of toxicity have been reported up to a plasma level of 1.4 pg/ml (Reynolds, 1971), very mild symptoms (slight numbness of eyelids) at 2.1 pg/ml (Jorfeldt et a 7., 1968) and more obvious although still mild symptoms (dizziness, numb lips, pressure in ears) at 2.6 to 4.5 pg/ml (Mather et al., 1971).

3.8 Choice of local anaesthetic for the present studies

The factors involved in the choice of a local anaesthetic to provide afferent neural blockade in the lung are different from those for most local anaesthetic procedures due to the high metabolic activity of the lung and the passage of virtually the entire cardiac output through the lung. The first of these factors supports the use of amide-linked agents since they would be expected to require to be cleared from the lung before they could be metabolized as opposed to ester-linked compounds which could be hydrolized within the lung. The second factor supports the use of agents with a low pKa (rapid onset), high degree of lipid solubility (reduced clearance) and large molecular size (reduced clearance). Further support for the requirement of a high degree of lipid solubility is provided by the general need for anaesthetic potency.

With these factors in mind, three local anaesthetics most likely to be useful in providing afferent neural blockade in the lung were chosen. These were lignocaine (lidocaine), bupivacaine and amethocaine (tetracaine); the first two are amide-linked, the last is an ester- linked local anaesthetic. Some of their chemical and biological properties are given in Table 3.1 along with those of procaine for comparison. Lignocaine is the most widely used local anaesthetic in clinical practice (British National Formulary, 1989) and has previously been used as an aerosol to block lung afferents both

59 clinically and experimentally. Bupivacaine is a potent, long acting agent, often used for lumbar epidural and spinal anaesthesia (British National Formulary, 1989) and has previously been used as an aerosol in a few studies examining the control of breathing (Jain et a l., 1973; Dain et al., 1975; Cross et al., 1976). Amethocaine is a rapidly acting compound commonly used for topical application especially in ophthalmology (British National Formulary, 1989) which, at the time of these studies, had not, to my knowledge, been used in aerosol form.

In clinical practice, a vasoconstrictor (usually adrenaline), is often used in combination with a local anaesthetic in order to reduce local blood flow and thus prolong the duration of anaesthesia. Although aerosols of lignocaine containing adrenaline were used in early studies reported in the literature (Petit and Delhez, 1970; Jain et al., 1973) there now seems to be no basis for the use of adrenaline in local anaesthetic aerosols. The sympathetic innervation to the pulmonary circulation includes a- and 3-adrenergic receptors on pulmonary vascular smooth muscle; vasoconstriction is produced by the former, vasodilatation by the latter in common with many other types of vascular smooth muscle (Fishman, 1985). Adrenaline, which is both an q- and 3- agonist, can produce pulmonary vasoconstriction or vasodilation, depending on the circumstances (Bergofsky, 1979). In any case to attempt to reduce blood flow either to the pulmonary circulation or to the airways does not seem desirable in a physiological study; nor does the potential for stimulation of cardiac 31 receptors seem warranted. For these reasons, adrenaline was not used in any study described in this thesis.

The choice from lignocaine, bupivacaine and amethocaine of one local anaesthetic to use in the studies described in this thesis, was made by testing their ability, when given as a large particle aerosol, to block a well described respiratory reflex. Since, in man, the Hering- Breuer inflation reflex can only be demonstrated during anaesthesia (Widdicombe, 1961) or sleep (Hamilton et al., 1988) and no certain test exists for the reflex effects of C-fibre stimulation (see Chapter 6), the cough reflex to citric acid aerosol was used; the method for testing this is described in detail in Chapter 5 (page 85).

60 These studies were carried out in the very early stages of the work for this thesis and were performed concurrently with the development of the aerosol generators (Chapter 2). They were, therefore, very much in the way of preliminary studies and the aerosols were delivered using a succession of large particle generators. Two factors which were constant throughout were the pattern of inhalation (the same as that described on page 35) and the testing of the cough reflex (by inhalation of citric acid aerosol as described on page 85).

In order to achieve the greatest degree of blockade in the shortest possible time, thereby minimizing drug clearance from or metabolism in the lung, I wanted to give the local anaesthetic in the highest concentration possible. For comparative purposes it was necessary to use similar concentrations of the three agents; bupivacaine, being the least water soluble of the three, dictated this concentration. An aqueous solution of bupivacaine is fully saturated at a concentration of approximately 6%. However, during the process of aerosol generation in a jet nebulizer, solvent evaporation increases the concentration of the remaining solution and decreases its temperature (Mercer, 1973); therefore the maximum useable concentration of bupivacaine is 5%. Similar concentrations of the other two local anaesthetics were used. The results of these studies are shown in Table 3.2.

In a given subject, amethocaine always abolished the cough reflex for a shorter duration than did bupivacaine. In general, lignocaine produced a shorter and much more variable duration of abolition than did bupivacaine. 5% bupivacaine was, therefore, chosen for use in the studies described in this thesis.

These results shown in Table 3.2 are consistent with studies in animals. Direct application of bupivacaine to the mucosal surface of the airway has been shown to abolish the cough reflex to mechanical stimulation in cats for a longer time than did the same concentration of lignocaine (Ford, et a 7., 1984). Camporesi, et al., (1979) found that both bupivacaine and amethocaine, when directly applied to the tracheal mucosa or given as an aerosol in dogs, blocked discharge in rapidly adapting receptors during mechanical stimulation of the airway at a lower concentration than did lignocaine; the duration of the

61 Table 3.2 Duration of blockade of the cough reflex to citric acid aerosol produced by a 10 min inhalation of three local anaesthetic agents as large particle aerosols in normal subjects.

Aerosol Subject Duration of Block (min) Generator Lignocaine Bupivacaine Amethocaine 4% 5% 4%

(A) 1 - 17 11 Wright’s 2 - 12,14 10 nebulizer 3 - 10 - 4 - - 20

(B) 2 3 21 4 DeVilbiss 35B 4 2,4 15 10 flowpast

(C) 1 19 31 - DeVilbiss 35B 2 2,15 16,16,20 - inspi ration 4 15,15,45,15,30 25,29,35,35 10 activated 5 12 17 -

Aerosol generators: (A) 2 Wright’s nebulizers run in parallel by air at 15 1/min as described by Jain (1975); (B) DeVilbiss 35B ultrasonic nebulizer with continuous flow of aerosol past mouthpiece at 10 1/min; (C) DeVilbiss 35B ultrasonic nebulizer with inspiratory and expiratory one-way valves as described on page 32-35. Aerosol was inhaled for 10 min to the pattern given on page 35. The cough reflex to citric acid aerosol was performed as described on page 85. The duration of block was the time in min after aerosol inhalation at which citric acid first produced cough.

62 blockade was not tested. Although, in that study, evidence was presented that the blocking concentration was lower for amethocaine than for bupivacaine when the local anaesthetic was directly applied to the mucosa (but not when given as an aerosol), this cannot be extrapolated to predict relative blocking times for these agents when given as aerosols since bupivacaine (amide-1inked) would be expected to require to be cleared from the lung before it could be metabolized whereas amethocaine (ester-linked) could be hydro!ized within the lung tissue. It is interesting to note in this context that Karlsson (1987) found the difference in duration of anaesthesia between amethocaine and lignocaine (amide-linked) to be significantly smaller when given as an aerosol to the airways than had been previously reported when applied to the tongue tip; this is perhaps because the lung is considerably more metabolically active, and therefore has a greater esterase activity, than the tip of the tongue.

It should be noted from Table 3.2 that improvements in the aerosol generator during the period of development finally resulted in a abolition of the cough reflex lasting between 16 and 35 minutes using bupivacaine; this provided sufficient time for physiological studies to be performed.

3.9 Determination of bupivacaine concentration in blood

After a venous or arterial blood sample had been taken, 10 ml of whole blood was mixed with lithium heparin, centrifuged at 800 G for 15 min at 4°C and the plasma drawn off and frozen for later analysis. The concentration of bupivacaine in plasma was determined using a modification of the technique described by Reynolds and Beckett (1968); bupivacaine was measured by gas chromatography (Perkin Elmer F33 gas chromatograph) following double alkaline extraction from plasma using ethyl pivacaine as an internal marker.

63 CHAPTER 4: DOGS - RESPIRATORY REFLEXES

4.1 Introduction

The initial studies in the work done for this thesis were performed in man. However, as will become evident later in this thesis, the results of these investigations showed that the inhalation of the small particle local anaesthetic aerosol had no significant effects on the pattern of breathing in man over a range of conditions. It was not known whether this was a genuine indication that information from receptors in the lung periphery was not involved in the control of breathing in such situations, or that such information was active but insufficient local anaesthetic was being deposited in the lung periphery to block its conduction. I therefore wished to test the ability of this aerosol to block a response which was known to be due to pulmonary C-fibre stimulation in man. The evidence for the existence of such a situation in man is weak and will be considered further in Chapter 6 (page 89-90). The only certain way to examine this question, therefore, appeared to be to test the ability of the small particle local anaesthetic aerosol to abolish a reflex believed to be mediated by afferent information from the lung periphery in animals. In order that the aerosol would deposit in a similar fashion to that in man, it was important to perform the study in a large animal species with a lung and airway size comparable to that of man. I therefore chose to study the pulmonary chemoreflex to capsaicin injection in dogs. This consists of apnoea, bradycardia and hypotension following central intravenous injection of capsaicin and is believed to be mediated by pulmonary C-fibre endings in the lung periphery (Coleridge et a l., 1965, Coleridge and Coleridge, 1984); the nature of the pulmonary chemoreflex is considered in more detail in Chapter 6 (page 87-101). Since this was to be the definitive test of the ability of a small particle local anaesthetic aerosol to block afferent information from the lung periphery, both the Turret/Optimist system and the fluidized bed generator were used in these studies.

The use of an anaesthetized animal also provided an opportunity to determine the ability of the large particle local anaesthetic aerosol to abolish reflex effects believed to be mediated by receptors located in the airways. The reflexes studied were the cough reflex to

64 mechanical stimulation of the airway (believed to be triggered by stimulation of rapidly adapting pulmonary stretch receptors) and the Hering-Breuer inflation reflex (thought to be mediated by slowly adapting pulmonary stretch receptors).

To determine whether any blockade of lung receptors produced by the large particle local anaesthetic aerosol was confined to the large airways, its ability to abolish the pulmonary chemoreflex to capsaicin injection was also examined. Similarly, to determine whether any blockade achieved by the small particle aerosol was confined to the lung periphery, its ability to block the cough and inflation reflexes was investigated.

In order to provide a perspective with which the other studies described in this thesis may be seen, the studies in dogs, although not chronologically the first to be performed, will be the first to be presented.

4.2 Methods

Animals and experimental setup

Eight mongrel dogs (18-28 kg; 6 male) were anaesthetized for surgery with 1.0-1.5% halothane in N20/02 (50/50) through an endotracheal tube following induction with intravenous sodium thiopentone (25 mg/kg). For the remainder of the experiment, anaesthesia was maintained with intravenous chloralose (40 mg/kg; Merck)given as required. Arterial blood gases were determined (Radiometer ABL1 Acid-Base Laboratory) at intervals throughout the experiment and remained within normal limits; acid-base balance was maintained with a sodium bicarbonate (1.26%) infusion. Body temperature was kept constant at 37°C with an electric blanket connected to a rectal probe (CFP Homeothermic Blanket Control 8142). The right femoral artery was cannulated and arterial blood pressure measured (Bell and Howell 4-442-001 pressure transducer). The right external jugular vein was cannulated with a 7 French gauge Cordis catheter; a blood pressure transducer was connected to the catheter which was advanced until the tip lay in the right ventricle, the position being confirmed by the pressure trace. To allow the vagi to be rapidly identified and cut at the end of the experiment, both

65 cervical vagus nerves were carefully exposed, a length of umbilical tape passed loosely around each and the site covered with saline- soaked swabs.

Airflow through the endotracheal tube was measured with a Fleisch No. 1 pneumotachograph connected to a Validyne DP45 pressure transducer and the signal integrated to give volume by a Severinghaus single breath computer. End-tidal PC02 was measured with a Beckman LB-2 Medical Gas Analyser. An electrocardiogram and beat-to-beat heart rate were recorded. Signals were displayed on an oscilloscope (LAN SCOPE II) and recorded on a rapidly responding chart recorder (Devices M19).

Reflexes

The pulmonary chemoreflex was elicited by a rapid injection of a small volume of capsaicin into the right ventricle. An event marker was depressed at the start of injection and released at the end; the time of injection was taken as the midpoint of this period. An apnoeic ratio was calculated as the duration of apnoea following capsaicin injection divided by the mean duration of the preceding breaths. In dogs 1 to 3, a stock solution of capsaicin (approximately 8% pure; McCarthy’s) made up to 1 mg/ml in 90% ethanol was diluted in 0.9% saline and injected in doubling doses until the pulmonary chemoreflex was evoked; this threshold dose consistently evoked the reflex and was used in all subsequent experiments in that animal. In dogs 4 to 8, a stock solution of capsaicin (80-85% pure; Sigma) made up to 10 mg/ml in 90% ethanol was diluted in 30% ethanol/70% saline and given as above. Volumes of diluent equal to the volume of the threshold dose of capsaicin were injected as controls.

The cough reflex was elicited by mechanical stimulation of the trachea and carina with a fine plastic catheter. To asses the Hering-Breuer inflation reflex, a water-sealed spirometer with rebreathing circuit and C02 absorber (P K Morgan), was connected to the endotracheal tube and, following a period during which baseline breathing was recorded, a weight was placed on the spirometer bell during inspiration. The duration of the resulting apnoea was measured and an inhibitory ratio calculated as the duration of apnoea divided by the mean breath

66 duration prior to the addition of the weight. In order to compare results for the inflation reflex, matched maximum inflation volumes before and after an intervention were selected and the inhibitory ratios produced by each compared.

Small particle local anaesthetic aerosols

Small particle aerosol produced by the Turret/Optimist system (page 48-52) and by the fluidized bed generator (page 45-48) were given at separate times to dogs 6,7 and 8. Aerosol was inhaled through the endotracheal tube by spontaneous breathing for 20 min. Lung reflexes were tested immediately before and after aerosol inhalation. Following recovery this procedure was repeated.

Large particle local anaesthetic aerosol

Large particle aerosol (page 32-35) was administered to spontaneously breathing dogs through the endotracheal tube which had been withdrawn until the tip lay just below the level of the vocal chords. In dogs 1 and 2, the aerosol was delivered for 10 min; during this procedure it was noticed that the pattern of breathing became progressively slower and deeper but that this had not reached a maximum by the end of 10 min. In dogs 3 to 7, therefore, aerosol delivery was continued until the increase in tidal volume had reached a plateau; this took about 20 min. Lung reflexes were tested immediately before and after aerosol inhalation. Following recovery this procedure was repeated. Bilateral vagotomy was performed in dogs 3 to 7 immediately after the last period of large particle aerosol inhalation and lung reflexes retested.

Local anaesthetic infusion

To exclude the possibility of a systemic effect of local anaesthetic following aerosol inhalation, the reflexes were tested before and after an intravenous infusion of bupivacaine (2.0-2.5 mg/kg) in dogs 3, 4 and 5.

67 Estimation of plasma bupivacaine

After each bupivacaine aerosol inhalation and infusion, an arterial blood sample was taken for the determination of plasma bupivacaine (page 63).

Statistical analysis

Statistical analyses were performed using Student’s t-test for paired data. The level of significance was taken as P<0.05 in a two-tailed test.

4.3 Results

Small particle local anaesthetic aerosols

The cardiovascular and respiratory effects of small particle bupivacaine aerosol inhalation are shown in Fig 4.1 A and B. Inhalation of aerosol from the Turret jet nebulizer (J) or fluidized bed nebulizer (FB) had no significant effect on heart rate or mean arterial blood pressure, but resulted in a small, though statistically significant, increase in VT (mean increase J = 23 ml (12%), P<0,01; mean increase FB = 27 ml (13%), P<0.02). Inhalation of aerosol from the fluidized bed also increased fR by 4 breaths/min (22%, P<0.05).

The rapid injection of capsaicin into the right ventricle produced the pulmonary chemoreflex consisting of apnoea, bradycardia and hypotension at a threshold dose of 40 pg/kg in dogs 1 to 3 (using the 8% pure capsaicin), 4 pg/kg in dogs 4, 5 and 8 (using the 80-85% pure capsaicin) and 8 pg/kg in dogs 6 and 7 (also using the 80-85% pure capsaicin). The mean latency for this was 1.9 + 0.4 sec (range 1.1 to 2.7 sec) in the 8 dogs. The mean time taken for injection was 0.9 + 0.5 sec. An example of the pulmonary chemoreflex is shown in Fig 4.2 A. The effect on the reflex of small particle bupivacaine aerosol inhalation is shown for dogs 6,7, and 8 in Fig 4.3 A and B; no significant effect was seen on any component of the reflex.

The cough reflex was always present before and after small particle bupivacaine aerosol inhalation. Fig 4.4 A and B show the matched

68 esrmn ff a o osbeatrbpvcie nuin in infusion bupivacaine after fRpossiblenotof was Measurement E, inhalation;aerosol of period secondthe after vagotomy with min eoo fo e () euie n liie e (B gnrtr C, (FB) generator;bed fluidized and (J) nebulizer jet from aerosol o 3.dog i . Crivsua n rsiaoyefcso eoo inhalation aerosolof effects respiratory and Cardiovascular 4.1Fig n itaeos nuino uiaan i os A&B sal particle smallB,& in A dogs. bupivacaine of infusion intravenous and nrvnu ifso. ahpit ersnsoeeprmn indog.one experiment one represents point Each infusion. intravenous fR (breaths/mln) V-j- (ml) MEAN BP (mmHg) fc (beats/mln) 20 for aerosolparticle large D, 10 formin; aerosol particle large os 6 7 8 os678 os12Dogs 3-7 Dogs 1,2 Dogs 6,7,8Dogs 6,7,8 69 Dogs 3,4,5 maximum inflation volumes and the associated inhibitory ratios for the Hering-Breuer inflation reflex before and after small particle aerosol inhalation. This reflex was unaffected by the aerosol.

Control injections of the diluent had no effect on resting cardiovascular or respiratory variables or on any reflex on any occasion. Plasma bupivacaine levels were 0.37 + 0.14 and 0.35 + 0.21 pg/ml in the three dogs immediately after jet and fluidized bed aerosol inhalations respectively.

Large particle local anaesthetic aerosol

Inhalation of large particle bupivacaine aerosol had no significant effect on heart rate or mean arterial blood pressure (Fig 4.1 C and D). However, it produced a progressive slowing and deepening of breathing which was maximal after approximately 20 min of aerosol inhalation (Fig 4.1 C and D). Mean VT increased by 35% after 10 min and 82% after 20 min of aerosol inhalation; mean fR decreased by 35% after 10 min and 48% after 20 min of aerosol. Subsequent bilateral vagotomy resulted in a further increase in mean VT of 15% and a decrease in mean fR of 13% (Fig 4.1 D) although these changes were not statistically significant.

An example of the effect of large particle bupivacaine aerosol on the pulmonary chemoreflex to capsaicin is shown in Fig 4.2; 20 min of aerosol inhalation resulted in complete abolition of the reflex with subsequent recovery by 20 min after the end of aerosol delivery. Fig 4.3 C and D shows data for this in dogs 1 to 7. Following 10 min of aerosol inhalation, the apnoeic ratio and maximum changes in systolic blood pressure and heart rate were reduced indicating attenuation of the reflex. After a 20 min inhalation, the reflex was abolished.

The cough reflex was elicited in 5 dogs and was always absent after large particle bupivacaine aerosol administration. Both 10 and 20 min of aerosol inhalation resulted in complete abolition of the inflation reflex (Fig 4.4 C and D); the inhibitory ratios were not significantly different from 1.0 for either time period. These inhibitory ratios were produced by inflation volumes which did not differ significantly from those before aerosol inhalation.

70 Fig 4.2 Pulmonary chemoreflex evoked by capsaicin injected into the right ventricle in dog 5. A, B & C, before, 3 min after and 20 min after a 20 min inhalation of large particle aerosol. The airflow traces show inspiration upwards. Capsaicin injection is shown by the event marker. The apnoea, hypotension and bradycardia seen before aerosol inhalation were absent immediately after aerosol but had returned 20 min later.

71 Dogs 6,7,8 Dogs 6,7,8 Dogs 1,2 Dogs 3-7 Dogs 3,4,5

-a ro

Fig 4.3 Pulmonary chemoreflex evoked by capsaicin injected into the right ventricle before and after aerosol inhalation and intravenous infusion of bupivacaine. A & B. smalL particle aerosol from jet (J) nebulizer and fluidized bed (FB) generator; C & D. large particle aerosol for 10 and 20 min; E, intravenous infusion. MAX A SYS BP . maximum percentage fall insystolic blood pressure; MAX A fc , maximum percentage fall in heart rate. Each point represents one experiment in one dog. An apnoeic ratio of 1.0 indicates abolition of that component of the reflex. Dogs 6,7-8 Dogs 6,7,8 Dogs 1,2 Dogs 3-7 Dogs 3,4,5

LU 300 2 z>mmi1 o > 200 z t o 100 1§ f z 0

Fig 4.4 Hering-Breuer inflation reflex before and after aerosol inhalation and intravenous infusion of bupivacaine. A & B, small particle aerosol from jet ( J ) nebulizer and fluidized bed (FB) generator; C & D. large particle aerosol for 10 and 20 min; E. intravenous infusion. Each point represents one experiment in one dog. An inhibitory ratio of 1.0 indicates abolition of the reflex. PULMONARY CHEMOREFLEX INFLATION REFLEX

--1------1------1 o 10 20 sec

Fig 4.5 Differential block of the pulmonary chemoreflex and the Hering-Breuer inflation reflex during recovery from airway anaesthesia induced by 20 min of large particle aerosol inhalation in dog 4. A, B & C, 5, 40, and 130 min after aerosol inhalation. All traces show airflow with inspiration upwards. The event marker indicates the point o capsaicin injection or the addition of a weight. Both reflexes were abolished by the aerosol; the chemoreflex had recovered at a time when the inflation reflex was s t i l l blocked. An example of the timecourse of the recovery of the pulmonary chemoreflex and the inflation reflex following a 20 min inhalation of large particle aerosol is presented in Fig 4.5. The chemoreflex had recovered at a time when the inflation reflex was still blocked. In all dogs, the three reflexes had recovered by approximately 1 hour after the end of aerosol delivery; the pulmonary chemoreflex was the first to return followed by the inflation reflex and then the cough reflex.

These reflexes were absent following bilateral vagotomy. Plasma bupivacaine levels immediately after 10 and 20 min of aerosol inhalation were 1.87 + 0.90 and 3.82 + 1.63 pg/ml respectively.

Intravenous infusion of local anaesthetic

Intravenous infusion of bupivacaine resulted in similar blood levels (3.28 + 2.14 M9/mD to those seen after 20 min of large particle aerosol administration. However, no significant effects were seen on the pattern of breathing, blood pressure or heart rate (Fig 4.1 E) nor on the pulmonary chemoreflex (Fig 4.3 E), cough reflex or inflation reflex (Fig 4.4 E).

4.4 Discussion

Some controversy exists in the literature regarding the location of the receptors responsible for the pulmonary chemoreflex. The difficulty arises because of concern about the relative vascular accessibility of bronchial and pulmonary C-fibre endings following the injection of chemicals into the right heart. On theoretical grounds it is possible that a substance injected into the right heart could rapidly reach receptors located in the airways since extensive anastomotic connections exist between the bronchial and pulmonary circulation in many species including the dog (Modell et a l. , 1981) and because a drug within a pulmonary capillary could diffuse the short distance through surrounding lung parenchyma to reach the inner airway wall (Giuntini and Fazio, 1979). Experimental evidence that this occurs has come from Sant’Ambrogio and Sant’Ambrogio (1982) who have demonstrated in the dog that slowly adapting and rapidly adapting receptors, localized in the conducting airways, can be stimulated more

75 rapidly by right atrial than left atrial injection of veratridine and benzonatate. However, in their study, some extrathoracic receptors were not stimulated at all by right heart injection and the mean delay between right heart injection and intrapulmonary receptor response was 6.1 sec for both slowly adapting and rapidly adapting receptors. With specific reference to C-fibre afferents, Coleridge and Coleridge (1977) demonstrated that pulmonary C-fibres are stimulated within a mean of 2.1 sec of right heart injection of capsaicin whereas bronchial C-fibres respond within a mean of 9.3 sec. This evidence indicates that the pulmonary chemoreflex in the present study, which occurred within a mean of 1.9 sec of right heart injection of capsaicin, results from stimulation of pulmonary C-fibre endings located "near the alveoli close to the pulmonary capillaries" as originally suggested by Paintal (1969).

