Mechanisms and clinical implications of the neuroendocrine … · mechanism by which the observed...

Mechanisms and clinical implications of the

neuroendocrine response to a novel carbon dioxide

stressor in man

Dr Joey Michael Kaye MBBS FRACP

This thesis is presented for the degree of Doctor of

Philosophy

The University of Western Australia

School of Medicine and Pharmacology

2005

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DEDICATION

To my wife - CMK

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ABSTRACT

Maintenance of normal health requires an intact stress system capable of

mounting the metabolic, autonomic, behavioural and motor responses required

for coping with or avoiding physiological and pathological challenges. The

neuroendocrine component of this response principally involves the

hypothalamic-pituitary-adrenal (HPA) and sympatho-adrenomedullary (SAM)

axes. Impaired regulation of these axes has been implicated in the pathogenesis

and expression of numerous disease states, however, it has proved very difficult

to reproducibly activate the HPA and SAM axes and no single test exists that can

reliably and safely be used to study these systems in man.

Carbon dioxide (CO2) is the principal regulator of respiration, acid-base balance

and behavioural-state arousal in humans. Paradigms of CO2 inhalation have been

used in psychiatric research to investigate panic and anxiety disorders, but

evaluation of other components of the stress response to CO2 has not previously

been performed. I hypothesised that a single breath of 35% CO2 would be a

simple and reliable tool for the evaluation of the stress response in humans. A

single breath of four doses of CO2 (5%, 25%, 35% and 50%) was administered to

9 healthy volunteers in a randomised, single blind fashion. Subjective symptoms

of anxiety increased in a dose-dependent manner. Inhalation of a single breath of

35% CO2 stimulated significant ACTH (p = 0.006), noradrenaline (p < 0.0001),

cortisol (p = 0.02) and prolactin (p = 0.002) release. It also provoked an acute

pressor response and an associated bradycardia (p < 0.0001 for both). No

significant habituation of psychological, HPA or cardiovascular responses was

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seen when this dose was repeated after one week (n = 10) or 6 months (n = 5). It

was apparent that a single breath of 35% CO2 reliably and safely produced SAM

and HPA axis activation and further studies were then undertaken to assess the

mechanism by which the observed responses occurred and its potential clinical

implications.

Administration of naltrexone (an opiate antagonist) to 10 normal volunteers

disinhibited the HPA axis (p < 0.0004), whilst administration of metyrapone (a

cortisol synthesis inhibitor) significantly reduced baseline cortisol (p < 0.03)

levels. However, this alteration in HPA axis activity had no effect on either

cardiovascular or psychological responses. Further, in a study of 8 breastfeeding

mothers (a state associated with physiological suppression of the HPA axis)

suckling significantly reduced plasma cortisol levels compared with control (p =

0.002) and bottle-feeders (p = 0.003). Despite this cortisol, systolic blood

pressure (SBP), heart rate and psychological responses to 35% CO2 were not

affected.

Continuous cardiovascular monitoring of subjects immediately following a single

breath of 35% CO2 identified bradycardia as the first response followed by an

acute pressor response and an increase in noradrenaline. In a study of 20 male

diabetic subjects, 11 with autonomic neuropathy (with early parasympathetic

dysfunction), the autonomic neuropathy subjects failed to demonstrate any CO2-

induced bradycardia (p < 0.0001) suggesting an early effect of autonomic

neuropathy on cardiac vagal innervation. Further studies of patients with

abnormalities of central CO2 chemoreceptor activation and integration identified

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dissociation between the sympathetic (pressor response) and parasympathetic

(bradycardic response) components of the response.

The role of central autonomic centres in determining the overall response to CO2

was addressed in a study of 9 patients with multiple systems atrophy (MSA) and

9 with pure autonomic failure (PAF) (central vs peripheral with autonomic

failure). Resting noradrenaline was significantly lower in PAF (p < 0.0001).

Following the CO2 challenge SBP increased in both PAF (p < 0.001) and MSA

(p = 0.002), although this increase was significantly blunted and delayed

compared with controls. Cortisol responses were lower in MSA patients who also

experienced fewer somatic symptoms of fear. Finally, control subjects showed a

reduction in skin blood flow that was blunted in MSA subjects, whilst PAF

subjects showed a striking increase in skin blood flow (p = 0.006). This increase

was due to non-neurally mediated vasodilation produced by CO2 that is normally

masked by the centrally-mediated vasoconstriction. In addition to providing

insight into the mechanism of CO2 mediated physiological changes this

difference may prove clinically important in differentiating the autonomic failure

syndromes.

In summary, it appears that the SAM response to the 35% CO2 challenge is

driven by activation of brainstem autonomic centres with secondary activation of

the HPA axis. The vagal response occurs independently whilst the psychological

response occurs probably as a result of both direct activation of limbic centres by

CO2 as well as indirect activation from brainstem sympathetic systems.

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The single breath 35% CO2 inhalation test has shown itself to be a safe, reliable

and reproducible means of generating a stress response in humans and should

prove a useful addition to currently available laboratory tests of the human stress

response.

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION……………………………………………...1

1.1 The neuroendocrine stress response in man…………………………..….2

1.1.1 Clinical importance………………………………………………...2

1.1.2 Background………………………………………………………...7

1.1.2.1 The HPA axis……………………………………………….12

1.1.2.2 AVP………………………………………………………...16

1.1.2.3 The sympatho-neural and sympatho-adrenomedullary

axis………………………………………………………….17

1.1.2.4 Prolactin…………………………………………………….19

1.1.2.5 Opiates…………………………………...…………………20

1.1.2.6 Serotonin (5-HT)……………………………………...……21

1.1.2.7 The growth, thyroid and reproductive axes…………...……22

1.1.2.8 Other neurohormonal systems…………….………………..23

1.2. Investigating the stress response………………………………………...25

1.2.1. Currently available tools and their limitations………………...….25

1.2.1.1. Psychological challenges…………………………………...26

1.2.1.2. Physiological challenges……………………………………28

1.2.1.3. Pharmacological challenges………………………………...30

1.2.2. CO2 as a potential neuroendocrine stressor……………………….33

1.3. Summary, aims and hypotheses………….………………………...……35

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CHAPTER 2: METHODOLOGY…………………………………..………..37

2.1 The single breath 35% CO2 model………………………………...……38

2.1.1 Design…………………………………………………………….42

2.1.2 Procedure…………………………………………………………45

2.1.3 Inclusion/Exclusion criteria………………………………………47

2.1.4 Monitoring…………………………….………………………….49

2.1.4.1 Ventilation………………………………………………….49

2.1.4.2 Psychology…………………………………………………50

2.1.4.3 Cardiovascular physiology…………………………………53

2.1.4.4 Biochemistry……………………………………………….55

2.2 Plasma catecholamine HPLC…………………………………………..63

2.3 Salivary amylase……………………………………………………….73

CHAPTER 3: THE 35% CO2 MODEL: INITIAL DESCRIPTION………78

3.1 Rationale for the use of CO2 as a neuroendocrine stressor……………..79

3.1.1 CO2 physiology…………………………………………………..80

3.1.2 CO2 as a psychological stressor…………………………………..81

3.1.3 CO2 as a cardiovascular stressor………………………………….83

3.1.4 CO2 as a neurohormonal stressor…………………………………84

3.2 Experimental plan……………………………………………………….86

3.3 Dose response study…………………………………………………….87

3.3.1 Introduction and methods…………………………………………87

3.3.2 Results…………………………………………………………….88

3.3.3 Discussion…………………………………………...……………97

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3.4 Reproducibility studies………………………………………………...103

3.4.1 Introduction and methods………………………………………..103

3.4.2 Results…………………………………………………………...104

3.4.3 Discussion…………………………………………...…………..112

3.5 Summary………………………………………………………………113

3.6 Conclusion…………………………………………………………….119

CHAPTER 4: THE 35% CO2 MODEL: MECHANISMS UNDERLYING

THE NEUROENDOCRINE RESPOSNE………………………………….121

4.1 Introduction……………………………………………………...…….122

4.2 Experimental plan……………………………………………………...126

4.3 Central neurotransmitter study………………………...………………127

4.3.1 Introduction and methods…………….………………………..127

4.3.2 Results…………………………………………………………129

4.3.3 Discussion…………………………………………...…………137

4.4 Mineralocorticoid/glucocorticoid study……………………………….143


4.4.2 Results…………………………………………………………146

4.4.3 Discussion…………………………………………...…………152

4.5 Lactation study……………………………………...…………………154


4.5.2 Results…………………………………………………………158

4.5.3 Discussion…………………………………………...…………165

4.6 Peripheral versus central autonomic nervous system effects………….167


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4.6.2 Results…………………………………………………………168

4.6.3 Discussion…………………………………………...…………172

4.7 Conclusion……………………………………………………………..176

CHAPTER 5: THE 35% CO2 MODEL: RESPONSES IN SPECIFIC

SUBPOPULATIONS – FURTHER MECHANISMS AND POTENTIAL

CLINICAL RELEVANCE…………………………………………………180

5.1 The role of the HPA axis …………………………………...…………181

5.2 Addison’s disease……………………………………………...………185


5.2.2 Results…………………………………………………………188

5.2.3 Discussion…………………………………………...…………193

5.3 The role of the autonomic nervous system…………………………….196

5.4 Experimental plan……………………………………………………...199

5.5 Diabetic autonomic neuropathy………………………………………..200


5.5.2 Results…………………………………………………………206

5.5.3 Discussion…………………………………………...…………212

5.6 Chronic autonomic failure syndromes…………………………………216


5.6.2 Results…………………………………………………………223

5.6.3 Discussion…………………………………………...…………235

5.7 Conclusion……………………………………………………………..241

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CHAPTER 6: SUMMARY, CONCLUSION AND FUTURE

DIRECTIONS………………………………………………………………...245

6.1 Summary …………………………………………………...………….246

6.2 Conclusions and future directions……………………………………..252

CHAPTER 7: REFERENCES………………………………………………254

APPENDICES……………………………………………………………..….273

Appendix 1 Visual Analogue Scale

Appendix 2 Somatic Symptom Questionnaire

Appendix 3 Edinburgh Post-Natal Depression Score Questionnaire

Appendix 4 Protocol for the extraction of catecholamines #1




LIST OF TABLES

Table 3.01 Dose response study - baseline characteristics……………….…91

Table 3.02 Diurnal variability study - baseline characteristics…………….109

Table 4.01 Neurotransmitter study - baseline characteristics…………...…131

Table 4.02 Lactation study - baseline characteristics………………….…..160

Table 5.01 Addison’s disease study - baseline characteristics………….…190

Table 5.02 Diabetic autonomic neuropathy study –

baseline characteristics……………………………………...…208

Table 5.03 Autonomic failure - baseline characteristics………………..…227

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Table 5.04 Autonomic failure – hormonal and cardiovascular

responses………………………………………………….……229

Table 5.05 Autonomic failure – Skin blood flow response…………….….232

LIST OF FIGURES

Figure 2.01 Schematic of the breathing circuit used to deliver

the CO2 breath………………………………………………..…44

Figure 2.02 Arrangement for patient monitoring……………………….……58

Figure 2.03 Arrangement for patient monitoring…………………………….59

Figure 2.04 Arrangement for patient monitoring…………………………….60

Figure 2.05 Arrangement for CO2 delivery………………………………….61

Figure 2.06 Arrangement for CO2 delivery………………………………….62

Figure 2.07 Chromatogram – unextracted standard………………...….…….67

Figure 2.08 Chromatogram – successful extraction………………………….68

Figure 2.09 Chromatogram – successful extraction………………………….69

Figure 2.10 Chromatogram – poor recovery……...………………………….70

Figure 2.11 Chromatogram – unsuccessful extraction……………………….71

Figure 2.12 Chromatogram – unsuccessful extraction……………………….72

Figure 3.01 Dose response study – psychological responses………………...92

Figure 3.02 Dose response study – cardiovascular responses…………...…..93

Figure 3.03 Dose response study – hormonal responses……………...……..94

Figure 3.04 Dose response study – hormonal responses……………...……..95

Figure 3.05 Dose response study – correlations……………………………..96

Figure 3.06 Repeatability study – responses at 1 week and at 6 months...…106

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Figure 3.07 Repeatability study – cardiovascular responses at 1 week

and at 6 months……………………………...…………………107

Figure 3.08 Salivary amylase response and correlations…………...………108

Figure 3.09 Diurnal variability study – morning

versus evening responses………………………………..……..110

Figure 3.10 Diurnal variability study – cardiovascular responses……...…..111

Figure 4.01 Overview of the neuroanatomical stress pathways……………125

Figure 4.02 Neurotransmitter study – baseline cortisol and prolactin……..132

Figure 4.03 Neurotransmitter study – individual hormonal responses….….133

Figure 4.04 Neurotransmitter study – peak hormonal responses………..….134

Figure 4.05 Neurotransmitter study – cardiovascular responses………..….135

Figure 4.06 Neurotransmitter study – psychological responses………...….136

Figure 4.07 Glucocorticoid/mineralocorticoid study – baseline cortisol.….148

Figure 4.08 Glucocorticoid/mineralocorticoid study – cortisol response….149

Figure 4.09 Glucocorticoid/mineralocorticoid study –

psychological responses…………………………………….…150

Figure 4.10 Glucocorticoid/mineralocorticoid study - cardiovascular

responses…………………………………………………….…151

Figure 4.11 Lactation study – baseline and peak cortisol response………...161

Figure 4.12 Lactation study – baseline and peak prolactin response……….162

Figure 4.13 Lactation study – cardiovascular responses……………………163

Figure 4.14 Lactation study – psychological responses……………….……164

Figure 4.15 Peripheral vs central neuropathy –

cortisol and prolactin response………………………..……….169

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Figure 4.16 Peripheral vs central neuropathy –

cardiovascular responses…………………………………..…..170

Figure 4.17 Peripheral vs central neuropathy - psychological responses…..171

Figure 4.18 Principal components of the CO2 mediated stress response…...179

Figure 5.01 Addison’s disease study - cardiovascular responses…………..191

Figure 5.02 Addison’s disease study - psychological responses………..….192

Figure 5.03 Diabetic autonomic neuropathy –

cortisol and prolactin response………………….……………..209

Figure 5.04 Diabetic autonomic neuropathy - cardiovascular responses…..210

Figure 5.05 Diabetic autonomic neuropathy - psychological responses…...211

Figure 5.06 Calculation for change in skin blood flow…………………….222

Figure 5.07 Autonomic failure - cardiovascular responses…………..…….228

Figure 5.08 Autonomic failure – hormone responses………………..…….230

Figure 5.09 Autonomic failure - psychological responses…………...…….231

Figure 5.10 Autonomic failure – skin blood flow response from single

subjects…………..…………………………………………….233

Figure 5.11 Autonomic failure – mean skin blood flow response………….234

Figure 5.12 Principal components of the CO2 mediated stress response -

revised………………………………………………………….244

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ACKNOWLEDGMENTS

The advice, support, wisdom and encouragement of many people has contributed

to the success of this project. Principally, Professor Stafford Lightman, for

giving me the opportunity to work and study alongside the extraordinary

members of the Henry Wellcome Laboratories for Integrative Neuroscience and

Endocrinology as well as the wonderful staff and patients of the Bristol Royal

Infirmary. His patience, encouragement and never-failing enthusiasm for this

project has ensured its success and has provided me with an appreciation of

research performed at the highest level. I am deeply grateful for all his support

and direction, but particularly for showing me that things are never quite so

difficult or frustrating if approached with just the right amount of humour, wit

and intelligence.

To Professor Peter Leedman for encouraging me to do this in the first place. To

Dr Chris Lowry, Phil Johnson, Jacob Hollis, David Knight and all the members

of the Integrative Neuroscience and Endocrinology laboratories for making me

feel at home and for their guidance, patience and direction particularly when not

everything went to plan. To Moira Hunt who gave so much of her time and

experience to assisting me with the clinical experiments.

I would also like to acknowledge the generous support of many members of the

University of Bristol and the Bristol Royal Infirmary whose time and expertise

has contributed greatly to this project.

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Professor David Nutt and Jayne Bailey for sharing their expertise and experience

with their CO2 model. Dr Adrian Kendrick and Fiona Buchanan for all their time

and assistance in modifying and optimising the CO2 delivery system. Professor

Peter Soothill and Dr Roger Corrall for their willingness to share their knowledge

and expertise. The nursing staff of the Antenatal Day Assessment Unit at St

Michael’s Hospital and the nursing staff of the Diabetes Outpatient Clinic at the

BRI for their advice, patience and good humour. Dr Rob Andrews who helped in

the design, organise and run the studies in patients with Addison’s disease.

To the staff and patients of the Autonomic Unit at the National Hospital for

Neurology and Neurosurgery, Queen Square, London, including Professor Chris

Mathias, Katherine Bleasedale-Barr and Laura Watson for your willingness and

support in expanding our work into your midst. I’m particularly grateful to Dr

Tim Young for his thoughtfulness and unselfish support of this project. For

dedicating himself to its success and for giving up so much of his time and

energy.

This project was generously supported by the University of Western Australia

through a FA Hadley Travelling Fellowship, the Neuroendocrinology Charitable

Trust, UK and the Charitable Trustees of the United Bristol Healthcare Trust.

To all the people of Bristol, who welcomed us, supported us and nurtured us,

thank you for giving us some of the most enjoyable years of our lives.

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Finally and most importantly to my wife, Carolyn, whose unerring love and

dedication through some of our very best and very worst times, has given my life

a sense of purpose and meaning and who, with our children, has made it all

worthwhile.

Joey Kaye

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PERSONAL CONTRIBUTION OF THE CANDIDATE The candidate was involved in the discussions and decision-making with the

other principal investigators regarding the development of the concept of using

inhaled CO2 as a challenge for the investigation of the human stress response.

The candidate undertook the design, development and modification of the

methodology for the administration of the CO2 as well as the protocols for

monitoring and recording of responses. With regard the test procedure, the

candidate was responsible for:

• Administration of all of the CO2 challenges (apart from the study performed

at the National Hospital for Neurology and Neurosurgery where Dr Tim

Young performed some challenges after instruction by the candidate).

• Collection, alliquoting and storage of all biochemical samples.

• Collection and recording of all physiological responses.

• Administration and recording of all psychological response questionnaires.

• Quality control, identifying and documenting any adverse responses for

safety and tolerability.

With regards the individual studies, the candidate was responsible for:

• The design of each study.

• All patient/subject recruitment (assisted by Dr Tim Young for the autonomic

neuropathy study and Dr Rob Andrews for the Addison’s study).

• Administration of all patient/subject information sheets and consent forms.

• Storage and dispensing all administered medication/placebo capsules.

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• All data collection, storage and statistical analysis with advice from Dr

Valerie Burke, statistician with the School of Medicine and Pharmacology,

UWA.

• The administration and interpretation of all the autonomic function studies in

the diabetic autonomic neuropathy study.

With regard biochemical analyses, the candidate was responsible for:

• Establishing and performing a high performance liquid chromatography

(HPLC) assay for plasma catecholamines.

• Performing all ACTH radioimmunoassays for the dose response study.

• Storing and arranging the analysis of other blood and saliva samples.

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ABBREVIATIONS

5-HT Serotonin ACEI Angiotensin Converting Enzyme Inhibitor ACTH Adrenocorticotropic Hormone AN Autonomic Neuropathy Ang II Antagonist Angiotensin II Receptor Antagonist ANOVA Analysis of Variance AVP Arginine Vasopressin BF Breast Feeding BO Bottle Feeding BP Blood Pressure C Control CCHS Congenital Central Hypoventilation Syndrome CCK-4 Cholecystokinin 4 CnA Central Nucleus of the Amygdala CNS Central Nervous System CO Cardiac Output CO2 Carbon Dioxide CRH Corticotropin Releasing Hormone CRH-R1 Corticotropin Releasing Hormone Receptor 1 CRH-R2 Corticotropin Releasing Hormone Receptor 2 CV Co-efficient of Variation DAN Diabetic Autonomic Neuropathy DBP Diastolic Blood Pressure DHBA 3,4 dihydroxybezylamine hydrogen bromide DMNX Dorsal Motor Nucleus of the Vagus E/I ratio Expiration / Inspiration ratio ECG Electrocardiogram EDTA Ethylenediaminetetraacetic Acid EGTA Ethylene Glycol-bis N,N,N,N-Tetraacetic Acid ELISA Enzyme Linked Immunoabsorbent Assay EPDS Edinburgh Post-natal Depression Score FSH Follicle Stimulating Hormone GABA Gamma Aminobutyric Acid GH Growth Hormone GR Glucocorticoid Receptor HbA1c Glycosolated Haemoglobin HPA Hypothalamic-Pituitary-Adrenal HPLC High Performance Liquid Chromatography HR Heart Rate IGF-1 Insulin-like Growth Factor 1 IL-1 Interleukin 1 IL-6 Interleukin 6 ITT Insulin Tolerance Test KH2PO4 Potassium dihydrogen orthophosphate dihydrite LC Locus Coeruleus LH Leutinising Hormone LiHep Lithium Heparin

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MHPG 3-Methoxy-4-Hydrophenylglycol MR Mineralocorticoid Receptor MRI Magnetic Resonance Image mRNA Messenger Ribonucleic Acid MSA Multiple Systems Atrophy NaCl Sodium Chloride NPY Neuropeptide Y NREM Non-Rapid Eye Movement O2 Oxygen PaCO2 Arterial Partial Pressure of carbon Dioxide PAF Pure Autonomic Failure PD Parkinson's Disease PNMT Phenoxyethanolamine-N-methyltransferase PNS Parasympathetic Nervous System PRA Plasma Renin Activity PU Perfusion units PVN Paraventricular Nucleus RIA Radioimmunoassay SAM Sympatho-adrenomedullay SBP Systolic Blood Pressure SEM Standard Error of the Mean SSQ Somatic Symptom Questionnaire SSRI Selective Serotonin Reuptake Inhibitor SV Stroke Volume T3 Tri-iodothyronine T4 Thyroxine TNF-α Tumour Necrosis Factor - alpha TPR Total Peripheral Resistance TRH Thyrotropin Releasing Hormone TSH Thyroid Stimulating Hormone TSST Trier Social Stress Test VAS Visual Analogue Scale VC Vital Capacity VLM Venterolateral Medulla VO2max Maximum Oxygen Consumption

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PUBLICATIONS ASSOCIATED WITH THIS THESIS

1. KAYE JM, Buchanan F, Kendrick A, Johnson P, Lowry C, Bailey J, Nutt D

and Lightman S. (2004) Acute carbon dioxide exposure in healthy adults:

Evaluation of a novel means of investigating the stress response. J

Neuroendocrinol 16 : 256-264

2. KAYE JM, Hunt M, Soothill, P and Lightman S. (2004) Responses to the

35% CO2 challenge in post-partum women. Clinical Endocrinology 61 :

582-588

3. KAYE JM, Corrall R and Lightman S. (2005) A new test for autonomic and

neuroendocrine responses in diabetes mellitus: evidence for early vagal

dysfunction. Diabetologia 48 (1) : 180-186

4. KAYE JM and Lightman S. Endocrine response to psychological stress.

(2003) In: An Introduction to Human Psychoneuroimmunology. (Vedhara K,

Ramakalawan T, Tallon D and Johnston M. Eds) Oxford University Press

2005

5. KAYE JM, Young TM, Mathias CJ, Watson L and Lightman SL. (2005)

Neuroendocrine and Behavioural Responses to CO2 Inhalation in Central

versus Peripheral Autonomic Failure. Neurology (in press)

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6. Young TM, KAYE JM, Lightman SL, Mathias CJ. Acute effects of CO2

inhalation on systemic and regional blood haemodynamics in two forms of

sympathetic denervation: multiple system atrophy and pure autonomic

failure. J Physiology (submitted July 2005)

CHAPTER 1

INTRODUCTION

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1.1 The neuroendocrine stress response in man

1.1.1. Clinical Importance

Throughout the history of medicine, reference has been made to the influence of stress,

particularly in the form of negative emotions and psychological distress, on physical

health [Sternberg 1997]. However, it has only been in the last few decades that clear

scientific evidence supporting such a notion has been forthcoming. In numerous recent

epidemiological surveys, stress and impaired psychological functioning have been

shown to be associated with an increased prevalence of serious medical conditions.

These have included psychiatric conditions such as depression and post-traumatic stress

disorder [Checkley 1996, Chrousos and Gold 1998, Gold et al 1998, Ehlert et al 2001],

vascular disease such as coronary heart disease [Rozanski et al 1999, Krantz and

McCeney 2002], immune-mediated conditions including asthma [Sternberg et al 1992a,

Wright et al 1998] and other conditions such as osteoporosis, diabetes, dementia and

premature death [Chrousos 2000, Mathe 2000]. Why some individuals manifest stress

as psychiatric illness, whilst others are more prone to physical disease and yet others

seem resistant to the effects of stress exposure is not well understood. The

neurobiological pathways that underlie these processes and the factors that contribute to

the expression of disease within an individual are still being explored. One of the most

important factors limiting the investigation of stress and its consequences is the lack of

a universally accepted scientific definition of stress [McEwen 1998]. Whilst stress as a

concept is understandable to most, an accepted definition remains elusive [Harbuz and

Lightman 1992]. Noble [2002], using Stedman’s Medical Dictionary [Stedman 1990],

defines stress as the ‘reactions of the body to forces of a deleterious nature, infection,

and various abnormal states that tend to disrupt normal physiologic equilibrium

2

(homeostasis)’. In practical terms, he then describes three methods that can be used to

measure the body’s response to stress. These include: questionnaires such as the Life

Stress Inventory [Boone and Christensen 1998] and the Perceived Stress Scale [Cohen

et al 1983]; biochemical measures particularly cortisol and catecholamines; and

cardiovascular responses including blood pressure, heart rate and heart rate variability.