The results of the present study indicate that a small particle local anaesthetic aerosol, which deposits primarily in the lung periphery in man (page 52-53) and is of a size which has been shown to deposit at alveolar level in dogs (Rizk et a/., 1984), fails to block the pulmonary chemoreflex to right heart injection of capsaicin in dogs. These results are in contrast to those reported in the rat where the tachypnoea produced by pulmonary emboli was abolished by inhalation of a small particle lignocaine aerosol (Stark et ah, 1985). This tachypnoea has been shown in the rabbit to be mediated by C-fibres (Guz and Trenchard, 1971). However Stark and colleagues used an aerosol with a mass median aerodynamic diameter of 2.5 pm which is likely to deposit at alveolar level in man, but is unlikely to do so in a small mammal such as the rat which has much smaller airways and a higher resting respiratory frequency than dog or man; both of these factors are likely to promote increased airway deposition. Indeed experiments in which guinea pigs inhaled aerosols with a mass median diameter less than 1 pm resulted in significant airway deposition of the aerosol (Wolff et ah, 1979). It seems likely, therefore, that the "small" particle aerosol of Stark et ah was acting in a similar fashion to the large particle aerosol used in the present work.

The inability, in the present study, of a small particle local anaesthetic aerosol produced by a modified jet nebulizer or by a fluidized bed generator to block a reflex believed to be mediated by

76 C-fibre endings located at alveolar level, may be due to insufficient drug deposition on the surface of the lung periphery to achieve adequate anaesthesia of C-fibre endings. The low plasma bupivacaine levels seen in the dogs after aerosol inhalation possibly reflect this. However, the possibility must also be considered that the nerve endings or receptors responsible for the pulmonary chemoreflex are not located in, or near, the alveoli. This is discussed further on pages 78 and 79. Whatever the reason for its lack of effect in blocking these receptors in dogs, it indicates that the aerosol is unlikely to be effective in blocking the same receptors in man.

In contrast to the results with the small particle aerosol, the large particle local anaesthetic aerosol attenuated the pulmonary chemoreflex after a 10 min inhalation and abolished it after a 20 min inhalation. To my knowledge this represents the first demonstration that a local anaesthetic aerosol can be used to block a reflex believed to originate from alveolar level. Jain et a 7. (1973) reported that the inhalation of a large particle 5% bupivacaine aerosol in the rabbit abolished the cough reflex but that the pulmonary chemoreflex to phenyl diguanide was never impaired and often enhanced. I have measured the particle size distribution, using cascade impaction, of the Wright’s nebulizer used by them and found it to have a mass median aerodynamic diameter of 4.1 pm; it would, therefore, be expected to deposit predominantly in the central airways in a similar fashion to the large particle aerosol used in the present study. The major difference between the studies is that the Wright’s nebulizer has a much lower mass output than the ultrasonic nebulizer used in the present study; the mass of drug per volume of aerosol was determined as 2.5 mg/1 for the Wright’s nebulizer by Jain et a l. (1973) and 5.46 mg/1 for the ultrasonic nebulizer by me. These figures are consistent with other reports of mass output for these nebulizers in the literature (Sterk et al., 1984). It is likely, therefore, that considerably more drug was deposited in the lung in the present study.

The finding that a large particle local anaesthetic aerosol abolishes a reflex believed to arise from nerve endings located at alveolar level is, at first, surprising. There are two mechanisms which could account for this. The first is that more drug is depositing at

77 alveolar level following large particle aerosol inhalation than following small particle aerosol inhalation. This is not an unreasonable suggestion since the large particle aerosol contains a considerable number of particles which could deposit in the alveoli (see Table 2.1 and Fig 2.2) and has a much larger mass of drug per volume of aerosol than does the small particle. However, the radio- labelled aerosol scans in man (Fig 2.4) demonstrate that the large particle aerosol produces considerably less peripheral deposition than does the small particle aerosol; if anything the somewhat smaller diameter airways and higher respiratory frequency of the dog would be expected further to enhance central deposition. The second mechanism is that local anaesthetic depositing in the airways may anaesthetize unmyelinated afferent fibres originating in the alveoli and running in the airway wall. This seems quite plausible since, especially in small airways, the mucus layer may be only 0.1 pm thick and the total diffusion path to the nerve fibre would be short. Furthermore it would seem easier to block one (or a small number) of afferent fibres in an airway than several nerve endings (which are likely to exist as a network) in the alveoli subserved by that airway. This leads once again to the question of the exact location of the receptors mediating the pulmonary chemoreflex. Perhaps in this context an absolute distinction between C-fibre endings located in alveoli and peripheral bronchioles is too rigid (and therefore unhelpful) since both may be rapidly accessible to chemicals injected into the right heart. Irrespective of which mechanism is responsible, the ability of the large particle to attenuate (after a 10 min inhalation) or block (after a 20 min inhalation) afferent information from peripheral C- fibre endings in the dog, indicates that it may be useful in studying the role of similar endings in man.

The ability of a large particle local anaesthetic aerosol to block airway reflexes seems to depend on the accessibility of the receptor to surface anaesthesia, the species tested and the amount of aerosol delivered. Previous authors have reported that the cough reflex can consistently be blocked by large particle 20% lignocaine or 5% bupivacaine aerosol in rabbits (Jain et al., 1973) and 5% bupivacaine aerosol in dogs (Dain et al., 1975; Cross et al., 1976). In contrast, the Hering-Breuer inflation reflex has been reported to be abolished in 11 of 26 rabbits with 20% lignocaine (Jain et al., 1973), 14 of 15

78 rabbits with 5% bupivacaine (Jain et a l., 1973), but only in 1 of 9 dogs studied by Dain et al. (1975) and 2 of 11 dogs studied by Cross et al. (1976) with 5% bupivacaine. In the present study the ability of the local anaesthetic aerosol to block completely both the cough and inflation reflexes is likely to be due to the use of an ultrasonic nebulizer with a higher mass output than the jet nebulizers used by previous workers. It has been reported that considerably higher concentrations of bupivacaine at the mucosal surface are required to abolish activity in slowly adapting as compared to rapidly adapting stretch receptors in the dog trachea (Camporesi et al., 1979). A further methodological difference in the present study was that the endotracheal tube was always withdrawn to just below the level of the vocal chords, allowing anaesthesia of the upper trachea where a significant proportion of stretch receptors lie (Miserocchi et al., 1973). The effect of the large particle local anaesthetic aerosol on the pattern of breathing in the dogs in the present study is also consistent with blockade of slowly adapting pulmonary stretch receptors.

The observation that blockade of the pulmonary chemoreflex required a greater duration of local anaesthetic aerosol inhalation than did blockade of the cough and inflation reflexes and that it returned before these reflexes is significant. These results make it unlikely that the nerve endings mediating the chemoreflex are located in the larger airways since, if that were the case, the inflation reflex (mediated by large myelinated fibres) would be expected to require a greater amount of local anaesthetic for its blockade and return more quickly than the chemoreflex (mediated by unmyelinated fibres). Since this was not the case, the results support the idea that the C-fibre endings mediating the pulmonary chemoreflex are located in the lung periphery. The greater duration of aerosol inhalation required for their blockade and the shorter time taken for return of their conduction presumably reflects the smaller mass of local anaesthetic deposited in the lung periphery. Although these results suggest that these endings are located in the lung periphery, they by no means indicate that they are located in the alveoli; they may equally well be located in peripheral bronchioles. Therefore, as mentioned above (page 78), an absolute distinction between C-fibre endings located in the alveoli and peripheral bronchioles may be too rigid.

79 These results in dogs indicate that the large particle bupivacaine aerosol is likely to be effective in blocking afferent information from rapidly adapting and slowly adapting pulmonary receptors in man.

80 CHAPTER 5: NORMAL MAN - RESTING STUDIES

5.1 Introduction

Although volume-related vagal afferent discharge is present in man at rest (Langrehr, 1964; Guz and Trenchard, 1971a), vagal blockade by injection of local anaesthetic around the nerves at the base of the skull has been reported to have no effect on resting breathing in anaesthetized (Guz et a h , 1964) or conscious (Guz et a h , 1966b) normal man. The inhalation of a large particle local anaesthetic aerosol in conscious normal subjects has been reported by two groups of investigators to have no effect on resting ventilation (Cross et a h , 1976; Chaudhary and Speir, 1979) and by one group to produce a small, but statistically significant, increase in VT and Tj (Savoy et ah, 1982).

However, important reservations exist concerning the methods of measuring ventilation and the conditions of resting wakefulness used in these studies. Considerable evidence now exists that in order to study resting breathing accurately, care must be taken both in the measurement of ventilation and in the definition of the conditions under which it is measured. The use of a mouthpiece and nosed ip has been shown to affect resting breathing pattern in conscious subjects by increasing VT and decreasing fR (Gilbert et ah, 1972; Rodenstein et ah, 1985). Resting breathing is also affected by visual and auditory stimuli (Asmussen, 1977; Shea et al., 1987b) and by mental concentration (Shea et ah, 1987b; Morgan and Cameron, 1985) with VE increasing due to an increase in fR. In the normal subjects studied by Chaudhary and Speir (1979) and Savoy et ah (1982), ventilation was measured using a mouthpiece, and in one of the two groups of subjects studied by Cross et ah (1976), it was measured via a tracheostomy; in their normal subjects, fR was determined from a PETC02 recording at the mouth and VT was not measured. Although Guz et ah (1966b) studied resting ventilation without the use of a mouthpiece and nose clip, this was performed only in two normal subjects and the pneumograph used, while accurately measuring fR, permitted only a crude analysis of VT. With respect to the conditions used for the study of resting breathing, no special conditions are described by Guz et ah (1966b), Chaudhary and Speir (1979), or Cross et ah (1976); the subjects of

81 Savoy et al. (1982) were seated comfortably but were not deprived of auditory or visual stimulation.

Consideration must also be given to the degree of airway anaesthesia, over the period of measurement of resting ventilation, achieved with the local anaesthetic aerosols used in previous studies. 4% lignocaine, used by Chaudhary and Speir (1979) and Savoy et al. (1982), is not as potent as 5% bupivacaine, used in the present work (see Tables 3.1 and 3.2). Although Cross et al. (1976) used 5% bupivacaine, the mass output of their aerosol was not as great as that of the large particle aerosol described in this thesis; in dogs, their aerosol did not consistently abolish the Hering-Breuer inflation reflex whereas this was always achieved with the aerosol used in the present study (page 70).

I wished to examine whether the large particle local anaesthetic aerosol developed for the present studies provided a degree of airway anaesthesia equal to or greater than that of the studies described above, and, if this were so, to examine its effect on ventilation, without the use of a mouthpiece and nosed ip, under carefully controlled conditions of rest.

Since these were the first studies performed in man using this aerosol, the first concern was the determination of its safety and an assessment of any central nervous system effects resulting from its inhalation. The effect of the aerosol on lung function and bronchial reactivity was also assessed.

A formal assessment was not made of the effects on resting breathing of the inhalation of a small particle local anaesthetic aerosol (see Discussion page 96 for reasons). However the general effects produced and blood levels achieved by inhalation of this aerosol were determined.

82 5.2 Methods

Large particle aerosol

Aerosol generation and administration

Large particle aerosols of 0.9% saline and, later on the same day, bupivacaine, were generated and administered to 5 normal subjects as previously described (page 32-35). In subjects 1 to 4, 5% bupivacaine was used. In subject 5, the use of this concentration on a previous occasion produced profound airway and oropharyngeal anaesthesia with abolition of the ability to swallow for 2 hours after the aerosol; 3% bupivacaine was therefore used in this subject for the present study. Subjects 2 and 3 only were aware of the purpose of the study.

Safety, general and central effects

During all aerosol inhalations a medical practitioner was present. Before bupivacaine aerosol administration, a short venous cannula (21G Butterfly) was introduced into a forearm vein to allow rapid access to the circulation if required; venous blood samples for the determination of plasma bupivacaine levels (page 63) were taken from this site 1 min before the end of bupivacaine aerosol administration aerosol and at 16 min and 30 min after the end of aerosol inhalation.

Immediately after aerosol inhalation subjects were questioned about the sensations associated with the anaesthesia and an assessment was made of any effects on the subject’s ability to swallow and the quality of their voice. To quantify possible central effects of the local anaesthetic, a set of visual analogue scales, divided in to four categories (1: mental sedation or intellectual impairment; 2: physical sedation or bodily impairment; 3: tranqui11ization or calming effects; 4: other types of feelings or attitudes; Norris, 1971) was completed by each subject before and after inhalation of saline and bupivacaine aerosol. The scales used are shown in Fig 5.1; subjects were asked to mark the scales as described in the instructions at the top of the form. In addition, reaction time and vigilance were assessed; a series of 20 vertical or horizontal bars were generated in random order by a microcomputer and presented to the subject on a visual display unit. The subject was instructed to respond to this by

83 Fig 5.1 A copy of the visual analogue scales used to assess central nervous system effects of saline and bupivacaine aerosol. The size has been reduced; in the original, the lines were 10 cm in length. The numbers on the left side indicate the groupings of Norris (1971) described in the text. Asterisks have been added to indicate the end of the scale from which scores were measured (i.e. the zero end of the scale).

Name ...... Oate ...... Time ...... Number ......

1. Please rate the way you feel in terms of the dimensions given below.

2. Regard the line as representing the full range of each dimension.

3. Rate your feelings as they are at the moment.

4. Mark clearly and perpendicularly across each line.

1 # Alert , nrowsy

3 # Calm Ftr.ited

2 * Strona Feeble

1 Muzzv C 1 ear-headed *

2 #Wel1-coordinated Clumsy

2 [etharaic Fnerqeti r. *

3 * Contented ni srnnt.ent.ed

3 T roubled T ranqui1 *

1 Mentally slow Oin r.k-wi t.t.ed *

3 Tense *

1 * Attentive

2 IncomDetent Proficient *

4 * Haopv Sad

4 Ant. aa o m stir. *

4 # Interested

4 Withdrawn Gregarious *

84 depressing either of two keys as quickly and as accurately as possible. The number of correct responses (vigilancet and the time taken for each correct response (reaction time) were recorded.

Resting ventil at ion

Measurements of resting ventilation were made on each subject for 5 min before and after inhalation of saline and bupivacaine aerosols. To minimize the effects of visual and auditory stimulation, measurements were made on subjects seated comfortably in a chair, blindfolded, wearing soundproof headphones, in a room separate from the investigator and recording equipment. Ventilation was measured without using a mouthpiece by respiratory inductance plethysmography (Respitrace). Abdominal and ribcage transducer bands were attached and kept in place by an elastic vest. The system was calibrated during quiet breathing in the standing and lying position (Cohn et ah, 1978) using simultaneous measurement of volume from a spirometer. The volume recorded from the calibrated respitrace was compared with that of a spirometer before each period of resting breathing was recorded and the difference used to provide a final correction of respitrace volume. PETC02 was measured at the nose with an infra-red gas analyser (Hewlett-Packard 47210 Capnometer). An electrocardiogram was recorded. All variables were displayed on an oscilloscope, recorded on a chart recorder and stored on FM tape. To ensure accurate breath detection, VT, Jz and TE were measured for each breath from the paper record; the data was transferred onto a microcomputer using a digitizing tablet (Summagraphics Bit Pad).

Adequacy of airway anaesthesia

The cough reflex was tested by inhalation of an aerosol of an aqueous solution of 5% (w/v) citric acid using an aerosol of 0.9% (w/v) sodium chloride as a control (modified from Bickerman and Barach, 1954). Both aerosols were generated by a Wright’s nebulizer at an airflow rate of 15 1/min; a flowpast system incorporating an aerosol reservoir ensured that aerosol was available throughout inspiration. Subjects were instructed to breathe deeply with their eyes closed; they were told that they were about to breath a substance which may or may not make them cough. Following a period of baseline breathing, a tap was

85 turned at the end of an expiration, without the subjects knowledge, so that for the next three breaths saline or citric acid aerosol was inhaled. The aerosols were administered in random order. The cough reflex was considered present if the subject coughed on any of the three breaths of citric acid aerosol and absent if no cough occurred. No subject coughed on inhalation of saline aerosol.

Protoco1

The order in which the investigations were carried out was the same for both aerosols: (1) completion of VAS for central effects; (2) measurement of vigilance and reaction time; (3) final correction of respitrace; (4) 1 min of rest before measurement; (5) 5 min recording resting breathing; (6) aerosol inhalation; (7) venous blood sampling 1 min before end of aerosol; (8-12) repeat (1-5); (13) testing of cough reflex; (14) venous blood sampling at 16 and 20 min after aerosol. The protocol for both aerosols was completed on the same day. Since the aerosols could not be given to the subjects in a "blind" fashion, due to the obvious sensations associated with bupivacaine inhalation, saline aerosol was always given first to avoid any problems due to persisting effects of bupivacaine.

Effect on lung function and bronchial reactivity

In a separate experiment on a different day, the effect of large particle bupivacaine aerosol on lung volume, specific airways conductance and bronchial reactivity was assessed in two subjects (3 and 5). Measurements were made of VTG and sGaw using a whole-body plethysmograph (Fenyves & Gut) immediately before and after bupivacaine aerosol inhaled as above. A cumulative methacholine challenge (Chung and Snashall, 1984) to determine the provocative dose of methacholine required to produce a 35% fall in sGaw (PD35) was also performed after bupivacaine aerosol; the baseline determination of PD35 was performed on a previous day.

Statistical analysis

The performance of the resting study before and after both saline and bupivacaine aerosol inhalation enabled data to be analysed using an

86 analysis of variance. This permitted any changes observed after inhalation of saline aerosol to be allowed for in determining the effect of bupivacaine. Analysis of variance was also used to assess changes in the visual analogue scales for central nervous system effects and for vigilance and reaction time. The variance-ratio test (F-test) was used to compare the degree of variability in the pattern of breathing. The level of significance was taken as P<0.05.

Small particle aerosol

An initial trial to assess the general effects, blood levels and safety of inhalation of a small particle local anaesthetic aerosol was performed in two normal subjects. After a short venous catheter had been introduced into a forearm vein, the subjects inhaled small particle bupivacaine aerosol produced by the Unicorn/Optimist system as previously described (page 48-52); a medical practitioner was present during the studies. Immediately on completion of aerosol inhalation, subjects were questioned about any sensations associated with the inhalation and the cough reflex to citric acid aerosol was assessed (page 85). Venous blood samples were taken for the determination of plasma bupivacaine concentration before the start of aerosol inhalation, at 9.5 and 19.5 min during the inhalation and at 10 min after its completion.

5.3 Results

Large particle aerosol

General effects of aerosol inhalation

After large particle saline aerosol inhalation, no subject reported any effect other than a slight salty taste in the mouth. Inhalation of bupivacaine aerosol produced initial irritation of the pharynx, which was sometimes intense, followed by profound oropharyngeal anaesthesia with huskiness of the voice and abolition of the ability to swallow. None of the subjects complained of any systemic side effect from bupivacaine aerosol inhalation. No changes in ECG were seen; heart rate averaged over each five min period did not change

87 (2-way analysis of variance) after saline (group mean (SD): pre 61.0 (8.3) beats/min; post 60.8 (8.2) beats/min) or bupivacaine (group mean (SD): pre 62.3 (6.9) beats/min; post 65.3 (3.4) beats/min) aerosol.

Plasma bupivacaine levels

Plasma bupivacaine levels following inhalation of this aerosol are given for each subject in Fig 5.2.

Central effects

No central nervous system effects of bupivacaine aerosol inhalation could be detected. The scores from the visual analogue scales used to assess possible central effects are shown in Table 5.1. No statistically significant differences were found between saline or bupivacaine aerosol inhalation using a 2-way analysis of variance performed for each scale. In order to increase the power of the analysis, the data was pooled into the four groups suggested by Norris (1971) (page 83) and a 3-way analysis of variance performed; no significant difference was seen after bupivacaine aerosol in the scores for any group.

Data for vigilance and reaction time before and after aerosol inhalation are shown for each subject in Table 5.2. No changes were seen in the number of correct responses or in the corresponding reaction times following saline or bupivacaine aerosol (2-way analysis of variance).

Resting ventilation

Bupivacaine aerosol inhalation had no effect on mean Vt ,Ti or TE but increased the variability in VT. Data for the pattern of breathing (VT, Tx and TE) over the 5 min periods recorded before and after inhalation of saline and bupivacaine aerosol are shown in Table 5.3. A 3-way analysis of variance was used to examine changes in each variable between the four time periods. Since this analysis required equal sample sizes in each group (balanced design), data from 40 sequential breaths (the smallest number recorded in a subject) from each time period were studied in each subject. No significant

88 Fig 5.2 Concentration of bupivacaine in plasma during and after a 10 min inhalation of large particle bupivacaine aerosol in five normal subjects. 5% bupivacaine was inhaled by four subjects and 3% bupivacaine by one subject (O). The times are given in min after the start of aerosol inhalation; the dashed line marks the end of the inhalation.

E CT> ©

* o o ’Q3 l m o E to _o CL

89 Table 5.1 Scores from visual analogue scales used to assess central nervous system effects of large particle saline and bupivacaine aerosol inhalation in normal subjects.

VAS Scale Pre Post Pre Post Saline Saline Bupivacaine Bupivacaim

Alert / Drowsy 47.6 38.4 38.6 27.8 (36.4) (25.3) (30.1) (16.7)

Clear-Headed / Muzzy 45.8 45.8 40.4 27.8 (25.5) (26.2) (27.6) (18.8)

Quick-Witted / Mentally slow 50.6 44.4 40.8 35.0 (29.7) (26.5) (30.6) (21.7)

Attentive / Dreamy 52.4 43.2 35.6 33.4 (26.3) (23.5) (19.3) (25.2)

Strong / Feeble 40.6 31.2 25.2 24.0 (9.8) (14.8) (13.7) 11.6)

Well-Coordinated / Clumsy 35.2 35.0 28.6 29.6 (25.2) (17.9) (20.2) (17.9)

Energetic / Lethargic 62.2 43.2 47.0 31.8 (27.6) (27.4) (38.4) (21.7)

Proficient / Incompetent 50.6 36.8 31.0 29.4 (29.7) (21.4) (29.4) (18.6)

Calm / Excited 19.2 25.6 15.2 32.6 (16.2) (14.5) (8.8) (21.8)

Contented / Discontented 22.6 30.0 23.4 32.8 (18.2) (6.0) (3.9) (20.6)

Tranqui1 / Troubled 29.2 26.8 19.2 31.8 (11.6) (6.8) (8.7) (21.9)

Relaxed / Tense 19.4 30.2 28.4 37.2 (13.7) (5.1) (8.9) (19.6)

Happy / Sad 33.4 27.8 27.6 29.4 (18.6) (4.0) (14.8) (11.5)

Amicable / Antagonistic 25.0 23.8 22.8 30.2 (10.0) (14.0) (11.8) (20.0)

Interested / Bored 55.0 47.6 40.8 36.4 (30.4) (30.7) (31.4) (27.9)

Gregarious / Withdrawn 43.8 49.4 38.2 44.2 (25.4) (22.1) (25.4) (21.9)

Data are the mean (and standard deviation) score for each scale in 5 subjects. The scales are grouped into the categories of Norris (1971).

90 Table 5.2 Vigilance and reaction time in normal subjects before and after large particle saline and bupivacaine aerosol.

Subj Variable Pre Post Pre Post Saline Saline Bupivacaine Bupivacaine

1 No. Correct 19 20 18 20 Mean Reaction Time 0.425 0.422 0.346 0.410 SD (0.149) (0.030) (0.010) (0.014)

2 No. Correct 16 19 18 19 Mean Reaction Time 0.515 0.408 0.456 0.458 SD (0.069) (0.100) (0.001) (0.016)

3 No. Correct 20 20 19 19 Mean Reaction Time 0.530 0.560 0.527 0.509 SD (0.088) (0.015) (0.037) (0.187)

4 No. Correct 20 19 20 20 Mean Reaction Time 0,640 0.566 0.608 0.560 SD (0.120) (0.058) (0.031) (0.030)

5 No. Correct 20 19 19 20 Mean Reaction Time 0.441 0.472 0.449 0.393 SD (0.050) (0.135) (0.040) (0.036)

Data are the number of correct responses and the mean and standard deviation for the correct reaction times (in sec) of responses to a total of 20 horizontal or vertical bars displayed at random on a computer display.

91 Table 5 •3 Effect of large particle saline and bupivacaine aerosol inhalation on the pattern of breathing in normal subjects at rest.

VT (ml) Tr (sec) te (sec)

Saline Bupivacaine Saline Bupivacaine Saline Bupivacaine

Subj Parameter Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post

1 mean 528.8 573.2 670.6 583.6 1.80 1.81 2.43 1.83 3.20 3.99 3.74 4.03 SD (85.5) (105.2) (74.4) (99.3) (0.33) (0.47) (0.45) (0.29) (0.55) (1.20) (0.50) (0.89) n 66 56 49 55 64 56 49 55 64 56 49 55

2 mean 368.7 344.9 378.9 258.4 1.58 1.70 1.75 1.63 2.80 2.97 3.10 2.76 SD (48.3) (44.0) (54.9) (73.2) (0.19) (0.14) (0.30) (0.33) (0.35) (0.30) (0.44) (0.71) n 74 69 56 67 74 69 56 67 74 69 56 67

3 mean 551.1 581.6 729.8 690.2 1.49 1.76 1.83 J . 69 3.90 3.49 3.35 3.12 SD (56.8) (57.9) (48.4) (52.6) (0.17) (0.21) (0.22) (0.15) (0.36) (0.30) (0.38) (0.34) n 68 62 68 63 68 62 68 63 68 62 68 63

4 mean 1033.8 739.0 677.4 727.7 3.20 2.25 2.04 2.31 (.. 24 4 . 36 3.77 4.66 SD (143.3) (132.8) (130.5) (204.5) (0.76) (0.45) (0.33) (0.41) (1.04) (1.50) (1.20) (1.18) n 40 48 49 40 40 48 49 4 0 4 0 4 8 49 4 0

5 mean 543.1 462.1 488.3 467.6 2.05 1.86 1.93 1.96 4.35 3.02 3.58 3.55 SD (82.2) (79.7) (64.4) (88.5) (0.24) (0.21) (0.20) (0.31) (0.66) (0.40) (0.58) (0.63) n 48 66 57 56 48 66 57 56 48 66 57 56

The table shows means, standard deviations and number of breaths recorded during min of resting breathing before ami after aerosol inhalation. differences were found in VT, Tz or TE after saline or bupivacaine aerosol. PETC02 averaged over each 5 min period did not change (2-way analysis of variance) after saline (group mean (SD): pre 37.7 (2.0) mmHg; post 39.4 (2.2) mmHg) or bupivacaine (group mean (SD): pre 39.2 (2.9) mmHg; post 38.4 (2.8) mmHg) aerosol.

To examine differences in the degree of variability in the pattern of breathing between the four periods, a variance ratio test (F-test), was used. The standard deviations from which the variances were calculated are given in Table 5.3. The variability in VT was unchanged by saline aerosol in all five subjects, but was increased after bupivacaine aerosol in all five subjects, the difference reaching statistical significance (P<0.05) in all except subject 3. Neither saline nor bupivacaine aerosol had any consistent effect on the variability of J z or TE.

Adequacy of airway anaesthesia

The cough reflex to citric acid was never impaired by saline aerosol. When tested at the end of the resting measurements after bupivacaine aerosol (14 to 18 min after aerosol), it was abolished in all subjects.

Effect on lung function and bronchial reactivity

The baseline values for VTG, sGaw and PD35 before bupivacaine aerosol inhalation were 4.64 1, 0.198 /sec/cmH20 and 27.2 pmol in subject 3 and 3.44 1, 0.151 /sec/cmH20 and 13.5 pmol in subject 5. After bupivacaine aerosol the values were 4.79 1, 0.187 /sec/cmH20 and 34.9 pmol in subject 3 and 3.35 1, 0.153 /sec/cmH20 and 20.6 pmol in subject 5. All these values are within the normal range for the subjects (Cotes, 1976; Chung and Snashall, 1984) and show no change after bupivacaine aerosol inhalation.

Small particle aerosol

When questioned at the end of small particle local anaesthetic aerosol inhalation both subjects reported that they had noticed nothing more than a little tickling at the back of the throat; this was in dramatic

93 Fig 5.3 Concentration of bupivacaine in plasma during and after a 20 min inhalation of small particle bupivacaine aerosol in six normal subjects. The times are given in min after the start of aerosol inhalation; the dashed line marks the end of the inhalation.

E cn 3 o Q. 3 m o cnE _□ Q_

94 contrast to the effects of large particle local anaesthetic aerosol inhalation. No other sensations associated with the inhalation were reported. The cough reflex to citric acid aerosol was unaltered by small particle local anaesthetic aerosol inhalation. The plasma bupivacaine levels produced by the inhalation are shown for both subjects in Fig 5.3.

The general effects of small particle local anaesthetic aerosol inhalation were assessed in a more formal manner in the study involving C02 rebreathing; this is presented in Chapter 8 (pages 144 and 152). The plasma bupivacaine levels produced by this inhalation of the aerosol are included in Fig 5.3.

5.4 Discussion

The inhalation of the large particle bupivacaine aerosol in this study produced effective airway anaesthesia (abolition of the cough reflex to citric acid aerosol) lasting for at least 14 min after the end of aerosol inhalation. Although the total duration of airway anaesthesia was not tested in these experiments, it was ample to allow resting ventilation to be measured. The procedure was found to be reliable and safe; no subject reported any effects other than those due directly to the local anaesthetic on the airway, no cardiovascular effects were observed, no changes were found in the visual analogue scales used to assess central nervous system effects and no changes were detected in vigilance or reaction time. No effect was found on lung volume, specific airways conductance or bronchial reactivity. The plasma bupivacaine levels were below-any published toxic level, although their magnitude reflects the degree of airway anaesthesia achieved.

The inhalation of the small particle bupivacaine aerosol was associated only with a slight sensation described as being in the back of the throat and consistent with minimal upper airway deposition. No unwanted side effects were reported. The plasma bupivacaine levels (including those from the subjects performing C02 rebreathing) were, with one exception, generally lower than that found after large particle aerosol inhalation, presumably reflecting the lower mass of drug believed to be depositing with the small particle aerosol. The

95 high levels found in the exceptional subject cannot be explained but may be an error since on a subsequent occasion when he inhaled the same aerosol, the maximum plasma level recorded was 0.69 pg/ml compared to the value of 1.61 reported on the present occasion. Although it would have made the study more complete to do so, the effect of the small particle local anaesthetic aerosol on the resting pattern of breathing was not determined since the large particle aerosol, which would be expected to be the more likely of the two to affect resting breathing, had already been found to have only a minor influence on this. Similarly, the effect of inhalation of small particle local anaesthetic aerosol on indicators of central nervous system side effects were not formally examined since the large particle aerosol, which was generally associated with higher plasma bupivacaine levels, had produced no detectable effects on this.

The results in dogs, presented in Chapter 4, indicate that the inhalation of the large particle aerosol provides an equivalent degree of blockade of rapidly adapting receptors but a greater degree of blockade of slowly adapting pulmonary stretch receptors and pulmonary C-fibres than the aerosols used by previous workers. It is difficult to compare the duration of airway anaesthesia achieved in the present study in man with that of studies reported in the literature because, in general, indices of airway anaesthesia have been tested immediately after aerosol administration only (Thomson, 1979; Savoy et ah, 1981; Chaudhary and Speir, 1979; Easton et a h , 1985) or they have not been tested at all (Chaudhary and Burki 1980; Gal, 1980; Savoy et ah, 1982; Sullivan and Yu, 1983; Sullivan et ah, 1986; Sullivan and DeWeese, 1985). In the exceptions to this, where airway anaesthesia has been confirmed at times after the end of aerosol inhalation, the cough reflex to citric acid aerosol was absent for 8 to 20 min (Cross et ah, 1976), about 15 min (Van Meerhaeghe et ah, 1986) and 14 min (Fennerty et ah, 1985). Therefore, the duration of anaesthesia using the bupivacaine aerosol in the present study is at least as great as any reported in the literature. It is unlikely, considering the blood levels produced by this aerosol in man, that a longer duration or greater degree of airway anaesthesia is possible without the risk of provoking central effects.

96 Inhalation of large particle local anaesthetic aerosol study had no effect on the mean levels of VT, JT, TE or PETC02 in normal subjects at rest when ventilation was measured by respiratory inductance plethysmography, without the use of a mouthpiece or nosed ip, and subjects were seated comfortably in an isolated room and deprived of auditory and visual stimulation.

This lack of effect on resting ventilation of local anaesthetic aerosol inhalation is in agreement with the studies of Cross et al. (1976) and Chaudhary and Speir (1979), also using local anaesthetic aerosol, and with the studies of Guz et al. (1964; 1966b), using direct injection of local anaesthetic to block the vagi. It is in contrast to those of Savoy et a l. (1982) who reported an increase in VT and Tr after inhalation of 4% lignocaine aerosol. However, the differences found in their study after local anaesthetic aerosol were small, especially when compared to the effects they found following saline aerosol inhalation: after lignocaine, VT, and 1T increased and f decreased by 50 ml, 0.20 sec and 1.9 breaths/min respectively; after saline, they changed in the same direction by 20 ml, 0.05 sec and 0.8 breaths/min. Their use of paired t-tests to compare the results before saline with those after saline and, separately, those before lignocaine with those after lignocaine does not permit the changes found after saline to be taken into account when examining the significance of the changes found after lignocaine. The analysis of variance used in the present study was designed to achieve this.

Although in the present study, inhalation of large particle local anaesthetic aerosol had no effect on the mean level of indices of resting ventilation, it increased the variability in VT. This finding has not previously been reported in normal man (Cross et a 1., 1976; Chaudhary and Speir, 1979: Savoy et al., 1982), but has been reported in awake dogs following vagal blockade (Kelsen et al., 1982). The receptors mediating this effect cannot be determined directly in the present study but it seems reasonable to propose that slowly adapting pulmonary stretch receptors are responsible since they are believed to be the main receptors involved in sensing changes in lung volume and a 10 min inhalation of this aerosol in the dog abolishes the inflation reflex believed to be mediated by them (page 70).

97 The large particle local anaesthetic aerosol was shown by this study to provide safe, effective airway anaesthesia of a duration sufficient for the performance of the other physiological studies planned for this thesis. The lack of any detectable central effects supports the conclusion that the effects of bupivacaine aerosol inhalation result directly from blockade of afferent neural information in the lung.

The results of this study indicate that afferent vagal information from the lung is not important in determining the mean level of ventilation in normal man at rest although it is involved in minimizing breath-to-breath fluctuations in depth. It seems likely that, in order to detect such subtle effects, the subjects must be studied under carefully defined conditions of resting wakefulness and non-invasive techniques must be used for the measurement of breathing.

98 CHAPTER 6: NORMAL MAN - SEARCH FOR PULMONARY CHEMOREFLEX

6.1 Introduction

As mentioned in Chapter 4, in order to determine whether the small particle local anaesthetic aerosol was able to block afferent information from C-fibres in the lung periphery, I wished to examine its ability to abolish a response which was known to be due to pulmonary C-fibre stimulation in man. It is the aim of this chapter to explore this topic.

Unmyelinated pulmonary vagal afferent fibres

The presence of unmyelinated fibres, thought from their structure to be sensory, has been demonstrated by electron microscopic studies in the alveolar walls of rats (Meyrick and Reid, 1971), mice (Hung et a l., 1973) and man (Fox et a l., 1980). The observation by these workers that the nerves were scarce may be due to the difficulty experienced in their recognition (Fox et al., 1980) or may be a true indication that not all alveoli are served by such fibres. In the lungs of the cat there are approximately 30 X 106 alveoli (Widdicombe, 1981) but only something in the order of 10,000 unmyelinated afferent vagal fibres (Agostoni et al., 1957; Jammes et al., 1982). Nonetheless, unmyelinated fibres are likely to have considerable importance since they greatly outnumber myelinated fibres in the vagal branches to the lung (Agostoni et al., 1957; Jammes et al., 1982).

Pulmonary chemoreflex

It is interesting to note that the histological search for these fibres resulted from physiological evidence for their existence. By the mid 1950’s (see review by Dawes and Comroe, 1954) a large body of work had reported that the inhalation of irritant gases and injection of a variety of chemicals into the pulmonary circulation produced a powerful reflex consisting of bradycardia, hypotension and apnoea followed by tachypnoea. The evidence indicated that this "pulmonary chemoreflex" was mediated by unmyelinated vagal fibres since it was not blocked until the vagi had been cooled to 2 to 3°C (Dawes and Comroe, 1954). Paintal (1955) first recorded impulses from these

99 fibres in the lungs of cats, work which was extended in dogs by Coleridge et a l. (1965). Paintal (1969) later suggested that, since the same fibres were stimulated within 3 sec of injection of phenyl diguanide into the right heart and within 0.5 sec of insufflation of halothane into the lungs, their endings were located close to the pulmonary capillaries and should be called juxta-pulmonary capillary receptors (type J receptors). Although this nomenclature is still used by Paintal, many others prefer the more general term C-fibres, defined as those fibres with conduction velocities below 2.5 m/sec (Iggo 1958), to refer to unmyelinated fibres. According to the schema of Coleridge and Coleridge (1977) these can be divided into two groups based on their vascular accessibility to chemicals: pulmonary C-fibres having endings which are rapidly accessible from the pulmonary circulation and bronchial C-fibres having endings rapidly accessible from the bronchial circulation; all 84 such fibres characterized by Coleridge and Coleridge (1977) had conduction velocities less than 2.5 m/sec. Although the term pulmonary C-fibre ending is now generally used as a synonym for J receptor (Coleridge and Coleridge, 1986), it should be remembered that, although 13 of the 15 fibres described by Paintal (1969) had conduction velocities characteristic of unmyelinated fibres, 2 had conduction velocities typical of small myelinated fibres. With the above arguments in mind, the term C-fibre has been used in this thesis.

Physiological and pathophysiological role

Since pulmonary C-fibres only occasionally fire with respiratory rhythm during eupnoea in dogs (Coleridge and Coleridge, 1977a, 1977b), they are thought to play little part in the normal control of breathing (Widdicombe, 1981). However, Paintal has proposed that they play a significant role in all conditions where pulmonary capillary pressure is increased. Indeed he has proposed that they must constitute an important source of dyspnoeic sensations from the lungs during exercise and may even, by reflex inhibition of limb muscles, terminate exercise (Paintal, 1970; Anand and Paintal, 1980). Their involvement is more certain, at least in animals, in the tachypnoea of several pathological states which affect the lung parenchyma including pulmonary congestion (Paintal, 1955,1969; Coleridge and Coleridge;

100 1977a), pulmonary embolism (Guz and Trenchard, 1971) and lung inflammation (Trenchard et a 7., 1972).

Search for pulmonary chemoreflex in man

In animals, the existence of the pulmonary chemoreflex has greatly facilitated the study of C-fibre function. However the chemicals eliciting the chemoreflex vary in different species: it is evoked in rats by capsaicin and phenyldiguanide (PDG) (Sapru et a 7.1981), in rabbits by PDG (Dawes et a l., 1951; Karczewski and Widdicombe, 1969), in cats by capsaicin (Toh et al., 1955) PDG (Dawes et al., 1951; Paintal, 1955) and 5-hydroxytryptamine (Paintal, 1955) and in dogs by capsaicin (Coleridge et al. 1964, 1965).

In man the classical chemoreflex has not been demonstrated, although it must be said that the search for it has not been extensive because the injection needs to be made into or near the right heart and the nature of the chemicals known to elicit it in animals is often unpleasant. Although the main effect of lobeline is to stimulate ventilation by carotid body excitation, it has also been used in attempts to elicit the pulmonary chemoreflex in man with variable results. Intravenous injection of lobeline produces hyperventilation, resulting from stimulation of carotid body chemoreceptors, preceded by cough (Eckenhoff and Comroe, 1951). Stern et al. (1966) reported that cough is produced when lobeline is injected into the extrapulmonary part of the pulmonary arteries, but not the intrapulmonary part of these arteries, nor into the left ventricle or descending aorta. Peripheral intravenous injection of lobeline has been reported to produce transient ventilatory depression in 17 of 20 subjects studied by Bevan and Murray (1963) and bradycardia and hypotension in 10 of these subjects within 4 - 9 sec of the time of injection; this was considered to be within the arm-lung circulation time. Coughing occurred in most of these subjects but coincided with the onset of hyperventilation 12 - 18 sec after injection; it was accompanied by a burning, flushing or irritating sensation localized to the mid-sternum or throat in some subjects.

The search for a lung chemoreflex in man was continued by Jain et al. ( 1972); they reported that injection of lobe line into the j^Tmorrarp^.^

101 artery produced a variable apnoea with or without cough within 3.2 sec followed by ventilatory stimulation from carotid body excitation. No circulatory effects were seen in any subject. 14 of the 18 subjects reported a sensation of fumes localized in the lower throat; 3 reported an accompanying burning sensation over the manubrium sterni. The time of onset of these transient sensations was not recorded.

The two chemicals most commonly used to elicit the chemoreflex in animals are capsaicin and PDG (Coleridge and Coleridge, 1984). Of these, only PDG has been tested in man, also by Jain et al. (1972). However, they found that injection of PDG into the pulmonary artery produced ventilatory stimulation, bradycardia and hypotension within 6.6 sec of pulmonary artery injection, a latency consistent with carotid body rather than pulmonary C-fibre stimulation. No sensations from anywhere in the body were produced.

The evidence was, therefore, that PDG, although a useful agent for stimulating pulmonary C-fibres in animals, did not do so in man. I was also unconvinced that lobeline could be used to stimulate these fibres in any reliable fashion in man. Therefore, in order to study the ability of local anaesthetic aerosol inhalation to block afferent information from pulmonary C-fibres in the lung in man, I first needed to establish a dependable consequence, if one existed, of their stimulation. The most likely candidate to achieve this seemed to be capsaicin.

Capsaicin

Capsaicin (8-methyl-/V-vani1lyl-6-nonamide) is the active principle in the fruit of various species of Capsicum and is the pungent agent in chili pepper and paprika. At a dose of 5 -20 pg/kg it produces acute activation of afferent C-fibres in dogs (Coleridge et al., 1965) and in doses 104 times greater, results in degeneration of these fibres in guinea pigs and rats (Lundberg et al., 1983). Such capsaicin pretreatment induces desensitization of the airway mucosa to a variety of mechanical and chemical stimulants (Lundberg and Saria, 1983).

Capsaicin is often referred to as a "selective" afferent C-fibre stimulant and any effects resulting from its administration are taken

102 to be evidence of C-fibre activation. It now seems that this view is somewhat simplistic although the exact mechanism by which capsaicin exerts its actions and the nature of the nerve fibres in which this occurs is the subject of intense investigation at present. A detailed examination of this fascinating topic is outside the scope of this thesis (see reviews by Lundberg and Saria, 1987; Holzer, 1988; Maggi and Meli, 1988) but some aspects are germane to the present work. The current view is that capsaicin selectively excites a subset of neuropeptide-containing primary afferent neurons (Maggi et a 7., 1988) by generating an influx of cations (especially Ca++) (Marsh et a 7., 1987) possibly via a specific capsaicin receptor site on the nerve terminal (Szolcsanyi and Jansco-Gabor, 1975). In adult animals the fibres stimulated are a specific group of C-fibre afferents but in immature animals capsaicin can destroy both C- and A-5 afferent fibres (Szolcsanyi, 1987).

The specificity of the neurons stimulated by capsaicin is important since it appears that although slowly and rapidly adapting pulmonary stretch receptors have myelinated fibres, they may have unmyelinated terminals (Widdicombe, 1981; Coleridge and Coleridge, 1986). The neuro-chemical nature of these endings, and therefore their sensitivity to capsaicin stimulation, remains to be determined. However, Coleridge et al. (1965) found that injection of 5 - 20 pg/kg of capsaicin into the right heart in dogs, a dose which caused massive discharge in all 55 pulmonary C-fibres studied, had no effect on 20 of 28 slowly adapting stretch receptors and produced a slight increase in the existing discharge of the remaining 8 such receptors. Similarly, Armstrong and Luck (1974) reported that injection of 20 pg/kg capsaicin into the right heart in cats, twice the dose which caused massive discharge in all 15 pulmonary C-fibres studied, had no effect on 15 of 24 rapidly adapting receptors and produced a slight potentiation of existing discharge in the remaining 9 such receptors.

Previous use of capsaicin in man

In man, capsaicin has been applied to the skin (Jansco et a l., 1968) and buccal mucosa (Szolcsanyi, 1977) where it causes pain and a burning sensation. Inhalation of capsaicin aerosol (MMAD 3.5 - 4.0 pm) produces dose-dependent coughing which is abolished by prior

103 application of lignocaine to the larynx (Collier and Fuller, 1984). It also results in a dose-dependent fall in airways conductance (Fuller et a l., 1985) but not FEV1 (Collier and Fuller, 1984).

The present studies

In the studies described in this chapter I have investigated whether the pulmonary chemoreflex could be elicited in normal man by central intravenous injection of capsaicin and determined whether the inhalation of a large or small particle local anaesthetic aerosol could block any effects resulting from such injection. I have also examined whether a peripheral intravenous injection of capsaicin produced similar effects in an attempt to obviate the need for a central venous catheter in future studies.

6.2 Methods

Subjects

The study was performed on three normal, healthy male subjects aged 29, 37 and 54 years. All subjects had normal lung function and none were taking regular medication. Since, to my knowledge, this represented the first occasion on which capsaicin had been intravenously administered to man, the study was performed on my PhD supervisor (subject 1), a medical colleague working with me on the study of local anaesthetic aerosols (subject 2) and myself (subject 3). Subjects were therefore fully conversant with the pharmacology of capsaicin and the purpose of the investigation. The protocol for the study was approved by the Ethical Committee of Charing Cross Hospital.

Central and peripheral intravenous catheters

To allow central intravenous injections to be made, a catheter was inserted under local anaesthesia into a vein in the antecubital fossa and advanced under fluoroscopic control until its tip lay in the superior vena cava (SVC). This was performed in subject 1 on two occasions on separate days and in subject 2 on a single occasion.

104 In order that further studies could be performed without the need for a central venous catheter, a short catheter was inserted on another day into a peripheral vein in the antecubital fossa. This was performed in subject 2 on two occasions and in subjects 1 and 3 on one occasion.

Capsaicin and control injections

A sterile stock solution of capsaicin (80-85% pure, Sigma) made up to 10 mg/ml in 90% ethanol was diluted in 30% ethanol/70% saline (150 mmol/1 NaCl) to provide capsaicin doses of 0.1, 0.5, 1.0, 2.0, and 4.0 pg/kg in 1 ml of solution. The lowest dose (0.1 pg/kg) was selected as a reasonable starting point since it was 40 times lower than the threshold dose found in dogs using the same capsaicin solution. At the beginning of the experiment, 0.25, 0.5 and 1.0 ml of 30% ethanol/70% saline were given as controls. However, due to concern about the cumulative dose of alcohol being given to subjects, 1 ml of normal saline was used as the control for the remainder of the experiment. Venous blood was taken from the other arm for the measurement of blood alcohol levels. Volumes of capsaicin or control solutions were rapidly injected into the catheters such that, allowing for the catheter deadspace, 1 ml was injected into the circulation. An event marker was depressed at the start and released at the end of injection to allow accurate timing of responses; all injections took less than 1 sec.

Capsaicin was injected twice at each dose level in increasing doses until the sensations resulting from the injection became intolerable; the maximum tolerable dose was then used for all subsequent capsaicin injections. Capsaicin and control solutions were given single blind in a randomized order.

Cardiovascular and respiratory measurements

All measurements were made on subjects supine, blindfolded, wearing soundproof headphones in a room separate from the recording apparatus. This ensured a minimum of visual and auditory stimulation and allowed injections to be made without the subjects’ knowledge.

105 Each injection of capsaicin or control solution was preceded by a control period during which baseline cardiovascular and respiratory measurements were made. The solution was then injected, without the subject’s knowledge, into the central or peripheral vein and measurements continued. VT, TI? TE were measured for each breath without a mouthpiece by respiratory inductance plethysmography (Respitrace). The volume signal was calibrated with a spirometer using a multiple linear regression technique (modified from Loveridge et a/., 1983). An ECG and fc for each beat were recorded. Arterial blood pressure was measured within 30 sec of injection with an ultrasonic blood pressure monitor and inflatable cuff (Arteriosonde 1225). All variables were displayed on an oscilloscope, recorded on a chart recorder and stored on FM tape with a time code for subsequent analysis.

Sensations

After each injection, when the period of cardiovascular and respiratory measurement had finished, the subjects were asked to describe what sensations, if any, they had experienced and the sequence in which they occurred. Additional experiments were performed to allow accurate timing of any such sensations. Subjects used a set of key words, derived on the basis of what they had previously reported, which were spoken as a sensation occurred to describe the anatomical origin of that sensation. This precluded any respiratory measurements during these studies. A video recording of the experiment, with sound and a time code, was made to allow accurate measurement of subject response times.

Local anaesthetic aerosol inhalation and local anaesthetic infusion

A peripheral venous injection of the maximum acceptable dose of capsaicin was repeated in subjects 1 and 2 following a 10 min inhalation of large particle bupivacaine aerosol (page 32-35). On another day, a central injection of a similar dose of capsaicin was given to subject 1 and a peripheral injection to subject 2 following a 20 min inhalation of small particle bupivacaine aerosol produced by the Turret/Optimist system (page 48-52); later on the same day capsaicin injection was repeated in these subjects following an

106 intravenous infusion (into the other arm) of 0.75 mg/kg bupivacaine over 10 min.

After each of these capsaicin injections, the timing of the associated sensations was performed as above. The cough reflex to citric acid aerosol was then tested (page 85) and a venous blood sample taken for the determination of plasma bupivacaine (page 63).