Chrousos [1998] also defines stress as ‘a state of threatened homeostasis’ with those

factors, both intrinsic and extrinsic, that are challenging this state termed stressors and

the complex adaptive physiologic, hormonal and behavioural responses that occur to

restore homeostasis is the stress response. By implication, stress is ultimately

damaging with negative consequences for the individual in whom it is occurring.

McEwen [2000] on the other hand, highlights the protective role of the stress response

in the short term, and the importance of associated learning and adaptation (a process

that requires plasticity of brain responses) following stress exposure to the longer term

health and survival of the individual. It is only when these responses occur in excess of

the body’s requirements, or continue for longer than is necessary then do damaging

effects result [McEwen 1994, McEwen 1998, McEwen 2000]. Moreover, stress

includes all of the many day-to-day events of normal life that result in activation of one

or more of the components of the stress response system. This process of continual

activity of the stress response system in order to maintain homeostatic equilibrium has

been referred to as allostasis [McEwen 2000]. The cost of this process, or allostatic

load, reflects a state of wear and tear for the individual. The extent of this load is

determined by genetic make-up, co-morbid illness, individual habit such as diet,

substance abuse and physical fitness and developmental experiences that set life-long

patterns of behavioural and physiological reactivity [McEwen 1998, McEwen 2000

Goldstein and McEwen 2002, McEwen 2002].

3

Repeated exposure to a particular stressor typically results in subsequent stress

responses that are smaller in amplitude and shorter in duration – a process of learning

and adaptation that minimises the impact of repeated exposure to the same stressor

[McEwen 2000]. Increasing allostatic load with its damaging consequences can result

from repeated stress; failure to undergo adaptation; prolonged or excessive responses; or

an inadequate initial response with or without a compensatory increase in the activity of

other systems [McEwen 2000]. For example, as described by McEwen [2000],

repeated stress that causes frequent surges in blood pressure and catecholamine release

is associated with accelerated atherosclerosis and an increased risk of myocardial

infarction [Rozanski et al 1999, Krantz and McCeney 2002].

Impaired adaptation or failure to appropriately terminate the stress response results in

chronic hyperactivity of the stress response systems. The most widely described

example of this is melancholic depression that is associated with overactivation of one

of the major stress response systems – the HPA axis [Chrousos 1998, Gold et al 1998,

Gold and Chrousos 1999]. This state of excessive cortisol exposure is associated with

marked hippocampal atrophy an area important in the formation of emotional memories

[McEwen 1998, McEwen 2002]. It is also increasingly apparent that HPA axis

dysregulation appears well before clinical symptomatology and is a predictor of

treatment resistance. Similarly, failure to normalise HPA axis responses with treatment

is a strong predictor of relapse [Holsboer 2000]. The hypercortisolaemic state also

appears to promote metabolic features seen more overtly in patients with Cushing’s

disease. Glucocorticoids regulate adipocyte differentiation and stress-induced excess

cortisol is associated with increased abdominal fat accumulation [Rosmond et al 1998,

Miller and O'Callaghan 2002]. Visceral adiposity is associated with the metabolic

syndrome and includes insulin resistance, hypertension, dyslipidaemia and premature

4

atherosclerosis [Chrousos 2000, Miller and O'Callaghan 2002]. Further, this syndrome

is epidemiologically linked to increased rates of cardiovascular and cerebrovascular

disease [Chrousos 1998, Chrousos 2000, Miller and O'Callaghan 2002]. Others have

also described increased bone loss with an increased predisposition to osteoporosis as

well as immune dysfunction and an increased risk of infectious and neoplastic disease

[Chrousos 2000, McEwen 2002, Miller and O'Callaghan 2002]. Indeed patients with

depression that is associated with chronic hyperactivity of the HPA axis have been

shown to have a reduced life expectancy predominantly as a result of an excess of

cardiovascular deaths [Sternberg et al 1992a, Chrousos 2000, Miller and O'Callaghan

2002] and an increased morbidity due to conditions associated with glucocorticoid

excess. These include osteoporosis, obesity, hypertension and immunosuppression

[Chrousos 1998, Webster et al 2002]. In addition to depression, hypercortisolism is

associated with other mood and affective disorders including anorexia nervosa, chronic

anxiety, obsessive-compulsive disorder, chronic alcoholism, and other situations such as

childhood sexual abuse [Chrousos 1998].

HPA axis activation with associated CRH hypersecretion has also been shown to

influence the activity of other regulatory systems in the body and may have a role in

producing some of the other clinical manifestations of stress. CRH hyperactivity is

associated with gastro-intestinal symptoms such as pain, increased gut motility and

diarrhoea – typical features of the irritable bowel syndrome that is commonly associated

with stress [Chrousos 1998, Fukudo et al 1998]. Similarly, glucocorticoids inhibit the

growth axis and it has been postulated that the severe growth retardation associated with

psychosocial abuse or deprivation during childhood is, in part, related to chronic HPA

axis activation [Chrousos 1998].

5

Chronic hypoactivation of the HPA axis in contrast is also associated with specific

disease states. Post-traumatic stress disorder, chronic fatigue syndrome and atypical

depression [Yehuda et al 1994, Chrousos 1998, Gold and Chrousos 2002, Miller and

O'Callaghan 2002] are associated with CRH hypoactivity and reduced cortisol

production. Similarly, immune dysregulation is an important consequence of altered

HPA axis activity. Relative CRH deficiency, as exemplified in the Lewis rat (in

comparison with the histocompatible Fischer rat), is associated with an enhanced

immune response and an increased resistance to infections and tumours, but an

increased susceptibility to some autoimmune conditions [Sternberg et al 1992b,

O’Connor et al 2000]. In humans studies, rheumatoid arthritis appears to be associated

with HPA axis hypoactivation [O’Connor et al 2000] with blunted cortisol diurnal

rhythms and reduced ACTH and cortisol levels [Templ et al 1996].

Dysregulation of another of the major stress response systems, the sympatho-

adrenomedullary axis, is also associated with significant clinical consequences.

Hyperactivity of the LC and other central noradrenergic centres have been shown to

influence anxiety and behavioural arousal, with dysregulation of this system postulated

as contributing to the pathogenesis of mood disorders [Gold and Chrousos 2002]

particularly depression [Leonard 1997, Aston-Jones et al 1999, Johnston et al 1999].

Further, the bidirectional relationship between this system and the HPA axis suggests

these systems reinforce and enhance each other thereby promoting and enhancing their

negative consequences [Kirschbaum et al 1993]. It is also apparent that hyperactivity of

the SAM system may also contribute to the somatic consequences of chronic stress

exposure [McEwen 2002] including the increased frequency of cardiovascular

complications associated with chronic stress [McEwen 1998, Yergani et al 2001].

6

Understanding the biological mechanisms that underlie these various disease states and

their long term sequelae is essential for the development of new and effective

therapeutic strategies and for the development of effective preventative practices.

Investigating these pathways, in the form of molecular and pharmacological studies,

animal models and human studies are all important in order to achieve these aims. For

example, understanding the role of CRH and its receptors in the pathogenesis of

depression has led to human studies with a CRH-receptor antagonist that is showing

early promise in the treatment of this disorder [Habib et al 2000, Zobel et al 2000,

Habib et al 2001]. The stress response is, however, complex and the tools available for

its study in humans are limited. The principal objective of this thesis is to describe a

model for use in the evaluation of the stress response in humans and its possible clinical

relevance.

1.1.2. Background

In the face of any threat or challenge, either real or perceived, an organism must mount

a series of coordinated and specific hormonal, autonomic, immune and behavioural

responses that allow it to escape or adapt to this threat [Chrousos 1998, McEwen 1998,

Habib et al 2001, Carrasco and Van Der Kar 2003]. In order to cope successfully with a

particular stressor, the characteristics and intensity of the stress response must match

that posed by the threat itself. Further, the duration of the response should be no longer

than is otherwise necessary for a successful outcome. [Kiecolt-Glaser et al 2002]. The

consequences of a response that is inadequate or excessive in terms of its specificity,

intensity or duration may be one or more of a multitude of psychological or physical

7

pathologies [Checkley 1996, Chrousos and Gold 1998, Gold et al 1998, McEwen 1998,

Rozanski et al 1999, Chrousos 2000, Ehlert et al 2001, Habib et al 2001, Krantz and

McCeney 2002, Vanltallie 2002].

In the early 1900’s, Walter Cannon introduced the concept of homeostasis - an ideal

steady state for all physiological processes, disruption of which initiated an emergency

‘fight or flight’ response typical of sympatho-adrenomedullary activation where

coordinated body processes would work together to restore this ideal balance [McCarty

1994]. Hans Selye [Selye 1956, Levine 2000] also emphasised the importance of

multiple, integrated systems that respond in a coordinated fashion to a particular stressor

[Carrasco and Van Der Kar 2003]. He highlighted the importance of the HPA axis in

the stress response when he described the General Alarm Reaction - an early response to

a noxious stimulus characterised by non-specific activation of the principal endocrine

response systems, the HPA and SAM axes. Continued stress exposure to the same

noxious agent had lasting effects on endocrine, immune and other systems characterised

by adrenal gland enlargement, gastric ulceration and lymphatic atrophy [McEwen 1998,

Koob 1999]. Seyle termed this the General Adaptation Syndrome and he noted that

recovery from this state was possible provided the stress was terminated. Continued or

repeated exposure, however, usually resulted in exhaustion and ultimately death [Koob

1999, Levine 2000].

In addition to noxious stimuli and other physical stressors such as exertion, physical

extremes, trauma and injury, early Freudian theory also suggested psychological stress

could produce a similar response. In the 1950’s Freud held that maternal influences and

early childhood trauma had profound and long lasting effects on the psychological and

physical health of the individual [Sternberg 1997, Levine 2000]. Animal studies by

Levine [2000], and many others [Brunson et al 2001] subsequently have shown long

8

term endocrine changes as a consequence of both psychological and physical stress

experienced in early development. Further work by Mason [1968] has demonstrated

that psychological stressors are some of the most potent stimuli of the endocrine stress

response particularly when they involve elements of novelty, uncertainty and

unpredictability [Ursin 1998]. This was highlighted by the observation that anticipating

an event can be as potent an activator of the stress response as the event itself [Levine

2000].

The notion that all stress is damaging and this damage occurs as a consequence of

excessive hormone production was initially implied by the observation that various

endocrine diseases were often associated with overt psychopathology and that this

corrected itself once the endocrine condition resolved [Brambilla 2000]. This led

several authors to postulate that single hormone abnormalities (either excesses or

deficiencies) were responsible for specific behavioural consequences such as depression

or schizophrenia [Brambilla 2000]. Whilst this notion is no longer held, it did lead on

to the idea that abnormalities of neurotransmitters and neurotransmitter systems are

more likely responsible for certain psychopathologies, and that pharmaceutical agents

that target these systems could produce successful treatments [Brambilla 2000]. It has

become apparent more recently, that the stress response is beneficial in protecting an

individual from a harmful situation and that the brain can learn, adapt and adjust its

future response to be more efficient and effective [McEwen 1998]. Problems arise

when the stress is sustained or becomes repetitive. The principal stress responsive

systems are energy expensive and failure to adequately deal with or recover from these

challenges may result in ill health. The ability to continually adapt to and recover from

stress (or allostasis [McEwen 2000]) is further diminished when we choose a lifestyle

consisting of a poor diet, little exercise, disturbed sleep and the consumption of

9

cigarettes and alcohol and so on. The consequences of not adequately recovering from

each stressful episode (allostatic load) is the promotion of ill-health through high blood

pressure, hypercortisolism, suppressed immune function and poor growth and

[Chrousos 1998, McEwen 1998, McEwen 2000, Kiecolt-Glaser et al 2002]. Ultimately,

the consequences for long-term physical and mental health are significant.

The stress response system has evolved as both an early warning system capable of

recognising potential or existing threats, and as a response system that can initiate and

drive the necessary processes required to escape or confront the threat. By its very

nature, the response must be dynamic, beginning rapidly with brain and behavioural

activation followed quickly by physiological activation [Richer et al 1996]. These

processes are characterised by positive-feedback and feed forward loops that enhance

and reinforce themselves as well as recruiting other arms of the stress response. Slower

acting hormone systems are recruited into the cascade providing checks and balances to

the already active, but energy expensive systems, putting a brake on the whole response

to ensure it is kept appropriate to the type of stress faced, to its intensity and duration,

and to ensure the response is switched off when the threat has been adequately dealt

with [Eriksen et al 1999, Sapolsky et al 2000].

Changes in the internal or external environment that represent either real or potential

threats are recognised with the parts of the brain responsible for receiving, integrating,

interpreting and then relaying this information on to those areas responsible for co-

ordinating the necessary response. This brain activation can be detected within

milliseconds and proceeds over seconds to minutes as the response continues to unfold.

Stereotypical orienting behaviour, initiated within seconds, gradually gives way to more

10

goal-directed behaviour that is specific to the stressor being faced and the environment

in which it is occurring [Eriksen et al 1999].

Activation of the autonomic nervous system occurs within seconds, mediated by the

release of catecholamines from sympathetic nerves and the adrenal medulla and

enhanced by the withdrawal of parasympathetic activity [McCarty 1994, Young and

Landsberg 1998]. These systems promote the immediate physiological, motor and

behavioural responses needed in the face of acute physical or psychological stress.

Within minutes of the onset of this cascade of events occurring, hypothalamic releasing

hormones stimulate the release of pituitary hormones with the appearance of ACTH

signalling the recruitment of the HPA axis into the process [Harbuz and Lightman 1992,

Chrousos 1998, Thorner et al 1998]. The cortisol response is much slower, with peak

levels not seen for 15-20 minutes after the onset of the stress. Early actions of the HPA

system provide additional energy resources for the stress response, whilst slower gene-

related effects over the next few minutes to hours serve to restrain ongoing actions of

the stress response which, if left unchecked, may prove to be unsustainable for the

individual [Chrousos 1998, Habib et al 2001].

The HPA and SAM axes are the principal endocrine effector arms of the stress

response. However, a number of other hormone axes and neurotransmitter systems are

either directly stress responsive themselves, or modulate these other hormone systems.

11

1.1.2.1. The HPA Axis

Corticotropin releasing hormone (CRH) was identified by Vale and others [Vale et al

1981] in 1981 as the 41-amino-acid peptide responsible for promoting the synthesis and

release of anterior pituitary ACTH. It is also widely distributed throughout the CNS,

including within the cortex where it has important effects on behaviour and cognitive

processing. Within the brainstem interactions with sympathetic and parasympathetic

centres influence autonomic functioning whilst within limbic and para-limbic regions

such as the amygdala, CRH influences the expression of mood and anxiety-type

behaviours [Owens and Nemeroff 1991, Harbuz and Lightman 1992, Thorner et al

1998, Carrasco and Van Der Kar 2003].

ACTH release from the anterior pituitary under the influence of CRH acts directly on

the adrenal cortex to promote the release of large amounts of adrenal glucocorticoids

into the circulation [Harbuz and Lightman 1992, Chrousos 1998, Habib et al 2001].

Glucocorticoids, in general, have two fundamental roles in the stress response. Firstly,

during stress free periods, basal levels have a role in preparing the organism for future

stress exposure. This involves energy storage and conservation by promoting glucose

and fat uptake and opposing energy utilisation. They also prime the immune system for

future activation and promote memory formation of previous stressors so that future

exposure to the same or similar stressor may facilitate a more rapid and efficient

response [Sapolsky et al 2000].

The second role is that of a modulating effect at the time of stress exposure itself.

Initially glucocorticoids enhance the cardiovascular effects of catecholamines and

vasopressin (AVP), promote energy provision and utilisation, influence and enhance

12

appropriate stress-related behaviours and stimulate certain aspects of the immune

response [O’Connor et al 2000, Sapolsky et al 2000, Tsigos and Chrousos 2002].

However, most of these responses would occur even in the absence of any circulating

glucocorticoids. It is perhaps even more important that once the stress response has

been initiated, some of the principal actions of glucocorticoids are to suppress and

restrain the activity of these systems, in particular the SAM and immune systems. In

doing so, glucocorticoids provide an essential regulatory balance to ensure the stress

response is appropriate in terms of both its intensity and duration and that all these

responses are ‘switched off’ when the stress has been successfully dealt with [Sapolsky

1994, McEwen 1998, Goldstein and McEwen 2002]

The functional role of CRH and its receptors within the brain has been extensively

researched in animals using anatomical lesion studies, CRH-receptor knockout mice,

and antibodies or CRH-like compounds with either inhibitory or stimulatory effects on

CRH receptors [Ma and Lightman 1998, Neumann et al 1998, Reichlin 1998, Bakshi

and Kalin 2000, Jessop et al 2001]. Overall, the addition of CRH seems to promote

anxiety-like behaviour and increase the physiological effects of stress, whilst inhibiting

CRH suppresses both stress-related behaviour and physiological changes. In several

studies the administration of CRH directly into the brains of rodents decreased

exploratory behaviour, increased fear and conflict behaviour, and suppressed feeding

and sexual activity [Jones et al 1998, Bakshi and Kalin 2000]. CRH also stimulated

stress-related physiological changes including increases in heart rate and blood pressure

[Jezova et al 1999]. At least two CRH receptors have been identified within the brain.

CRH-R1 is widely distributed in the pituitary, brainstem, cerebellum and amygdala and

appears to mediate stress induced HPA axis activation as well as stress related (and

possibly spontaneous) anxiety behaviour. Some evidence for this includes the

13

demonstration that CRH-R1 knockout mice display increased behavioural activity

consistent with reduced anxiety in situations where normal mice display increased

anxiety. Additionally, following an acute stress, these animals show a blunted ACTH

and glucocorticoid response [Owens and Nemeroff 1991]. Similarly, studies of

compounds that block CRH-R1 suggest treated animals display reduced anxiety to

some, but not all, anxiety provoking situations as well as displaying a reduced HPA

hormone response to high intensity stressors [Bakshi and Kalin 2000].

CRH-R2 has a more restricted distribution and a much higher affinity for CRH-like

peptides, particularly urocortin II and III, than for CRH. Studies of CRH-R2 knockout

mice as well as CRH-R2 inhibitors have yielded less consistent results with animals

displaying a mix of both increased and decreased anxiety behaviour and mixed

hormonal responses to stress that were dependent on such factors as the gender of the

animal and the type of provoking stimulus [Bakshi and Kalin 2000].

In addition to driving both HPA and behavioural responses to stress, CRH also

modulates the autonomic, immune and other endocrine responses to stress. CRH

activity in the brain activates central sympathetic systems with subsequent increases in

adrenaline and noradrenaline release, decreased parasympathetic outflow and an overall

increase in sympathetic tone with increased heart rate, blood pressure and cardiac output

[Jezova et al 1999, Habib et al 2001, Gammatopoulos and Chrousos 2002].

Bidirectional influences between CRH and the immune system result in cytokine (IL-1,

IL-6 and TNF-α) mediated stimulation of the HPA axis with increased cortisol release.

Cortisol itself has widespread anti-inflammatory actions, although there is evidence that

locally produced CRH and autonomic-immune interactions stimulated by CRH can

have pro-inflammatory effects [O’Connor et al 2000, Habib et al 2001].

14

Whilst much of the understanding of the biology of CRH is derived from studies in

rodents, human studies have indicated that dysregulation of CRH function contributes to

the pathophysiology of several disorders including: depression, post-traumatic stress

disorder, disorders of sleep, stress-related menstrual irregularity and infertility,

manifestations of the metabolic syndrome and functional gastro-intestinal disorders

[Chrousos 1998, Koob 1999, Gammatopoulos and Chrousos 2002]. Further, targeting

CRH peptides and their receptors are increasingly seen as an effective means of

managing these various conditions and a number of CRH agonists and antagonists are

currently under evaluation in clinical and pre-clinical studies. Oral CRH-R1 antagonists

have been shown to decrease anxiety-related endocrine, autonomic and behavioural

responses in rat and primate models [Habib et al 2000, O’Connor et al 2000]. They

have also been safely trialed in a human phase II clinical study of patients with major

depression where patients showed an improvement in depression and anxiety symptoms

on treatment with a significant worsening following treatment discontinuation [Zobel et

al 2000].

Cortisol secretion is precisely controlled by the complex feedback system that regulates

the HPA axis. Whilst significant variability exists between individuals, within an

individual both the circadian and ultradian rhythms of cortisol are tightly controlled and

highly stable. Repeated or chronic stress with consequent CRH hyperactivity and

excessive or unrestrained cortisol exposure has been shown to result in hippocampal

neuronal damage and impaired hipppocampal function [McEwen 1998]. The

hippocampus is involved with contextual memory formation and is also an essential part

of the cortisol negative feedback system. Impaired memory formation particularly for

emotional events may exacerbate future stress responses under similar circumstances,

15

whilst impaired negative feedback further exacerbates the hypercortisolism. Rosmond

et al [Rosmond et al 1998] was able to demonstrate that the HPA axis in non-stressed

individuals is characterised by a wide circadian rhythm (with distant morning zeniths

and evening nadirs), a discrete but small response to an acute stress and appropriate

suppression of cortisol levels following the administration of an exogenous

glucocorticoid indicating a normal pattern of secretion with an intact feedback

mechanism. In contrast, chronically stressed individuals displayed a decreased

circadian variability (due to decreased morning zeniths and increased evening nadirs), a

large acute stress response and inadequate cortisol suppression indicating an altered

pattern of secretion and impaired negative feedback with resultant cortisol

hypersecretion. The effect of this hypercortisolism in the brain includes depression and

anxiety [Chrousos and Gold 1998], whilst in the periphery it is associated with bone

loss, obesity, hypertension, insulin resistance and other features of the metabolic

syndrome [Chrousos and Gold 1998]. Further, some patients with cancer have been

shown to have alterations to their neuroendocrine and immune axes that may have

implications for their disease progression and outcome [Speigel et al 1998, Sephton et

al 2000].

1.1.2.2. Arginine Vasopressin

AVP released from magnocellular cells of the PVN and the supraoptic nucleus in

response to osmotic and haemodynamic stimuli promotes water and electrolyte

retention in the kidneys [Carrasco and Van Der Kar 2003] whilst AVP derived from

parvocellular cells of the PVN acts synergistically with CRH to stimulate the release of

ACTH [Ma et al 1997, Sephton et al 2000]. In animal studies, AVP appears to be an

16

important mediator of ACTH release during chronic stress. Chronic stress increases

AVP expression within the PVN [Ma et al 1997, Aguilera and Rabadan-Diehl. 2000],

whilst inhibitors of AVP impair the HPA response to various stressors including

insulin-induced hypoglycaemia and restraint stress [Linton et al 1985]. As mentioned,

during chronic or repeated stress, glucocorticoid levels are initially high with negative

feedback downregulating CRH receptors and subsequently suppressing CRH and

ACTH responses. As a result, CRH responses often decline with time in the face of

high cortisol levels (habituation). However, exposure to a novel stress during this time

is associated with a normal HPA response and this restored response is mediated by

AVP whose receptors that don’t show the same downregulation (and in some

circumstances may be upregulated) [Ma et al 1999, Aguilera and Rabadan-Diehl. 2000].

Recently, Scott and Dinan [1998] have suggested the impaired HPA axis regulation that

commonly accompanies major depression in humans reflects AVP activity rather than

CRH. AVP may therefore be an important target in the design of therapeutic agents for

this condition. AVP, it appears, sensitises the pituitary to the effects of a superimposed

novel stressor suggesting AVP and CRH are regulated independently.

1.1.2.3. The Sympatho-Neural and Sympatho-Adrenomedullary Axis

The hallmark sympathetic ‘fight or flight’ response, as described by Cannon [McCarty

1994], is characterised by global activation of the SAM system and features typical

physiological and behavioural activation including heart rate quickening, increased

blood pressure and rapid breathing. Fear, vigilance, sensory arousal and motor

activation often with trembling, goose bumps and piloerection (hair standing on end)

also occur. Release of glucose stores, immune activation and increased blood flow to

17

essential organs such as the brain occur whilst non-essential activity like digestion is

inhibited. This produces a state ‘of emergency’ which can rapidly attend to a sudden

change in physiological balance [Young and Landsberg 1998, Habib et al 2001]. This

response is characterised by its speed of onset, its ability to begin in anticipation of an

event being stressful, and by its interaction with other stress-responsive systems [Young

and Landsberg 1998]. This interaction can occur either through neural connections or

through increased blood flow that transports other messengers (such as hormones and

cytokines) more rapidly to their respective sites of action [Young and Landsberg 1998].

Whilst the ‘fight or flight’ reaction is a useful way of describing the global SAM

response to various stressors, it is clear from several studies that the adrenomedullary

and sympathetic nerve responses to stress are regulated independently and the

components of each vary in their response to different stressors [Bornstein and

Chrousos 1999]. In humans, for example, the physical stress of cold or exercise is

associated with both adrenaline and noradrenaline responses, whereas the response to

insulin-induced hypoglycaemia is mediated predominantly by adrenaline alone [Young

and Landsberg 1998]. Psychological stress is also a potent stimulus of the SAM axis,

with activation occurring at generally lower levels of stress than that required to

generate an HPA response [Singh et al 1999, Habib et al 2000, Zobel et al 2000].

Unlike cortisol and the HPA axis, SAM (catecholamine, heart rate and BP) responses to

either exercise or psychological stress do not show the same continuum of responses,

although differences between individuals are apparent when anticipating a stress, or in

response to cold stress [Cacioppo et al 1995, Kirschbaum et al 1995, Negrao et al

2000]. Concordance between high anticipatory or cold-induced heart rate responses and

high stress-induced cortisol responses has allowed the classification of individuals as

18

high or low responders, with the hypothesis that either hypo- or hyper-reactivity of the

stress response will influence an individual’s susceptibility to developing various

psychological, metabolic or immune-related disorders [Biondi and Picardi 1996].

Concordance between SAM responses and the HPA axis has its foundation in a large

number of neuroanatomical and behavioural studies indicating the importance of strong,

bi-directional neuronal influences of the brainstem catecholamine centres and CRH

mediated pathways including the PVN [Kvetnansky et al 1995, Koob 1999, Zeigler et al

1999, Gerra et al 2001]. This translates into a powerful feed-forward system where

stress-induced activation of catecholaminergic systems activates, and in turn is further

activated by, stress-responsive CRH neurones [Neumann et al 1998, Carrasco and Van

Der Kar 2003]. Such an interaction would serve to appropriately reinforce the principal

early stress response systems following an acute threat, but altered sensitivity of one or

other may significantly contribute to the disordered activity of the endocrine stress

response that underlies many psychological conditions. Hyperactivity of the brainstem

catecholamine system with subsequent CRH hyperactivity and reduced feedback

sensitivity, for example, has been postulated as a likely mechanism for the endocrine

changes seen in depression and anxiety states [Kvetnansky et al 1995, Gold et al 1998,

Koob 1999, Ressler and Nemeroff 1999].

1.1.2.4. Prolactin

Prolactin, released from the anterior pituitary under tonic inhibitory control from

dopamine neurons is required predominantly for milk production during lactation

[Reichlin 1998.]. It has, however, been shown in both animal and human experiments

19

to be released in response to acute stress, although its role is yet to be fully determined

[Chrousos 1998, Biondi and Picardi 1999, Habib et al 2001]. High prolactin levels in

rodents, such as those occurring during lactation, are associated with reduced expression

of anxiety behaviour and with HPA axis suppression [Torner et al 2001]. Further,

intracerebroventricular administration of prolactin in rats reduced both behavioural

anxiety and corticosterone responses to a superimposed stress, whilst a prolactin

receptor antagonist given by the same route enhanced anxiety behaviour [Torner et al

2002]. A few studies of physical and psychological stress have measured prolactin

responses in humans, but little consistency between these responses has been shown

[Richer et al 1996, Biondi and Picardi 1999]. In some studies prolactin levels have

been shown to increase, whilst in others, prolactin has either not changed or decreased

[Richer et al 1996, Biondi and Picardi 1999].

1.1.2.5. Opiates

Endogenous opiates and their receptors are ubiquitously distributed throughout the

central, peripheral and autonomic nervous systems. Opiate pathways have been shown

to influence a broad range of functions and behaviours related to stress including the

regulation of pain, reinforcement and reward, and the modulation of autonomic and

neuroendocrine axes [Olson et al 1996, Drolet et al 2001]. At least three families of

endogenous opiates have been identified (enkephalin, endorphin and dynorphin) which

act through three major opiate receptors – mu (µ), delta (δ) and kappa (κ) [Drolet et al

2001]. Each receptor subtype maintains a distinct anatomical distribution within the

brain, with µ and κ receptors in particular being associated with structures involved in

the neurobiology of stress. Sites include the hippocampus, the hypothalamic

20

paraventricular and arcuate nuclei, the central nucleus of the amygdala, locus coeruleus,

ventral lateral medulla and dorsal motor nucleus of the vagus [Mansour et al 1995,

Drolet et al 2001, Habib et al 2001]. Reciprocal innervation between CRH producing

neurones of the PVN, brainstem noradrenergic centres, the amygdala and limbic system

and the opiate system suggests a significant role for opiate pathways in the modulation

and regulation of the principal stress response centres [Drolet et al 2001, Habib et al

2001]. Acute stress exposure, for example, activates the HPA and SAM axes and

reciprocal innervation increases opiate expression and action within the PVN and other

stress responsive centres [Young and Lightman 1992, Mansi et al 2000, Drolet et al

2001]. Opiate pathway stimulation inhibits the activity of the central components of the

stress system thereby dampening their output. Further, projections to the hindbrain and

spinal cord produce analgesia [Habib et al 2001]. In other words, the opiate system

seems to act to diminish the impact of the stress response by dampening the physiologic

and behavioural responses that, if left unchecked, would be ultimately harmful to the

organism [McCubbin 1993, Drolet et al 2001].

1.1.2.6. Serotonin (5-HT)

Serotonergic systems are important regulators of the behavioural, autonomic and

endocrine responses to stress [Dinan 1996, Lowry 2002]. Originating primarily from

the brainstem raphe complex, serotonergic neurones project to multiple forebrain

centres including the central nucleus of the amygdala, hippocampus and medial septum,

areas involved in the emotional fear response [Lowry 2002]. 5–HT projections have

been shown to influence HPA axis responses through their regulation of CRH output at

the level of the PVN, but recent evidence also indicates an additional influence of 5-HT

21

on anterior pituitary ACTH release and on adrenal cortical glucocorticoid release

[Dinan 1996]. Projections to the locus coeruleus and other autonomic brainstem centres

influence autonomic ‘fight-and-flight’ responses whilst behavioural motor responses are

influenced by projections to areas such as the substantia nigra [Lowry 2002].

The influence of these serotonergic systems on the stress response is, however, complex

as they can either facilitate or inhibit responses. The complexity of these systems arises

in part by the vast array of 5-HT receptors within the brain [Chaouloff 2000] and in part

from evidence that the serotonergic system itself is made up of separate systems that are

also anatomically and functionally discrete [Lowry 2002]. Activation of serotonergic

systems on the one hand are associated with enhanced autonomic arousal, anxiety and

fear, whilst on the other hand stress-induced activation via separate serotonergic

pathways results in inhibition of HPA and SAM responses [Lowry 2002].

1.1.2.7. The Growth, Thyroid and Reproductive Axes

Anterior pituitary growth hormone (GH) release is stimulated following some acute

stressors [Chrousos 1998]. GH itself stimulates the release of IGF-I from the liver that

acts on many different tissues, but whose main role in acute stress is the release of

energy stores from the liver [Thorner et al 1998]. Physical stressors including exercise

are generally associated with an increase in GH levels [Richer et al 1996, Thorner et al

1998], although most psychological stressors are not [Negrao et al 2000]. Chronic

emotional disorders such as anxiety or depression are associated with suppressed GH

levels through the combined influence of CRH, which stimulates GH inhibitory

22

peptides such as somatostatin, and through a direct action of glucocorticoids on the GH

gene [Habib et al 2001].

Similarly, the thyroid axis is also suppressed in response to chronic stress-induced HPA

axis activation and thyroid hormone (T4) levels seem to follow a similar pattern to GH

levels in response to acute stress [Biondi and Picardi 1999, Habib et al 2001].

Hypothalamic TRH is inhibited by CRH and somatostatin, whilst glucocorticoids inhibit

both TSH release from the pituitary and T4 release from the thyroid gland [Chrousos

1998, Thorner et al 1998]. Further, they also reduce the conversion of T4 to its more

active form, triiodothyronine (T3), in peripheral tissues [Thorner et al 1998].

The reproductive axis is also very sensitive to the inhibitory influences of CRH,

glucocorticoids and components of other stress responsive pathways such as

inflammatory cytokines and endorphins [Habib et al 2001]. Chronic HPA axis

activation from physical or psychological stress, as in highly trained athletes or people

with anorexia nervosa, is commonly associated with suppression of reproductive

hormone release, and particularly with menstrual cycle inhibition in women [Knol 1991,

Chrousos 1998, Biondi and Picardi 1999].

1.1.2.8. Other Neurohormonal Systems

Oxytocin, a posterior pituitary hormone required for the induction of labour, is also

released during stress [Van de Kar and Blair 1999]. Currently its precise function

during stress is unclear, but some recent evidence suggests it acts to oppose or modulate

the action of AVP [Van de Kar and Blair 1999].

23

The renin-angiotensin system is an important hormonal system that regulates

circulating blood volume and blood pressure. It is activated in response to

haemodynamic stressors such as blood loss, but renin is also released during acute

psychological stress as part of the activation of the sympathetic nervous system and may

have a role in the anticipatory phase of the stress response [Van de Kar and Blair 1999].

Substance P is activated during pain and chronic inflammatory stress and appears to

add to the inhibitory influence some inflammatory mediators have on CRH and the

HPA axis. In addition, substance P increases sympathetic activation in response to pain

[Habib et al 2001].

Neuropeptide Y (NPY), a 36-amino acid peptide, is an additional important component

of the sympathetic nervous system. It is found within sympathetic brain centres where it

has important regulatory effects on both the HPA and SAM axes, influences appetite

and feeding behaviour and may also have anxiolytic properties [Habib et al 2001]. In

the periphery, it is found within sympathetic nerve fibres associated with blood vessels

and immune cells. Released with noradrenaline, neuropeptide Y contributes to the

control of blood pressure, blood flow and lymphocyte traffic [Elenkov et al 2000].

24

1.2 Investigating the stress response

1.2.1 Currently available tools and their limitations

Survival of an individual depends on the ability of the stress response systems to mount

a biologically appropriate response when threatened. Failure to do so is likely to be

deleterious and result in significant harm. Normal baseline or resting cortisol and other

stress hormone levels are not necessarily indicative of a system that is capable of

responding dynamically to perceived or actual threats. Partial or even complete ACTH

deficiency, for example, is often associated with normal resting cortisol levels and is

typically asymptomatic during non-stressed conditions. However, when stressed these

individuals are often unable to mount an appropriate cortisol response, a state that can

be life threatening. It is well established that baseline or even diurnal cortisol profiles

do not reflect the dynamic integrity of the HPA axis and are therefore not particularly

useful for its assessment [Erturk et al 1998]. Rather, it is necessary to use challenges

that assess the dynamic ‘responsiveness’ of the various components of the stress system.

Numerous laboratory based challenges have been developed and used for the study of

the stress response in humans. In addition, real life events and scenarios have also been

examined in order to try and predict the impact of stressful life events on responses and

disease outcomes. Laboratory paradigms that have been described have used a variety

of psychological, physiological or pharmacological challenges.

25

1.2.1.1 Psychological challenges

Psychological challenges will activate stress response systems if the challenge is

threatening or demanding without adequate perceived resources being available for

coping with the particular challenge [Ehlert et al 2001]. In other words, these

challenges are usually characterised as being novel, unpredictable and uncontrollable

[Ehlert et al 2001]. Typical challenges have involved performance tasks such as mental

arithmetic and the Stroop colour-word conflict test, speech or interview tasks, video

games and films or videos with a strong emotional content [Biondi and Picardi 1999].

Combined tasks are also commonly used, the Trier Social Stress Test (TSST) being the

most widely used example [Kirschbaum et al 1993, Ehlert et al 2001]. This task

involves a 10 minute stress anticipation period, followed by a speaking task and a serial

subtraction task in front of at least three unfamiliar observers that is videotaped and

recorded [Kirschbaum et al 1993]. Whilst cortisol levels in response to this task are

reproducibly elevated, albeit with a wide inter-individual variability, repeated

challenges do not stimulate the HPA axis in most individuals as the task, when repeated,

loses its novelty and unpredictability [Kirschbaum et al 1995].

Mental arithmetic alone and most other public speaking or performance tasks are not

reliably associated with a significant cortisol response although most do produce

significant increases in catecholamine release with expected increases in blood pressure

and heart rate [Jorgensen et al 1990, Sgoutas-Emch et al 1994, Biondi and Picardi

1999]. Combined tasks, including the TSST, consistently increase SAM responses that

are usually reproducible when the task is repeated [Al’Absi et al 1997, Biondi and

Picardi 1999]. Cortisol responses on the first occasion show wide inter-individual

variation with a gradual decline in responses with repeated exposure in most individuals

[Kirschbaum et al 1995]. As will be discussed later, a group of individuals will continue

26

to demonstrate HPA axis activation with repeated challenges. These individuals,

termed ‘high responders’ may be more prone to the adverse effects of chronic activation

of the stress response systems [Cacioppo et al 1995].

A few studies of both laboratory and real life psychological stress have measured

prolactin responses, but little consistency between responses has been shown [Richer et

al 1996, Biondi and Picardi 1999]. In some studies of academic exam stress and

parachute jumping, prolactin levels have been shown to increase, whilst other forms of

psychological stress have shown either no change or even decreases in prolactin levels

[Richer et al 1996, Biondi and Picardi 1999]. Similarly, laboratory psychological

stressors are generally not associated with a change in GH or thyroid hormone levels

(TSH or T4) [Biondi and Picardi 1999], although some studies of parachute jumping or

bereavement recall have shown a response [Richer et al 1996, Biondi and Picardi 1999,

Ehlert et al 2001].

Films, videotapes and some interview protocols have been inconsistent in the

production of both HPA and SAM responses and it is apparent that a response is

dependent on the subject matter being viewed or discussed [Biondi and Picardi 1999]

and the duration of exposure. As might be expected, challenges that involve exposure

to subject matter that the individual does not perceive as being emotionally relevant to

themselves is not likely to provoke a response [Biondi and Picardi 1999].

Whilst psychological stressors are widely used for the assessment of stress responses in

the laboratory setting, they have a number of specific limitations. Firstly, not all

challenges will reliably provoke an HPA axis response (for example mental arithmetic

or other performance tasks alone). Secondly, HPA responses rapidly decline with

repeated exposure making them unreliable for experiments evaluating different stress

27

conditions or interventions in the same individual. Thirdly, the challenges are often

labour intensive (requiring several investigators per participant) and time consuming

(with many typically lasting several hours). Finally, standardising the challenge

between individuals can be difficult as the success of the challenge depends on its

relevance to the subject and individuals vary considerably according to such factors as

their level of experience with public speaking, education and so on.

Parachute jumping, for example, is considered to be a psychological as opposed to a

physical challenge and has been studied previously as a paradigm that produces a

significant HPA, SAM, GH and TSH response [Richer et al 1996]. This paradigm is,

however, illustrative of some of the difficulties faced. The HPA response only occurred

in first-time jumpers. The challenge is inherently dangerous and is expensive with a

very restricted pool of potential volunteers. Sampling was also very difficult, requiring

a complex arrangement required for samples to be taken automatically from a brachial

arterial line (which itself has significant ethical, safety, cost and practicality issues)

[Richer et al 1996].

1.2.1.2 Physical challenges

Physical exertion (for example from treadmill running or cycling, knee bends or

handgrip) and pain (cold, venepuncture) have been the principal paradigms used as

physical challenges. Intense exercise, usually as running or cycling, has been

extensively studied as a means of generating a stress response. These challenges require

the subject to exercise to exhaustion, based on measuring maximum oxygen

consumption (VO2max) and reliably produce activation of the HPA, SAM, GH and in

28

some cases prolactin systems [Petrides et al 1994, Singh et al 1999, de Vries et al

2000]. HPA responses do, however, show the same continuum as are seen in response

to psychological challenges and there is a good correlation between high responders to

exercise stress and high responders to psychological stress [Singh et al 1999]. It is

postulated that there is a dissociation in the neuroendocrine (HPA and SAM) response

to exercise stress with SAM responses reflecting work-load and HPA responses

reflecting the mental effort or distress associated with exercising to exhaustion [Singh et

al 1999].

Several potential limitations to the use of this challenge exist. Principally, the

equipment is expensive and the challenge labour intensive usually requiring several

operators including someone specifically skilled in exercise physiology and the use of

the equipment. The equipment itself is not portable, hence limiting where it can be

performed and there may be significant safety issues associated with this level of

exercise. The test would also be restricted to those physically capable of performing

this type of exercise to this intensity. Finally, there are a number of confounding

variables influencing neurohormonal responses that make standardising exercise

protocols difficult. These include static vs dynamic exercise; position (supine vs erect);

the mass of muscle engaged in the exercise; the intensity and duration of the exercise;

and importantly an individuals level of training [de Vries et al 2000, O'Sullivan et al

2001].

The cold pressor test is a relatively well tolerated stressor that has been shown to

increase both SAM and HPA axis activity through combined effects on temperature and

pain sensing afferents [Bullinger et al 1984, Durel et al 1993, Pascualy et al 2000]. The

test requires the subject to immerse their hand in an ice bath (4°C) for a brief period (1-

29

3 minutes or longer depending on the protocol). Its principal use has been in examining

the pressor response as a predictor of risk of hypertension [Velasco et al 1997] and in

pain research [Washington et al 2000]. Little literature exists on the HPA response and,

in particular, stress responses to repeated exposure in the same individual. Whilst this

challenge is simple to perform and appears to produce reliable SAM and HPA

responses, doubts remain over the response to repeated exposure, and as with other

types of painful stimuli such as venepuncture, ethical difficulties exist related to

repeated exposure.

1.2.1.3 Pharmacological challenges

The administration of numerous pharmacological agents have been used in the

evaluation of various components of the stress response with the insulin tolerance test

considered to be the ‘gold standard’ for the evaluation of the integrity of the HPA axis.

In psychiatric research, pharmacological agents have been widely used in the study of

anxiety, fear and panic and there is extensive crossover between the components of the

fear/anxiety response and the general stress response.

Insulin induced hypoglycaemia or the insulin tolerance test (ITT) is considered the

‘gold standard’ test for the dynamic assessment of HPA axis integrity [Erturk et al

1998, Lange et al 2002]. The test involves the intravenous administration of a bolus

dose of insulin with regular blood glucose monitoring. Subjects must develop

symptoms consistent with hypoglycaemia (sweating, hunger, tachycardia and

confusion) and must have a recorded blood glucose level <2.5 mmol/l [Erturk et al

1998]. A test is considered normal if the peak cortisol response is >550 nmol/l at 60

30

minutes, and abnormal if lower than this. ACTH responses correlate poorly with

cortisol responses and are not useful in evaluating the axis [Borm et al 2003]. The test

has a high positive predictive value for determining a normal HPA response, but the

result can only be interpreted as either normal or abnormal. Unlike other dynamic tests

of the HPA axis that produce a continuum of cortisol values with the degree of cortisol

elevation predictive of their susceptibility to responses to other stressful conditions and

to disease outcomes [Greiz et al 1990a, Cacioppo et al 1995, Kirschbaum et al 1995,

Singh et al 1999], the ITT is unable to provide this sort of information. Further, the ITT

represents a well established test of HPA and GH axis integrity, but is not a well

established test of other components of the stress response particularly the SAM axis

and emotional arousal. Finally, the complexity and safety of the ITT has often been

questioned in its use as a routine clinical test [Erturk et al 1998]. A study by Nye et al

[Nye et al 2001] suggested that for reliability, the glucose nadir should be <1.6 nmol/l,

significantly increasing the potential for hypoglycaemic-associated complications such

as seizures.

Other pharmacological tests of HPA axis activity that have been employed include the

dexamethasone suppression test, the naloxone test, metyrapone administration, and

CRH with or without dexamethasone administration. These agents all act on different

levels of the HPA axis either directly stimulating anterior pituitary ACTH release

(CRH), interfering with cortisol synthesis (metyrapone) or altering negative feedback

(dexamethasone and naloxone) and specific response criteria have been set [Inder et al

1995, Checkley 1996, Gold et al 1998, Thorner et al 1998, Ehlert et al 2001]. All these

tests suffer from low sensitivity and specificity with considerable inter-individual

variation and significant numbers of non-responders. In addition they provide little if

any information on non-HPA components of the stress response.

31

The physiological and psychological features of fear, anxiety and at their extreme,

panic, in many ways resembles that of the generalised stress response, including

emotional arousal, cardiorespiratory and autonomic activation and HPA axis stimulation

[Gorman et al 2000]. Numerous pharmacological agents have been used to generate

fear responses. Yohimbine (an α2-adrenergic antagonist), cholecystokinin-β receptor

antagonists (CCK), fenfluramine (a 5-HT releasing agent), meta-chlorophenylpiperizine

(a mixed 5-HT receptor agonist), caffeine and β-carboline esters have all been used to

generate panic attacks that are associated with significant HPA axis activation as well as

cardiovascular stimulation [Bourin et al 1998, Sinha 1999, Argyropoulos et al 2002].

Not all are respiratory stimulants and catecholamine responses have been inconsistent

and variable [Bourin et al 1998]. Sodium lactate, bicarbonate, isoproterenol and

doxapram on the other hand will all induce panic attacks but without any significant

HPA axis activation [Bourin et al 1998, Sinha 1999, Argyropoulos et al 2002]. Carbon

dioxide exposure, in a number of different protocols, will produce emotional arousal,

autonomic activation and in some, but not all cases, HPA axis activation [Hardgrove et

al 1938, Sechzer et al 1960, Cross and Silver 1962, Gorman et al 1988, Woods et al

1988, Greiz et al 1990a, Greiz et al 1990b, Perna et al 1994, Papp et al 1997, Bourin et

al 1998, Verberg et al 1998, Sinha 1999, Argyropoulos et al 2002, Bailey et al 2002].

The rationale for choosing to evaluate this in more detail as a model of the stress

response is outlined below. Of the other agents, CCK-4 has been considered safest and

most reliable in terms of generating panic, autonomic and HPA axis activation

[Bradwejn et al 1991a, Bradwejn et al 1991b, Bourin et al 1998,]. Panic rates are at

least equivalent to a single breath of 35% CO2 [Koszycki et al 1991] and its major

drawbacks are its need to be given by intravenous infusion and cost [Bourin et al 1998].

32

In summary, the number of different challenges that have been used previously are

indicative of the lack of a single safe, simple and reliable test of the neuroendocrine

stress response. Many paradigms only target specific components of the stress response

system such as the behavioural response or the HPA axis, whilst others are either

difficult to perform and reproduce, costly or potentially dangerous. There was a

perceived need, therefore, to develop a paradigm that could safely and easily produce

reliable stress responses that could then be used to investigate the importance of these

systems in health and disease.

1.2.2. CO2 as a potential neuroendocrine stressor

Carbon dioxide is known to be the principal regulator of respiration, acid-base balance

and behavioural-state arousal in humans [West 1974]. Acting through central

chemoreceptors located predominantly within the ventrolateral medulla (VLM) of the

brainstem, small incremental changes in CO2 produce large changes in minute

ventilation (respiratory rate x tidal volume) to provide fine control over acid-base

homeostasis [West 1974]. In addition to regulating ventilation, changes in CO2

concentration are also associated with behavioural state arousal [Woods et al 1988]. In

particular, a rise in CO2 concentration is associated with increased arousal and a

perception of breathlessness or air hunger as well as sympathetic activation (see below).

This arousal is associated with activation of a number of different brain centres as

evidenced by functional MRI imaging during CO2 exposure. These brain centres are

also associated with other primitive feelings and behaviours including hunger, thirst and

pain [Brannan et al 2001, Liotti et al 2001]. It is likely this arousal, sympathetic

activation and fear produced by CO2 exposure has evolved as an instinctive protective

33

mechanism to warn and escape from an impending threat to respiratory homeostasis,

such as may occur with suffocation [Brannan et al 2001, Liotti et al 2001].

As a challenge paradigm, CO2 has been studied extensively in relation to its respiratory

physiology, its autonomic response (particularly the cardiovascular response to CO2

when it was used as an anaesthetic induction agent) and more recently its emotional and

behavioural response in anxiety research. There is also extensive literature on its safety

and methods of administration. A detailed review of the literature regarding CO2 and

the rationale for its choice in the development of a novel neuroendocrine stress

paradigm is provided in Chapters 2 and 3. Most importantly there was local expertise

within the University of Bristol on its use in psychiatric research involving human

subjects. Whilst formal neuroendocrine studies were limited and inconsistencies existed

in the described autonomic and cortisol responses to CO2 exposure, the current literature

did support the potential for CO2 as useful challenge in the investigation of the stress

response.

34

1.3 Summary, aims and hypotheses

At present, there is no good single test that can safely and reliably assess the activity of

the HPA, autonomic and prolactin stress response systems in man in either a clinical or

research setting. The aim of this thesis therefore, was to develop a novel stress test

based on the detailed study of the response to CO2 inhalation. A further aim was to use

this test to evaluate the stress response in normal individuals and selected patient

groups. In particular, the thesis aimed to:

i. evaluate the safety, reliability and reproducibility of a single breath of

35% CO2 as a model of the neuroendocrine stress response,

ii. evaluate the mechanisms by which the physiological and

neurohormonal responses occur and are regulated,

iii. evaluate various clinical populations using this model to determine the

influence of disease pathophysiology on the performance of the stress

response, and

iv. determine whether the characteristics of the stress response in specific

disease states are predictive of clinical outcome.

The principal hypothesis that this thesis sought to evaluate, therefore, was that a single

breath of 35% CO2 would reliably produce activation of the HPA and SAM axes.

Activation of other neurohormonal and autonomic components of the stress response

system may also occur and all of these responses could be readily measured in a clinical

setting. Further, it was hyopthesised that activation of these axes was dependent on

initial activation of brainstem noradrenergic centres, but that manipulation of the HPA

35

axis would either inhibit or augment other responses. Finally, it was also hypothesised

that the pattern of response to the challenge would vary predictably in circumstances

where underlying disease processes impacted on the function of the various components

of the stress response and that this information would have clinical relevance.

36

CHAPTER 2

METHODOLOGY

37

2.1. The single breath CO2 model

Existing tools available for the laboratory or clinical study of the neuroendocrine stress

response lack the necessary simplicity, safety, practicality and reproducibility that

would make any one test ideal. One of the principal objectives of this project was the

development of a test that would more closely meet these requirements and would

therefore be a more useful and applicable model for the study of the neuroendocrine

response to stress in man. The preferred model, a single breath of 35% CO2, was

chosen based on recent experience with various CO2 challenges as research tools for the

investigation of panic and anxiety. This included some experience with the

psychological responses to these challenges in experiments performed within the

Neuroendocrinology Research Centre and the Department of Psychopharmacology at

the University of Bristol.