Circulation time

The circulation times from the sites of injection to the ear lobe were determined by injecting a 1 ml bolus of cardiogreen dye (1 mg/ml; HW &D) into the central or peripheral catheters and detecting its arrival time at the ear using an ear oximeter (Hewlett-Packard 47201A). The SVC-ear lobe or arm-ear lobe circulation time was determined in this way at the end of every study in all subjects.

Statistical analysis

Balanced three-way analysis of variance was performed for fc averaged over a breath, VT, Tx and TE for the five breaths before and after central injections of capsaicin and saline solutions.

6.3 Results

General effects of capsaicin injection

Central intravenous injections (subjects 1 and 2) of capsaicin but not control solutions produced dose-dependent sensations which occurred sequentially in the chest, face, rectum and hands and feet. Neither subject was aware of any sensation at the moment of injection. The threshold dose for the sensations was 0.5 pg/kg in both subjects; the maximum tolerable dose was 2 pg/kg in subject 1 and 4 pg/kg in subject 2.

Peripheral intravenous injections (subjects 1, 2 and 3) of capsaicin but not control solutions produced a similar, though less intense, series of sensations. In all subjects this was preceded by a local

107 pain in the arm as the capsaicin was injected, which increased in intensity as the dose increased. The maximum tolerable dose was the same for both peripheral and central injections in subjects 1 and 2; in subject 3 it was 4 pg/kg.

Blood alcohol levels for all three subjects were less than 10 mg/100 ml. No subjects complained of chest tightness, wheezing or any other effects of capsaicin or control injection. No cardiac arrhythmia or systemic hypotension was observed during the study and all subjects have subsequently remained well.

Cardiovascular and respiratory effects of central injection of capsaicin

Central injection of capsaicin at 4 pg/kg, the highest dose tolerated, produced cough with a latency consistent with pulmonary C-fibre stimulation in one of the subjects. Below this dose, it had no discernable effect on the pattern of breathing, heart rate or arterial blood pressure within the circulation time to the earlobe in either subject. An example of the effect of a central capsaicin injection (at the maximum dose which did not produce coughing) on the pattern of breathing and ECG is shown in Fig 6.1 A. The data for fc averaged over a breath VT, TI7 and TE for three such injections each in subjects 1 and 2 are shown in Figs 6.2 A and 6.2 B respectively. A balanced three-way analysis of variance was used to compare this data before and after saline and capsaicin injection. There was no significant change in the mean level of any variable after an i njection: fc (saline pre 69.5,post 69.1; capsaicinpre 71.4,post 74.1 b/min), VT (saline pre 484,post 510; capsaicin pre 554, post 536 ml), Tx (saline pre 1.93,post 2.10; capsaicinpre 2.04, post 2.04 sec),

Te (saline pre 2.53,post 3.27; capsaicinpre 3.27, post 3.27 sec). This analysis was also used to examine whether data from individual breaths after an injection were significantly different from baseline values; no such differences were found.

Values for systolic and diastolic blood pressure obtained before and after saline and capsaicin injection were compared using a two-way

108 Fig 6.1 Effect of central and peripheral intravenous injection of capsaicin in subject 2. a, 2 pg/kg injected into the SVC; b, 4 pg/kg injected into the SVC; c, 4 pg/kg injected into an arm vein. The arrows indicate the point of capsaicin injection (CAPS) and the circulation time of cardiogreen dye (CT) from the site of injection to the ear lobe. The volume trace shows inspiration upwards.

VT 0.5 £

ECG

______I ^ t 10 sec CAPS CT

i

c.

109 Fig 6.2 A Effect of central injections of saline (open symbols) and capsaicin (filled symbols) on fc averaged over a breath, VT, Tj, and TE for the five breaths before and after an injection in subject 1. Each of the data sets corresponds to one injection of saline or 2 pg/kg capsaicin.

SALINE CAPSAICIN

5

A.

,A ---- A \ \A ^ y S T T I ■

110 Fig 6.2 B Effect of central injections of saline (open symbols) and capsaicin (filled symbols) on fc averaged over a breath, VT, Tj, and TE for the five breaths before and after an injection in subject 2. Each of the data sets corresponds to one injection of saline or 2 pg/kg capsaicin.

SALINE CAPSAICIN

Breath Number Breath Number

111 analysis of variance. No differences were found in either blood pressure before and after an injection: systolic (saline pre 122, post 126; capsaicin pre 127, post 128 mmHg), diastolic (saline pre 83, post 84; capsaicin pre 88, post 89 mmHg).

In subject 2, the single central injection made of 4 pg/kg capsaicin produced paroxysmal coughing 3.9 sec after the end of injection (Fig 6.1 B).

Cardiovascular and respiratory effects of peripheral injection of capsaicin

The occurrence of local arm pain associated with peripheral intravenous injections of capsaicin often produced changes in the pattern of breathing accompanying the pain; this made the analysis of ventilatory records following such injections difficult and of dubious value. However, where this analysis was possible, no cardiovascular or respiratory effects were seen within the arm-ear lobe circulation time. An example of the pattern of ventilation and ECG after a peripheral injection of capsaicin is shown in Fig. 6.1 c.

Sensations produced by capsaicin injection

Central and peripheral intravenous injections of capsaicin above the threshold dose produced transient "hot, flushing" sensations in the chest, face, rectum and extremities. These were discrete, sequential sensations each lasting for several seconds. An additional characteristic quality of the chest sensation alone was a "raw, burning feeling"; it was the intensity of this sensation that limited the dose of capsaicin which could be injected centrally. With peripheral injections of capsaicin, a combination of this sensation and the local arm pain associated with peripheral injections limited the dose which could given. Repeated capsaicin injection did not result in tachyphylaxis of any of these sensations. No subject reported feeling breathless.

The key words used to time this sequence of sensations were "chest", "face", "rectum", "hands" and "feet". The timing of the sensations after capsaicin injections in any subject was highly reproducible.

112 The time of onset of the chest sensation and the circulation time to the* ear of cardiogreen dye for each injection site are shown for the three subjects in Table 6.1. The sensation in the face occurred a mean of 2.5 sec after the circulation time to the ear. On no occasion did control injections result in any sensations.

Table 6.1 Time of onset of the chest sensation after central and peripheral intravenous injections of capsaicin and the corresponding circulation time to the ear of cardiogreen dye.

Subjects Central Injection Peripheral Injection

Chest Circulation Chest Ci rculation sensation time to ear sensation time to ear (sec) (sec) (sec) (sec)

1 4,4 8 8,7,8 11 2 3,3 9 16,12,14 26 3 — 13,13,14 20

Central injections were made into the superior vena cava and peripheral injections into an arm vein Each value represents one determination.

Effects of local anaesthetic aerosol inhalation and local anaesthetic infusion

Inhalation of large particle aerosol (subjects 1 and 2) abolished chest sensation normally produced by capsaicin injection; all other sensations were unaffected in intensity or timing. Plasma bupivacaine concentrations at the end of capsaicin injection were 0.96 and 1.01 pg/ml in subjects 1 and 2 respectively. The cough reflex to citric acid aerosol was abolished when tested at this time.

In contrast, inhalation of small particle local anaesthetic aerosol (subjects 1 and 2) had no effect on the timing or intensity of the chest sensation or any other sensation produced by capsaicin injection. Plasma bupivacaine concentrations at the end of local anaesthetic aerosol inhalation were 0.66 and 0.60 pg/ml in subjects 1 and 2 respectively.

113 Intravenous infusion of bupivacaine also had no effect on any sensation produced by capsaicin injection. Plasma bupivacaine concentrations at the end of bupivacaine infusion were 1.94 and 1.41 pg/ml in subjects 1 and 2 respectively.

6.4 Discussion

The studies described in this chapter demonstrate that intravenous capsaicin injection to the maximum tolerable dose (2-4 pg/kg) does not elicit any component of the pulmonary chemoreflex in conscious man at a time when it produces an intense chest sensation. These results are in contrast to those in anaesthetized animals where other workers have found marked apnoea, bradycardia and hypotension following central intravenous injection of capsaicin at 0.2 pg/kg in rats (Sapru et a l., 1981), 5-15 pg/kg in cats (Tatar et a 7., 1988) and 5-20 pg/kg in dogs (Coleridge et a l., 1964). Moreover, in my hands 4-8 pg/kg of the same capsaicin preparation also produces this triad of effects in anaesthetized dogs (Chapter 4). However it can be seen that the dose of capsaicin used in man lies at the threshold required to elicit the reflex in large animals and the anaesthesia used in studies on animals may potentiate the effect of capsaicin. Evidence supporting both these points comes from a study in awake dogs by Clifford et al. (1987). They found that peripheral injection of capsaicin at 5 pg/kg, a dose which they expected to produce the pulmonary chemoreflex, failed to elicit a response; 10 pg/kg produced an equivocal response and 20 pg/kg a reproducible response. The possibility therefore exists that a the same dose as used in conscious man in the present study may elicit the reflex in anaesthetized man. It is unlikely that a higher dose than that used in the present study could be given to conscious man.

Because of the nature of this study, the number of subjects was limited. I was, therefore, concerned to ensure that a small effect of capsaicin was not missed either by the method of measurement or data analysis. The subjects were studied under conditions of minimal visual and auditory input (as discussed in Chapter 5, page 81) and injections were made without the subject’s knowledge. Respiratory inductance plethysmography was chosen because it does not affect the pattern of breathing and is an accurate and sensitive technique for

114 detecting changes in the pattern of resting breathing (Loveridge et al., 1983; Shea, 1988). However considerable variability existed in the baseline measurements of VT, Tx and TE, especially in subject 1. Such variability has been well described (Newsom Davis and Stagg, 1975; Gribben et a 7., 1985; Shea et a/., 1987a). The smallest significant change in a variable that would be detected by the analysis of variance is given by Fisher’s least significant difference (Fisher, 1935) at the P<0.05 level. In this study this was 100 ml for VT, 0.38 sec for Tz and 1.11 sec for TE; changes smaller than these values would not be detected as significant. In contrast, the variation in fc was small indicating stability of this variable. Since Fisher’s least significant difference for fc was 2.4 beats/min, any significant change after injection would have been readily detected. So, although it is possible that a small change in the pattern of breathing could have been missed in this study, it is unlikely that any change in heart rate would have gone undetected.

Although an intra-arterial method of measuring blood pressure is essential for the detection of transient changes in this variable, the use of such a technique was considered unjustified in this study. It is, therefore possible that a transient change in blood pressure might have been missed by the non-invasive technique used in the present study. However, a decrease in arterial blood pressure after capsaicin injection without a concomitant decrease in heart rate has never, to my knowledge, been reported in animals (Toh et a 1., 1955; Coleridge et al., 1964; Sapru et al., 1981; Coleridge and Coleridge, 1984).

Evidence for the existence in man of a reflex mediated by receptors rapidly accessible from the pulmonary circulation has come from previous studies using lobeline. Though Bevan and Murray (1963) described transient bradycardia and hypotension within the arm-lung circulation time in some of their subjects, the evidence for the accompanying changes in breathing pattern is unconvincing. In contrast, Jain et al. (1972) reported the occurrence of apnoea in the majority of their subjects but found no accompanying cardiovascular effects.

Although I was unable to demonstrate the pulmonary chemoreflex in my subjects, central injection of capsaicin produced paroxysmal coughing

115 within 3.9 sec of injection in one of the subjects. This is an interesting result since some controversy has recently arisen as to the type and location of receptors responsible for cough. Although Widdicombe has proposed that cough is mediated by rapidly adapting receptors in the upper airway (Widdicombe, 1954c; 1981; 1982), recent evidence indicates that C-fibres located in the larynx and lungs may also mediate cough. Much of this evidence comes from studies using capsaicin.

Inhalation of capsaicin aerosol in conscious man (Collier and Fuller, 1984) and guinea-pig (Forsberg and Karlsson, 1986) produces coughing thought to be mediated by stimulation of laryngeal C-fibre afferents (Sant’Ambrogio, 1987a). Several groups of workers (Coleridge and Coleridge, 1986; Sant’Ambrogio et a 7., 1984; Sant’Ambrogio, 1987a; Karlsson et a 7., 1988) have also argued that bronchial and pulmonary C-fibres may be involved in the genesis of cough. Despite this, intravenous injection of capsaicin has not been reported to produce coughing in animals whether anaesthetized (Coleridge and Coleridge, 1986) or awake (Clifford et a 7., 1987a, 1987b). However in man, cough has been reported after intravenous or right heart injection of lobeline (Eckenhoff and Comroe, 1951; Stern et a 7., 1966; Jain et a l., 1972) within a response time consistent with an origin from pulmonary receptors. Nevertheless, Widdicombe and colleagues (Tatar et a l., 1988) have recently argued that pulmonary C-fibre stimulation does not produce cough but reiterate the view that rapidly adapting receptors are responsible. In the present study, the production of cough 3.9 sec (4 heart-beats) after injection of capsaicin into the SVC suggests that the receptors responsible are located close to the pulmonary capillaries. Taken in conjunction with the relative selectivity of capsaicin for C-fibre afferents, this indicates that the receptors mediating the cough are pulmonary C-fibre afferents. Unfortunately, because of the violent nature of the coughing induced by this concentration of capsaicin, I was reluctant to repeat the injection after local anaesthetic aerosol inhalation.

Although methods to quantify the intensity of sensations are well described (Baird and Noma, 1978) and indeed have been used by me in work described in other chapters of this thesis, the sensations produced by capsaicin were transient and unfamiliar. Therefore, no

116 attempt was made to quantify their intensity. The sensations resulting from capsaicin injection are similar to those previously described to occur in the chest and throat after lobeline injection (Bevan and Murray, 1963; Jain et al., 1972). However, precise timing of these sensations has not been previously reported. The short latency of the chest sensation suggests that it originates from stimulation of receptors rapidly accessible from the pulmonary circulation; these receptors are likely to be pulmonary C-fibre endings. Since Paintal (1970) has proposed that these receptors may be an important source of dyspnoeic sensation in the lung, it is significant that the intense stimulation of these receptors in the present study never produced the sensation of breathlessness in any subject. However the intense chest discomfort reported following capsaicin injection does provide evidence in man that pulmonary C-fibre endings act as nociceptors as proposed by Widdicombe (1981). This may account for the similar sensations experienced after inhalation of irritant gases or aerosols. Although these receptors are believed to be primarily activated by tissue damage, the accumulation of interstitial fluid, and by release of mediators (Widdicombe, 1981), I am unaware of any disease state which produces a chest sensation similar to that found in the present study. Perhaps the distinction lies in the difference between a mass activation of pulmonary C-fibre endings following capsaicin injection and a less intense and perhaps less extensive stimulation of these endings by disease states.

The observation that the inhalation of a local anaesthetic aerosol can abolish the chest sensation produced by capsaicin injection indicates that the receptors responsible are located not in the pulmonary artery but in the lung. This taken together with the short latency of the response provides further evidence that the sensation is due to pulmonary C-fibre stimulation. As tachyphylaxis was not observed, this result was not due to repeated administration of capsaicin nor was it due to a central effect of the local anaesthetic since bupivacaine infusion had no effect on any sensation. It is perhaps surprising that this sensation was abolished by the large particle, but not small particle, local anaesthetic aerosol. This observation seems analogous to the abolition of the pulmonary chemoreflex in the dog (Chapter 4) and the reasons underlying it are, therefore, the same as those discussed previously (page 77-78).

117 The results of the studies described in this chapter provide no evidence for the existence of a pulmonary chemoreflex in conscious man but this lack of effect may reflect the relatively low concentrations of capsaicin which could be used. However the results demonstrate that stimulation of receptors which are rapidly accessible from the pulmonary circulation produces paroxysmal coughing; it seems likely that the receptors responsible are pulmonary C~fibre endings. In addition, the results provide evidence for the existence of a nociceptive system of nerve endings in the human lung which can be blocked by the inhalation of a large particle local anaesthetic aerosol.

118 CHAPTER 7: NORMAL MAN - EXERCISE

7.1 Introduction

Although afferent vagal information from the lungs does not appear to be important in the control of breathing in man at rest (Chapter 5) it may play a role at larger tidal volumes (and therefore a greater degree of stretch). The Hering-Breuer inflation reflex, mediated by slowly adapting pulmonary stretch receptors in animals, has been demonstrated in man under general anaesthesia (Widdicombe, 1961; Guz et al., 1964) and during non-rapid eye movement sleep (Hamilton et a/., 1988); however the reflex was only apparent at inflation volumes greater than 1 litre above FRC. I wished to investigate whether such vagal feedback from the lungs was involved in determining the pattern of breathing when ventilation was increased by exercise. This was achieved by examining the ventilatory response to exercise in normal subjects following inhalation of a large particle local anaesthetic aerosol.

A further role for pulmonary vagal afferent receptors during exercise has been suggested by Paintal (1969, 1970) who has proposed that pulmonary C-fibre endings are stimulated by any condition that leads to an increase in pulmonary capillary pressure including exercise. According to him this would limit exercise in two ways: firstly by reflex inhibition of muscular activity (Paintal, 1969) and secondly by producing the sensation of breathlessness (Paintal, 1970). Evidence in support of the first mechanism (the J-reflex; Paintal, 1970) comes from the observation that central injection of PDG inhibits flexor and extensor monosynaptic reflexes of hind limb muscles in the cat (Deshpande and Devanandan, 1970) and from a somewhat convoluted experiment where inhibition of the knee jerk reflex in cats was produced by central injection of PDG at a concentration which stimulated pulmonary C-fibres to the same extent as did increasing pulmonary blood flow by 133% ( Anand and Paintal, 1980). Transient loss of motor function in addition to the pulmonary chemoreflex has been described in conscious cats (Kalia et a l., 1973) and fish (Satchel!, 1977) in response to intravenous injection of PDG and in conscious dogs (Clifford et a l., 1987b) after capsaicin injection.

119 Evidence for Paintal’s second proposal, that pulmonary C-fibre stimulation is involved in the genesis of breathlessness, remains to be provided. In an attempt to investigate whether such mechanisms were operative in normal man, I also studied the ventilatory response to exercise following inhalation of a small particle local anaesthetic aerosol since, at the time these experiments were performed, it was still proposed that this aerosol could block afferent information from receptors in the lung periphery.

7.2 Methods

Subjects

All studies were performed on healthy volunteers who had given informed consent after full explanation of the nature of the study. None of the subjects had a history of asthma or any other atopic condition. The protocol for the studies was approved by the Ethical Committee of Charing Cross Hospital. A medically qualified colleague was present during all exercise tests.

Design of studies

Eight subjects (aged 24 - 54 years; 2 female) performed exercise tests immediately after large particle saline aerosol inhalation and, after a rest period of approximately 3 hours, large particle bupivacaine aerosol inhalation (page 35). The cough reflex to citric acid aerosol (page 85) was tested on completion of each exercise test. Plasma bupivacaine levels were measured at the end of exercise in 2 subjects.

In two subjects exercise tests were performed immediately after intravenous infusion of 20 ml of 0.9% (w/v) saline and, 3 hours later, 0.25% (w/v) bupivacaine hydrochloride. The bupivacaine (0.75 mg/kg) was infused over 10 - 20 min to achieve blood levels comparable with those measured after large particle bupivacaine aerosol inhalation.

Because of concern that blood lactate, residual from the first exercise test, may have affected the results of the second test, six subjects (aged 23 - 35 years; 1 female) performed two exercise tests 3 hours apart without aerosol inhalation. In two of these subjects,

120 venous blood samples were taken from a cannula in an arm vein before and at 1 min, 1 hr, 2 hr and 3 hr after the first exercise test. Lactic acid concentration in these samples was determined by a commercially available enzymatic technique (Sigma, UK).

A similar study to that involving large particle aerosols was performed in eight subjects (aged 24 - 32 years; 2 female) immediately after inhalation of small particle saline and bupivacaine aerosol generated by the Unicorn/Optimist system (page 48-52). Because the results of the control study without aerosol indicated that small alterations in the pattern of breathing could be detected during an exercise test performed 3 hours after a test earlier the same day, the exercise tests involving small particle aerosols were performed at the same time of day on consecutive days.

A total of sixteen normal subjects was used for these studies. Where a subject performed more than one type of study, these were done on separate days. In an ideal study design, saline and bupivacaine aerosols and infusions would be given in a double-blind fashion and in random order to each subject. However this was not possible for the large particle aerosol inhalation since the subjects were instantly aware of the difference between saline and bupivacaine aerosols. Therefore, since the subjects could not be blinded to the order of aerosol administration, it seemed prudent always to give saline first in order to avoid any potential contamination by residual bupivacaine of the results of the second exercise test. Since the studies involving intravenous infusion were to act as a control for these experiments, they were also performed by giving saline first. Since the small particle saline and bupivacaine aerosols were given on separate days and since, from previous experience, most subjects could not detect a difference between the two aerosols, these were given in a double-blind, randomized fashion.

Exercise

All exercise tests were performed on a treadmill with variable speed and elevation (Marquette 1800). This was chosen in preference to a cycle ergometer since, at least in normal subjects, a greater maximal oxygen consumption can be achieved due to the greater muscle mass used

121 in exercise on a treadmill (Jones, 1988a). The tests consisted of 1 min of rest followed by a 1 min 'warm-up’ period at minimum workload (1 m.p.h.; 0% elevation) after which the workload was increased by a constant amount at 1 min intervals until the subject was exhausted. The workload increment was 30 W for males and 25 W for females. The speed and elevation required to achieve each workload were determined for each subject on the basis of their weight and stride-length.

Values for fR, VT, VE, V02 and VC02 averaged over each 30 sec period during exercise were measured in subjects breathing via a mouthpiece using a computer-assisted exercise system (Ergostar, Fenyves & Gut). In this system, flow was measured by a pneumotachograph (Fleisch No.3) and integrated to give volume; gas exchange was measured from a flow- weighted sample of mixed-expired gas; PETC02 was measured at the mouth using a mass spectrometer (MGA 200, Centronics). The pneumotachograph was calibrated using a 1 litre syringe, the gas analysers calibrated with gases whose 02 and C02 concentrations had been determined previously by LLoyd-Haldane analysis, and the ability of the complete system to determine respiratory variables confirmed. Values for

PetC02 were corrected using the equation of Jones et aL (1979) to provide a better estimate of arterial PC02. An ECG was recorded from three bipolar chest leads (CASE, Marquette Electronics). Airflow, volume, PC02 at the mouth and ECG were recorded on a chart recorder. Tj and TE were measured for each breath from the airflow record.

The subjects quantified their sensation of breathlessness using a visual analogue scale. This consisted of a 10 cm line, placed before the subject, and marked with "not at all breathless" at one end and "extremely breathless" at the other. Subjects could control the position of a light along this line by the use of a linear potentiometer mounted on the handrail of the treadmill. The subjects indicated the onset of breathlessness using the VAS and, at 30 sec intervals thereafter, a VAS response was summoned by the illumination of a small light; responses were recorded on a chart recorder. All subjects were trained in the use of the VAS before the study and understood that it was their sensation of breathlessness specifically that they were to record. The validity of the VAS for measuring breathlessness has previously been described (Stark et a L, 1981; Adams et aL , 1985).

122 Statistical Analysis

Statistical analyses were performed using two-way analysis of variance and Student’s t-test for paired data. The level of significance was taken as P<0.05 in a two-tailed test.

7.3 Results

Large particle local anaesthetic aerosol

The cough reflex to citric acid aerosol was present when tested at the end of exercise after saline aerosol inhalation and absent at the end of exercise after bupivacaine aerosol in all eight subjects. (A ninth subject, initially included in the study, coughed at this time and was therefore excluded from analysis). Plasma bupivacaine levels at the end of exercise were 1.02 and 1.54 pg/ml in subjects 5 and 8 respectively. (It is noteworthy that the plasma bupivacaine at the end of exercise was 0.62 pg/ml in the subject whose cough reflex was not blocked presumably reflecting a problem with the initial aerosol inhalation in this subject).

There were no differences in the maximum exercise ability of subjects after large particle bupivacaine compared with saline aerosol inhalation (paired t-test) as indicated by maximum workload (mean [SD] after saline 191 [52.3] W; after bupivacaine 195 [52.4] W), maximum V02 (mean [SD] after saline 2780 [604] ml/min; after bupivacaine 2780 [796] ml/min) or maximum VC02 (mean [SD] after saline 3460 [725] ml/min; after bupivacaine 3530 [915] ml/min).

Inhalation of large particle local anaesthetic aerosol produced a slower deeper pattern of breathing during exercise. The ventilatory response to exercise following saline and bupivacaine aerosol inhalation is shown for all eight subjects in Fig 7.1. It can be seen that, in the majority of subjects, fR was decreased and VT increased following large particle local anaesthetic aerosol. This slower and deeper pattern of breathing tended to result in an increase in VE.

123 Fig 7.1 (Part 1) Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 1 to 4. Exercise was preceded by a rest period (REST) and a warm-up period (W).

\\\ 'b 2 5

\

( u j u i ) S V A (uiLU/cm09jq) (0 ^ (U|Ui/|LU) 3/\ (U|UJ/|LU) 20A

124 Fig 7.1 (Part 2) Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 5 to 8. Exercise was preceded by a rest period (REST) and a warm-up period (W). Workload (W) (W) Workload (W) Workload (W) Workload (W) Workload REST REST W 30 60 90 120 190 180 210 REST W 29 90 79 100 129 REST W 29 90 79 100 129 190 REST W 30 SO 90 121

125 et 3 or aatwtot eoo ihlto (is:; second:#) (first:0; inhalation aerosolwithout apart 3 hours tests o ujcs efrigtre tde: , xrie fe large after exercise A, studies:three performing subjectsfor eoo (=) Ec pit stema vle o l subjectsall for value is meanthe point Each (n=8).aerosol particle saline (0) and bupivacaine (•) aerosol (n=8); B, two exercise (n=8); B, two (•) aerosol (0) bupivacaine and salineparticle exercise incremental ofmaximal last 5 min the over data Mean 7.2Fig significance. necessary distance vertical the indicates and bar a as is shown for P=0.05 difference least significant Fisher’s study. given a performing ewe w pit frte o e ifrn tti level ofthis at different be tothem for points twobetween V02 (ml/min) PETC02 (mm Hg) VE (l/mln) Vj (I) fR (breaths/mln) (•) (0) bupivacaine and saline particle small after (n=6); C, exercise : ag Pril Arsl B Rpa Eecs - N Aerosols No - Exercise Repeat B: Aerosols Particle Large A: 126 : ml Pril Aerosols Particle Small C: A statistical analysis of the data for the group as a whole was performed using two-way analysis of variance; data for V02, VC02, fc,

fr , VT, VE, PetC02, Tj and TE were examined by this method. Since the analysis required equal sample sizes in each group, data from the last 5 min of exercise, where changes in the pattern of breathing may be expected to be greatest, were analysed. The mean values obtained from this analysis are plotted in Fig 7.2 A and Fig 7.3. Fisher’s least significant difference at the P=0.05 level is included for each variable in these figures; this allows data obtained at specific times throughout the exercise test to be compared. There was no difference in V02 or fc during exercise following saline and bupivacaine aerosol inhalation confirming that the work performed at each level of exercise was the same for both tests (Fig 7.2 A). However, after bupivacaine aerosol inhalation, fR decreased (mean fall 9.2%; P<0.05) and VT increased (mean rise 18.3%; P<0.001). This resulted in an mean increase in overall ventilation (VE) of 7.7% which just failed to reach statistical significance (P=0.07) although by the end of exercise values for VE after the two aerosols were significantly different as indicated by Fisher’s least significant difference and analysis of variance (P<0.05). Although there was no change in VC02, the PETC02, reflecting alveolar ventilation, decreased significantly following large particle bupivacaine aerosol inhalation (mean fall 6.0%; P<0.05). The reduction in fR during exercise following bupivacaine aerosol inhalation resulted from increases in both Tx and

Te (Fig 7.3) although neither difference reached statistical significance.

Fig 7.3 Mean data for T: and TE over the last 5 min of maximal incremental exercise for subjects performing exercise after large particle saline (0) and bupivacaine (•) aerosol inhalation. Each point is the mean value for 8 subjects. Fisher’s least significant difference for P=0.05 is shown as a bar and indicates the vertical distance necessary between two points for them to be different at this level of significance.

3[ I

0 ------■------i------■------L_ 0.0 1.0 2.0 3.0 X.O 5.0 Time (min)

127 Although the main aim of this study was to examine changes in the frequency and tidal volume produced by local anaesthetic aerosol inhalation, it is of interest to note that the increase in fR which occurred as exercise progressed after both aerosols was achieved by decreases in both Jz and TE (Fig 7.3; P<0.001) although the contribution from TE was greater.

When questioned at the end of exercise, seven of the eight subjects reported that they felt less breathless and six of these felt the sensation come on later during exercise after large particle bupivacaine as compared to saline aerosol. Four subjects volunteered that, when the sensation of breathlessness did arise, it increased in intensity more rapidly after bupivacaine aerosol. However, using the VAS for breathlessness (Fig 7.1) the results were less clear. There was no significant difference in the point at which the threshold VAS was recorded either with respect to time (mean [SD] after saline 5.3 [1.9] min; after bupivacaine 5.9 [2.5] min) or VE (mean [SD] after saline 43.2 [20.7] 1/min; after bupivacaine 49.9 [31.8] 1/min). To examine the VAS at an intermediate value of VE, the VAS scores, at the values for VE which were closest to 50 1/min, were compared; no significant difference was found (paired t-test) in the VAS scores at this level of VE (mean [SD] after saline 19.5 [16.9] mm; after bupivacaine 19.8 [19.4] mm). To examine the VAS at the end of exercise with respect to VE, the VAS scores, at the maximum values for • VE which could be matched within 10% between the two runs, were compared. No significant difference was found in the VAS scores at matched maximum VE (mean [SD] after saline 74.4 [23.7] mm; after bupivacaine 60.4 [40.3] mm). When compared over the last 5 min of exercise using analysis of variance, no significant difference was found between the VAS scores following saline and bupivacaine aerosols.

Intravenous infusions

Intravenous infusion of bupivacaine resulted in blood levels of 0.57 and 1.22 pg/ml immediately before exercise and 0.55 and 0.57 pg/ml immediately after exercise in subjects 3 and 9 respectively.

128 Fig 7.4 Effect of intravenous saline (0) and bupivacaine (§) infusion on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 3 and 9. Exercise was preceded by a rest period (REST) and a warm-up period (W).

100 Sub| 9 U 28yr* 75

1 /

/ //

0 3.0

✓ 7 ^

100

DEST W 30 60 90 120 ISO 160 Workload (W) Workload (W)

129 The ventilatory response to exercise following intravenous infusions of saline and bupivacaine is shown for both subjects in Fig 7.4. No differences were apparent in any variable following bupivacaine compared with saline infusion. When questioned at the end of exercise, both subjects reported that their sensation of breathlessness was the same during both exercise tests. No differences were detected in the VAS for breathlessness during exercise after saline and bupivacaine infusion.

Repeat exercise tests without aerosols

The ventilatory response to exercise during the two tests performed 3 hours apart is shown for all subjects in Fig 7.5. No consistent differences were seen in the pattern of breathing between the two tests although some subjects appeared to show an increase in fR during the second test. Analysis of variance performed as above on data for the last 5 min of exercise (Fig 7.2 B) confirmed that there was no significant difference between the two tests for V02, VT, VE or PETC02 although fR was significantly increased (mean rise 5.8%; P=0.04). No differences between the two tests were seen in the VAS scores for breathlessness (Fig 7.5).

The concentration of lactic acid in venous blood was 1.0 and 0.7 mmol/1 before the first exercise test in subjects 3 and 9 respectively. In the same subjects after the end of this test it was 6.9 and 7.1 mmol at 1 min after, 1.4 and 1.1 mmol at 1 hr after, 1.7 and 0.7 at 2 hr after and 1.4 and 0.6 at 3 hr after exercise. The reported normal range in venous blood from fasting subjects at rest is 0.4-1.42 mmol/1 (Marbach & Weil, 1967).

130 Fig 7.5 (Part 1) Effect of performing two exercise tests 3 hours apart without aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 1, 3 and 9. (0), first test; (•), second test. Exercise was preceded by a rest period (REST) and a warm-up period (W).

131 Fig 7.5 (Part 2) Effect of performing two exercise tests 3 hours apart without aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 10, 11 and 12. (0), first test; (•), second test. Exercise was preceded by a rest period (REST) and a warm-up period (W). Workload (W) Workload (W) Workload (W) REST REST W 30 60 SO 120 130 180 210 240 270 REST W 23 30 73 100 123 130 REST W 30 CO SO 120 ISO 180 210 240 270

( ujui) SVA (u|Ui/smo»jq) H) (I) M ( uiuj/ i) 3/\ ( uiuj/ | iu) Zq\

132 Small particle local anaesthetic aerosol

Subjects 8, 11, 13 and 15 inhaled small particle saline aerosol on the first study day and small particle bupivacaine aerosol on the following day; this order was reversed in subjects 1, 5, 14 and 16. When questioned at the end of the second test, subjects 5, 8, 11 and 13 could not discern any difference during aerosol inhalation between the two aerosols; the other four correctly distinguished which was the local anaesthetic aerosol due to a very slight taste in the mouth and back of the throat. There were no differences in the maximum exercise capacity of subjects after small particle bupivacaine compared with saline aerosol inhalation (paired t-test) as indicated by maximum workload (mean [SD] after saline 218 [66.5] W; after bupivacaine 221 [62.9] W), maximum V02 (mean [SD] after saline 3201 [889] ml/min; after bupivacaine 3165 [790] ml/min) or maximum VC02 (mean [SD] after saline 4028 [1127] ml/min; after bupivacaine 3909 [990] ml/min).

No changes were found in the pattern of breathing during exercise following small particle local anaesthetic aerosol. The ventilatory response to exercise following saline and bupivacaine aerosol inhalation is shown for all eight subjects in Fig 7.6. No consistent differences could be seen during exercise in any variable.

The data for the group as a whole were analysed by analysis of variance in the same way as for data following large particle aerosol inhalation. The mean values obtained by this analysis are shown, along with Fisher’s least significant difference at the P=0.05 level, in Fig 7.2 C. There was no significant difference in V02 or fc during exercise following saline and bupivacaine aerosol inhalation confirming that the work performed at each level of exercise was the same for both tests (Fig 7.2 C). There was also no significant difference in fR, VT, VE or PETC02 during exercise following bupivacaine aerosol. Furthermore, neither Fisher’s least significant difference nor the analysis of variance gave any indication that a significant difference existed in any variable at individual points during the test.

133 Fig 7.6 (Part 1) Effect of small particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 1, 5, 8 and 11. Exercise was preceded by a rest period (REST) and a warm-up period (W).

134 Fig 7.6 (Part 2) Effect of small particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in subjects 13, 14, 15 and 16. Exercise was preceded by a rest period (REST) and a warm-up period (W). Workload (W) Workload (W) Workload (W) Workload (W)

(turn) SVA (u|iu/«mD*jq) tij 0) (u[uj/|ui) a3 (uiiu/|tu)Z qa When questioned at the end of exercise, subjects 1, 11, 13 and 16 reported feeling less breathless during exercise after small particle bupivacaine aerosol inhalation; the others noticed no difference. As a consequence of the discrepancy between the verbal report of breathlessness and that indicated by the VAS during the study using large particle aerosols (page 128), all subjects performing the present study were asked, after each test, whether they believed that they had used the VAS to record their sensation of breathlessness accurately throughout the test; all subjects confirmed that their use of the VAS was reliable. However, using the VAS for breathlessness (Fig 7.6) only subject 16 recorded any notable difference between the two tests; this subject, who had verbally reported feeling less breathless during exercise after small particle bupivacaine aerosol, produced slightly higher VAS scores after bupivacaine. There was no significant difference for the group (paired t-test) in the point at which the threshold VAS was recorded either with respect to time (mean [SD] after saline 4.5 [2.2] min; after bupivacaine 4.6 [2.3] min) or VE (mean [SD] after saline 33.5 [24.4] 1/min; after bupivacaine 34.1 [25.1] 1/min). No significant difference (paired t-test) was found in ft the VAS scores at the values of VE closest to 50 1/min (mean [SD] after saline 30.1 [17.7] mm; after bupivacaine 33.5 [21.3] mm). No differences in VAS scores were found at the maximum values of VE matched to within 10% between the two runs (mean [SD] after saline 74.4 [23.7] mm; after bupivacaine 60.4 [40.3] mm). When VAS scores for the group were compared over the last 5 min of exercise using analysis of variance, no significant difference was found.

7.4 Discussion

The main result of the present study is that the inhalation of a large particle local anaesthetic aerosol resulted in slower and deeper breathing during exercise. There was no change in the pattern of breathing during similar exercise following local anaesthetic infusion indicating that the differences after local anaesthetic aerosol inhalation were not due to a systemic effect but were due to direct blockade of afferent information from receptors in the lung. The results were also not due to the experimental protocol since repeat exercise testing 3 hours apart without aerosol inhalation showed no effect on tidal volume and resulted in a small increase in respiratory

136 frequency. The reasons for this last observation remain unclear since normal subjects would be expected to have recovered (V02 and blood lactate returned to pre-test levels) within 60-90 min of maximal exercise (Astrand and Rodahl, 1986). This was indeed the case for the subjects in the present study whose V02 had returned to the pre-test level by the start of the second test; moreover, there was no difference in V02 throughout the second period of exercise. In both subjects in whom it was measured, blood lactate had returned to within the normal range by 3 hours after the first exercise test. In any case, the reduction in respiratory frequency during exercise after local anaesthetic aerosol inhalation occurred despite this tendency to increase fR on repeated exercise.

In contrast to this, small particle local anaesthetic aerosol inhalation had no effect on the pattern of breathing during exercise. Problems due to repeated exercise were avoided in this study by performing the tests after saline and local anaesthetic aerosols on consecutive days. In the light of the lack of effect of such an aerosol on the pulmonary chemoreflex to capsaicin in the dog (page 68) and on the chest sensation after capsaicin injection in man (page 113) the meaning of this result is uncertain. Since the small particle aerosol did not block these effects (when the large particle local anaesthetic aerosol did) its lack of effect on the pattern of breathing during exercise may be due to a lack of effective anaesthesia of lung receptors.

Perhaps the most obvious interpretation of the results of the study involving large particle local anaesthetic aerosol is that the local anaesthetic blocked afferent information from pulmonary stretch receptors allowing inspiration to continue and produce slower, deeper breathing. Previous experiments in conscious dogs have demonstrated slower, deeper breathing on exercise following bilateral vagal blockade (Phillipson et al., 1970) and chronic pulmonary denervation (Clifford et a l., 1986). However, by their nature, such studies do not provide information as to which type/types of receptor is/are responsible for the changes in the pattern of breathing. It is also possible that inhibition of afferent activity from pulmonary C-fibres may play a part in the change in the pattern of breathing after local anaesthetic aerosol; Phillipson and colleagues have performed

137 experiments in conscious dogs where the exposed vagi were progressively cooled and reported (albeit only in abstract form: Phillipson et al., 1975a) that slower, deeper breathing occurred on exercise when the responses to lung inflation (presumably due to slowly adapting pulmonary stretch receptors) and inhalation - cigarette smoke (believed to be due to rapidly adapting receptors) were blocked but that the effect was maximal when the response to capsaicin injection (due to pulmonary C-fibre stimulation) had also been blocked.

Although, in the present study, the pattern of breathing was altered during exercise after large particle local anaesthetic aerosol inhalation, the overall level of VE did not change. However, the PetC02 on exercise after bupivacaine aerosol was lower than that during exercise after saline despite the VC02 being unchanged. Although considerable caution must be exercised in equating the PETC02 with arterial PC02 (even after the application of the correction of Jones et al. (1979)) it is reasonable to use the PETC02 as an accurate reflection of the relative level of arterial PC02. Therefore, the decrease in PETC02 seen after local anaesthetic aerosol reflects an increase in alveolar ventilation due to the increase in VT. This indicates that, when afferent information from the lung in man is removed, the system responsible for ventilatory control will accept a lower PC02 than under normal circumstances.

The situation in animals with respect to this is somewhat unclear partly because of the different techniques used to remove afferent information from the lungs. Thus, studies with vagal cooling will, to a certain extent, allow selective removal of information carried by different fibre types while studies involving nerve section may remove information from receptors located at different levels in the lung depending on where the nerves are sectioned. A further problem with the latter studies is that reinervation of stretch receptors can occur after lung denervation in dogs (Clifford et a l., 1987c). With these considerations in mind the results of previous work in animals can be interpreted as follows. In conscious dogs at rest (Phillipson et a l., 1973), vagal cooling to 4-8°C, which abolished the Hering-Breuer inflation reflex, produced an increase in VE and a decrease in PaC02; with further cooling below 4°C, the PaC02 returned to normal. This

138 latter result is consistent with that reported at rest in dogs following complete cervical vagal blockade (Phillipson et al., 1970) and in dogs (Clifford et a l., 1986) and ponies (Flynn et a l., 1985) after chronic vagal denervation at the hilum; in all these studies the PaC02 was unchanged despite slower, deeper breathing. On exercise, the PaC02 was unchanged although the pattern of slower, deeper breathing was magnified in dogs (Clifford et a l. , 1986) and ponies (Flynn et a l., 1985) after chronic vagal denervation at the hilum. The results of these studies imply that complete vagal blockade was not achieved after inhalation of large particle local anaesthetic aerosol in the present study but that pulmonary stretch receptor blockade was responsible for the slow, deep breathing with a decrease in PetC02.

The only study in man which is similar to the present work is that of Van Meerhaeghe et al. (1986). These investigators studied normal subjects during 5 min of steady-state exercise at 100 W after inhalation of a large particle aerosol of 4% lignocaine; this was compared to a similar exercise test without aerosol inhalation performed about 35 min previously. They found no changes in the pattern of breathing after lignocaine aerosol. The two main differences between their study and the present work is that the 4% lignocaine aerosol used by them may not have resulted in as effective anaesthesia as the 5% bupivacaine used in the present study (they did not confirm block of the cough reflex at the end of exercise) and the performance of a second exercise test 35 min after the control test may have obscured any changes in the pattern of breathing.

Since the studies using local anaesthetic aerosol inhalation were performed, the ventilatory response to exercise has been examined in patients who have had combined heart-lung transplantation. The lungs of such patients are completely denervated although the innervation of most of the trachea remains intact. Such patients show an increase in VT on exercise resulting in an increase in VE and a reduction in PETC02 compared to matched heart transplant patients (Higenbottam et a l., 1986; Banner et al., 1989). In these studies the changes were accompanied by little or no difference in fR although more recent observations suggest that fR may be increased on exercise in these

139 patients (Banner, Lloyd, Hamilton, Innes, Guz & Yacoub, unpublished results).

No obvious explanation can be found for the discrepancy between the reported sensation of breathlessness and that indicated using the VAS during exercise following large particle aerosol inhalation; the only reasonable interpretation is that the reduction in breathlessness reported verbally after local anaesthetic was too small to be detected by the VAS. The subjects were well trained in the use of the VAS - indeed several were also subjects for other studies performed by my colleagues confirming the validity of the VAS for scaling breathlessness (Adams et a l., 1985). In the studies after local anaesthetic infusion, small particle local anaesthetic aerosol inhalation and those involving repeat exercise without local anaesthetic, no differences in breathlessness were recorded using the VAS or by verbal report. Therefore, the reduction in reported breathlessness after large particle aerosol, although probably small in magnitude, does seem significant. It is, however, not possible to distinguish whether this decrease in breathlessness is due to a reduction in afferent activity from lung receptors being perceived centrally as less breathlessness or whether it is an indirect result of the change in the pattern of breathing. The observation that there is no difference in breathlessness during exercise in heart-lung as compared with heart transplant recipients (Banner et al., 1989), may indicate that the reduction in breathlessness following local anaesthetic aerosol inhalation is linked to the reduction in fR by which it is accompanied.

The results of these studies indicate that, when tidal volume is increased in man by a natural stimulus such as exercise, afferent information from slowly adapting pulmonary stretch receptors plays a small part in limiting inspiration.

140 CHAPTER 8: NORMAL AND LARYNGECTOMIZED MAN - C02 REBREATHING

8.1 Introduction

The results presented in this thesis so far indicate that, although afferent information from the lung in man does not appear to be important in determining the pattern of breathing at rest (Chapter 5), it is involved to a small extent during exercise when ventilation is increased (Chapter 7). Another method commonly used to increase ventilation is that of C02 inhalation. In awake animals the ventilatory response to hypercapnia, especially the increase in respiratory frequency, is reduced after vagal section (Scott, 1908; Richardson & Widdicombe, 1969; Flynn et a l., 1985a) or blockade (Phillipson et a l., 1970). A similar result has been reported in conscious man after blockade of the vagus and glossopharyngeal nerves by injection of local anaesthetic at the base of the skull (Guz et a 7., 1966a; Guz and Widdicombe, 1970); the uncomfortable need to breathe which is usually associated with C02 rebreathing, was also absent after nerve blockade. However, following inhalation of a large particle local anaesthetic aerosol, an increase in the ventilatory response to hypercapnia is found in normal subjects (Cross et al., 1976; Sullivan and Yu, 1983); this is associated with an increase in breathlessness. In the first study described in this chapter, I wanted to reexamine this observation using the large particle bupivacaine aerosol which I had developed.

When I found that the ventilatory response was increased after inhalation of the large particle aerosol, a second study was performed in an attempt to elucidate the mechanism responsible. The recent demonstration in the dog of receptors in the larynx responding specifically to transmural pressure and airflow (Sant’Ambrogio et al., 1983; Mathew et al., 1984) has focused attention on the importance of the larynx in the regulation of the pattern of breathing and airway patency. Previous studies in cats reported the presence of laryngeal receptors which were inhibited by 5 and 10% C02 (Boushey et al., 1974); passing C02 through the isolated larynx decreased VE by reducing fR (Boushey & Richardson, 1973). Since, during inhalation of a large particle aerosol, a considerable mass deposits in the laryngeal region, it seemed possible that the increased ventilatory

141 response seen after inhalation of a large particle local anaesthetic aerosol could be due to the removal of inhibitory afferent information from laryngeal receptors. The second study described in this chapter examines the effect of large particle local anaesthetic aerosol inhalation on the ventilatory response to hypercapnia in laryngectomized man.

In rabbits, pulmonary C-fibres have been reported to mediate the increase in fR during C02 rebreathing (Russell et a/., 1984). A third study was therefore performed to determine the effect of the small particle local anaesthetic aerosol on the ventilatory response to C02.

8.2 Methods

Subjects

The first study, which examined the ventilatory response to C02 after inhalation of large particle saline and bupivacaine aerosols, and the third study, which examined this response after small particle aerosol inhalation, were performed on normal healthy volunteers. The second study examined the ventilatory response to C02 after inhalation of large particle aerosol in subjects who had permanent tracheal stomas after surgery for laryngeal carcinoma. Details of these laryngectomized subjects are given in Table 8.1; all subjects were healthy at the time of study and had no evidence of recurrence of carcinoma on physical examination or chest radiograph.

All subjects gave their consent after full explanation of the nature of the study. The protocols for the studies were approved by the Ethical Committee of Charing Cross Hospital.

Design of studies

Four normal subjects (aged 23-35 years; 1 female) performed a hypercapnic rebreathing test immediately after inhalation of large particle saline aerosol and, after a recovery period of at least 45 min, after large particle bupivacaine aerosol inhalation (page 32-35). The cough reflex to citric acid aerosol (page 85) was tested on completion of each rebreathe.

142 An identical study was performed in 7 laryngectomized subjects (aged 61-77 years; all male) after inhalation of large particle saline and bupivacaine aerosols. To allow the inhalation of aerosols and to permit the ventilatory response to C02 to be assessed, the area around a subject’s stoma was cleaned with 70% isopropyl alcohol and a plastic tracheostoma ring (V Mueller, Chicago) attached with silicone skin adhesive (V, Meuller, Chicago). This allowed the connection of standard 22mm-diameter tubing. The adequacy of this connection was tested by checking for C02 leaks around the stoma with a mass spectrometer probe (MGA 200, Centronics). The cough reflex in these subjects was elicited by passing a polythene catheter into the airway until the subject coughed or until the catheter could go no further. The catheter length at which this occurred was measured. The reflex was measured before bupivacaine aerosol inhalation and at the end of the rebreathe after bupivacaine inhalation. Venous blood samples for the determination of plasma bupivacaine were taken from each subject at the end of rebreathing after bupivacaine aerosol.

Table 8.1 Details of laryngectomized subjects.

Subj. Age Time Cigarette FEV1 FEV, FVC FVC FRC FRC Kco Kco No. Postop Consumption (yr) (yr) (pack-yr) (1) (%) (1) (%) (1) (%) (%)

L1 61 8.7 0 3.2 115 4.2 109 3.4 99 1.3 93

L2 59 15.0 29 2.3 86 3.0 85 2.2 66 1.6 108

L3 73 0.2 59 1.9 68 3.5 85 4.6 114 1.3 102

L4 70 14.0 84 2.6 92 4.0 98 4.5 122 1.1 83

L5 73 6.8 3 3.0 118 3.7 101 2.9 79 1.8 142

L6 77 11.0 35 2.0 89 2.9 89 3.1 86 1.3 110

L7 63 2.1 94 2.6 84 3.9 92 2.7 79 1.4 104

'ime postop, time between laryngectomy and study; pack--yr, product of daily cigarette pack consumption and the number of years smoked; FEV1? forced expiratory volume in 1 sec; FVC forced vital capacity; FRC, functional residual capacity; Kco, carbon monoxide transfer coefficient (mmol/min/kPa/1); %, percent of normal predicted value (Cotes, 1975).