The ability of CO2 to induce anxiety and the increased sensitivity of anxiety sufferers to

CO2 exposure was recognised in the early twentieth century [Drury 1919] and

subsequently during evaluation of its physiological and clinical properties, particularly

as an anaesthetic induction agent [Cohen and White 1951, La Verne 1953, Sechzer et al

1960, Davy 1972, Woods et al 1988, Rassovsky and Kushner 2003]. More recently,

researchers in psychiatry evaluated carbon dioxide inhalation as a potential treatment of

people suffering panic and anxiety disorders [Gorman et al 1984, Van den Hout MA

and Griez 1984, Verberg et al 2001]. Their demonstration that panic sufferers are, in

fact, more sensitive to the effects of CO2 administration led to the more detailed

evaluation of CO2 inhalation and the neurobiological pathways mediating this increased

susceptibility. Since then, several methods have been developed for the administration

of CO2 and the assessment of its response. There has, however, been little in the way of

38

standardisation of models in terms of equipment used, procedure and order of

administration, instruction given and response monitoring [Cohen and White 1951,

Lejuez et al 1998, Verberg et al 2001]. Most response monitoring has been in relation

to the induction of panic attacks the definition of which and its means of assessment has

also varied widely [Lejuez et al 1998].

Amongst anxiety researchers, the CO2 inhalation challenge has become the principal

tool for investigating panic as it has allowed the study of the neurobiological

mechanisms that underlie anxiety, and has also been useful in evaluating panic in the

clinical setting including the evaluation of pharmacological and cognitive therapies. In

contrast to other panic-inducing challenges such as the infusion of sodium lactate,

noradrenaline, CCK-4 and isoproterenol, or the administration of oral yohimbine or

caffeine [Cohen and White 1951], CO2 has proven to be a more efficient and more

sensitive challenge for provoking anxiety [Greiz et al 1990b, Sapolsky 1994, Perna et al

1995a, Lejuez et al 1998, Verberg et al 1998]. CO2 inhalation has also been described

as being a safer challenge with intense, but short-lived symptoms of anxiety [Cohen and

White 1951], with no significant adverse effects [Verberg et al 2001] that was relatively

easy to administer and non-invasive [Lejuez et al 1998].

In general, there exist three separate approaches to the administration of CO2 in humans

for the investigation of panic. Each method has been used widely by several groups and

a degree of variation in the approach to each exists. The three methods, as outlined

below, vary considerably in the equipment used for CO2 delivery, the relative

concentration of CO2 used and the duration of the exposure. The three methods, and

their respective advantages and disadvantages will be described briefly as these

significantly influenced the choice of method used for our studies.

39

i. The steady-state method.

This technique involves the delivery of a fixed concentration of CO2, typically 5 - 7.5%

CO2, for approximately 10-20 minutes or until a panic attack occurs. The subject either

wears a plastic hood placed over the head into which the CO2 is vented, or they breath

through a mask fitted over the nose and mouth into which a mixture of air and the

required CO2 concentration is delivered. The principal advantage of this technique is

that the timing of the CO2 exposure can be carefully controlled thereby reducing the

confounding effects of anticipatory anxiety [Lejuez et al 1998]. Disadvantages are the

significant anxiogenic effects of wearing the respiratory canopy which may also

stimulate other components of the stress response system [Sanderson and Wetzler

1990], the length of time each test takes, the variability in dose received [Gorman et al

1988] and the prolonged length of time subjects are experiencing significant anxiety

symptoms [Lejuez et al 1998].

ii. The Read re-breathing method.

Introduced in 1967 by Read [Read 1967], this technique requires the subject to breath

within a closed system attached to a mouth-piece whilst the nose is occluded with a

nose clip. The CO2, typically 5-7%, is combined with O2, however, as re-breathing of

expired air proceeds, the inspired CO2 concentration increases progressively.

Significant anxiety usually occurs after about 15 minutes. Problems with this method

include the length of time the test takes [Lejuez et al 1998], the variability in CO2

concentration [Gorman et al 1988] and the confounding anxiogenic effect of wearing a

mask and nose-clip for a considerable length of time [Askanazi et al 1980]. Its main

advantage is that it has been the most widely used technique historically.

40

iii. Single or double breath inhalation.

This method involves one or two full vital capacity inhalations of 35% CO2 combined

with either 65% O2 or air [Lejuez et al 1998]. Most commonly the gas mixture is

breathed in through a mask that covers the mouth and nose. The mask is connected to a

gas reservoir via a 3-way tap that is manually opened and closed as the subject receives

specific instructions on when to breathe. Its main advantage is its speed of onset and

very short duration of action. Thus it produces the highest level of systemic CO2 among

the three methods, but only lasts a very short amount of time. Anxiety symptoms are at

least as intense as the other methods (at their peak), but are extremely short lived. Its

main disadvantage is the potential confounding effect of anticipatory anxiety since the

onset of the breath (and therefore the CO2 exposure) is obvious to the patient. In

addition, some subjects find taking a second full vital capacity breath difficult.

Very little work has been done to directly compare the efficacy, safety and reliability of

the three methods. In a small study by Woods et al [1988] the panic rates produced by a

5% CO2 steady-state challenge compared to the Read re-breathing technique were

equivalent (67% vs 75%, respectively). Gorman et al [1994] and Papp et al [1997] both

administered 5% and 7% CO2 using the respiratory canopy method and demonstrated

increased panic rates with the higher dose compared to the lower dose. Further, it is

also not clear whether the three methods represent biologically similar or distinct

challenges based on their mechanism of action. Continuous exposure to low dose CO2

produces hypercapnia and respiratory acidosis [Sanderson and Wetzler 1990, Lejuez et

al 1998], whilst it has been suggested that a single breath of high dose CO2 produces an

initial brief hypercapnia that stimulates ventilation with the resultant hyperventilation

causing hypocapnia (as CO2 is blown) off with subsequent respiratory alkalosis

[Zandbergen et al 1989]. In the former situation panic is caused by the acidosis,

41

whereas in the latter situation it is caused by the alkalosis akin to the infusion of sodium

lactate [Peskind et al 1998].

The simplicity of the 35% single breath method and the rapidity of its apparent response

suggested this method was more suited to our stated test requirements. Additional

evidence that this method would be advantageous came from two studies of the HPA

response to CO2 exposure. As will be discussed in more detail, few studies have

examined stress, particularly HPA, responses to CO2. Results from studies of low dose

continuous CO2 exposure were contradictory with the most recent study and general

consensus favouring no significant HPA response to low dose CO2, despite the

induction of panic in panic disorder patients [Sinha 1999]. On the other hand, a pilot

study of a single breath of 35% CO2 in healthy volunteers performed at the University

of Bristol [Argyropoulos et al 2002] did demonstrate a cortisol response. No detailed

study of the neuroendocrine stress response to 35% CO2 has been conducted. Based on

the evidence above, there appeared a pressing need to evaluate the potential for a single

breath CO2 test to perform reliably in this setting. Consequently, I performed a

systematic and detailed review of the response to a single breath of 35% CO2 was

undertaken with particular regard to developing this as a potentially useful test in

evaluating the role of neuroendocrine stress response systems in health and disease in

man.

2.1.1. Design

Several models of CO2 administration have been described for both experimental and

clinical use. Most designs have relied on some form of manual administration of CO2

42

using a mask or mouthpiece, a 3-way valve and a balloon reservoir. Some groups have

developed computer-controlled automated delivery models [Lejuez et al 1998] that have

greater precision in gas delivery but are significantly more expensive and are useful

particularly for the delivery of low dose CO2 over many minutes. Manually controlled

delivery systems are more suited to and have been used more extensively in single

breath experiments. This was felt to be more appropriate for our model. Pre-mixed 10

L cylinders of medical grade CO2 and O2 were obtained from a commercial source

(BOC Gases, Guildford, Surrey, UK). For most experiments a concentration of 35%

CO2 / 65% O2 was obtained. A 10 L silicone reservoir (Douglas) bag (Hans Rudolf

Inc., Kansas City, Missouri, USA) was connected to the cylinder with silicone tubing

and could be filled directly from the source cylinder. Outflow from the Douglas bag

was through an analogue flow meter (Ohmeda Medical, Columbia, Maryland, USA)

and was controlled manually by a 3-way tap (Hans Rudolf). One port of the 3-way tap

received gas from the Douglas bag via the flow meter, with the opposite port connected

to either a silicone mouthpiece or face mask. The third port remained open to air. With

the subject breathing through the mouthpiece or facemask, the 3-way tap allowed the

investigator to control whether the subject was breathing normal air or the gas from the

Douglas bag. A simplified schematic diagram of the breathing circuit is given in Figure

2.01.

The flow meter allowed inspired vital capacity to be recorded. Based on previous

literature of the 35% CO2 breath test in panic, recording vital capacity is necessary as it

had been suggested that the challenge is not likely to succeed if an inadequate intake of

the gas is taken [Verberg et al 2001]. As such, vital capacity was recorded and a test

breath was considered adequate if it was at least 80% of a baseline air vital capacity

breath.

43

Subject Mouth- 3-way Flow Douglas CO2 piece tap meter bag cylinder

ii. CO2

i. Normal air

Figure 2.01. Schematic diagram of the breathing circuit used to deliver a

single breath of 35% CO2. Subjects breathe through a mouthpiece or facemask. With the tap vertical they would inhale and exhale room air only (i - top panel). With the tap turned horizontally, inhalation would be from the pre-filled Douglas bag containing the CO2/O2 mixture. Flow through the flow meter records inspired vital capacity (ii -lower panel). The tap would be manually returned to the vertical position before expiration so that exhaled gases would be to room air.

44

2.1.2. Procedure

Most tests were performed in a dedicated area of the University of Bristol’s Research

Centre for Neuroendocinology’s Clinical Investigation Unit. As will be discussed, for

the collaborative study with the Autonomic Unit at the National Hospital for Neurology

and Neurosurgery, Queen’s Square, London and St Mary’s Hospital, London, testing

was performed in the Clinical Investigation Unit of the National Hospital for Neurology

and Neurosurgery. For the study involving patients with Addison’s disease, testing was

performed in a dedicated room within the University of Bristol Exercise Physiology

department.

Each unit contained a comfortable chair and a bed and subjects could either remain

seated or resting on the bed during cannula insertion and for the time prior to the test

commencing. All tests were performed with the subject in the seated position.

Written informed consent was provided in all cases. Studies were approved by the local

ethics committee of the North Bristol United Healthcare Trust and where appropriate by

the ethics committees of the National Hospital for Neurology and Neurosurgery,

Queen’s Square, London and St Mary’s Hospital, Praed St, London.

For the initial study, which was essentially a descriptive pilot study, a sample size

power calculation was not performed. For subsequent studies where group comparisons

were being made, a power calculation was performed based on the size needed to detect

cortisol responders from non-responders. This was determined from the difference

between the cortisol response to 35% CO2 (responders) compared to 5% CO2 (non-

responders). Based on an approximate 30% increase in cortisol over baseline with a

standard deviation of the difference between responders and non-responders of 29.4

45

mmol/l, the calculated sample size was 10-12. Based on this, future studies aimed for a

sample size of 10 in each group.

Upon arrival subjects underwent a full clinical interview and physical examination

including baseline blood pressure and had an ECG performed. A 20 gauge intravenous

cannula (Venflon, Viggo Spectramed, Helingsborg, Sweden) was inserted into the

subjects antecubital vein and kept patent with 0.9% saline. Subjects then rested quietly

for 30 minutes and were allowed access to neutral reading material during this time. At

the end of the rest period, patients where shown the device used for the delivery of the

CO2 and were instructed in its use. All subjects received the same instructions.

Subjects where told that they would have their nose occluded with a nose-clip whilst

they breathed through the mouthpiece. The 3-way tap was demonstrated and it was

explained to the subjects that the tap would be held in such a position that they would

only be breathing room air. When instructed they would be asked to take a deep breath

in and exhale fully. The operator would then turn the tap to the open the Douglas bag

port, and the subject would be asked to take one full deep inspiration and hold it for

slow count of 4. The operator would then turn the tap back to the original position and

the subjects allowed to exhale and then asked to breathe normally. After 3 or 4 breaths

of room air through the mouthpiece, the device and nose-clip would be removed from

the subject.

In addition to instruction regarding the procedure, subjects were also given a brief

description of what they might experience from the challenge. Descriptions were

always the same with subjects being told that “the CO2 is harmless but they may feel

some very transient and short-lived feelings that might include feeling breathless and an

some cases anxious. The intensity of these feelings are variable with some people

46

experiencing very little whilst others may experience them more intensely”. Panic

attacks were not referred to at any stage.

At the end of the rest period, with the Douglas bag removed and using the mouth-piece,

3 way tap and flow meter assembly only, subjects performed a number of practice

breaths in order to get used to using the device, understand the instructions and to

record baseline vital capacity. The Douglas bag was then reconnected and filled with

the CO2 / O2 mixture. Physiological monitoring commenced and baseline blood samples

and questionnaires where obtained. Baseline cardiovascular monitoring continued for 5

minutes and at the end of this period the test breath was taken. As soon as the device

was removed, subjects were asked to complete a ‘peak response’ questionnaire and at 2

minutes post-exposure the first blood sample was taken. Further sampling and

questionnaires were collected at 10, 20, 30 and in some cases 40 and 60 minutes

following exposure. Cardiovascular monitoring continued for 5 to 6 minutes following

exposure.

2.1.3. Inclusion/exclusion criteria CO2 challenge tests have been used in psychiatric research for many years and standard

exclusion criteria have been published [Verberg et al 2001]. These criteria are not

based on actual occurrences of harm, but have been surmised based on the known

physiological effects of CO2. Challenge tests to date have been performed without

evidence of any significant adverse events [Lejuez et al 1998, Verberg et al 2001]. The

most likely side effect and therefore potential risk, is the generation of acute anxiety and

panic attacks. Unlike psychiatry research where the frequency of panic attacks is often

the principal outcome measure, panic provocation was not an intended objective of the

47

experiments conducted in this thesis. Indeed, whilst anxiety reactions and emotional

arousal were recorded, all attempts were made to exclude subjects who were felt to be at

significant risk of CO2-induced panic. As described earlier, anxiety sensitivity to 35%

CO2 varies considerably with panic disorder patients being most susceptible even

compared to sufferers of other forms of anxiety disorders such as obsessive-compulsive

disorder, social phobias and generalised anxiety disorder [Greiz et al 1990a, Greiz et al

1990b, Perna et al 1994, Perna et al 1995a, Perna et al 1995b, Verburg et al 1995,

Caldirola et al 1997]. However, some patients with anxiety disorders did respond in a

similar fashion to those with panic disorder [Caldirola et al 1997]. Healthy volunteers

have very low rates of panic in response to the 35% CO2 challenge. Depending on how

panic is defined, panic rates in healthy volunteers have been reported to range from 0%

to 2% [Griez et al 1987, Greiz et al 1990a, Perna et al 1995c]. First degree relatives of

patients with panic disorder, who themselves have never previously experienced panic

attacks, are however, more susceptible to panic in response to 35% CO2 compared to

normal individuals without a family history implying a genetic predisposition and

familial clustering of panic sensitivity [Perna et al 1995b, Perna et al 1995c, Bellodi et

al 1998, Verberg et al 2001]. Since the generation of panic attacks was not a desired or

necessary outcome of these studies, it was felt that subjects with a personal or family

history of panic disorder or other severe anxiety disorder who are more likely to be at

risk of panic attacks in response to the CO2 challenge should be excluded from

participation. Further, it should be noted that even in susceptible individuals, the

experience of experimentally induced panic with CO2 did not increase the likelihood of

spontaneous panic or the risk of panic in response to other panicogens in the weeks and

months following the original exposure [Harrington et al 1996, Perna et al 1999,

Verberg et al 2001].

48

In view of the cardiovascular changes that have been reported to occur in response to

CO2 exposure, particularly an increase in blood pressure, subjects with uncontrolled

hypertension, or those with a history of previous stroke, transient cerebral ischaemia,

cerebrovascular disease, angina, ischaemic heart disease or previous cardiac

arrhythmias were excluded. Severe chronic airways disease and asthma were also

exclusions on the grounds that high dose CO2 may be irritant to the airways and induce

bronchospasm. Other medical exclusion criteria included cerebral aneurysm, epilepsy

and pregnancy.

Normal volunteers were required to be in good physical health without any present or

past psychiatric history and to be medication free for the 2 weeks prior to participation.

Incidental use of simple analgesics was permitted up to the day before the test, but

inhaled or oral glucocorticoids or β-agonists were specifically excluded. As will be

explained in detail, clinical studies involving patients with diabetes, Addison’s disease,

autonomic neuropathy or post-lung transplantation were permitted to remain on their

usual treatments as long as this did not include α or β - agonists or antagonists, anti-

depressants or psychotropic medications or opiates. All subjects were asked to refrain

from alcohol, caffeine or nicotine consumption for 12 hours prior to undergoing testing.

Other inclusion/exclusion criteria that relate to the specific experiments of the clinical

population being tested will be detailed in the description and discussion of these

studies in subsequent chapters.

49

2.1.4. Monitoring

2.1.4.1. Ventilation

CO2 is the principal regulator of ventilation and small increases in PaCO2 significantly

increase ventilatory responses including respiratory rate, tidal volume and minute

ventilation. Several studies have examined changes in these respiratory variables in

panic disorder patients in response to the low dose CO2 challenge [Gorman et al 1988,

Papp et al 1997, Lejuez et al 1998]. Whilst results vary, it has been suggested that

disordered ventilatory responses (baseline hyperventilation, more rapid rises in

respiratory rate and minute ventilation and irregular breathing patterns) were more

common in panic disorder patients and were more likely to predict a panic attack in any

one individual [Gorman et al 1994, Papp et al 1997, Lejuez et al 1998]. Recording

ventilatory responses, however, requires additional equipment including a

pneumotachograph arrangement that can measure both inspiratory and expiratory flow

rates as well as calculate minute ventilation. This needs to be built into the breathing

circuit and requires the subject to be breathing through the circuit at all times.

Given minute ventilation (respiratory rate x tidal volume) is the principal respiratory

variable that changes in response to CO2 exposure, the speed of onset and recovery of

ventilatory responses to a single breath 35% CO2 makes this a less useful physiological

marker of this challenge. This is in contrast to challenges involving prolonged exposure

to low dose CO2. In addition, the additional equipment expense and the lack of

portability of the circuit further limit the utility of measuring respiratory variables if it

includes a pneumotachograph and the necessary amplifiers. Finally, many subjects find

it difficult breathing through a mouth-piece for more that a few breaths increasing the

50

potential for confounding from anxiety provocation associated with the use of a

pneumotachograph.

In view of this, it was felt that recording respiratory variables in this particular challenge

would add unnecessary expense, limit the portability of the test and add little in the way

additional useful information.

2.1.4.2. Psychology

Traditionally, the CO2 challenge has been used as a stimulus of panic, and panic

response rates have been the principal outcome measure of most challenge test to date.

Despite this, there is significant variability in the tools used by different groups to

measure panic or anxiety responses and little consistency between them in the definition

of panic attacks. Studies vary depending on whether they used a behavioural definition

of panic, observation of the subject by an experimenter or self-report measure [Lejuez et

al 1998]. A behavioural definition of panic, that is a request by the subject to terminate

the procedure, and observation of the subject are methods specifically designed to

record panic ‘frequency’. Self-report methods, on the other hand, are designed more

specifically to measure the ‘reactivity’ of an individual to the challenge and may also

allow the intensity of the reaction to be quantified [Lejuez et al 1998, Verberg et al

2001]. In the development of the 35% CO2 challenge, we were more interested in

assessing the degree of emotional arousal generated rather that in inducing panic

attacks, hence self-report methods were preferred.

Self-report tools are essentially pen-and-paper questionnaires that contain specific

symptom checklists. Each symptom has its own analogue scale whereby the subject can

51

rate their experience of that symptom on a continuum from ‘0 – I don’t feel this

symptom at all at the moment’ through to ‘100 – This is the worst I’ve ever experienced

this particular symptom’. These self-report questionnaires, also termed visual analogue

scales, are usually divided into two. The first set of questions relates to subjective global

feelings of anxiety. Questions, for example, include ‘To what degree do you feel

anxious’; ‘to what degree do you feel happy’; ‘to what degree do you feel fearful’ and

‘to what degree do you feel relaxed?’ The second set of questions includes a list of

somatic symptoms that are typically associated with acute anxiety or a panic attack.

Each symptom is rated on a similar scale. Several validated somatic symptom

questionnaires of panic exist including the one used in our studies developed by Nutt et

al [1990]. Questionnaires are administered to assess baseline (immediately before

exposure) and peak (immediately after exposure) experiences, the calculated difference

being the effect of the challenge itself. Examples of the VAS used in the subsequent

studies are attached as appendix 1 and 2.

One problem with this technique is the ‘ceiling effect’. That is responses greater than

100 are not possible therefore for an individual with baseline score of 90, an increase of

only 10 is possible. In other words whether a change from 90 to 100 is the same as a

change from 0 to 10 can be argued.

Some groups have used similar scales to arbitrarily define a panic attack in response to

CO2. In one study [Rapee et al 1992] for example, panic was defined as a score of 1 out

of a maximum of 8 (a score equivalent to 13 out of 100 on the VAS we used) for feeling

fear or panic. Using this criterion, they found panic rates of 65%. The same authors

then altered the criteria to a score of at least 5 (equivalent to at least 63 out of 100) and

found panic rates of 41%. Other groups required a similar a score (50-60 out of 100) in

4 of 13 panic-like symptoms to define a panic attack [Verberg et al 2001]. Neither of

52

these criteria, however, took into account the duration of symptoms, that is whether they

were felt just fleetingly or for several minutes. In the studies described herein, self-

reporting scales were used to rate individual reactivity to the challenge and were not

used to define panic. Panic attacks were defined according to a combination of

observation, behavioural responses and self-reporting whereby subjects who described

severe anxiety with panic-like symptoms that lasted for at least several minutes were

considered to have had a panic attack. As will be detailed for each experiment, all

responses to the 35% CO2 challenge were transient and well tolerated. No subject

described or was observed to have panicked in response to the challenge.

2.1.4.3. Cardiovascular physiology

Cardiovascular responses to CO2 exposure have been studied for many years. Rodents,

mammals and non-human primates have been extensively studied. In addition, many

human studies have been performed, although most have involved chronic or sub-acute

exposure. Studies of acute exposure (single breath) are more limited particularly

recently. As has been described in more detail, results have varied depending on the

method and concentration of CO2 administration, but in general responses have

included an increase in systolic blood pressure and tachycardia [Hardgrove et al 1938,

La Verne 1953, Tenney 1956, Sechzer et al 1960, Tenney 1960, Cross and Silver 1962,

Cullen and Edgar 1974, Woods et al 1988, Argyropoulos et al 2002, Bailey et al 2002].

Increased diastolic blood pressure, cardiac output and stroke volume have been reported

in some, but not all studies in which they were measured [Tenney 1956, Sechzer et al

1960, Tenney 1960, Woods et al 1988]. Similarly, catecholamine release, particularly

noradrenaline and on some occasions adrenaline, has been reported in some but not all

53

studies [Hardgrove et al 1938, Tenney 1956, Sechzer et al 1960, Tenney 1960, Cross

and Silver 1962, Woods et al 1988, Krystal et al 1989]. Reports of changes in total

peripheral resistance have been variable [Hardgrove et al 1938, Cullen and Edgar 1974,

Woods et al 1988].

Techniques for non-invasive measurement of cardiovascular parameters including

systolic and diastolic blood pressure, heart rate, cardiac output, stroke volume, total

peripheral resistance and the pre-ejection period have increased substantially in recent

years. The first experiment described herein was performed using a Finapress®

(Ohmeda) device that measures beat-to-beat blood pressure and heart rate responses

(see Chapter 3). Subjects wear a self-inflating finger cuff that contains a photosensitive

cell connected via a servo-controlled pump that inflates the cuff to maintain a constant

pressure. The cuff is worn on the opposite hand to the arm in which the cannula is

inserted so as to avoid problems with blood sampling. The hand must be held still,

approximately at the level of the heart. This recording device was a very useful tool for

recording continuous changes in cardiovascular parameters. The main problem that

arose with the use of the device was interference related to hand movement. Any

movement of the hand on which the cuff was worn caused significant variation in

readings causing falsely elevated or reduced readings. This was a particular problem at

the time when the CO2 was being inhaled as subjects found it difficult to keep from

moving their hands. The Finapress® proved difficult to use with several results

uninterpretable. Instead the simpler Dynamap (Critikon, Tampa, Florida, USA)

automated blood pressure recording device was used for the majority of studies. With

this device, a self-inflating upper arm blood pressure cuff that measures heart rate and

mean arterial pressure is worn. The device then derives systolic and diastolic blood

54

pressures. It can be set to record values every minute. The device is not sensitive to

the effects of movement in the hand.

A collaborative study performed at the University College of London’s Autonomic Unit

(located within the National Hospital for Neurology and Neurosurgery) took advantage

of the availability of more sophisticated cardiovascular monitoring equipment (see

Chapter 5). In this study, a more advanced form of the Finapress®, the Finometer®

(Ohmeda) was used. This monitor also used the self-inflating finger cuff principal but

was able to also self-correct for hand position relative to the heart and was more

insensitive to interference from hand movement. The Finometer® was also used to

obtain other cardiovascular parameters including cardiac output, stroke volume, total

peripheral resistance and the pre-ejection period.

Also available in this collaboration was a laser flow doppler (Perimed) that was used to

measure skin blood flow responses in the hand (see Chapter 5). The laser doppler uses

two laser probes to measure changes in blood flow in very small blood vessels of the

hand compared to the forearm. Flow changes in these blood vessels of the hand

represent sympathetically mediated vasoconstriction or vasodilatation whilst the

forearm measures correct for the effect of movement.

Samples for plasma catecholamines were taken from most subjects and a High

Performance Liquid Chromatography (HPLC) assay was developed within the

University Research Centre for Neuroendocrinology (see below) for their measurement.

For technical reasons only samples from the first experiment were successfully analysed

with this assay. The Autonomic Unit, National Hospital for Neurology and

Neurosurgery, Queen Square, London, using an established HPLC method, performed

55

the assays on samples from the collaborative study. This group has published

exensively using this assay [see Kimber et al 2000; 2001 as examples]. Intra- and inter-

assay coefficients of variation were 4.7 and 4.3% respectively for noradrenaline, 4.6 and

5.1% respectively for adrenaline.

2.1.4.4. Biochemistry

Blood samples for hormone levels were taken at various time points before and after the

CO2 exposure through the intravenous cannula. For the first experiment, on each

sampling occasion, 15 ml of venous blood was taken in 3 x 5 ml aliquots. One aliquot

was collected into a tube containing SST and assayed within 24 hours for cortisol,

prolactin, GH, TSH, FSH and LH. The remaining 2 aliquots were collected into pre-

chilled tubes containing lithium heparin (LiHep) and EDTA respectively. These

samples were then immediately cold centrifuged at 4°C, the plasma separated and stored

at –50°C until assayed for plasma renin activity, AVP (LiHep) and for ACTH (EDTA).

In the studies where plasma catecholamines were measured, samples were collected into

pre-chilled LiHep tubes to which 1ml of EGTA (anti-oxidant) was added. Samples

were immediately cold centrifuged at 4°C, the plasma separated and stored at –80°C

until assayed. Saliva samples collected using salivettes (Sarstedt) were stored at –20°C

until assayed for cortisol and salivary amylase.

Standard immunometric assays (Immulite 2000, DPC, Los Angeles, California, USA)

were used for plasma cortisol, prolactin, GH, TSH, FSH, LH and salivary amylase.

These assays were performed at cost by the Department of Chemical Pathology, Bristol

Royal Infirmary. Intra- and inter-assay coefficients of variation (CV’s) were 5.3% and

56

7.2%; 2.5% and 6.9%; 3.4% and 5.5%; 2.1% and 4.3%; 3.6% and 6.7%; 3.7% and

5.3%; 2.0 and 3.4%, respectively.

Salivary cortisol assays were performed at cost by Cultech Ltd (Swansea, UK) using the

Neogen cortisol ELISA kits (Neogen, Lamsing, Missouri, USA). Renin was assayed at

cost by the Blood Pressure Unit, Western Infirmary (Glasgow, UK) according to the

method of Miller et al [Miller et al 1980].

ACTH assays were performed by myself within the URCN using a commercially

available radioimmunoassay (DSL, Webster, Texas, USA). Samples were analysed in

duplicate and the CV’s for the assay were 5.9% and 7.3% respectively.

AVP assays were kindly performed by Dr Mary Forsling, King’s College, London using

a double antibody radioimmunoassay following separation from plasma proteins by

methanol extraction and chromatography.

Figures 2.02, 2.03 and 2.04 demonstrate how physiological monitoring equipment was

applied and Figures 2.05 and 2.06 demonstrate the administration of the CO2.

57

a

bc

Figure 2.02. Arrangement used for patient monitoring. a: Laser doppler probes b: Intravenous cannula c: Finometer® finger probe and transducer

58

c

b

a

Figure 2.03. Arrangement used for patient monitoring. a: Laser doppler probes b: Intravenous cannula c: Finometer® finger probe d: Laser doppler

d

59

c

b

a

Figure 2.04. Arrangement used for patient monitoring. a: Finometer® monitor b: Laser doppler c: Laser doppler monitor

60

a

b

c d

e

Figure 2.05. Arrangement for CO2 delivery.

a: Nose-clip b: Mouth-piecec: Manual control valve (3-way tap) d: Flow meter e: Douglas bag

61

a c d

be

f

Figure 2.06. Arrangement for CO2 delivery.

a: Nose-clip b: Mouth-piecec: Manual control valve (3-way tap) d: Flow meter e: Douglas bag f: CO2 cylinder

62

2.2. Plasma catecholamine HPLC

A number of methods have been developed for the measurement of catecholamines

(adrenaline, noradrenaline and dopamine) in human plasma. HPLC with

electrochemical detection being the most commonly used technique as it provides the

greatest sensitivity and specificity. Since its initial description by Kissinger et al

[Refshauge et al 1974] in 1973, several modifications and variations have been

described, however, it remains a difficult assay to perform [Forster and Macdonald

1999, Raggi et al 1999].

The principal of electrochemical detection is based on the detection of a change in an

electrical current maintained between two electrodes that occurs as the result of

oxidation or reduction of the substance of interest [Raggi et al 1999]. The electrical

potential needed to oxidise or reduce a particular substance (polarising potential) is

maintained between two electrodes. An electrochemical substance passing between the

electrodes will be oxidised or reduced leading to a gain or loss of electrodes. The

resulting current is detected by a measuring instrument, amplified and displayed as a

chromatographic signal. Since only a limited number of substances have the same

polarising potential, electrochemical detection typically has a high sensitivity and

specificity for identification of the substance of interest. Specificity, however, is

diminished if several compounds are oxidised or reduced at a similar polarising

potential. Interference from catecholamine metabolites and other substances within the

plasma or derived from the extraction procedure (see below) or mobile phase is one of

the major problems with plasma catecholamine HPLC assays [Forster and Macdonald

1999]. The second major problem is the low yield of plasma catecholamines in the

plasma (especially under resting conditions) [Raggi et al 1999]. Specificity and

63

sensitivity of the assay can be improved by extracting catecholamines from the plasma

first (purification step) in order to remove interfering substances and to increase the

yield of catecholamines within the sample as much as possible. This purification step

involves binding the catecholamines to alumina [Benedict 1987, Raggi et al 1999], a

boric acid gel [Imai et al 1988] or organic solvents [Smedes et al 1982, Forster and

Macdonald 1999] under alkaline conditions, washing or removing the remaining plasma

and as many impurities as possible and then, under acidic conditions, unbinding and

collecting the catecholamines prior to undertaking chromatographic analysis [Raggi et

al 1999].

Sample collection is equally important, as catecholamines are rapidly oxidised at room

temperature. Sample collection requires pre-chilled heparinised tubes, with the

immediate addition of an anti-oxidant such as EGTA, cold centrifugation at 4°C and

freezing at –20 to –80°C prior to analysis.

Commercially available kits are available for the extraction phase of the assay.

However, we initially chose to develop an assay based on published protocols for the

extraction of catecholamines from human plasma [Forster and Macdonald 1999, Raggi

et al 1999] and advice from Dr Ian MacDonald, School of Biomedical Sciences,

Nottingham University Medical School, Queen’s Medical Centre, Nottingham, UK.

These protocols used the addition of alumina to a plasma sample for catecholamine

binding, washing of the alumina to remove impurities and the unbinding the

catecholamines with perchloric acid. Protocols used in the development of the assay are

given in appendices 3, 4 and 5.

Once extracted, catecholamines were run through a 15 x 3.9 mm reverse phase

Bondapak column (Waters, Milford, Massachusets, USA) with a Waters 510 pump and

64

a programmable sample processor (Waters 710-B WISP). Quantification was on a

Waters M-460 dual electrochemical detector with a working potential of +0.74 V.

Mobile phase was HPLC grade water with 130 ml methanol, 35 mg EDTA, 9.53 g

potassium dihydrogen orthophosphate dihydrate (KH2PO4) and 200 g octanesulphonic

acid to a pH of 3.4.

Standard solutions of adrenaline and noradrenaline with 3,4-dihydroxybenzylamine

hydrogen bromide (DHBA) as internal standard were made and standard curves created.

Sample concentrations were estimated from comparisons of their peak heights with

known concentrations of pure standards. Calculated catecholamine concentrations were

expressed as pg/ml plasma. Examples of obtained chromatograms are shown in Figures

2.07, 2.08 and 2.09. To determine the sensitivity of the assay, a lower limit of

dectection study was performed. This involved serial dilutions of known concentrations

of stantards (noradrenaline and adrenaline). Concentrations tested were 1000, 500, 250,

25 and 2.5 pg/ml. Serial dilutions of plasma samples spiked with known concentrations

of standards were also performed to assess recovery rates. Technical difficulties (as

described below) hampered the interpretation of these procedures and further attempts

to optimise the essay were not pursued.

Sample recovery using this technique varied from 50 – 65%, but the technique was used

to measure catecholamines from the first CO2 dose response study performed. Shortly

after this, however, a laboratory fire damaged several pieces of equipment including the

HPLC system. It was decided by the laboratory to replace the system with the more

up-to-date Coulochem II® system (ESA Inc, Chelmsford, Massachusets, USA). This

system combined the Coulochem II® Electrochemical detector with a 582 solvent

delivery module (ESA) and an ESA 542 Autosampler. The system also used the EZ

Chrom Elite™ (ESA) software system for chromatogram display and calculations.

65

With this system, it was also decided to obtain catecholamine extraction kits from the

same company. These kits used syringes pre-loaded with an alumina matrix (PCAT

analysis kits, ESA) and a vacuum manifold (ESA) in order to run plasma samples

through the alumina. Once bound, a supplied acidic eluting solution was run through

the same matrix to extract the catecholamines. Samples were then run through an MD-

150, 3 µm, 3.2 x 15 mm reverse phase column (ESA) and quantified with a dual

electrode detector (ESA Coulochem II®). Internal standard was again DHBA and

calculated catecholamine concentrations were expressed as pg/ml plasma. Mobile

phase was the Cat-A-Phase® II Mobile Phase (ESA) provided as part of the kit. Pure

standards provided with the kit were used to create a standard curve. Recovery using

this method varied from 30 – 75% and was often inconsistent. Examples of

chromatograms are shown in Figures 2.10, 2.11 and 2.12. Several attempts using

spiked plasma and experimental samples were made using this new system and

methodology were made but results were inconsistent and unreliable and have not been

reported in any of the studies where this methodology was used.

66

Figure 2.07. HPLC chromatogram of a standard mixture of

catecholamines (noradrenaline, adrenaline, DHBA (internal standard) and dopamine.

67

Figure 2.08. HPLC chromatogram following successful extraction of catecholamines from a plasma sample.

68

Figure 2.09. HPLC chromatogram following successful extraction of catecholamines from a plasma sample.

69

Figure 2.10. HPLC chromatogram demonstrating poor recovery of

catecholamines (including internal standard) from a plasma sample.

70

Figure 2.11. HPLC chromatogram following unsuccessful extraction of catecholamines from a plasma sample.

71

Figure 2.12. HPLC chromatogram following unsuccessful extraction of catecholamines from a plasma sample.

72

2.3. Salivary amylase

In 1996 Chatterton and others [Chatterton et al 1996] described the use of salivary α-

amylase as a measure of the plasma catecholamine response to various stressors in

humans. The physiological role of salivary amylase is principally in the initiation of

starch digestion, but it also has a role in the maintenance of tooth integrity, inhibition of

bacterial adherence and colonisation and reducing available sugar substrate for bacterial

growth [Pedersen et al 2002]. The overall composition of saliva, including the

concentration of α-amylase, is strongly dependent on salivary flow rate [Pedersen et al

2002]. This was thought to be the likely mechanism by which α-amylase levels reflect

plasma catecholamine levels. With sympathetic stimulation, salivary flow and therefore

α-amylase levels, increase proportionally. However, salivary flow rate is dependent on

many different factors, including the type and size of the principal salivary gland from

which the saliva is being secreted as these differ in their response to stimulation and in

their secretory composition [Busch et al 2002, Pedersen et al 2002]. Other factors

include the state of hydration; nutritional state; the time of day as salivary flow follows

a circadian rhythm; the nature and duration of the stimulus; emotional state and gender

[Pedersen et al 2002].

In the study by Chatterton et al [1996], salivary amylase and plasma noradrenaline and

adrenaline levels were measure following exposure of normal individuals to a variety of

stressors including exercise, written examination and thermal stress. They found a

significant correlation between α-amylase and noradrenaline following both exercise

and examination stress. Neither adrenaline nor salivary cortisol correlated with α-

amylase. The α-amylase level also increased with both heat and cold, although

corresponding catecholamine levels were not measured. Based on these results, it was

73

concluded that salivary α-amylase levels reflected plasma catecholamine levels

particularly noradrenaline [Chatterton et al 1996].

Several animal studies exist that have shown sympathetic stimulation increases both

salivary flow and α-amylase levels, although this is dependent on both the stimulus and

the gland being studied. Busch et al [2002] studied α-amylase release from rat parotid

and sub-mandibular glands following administration of isoproterenol (a β-adrenergic

agonist). In this study, β-adrenergic stimulation increased α-amylase release from the

parotid but not the sub-mandibular glands. Similar studies of the effect of sympathetic

stimulation and inhibition on salivary flow and α-amylase release have not been

performed.

Following the initial study by Chatterton et al [1996], he and several other groups have

published studies that have reported salivary α-amylase as a surrogate marker of plasma

catecholamine/noradrenaline release. In a study of the hormonal response to skydiving,

Chatterton et al [1997] identified significantly elevated α-amylase levels on the day of

the jump compared to a rest day, with a significant rise in α-amylase levels from

baseline following the jump. This pattern was similar to noradrenaline levels measured

in other parachute jump experiments [Richer et al 1996] and the authors conclude this is

further evidence validating the use of salivary amylase as a marker of catecholamine

activation [Chatterton et al 1997]. Interestingly, however, in the first parachute jumping

study [Chatterton et al 1996], there is no mention of other features of sympathetic

stimulation including heart rate and blood pressure changes. In the second study,

[Richer et al 1996], heart rate responses correlated with adrenaline release but not with

noradrenaline.

74

Nater et al [2002] measured salivary α-amylase and plasma catecholamine responses to

a psychological stressor (the TSST). Whilst the test increased salivary α-amylase,

noradrenaline and adrenaline significantly, there was no correlation between the α-

amylase response and the noradrenaline or the adrenaline response.

Several other groups have reported salivary α-amylase responses to a variety of

stressors as a surrogate of plasma catecholamines. Xiao et al [2000] examined the

salivary α-amylase response to three simulated trauma cases in 10 anaesthetists. The

first case simulated routine management (and therefore low stress), whilst the 2nd and

3rd cases were designed to contain stressful management events. Noradrenaline, blood

pressure and heart rate responses were not measured. Salivary amylase levels were high

before starting and after the first 2 cases (routine and stressful), but not after the third

(stressful) case indicating a lack of correlation between the degree of perceived stress

and α-amylase responses.

Hojo et al [2003] measured α-amylase as well as blood pressure, heart rate and cortisol

responses to a distressing video challenge in healthy volunteers. Their results showed a

significant increase in both α-amylase and cortisol in response to the challenge, but no

change in either blood pressure or heart rate. Similarly, Morrison et al [2003] studied

intensive care nurses during real life working stress. They found a significant

correlation between noise stress, subjective annoyance and heart rate changes. Salivary

amylase levels showed large inter-individual variation and did not correlate with heart

rate or subjective measures of stress.

In summary, following a single study showing a correlation between salivary α-amylase

and noradrenaline during an acute stress paradigm [Chatterton et al 1996], several

75

different authors have accepted this as a surrogate marker of catecholamine (particularly

noradrenaline) production. However, one study failed to show a significant correlation

between salivary α-amylase and either noradrenaline or adrenaline, whilst others have

failed to measure or show a relationship between α-amylase and other markers of

sympathetic activation including heart rate and tachycardia. Given the difficulty in

collecting and measuring plasma catecholamines, a simple surrogate marker would be

extremely useful in stress research. The simplicity of measuring α-amylase and the

apparent initial correlation makes this test extremely attractive, however, the evidence

as summarised above appears conflicting.

As part of the studies described in this thesis, salivary amylase levels were measured in

both healthy subjects undergoing the 35% CO2 challenge, as well as several of the

clinical populations. The further evaluation and validation of salivary α-amylase as a

non-invasive surrogate marker of sympathetic activation is due to be further

investigated by other researchers at the University of Bristol. Planned studies include

the measurement of α-amylase in response to the insulin hypoglycaemia challenge;

further studies of the 35% CO2 challenge correlating α-amylase levels with

catecholamine responses; and studies of α-amylase in response to α and β-adrenergic

stimulation and inhibition in humans.

In this thesis, for each individual study where α-amylase was measured, the effect of the

CO2 challenge will be described in detail. However, in summary, there appeared to be

marked inter-individual variation in the α-amylase response to the challenge. This

variation included both the intensity and the timing of the response. Further, in healthy

individuals, there was no correlation between amylase responses and noradrenaline,

SBP, DBP, HR, cortisol, prolactin or any of the psychological parameters measured.

76

Similarly, despite some controls and patients groups differing significantly in baseline

noradrenaline, cortisol, SBP or heart rate, there was no difference in their resting

amylase levels.

It appears therefore that salivary α-amylase may not be a good surrogate marker of

sympathetic stimulation, at least in this challenge paradigm. The further studies

mentioned above should help to clarify its role as a non-invasive measure of activation

of the sympathetic stress system.

77

CHAPTER 3

THE 35% CO2 MODEL: INITIAL DESCRIPTION

78

3.1. Rationale for the use of CO2 as a neuroendocrine stressor

There were several reasons for thinking that CO2 exposure would be an appropriate

challenge for the investigation of the stress response. Ideally, the most useful challenge

paradigm would be one that:

i. Is safe for use in humans,

ii. Reliably produces emotional, hormonal and autonomic responses akin to those

described for the general stress response,

iii. Produces responses that are consistent, reproducible and occur in a dose dependent

manner [Bourin et al 1998],

iv. Is simple to administer and produces responses that are easily recorded,

v. Is inexpensive and portable.

As mentioned, several laboratory based paradigms for the evaluation of the stress

response exist and the specific pros and cons of these existing tests have already been

detailed. Existing paradigms have used a variety of psychological, physical and

pharmacological stressors. However, all of these tests have their own specific

limitations, risks and benefits and as yet none fulfils all the criteria listed above. The

lack of an existing suitable neuroendocrine stressor has led us to evaluate CO2 as a

potential model of the stress response in order to develop a test that might have broad

use in both a research and clinical setting.

79

3.1.1. CO2 physiology

Spontaneous respiration is an automatic function that originates in the brainstem and

relies on the rhythmic firing of ‘inspiratory’ and ‘expiratory’ neurons [Nattie 1999].

Control of breathing relies on several chemical (O2, CO2 and pH) and mechanical

factors (lung volume changes, respiratory and other muscle and joint inputs) [Thews

1983, Nattie 1999]. Central chemoreceptors, located predominantly on the ventral

medullary surface of the brainstem are sensitive particularly to changes in arterial and

alveolar CO2 concentrations and provide the principal means of regulating metabolic

acid-base balance [West 1974, Nattie 1999]. Small changes in CO2 levels produce

rapid and dramatic changes in minute ventilation (the product of respiratory rate and

tidal volume). Ventilation, for example, doubles in response to a 2 mmHg rise in

PaCO2 [Nattie 1999]. This is in stark contrast to changes in O2 levels which, sensed

exclusively through peripheral chemoreceptors, have little effect on minute ventilation

until PaO2 levels have fallen from normal values (100 mmHg) to around 60-70 mmHg

[Thews 1983]. More recently, evidence has accumulated indicating that central CO2

chemoreceptors are more widely distributed than previously thought. In addition to the

ventral medullary surface, CO2 chemoreceptors are present throughout the brainstem,

including such areas as the locus coeruleus, raphe complex and pons and are also

present within the cerebellum [Nattie 1999]. In addition to their anatomical

distribution, these chemoreceptors also appear to vary in their sensitivity and thresholds

to activation by CO2 and it has been postulated that this reflects the importance of CO2

in the regulation of other homeostatic functions beyond ventilation [Nattie 1999]. Some

of these functions include sleep and sleep arousal, the regulation of metabolic rates for

temperature control and change in ventilation in order to sustain the demands of

exercise [Nattie 1999].

80

From an evolutionary perspective, it has been suggested that hypercapnia (such as may

occur in impending suffocation) is perceived by the organism as an immediate threat to

life, and the response demands not only a change in ventilation, but also behavioural

arousal, autonomic cardiovascular activation and a motor response in order to escape

from this threat [Nattie 1999, Brannan et al 2001, Liotti et al 2001]. It is not surprising

therefore, that exposure to CO2 will consistently produce not only hyperventilation and

an increase in minute ventilation, but will also produce cardiovascular activation

(including tachycardia and hypertension) as well as marked behavioural and emotional

arousal [Hardgrove et al 1938, Davy 1972, Sechzer et al 1960, Cross and Silver 1962,

Gorman et al 1988, Woods et al 1988, Greiz et al 1990a, Greiz et al 1990b, Perna et al

1994, Papp et al 1997, Bourin et al 1998, Verberg et al 1998, Sinha 1999, Argyropoulos

et al 2002, Bailey et al 2002].

3.1.2. CO2 as a psychological stressor

The feelings generated when inhaling high doses of CO2 were noted as far back as 1800

[Davy 1972], and it was first postulated as a trigger for anxiety in 1951 by Cohen and

White [Cohen and White 1951]. Detailed experiments, however, were only first

conducted in the early 1980’s. Gorman and others [Gorman et al 1984, Verberg et al

2001], based on the association of panic with hyperventilation (with resultant

hypocapnia) and the observation that attacks could be prevented by breathing into a

paper bag (to raise PaCO2), hypothesised that breathing low doses of CO2 (5%) should

reduce anxiety sensitivity. Paradoxically, however, it was found that more subjects

panicked in response to CO2 than did in response to hyperventilation [Gorman et al

81

1984]. Subsequent to this, several groups have studied the anxiogenic effects of CO2

exposure in more detail. As has been discussed, several different paradigms have been

used to administer CO2 to both healthy volunteers and patients with a variety of anxiety

related disorders [Read 1967, Sanderson and Wetzler 1990, Lejuez et al 1998]. Low

dose CO2 exposure (for example, breathing 5-7.5% CO2 over 10 – 15 minutes) will

provoke the gradual onset of hyperventilation and feelings of breathlessness or air

hunger [Gorman et al 1988, Woods et al 1988, Papp et al 1997, Brannan et al 2001,

Liotti et al 2001]. Additionally, it is associated with many of the somatic symptoms of

acute anxiety including dizziness, blurred vision, feeling hot or flushed and feeling that

the heart is racing. In addition, in some individuals low dose CO2 will provoke panic

attacks [Gorman et al 1984, Van den Hout MA and Griez 1984, Rapee et al 1992,

Gorman et al 1994, Rassovsky and Kushner 2003].

A single breath of 35% CO2, based on a model used by Wolpe [1958, Verberg et al

2001] in 1954 for the treatment of anxiety, was investigated further by several groups

including Van den Hout and Griez [1984]. Studies by these groups found that this

challenge provoked strong somatic symptoms associated with anxiety as well as

autonomic activation in most subjects, but induced specific feelings of anxiety and

panic only in subjects with an underlying diagnosis of panic disorder [Van den Hout

MA and Griez 1984, Griez et al 1987, Greiz et al 1990a, Verberg et al 2001]. Since

then, numerous groups performing similar studies have reinforced the reliability and

safety of CO2 as a biological challenge [Greiz et al 1990b, Perna et al 1994, Perna et al

1995a, Verburg et al 1995, Verberg et al 1998]. These groups have also identified

panic disorder patients, as having a specific sensitivity to CO2 exposure, as opposed to

patients with other forms of anxiety disorders [Perna et al 1995a, Verburg et al 1995,

Caldirola et al 1997]. Further, they have also indicated an inherited tendency towards

increased CO2 sensitivity in first degree relatives of panic disorder patients [Perna et al

82

1995b, Perna et al 1995c, Harrington et al 1996, Perna et al 1999] and the importance

of specific pharmacological agents in treatment of panic attacks [Van den Hout et al

1987, Pols et al 1991, Pols et al 1993, Pols et al 1996a, Pols 1996b, Bertani et al 1997,

Perna et al 1997, Nardi et al 2000, Battaglia et al 2001, Meiri et al 2001, Perna et al

2002, Bertani et al 2003].

3.1.3. CO2 as a cardiovascular stressor

Much of the existing literature on autonomic cardiovascular responses to CO2 exposure

stems from physiological studies of low dose CO2 related to the use of CO2 in

anaesthesia and mild to moderate hypercapnia as it occurs particularly in respiratory

disease and artificial ventilation. The commonly used models of both low and high

dose CO2 exposure in psychiatric research have only very infrequently recorded

autonomic responses.

Studies of chronic low dose (5-10%) CO2 exposure in animals and non-human primates

have described sympathetic nervous system stimulation with tachycardia, increased

systolic and diastolic blood pressures and an increase in catecholamine metabolites

[Tenney 1956, Woods et al 1988, Krystal et al 1989]. In one animal study [Witzleb

1983], CO2 exposure during artificial ventilation (with respiratory rate held constant)

resulted in a significant pressor response but with bradycardia.

In humans, similar CO2 concentrations have consistently reported increases in systolic

blood pressure and most have reported tachycardia [Hardgrove et al 1938, Sechzer et al

1960, Tenney 1960, Rammana Reddy et al 1986, Gorman et al 1988, Woods et al

1988]. Studies vary, however, in the observed changes in other cardiovascular

83

parameters, with some showing increases in cardiac output, stroke volume and

decreases in total peripheral resistance [Sechzer et al 1960, Gorman et al 1988]. Other

studies in contrast have shown no change in cardiac output or stroke volume and

increases in total peripheral resistance [Kety and Schmidt 1948, Tenney 1960, Witzleb

1983]. Similarly, despite the consistency of blood pressure responses, catecholamine

production has been inconsistent with some studies showing an increase in both

adrenaline and noradrenaline [Sechzer et al 1960, Tenney 1960] with others showing

either an increase in noradrenaline only [Rammana Reddy et al 1986] or no

catecholamine response at all [Woods et al 1988]. The variance in responses between

the studies has been attributed to the significant variation in CO2 dose and duration of

exposure as well as differences in monitoring techniques [Tenney 1960].

A single study of brief exposure (25 seconds) to 20% CO2 [Zvolensky et al 2001]

reported a significant increase in heart rate, but did not examine blood pressure or other

physiological responses. Very few of the 35% CO2 challenge studies have evaluated

cardiovascular responses. One study by Mieri et al [2001] showed an increase in heart

rate with a trend towards increased noradrenaline levels, whilst the recent study by

Argyropoulos et al [2002] showed a significant increase in systolic blood pressure that

was associated with a significant fall in heart rate.