143 A third identical study was performed in eight normal subjects (aged 24-33 years; 2 female) after inhalation of small particle saline and bupivacaine aerosol from the Unicorn/Optimist system (page 48-52). The cough reflex to citric acid aerosol was tested on completion of each rebreathe. Blood samples for the determination of plasma bupivacaine were taken before and at 9.5, 19.5 and 30 min after inhalation of small particle bupivacaine aerosol in four subjects. In addition, since this was the first physiological study performed involving the inhalation of the small particle aerosol (antedating the studies described in Chapter 7), the subjects were questioned formally about the general effects associated with the aerosol inhalations.

Ventilatory response to C02

The ventilatory response to hypercapnia was determined by a modification of the Read technique (Read, 1967). Subjects rebreathed into a 6 1 bag which initially contained 6% C02 in air; oxygen was automatically added throughout the test to keep the subject’s end- tidal P02 constant at the resting level (approximately 100 mm Hg). Airflow from this bag-in-box system was measured with a pneumotachograph (Fleish No.3) and integrated to give volume. End- tidal PC02 and P02 were continuously sampled at the mouth or stoma and analysed with a mass spectrometer (MGA 200, Centronics). The pneumotachograph was calibrated using a 1 litre syringe and the mass spectrometer calibrated with gases whose C02 and 02 concentrations had previously been determined with a Lloyd-Haldane apparatus. During rebreathing subjects quantified their sensation of breathlessness using a visual analogue scale (page 122). The test was continued until the PETC02 had reached 60-65 mm Hg or until the subject had reached their limit of tolerance. Airflow, PETC02, PET02, and VAS for breathlessness were recorded on a chart recorder and stored on magnetic tape for subsequent analysis. The response was analysed to give values of VT, fR, TI? TE and VE for each breath.

Linear regression analysis was performed on the relationships Ve/PEtC02, fR/PETC02 and VT/PETC02 for each test in each subject; the analysis was confined to that part of the response where a linear increase in VE/PETC02 was observed (i.e. after the "dog-leg"). The VAS scores for breathlessness during rebreathing were analysed in a

144 * similar fashion to that performed for exercise (page 128); the VE at threshold VAS, the VAS at an intermediate level of VE (40 1/min) and the VAS at the maximum values for VE which could be matched to within 10% between the two rebreathes were determined. The VAS scores were also examined in an analogous way with respect to PETC02; the PETC02 at threshold VAS, the VAS at an intermediate level of PETC02 (55 mmHg for the normal subjects and 50 mmHg for the laryngectomized subjects) and the VAS at the maximum values for PETC02 which could be matched to within 5% between the two tests were determined.

Statistical analysis

Statistical analysis was performed using Student’s t-test for paired data. The level of significance was taken as P<0.05 in a two tailed test.

8.3 Results

Large particle local anaesthetic aerosol in normal subjects

The cough reflex to citric acid aerosol was present at the end of the hypercapnic rebreathe following large particle saline aerosol inhalation and absent at the end of the rebreathe following bupivacaine aerosol inhalation in all four subjects.

An example of the ventilatory response to C02 is shown for one subject in Fig 8.1. The slopes of the relationships of VE, VT and fR with PeTC02 during rebreathing after large particle saline and bupivacaine aerosol are plotted for each subject on the same figure. Ventilation was increased with respect to PETC02 in all four subjects; the overall increase in VE/PETC02 slope of 58.6% just failed to reach statistical significance (P=0.086; paired t-test). There was no significant difference in the intercept of the regression line with the PETC02 axis (mean [SD] after saline 43.7 (5.5] mmHg; after bupivacaine 45.5 [5.4] mmHg). The increase in VE was achieved by an increase in fR alone in two subjects, an increase in VT alone in one subject and by increases in both fR and VT in one subject; the changes in the slopes of ^r/PstCO^ and VT/PETC02 were not statistically significant for the

145 Fig 8.1 Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response to normoxic hypercapnic rebreathing in normal subjects. The left-hand panel shows the response in subject 1; each point represents data for one breath. The right-hand panel shows the slopes from linear regression of the relationships between minute ventilation and its components and end- tidal PC02; the analysis was confined to that part of the response where a linear increase in VE/PETC02 was observed. Mean values for the group are shown as bars; data from subject 1 is indicated by an asterisk.

30 r

4 ? 20 - *JP • s°g° n

0.0

SAL BUP p£jC02 (mm Hg) Fig 8.2 Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the VAS scores for breathlessness recorded during normoxic hypercapnic rebreathing in normal subjects. The left hand panel shows the ventilation at which the threshold VAS was recorded, the VAS score at a ventilation of 40 1/min and the VAS score at the maximum ventilation which could be matched to within 10% between the two rebreathes. The right-hand panel shows analogous data for VAS with respect to end-tidal PC02. Mean values for the group are shown as bars.

SAL BUP SAL BUP

147 group as a whole. In those subjects where fR/PETC02 was increased, it was achieved by decreases in both and TE.

When questioned at the end of each test, one subject denied feeling breathless during rebreathing following inhalation of large particle saline or bupivacaine aerosol. Of the other three subjects, one felt more breathless during rebreathing after bupivacaine, one felt very distressed ("couldn’t expand lungs") but did not describe this as feeling more breathless and one reported no difference in breathlessness after bupivacaine aerosol inhalation. Using the VAS for breathlessness, these three subjects tended to indicate that they were less breathless for a given level of ventilation during rebreathing after large particle bupivacaine aerosol (Fig 8.2). The VE at threshold VAS was higher during rebreathing after large particle bupivacaine inhalation in all three subjects although this failed to reach statistical significance for these subjects taken as a group (P=0.060). Similarly, although the VAS at an intermediate VE and at m the maximum VE was decreased in all three subjects, the difference was not statistically significant. There were no differences in VAS with respect to PETC02 (Fig 8.2).

Large particle local anaesthetic aerosol in laryngectomized subjects

None of the subjects reported any effect of inhalation of large particle saline aerosol; some found the inhalation of large particle bupivacaine aerosol produced an initial irritation at the tracheal stoma followed by numbness of this area. No subject reported wheeze or any other side effect following bupivacaine aerosol inhalation. The duration of the rebreathe was reduced and a lower PETC02 was achieved after bupivacaine aerosol (mean [SD] PETC02 after saline 59.1 mmHg; after bupivacaine 53.0 mmHg; P<0.01) since all subjects were more breathless.

Before bupivacaine aerosol inhalation, the cough reflex was always present on mechanical stimulation of the airway with a catheter probe; the catheter length required to elicit cough is given for each subject in Table 8.2. At the end of C02 rebreathing after bupivacaine aerosol inhalation, probing with the catheter produced cough in three subjects but the distance to which it had to be advanced was increased

148 considerably from that prior to aerosol inhalation (Table 8.2). In the remaining four subjects no cough was elicited at the maximum length to which the catheter could be advanced (Table 8.2).

Table 8.2 Cough reflex to mechanical probing in laryngectomized subjects before large particle bupivacaine aerosol inhalation and at the end of hypercapnic rebreathing after bupivacaine aerosol.

Subj Distance probe Response Distance probe Response No. advanced before aerosol advanced after aerosol before aerosol after aerosol (cm) (cm)

L1 not measured cough 9 no cough

L2 not measured cough 26 no cough

L3 4 cough 25 cough

L4 3 cough 30 no cough

L5 8 cough 28 no cough

L6 5 cough 17 cough

L7 5 cough 9 cough

The cough reflex was tested by passing a polythene catheter probe through the stoma into the airway until the subject coughed or until the catheter could go no further. The catheter length at which this occurred was measured.

The plasma bupivacaine concentrations at the end of the rebreathe following large particle local anaesthetic inhalation in subjects 1 to 7 were 1.31, 1.30, 0.99, 0.50, 1.10, 1.28 and 1.11 pg/ml respectively.

Data for the ventilatory response to C02 after large particle aerosol inhalation in laryngectomized subjects is presented in Fig 8.3 in the same way as for the normal subjects. The slope of the relationship between VE and PETC02 was increased during rebreathing after large particle bupivacaine aerosol inhalation in six of the seven subjects (Fig 8.3) resulting in a mean increase for the group of 69.8% (P=0.019; paired t-test). The intercept of the VE/PETC02 regression

149 Fig 8.3 Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response to normoxic hypercapnic rebreathing in laryngectomized subjects. The left-hand panel shows the response in subject L1; each point represents data for one breath. The right-hand panel shows the slopes from linear regression of the relationships between minute ventilation and its components and end- tidal PC02; the analysis was confined to that part of the response where a linear increase in VE/PETC02 was observed. Mean values for the group are shown as bars; data from subject L1 is indicated by an asterisk.

150 Fig 8.4 Effect of large particle saline (0) and bupivacaine (t) aerosol inhalation on the VAS scores for breathlessness recorded during normoxic hypercapnic rebreathing in laryngectomized subjects. The left hand panel shows the ventilation at which the threshold VAS was recorded, the VAS score at a ventilation of 40 1/min and the VAS score at the maximum ventilation which could be matched to within 10% between the two rebreathes. The right-hand panel shows analogous data for VAS with respect to end-tidal PC02. Mean values for the group are shown as bars.

151 line with the PETC02 axis was unchanged by bupivacaine aerosol (mean [SD] after saline 37.1 [7.1] mmHg; after bupivacaine 38.2 [7.2] mmHg). The increase in VE/PETC02 was achieved by increases in the slope of both fR/PETC02 (mean increase of 60.0%) and VT/PETC02 (mean increase of 123.1%) although these differences failed to reach statistical significance (P=0.07 and P=0.08 respectively; paired t-test). In those subjects who demonstrated an increase in fR at a given PETC02, it was due to a decrease in both Tx and TE.

On questioning at the end of rebreathing all subjects reported that they were more breathless or that it was harder to breathe during the test after large particle bupivacaine compared with that after saline aerosol. This result was confirmed using the VAS for breathlessness. However, using the VAS, subjects L3 and L5 did not record feeling breathless during rebreathing after saline aerosol. Subject L5 continued to record VAS scores of zero on rebreathing after bupivacaine while subject L3 used the VAS to indicate breathlessness during this test. The results using the VAS are shown for each subject in Fig 8.4; for subject L3 after saline aerosol, the VE and the PetC02 at threshold VAS were taken to be the maximum values achieved for these variables. The onset of breathlessness occurred at a lower VE (P=0.017; paired t-test) and the VAS scores at matched % maximum levels of VE were greater (P=0.014; paired t-test) during hypercapnia after bupivacaine aerosol; the VAS at an intermediate level of VE was also increased but this failed to reach statistical significance (P=0.077; paired t-test). The results were similar when VAS was examined with respect to PETC02; breathlessness was significantly greater after bupivacaine aerosol for all three comparisons (P<0.02; paired t-test).

Small particle local anaesthetic aerosol in normal subjects

In order to determine the general effects of small particle aerosol inhalation, all subjects were asked a series of questions after inhalation of small particle saline and bupivacaine aerosols. These we re: "Can you swallow normally?" "Is your voice normal?" "Have you any sensations in your mouth or throat?"

152 "Do you feel anything else?"

After saline aerosol, seven subjects reported that they could swallow normally, the other subject reported that it was more difficult; after bupivacaine aerosol, six subjects reported that their ability to swallow was unchanged, the other two felt that it was slightly reduced. No subject reported any change in their voice after either aerosol. Following saline aerosol inhalation, five subjects reported no change in sensation in their mouth or throat, two felt that their mouth was dry (one of these volunteered that it felt anaesthetized) and the other subject described her throat as feeling smooth; after bupivacaine aerosol, three subjects said the sensations from their mouth and throat were unchanged, one reported that his tongue felt slightly odd and the other four noted a slight lump, tickle or numbness at the back of the throat.

The cough reflex to citric acid aerosol was present at the end of the C02 rebreathe following both small particle saline and bupivacaine aerosol inhalation in all eight subjects. The plasma bupivacaine concentrations in four of these subjects after inhalation of small particle local anaesthetic aerosol are presented in Chapter 5 (Fig 5.3; page 94).

Data for the ventilatory response to C02 after small particle aerosol inhalation is presented in Fig 8.5 in the same way as for the large particle aerosol. There was no change in the relationship between VE and PetC02 after small particle bupivacaine as compared to saline aerosol (Fig 8.5); both the slope of the linear regression line and its intercept with the PETC02 axis were unchanged. There was also no change in the slope of the VT/PETC02 relationship. It is perhaps worthy of mention that, although there was no statistically significant difference in the slope of the fR/PETC02 relationship after inhalation of small particle bupivacaine (P=0.14; paired t-test), the slope was decreased in six of the eight subjects giving a reduction of 19.0% for the group as a whole.

When questioned at the end of the rebreathe after inhalation of small particle saline aerosol, all eight subjects reported feeling breathless. At the end of the test after bupivacaine aerosol, all

153 Fig 8.5 Effect of small particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response to normoxic hypercapnic rebreathing in normal subjects. The left-hand panel shows the response in subject 6; each point represents data for one breath. The right-hand panel shows the slopes from linear regression of the relationships between minute ventilation and its components and end- tidal PC02; the analysis was confined to that part of the response where a linear increase in ^E/PETC02 was observed. Mean values for the group are shown as bars; data from subject 6 is indicated by an asterisk.

SAL BUP PETCO2 (mm Hg)

154 Fig 8.6 Effect of small particle saline (0) and bupivacaine (•) aerosol inhalation on the VAS scores for breathlessness recorded during normoxic hypercapnic rebreathing in normal subjects. The left hand panel shows the ventilation at which the threshold VAS was recorded, the VAS score at a ventilation of 40 1/min and the VAS score at the maximum ventilation which could be matched to within 10% between the two rebreathes. The right-hand panel shows analogous data for VAS with respect to end-tidal PC02. Mean values for the group are shown as bars.

155 subjects reported feeling less breathless than during the test after saline aerosol. The results using the VAS for breathlessness confirmed this finding (Fig 8.6). In all eight subjects, the VE at threshold VAS, VAS at an intermediate VE and VAS at maximum VE were increased (Fig 8.6) although the mean levels for the group were statistical significantly different only for the last two of these (P=0.003, P=0.006 respectively; paired t-test). A similar pattern was seen when the VAS was considered with respect to PETC02 (Fig 8.6) although the mean levels for the group were significantly different only for PETC02 at threshold VAS (P=0.006; paired t-test).

8.4 Discussion

The results of the studies described in this chapter confirm previous observations (Cross et al., 1976; Sullivan and Yu, 1983) that the inhalation of a large particle local anaesthetic aerosol increases the ventilatory response to C02 and indicate that this effect is not mediated by laryngeal receptors. Changes in the sensation of breathlessness during hypercapnia after large particle local anaesthetic aerosol inhalation in normal subjects were equivocal but inhalation of this aerosol in laryngectomized subjects greatly increased breathlessness. In contrast, the inhalation of a small particle local anaesthetic aerosol had no significant effect on the pattern of breathing during hypercapnia but reduced breathlessness.

Before discussing the results of this study further, I feel it is important to note that, although C02 rebreathing is a useful technique for the investigation of the control of breathing, it has at least two unusual features which influence the interpretation of the results obtained from its use. The first is a point of general significance; the second is important if the role of lung receptors in the control of breathing is to be examined. Firstly, during C02 rebreathing, the chemoreceptors are stimulated to increase ventilation by arterial PC02 levels which are not present under normal physiological conditions; secondly, lung receptors are exposed to C02 during inspiration as well as expiration and at higher levels than are experienced under normal conditions. Nevertheless, despite its being unphysiological, or rather, because it is unphysiological, the presence of high levels of

156 C02 in the lungs throughout the breathing cycle may provide insights into receptor physiology not given by other interventions.

The exaggerated ventilatory response to C02 rebreathing seen after large particle local anaesthetic aerosol in the normal subjects in the present study was similar to that found by Cross et al. (1976). In their study, the mean VE/PETC02 slope rose by 71% from a control value of 3.1 1/min/mmHg; in the few subjects tested in the present study this increased by 58% from a control value of 4.2 1/min/mmHg. In both studies the increase in VE was achieved by increases in fR and/or VT. An increase in the ventilatory response to C02 has also been reported by Sullivan and Yu (1983) although differences in their technique (a step increase to an inspired C02 of 7%) exclude a quantitative comparison of their results with those of the present study.

In contrast to the results of these studies, Easton et al. (1985), • found no significant difference in VE/PETC02 in normal subjects during C02 rebreathing after inhalation of large particle lignocaine compared with a control rebreathe. They did, however report a small change in the pattern of breathing such that fR was decreased by a mean of 1.48 breaths/min and VT was increased by a mean of 0.19 1 at a VE of 25 1/min. The reason for their failure to demonstrate an increase in the ventilatory response to C02 is likely to lie in the type and dose of the local anaesthetic used by them; in their study, 5 ml of 4% lignocaine was nebulized whereas in the present study, 5 ml of 5% bupivacaine was nebulized. As mentioned previously (Chapter 3, page 61-63) lignocaine is not as potent and does not provide the same duration of anaesthesia as does an equal amount of bupivacaine. In the present study, airway anaesthesia, as measured by the abolition of the cough reflex, was confirmed in each subject at the end of the rebreathe following large particle bupivacaine aerosol inhalation; unfortunately, Easton et al. (1985) did not test any index of airway anaesthesia at the end of the rebreathe after lignocaine aerosol but demonstrated that, immediately after lignocaine aerosol inhalation, the threshold of the cough reflex to citric acid aerosol was increased and the gag response was decreased but not uniformly extinguished. Easton et al. (1985) themselves have considered this point and concluded that lower doses of a local anaesthetic such as lignocaine may produce changes in breathing pattern without affecting VE/PETC02

157 whereas higher doses or the use of a more potent local anaesthetic such as bupivacaine may increase VE/PETC02. While I certainly agree with the latter part of this conclusion, the magnitude of the changes in the pattern of breathing reported in their study leaves me in some doubt as to the strength of the evidence supporting the first part of their proposal.

Before considering the effect of large particle local anaesthetic aerosol inhalation on the ventilatory response to C02 in laryngectomized subjects, it is important to compare the baseline response without local anaesthetic aerosol in these subjects with that found in normal subjects. The values for the slope of VE/PETC02 after saline aerosol in the laryngectomized subjects (1.6-3.2 1/min/mmHg) are within the range for normal subjects previously reported by others (1.16-6.18 1/min/mmHg by Read (1967); 0.47-6.23 1/min/mmHg by Irsigler, (1976)) and determined in this laboratory (1.6-9.7 1/min/mmHg by Adams et ah, (1985)). That they lie toward the lower end of this range is expected since the laryngectomized subjects studied were aged between 59 and 77 years and the slope of VE/PETC02 is inversely related to age (Molho et al., 1986). The only previous study (to my knowledge) on the ventilatory response to C02 in laryngectomized subjects (Gardner, 1983) used a steady-state method to produce mild hypercapnia and found no difference in the slope of

Ve/PetC02 in laryngectomized subjects compared with normal age-matched controls.

The principal result of the present study in laryngectomized subjects is the demonstration that the ventilatory response to C02 is increased after local anaesthetic aerosol inhalation; furthermore the magnitude of this increase and the changes in the components of VE responsible for it are similar to that found in normal subjects with an intact larynx. The mean increase in the slope of VE/PETC02 of 70% from a control value of 2.4 1/min/mmHg is very similar to that reported by Cross et al. ( 1976) and found in the normal subjects in the present studies. Also in common with the results of those studies is the observation that the increase in VE/PETC02 in laryngectomized subjects was brought about by increases in fR and/or VT.

158 The results concerning the cough reflex in laryngectomized subjects after local anaesthetic aerosol inhalation are interesting because, although no subject coughed with mechanical stimulation to the same catheter length as they did before local anaesthetic aerosol inhalation (indicating a good degree of airway anaesthesia), two subjects coughed during mechanical stimulation at a distance of 17 and 25 cm from the stoma. Although it is well established that coughing in animals is evoked by stimulation of rapidly adapting receptors located in the large airways (Widdicombe, 1954c), mechanical stimulation of similar receptors located deeper in the lungs is not believed to induce cough (Mills et ah, 1969; Widdicombe, 1982). By contrast - as mentioned previously (page 116) - several authors (Coleridge and Coleridge, 1986; Sant’Ambrogio et a 7., 1984; Sant’Ambrogio, 1987a) believe that coughing can be produced by stimulation of bronchial and/or pulmonary C-fibre endings. The results of the present study provide further evidence for the conclusions of the experiments involving capsaicin injection (page 116) that stimulation of receptors located in the lung periphery can induce cough; the receptors responsible may be C-fibre endings.

In the present study, the inhalation of a small particle local anaesthetic aerosol had no significant effect on the ventilatory response to C02. In view of the fact that, in the other experiments described in this thesis, this aerosol has consistently failed to effect either the level of ventilation or the pattern of breathing, the lack of effect on the ventilatory response to C02 cannot readily be interpreted. It is not possible to decide from this whether pulmonary C-fibre afferents are involved in the ventilatory response to C02 since it seems likely that insufficient local anaesthetic has reached these endings to block their conduction.

Concern that the increased ventilatory response to C02 after large particle local anaesthetic aerosol inhalation may be due to a central effect of absorbed local anaesthetic, led Cross et ah (1976) and Sullivan et ah (1986) to examine the effect of intravenous infusion of bupivacaine and lignocaine respectively on hypercapnia. Despite achieving similar blood levels of local anaesthetic to those achieved after aerosol inhalation, no difference was found in the ventilatory response to C02. In addition, in the studies described in Chapter 5,

159 no evidence was found of a central effect after large particle bupivacaine aerosol inhalation. The evidence is, therefore, that the increased ventilatory response found after large particle local anaesthetic aerosol inhalation results from blockade of afferent information from lung receptors. Since the laryngectomized subjects have a normal ventilatory response to C02 after saline aerosol and the increase in the hypercapnic response after bupivacaine aerosol is similar to that seen in normal subjects, laryngeal receptors cannot be responsible for the abnormal ventilatory response observed after local anaesthetic aerosol; the receptors involved must lie lower in the respiratory tract.

The increased ventilatory response after local anaesthetic aerosol inhalation could be due to an indirect or a direct effect of C02 on lung receptors during hypercapnia: there may be a change in the response of receptors to the increase in VT (and therefore the increase in lung stretch) which occurs when ventilation is increased by hypercapnia, or there may be a change in the response of receptors directly sensitive to C02. If the first mechanism were responsible, it may be expected that a similar increase in the ventilatory response would be seen after local anaesthetic aerosol during all forms of stimulated breathing. The results of Sullivan and DeWeese (1985) indicate that this is not so. They showed in normal subjects that ventilation was unchanged during hypoxic breathing after lignocaine aerosol inhalation in contrast to their previous results where ventilation was increased during hypercapnic breathing after inhalation of the same aerosol (Sullivan and Yu, 1983; Sullivan and DeWeese, 1985). Furthermore, during exercise (this thesis Chapter 7), slower, deeper breathing with little change in VE was produced by large particle local anaesthetic aerosol inhalation in comparison to the faster, deeper breathing with a greatly increased VE seen during hypercapnia after the same aerosol.

To elucidate the details of the mechanism by which the increased ventilatory response is achieved, it is necessary to consider the reported effects of C02 on lung receptors in animals and the degree to which the large particle aerosol used in the present study is likely to block each of these in man. Although it is generally agreed that slowly adapting pulmonary stretch receptors are inhibited by C02 in

160 mammals (Coleridge and Coleridge, 1986; Sant’Ambrogio, 1987b) the nature of this effect is not straightforward. The debate about it has two main foci: the effect of C02 when delivered to animals when they are below normocapnic levels and the effect of C02 when delivered above normocapnic levels. With respect to the first of these, many authors now believe (Coleridge and Coleridge, 1986; Widdicombe, 1982) that hypocapnia stimulates stretch receptors (decreasing ventilation) and that giving C02 via the airways simply returns the C02 to its original level and therefore reduces stretch receptor firing to normal (producing an apparent increase in ventilation). However, it is the effect of C02 on stretch receptors above normocapnic levels which is germane to the present study. Coleridge et a 7. (1978) reported that, in the dog when the lung was vascularly isolated, increasing the inspired C02 from 4.5% (lung PC02 of 30 mmHg) to 7.2% (lung PC02 of 50 mmHg) decreased activity in 10 of 18 stretch receptors fibres, although the effect for the group of fibres as a whole was small. In a more elaborate series of experiments on dogs in which gas tensions in the pulmonary and systemic circulations were controlled separately, Green et al. (1986) showed that increasing the pulmonary arterial PC02 from 25 to 50 mmHg and from 50 to 80 mmHg reduced activity by 15 and 9% respectively in 26 pulmonary stretch receptors. Although the significance of such inhibition under normal physiological conditions is debatable, it may be relevant under the physiologically abnormal circumstances of C02 rebreathing.

Increases in inspired PC02 are reported to have no significant effect on the discharge of rapidly adapting stretch receptors in the rabbit (Sellick and Widdicombe, 1969) or dog (Coleridge et al., 1978; Sampson and Vidruk, 1978).

The sensitivity of C-fibre endings in the lungs to C02 may be species- dependent. Coleridge et al. (1978) found no change in the discharge frequency in 13 bronchial and 9 pulmonary C-fibres when lung PC02 was increased from 30 to 46 mmHg in dogs, whereas Delpierre et al. (1981) reported that the discharge frequency was doubled in 26 of 85 pulmonary and bronchial C-fibres when PETC02 was increased from 5% (approximately 36 mmHg) to 10% (approximately 71 mmHg) in cats. In rabbits, pulmonary C-fibre activity was increased by injection of sodium dithionite into the right atrium; it was proposed that this was

161 due to C-fibre stimulation by C02 or H+ released by the dithionite (Trenchard et al., 1984). Similar injects of dithionite in the same animals increased fR without significantly changing VT (Trenchard et al., 1984).

With respect to the degree to which the large particle local anaesthetic aerosol produces blockade of these receptors in man, the evidence accumulated in this thesis so far suggests that the inhalation of this aerosol will produce blockade of rapidly adapting receptors and a majority of slowly adapting receptors in the larger airways; conduction is likely to be intact in a number of C-fibre endings in the lung periphery. Combining this with the reported effects of hypercapnia on pulmonary receptors in other mammals presented above does not allow the deduction of a clear mechanism to explain the increased ventilatory response to C02 seen in the present study. It seems that a clear understanding could only be provided by recording afferent information in single vagal fibres from each receptor type during hypercapnia with and without local anaesthetic aerosol inhalation.

However, it is of interest that an increased ventilatory response to hypercapnia has been reported in anaesthetized rabbits when stretch receptors were blocked with sulphur dioxide (Davies et al., 1978) and in awake dogs when the inflation reflex was blocked by vagal cooling to 4-8°C (Phillipson et al., 1973). Evidence that C-fibres mediate this effect has been reported by Russell et al. (1984); in anaesthetized rabbits, anodal blockade of conduction in myelinated vagal fibres resulted in an increased ventilatory response due to increases in VT and an increased frequency response; when the conduction in C-fibres was also blocked the frequency response was abolished but VT remained increased.

It seems possible that, in the present studies the inhalation of the large particle aerosol results in a removal of a inhibitory influence on breathing from receptors in the larger airways while preserving activity from C-fibre endings in the lung periphery. Such an interpretation would account for the differences seen in the ventilatory response to C02 after large particle local anaesthetic aerosol inhalation in the present study and after complete vagus (and

162 glossopharyngeal) nerve blockade by percutaneous injection of local anaesthetic at the base of the skull (Guz et al. 1966a, 1970). In the latter studies the ventilatory response was reduced mainly by decreases in fR. The assumption that peripheral chemoreceptors were functionally denervated by the concomitant hyperoxia provided in those studies may be incorrect since, at least in the cat, hyperoxia diminishes but does not abolish activity from carotid body chemoreceptors during hypercapnia (Fitzgerald and Parks, 1971). However, a similar reduction in the frequency response to hypercapnia has been reported in patients with combined heart-lung transplantation compared to normal subjects of similar age and height (Sanders et a/., 1989); no changes were found in the ventilatory response to hypoxia.

The results concerning the sensation of breathlessness during C02 rebreathing after large particle local anaesthetic aerosol in normal subjects in the present study are equivocal, perhaps because of the small number of subjects involved. However, an exaggerated sensation of breathlessness during rebreathing after local anaesthetic aerosol has previously been reported in normal subjects (Cross et al., 1976). The results in the laryngectomized subjects clearly indicate that all subjects were more breathless during rebreathing after large particle local anaesthetic aerosol; indeed the severity of the sensation was such that the duration of the test was reduced after this aerosol. The mechanism responsible for the increased breathlessness is not immediately obvious. It is unlikely to result from an increase in the work of breathing since bupivacaine aerosol has no effect on lung volumes or airways resistance in normal subjects (Cross et al., 1976; this thesis page 93); the laryngectomized subjects in the present study had remarkably normal lung function (Table 8.1) and no subject reported wheeze after bupivacaine aerosol.

Work directed at understanding the mechanisms responsible for breathlessness has been performed by Guz and colleagues (Freedman et al., 1987; Chronos et al., 1988; Cockcroft and Adams, 1986). Experiments in patients with severe chronic airflow obstruction demonstrated that breathlessness was greatly reduced when ventilation was increased voluntarily rather than when it was reflexly increased by C02 rebreathing (Freedman et al., 1987). When normal subjects were suddenly made hypoxic during exercise, breathlessness increased

163 slightly before an increase in ventilation (Chronos et a 7., 1988). Taken together, these results suggest that breathlessness is not simply due to the awareness of an increased ventilation or to sensing of afferent information from lung or chest wall receptors, but is perceived centrally in situations where a reflexly increased drive to breathe causes an increase in motor respiratory activity (Cockcroft and Adams, 1986). It therefore seems possible that the increase in breathlessness during hypercapnia after large particle local anaesthetic aerosol could result from an increase in the reflex drive to breathe from C-fibres in the lung periphery unopposed by an inhibitory influence from receptors in the airways. The abolition of the sensation described as "a need to expand the chest more than was in fact possible" during hypercapnia after complete vagal blockade (Guz et a 7., 1966a) would then be due to the removal of all afferent information from the lung including that carrying information from C- fibres.

Although strong evidence exists that breathlessness was decreased during hypercapnia after small particle local anaesthetic aerosol inhalation in the present study, it was not associated with any significant changes in the ventilatory response or its components. The mechanism responsible for the decrease in breathlessness is, therefore, unclear. It is conceivable that the local anaesthetic aerosol removes some afferent information from C-fibres resulting in a decreased drive to breathe which, although too small to be expressed as a significant reduction in fR, is sufficient to reduce breathlessness.

In conclusion, the increased ventilatory response to hypercapnia previously reported after inhalation of a large particle local anaesthetic aerosol in normal subjects as been confirmed. The discovery of a similar result in 1aryngectomized subjects indicates that the removal of an inhibitory influence on breathing from the larynx is not responsible for this phenomenon. The mechanism which is responsible may involve the removal of an inhibitory influence on breathing from receptors in the larger airways while preserving activity from C-fibre endings in the lung periphery. The results of these studies therefore indicate that altering pulmonary receptor

164 discharge when ventilation is stimulated by hypercapnia, can have a marked effect on the control of breathing.

The results of the studies during C02 rebreathing after inhalation of large and small particle local anaesthetic aerosols indicate that blockade of afferent information from lung receptors can alter the sensation of breathlessness. The results after large particle aerosol provide strong support and those after small particle aerosol provide weak support for the concept that breathlessness changes pari passu with the central perception of changes in the reflex drive to breathe.

165 CHAPTER 9: PATIENTS WITH INTERSTITIAL LUNG DISEASE - EXERCISE

9.1 Introduction

Up to this point, the investigations presented in this thesis have examined the control of breathing in healthy subjects and found that afferent information from the lung is involved, though not in a major way, in determining the pattern of breathing in man at rest (Chapter 5) and has only a small influence on ventilatory control when breathing is increased by a natural stimulus such as exercise (Chapter 7). However, the results of the studies involving C02 rebreathing (Chapter 8) indicate that disturbing the discharge of lung receptors (with a large particle local anaesthetic aerosol) when ventilation is stimulated, can have a marked effect on the control of breathing and the sensation of breathlessness. It is possible, therefore, that afferent vagal information may be important in generating the changes in breathing pattern seen in certain disease states; this suggestion was first made by Breuer (1868). The diseases most often implicated in this proposal are those involving a disturbance, especially inflammation, of the lung parenchyma since such diseases are generally characterised by tachypnoea and/or breathlessness (Jackson and Fulmer, 1988; Turner-Warwick, 1988).

Studies in animals have confirmed that the vagus is involved in mediating the tachypnoea produced by a variety of experimentally induced situations which alter the lung parenchyma; such interventions include pneumonia produced by Friedlander’s bacillus in the dog (Porter and Newburgh, 1917), pneumonia produced by injection of hot water into one lung in the cat (Frankstein and Sergeeva, 1966; Frankstein, 1970), pulmonary embolism after intravenous injection of inert micro-spheres in the rabbit (Guz and Trenchard, 1971b), inflammation produced by instillation of carageenin into one lung in anaesthetized rabbits and conscious cats (Trenchard et al., 1972) and diffuse pneumonitis produced by intravenous injection of complete Freund’s adjuvant in awake dogs (Phillipson et al., 1975b). The results of several of these studies (Frankstein, 1970; Guz and Trenchard, 1971b; Trenchard et al., 1972) indicate that C-fibre endings are stimulated by these processes and mediate the tachypnoeic response. In addition, Paintal (1969; 1970) has proposed that

166 pulmonary C-fibres are stimulated to produce breathlessness by situations involving pulmonary congestion.

To examine whether these receptors are involved in lung disease in man, I studied patients with diffuse interstitial lung disease but no airflow obstruction; ideally, I would like to have studies patients with active infiltrations and no changes in lung mechanics, but such patients are generally too ill to participate in this research. Diseases classified under the general heading of interstitial lung disease have a common pathogenesis: an initial injury of the alveolar wall results in the accumulation of inflammatory cells in the pulmonary interstitium (alveolitis) which may lead to structural changes due to the deposition of connective tissue (fibrosis) (Crystal and Ferrans, 1988). Patients with interstitial lung disease characteristically have an abnormally high fR and low VT with an exaggerated sensation of breathlessness on exercise and, in more severe cases, at rest (Kaltreider and McCann, 1937; Holland and Blacket, 1960; Lourenco et al., 1965; Bradley and Crawford, 1976; Spiro et al., 1981; Burdon et al.,1983). These changes are generally not reduced, or are only partially reduced, when the alterations in arterial blood gases resulting from ventilation/perfusion mismatching are corrected (Turino et al., 1963; Lourenco et al., 1965; Bye et al., 1982). This has led to the proposal that the tachypnoea of interstitial lung disease is due to altered discharge of receptors in the lungs (Turino et al., 1963) and/or chest wall (Lourenco et al., 1965; Bradley and Crawford, 1976; Van Meerhaeghe et al., 1981). I therefore examined the ventilatory response and degree of breathlessness in these patients during exercise after inhalation of a local anaesthetic aerosol.

Since the evidence suggested that the lung receptors most likely to be involved in the changes in the pattern of breathing were pulmonary C- fibre endings, it was discharge from these endings that I wished to block. However, since the previous studies described in this thesis had indicated that the large particle local anaesthetic aerosol was more effective than the small particle aerosol at blockade of neural information believed to arise from pulmonary C-fibres, the former aerosol was chosen for use in the present study.

167 9.2 Methods

Subjects

The study was performed on six subjects (all male) with diffuse interstitial lung disease who gave written consent after a full explanation of the nature, but not the purpose, of the study. The protocol for the investigation was approved by the Ethical Committee of Charing Cross Hospital. A medically qualified colleague was present during all exercise tests.

Patients who were potentially suitable for inclusion in the study on the basis of a provisional diagnosis of interstitial lung disease, were referred by clinical colleagues at Charing Cross Hospital. The details of the subjects selected are given in Table 9.1.

Table 9.1 Detai1s of patients with i nterstit'ial lung disease.

Subj . Age FEV 1 FVC TLC ^co Sa°2(%) Diagnosis

No. (yr) (1) (%) (1) (

1 53 1 . 92 60 2.58 61 3.85 59 0.69 46 88 65 Sarcoidosis

2 45 1.29 41 1.88 48 2.76 49 1.50 93 96 77 Sarcoidosis

o0 67 2.13 76 2.64 67 4.47 68 1.10 83 96 88 CFA

4 74 1 . 64 60 2.03 50 3.68 53 0.62 50 96 91 CFA

t; 67 1 .81 73 1.91 55 2.90 50 0.74 56 39 34 CFA

6 62 3.05 115 4.52 125 6.47 111 0.45 32 93 69 EAA

FEV1, forced expiratory volume in 1I sec; FVC, forced vital capacity; TLC, total lung capacity; Kco, carbon monoxide transfer coefficient (mmol/min/kPa/1); %, percent of normal predicted value (Cotes, 1975); CFA, cryptogenic fibrosing alveolitis; EAA, extrinsic allergic alveolitis; Sa02 Rest, Sa02 breathing air at rest on the familiarization day; Sa02 End ex, Sa02 breathing air at the end of exercise on the familiarization day (the fall in Sa02 was prevented by adding 02 during exercise on subsequent tests)

168 Lung function tests in all subjects demonstrated a reduction in lung volumes and/or a defect in gas transfer in the absence of airflow obstruction. All subjects had chest radiographs showing widespread bilateral shadowing characteristic of interstitial lung disease. Subjects 1 and 2 had a positive Kveim test for sarcoidosis and a liver biopsy showing granulomatous inflammation. Subject 3 had a clinical course consistent with cryptogenic fibrosing alveolitis. In subjects 4, 5 and 6, histological confirmation of the diagnosis was obtained following open lung biopsy. No subject had evidence of chest infection or was in respiratory failure at the time of study.

During the study subjects continued their normal medication if any. Subjects 1 and 2 took 30 mg and 10 mg respectively of prednisolone (Deltacortri1 Enteric, Pfizer) orally as a single morning dose; subject 5 took 30 mg of mianserin (Bolvidon, Organon) and 10 mg of temazepam (Normison, Wyeth) orally at night. The other subjects were not taking any medication.

Design of studies

The six patients performed an exercise test at the same time of day on each of three days. The first test was used to familiarize each patient with the procedure, train and give them practice in the use of the VAS for breathlessness and to assess the degree of arterial oxygen desaturation which they had on exercise. During the two subsequent tests, 100% oxygen was added to the inspired air to keep the Sa02 at, or above, 95%.

On the second and third test day, subjects performed an exercise test immediately after the inhalation of large particle saline or bupivacaine aerosol (page 32-35). Five subjects inhaled saline aerosol on the second day and bupivacaine on the third; in the other subject (subject 4) the order was reversed. The cough reflex to citric acid aerosol was tested before the administration of bupivacaine aerosol and at the end of the exercise test after this aerosol. A venous blood sample was taken at this time in five of the subjects for the determination of plasma bupivacaine.

169 Exercise

All exercise tests were performed on an electronically-braked cycle ergometer (Lanooy, Lode). This was chosen in preference to the treadmill used in the studies in normal subjects since many patients find it difficult to walk on the moving belt of a treadmill especially when breathing on a mouthpiece attached to respiratory equipment (personal experience and Jones, 1988a). The tests consisted of 1 min at rest followed by a 4 min 'warm-up5 period at minimum workload (5 W) after which the workload was increased in 15 W increments at constant intervals to maximum exercise. A 4 min period at minimum workload was chosen instead of the 1 min period used in normal subjects because many patients will only be able to perform a small amount of exercise even with the 15 W workload increments used in these tests; since many patients with severe respiratory disease find 5 W to be a significant level of work, the use of a longer period at this workload allowed the collection of more exercise data in these subjects. Values for fR, VT, VE, V02 and VC02 averaged over each 30 sec period during exercise were measured in subjects breathing via a mouthpiece connected to a Fleisch No.2 pneumotachograph using the computer-assisted system (Ergostar, Fenyves & Gut) previously described for the studies in normal subjects (page 122). PETC02 and an ECG were also recorded as previously described (page 122). Arterial oxygen saturation was measured continuously with an ear oximeter (Hewlett-Packard, 47201A). Subjects quantified their sensation of breathlessness every 30 sec from the beginning of exercise using a visual analogue scale.

The addition of oxygen to the inspired air will affect the measurement of flow, and therefore volume, by a pneumotachograph. The pneumotachograph operates on the principle that the pressure drop across a resistance is proportional to the flow rate of gas through it. The resistance in the Fleisch pneumotachograph is provided by a series of fine, parallel tubes in order that the flow through it should be laminar. The pressure drop therefore follows Poiseuille’s law ( A P = SVrjL/nr4), where A P is the pressure drop, V is the flow rate, r^ is the viscosity of the gas, L is the length of the tube and r is its radius. It follows from this that the pressure drop measured by the pneumotachograph is directly proportional to gas flow and the output of the system is, therefore, calibrated directly in terms of

170 flow. However, it is also apparent from Poiseuilie’s law that the pressure drop is directly proportional to the viscosity of the gas concerned; this has been confirmed experimentally (Green, 1965). The pneumotachograph should, therefore, be calibrated with the inspired gas with which it is to be used. This was not possible in the present study since the inspired oxygen concentration was increased throughout the period of exercise. To allow for this, the pneumotachograph was calibrated with air in the standard way, and all values for the t volume-related variables measured by it during exercise (VT, VE and * VC02) were subsequently corrected to allow for increases in gas viscosity produced by the addition of oxygen (Turney and Blumenfeld, 1973; Handbook o f Chemistry and P h y sics, 1975). It should be mentioned that the magnitude of this correction was very small; the maximum correction of volume due to the addition oxygen was 2.0, 2.8, 1.1, 0.8, 2.0 and 2.6 % in subjects 1 to 6 respectively.

Statistical analysis

Statistical analyses were performed using two-way analysis of variance and Student’s t-test for paired data. The level of significance was taken as P<0.05 in a two-tailed test.

9.3 Results

No subject reported any effect after large particle saline aerosol inhalation. The effects after large particle bupivacaine aerosol were similar to those described by normal subjects (Chapter 5; page 87) including profound oropharyngeal anaesthesia and impairment of the ability to swallow. None of the subjects noted wheeze or reported any other side effect of the local anaesthetic. The cough reflex to citric acid aerosol was present in all six subjects before bupivacaine aerosol inhalation. In five of the subjects it was completely abolished at the end of exercise following bupivacaine aerosol; the other subject (subject 3) coughed on the last of three breaths of citric acid aerosol indicating considerable impairment but not absence of the reflex.

171 The plasma bupivacaine levels at the end of exercise after large particle bupivacaine aerosol inhalation were 1.63, 1.49, 0.63, 1.34 and 0.82 pg/ml in subjects 1, 2, 3, 4, and 6 respectively.

The Sa02 fell in all subjects during exercise on the familiarization day (Table 9.1). As a consequence, supplementary oxygen was added to the inspiratory airflow during exercise after each aerosol inhalation in all subjects; the Sa02 levels measured during these tests are shown in Fig 9.1. It can be seen that the attempt to keep Sa02 at 95% or greater was generally successful (especially in view of the degree of desaturation found during the familiarization day); only in subject 6 for the last half-minute of exercise after saline aerosol did the saturation fall below 94%. The addition of varying amounts of oxygen * to the inspired air during these tests made the measurements of V02 invalid. Although it was not formally analysed, no notable change was seen in the ventilatory response to exercise after saline aerosol (during which oxygen was given) as compared to the familiarization test (performed without oxygen). All patients exhibited a rapid, shallow pattern of breathing during exercise after saline aerosol similar to that previously reported for patients with this condition and considerably different from that reported for normal subjects (Bradley and Crawford, 1976; Spiro et al., 1981; Van Meerhaeghe et a 7., 1981).

There were no differences in the maximum exercise ability of subjects after large particle bupivacaine compared with saline aerosol (paired t-test) as indicated by maximum workload (mean [SD] after saline 88 [40] W; after bupivacaine 80 [32] W), maximum heart rate (mean [SD] after saline 124 [19] beats/min; after bupivacaine 121 [17] beats/min) « or maximum VC02 (mean [SD] after saline 1287 [620] ml/min; after bupivacaine 1141 [475] ml/min).

Inhalation of large particle local anaesthetic aerosol produced no statistically significant changes in the pattern of breathing for the subjects as a group. The ventilatory response to exercise following saline and bupivacaine aerosol inhalation is shown for all six subjects in Fig 9.1. Although the pattern of breathing was altered in some subjects after bupivacaine as compared to saline aerosol, no consistency in the nature of the change existed between subjects.

172 ,1 (Part 1) Effect of large particle saline (0) and bupivacaine ire sol inhalation on the ventilatory response and breathlessness di ring maximal incremental exercise in patients with stitial lung disease (subjects 1 and 2). Exercise was preceded by in rest period (REST) and a 4 min warm-up period (W). 100

BO

60

40

20

0 40

33

30

23

20 40

30

20

10

0 2.0 t.S

1.0

0.5

0.0

60

40

20

0 100

93

90

83

80 500

000

500

0

REST W 13 30 43 10 73 Workload (W)

173 Fig 9.1 (Part 2) Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in patients with interstitial lung disease (subjects 3 and 4). Exercise was preceded by a 1 min rest period (REST) and a 4 min warm-up period (W).

13 30 43 SO 73 >0 103 120 133 REST W IS 30 43 60 Workload (W) W orkload (W)

174 Fig 9.1 (Part 3) Effect of large particle saline (0) and bupivacaine (•) aerosol inhalation on the ventilatory response and breathlessness (VAS) during maximal incremental exercise in patients with interstitial lung disease (subjects 5 and 6). Exercise was preceded by a 1 min rest period (REST) and a 4 min warm-up period (W).

100 Sub) 6 80 60 40 •V 20 0

2.5

2.0 1.5 1.0 0.5 0.0 too 80 o-o

60

40

20 0 100

80 2000

REST w ts 30 45 60 75 90 105 120 Workload (W) Workload (W)

175 Data for the group as a whole was analysed using two-way analysis of variance as in the same way as for data obtained during exercise in normal subjects (page 127); VC02, fc, fR, VT, VE, PETC02 and Sa02 were examined by this method. Since the analysis required equal sample sizes in each group, data from the last 5.5 min of exercise, where changes in the pattern of breathing may be expected to be greatest, were analysed. Mean values obtained from this analysis are plotted in Fig 9.2 with values for Fisher’s least significant difference at the P=0.05 level. There was no significant difference in VC02 or fc during exercise following saline and bupivacaine aerosol inhalation confirming that the work performed by the group at each level of exercise was the same for both tests (Fig 9.2). There was also no significant difference in Sa02 between the two tests. No significant differences were found in fR, VT, VE or PETC02 during exercise following bupivacaine aerosol.

Although there was no difference in the VC02 between the two tests for the group as a whole, some individuals showed a difference at some levels of exercise (Fig 9.1); the reason for this is not clear but presumably reflects day-to-day variation in these patients. To ensure that a change in the pattern of breathing was not being obscured by these differences, values for fR, VT and VE with respect to VC02 were examined over the last 5.5 min of exercise by analysis of variance. • « • • No significant differences were found in fR/VC02, VT/VC02 or VE/VC02 after bupivacaine as compared with saline aerosol.

When questioned at the end of exercise, subjects 2, 5 and 6 reported feeling more breathless during exercise after large particle bupivacaine aerosol inhalation, subject 3 felt less breathless, and subjects 1 and 4 noted no difference. The results using the VAS for breathlessness are shown for each subject in Fig 9.1. No significant difference was observed for the group (paired t-test) in the point at which breathlessness was first recorded either with respect to time (mean [SD] after saline 3.8 [4.4] min; after bupivacaine 1.8 [2.4] min) or VE (mean [SD] after saline 28.3 [11.2] 1/min; after bupivacaine 19.8 [2.6] 1/min). No significant difference was found in the VAS scores at the values of VE closest to 40 1/min (mean [SD] after saline 50.7 [25.5] mm; after bupivacaine 61.5 [20.0] mm) or at the maximum values of VE matched to within 2 1/min between the two

176 Fig 9.2 Mean data over the last 5.5 min of maximal incremental exercise in patients with interstitial lung disease after large particle saline (0) and bupivacaine (i) aerosol inhalation. Each point is the mean value for 6 subjects. Fisher’s least significant difference for P=0.05 is shown as a bar and indicates the vertical distance necessary between two points for them to be different at this level of significance. 50 r I Z'—N c 40 o— 8=«= E \ 30 ■ _c "5 k.a) 20 : V-/-Q O' 10 ■ 0 1.5 r

„o— o 1.0 c O— O' 1— > 0.5

0 .0 L 60 f F I 50 \ t c 40 [■ 0^ 8' E t 30 t :8^

LJ 20 [ > i

, ° i 0 L 40 r [I i cn X 35 l E E 30 (■ O----O----Q-----O-----Q----Q___^ ___£ CM f •-•-----. _____| o f (J1— UJ 25 l CL 20 1500 r

1000 h

CM 8^ O 500 L 8= o >

0.0 1.0 2.0 3.0 4.0 5.0 6.0 Time (min) 177 tests (mean [SD] after saline 75.7 [23.5] mm; after bupivacaine 86.2 [14.3] mm). When VAS scores were compared over the last 5.5 min of exercise using analysis of variance, no significant difference was found after large particle bupivacaine as compared with saline aerosol inhalation.

9.4 Discussion

The results of the present study demonstrate that the inhalation of a large particle local anaesthetic aerosol has no effect on the ventilatory response or breathlessness during exercise in patients with interstitial lung disease. In previous studies in this thesis, this aerosol has been shown to block reflex effects on breathing thought to arise from receptors located not only in the airways, but also from those in the lung periphery. In view of the lack of effect of the large particle aerosol in the present study, it was felt to be inappropriate to subject the patients to a further series of exercise tests following inhalation of the small particle aerosol when it has had no effect on the reflex control of breathing in any study previously reported in this thesis.