3.1.4. CO2 as a neurohormonal stressor

Studies of the neurohormonal response to CO2 exposure are limited and the results

conflicting. In a dose ranging study of prolonged CO2 exposure in rhesus monkeys

(5%, 7.5% and 10% CO2 for up to 180 minutes), Krystal et al [1989] showed a dose

84

dependent increase in cortisol, GH and prolactin. Similarly, Woods et al [1988] showed

a dose dependent effect of low dose CO2 exposure in humans. In this study, 15 minutes

of 5% CO2 had no effect on cortisol levels, whilst there was a small increase in cortisol

following the same duration of 7.5% CO2. Neither dose produced a significant change

in GH or prolactin levels. Other similar low dose exposure studies have also shown a

significant increase in cortisol levels [Sechzer et al 1960, Tenney 1960]. In contrast,

Sinha et al [1999] did not demonstrate cortisol release in panic disorder patients with a

5% CO2 paradigm despite generating significant panic attacks in this subgroup.

In response to a single breath of 35% CO2 Argyropoulos et al [2002] showed a

significant cortisol rise, although in contrast, no cortisol increases where seen with this

challenge in studies by Meiri et al [2001] or Van Duinen et al [2004, Griez and

Schreurs 2003]. Meiri et al [2001] was also unable to demonstrate a prolactin response

despite significant anxiety responses. Differences in responses have again been

attributed to the small number of studies done with varying methodologies.

In summary, the existing literature regarding CO2 is extensive with a large amount of

data available on its use and safety in humans. Models of CO2 exposure used for the

investigation of anxiety disorders are well described and have been shown to be safe

and reliable. Data regarding autonomic and neurohormonal responses are more limited

but the existing literature supports the need for further evaluation of this model as a

potential means of investigating the stress response.

85

3.2. Experimental plan

The specific aim of this first series of experiments was to determine the ease, safety and

reliability of a single breath of carbon dioxide in producing a stereotypical

neuroendocrine stress response. In order to do this, a dose response study incorporating

four increasing concentrations of CO2 was undertaken so as to determine the optimum

concentration that would generate a stress response without significant adverse effects.

The four concentrations tested were 5%, 25%, 35% and 50%. All were combined with

pure oxygen to create mixtures that were all hyperoxic relative to air.

Once an optimal dose had been identified, the reliability and reproducibility of this dose

was assessed in three separate experiments. Firstly, reproducibility was assessed by

comparing responses to one exposure to 35% CO2 that was then repeated in the same

individual after an interval of either one week or 6 months. In a subsequent experiment,

responses at different times of the cortisol circadian cycle were evaluated.

86

3.3. Dose response study

3.3.1. Introduction and methods

Healthy adult volunteers were recruited by advertisement from within the University of

Bristol and were paid for participation in the studies. All potential participants

underwent a full clinical interview, examination and electrocardiogram. Exclusion

criteria, monitoring and procedural details were as described in the methodology section

(Chapter 2). The ethics committee of the United Bristol Healthcare Trust approved the

study and all subjects provided written informed consent prior to participation.

Nine subjects (5 male) with a mean age of 27.2 years (range 24 – 36) were enrolled and

all completed the study. Identical cylinders of pre-mixed CO2 and O2 (BOC gases)

containing each of the four concentrations to be tested were obtained and colour coded

to maintain blinding. Subjects attended once a week for four weeks and received each

of the four concentrations once, in random order. Visual analogue scale questionnaires

and blood samples were taken simultaneously every 15 minutes, commencing 30

minutes before exposure and continuing for 60 minutes following. An additional

sample was taken at 2 minutes following exposure. Blood and saliva sampling for

cortisol, prolactin, plasma renin activity (PRA), GH, TSH, FSH, LH, ACTH and plasma

catecholamines was as according to the protocol already described.

Continuous pulse and blood pressure recordings were made with the Finapress recorder

(Ohmeda). Cardiovascular responses to each concentration of CO2 were determined by

calculating the mean of every 5 beats derived from the continuous beat-to-beat monitor.

The maximum change from a baseline mean 30 seconds prior to exposure was then

87

calculated for both systolic blood pressure and heart rate. Similarly, for each specific

hormone, the absolute maximum change from baseline was also determined. These

were then subject to a mixed model analysis in order to account for the presence of both

fixed (dose) and random (subject) effects at each time point. Correlations were

determined using Pearson’s correlation coefficient. All data are presented as mean +/-

SEM. A p-value of less than 0.05 was considered significant.

3.3.2. Results

Anxiety symptoms were transient and well tolerated. There were no other significant

adverse effects from the CO2 exposure and no subjects described or were observed to

have signs or symptoms suggestive of panic.

Five of the 9 subjects were unable to take a full vital capacity breath of 50% CO2

(inspired VC < 80% of baseline). It has been suggested this may be related to

oropharyngeal spasm [146] induced by the high CO2 concentration. Since more than

half the subjects were unable to achieve an adequate breath of this dose, their results

have been not been included in the statistical analysis. The test breath from all other

exposures was considered adequate (VC > 80% of baseline).

Psychological response

Prior to exposure, subjective feelings of anxiety, fear and breathlessness were minimal

(Table 3.01) and did not change significantly in anticipation of the test. Peak increases

in these three symptoms occurred immediately following the exposure and had returned

to baseline within minutes. The peak increase in subjective feelings of anxiety,

breathlessness and in the 5 most commonly experienced somatic symptoms was

88

significantly greater following 25% than after 5% CO2 (p < 0.05), and similarly, these

increased further following 35% compared to 25% CO2 (p < 0.05). Subjective feelings

of fear increased significantly after 25% compared to 5% (p < 0.05), but did not

significantly increase further after 35% CO2. The symptoms experienced consistently

by most subjects and which accounted for most of the difference in experience between

the various CO2 doses included: feeling short of breath; feeling hot; dizziness; feeling

light-headed or faint, blurred or narrowed vision and having difficulty concentrating

(Figure 3.01).

Physiological response

Cardiovascular responses following CO2 exposure are shown in Figure 3.02. Baseline

cardiovascular parameters were not significantly different (Table 3.01). From the

continuous beat-to-beat recording, the mean of every 5 beats was calculated to give a

single reading from which peak changes were calculated. SBP and HR increased

slightly in all subjects immediately prior to all doses, however, no significant change in

blood pressure or heart rate occurred following exposure to either 5% or 25% CO2. In

contrast, following exposure to 35% CO2, a significant bradycardic effect occurred that

persisted for up to 90 seconds (mean peak change in heart rate –22.1 +/- 5.4 b/m

compared to 7.6 +/- 4.2 b/m following 5% CO2 and 0.5 +/- 1.9 b/m following 25% CO2;

p < 0.001). The bradycardia associated with 35% CO2 was followed by a significant

rise in SBP (mean peak change in SBP 28.5 +/- 6.7 mmHg following 35% CO2

compared to 0.2 +/- 3.2 mmHg following 5% CO2 and –7.1 +/- 4.6 mmHg following

25% CO2; p < 0.001).

89

Biochemical response

Baseline concentrations of measured hormones were equivalent prior to each CO2

exposure (Table 3.01). Neither 5% nor 25% CO2 had a significant effect on any of the

measured hormones. 35% CO2 on the other hand produced a significant increase in

ACTH, plasma and salivary cortisol, prolactin and noradrenaline but had no effect on

adrenaline, AVP, PRA, TSH, GH, LH or FSH. The peak change in ACTH and

noradrenaline occurred at 2 minutes post 35% CO2 exposure, whilst the peak effect on

plasma and salivary cortisol and prolactin occurred at 15 minutes. Figures 3.03 and 3.04

show peak individual and mean responses for these hormones.

Correlations

Following exposure to 35% CO2, there was a significant positive correlation between

the peak change in plasma cortisol and the peak change in SBP (p = 0.02, R2 = 0.607)

(Figure 3.05) but not with the peak HR change. A significant positive relationship

between plasma cortisol change and the peak change in anxiety scores was also present

(p = 0.04, R2 = 0.466). No other significant correlations were present.

90

Table 3.01 Baseline physiological, psychological and hormonal characteristics prior to each CO2 exposure.

5% 25% 35%

Physiology Systolic BP (mmHg) 129.3 +/- 11.4 135.8 +/- 9.0 135.9 +/- 8.9 Diastolic BP (mmHg) 79.6 +/- 8.7 75.1 +/- 5.5 77.1 +/- 4.2 HR (beats/min) 75.1 +/- 4.2 79.1 +/- 5.1 72.1 +/- 1.7 Temperature (°C)

36.5+/-0.2 36.5+/-0.1 36.3+/-0.2

Psychology Anxiety (mm) 8.3+/-2.2 10.0+/-3.1 12.2+/-3.1 Fear (mm) 5.0+/-2.7 4.4+/-1.7 5.0+/-1.8 Breathlessness (mm) 1.1+/-0.7 1.1+/-0.8 1.6+/-1.1 Relaxed (mm)

75.0+/-5.5 75.0+/-6.0 73.8+/-8.4

Endocrinology Plasma cortisol (nmol/l) 285.8+/-43.4 300.2+/-35.9 318.7+/-43.8 Salivary cortisol (nmol/l) 90.4+/-31.1 102.3+/-23.4 126.8+/-33.6 ACTH (pg/ml) 46+/-0.3 49+/-0.6 45+/-0.2 PRL (nmol/l) 128.1+/-11.6 156.7+/-15.3 155.4+/-16.1 AVP (pg/ml) 1.4+/-0.2 1.1+/-0.3 1.0+/-0.6 FSH (IU/l) 3.2+/-0.8 2.8+/-0.6 2.4+/-0.4 LH (IU/l) 2.8+/-0.7 2.1+/-0.3 2.0+/-0.5 GH (mU/l) 4.6+/-2.5 5.5+/-2.1 8.2+/-4.2 TSH (mU/l) 1.6+/-0.3 1.4+/-0.4 1.5+/-0.3 Renin (fmol/l) 16.7+/-3.1 17.0+/-3.0 12.1+/-2.8 Noradrenaline (pg/ml) 359.1+/-53.2 N/A 431.0+/-68.8 Adrenaline (pg/ml) 92.4+/-32.1 N/A 95.5+/-44.2 n=9 (all subjects received all three doses in random order) All differences are non-significant. BP = blood pressure; HR = heart rate.

91

Anxiety, fear andbreathlessness

anx0

25

50

75

1005%25%35%

mm

b

a

Con

cent

ratin

g0

25

50

75

mm

b

a

Diff

icul

ty

Figure 3.01. Subjspecific somatic sya:p<0.05 5% vs 25b:p<0.05 25% vs 3

a

iety fe

b

Somatic sy

Feel

ing

hot

b b

a

ective anxiety,mptoms (botto% CO25% CO2

a

ar breathle

mptoms

Diz

zine

ss

Ligh

thea

ded

b

a a

fear and breathm) following C

ssness

Blu

rred

vis

ion

b

a

lessness (top) and O2 exposure.

92

Systolic Blood Pressure

70 90 110 130 150

100

120

140

160

1805%25%35%

beat

mm

HgSystolic Blood Pressure

5% 25% 35%

-50

0

50

100

∆m

ax S

BP(

mm

Hg)

Heart Rate

70 90 110 130 150

50

60

70

80

90

1005%25%35%

beat

bea

ts/m

in

Heart Rate

5% 25% 35%

-50

-25

0

25

50

∆m

ax H

eart

Rat

e(b

eats

/min

)

a

b

Figure 3.02. Systolic blood pressure and heart rate responses to a single breath of 5%, 25% and 35% CO2 represented as mean response over time (left hand panels with exposure represented by hashed bar) and as maximum response from baseline for each individual (right hand panels). a and b: p<0.001

93

ACTH

5% 25% 35%-25

0

25

50

75∆

max

AC

TH (p

g/m

l)Plasma Cortisol

5% 25% 35%-200

-100

0

100

200

∆m

ax p

lasm

a co

rtis

ol(n

mol

/l)

Salivary Cortisol

5% 25% 35%-100

0

100

200

300

400

∆m

ax S

aliv

ary

Cor

tisol

(nm

ol/l)

Prolactin

5% 25% 35%-100

0

100

200

∆m

ax P

rola

ctin

(nm

ol/l)

Noradrenaline

5% 35%-250

0

250

500

750

∆m

ax N

orad

rena

line

(pg/

ml)

a b

c d

e

Figure 3.03. Individual and mean peak ACTH, plasma cortisol, salivary cortisol, prolactin and noradrenaline responses to a single breath of 5%, 25% and 35% CO2. a: p=0.006; b: p=0.02; c: p=0.02; d: p<0.001; e: p=ns

94

FSH

5% 25% 35%

-0.5

0.0

0.5

1.0

∆m

ax F

SH (I

U/l)

LH

5% 25% 35%

-1

0

1

2

∆m

ax L

H (IU

/l)

GH

5% 25% 35%

-30

-20

-10

0

10

20

∆m

ax G

H (m

U/l)

TSH

5% 25% 35%

-0.5

0.0

0.5

1.0

∆m

ax T

SH (m

U/l)

RENIN

5% 25% 35%

-10

0

10

20

∆m

ax R

enin

(fm

ol/l)

AVP

5% 25% 35%

-1

0

1

∆m

ax A

VP(p

g/m

l)

Figure 3.04. Individual and mean peak FSH, LH, GH, TSH, Renin and AVP responses to a single breath of 5%, 25% and 35% CO2. Differences between responses for each dose were not significant.

95

Plasma Cortisol vs Anxiety

0 50 100 150 200

0

50

100

p=0.04R2=0.4667

∆max Plasma cortisol (nmol/l)

∆m

ax A

nxie

ty V

AS

(mm

)

Plasma Cortisol vs SBP

0 50 100 150 200

0

25

50

75

p=0.02R2=0.607

∆max Plasma Cortisol (nmol/l)

∆m

ax S

BP

(mm

Hg)

Figure 3.05. Correlations between peak change plasma cortisol following exposure to a single breath of 35% CO2 and peak change in systolic blood pressure (upper panel) and peak change in anxiety score (lower panel).

96

3.3.3. Discussion

The principal aim of this first study was to detail the response to CO2 inhalation and

identify an optimum dose that would serve as the basis for future studies.

50% CO2 seemed to induce an involuntary inhibition of inspiration that prevented a

subject from taking a full vital capacity breath of this mixture. Whether this is due to

CO2 induced laryngeal spasm secondary to the action of CO2 chemoreceptors in the

nasopharynx as Davey [1972] suggests is uncertain. However, from volunteers

descriptions is was clearly evident that the bitter taste associated with CO2 was much

more obvious when breathing through a nasal-oral mask (when nasal chemosensitive

taste receptors [Coates 2001] are stimulated) as compared to a mouth-piece and nose-

clip. As a result, the frequency if inadequate breaths (< 80% VC), was far greater with

50% CO2 or when the nasal-oral mask was used.

Psychological response

The principal psychological changes seen in this study were a dose dependent increase

in subjective feelings of anxiety, breathlessness and a few specific somatic symptoms of

fear namely difficulty concentrating, dizziness or lightheadedness, blurred or narrowed

vision, a feeling of the heart racing or pounding and feeling hot or flushed. Other

symptoms were experienced rarely. For example few, if any subjects described nausea,

chest pain, butterflies or feeling tremulous. This is consistent with the data of Verberg

et al [1988] who has described the CO2 challenge as producing a narrow spectrum of

very specific somatic symptoms in both normal individuals and panic disorder patients.

In this study, no subject panicked in response to any of the exposures. This is again

consistent with published literature suggesting the CO2-induced panic rate, from either

low-dose chronic exposure or acute high dose (single breath of 35% CO2), in healthy

97

individuals is low [Rassovsky and Kushner 2003]. Whilst considerable variation exists

in the methods of administering CO2 and in the definition and recording of panic

attacks, a number of studies have examined the anxiety response to a single breath of

35% CO2 in both susceptible panic disorder patients, their relatives and healthy controls

[Battaglia and Perna 1995, Perna et al 1995a, Perna et al 1995b, Perna et al 1995c,

Perna et al 1995d, Perna 1995e, Verburg et al 1995]. Overall in susceptible individuals

panic attack rates occur in about 50-75%, whilst normal controls panicked in only 0-5%

of cases. First degree relatives panicked in about 22% of cases [Perna et al 1995b].

Further, even with the higher doses of CO2, anxiety symptoms were only moderate in

intensity and transient, resolving completely within 2-3 minutes. Subjects participating

in this study had, however, been carefully selected to exclude a personal or familial

predisposition to panic or anxiety.

Sympatho-neural and sympatho-adrenomedullary response

A single breath of 35% CO2 produced a marked systolic pressor response that was

preceded by a significant and persistent bradycardia. Five and 25% CO2 did not have

any significant effect on cardiovascular parameters or catecholamine release. The

pressor response to 35% CO2 was paralleled by a significant release of noradrenaline at

2 minutes following exposure. No significant adrenaline response was seen and this

most likely reflects the differential regulation of sympatho-neural and sympatho-

adrenomedullary systems with CO2 preferentially stimulating sympatho-neural

pathways but not affecting adrenaline release from the adrenal medulla [Young and

Landsberg 1998]. Cardiac output, stroke volume, peripheral resistance and other indices

of cardiovascular function were not assessed in these initial studies, however, as will be

described in more detail in Chapter 5, detailed cardiovascular responses have been

evaluated using non-invasive monitoring techniques. Results of these indicate the

98

pressor response is associated with a marked increase in total peripheral resistance

without a significant change in either stroke volume or cardiac output. This would be

consistent with noradrenaline mediated peripheral vasoconstriction without a significant

adrenaline mediated inotropic effect. Several groups, including Cullen and Edgar

[1974] have shown CO2-mediated peripheral vasoconstriction and increased total

peripheral resistance with an associated increase in blood pressure that was, in part,

mediated through peripheral chemoreceptors.

Parasympathetic response

It was initially thought that reflex baroreceptor activity in response to the increase in

blood pressure produced the bradycardia. Examination of the continuous beat-to-beat

monitoring of heart rate and blood pressure clearly shows the onset of the bradycardia

occurring before the pressor response, making reflex bradycardia unlikely. Three

possible mechanisms producing bradycardia can be postulated. Firstly, the bradycardia

may simply reflect the respiratory gasp reflex that occurs with a single vital capacity

breath through the mouth. The bradycardia associated with this reflex is, however, brief

and less marked than was observed. Indeed, continuous physiological monitoring as

performed in studies described in Chapter 5 clearly show a transient small gasp reflex

bradycardia followed by a more intense sustained bradycardia associated with CO2

inhalation. Secondly, this response might represent a negative chronotropic effect of

CO2 on the myocardium. Tenney [1956, 1960] has shown in isolated heart muscle

preparations a direct effect of CO2 producing a slowing of spontaneous heart muscle

contractions. Several experiments described fully in Chapters 4 and 5 have shown no

effect of CO2 on heart rate when the heart has been either surgically (as in heart-lung

transplantation) or pathologically (as in various forms of autonomic neuropathy)

denervated. This suggests CO2 is acting through neural mechanisms rather than directly

99

on the myocardium. This leaves the most likely mechanism – direct vagal action either

through CO2 stimulation of brainstem vagal centres or through activation of reflex arcs

between peripheral chemoreceptors (within the oropharynx or carotid bodies) and the

vagus nerve as have been described by Henry et al [1998].

Hypothalamic-pituitary-adrenocortical response

A single breath of 35% CO2 produced a significant release of ACTH at 2 minutes and

plasma cortisol at 15 minutes. A single breath of 5% or 25% CO2 had no effect on the

HPA axis. Plasma cortisol release was reproduced following a second exposure to 35%

CO2 after one week and after 6 months. Most psychological stress paradigms show a

significant inter-individual variability in the magnitude of the cortisol response

[Sapolsky 1994] and this was similarly seen in these studies. Repeated exposure to both

physical and psychological stressors typically results in a decline in cortisol responses

(habituation) [Singh et al 1999] making many of these laboratory stress paradigms

unsuitable for repeated evaluation of HPA axis activity in the same individual. Whilst

no significant habituation of cortisol responses was seen on repeated exposure, there

was a trend toward smaller cortisol responses in the group that returned after 6 months.

This was associated with overall smaller vital capacity test breaths and may reflect

reduced CO2 exposure rather than habituation. Sub-optimal vital capacity (< 80%

baseline) resulted in little or no cortisol response and this reinforces the importance of

achieving an adequate test breath. However, from this series of experiments we can’t be

sure that more frequent exposure would not result in a more significant reduction in

cortisol responses.

100

Other endocrine responses

Release of both prolactin and GH may occur as part of the endocrine stress response and

an increase of both has previously been demonstrated following hypercapnia [Woods et

al 1988]. Prolactin has been shown to have anxiolytic properties and it may play a role

in modifying anxiety-related behaviour [Torner et al 2001]. Whilst a significant

prolactin response did occur with 35% CO2, the anticipated rise in GH did not. Neither

was there a significant release of other anterior pituitary hormones (TSH, LH, or FSH).

AVP is a stress responsive neuropeptide capable of stimulating pituitary ACTH release

and plays an important role in the HPA response to both acute [Scott and Dinan 1998]

and chronic [Ma and Lightman 1998] stress. Hypercapnia induces marked increases in

c-fos expression in AVP containing neurones of the supraoptic and paraventricular

nuclei [Kc et al 2002], although, this was probably secondary to activation of pathways

projecting from the brainstem. In this study we did not observe any significant change

in AVP levels, although it is important to note that peripheral blood levels of AVP may

not necessarily reflect AVP levels within the hypophyseal portal blood.

The renin-aldosterone system has a role in maintaining cardiovascular integrity

particularly in response to haemodynamic challenges and is regulated by sympathetic β-

adrenergic innervations [DiBona and Kopp 1997]. Despite this there was no change in

plasma rennin activity in keeping with the known specificity of activation of different

parts of the sympathetic nervous system.

Correlations

As mentioned, wide inter-individual variation in cortisol responses to 35% CO2 was

seen in all these studies and this is consistent with most laboratory stress paradigms,

101

particularly those involving psychological stressors. Similarly SAM responses

including noradrenaline, blood pressure and heart rate changes showed wide inter-

individual variation. This variation occurs through the interplay of numerous influences

including genetic, developmental, social and individual personality characteristics

[Kirschbaum et al 1995, Gerra et al 2001, Levine 2000]. Repeated exposure,

particularly to psychological stress, is associated with a significant decline in ACTH

and cortisol responses (habituation), but not in catecholamine or blood pressure

responses. Individuals who show reduced habituation (termed high responders) tend to

perceive more stress and express higher rates of symptoms of anxiety and depression

[Kirschbaum et al 1995, Van Eck et al 1996, Gerra et al 2001]. These individuals also

appear to display reduced habituation to physical stressors as well as psychological

stressors [Singh et al 1999]. Cacioppo et al [1995] have also suggested that high

responders are characterised by higher levels of neuroticism, higher anticipatory anxiety

and poor psychological defence and these individuals typically show larger and longer

lasting cortisol and SAM stress responses to psychological stress. The authors imply

these individuals are more susceptible to the negative health consequences of an

impaired neuroendocrine stress response system.

102

3.4. Reproducibility studies


From the previous study, a single breath of 35% CO2 had been identified as the

optimum dose to use for ongoing studies. This was based on its ability to generate an

HPA, prolactin, SAM and parasympathetic response, whilst being well tolerated with

little adverse effect. Further studies were then undertaken to evaluate the

reproducibility of 35% CO2 exposure. In the first of these, ten male subjects with a

mean age of 24.4 (+/- 1.2) years (range 22-35) received a single breath of 35% CO2 on

two occasions separated by one week. Results from three were excluded because one of

their two test breaths was < 80% of their expected VC. Blood samples for cortisol and

prolactin were collected in the same manner as described for the dose response study at

the same intervals. Saliva, for α-amylase, was also obtained from these 10 subjects

using Salivettes (Sarstedt) and collected at the same times as blood samples were taken.

VAS questionnaires were administered immediately before and immediately following

the exposure only. Cardiovascular monitoring in these studies where performed with

the automated blood pressure cuff (Dynamap) that recorded responses every minute.

Since only one concentration was administered to the same individual on two occasions,

a paired t-test analysis was used to determine the difference in the maximum change for

each parameter following each exposure.

A separate group of 5 male subjects (21.1 +/- 1.4 years) received a single breath of 35%

CO2 according to the same protocol. These subjects then returned after 6 months when

the procedure was repeated. The CO2 administration occurred at the same time of the

day (between 1100 – 1300hours) for both visits in both of the above studies.

103

In order to assess the effect of the natural diurnal variation in plasma cortisol levels on

reproducibility, a fourth group of 10 healthy volunteers (4 male) aged 24.3 +/- 0.7 years

(range 22-30) was studied. Each subject attended twice, one week apart. One of the

visits was scheduled at 7 o’clock in the morning (peak baseline cortisol levels) and the

other at 8 o’clock in the evening (trough baseline cortisol levels). The time of the first

visit was assigned randomly. Each subject received a single breath 35% CO2 at each

visit according to the same protocol already described, however, instead of using a

mouth-piece and nose clip, this experiment was conducted using a nasal-oral face mask

that allowed the subject to breath through both the mouth and nose. Cardiovascular

monitoring using the Dynamap recorder, blood sampling for plasma cortisol and

prolactin and administration VAS questionnaires were as for the repeatability studies

described above. Data from 3 individuals was excluded because at least one of their test

breaths was < 80% of baseline VC.

3.4.2. Results

As shown in Figures 3.06 and 3.07, the increase in subjective feelings of anxiety,

plasma cortisol and SBP with the corresponding fall in HR that occurred following the

initial breath of 35% CO2 was not significantly different when the exposure was

repeated after 1 week. Similarly, following a single breath of 35% CO2 repeated after 6

months, similar increases in plasma cortisol, SBP, anxiety and breathlessness where

seen, with the same corresponding fall in heart rate (Figures 3.06 and 3.07). Whilst

differences in cortisol responses where not significant, there was a trend toward a lower

cortisol response at the 6 month visit, although measured VC for this visit also tended to

104

be lower compared to baseline (5.6 +/- 0.5 L at 0 months compared to 5.2 +/- 0.2 L at 6

months).