The addition of oxygen to the inspired air during both exercise tests after aerosol inhalation maintained the patients’ Sa02 at normal levels throughout exercise and thereby prevented a change in the hypoxic drive to breathe which may have made the results of the study difficult to interpret; it did not, however, resolve the rapid, shallow breathing and breathlessness exhibited by the patients on exercise.

Although the results of this study indicate that altered discharge from slowly adapting or rapidly adapting receptors in the airways is not responsible for the tachypnoea of interstitial lung disease, they do not exclude a role for pulmonary C-fibres or other receptors located in the lung periphery since it is possible that the large particle local anaesthetic aerosol did not adequately block afferent impulses from such receptors. Nevertheless, if such fibres were important one might have expected at least a small decrease in fR after large particle aerosol inhalation.

178 It has been reported that blockade of the vagus nerves by injection of lignocaine at the base of the skull or by direct application to the exposed nerves in the neck reduced fR in four of five patients with infiltrative lung disease (Guz et a7., 1970). It is possible that the discrepancy between these results and those of the present study is due to the persistence of residual activity in vagal afferent fibres in patients in the present study. Other workers have reported that the inhalation of a large particle aerosol of 4% lignocaine has no effect on the pattern of breathing at rest in patients with interstitial lung disease (Savoy et al., 1981) although, as previously mentioned (pages 139 and 157), the efficacy of such blockade is questionable.

It is significant to note that in the study of Guz et al. (1970) the respiratory frequency, although reduced in four patients with infiltrations in the lung after bilateral vagal blockade, was still considerably elevated above the normal level; the mean fR before blockade was approximately 43 breaths/min, after vagal blockade it was 27 breaths/min. A similar result was seen in the study of Phillipson et al. (1975b) in dogs with experimental pneumonitis. They found that, although complete, reversible vagal blockade decreased fR and increased VT during exercise, these variables did not return to normal levels. In that study, the ventilatory response to exercise was performed at intervals throughout the development of the pneumonitis; it may be of considerable significance to note that vagal blockade appears to have had the greatest impact on reversing the changes in fR and VT during the early inflammatory stage of the disease as compared to the later granulomatous stage (Phillipson et al., 1975b, Fig. 7). Although these authors did not discuss this aspect of their results, the observations may support the proposal of Cotes et al. (1970) that in the proliferative stage of parenchymal lung disease, respiratory frequency is increased out of proportion to any reduction in vital capacity due to activation of lung receptors by the disease process, while in the fibrotic stage it is due to the alterations in lung mechanics. It is possible that this could explain the lack of effect of the local anaesthetic aerosol in the present study since all but one of the subjects had longstanding restrictive lung disease. However, the ventilatory response to exercise after local anaesthetic

179 aerosol in the other subject (subject 6) was not notably different from the results in the remainder of the group.

Evidence that vagal afferent stimulation can fully account for tachypnoea and breathlessness in at least one condition is provided by a study in one patient reported by Davies at al. (1987). These authors studied the ventilatory response to exercise in a patient who had pulmonary venous obstruction of the right lung following a failed surgical procedure. The patient had normal lung function but became grossly tachypnoeic and breathless, although her tidal volume remained normal, during exercise. Her Sa02 was normal during exercise. The main abnormality was the presence of mild pulmonary hypertension at rest exacerbated by exercise. Following section of the right vagus nerve, her respiratory frequency on exercise fell to within normal limits and her breathlessness was abolished. This study specifically supports Paintal’s original proposal (Paintal, 1969; 1970) that pulmonary congestion, especially on exercise, is responsible for breathlessness due to stimulation of pulmonary C-fibre endings; however it is not possible to know whether the change in breathlessness occurs as a result of the change in the pattern of breathing or vice versa.

What then is the mechanism by which an increase in the stiffness of the lung produces a change in the pattern of breathing in patients with interstitial lung disease? Although a review of the considerable body of work on this topic is outside the scope of this thesis, a short discussion is relevant. It has been proposed that, since the frequency and depth of breathing are normally adopted to minimize respiratory muscle work (Otis et al., 1950) and force (Mead, 1960), patients with interstitial lung disease, when faced with the increased ventilatory demands of exercise, minimize the peak intensity of force (decreased VT) and the duration of force development (increased fR) (Burdon et al., 1983). Killian et al., (1982) have reported that during the addition of an elastic load to breathing in normal subjects (used as a model for the increased elastance of patients with interstitial lung disease) the perceived magnitude of the load is directly related to the intensity and duration of the inspiratory muscle force developed. Evidence that consciousness is involved in determining the pattern of breathing in such situations is provided by

180 studies in which the tachypnoea which follows the addition of an elastic load in conscious subjects is absent during general anaesthesia (Freedman and Campbell, 1970; Margaria et a l., 1973). It seems that consciousness is also involved in determining the pattern of breathing in patients with interstitial lung disease since the tachypnoea seen in such patients when awake is reduced virtually to normal during deep, non-rapid eye movement sleep (Shea et a l., 1989). The nature of the receptors involved in optimizing the breathing pattern is not known. It seems unlikely that they are located solely in the airways since the detection and scaling of elastic loads is unaffected by the inhalation of a local anaesthetic aerosol (Chaudhary and Burki, 1980; Burki et a l., 1983); moreover the results of the present study do not support the idea that airway receptors are essential to the determination of the pattern of breathing in patients with interstitial lung disease on exercise. Although this does not rule out the possibility that the information can come from receptors in the lungs it strongly suggests that it must also come from another source; receptors in the intercostal joints, intercostal muscles, diaphragm, accessory muscles are all possible candidates.

Although Christie (1938) proposed that dyspnoea in patients with lung disease resulted from changes in afferent vagal information, he believed this to come from alterations in the Hering-Breuer reflex. Paintal (1970) subsequently suggested that pulmonary C-fibre endings are an important source of dyspnoeic sensation from the lung and that they are stimulated by changes in interstitial volume or pressure. The results of Guz et al. (1970) have been used as support for this belief since they document that vagal blockade reduces breathlessness in patients with lung infiltrations at rest. However, an examination of the results of their study reveals that two of the patients were not breathless before vagal blockade and another was unable to comment on any change in her sensation after blockade; of the two remaining patients, one reported the abolition of a tight feelingin his chest and the other that she had "ceased to feel her breathing". These results cannot, therefore, be taken as firm evidence that vagal afferent discharge from the lung is directly involved in the genesis of the sensation of breathlessness. The results of the present study do not indicate that breathlessness in patients with interstitial lung disease is altered during exercise by the inhalation of a large

181 particle local anaesthetic aerosol. This, taken with the observation that the pattern of breathing was also unchanged after local anaesthetic aerosol, is consistent with the view that breathlessness is perceived centrally in situations where a reflexly increased drive to breathe causes an increases in motor respiratory output (Chapter 8, page 164); since the reflex drive to breathe does not appear to have been altered by inhalation of the aerosol, the breathlessness would not be expected to alter. Once again it must be mentioned that all but one of the patients in this study had established restrictive lung disease and the situation may be different in the early inflammatory stage; however breathlessness was also unchanged by local anaesthetic aerosol inhalation in the other patient (subject 6).

The results of this study provide no evidence that altered vagal afferent discharge is responsible for the abnormal ventilatory response to exercise or the accompanying sensation of breathlessness in patients with interstitial lung disease. They are consistent with the view that the pattern of breathing in these patients is a behavioural response to minimize a sensation of discomfort arising from receptors in the chest wall or respiratory muscles (and possibly airway and/or lung receptors). However, the results in patients with interstitial lung disease do not exclude the possibility that altered vagal discharge could affect the pattern of breathing and increase breathlessness in processes where a disturbance of the lung parenchyma occurs without a change in lung mechanics.

182 CHAPTER 10: CONCLUSIONS AND PLANS FOR FUTURE WORK

10.1 Conclusions

The use of local anaesthetic aerosols in the studies described in this thesis has provided a method by which safe, reversible blockade of airway and lung receptors can be achieved and has produced a good deal of information concerning the part played by these receptors in the control of breathing in man.

Successes and failu res with local anaesthetic blockade

The results of the studies in the dog demonstrate that complete, reversible abolition of reflexes believed to be mediated by slowly adapting stretch receptors, rapidly adapting stretch receptors and pulmonary C-fibre endings, can be achieved by the inhalation of a large particle local anaesthetic aerosol. Reliable blockade of the inflation reflex and the pulmonary chemoreflex in these studies, in comparison to that reported in previous work, is believed to reflect the use of an aerosol generator with a high mass output. The duration of aerosol inhalation required for the blockade of the different reflexes and the order in which they reappear indicate that rapidly adapting receptors were blocked most readily and pulmonary C-fibres least readily. The return of the three reflexes at different times confirms that different receptors are responsible for their generation; the return of the inflation reflex at a time when the cough reflex was still blocked is consistent with a superficial location in the airway wall of the receptors responsible for cough during mechanical stimulation of the airway. In contrast, inhalation of a small particle local anaesthetic aerosol in the dog had no effect on any of these reflexes.

The rationale for the development and use of two local anaesthetic aerosols of different particle size in the studies performed for this thesis was that their inhalation may allow blockade, in a selective fashion, of afferent activity from receptors located in the lung periphery (pulmonary C-fibre endings) and in the airways (rapidly adapting and slowly adapting stretch receptors). Implicit in this, is the proposal that C-fibre endings in the lung periphery can mediate

183 changes in the pattern of breathing in response to a variety of natural and experimental stimuli. However, the studies in dogs showed that a large particle local anaesthetic aerosol (depositing predominantly in the larger airways) was able to block the pulmonary chemoreflex to capsaicin injection while a small particle local anaesthetic aerosol (depositing in the lung periphery) was not.

Perhaps the most obvious interpretation of these results is that the nerve endings responsible for the pulmonary chemoreflex are located, not in the lung periphery, but in the larger airways. Such a conclusion would imply that the conventional distinction between 'bronchial’ and 'pulmonary’ C-fibre endings may not be as rigid as has been previously believed. The results of the studies in man are also consistent with this interpretation; the burning sensation in the chest following capsaicin injection, believed to be due to C-fibre stimulation, was abolished by the large particle, but not the small particle, local anaesthetic aerosol.

However, the results of these studies could also be explained in terms of the more conventional view of a peripheral location of pulmonary C- fibre endings. Thus, although the radioisotope scans in man clearly show that the majority of the large particle aerosol deposits in the larger airways, they also indicate that some aerosol deposits in the lung periphery. The studies in the dog demonstrate that, while the inflation reflex and the cough reflex are abolished by a 10 min inhalation of the large particle local anaesthetic aerosol, the pulmonary chemoreflex to capsaicin injection requires a longer period of aerosol administration for its complete blockade and returns before the other reflexes. The most likely mechanism to account for this would be that sufficient large particle local anaesthetic aerosol deposits in the smaller airways to block afferent fibres from the lung periphery running in the airway wall. The longer duration of aerosol administration required to abolish the pulmonary chemoreflex and its return before the other reflexes, would reflect the lower concentration of local anaesthetic in the lung periphery. Another explanation is that local anaesthetic aerosol depositing in the large airways diffuses through the airway wall and blocks afferent C-fibres running in nerve bundles. The greater difficulty in blocking the pulmonary chemoreflex would indicate a lower concentration of local

184 anaesthetic in the nerve bundles than in slowly and rapidly adapting stretch receptors located more superficially in the airway wall.

If the conventional view of a peripheral location for pulmonary C- fibre endings is true, the failure of the small particle aerosol to block afferent information from them would indicate a low concentration of drug deposited in the lung periphery. Although the mass of aerosol deposited with the small particle aerosol in man was of the same order as that achieved with the large particle aerosol (Chapter 2), the surface area of the lung periphery is much greater than that of the large airways; the alveolar surface area in man has been estimated to be 70-100 m2 (Weibel, 1963; Gehr et a l., 1978) while that of the airways, from trachea to respiratory bronchioles inclusive, is calculated to be 0.2-0.6 m2 (Weibel, 1963; Horsfield and Cumming, 1968; Horsfield et a 7., 1971). The concentration at the alveolar surface produced by the small particle aerosol would therefore, be much lower than that achieved on the airway surface by the large particle aerosol. (As an exercise, the possible molar concentrations of local anaesthetic at the epithelial surface in the two situations have been calculated and are shown in Appendix 2. It must be stressed that many unfounded assumptions must necessarily be made in this process and it is performed purely as an illustration of the two situations).

In considering these results, it is felt that an absolute distinction between C-fibre endings located in alveoli and smaller airways is perhaps too rigid (and therefore unhelpful) since both may be rapidly accessible to chemicals injected into the right heart.

Involvement o f vagal receptors in the control o f breathing in normal man

The studies in man at rest indicate that afferent vagal information from the lung is not important in determining the mean level of ventilation at rest although it is involved in minimizing breath-to- breath fluctuations in tidal volume; it is suggested that slowly adapting pulmonary stretch receptors are responsible for this. However, when tidal volume is increased by a natural stimulus such as exercise, afferent information, once again believed to come from

185 slowly adapting stretch receptors, plays a small part in limiting inspiration.

Although it does not seem to be important in the control of breathing under normal circumstances, the presence of C-fibre activity in the lungs of man is indicated by the occurrence of a burning sensation in the chest following intravenous injection of capsaicin. Confirmation that this is mediated by receptors in the lung is provided by its abolition by prior inhalation of a local anaesthetic aerosol. Since the sensation appears within 4 seconds of injection of capsaicin into the superior vena cava, it is thought to be due to C-fibre endings in the lung periphery. No evidence was found for the existence of the pulmonary chemoreflex at the concentrations of capsaicin which could be used. However, in one subject, paroxysmal coughing occurred within 4 seconds of injection of the highest dose of capsaicin used; this is taken as evidence that C-fibres in the lung can be involved in the genesis of cough.

The increased ventilatory response to carbon dioxide rebreathing after inhalation of a large particle local anaesthetic aerosol in normal subjects has been confirmed; the discovery of a similar result in laryngectomized subjects indicates that the removal of an inhibitory influence on breathing from the larynx is not responsible for this phenomenon. The mechanism which is responsible may involve the removal of an inhibitory influence on breathing from receptors in the larger airways while preserving activity from C-fibre endings in the lung periphery. The results of these studies therefore indicate that altering pulmonary receptor discharge when ventilation is stimulated by hypercapnia, can have a marked effect on the control of breathing.

The results of all studies in normal subjects are consistent with the view that breathlessness is perceived in situations where a reflexely increased drive to breathe causes an increase in motor respiratory activity.

186 Involvement o f vagal receptors in the control o f breathing in patients with lung disease

The study performed in patients with interstitial lung disease provides no evidence that an altered afferent vagal discharge from lung receptors is responsible for the abnormal ventilatory response or the increased sensation of breathlessness experienced by these patients during exercise. Rather, the results are compatible with the proposal that the pattern of breathing in these patients is a behavioural response to minimize a sensation of discomfort arising from receptors in the chest wall or respiratory muscles. However, since the majority of patients studied had established restrictive lung disease, these observations cannot be used to exclude the involvement of afferent vagal information from the lung in determining the pattern of breathing and in producing breathlessness in processes where a disturbance of the lung parenchyma occurs with active inflammation.

10.2 Proposals for future studies

Aerosols

In recent years there has been dramatic increase of interest in, and therefore knowledge about, aerosols as they relate to the lungs. Some of this interest has been aimed at selective deposition of aerosols at different levels of the tracheobronchial tree and has recently provided an inhalation technique which considerably enhances central airway deposition of aerosol (Mortensen et a l., 1988; Bennett and Ilowite, 1989). With respect to methods for increasing peripheral deposition, no improvements appear to have been made over those used in this thesis. However, interest in achieving this is high, largely because of the increased desire to deliver therapeutic agents to the lung periphery, and further development seems probable.

Although no significant improvement appears to be on the horizon with respect to better local anaesthetics with which to achieve neural blockade, an unrelated agent has emerged which has considerable potential in this area. Ruthenium Red is believed to provide selective blockade of capsaicin-sensitive neurons (Maggi et al., 1988;

187 Amann and Lembeck, 1989) but does not yet appear to have been used to study the control of breathing.

With respect to studies involving the use of aerosols for purposes other than in the study of the control of breathing, I am certain that aerosols will be used with increasing frequency to target pharmacologically active agents at specific sites in the lung. These will not only include clinically useful drugs such as pentamidine (recently delivered to the alveoli in immunosupressed patients to prevent lung infection; Montgomery et a 7., 1988) but will also include investigative agents such as cytokines (Debs et a7., 1988) which are involved in cell-cell interactions in the lung.

Control of breathing

The results of this work indicate that afferent information from the lung is not essential to the control of breathing in man either at rest or during exercise. One of the most obvious teleological reasons for this is that man has a greater number of behavioural requirements which involve breathing than do lower mammals; the most important of these must be speech. This has crucial consequences for studies examining reflex pathways in man; unless the reflex activation of the respiratory muscles is extremely intense, it can be overcome by volitional control. An example of a high degree of reflex control of breathing is provided by the cough reflex; this usually produces an extremely high level of respiratory muscle activation, which can only sometimes be overcome volitionally (for example by the desire to speak). An example of a low degree of reflex control is provided by the Hering-Breuer inflation reflex; although it has been reported that this can be demonstrated in conscious respiratory physiologists (Christiansen and Haldane, 1914) it is not possible to do so in conscious naive subjects (Widdicombe, 1961; Hamilton et a l ., 1988). It is interesting to note that even such stimuli as exercise (Jones, 1988b) and hypercapnia (Murphy et a l., 1990) are now considered in some quarters to have a behavioural component.

In the light of such considerations it now seems to me untenable to perform any studies on the control of breathing in man without at least considering the relative contributions of behavioural and reflex

188 drives to breathe. The experimental situations in which the relative contributions of these drives could be examined are those in which either a reflex drive or a behavioural drive, is at a maximum or at a minimum.

Whereas local anaesthesia provides a method by which reflex influences on breathing can be minimized, the main natural condition under which behavioural control is minimal is that of sleep. Indeed it is for precisely this reason that this state has been used in recent years to study the control of breathing in man; the Hering-Breuer inflation reflex can, for example, be demonstrated in man during deep non-rapid eye movement sleep (Hamilton et a 7., 1988). Once the existence of a reflex effect on breathing has been demonstrated during sleep, its abolition, also during sleep, by a local anaesthetic aerosol would confirm that the receptors involved were in the lung; this remains to be done.

By using these two techniques to complement each other, the relative roles of reflex and behavioural drives to breathe may be determined for a given situation. For example the altered pattern of breathing in patients with lung disease may be due to changes in the reflex drive to breathe or may be a behavioural response. The study of such patients in a situation which reduces one of these may allow the underlying cause to be determined. Thus, if the pattern of breathing is returned toward normal during sleep (as in the case of patients with interstitial lung, disease; Shea et a7., 1989), a behavioural response is implicated; if, on the other hand, the pattern of breathing is ameliorated by a local anaesthetic aerosol, altered discharge from lung receptors would be expected to be responsible.

Obviously interventions other than the use of local anaesthetic aerosols and sleep would be required to provide situations in which a reflex drive or behavioural drive to breathe was maximal. Although I am not aware of any situations which may be regarded as maximal in this context, experimentally induced cough may have considerable reflex effects on breathing and magnetic stimulation of the cortex is currently in use to study the behavioural drive to breathe.

189 APPENDIX 1

Example of the calculation of mass of aerosol bupivacaine deposited in the lungs using an individual’s perfusion (Q) scan as a calibrating phantom.

1) To calculate activity of Tc in the Q scan

Activity in syringe when Tc drawn up (time 1702) = 1.63 mCi Residue in syringe when Q scan done (time 1756) = 0.044 mCi Time difference = 54 min £ life of Tc = 6.00 hr (Lederer et a 7., 1967) so decay factor for 54 min = 0.90127 Therefore activity of Tc-labelled albumin injected (corrected to time of Q scan) = (1.63 X 0.90127) - 0.044 = 1.425 mCi

2) To calculate calibration factor in this individual

Gamma counts from a 2 min Q scan after injection of Tc-labelled albumin = 221690 counts Therefore calibrating factor = 221690 / 1.425 = 155572 counts/mCi

4) To calculate activity of Tc in the aerosol scan

Cannot use counts in left lung because of ’contamination’ by counts from the stomach so use right lung. Counts in right lung from 2 min scan after aerosol inhalation = 15399 counts

Therefore Tc activity = 15399 / 155572 = 0.099 mCi

From the Q scan we see that the counts from the right lung comprise 48.6% of the total counts. Therefore Tc activity in both lungs = 0.099 X 100 / 48.6 = 0.204 mCi

5) To convert activity of Tc to mass of bupivacaine

Activity of Tc in bupivacaine solution to be nebulized at time Tc was drawn up = 1.75 mCi/ml Time difference to aerosol scan = 54 min Decay factor = 0.90127

Therefore activity in bupivacaine solution at time of scan = 1.75 X 0.90127 = 1.58 mCi/ml

Bupivacaine concentration in solution to be nebulized = 45.5 mg/ml So specific activity of solution = 1.58 / 45.5 = 0.0347 mCi/mg

Therefore mass of bupivacaine in both lungs after 2 min of aerosol inhalation = 5.87 mg So from a 10 min aerosol inhalation the mass of bupivacaine deposited in both lungs = 29.3 mg

190 APPENDIX 2

Theoretical exercise to estimate the concentration of local anaesthetic in the epithelial and alveolar lining fluid following inhalation of large and small particle aerosol in man.

1) Large particle aerosol

Assumptions

Total airway surface area = 0.6 m2 (Weibel, 1963) Thickness of epithelial lining fluid = 2 pm Mass of bupivacaine deposited = 28 mg (Chapter 2) Volume of aerosol deposited = 0.56 ml (5% aqueous solution) Molecular Weight of bupivacaine HC1 = 324.9

Calculations

Volume of epithelial lining fluid = 0.6 x 2 x 10‘6 x 103 litres = 1.2 x 10“3 litres Molar amount of bupivacaine deposited = 28 / 342.9 mmoles = 0.081 mmoles Concentration on the epithelium = 0.081 / ((1.2+0.56)x10"3) mM = 67.5 mM

2) Small particle aerosol

Assumptions

Total alveolar surface area = 80 m2 (Weibel, 1963) Thickness of alveolar lining fluid = 0.5 pm Mass of bupivacaine deposited = 21 mg (Chapter 2) Volume of aerosol deposited = 0.42 ml (5% aqueous solution)

Calculations

Volume of alveolar lining fluid = 80 x 0.5 x 10“6 x 103 litres = 40 x 10-3 litres Molar amount of bupivacaine deposited = 21 / 342.9 mmoles = 0.061 mmoles Concentration on the epithelium = 0.061 / ((40+0.42)x10“3) mM = 1.5 mM

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217 PUBLISHED WORK WITH RESULTS IN COMMON WITH THIS THESIS

GUZ, A., HAMILTON, R.D. & WINNING, A.J. (1985). The effects of local anaesthetic aerosols of different particle size on the response to C02 rebreathing in man. Journal of Physiology (London). 358, 94P. HAMILTON, R.D., WINNING, A.J. & GUZ, A. (1985). Maximal exercise in normal man - effects of inhaled local anaesthetic aerosol depositing at alveolar level. C lin ic a l Science. 68, Suppl 11., 46P. HAMILTON, R.D. & WINNING, A.J. (1987). Dry-particle aerosol generation with a fluidized bed: a method of delivering drug to the lung periphery. Journal of Physiology (London). 387, 25P. HAMILTON, R.D., WINNING, A.J. & GUZ, A. (1987). Blockade of 'alveolar’ and airway reflexes by local anaesthetic aerosol in dogs. Respiration Physiology. 67, 159-170. HAMILTON, R.D., WINNING, A.J., PERRY, A. & GUZ, A. (1987). Aerosol anaesthesia increases hypercapnic ventilation and breathlessness in laryngectomized humans. Journal of Applied Physiology. 63, 2286-2292. WINNING, A .J ., HAMILTON, R.D., SHEA, S.A. & GUZ, A. (1985). Vagal influence on the breathing pattern of resting normal subjects. Thorax. 40, 234. WINNING, A.J., HAMILTON, R.D., SHEA, S.A., KNOTT, C. & GUZ, A. (1985). The effect of airway anaesthesia on the control of breathing and the sensation of breathlessness in man. Clinical Science. 68, 215-225. WINNING, A .J., HAMILTON, R.D., SHEA, S.A. & GUZ, A. (1986). Respiratory and cardiovascular effects of central and peripheral intravenous injections of capsaicin in man: evidence for pulmonary chemosensitivity. Clinical Science. 71, 519-526. WINNING, A.J., HAMILTON, R.D. & GUZ, A. (1988). Ventilation and breathlessness on maximal exercise in patients with interstitial lung disease after local anaesthetic aerosol inhalation. Clinical Science. 74, 275-281. WINNING, A.J., HAMILTON, R.D., SHEA, S.A. & GUZ, A. (1985). Vagal influence on the breathing pattern of resting normal subjects. Thorax. 40, 234.

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