As shown in Figure 3.08, there was wide inter-individual variation in salivary amylase

levels both at rest and in following CO2 exposure. There was no significant increase in

amylase following the test. Further, as is also shown in Figure 3.08, there was no

significant correlation in the change in amylase levels with the SBP, DBP or heart rate

response in these subjects.

Table 3.02 shows baseline values for cardiovascular, hormonal and psychological

parameters at different times of the day. Baseline cortisol levels were significantly

higher in the morning compared to the evening (561.1 +/- 41.3 compared to 199.6 +/-

28.9 nmol/l respectively, p < 0.0001). There was no significant difference in other

parameters prior to exposure. A significant pressor response with associated

bradycardia was seen at both times of the day (Figures 3.09 and 3.10). Similarly, the

pattern of anxiety, breathlessness and other somatic symptom responses was the same as

in previous experiments, and there was no difference between morning and evening.

There was no statistically significant difference between the maximum change in

cortisol levels following CO2 exposure, although there was a trend toward lower

responses in the morning (higher baseline cortisol levels). No significant increase in

prolactin release was seen following either CO2 exposure (Figure 3.09). Whilst test

breaths were considered adequate, (> 80% of baseline) the average test breath using the

nasal-oral mask appeared lower that in the dose response study (2.8 +/- 0.3 compared to

4.0 +/- 0.3 L respectively) which had a similar sex distribution.

105

Response to 35% CO2 atbaseline and repeated after 1

week

Anx

iety

(mm

)

Bre

athl

essn

ess

(mm

)

Cor

tisol

(nm

ol/l)

SB

P (m

mH

g)

HR

(b/m

)-25

0

25

50

75Baseline1 week

mm

Response to 35% CO2 atbaseline and repeated after 6

months

Anx

iety

(mm

)

Bre

athl

essn

ess

(mm

)

Cor

tisol

(nm

ol/l)

SB

P (m

mH

g)

HR

(bpm

)-25

25

75

125Baseline6 months

mm

Figure 3.06. Psychological, cardiovascular and cortisol responses to a single breath of 35% CO2 administered at baseline and then repeated after 1 week (n=7, top panel) or, in a separate group of subjects, repeated after 6 months (n=5, lower panel). Differences between responses for each parameter were not significant.

106

Systolic blood pressureresponse to repeated 35% CO2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

100

110

120

130

140

Time (minutes)

mm

Hg

Pulse rate response torepeated 35% CO2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

80

Time (minutes)

bpm

O baseline • 1 week

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

60

70

80

90

Time (minutes)

bpm

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

110

120

130

140

150

160

Time (minutes)

mm

Hg

O baseline • 6 months

Figure 3.07. Cardiovascular responses for the 5 minutes before and 5 minutes after a single breath of 35% CO2 given at baseline and then repeated after 1 week (n=7, top panels) and in a separate group, repeated after 6 months (n=5, lower panels). Left hand panels represent systolic blood pressure. Right hand panels represent heart rate. Exposure is represented by hashed line.

107

Salivary amylase vs SBP

0.0 2.5 5.0 7.5 10.0 12.5

-25

0

25

50

R2=0.2371p=ns

∆max Amylase (U/l)

∆m

ax S

BP

(mm

Hg)

Salivary amylase vs DBP

0.0 2.5 5.0 7.5 10.0 12.5

-20

-10

0

10

R2=0.2695p=ns


∆m

ax D

BP

(mm

Hg)

Salivary amylase vs HR

0.0 2.5 5.0 7.5 10.0 12.5

-30

-20

-10

0

10

R2=0.0627p=ns


∆m

ax H

R(b

/m)

Salivary amylase response to35% CO2

Baseline Peak

0

100000

200000

300000

U/l

Figure 3.08. Salivary amylase response to 35% CO2 in 10 healthy volunteers including peak response and correlations between change in salivary amylase and change in SBP, DBP or HR.

108

Table 3.02. Baseline physiology, psychology and hormonal characteristics for the morning compared to evening visits.

am visit pm visit p

Physiology

SBP (mmHg) 107.9+/-4.9 118.7+/-4.0 ns

Heart Rate (bpm) 66.1+/-2.9 66.2+/-2.9 ns

Test Vital Capacity (L) 3.0+/-0.4 2.8+/-0.3 ns

Psychology

Anxiety (mm) 27.0+/-6.1 35.5+/-8.0 ns

Breathlessness (mm) 57.5+/-8.2 66.0+/-6.2 ns

Endocrinology

Plasma cortisol (nmol/l) 561.1+/-41.3 199.6+/-28.9 <0.0001

Prolactin (mU/l) 230.0+/-32.7 186.3+/-28.9 ns

109

Response to 35% CO2according to time of

administration

Anxi

ety

(mm

)

Brea

thle

ssne

ss (m

m)

Cor

tisol

(nm

ol/l)

Pro

lact

in (m

iu/l)

SBP

(mm

Hg)

HR

(bpm

)-25

0

25

50

75ampm

mm

Figure 3.09. Change in plasma cortisol, prolactin, cardiovascular and subjective anxiety responses following a morning (am) compared to evening (pm) breath of 35% CO2 (top panel) and in the lower panels is the cardiovascular response for the 5 minutes before and after exposure (represented by the hashed line). n=7. Differences between responses for each parameter were not significant.

110

Systolic blood pressureresponse according to time of

administration of CO2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

90

100

110

120

130

140

Time (minutes)

mm

Hg

Pulse rate response accordingto time of administration of CO2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

80

90ampm

Time (minutes)

bpm

Figure 3.10. Cardiovascular responses following a single breath of 35% CO2 given at 7 am compared to 8 pm (n=7). Upper panel represents systolic blood pressure. Lower panel represents heart rate. Exposure is represented by hashed line.

111

3.4.3. Discussion

Significant psychological habituation following repeated 35% CO2 exposure either at

one week or at 6 months did not occur. A similar persistence of responses has been

seen in other studies of 35% CO2. Anxiety responses were equivalent when exposure

was repeated after 1 [Van den Hout et al 1987], 2 or 6 days [Perna et al 1994, Perna et

al 1997]. This is in marked distinction to pure psychological stressors such as the Trier

Social Stress Test (TSST), which is usually only effective the first time it is performed

[Gerra et al 2001].

Repeat exposure, whether at 1 week or at 6 months, showed the cardiovascular response

to be robust and easily reproducible. No evidence of habituation or a decline in

responses was apparent event at 6 months when the mean vital capacity breath was

somewhat smaller compared to the initial visit. The study examining the effects of

diurnal variation showed the typical circadian variation in blood pressure was evident in

these normal subjects with baseline blood pressure tending to be lower in the morning

compared to the evening [Witzleb 1983]. Despite this, and the significantly different

baseline cortisol levels at these times, both systolic blood pressure and heart rate

responses were no different following 35% CO2 exposure. Similarly, baseline cortisol

levels did not have any significant effect on cortisol, prolactin or psychological

responsiveness following 35% CO2 exposure.

112

3.5. Summary

Carbon dioxide-induced anxiety has been extensively researched for several decades,

but the mechanism of CO2-anxiogenesis is still unclear. At present, the two commonly

held hypotheses regarding CO2-mediated panic induction refer to hypersensitivity of

either the brainstem locus coeruleus or the central nucleus of the amygdala as the

triggers of panic [Gorman 2003]. The former theory holds that panic is a form of

severe anticipatory anxiety whereby autonomic sensations (such as breathlessness or

suffocation-like symptoms produced by CO2) are misinterpreted by the individual as

representing a more serious and imminent threat. This then triggers the LC/sympathetic

response system with subsequent stimulation of limbic and HPA axes [Gorman 2003,

Griez and Schreurs 2003]. Evidence supporting this includes yohimbine (a stimulus of

brainstem noradrenergic centres) mediated panic attacks are inhibited by blocking

noradrenergic brainstem activity with clonidine [Gorman 2003]. Further, in animal

studies, lesions of the LC inhibit anxiety behaviour [Gorman 2003].

Opposing this view, the latter hypothesis describes a more complex view of anxiety and

CO2-induced panic as arising from a hypersensitivity of the alarm system that has

evolved to regulate CO2 and lactate levels [Klein 1993]. This system involves the

central nucleus of the amygdala, centres involved in emotional memory as well as

centres involved in cognitive processing of environmental cues [Gorman 2003].

Neuroimaging has clearly defined activation of these areas in response to a number of

fear inducing stimuli [Brannan et al 2001, Liotti et al 2001]. It is also argued that the

complexity of the panic response is such that it is more likely to result from stimulation

of these limbic centres with subsequent activation of the HPA and noradrenergic centres

[Gorman 2003].

113

Many different substances (for example lactate, doxopram, yohimbine, cholecystokinin-

B receptor agonists, caffeine and CO2), each with vastly different mechanisms of action,

can produce panic attacks [Sinha 1999] in humans. Similarly, both cognitive

behavioural therapy [Gorman et al 2000] and a diverse range of neurotransmitter

agonists [Bertani et al 1997, Nardi et al 2000], and antagonists [Battaglia et al 2001]

can inhibit such attacks. It is likely, therefore, that both pathways have important roles

in mediating some or all of the features of CO2-induced anxiogenesis and the

predominant mechanism are yet to be determined. It is also likely that the pathways

involved will vary between panic disorder patients and normal volunteers and between

the type of stimulus applied (low dose versus high dose CO2 for example) as well as

other factors.

Most studies of hypercapnia, however, demonstrate activation of central chemoreceptor

sites particularly noradrenergic centres within the brainstem with subsequent

sympathetic nervous system stimulation. In animal models, CO2-induced activation of

the LC increases respiratory drive, induces hypertension and tachycardia, increases

cerebral blood flow and is partly responsible for regulating arousal responses [Krystal et

al 1989, Haxhiu et al 2001]. Bilateral LC lesions in cats impair this response

[Rammana Reddy et al 1986]. In humans, cardiovascular stimulation is a feature of

CO2–induced anxiety in both healthy volunteers and panic disorder patients [Gorman et

al 1988]. Systolic hypertension, tachycardia and decreased total peripheral resistance

have been the most common features described, although catecholamine release has not

been a consistent finding. Sechzer et al [1960] and Tenney [1960] describe both

adrenaline and noradrenaline release, while Woods et al [1988] failed to show an

increase in the noradrenaline metabolite MHPG in response to prolonged exposure to

low dose CO2. All these studies involved CO2 concentrations of between 5 – 15%

114

administered over minutes to hours, and this, plus differences in sampling times may

account for some of the observed variation in catecholamine release.

HPA axis activation following CO2 exposure in other paradigms has been inconsistent

and variable, with some studies demonstrating cortisol release [Sechzer et al [1960,

Woods et al 1988, Krystal et al 1989, Argyropoulos et al 2002], whilst others have

failed to demonstrate a response despite some of them generating acute anxiety and

even panic [Van den Hout et al 1987, Sinha 1999, Coplan et al 2002, Van Duinen et al

2004]. The reason for this difference is not immediately clear, although, it appears the

threshold to produce behavioural arousal is significantly less than that required to

generate an HPA response. This threshold difference has been recognised in a number

of laboratory stress paradigms as well as in real life stressors [Biondi and Picardi 1999].

Similarly, numerous studies, including those by Pacak et al [1998, Pacak and Miklos

2001], have shown that varying intensities of a particular stressor will produce different

degrees or patterns of HPA and SAM stimulation. It is possible that differences in

intensity of the CO2-specific stress response are responsible for the behavioural arousal

(ie anxiety) seen with low doses, whilst HPA axis activation only occurs with much

higher doses.

An alternative explanation, however, is that acute and chronic hypercapnia might

represent qualitatively distinct stressors. Herman et al [1997] suggested stressors that

represent an immediate threat to life (such as hypoxia or hypotension) directly activate

brainstem and hypothalamic nuclei generating immediate physiological responses

required to counter these threats. In contrast, during situations such as novelty or

conditioned fear, there is a need for some form of higher cortical processing of the

challenge relative to past experience before the situation is perceived as stressful. Acute

115

hypercapnia from a single breath of 35% CO2 may be an example of the former

situation, being an immediate threat to respiratory homeostasis whereas the somatic

symptoms associated with low dose CO2 may first require interpretation as distressing

before a neuroendocrine response is generated [Klein 1993, Gorman et al 2000].

It is clear from studies of the biological basis of fear and panic that activation of a

number of different brain centres can result in anxiety and panic attacks that appear

outwardly similar. Differences do exist, however, particularly in whether or not HPA

axis activation is a feature of the attack. Exposure to some pharmacological agents

including yohimbine, cholecystokinin-B receptor agonists and caffeine will produce

acute panic without HPA axis activation, whereas lactate, isoproterenol and doxopram

all produce both panic and HPA axis activation [Sinha 1999]. Nattie [1999] has shown

that central CO2 chemoreceptors are distributed widely throughout the brainstem,

cerebellum and midbrain and these chemoreceptors retain discrete neuroanatomical and

functional properties. Further, CO2 sensitive receptors in different brain regions respond

selectively to a narrow range of CO2 concentration and it is possible that the different

responses seen to acute and chronic CO2 exposure could reflect activity of different

neuroanatomical pathways or different thresholds of activity of the same pathway.

There are several possible pathways that may mediate HPA axis activation in response

to CO2 exposure. Immediate early gene (c-fos) expression studies in rodents [Haxhiu et

al 2001] have demonstrated activation of the hypothalamic paraventricular nucleus

(PVN) following CO2, although the presence of CO2/pH sensitive chemoreceptors

within the PVN itself have not been clearly demonstrated. It seems much more likely

that the PVN is activated either in response to activation of brainstem noradrenergic

centres, or from limbic centres mediating the psychological response, or both. In

116

addition, other neurotransmitter pathways may be important in modifying the HPA axis

response to this challenge. Such possibilities include serotonergic pathways that are

involved in respiration, arousal [Haxhiu et al 2001] and CO2–induced anxiety [Klaasen

et al 1998, Kc et al 2002]; AVP, which is important in HPA responses to stress and has

been shown in animal studies to be activated by hypercapnia [Bornstein and Chrousos

1999], although did not appear to be affected by CO2 in this study (see below); and

finally non-ACTH mediated release of cortisol from the adrenal cortex secondary to

SAM activation [Bornstein and Chrousos 1999] is also possible.

Consistent with the literature, a significant correlation between change in cortisol and

change in anxiety was observed, as was a significant correlation between change in

cortisol and change in SBP. This suggests low responders to the CO2 stress are

characterised by low cortisol, anxiety and blood pressure responses as opposed to high

responders. Gerra et al [2001] attempted to identify whether significant SAM

habituation could occur from repeated exposure to psychological stress. Their studies

confirmed cortisol habituation in some, but not all subjects, but no habituation of SAM

responses. This dissociation of responses was unexpected in view of the links between

brainstem sympathetic centres and the HPA axis [Bugajski et al 1995, Gerra et al 2001],

nevertheless, similar dissociations have been reported elsewhere [Malarkey et al 1995].

In the above studies of repeated CO2 exposure, there was a non-significant trend

towards reduced cortisol responses when the dose was repeated either at 1 week or at 6

months, although at least in the 6 month study this may have been related to smaller

inspired vital capacity volumes. ACTH and noradrenaline responses were not

measured after repeated exposure, but blood pressure and heart rate responses were

robust and easily reproducible. Individual cortisol responses varied markedly with

some responses declining after the second exposure, some increasing and others

117

remaining relatively constant. The small sample sizes used precludes in conclusions

being drawn about the likelihood of habituation occurring or the proportion of

individuals who are likely to be low or high responders. Similarly, in these studies an

attempt to identify subjects as high or low responders prior to exposure was not made.

Thus whether cortisol responses to this model are predictive of personality type,

baseline anxiety or future risk is not known.

118

3.6. Conclusion

The aim of this first series of experiments was to explore the neuroendocrine,

psychological and cardiovascular effects of acute hypercapnia in normal individuals

with the intention of detailing the response and assessing the validity of using 35% CO2

as a simple means of evoking sympathetic and HPA axis responses. The results confirm

that the anxiogenic response to hypercapnia in normal individuals is dose dependent,

but anxiety is transient and 35% CO2 is well tolerated. Cardiovascular and cortisol

responses, on the other hand, were only activated by 35% CO2 but were reproducible

when repeated after short (1 week) and long (6 month) intervals. Haemodynamic

responses in particular were robust, easily measured, and unaffected by intervals

between doses or by time of exposure.

The mechanisms and neuroanatomical pathways behind the observed responses are still

to be determined and some of the experiments detailed in the subsequent chapters

address some of the possibilities. In summary, however, the description above suggests

that the initial response to a single breath of 35% CO2 is vagally mediated bradycardia

followed by noradrenaline mediated peripheral vasoconstriction producing an acute

pressor response as a result of stimulation of brainstem noradrenergic centres. HPA

axis activation is a feature of acute (35% CO2) exposure but is likely to occur indirectly

as a result of either brainstem noradrenergic projections to the PVN and/or from

projections from the limbic fear centre (including the CnA). Stimulation of the CnA

with subsequent involvement of the cortex and limbic areas associated with this fear

circuit is likely to be responsible for the observed emotional arousal. However, whether

this is a direct CO2 effect, as suggested by Gorman et al [2003], or an indirect effect of

119

brainstem activation, as suggested by Bailey et al [2003], or both, is yet to be

determined.

120

CHAPTER 4

THE 35% CO2 MODEL:

MECHANISMS UNDERLYING THE

NEUROENDOCRINE RESPOSNE

121

4.1. Introduction

The principal observations from the initial studies performed with 35% CO2 were

emotional arousal, initial bradycardia and a significant pressor response that was

associated with noradrenaline release. Hormonal responses included prolactin release

and HPA activation with ACTH and cortisol release. Further, cortisol responses were

well correlated with both the pressor response and with aspects of emotional arousal,

but not with heart rate changes.

As mentioned, studies of CO2 challenges and panic induction in the literature have

yielded two potential hypotheses regarding the likely mechanisms that mediate CO2

responses. The ‘false suffocation’ theory of Klein [1993] suggests that in panic

disorder, panic attacks originate from an increased sensitivity of the CnA and its

associated fear circuit as described by Gorman et al [2000]. Components of this fear

circuit are responsible for the various features of panic including anticipation of the

event, the acute attack and phobic avoidance. The circuit can be activated at different

regions explaining how panicogens with vastly different mechanisms of action can all

produce similar panic attacks and how different treatments can all be effective [Van den

Hout et al 1987, Greiz et al 1990b, Nutt et al 1990, Bertani et al 1997, Perna et al 1997,

Sinha 1999, Gorman et al 2000, Nardi et al 2000, Battaglia et al 2001].

Communication between the CnA and other components of this circuit with brainstem

noradrenergic centres would be responsible for the sympathoadrenal activation seen in

fear and panic. Projections between the CnA and brainstem with the hypothalamic

PVN, on the other hand, would be responsible for the HPA axis activation associated

with some, but not all panicogens [Sinha 1999, Gorman et al 2000].

122

More recently, an alternative hypothesis has been proposed based on studies that have

shown significant positive correlations between the emotional arousal and the cortisol

response and with sympathetic activation [Argyropoulos et al 2002, Bailey et al 2002].

Based on their work, these authors have proposed that stimulation of brainstem

noradrenergic centres (particularly the LC) is the initiating event following CO2

exposure and that a feed forward loop involving the CnA and the PVN results in

subsequent psychological responses and HPA axis activation respectively. In addition,

activation of CRH pathways that project back to the LC and brainstem as well as to the

CnA enhance these same responses. Sensitivity of these and other modulating

neurotransmitter pathways are then responsible for individual variability and the clinical

susceptibility of some individuals to anxiety and panic disorders [Bailey et al 2002].

Other neurotransmitter pathways such as serotonin [Dinan 1996, Klaasen et al 1998,

Ben Zion et al 1999, Miller et al 2000, Schruers et al 2000, Lowry 2002, Schruers et al

2002], endogenous opiates [Gritz et al 1976, Vythilingam et al 2000, Drolet et al 2001],

GABA, acetylcholine, substance P and neuropeptide Y [Harbuz and Lightman 1992,

Chrousos 1998, Habib et al 2001] have been implicated as regulators or modifiers of the

observed response to CO2 inhalation.

Neuroanatomical and immunohistochemistry studies in animals and neuroimaging

studies in humans lend support to both hypotheses. It would seem likely that rather than

being mutually exclusive, both hypotheses are complementary with one or other being

dominant depending on the nature of the hypercapnoeic stressor and the circumstances

in which exposure is occurring. Specifically, there is evidence indicating the presence

of CO2/pH sensitive chemoreceptors in the region of the VLM, LC and other

noradrenergic brainstem sites [Nattie 1999, Haxhiu et al 2001]; in association with 5-

HT neurons of the raphe complex [Wang et al 2001, Wang et al 2002]; and within

123

limbic centres [Nattie 1999]; but not within the PVN or pituitary gland [Haxhiu et al

2001, Kc et al 2002]. Functional neuroimaging in humans breathing low dose CO2 has

confirmed activation of multiple brain centres including the VLM, LC, CnA, as well as

other limbic and para-limbic centres, frontal lobes and the cerebellum [Brannan et al

2001, Liotti et al 2001].

A simplified overview of the principal neuroanatomical centres and their

interconnections likely to be involved in mediating the various components of the CO2

stress response is given in Figure 4.01. It is not yet clear, however, whether the

initiating event is activation of brainstem noradrenaline with subsequent psychological

or HPA responses, or whether the ‘central fear circuit’ (CnA) is activated first with

subsequent brainstem and HPA activation.

124

VLMLC

CnA

Pit

Hipp

V

PVN

Pit

CRH

ACTH

Cortisol

Vagus

SNS PNS

VLM

LC

CnA

Hipp

5-HT

opioid

Figure 4.01. Simplified overview of the principal centres likely to be involved ingenerating the psychological, HPA and SAM responses to acute hypercapnia. CnA: Central nucleus of amygdala; Pit: Pituitary; PVN: Hypothalamicparaventricular nucleus; Hipp: Hippocampus; VLM: Ventrolateral medulla; LC:Locus coeruleus; SNS: Sympathetic nervous system; PNS: Parasympathetic nervoussystem; 5-HT: Serotonin network

125


In order to evaluate some of the mechanisms and pathways mediating the various

responses to CO2, a series of experiments were conducted to examine the specific roles

of the HPA axis as well as the central and peripheral components of the autonomic

nervous system. The contribution of three central neurotransmitter pathways

(serotonergic, opiate and noradrenergic) was examined in the first study, whilst the

impact of a suppressed HPA axis on the psychological, cardiovascular and prolactin

response to CO2 was assessed in two further studies. One study involved the

administration of a glucocorticoid synthesis inhibitor (metyrapone) or a

mineralocorticoid receptor antagonist (spironolactone) or a combination of both in

normal volunteers. The other experiment involved the assessment of breastfeeding

mothers who, during the lactation phase, are thought to have a physiologically

suppressed HPA axis.

Finally, examining two individuals with functional alterations in their baseline

autonomic nervous system assessed the role of the autonomic nervous system. The

first individual was an adult male with the idiopathic congenital central hypoventilation

syndrome (CCHS), a rare inherited condition of central CO2 insensitivity associated

with hypoventilation and other features of impaired cental autonomic integration. The

second individual studied, an adult male following dual lung-heart transplant,

represented a clinical model of peripheral cardio-pulmonary denervation.

126

4.3. Central neurotransmitter study


Central neurotransmitter concentrations were altered by the administration of

paroxetine, naltrexone or nortriptyline. Paroxetine, a potent selective serotonin re-

uptake inhibitor (SSRI), is used in clinical practice predominantly as an anti-depressant.

It acts acutely to inhibit the pre-synaptic re-uptake and storage of serotonin thereby

increasing intra-synaptic serotonin concentrations [Stahl 1998]. However, the initial

administration of this drug is often associated with a worsening of clinical symptoms

(especially anxiety), with a delay of several days to weeks before clinical anti-

depressant or anti-anxiety effects are seen. The delayed response is thought to be

related to modification of 5-HT receptor number and function [Stahl 1996, Stahl 1998].

The mu and kappa opiate receptors are the principal receptors responsible for opiate

regulation of HPA axis activity [Drolet et al 2001]. Naltrexone, an orally available

opiate antagonist is selective for mu receptors at low doses, but at higher doses will also

antagonise kappa receptors [Gritz et al 1976]. Nortriptyline is a tricyclic anti-

depressant and noradrenaline re-uptake inhibitor. In this study it was used in preference

to Reboxetine a more potent and more selective noradrenaline re-uptake inhibitor

because Reboxetine has been associated with causing significant hypertension and this

was felt to be potentially dangerous to use given the significant pressor response

associated with the CO2 challenge [Stahl 1996, Penttila et al 2001].

Twenty seven healthy volunteers who had not participated in any previous CO2 related

studies were recruited from within the University of Bristol. Volunteers were assigned

to one of three treatments in a randomised single blinded fashion. Treatment groups

127

were either placebo and paroxetine 20 mg; or placebo and nortriptyline 25 mg; or

placebo and naltrexone 50 mg. Identical capsules containing either a single dose of

placebo or the active drug were administered between 7 and 8 am the morning of the

test. All tests were conducted between 1 and 2 pm that same day. Each subject

attended for two visits one week apart and the order (placebo vs active drug) was

randomised and double blind. At each visit a single breath of 35% CO2 was

administered according to the same protocol as described above.

All subjects attended all scheduled visits. The administered medications were well

tolerated without significant side effect apart from naltrexone that caused mild dizziness

and occasional nausea in most subjects assigned to this treatment. One subject in this

group vomited the morning of the test. The test protocol was as described previously

and for all visits inspired VC was measured and test breaths were all considered

adequate, being at least 80% of baseline. The CO2 itself was well tolerated without

significant adverse effect.

Each group had 9 subjects. In the paroxetine group mean age was 24 years (range 22-

27) with 4 males. The mean age in the naltrexone group was 23 years (range 22-25)

with 4 males, whilst in the nortriptyline group, mean age was 23 years (range 22-25)

with 4 males.

Repeated measures analysis of variance was used to determine the effects of CO2

exposure and the differences between the groups with paired t-test analysis used to

determine differences between single time points including baseline measures for the

placebo and active treatment visits for each subject.

128

4.3.2. Results

Baseline hormone, cardiovascular and psychological measures are given in Table 4.01

with baseline hormone measures also represented graphically in Figure 4.02. As

illustrated, there were no differences in any baseline measures in the groups taking

either paroxetine or nortriptyline, whilst the group taking naltrexone had, on their

naltrexone visit, significantly higher basal cortisol and prolactin levels (p = 0.0004 and

p = 0.03 respectively). In addition, for the naltrexone visit, subjective VAS scores for

dizziness and nausea were significantly elevated at baseline compared to their placebo

visit (p = 0.02 for both dizziness and nausea). Similarly, cardiovascular measures in the

naltrexone group indicated a significantly higher baseline SBP (p = 0.003) compared to

the placebo visit, with no difference in baseline heart rates.

A single breath of 35% CO2 produced a significant increase from baseline in cortisol,

prolactin, SBP, anxiety fear, breathlessness and the somatic symptoms of ‘awareness of

heartbeat’, ‘feeling hot’, ‘blurred vision’ and ‘dizziness’, with a significant fall in HR (p

< 0.05 for all). Within each group, following exposure to a single breath of 35% CO2,

individuals treated with paroxetine showed no difference in their maximum change in

both cortisol and prolactin compared to their placebo visit. Naltrexone and nortriptyline

treated individuals showed no difference in their cortisol response but did show a non-

significant trend towards a smaller prolactin response. (Figure 4.03. and Figure 4.04.).

Between each group, there was no difference in cortisol or prolactin responsiveness.

The cardiovascular response (both SBP and HR) to 35% CO2, both within each group

(treatment visit compared to placebo visit) and between the three treatments, was no

different. Maximum change from baseline for SBP and HR as well as a temporal

129

profile of cardiovascular response to CO2 are shown in Figure 4.05. Psychological

responses, both within and between groups, were also equivalent, although there was a

non-significant trend towards a greater fear response after taking paroxetine, and a

smaller change in somatic symptoms (apart from dizziness) in those treated with

nortriptyline (Figure 4.06.).

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Table 4.01. Baseline hormonal, cardiovascular and psychological parameters for each

visit: placebo vs paroxetine; placebo vs naltrexone; placebo vs nortriptyline.

Placebo Paroxetine Placebo Naltrexone Placebo Nortriptyline

Cortisol

(nmol/l)

358.8+/-41.2 386.3+/-54.9 288.1+/-27.1 675.4+/72.4* 371.6+/-34.9 365.7+/-57.3

Prolactin

(nmol/l)

156.1+/-25.6 168.2+/-24.4 124.3+/-16.5 249.8+/-45.7† 138.1+/-20.8 202.0+/-37.1

SBP

(mmHg)

116.0+/-2.3 112.4+/-1.6 111.9+/-4.0 117.6+/-3.1† 111.9+/-4.0 113.6+/-2.2

HR

(b/m)

72.0+/-2.5 68.3+/-2.8 71.6+/-3.3

69.7+/-2.4 74.4+/-7.2 76.1+/-2.5

Dizzines

s (mm)

0.0+/-0.0 0.5+/-1.6 1.1+/-3.3 15.0+/-17.6‡ 0.0+/-0.0 1.6+/-3.5

Nausea

(mm)

0.0+/-0.0 1.1+/-0.7 1.1+/-1.1 23.8+/-6.9‡ 0.0+/-0.0 2.2+/-1.2

For each group n=9. *p=0.0004; †p=0.03; ‡ p=0.02

131

Baseline cortisol

plac

ebo

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xetin

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Baseline prolactin

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ipty

line0

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/l

†

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Figure 4.02. Baseline cortisol (upper panel) and prolactin (lower panel) levels for the placebo/paroxetine; placebo/naltexone and placebo/nortriptyline visits. *p=0.0004, †p=0.03

Maximum change in cortisol -placebo vs paroxetine

Placebo Paroxetine

-200

-100

0

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300nm

ol/l

Maximum change in prolactin -placebo vs paroxetine

Placebo Paroxetine

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Maximum change in cortisol -placebo vs naltrexone

Placebo Naltrexone

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ol.l

Maximum change in cortisol -placebo vs nortriptyline

Placebo Nortryptiline

-100

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nmol

/l

Maximum change in prolactin -placebo vs nortriptyline

Placebo Nortriptyline

-25

0

25

50

75

nmol

/l

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Figure 4.03. Individual and mean cortisol and prolactin responses to a single breath of 35% CO2 in each treatment group. Responses are maximum change from baseline and comparisons are made between an individuals placebo visit and their treatment visit. Individuals taking paroxetine are shown in the upper panels, whilst those taking naltrexone are shown in the middle panels, with those taking nortriptyline shown in the lower panels. Graphs on the left represent cortisol responses, with those on the right prolactin responses. All differences are non-significant by paired t-test.

Cortisol

Pla

cebo

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oxet

ine

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trexo

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cebo

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iline0

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oxet

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cebo

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trexo

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cebo

Nor

trypt

iline0

10

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40

50

nmol

/l

Figue 4.04. Mean peak cortisol and prolactin responses to a single breath of 35% CO2 for the placebo/paroxetine; placebo/naltrexone and placebo/nortriptyline visits. All differences are non-significant.

134

SBP

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

100

110

120

130

140

150placeboparoxetinenaltrexonenortriptyline

Time (mins)

mm

Hg

SBP

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xetin

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line

-10

0

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Hg

HR

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

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100placeboparoxetinenaltrexonenortiptyline

Time (mins)

bpm

HRpl

aceb

o

paro

xetin

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naltr

exon

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nortr

ipty

line

-75

-50

-25

0

25

bpm

Figure 4.05. Cardiovascular responses to a single breath of 35%CO2 for the placebo (combined), paroxetine, naltrexone and nortriptyline groups. Differences are all non-significant.

135

Anxiety

plac

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Fear

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Breathlessness

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Heart beat

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Feeling hot

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Blurred vision

plac

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Dizziness

plac

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xetin

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plac

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exon

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line0

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50

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mm

Figure 4.06. Mean psychological (anxiety, fear, breathlessness, awareness of heart beat, feeling hot, dizziness and blurred vision) responses to a single breath of 35% CO2 for the placebo/paroxetine; placebo/naltexone and placebo/nortriptyline visits. All differences are non-significant.

136

4.3.3. Discussion

The administration of a single dose of paroxetine (20mg) or nortriptyline (25mg)

approximately 6 hours prior to testing had no significant effect on baseline cortisol,

prolactin or cardiovascular measurements. Similarly, neither of these agents produced

any significant side effect that volunteers were aware of. A single dose of naltrexone

(50 mg), administered at a similar time produced significant dizziness and some nausea

in most volunteers and was associated with elevated baseline cortisol, prolactin and SBP

measurements. Naltrexone is used clinically in the management of drug and alcohol

dependence [Hersh et al 1998, Anton et al 1999], however, several studies have

reported variable success depending on treatment compliance. One of the most

important factors determining compliance with naltrexone has been its side effect

profile. In a review of several studies examining naltrexone side effects and

compliance, Oncken et al [2001] classed the principal side effects as either

gastrointestinal (particularly nausea and vomiting) and neuropsychiatric (including

headache, dizziness, nervousness and fatigue). The specific side effects and the

frequency with which they occurred in the naltrexone treated group in this study is

consistent with the above published literature.

As already mentioned, endogenous opioids have an important role in modulating the

HPA axis response to stress. At rest, endogenous opioids act predominantly at the level

of the hypothalamic PVN to provide a tonic inhibition of ACTH and cortisol release.

They are probably responsible for dampening and restraining the stress response as a

means of protecting the organism from the detrimental effects of excessive activation of

the stress response [Drolet et al 2001]. Opioid antagonists, including a single dose of

naltrexone in non-human primates and humans, disinhibit the HPA axis resulting in a

137

significant increase in ACTH and cortisol release [King et al 2002, Williams et al

2003]. This increase occurs from about 2-4 hours after ingestion of the dose, and the

significantly elevated baseline cortisol seen in this study would be consistent with this

effect.

The cortisol response to CO2 following naltrexone pre-treatment was not significantly

altered. Whilst it was hypothesised that opioid antagonism would result in an enhanced

HPA response this has not been commonly observed with other stress paradigms.

Yohimbine, an α-2 adrenoreceptor antagonist, for example activates the HPA axis

through enhancement of LC and brainstem noradrenergic projections to the PVN

[Vythilingam et al 2000]. Increased cortisol responses to a yohimbine/opioid

antagonist combination were seen in one study when yohimbine was given to

individuals pre-treated with an extremely high dose of intravenous naloxone (1mg/kg)

[Charney and Heninger 1986]. In contrast, there was no synergistic effect on cortisol

release when yohimbine was given to individuals pre-treated with oral naltrexone for 8

days [Rosen et al 1999].

Opioid antagonists, however, do appear to alter emotional and behavioural responses to

stress. Principally, their clinical use stems from the decreased cravings and alcohol-

induced ‘high’ experienced by those dependent on alcohol [McCaul et al 2001].

Administered to patients with anxiety disorders, however, naltrexone and naloxone have

been shown to exacerbate feelings of anxiety and to increase physiological responses

when faced with an acute anxiety provoking situation [Rosen et al 1999]. Similarly,

naltrexone pre-treatment antagonised the anti-anxiety effects of benzodiazepines in rats

[Billingsley and Kubena 1978] and significantly increased anxiety responses to

138

yohimbine in humans [Rosen et al 1999]. Unlike these studies, however, naltrexone did

not appear to affect psychological responses to CO2 exposure.

Opiate peptides also have a role in cardiovascular homeostasis mediated both centrally

and peripherally through both receptor-dependent and receptor-independent pathways

[Gritz et al 1976]. Responses are complex, however, in general opiate agonists produce

a fall in systolic blood pressure with a more variable effect on heart rate. It might be

expected therefore that opiate antagonism would result in blood pressure increases, but

this has not been universally demonstrated particularly with acute dosing. Gritz et al

[1976] studied single dose effects of naltrexone and did not demonstrate blood pressure

or heart rate differences. McCubbin et al [1988] on the other hand has suggested opiate

antagonism is associated with exaggerated SAM and circulatory reactivity characteristic

of early stages of hypertension. In his study, naloxone pre-treatment, however, did not

affect resting blood pressure, but did exacerbate SBP responses to an acute

psychological stressor in individuals with normal resting blood pressures [McCubbin et

al 1988]. Naltrexone pre-treatment in this study increased baseline SBP, however,

blood pressure and heart rate changes were no different compared with placebo

following CO2 exposure. Alternatively, the higher resting blood pressures may simply

reflect the increased distress associated with naltrexone side effects that were not

present with either paroxetine, nortriptyline or any of the placebo visits.

Opiate peptides are also involved in the regulation of prolactin secretion predominantly

through their action on tuberoinfundibular dopamine levels. Dopamine pathways,

which provide tonic inhibition of prolactin secretion, are inhibited by opiate agonists

thereby increasing prolactin levels [Ellingboe et al 1980, Gilbeau et al 1985, McCubbin

et al 1988]. This regulation is mediated by µ and κ receptors [Kreek et al 1999,

Butelman and Kreek 2001, Andrews and Grattan 2003], antagonism of which would be

139

expected to produce a reduction in prolactin secretion. Several studies have confirmed

that opiate antagonists including naltrexone either decrease or have no effect on plasma

prolactin levels [Gold et al 1979, Rubin et al 1979, Volavka et al 1979, Mello et al

1989]. Paradoxically, baseline prolactin levels in this study were elevated in the

naltrexone group, although there was a non-significant trend towards a smaller prolactin

response to CO2 in this group. The reason for the difference in baseline prolactin is not

immediately clear, although opiate antagonism does vary in its effect on prolactin levels

according to diurnal rhythms (with smaller effects in the morning compared to the

evening) [Frecska et al 1988].

A single dose of the SSRI paroxetine had no significant effect on baseline or CO2

stimulated hormonal, cardiovascular or psychological parameters. Serotonin (5-HT)

pathways regulate a number of central functions including HPA, autonomic and

behavioural responses to stress and 5-HT can either facilitate or inhibit the activity of

these response systems [Lowry 2002]. Clinically, SSRI’s are used in the treatment of

anxiety, mood and affective disorders, although their precise mechanism of action is not

yet fully understood. Initially, SSRI’s increase 5-HT levels within the neuronal synapse

by reducing 5-HT re-uptake and storage [Barker and Blakely 1995, Stahl 1998]. This

early administration is often associated with a worsening of anxiety symptoms. Over

the next few days, anxiety symptoms improve and this is thought to be related to effects

on receptor number and function including receptor desensitisation and to effects on

other systems such as alterations in neurosteroid activity [Stahl 1996, Stahl 1998,

Nechmad et al 2003]. Studies of tryptophan depletion in both healthy volunteers and

panic disorder patients have shown a significant increase in anxiety symptoms with an

acute reduction in brain 5-HT following both high (35%) [Klaasen et al 1998, Schruers

et al 2000a, Schruers et al 2002] and low (5%) [Miller et al 2000] dose CO2 challenges.

140

Similarly, treatment with the 5-HT antagonist metergoline exacerbated CO2 induced

anxiety [Ben Zion et al 1999].

Conversely the tryptophan replete state appeared to protect against anxiety provocation

from CO2 [Klaasen et al 1998, Schruers et al 2000a]. Pre-treatment with d-

fenfluramine, a 5-HT releasing agent, in patients with panic disorder caused increased

baseline anxiety levels but a blunted anxiety response to 7% CO2 [Mortimore and

Anderson 2000]. Cortisol and cardiovascular measures before and after this low dose

CO2 challenge were no different with d-fenfluramine pre-treatment, although some

more severely affected patients had higher baseline and stimulated prolactin levels.

Chronic treatment of panic disorder patients with SSRI’s (1 week to 1 month)

significantly reduced their anxiety response to both high (35%) and low (5%) dose CO2

challenges [Bertani et al 1997, Perna et al 2002, Bertani et al 2003]. Acute

administration of paroxetine, on the other hand, could be postulated to be analogous to

d-fenfluramine use, although in our normal volunteer study no effects from paroxetine

were seen. The reasons for this are unclear, but may relate to the higher anxiety

responsiveness and sensitivity of panic disorder patients compared to healthy

volunteers. Alternatively, it is possible that the dose was insufficient to produce a

significant change in central 5-HT levels, although it is worth noting that a single dose

of 20 mg paroxetine was sufficient to enhance motor output in patients following acute

stroke [Loubinoux et al 2002].

Nortriptyline, a tricyclic antidepressant and noradrenergic re-uptake inhibitor, increases

central noradrenergic neurotransmission. A related agent, desipramine, has been shown

to increase baseline ACTH and cortisol levels in humans [Pomara et al 2001]

presumably through reciprocal pathways between brainstem noradrenergic centres and

141

the HPA axis. Acute nortriptyline administration (single dose of 50 mg) has also been

shown to increase resting systolic blood pressure [Torpy et al 1995]. Treatment with

imipramine, another related tricyclic antidepressant, for 7 days was noted to decrease

anxiety responses to 35% CO2 in subjects with panic disorder [Bertani et al 1997]. In

this study, however, a single dose of 25 mg nortriptyline did not affect baseline or

stimulated hormonal, cardiovascular or psychological measures.

Whilst is was anticipated that pre-treatment with naltrexone and perhaps paroxetine

might increase anxiety symptomatology and possibly hormonal and cardiovascular

responses to CO2 inhalation, it is interesting to note that other studies of pre-treatment

with anxiogenic agents either had no effect or in fact reduced anxiety responses to 35%

CO2. Pols et al [1994], for example, pre-treated healthy volunteers with yohimbine

(20mg) or placebo before giving 35% CO2 in a randomised cross-over study.

Yohimbine pre-treatment did not produce the anticipated increase in anxiety responses

to CO2 compared to placebo. Schruers et al [2000b], pre-treated healthy volunteers

with 10 µg of the anxiogenic agent CCK-4 in a randomised, double-blind, placebo

controlled manner. Following 35% CO2, instead of the anticipated increase in anxiety

responses, anxiety was in fact reduced compared to placebo. The authors postulate that

CCK-4 and CO2 acted through different neurobiological systems to inhibit rather than

enhance each other.

142

4.4. Mineralocorticoid/glucocorticoid study


The relationship between the two major effector limbs of the stress response (the HPA

and SAM axes) is a complex one that is maintained through direct and indirect

reciprocal connections at both central and peripheral levels. However, the importance

of these systems in the maintenance of normal health requires their regulation to be

finely controlled with multiple, multi-level inputs that have extensive redundancy and

plasticity. Stress responsive noradrenergic neurones from the LC, the caudal nucleus of

the solitary tract (A2 cell group) and from the medullary A1 group all project directly to

the hypothalamic PVN where they influence both CRH and ACTH activity [Zeigler et

al 1999, Habib et al 2001, Carrasco and Van Der Kar 2003]. Brainstem noradrenergic

neurones also project directly to the forebrain and amygdala which indirectly influence

HPA activity [Carrasco and Van Der Kar 2003]. CRH releasing neurones, in addition to

regulating ACTH release from the anterior pituitary, also project to brainstem

noradrenergic centres where they influence activity particularly of the LC [Pacak et al

1995, Koob 1999, Carrasco and Van Der Kar 2003].

Glucocorticoids, the final effectors of the HPA axis, regulate the stress response through

their action on two receptor subtypes. Within the brain, high affinity, type I or

mineralocorticoid (MR) receptors have a limited distribution, being found particularly

in the hippocampus, where they are involved in feedback processes important for the

normal day-to-day variation in cortisol levels. In contrast, low affinity type II

glucocorticoid (GR) receptors have a more widespread distribution and are important

regulators of the glucocorticoid response to stress [de Kloet 1991, Harbuz and Lightman

1992, Chrousos 1998, Young et al 1998, Sapolsky et al 2000]. Activation of

143

mineralocorticoid receptors in the hippocampus, for example, promotes GABA-ergic

activity that in turn inhibits the activity of the HPA axis [Carrasco and Van Der Kar

2003]. Peripherally, the adrenal cortex (responsible for glucocorticoid production) and

the adrenal medulla (responsible for catecholamine, particularly adrenaline, production)

is regulated by multiple extra- and intra-adrenal inputs. ACTH is the principal regulator

of cortisol biosynthesis and release from the adrenal cortex, however, steroidogenesis is

also influenced by the SAM axis, with stimulation of neural (noradrenergic) inputs to

the adrenal cortex increasing glucocorticoid synthesis and release [Bornstein and

Chrousos 1999]. Similarly, a wide variety of other peptides released from sympathetic

and other nerves, including adrenaline, serotonin, neuropeptide Y and substance P, have

also been shown to modulate adrenocortical activity [Bornstein and Chrousos 1999].

Glucocorticoids, on the other hand, are also important in the regulation of adrenaline

release from the adrenal medulla. The medullary enzyme phenylethanolamine-N-

methyltransferase (PNMT) controls the synthesis of adrenaline from noradrenaline

within the adrenal medulla. The activity of this enzyme is dependent on ACTH and

high intra-medullary concentrations of glucocorticoids [Wurtman 2002], with a number

of studies demonstrating an impaired adrenaline response to various stressors in the

setting of ACTH and glucocorticoid deficiency [Kvetnansky et al 1995, Jeong et al

2000, Wurtman 2002]. On the basis of this interaction, glucocorticoid deficiency (as

produced by metyrapone administration) might be expected to impair the SAM response

to stress.

On the other hand, MR antagonists (such as spironolactone) would be expected to

inhibit HPA axis negative feedback and result in increased cortisol secretion. This is

indeed the case in rodents [Bradbury et al 1994, Young et al 1998] and has been shown

in one human study. Young et al [1998] administered a high dose of spironolactone

144

(400 mg) to healthy volunteers and demonstrated a transient (< 2 hour) elevation of

cortisol. Two doses of spironolactone (400 mg 5 hours apart) produced a more

sustained cortisol elevation, but did not increase ACTH levels.

The effect of glucocorticoid deficiency and mineralocorticoid antagonism in normal

volunteers was assessed using the standard 35% CO2 challenge as previously described.

Metyrapone and spironolactone were chosen because of their known safety, selectivity

and ease of administration (both are available orally).

Nine (5 male) healthy volunteers mean age 22 years (range 20 – 39) who had not

participated in any previous CO2 related studies were recruited from within the

University of Bristol. Volunteers attended on four occasions one week apart. At each

visit they received a single dose of 35% CO2 according to the same protocol as

described above. For each visit volunteers were pre-treated on one occasion with both

spironolactone and metyrapone, once with metyrapone only, once with spironolactone

only and once with placebo only. Visits were randomised, and subjects were blinded to

the treatment condition on each occasion. Doses used were spironolactone 50 mg,

metyrapone 750 mg or placebo in an equivalent number of capsules. For each visit, a

dose was taken the evening before the test with a second dose the morning of the test.

The treatments were all well tolerated with no significant side effects from either drug

reported. All the tests were conducted between 1 and 3 pm and all subjects completed

all the tests. Baseline and test VC was recorded and all test breaths were within 80% of

the baseline breath. Cardiovascular measures were recorded as before with the

Dynamap monitor recording pulse rate and blood pressure every minute for 5 minutes

before and 5 minutes after CO2 exposure. Psychological responses were recorded using

the same visual analogue scales. An intravenous line for blood sampling was placed 30

145

minutes before testing commenced and samples for cortisol levels were taken at

baseline, 10, 20 and 30 minutes after exposure. Blood samples were not collected on 2

occasions (once during a spironolactone only visit and once during a

spironolactone/metyrapone visit) because of failed intravenous access at the time of

testing.


exposure and the differences between the groups with paired t-test analysis used to

determine differences between single time points including baseline measures for the

placebo and active treatment visits for each subject.

4.4.2. Results

There was no difference in baseline psychological or cardiovascular parameters for any

of the visits. Baseline cortisol levels, shown in Figure 4.07, were significantly reduced

on the two occasions subjects took metyrapone. There was no independent or

additional effect of spironolactone on cortisol levels.

A single breath of 35% CO2 produced a significant increase from baseline in cortisol,

prolactin, SBP, anxiety, fear and breathlessness with a significant fall in HR (p < 0.05

for all). Cortisol responses to 35% CO2 were equivalent in the control, spironolactone

only and metyrapone only conditions, but there was no significant cortisol response in

the combined spironolactone/metyrapone condition (p = 0.03 control vs

spironolactone/metyrapone) (Figure 4.08). Psychological responses, including anxiety,

fear, breathlessness (shown in Figure 4.09) and various somatic symptoms (not shown)

146

were no different between any of the treatment or control groups. Similarly, as shown

in Figure 4.10, SBP and HR responses were equivalent in all groups.

147

Baseline Cortisol

C S M S/M

0

100

200

300

400

500

nmol

/l *† **‡

Figure 4.07. Baseline cortisol levels in the control (C), spironolactone (S), metyrapone (M) and combined (S/M) groups. *p=0.03 C v M; †p=0.006 C v SM **p=0.0008 S v M; ‡p=0.0005 S v SM

148

Cortisol response to 35% CO2

C S M S/M

-100

0

100

200

nmol

/l


C S M S/M

0

25

50

75

100

nmol

/l

Figure 4.08. Peak cortisol response to 35% CO2 in the control (C), spironolactone (S), metyrapone (M) and combined (S/M) groups. *p=0.03 C v SM; †p=0.009 S v M

149

Psychological responses to35%CO2

Anx

iety

Fear

Bre

athl

essn

ess

0

25

50

75CSMS/Mm

m

Figure 4.09. Psychological responses to 35% CO2 in the control (C), spironolactone (S), metyrapone (M) and combined (S/M) groups. Differences between groups are all non-significant.

150

SBP

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

100

110

120

130CSMSM

Time (mins)

mm

Hg

SBP

C S M S/M

-10

0

10

20

30

mm

Hg

HR

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

80CSMSM

Time (mins)

bpm

HR

C S M S/M

-50

-25

0

25

bpm

Figure 4.10. Cardiovascular responses to 35%CO2 in thecontrol (C), spironolactone (S), metyrapone (M) and combined (S/M) groups. Differences between groups are all non-significant.

151

4.4.3. Discussion

Two doses of spironolactone alone (50 mg 12 hours apart) had no effect on baseline

cortisol levels 4-5 hours after the second dose compared to placebo. Further,

spironolactone alone had no effect on CO2 stimulated cortisol levels. This may well

have been due to the smaller doses and longer intervals used in this study, however, as

has been seen previously baseline cortisol levels have not affected cortisol

responsiveness to a single breath of 35% CO2. Spironolactone alone also had no effect

on cardiovascular or psychological responses to CO2.

Metyrapone, a specific inhibitor of the 11β-hydroxylase enzyme, prevents cortisol

synthesis and reduces circulating cortisol levels [Laborie et al 2003]. Effects on other

aspects of the HPA and SAM axes are complex, with acute dosing likely to reduce

cortisol levels and possibly impair adrenergic adrenomedullary responses. Longer term

dosing, on the other hand, as has been shown by Laborie et al [2003] to produce

positive feedback with increased CRH neurotransmission and increased ACTH. This is

likely to enhance the effect of CRH on brainstem noradrenergic activity and increase

adrenal intra-medullary adrenaline production thereby enhancing the sympathetic

behavioural and cardiovascular responses to stress. Consistent with this, cortisol levels

in those individuals taking metyrapone were significantly reduced compared to the

placebo and spironolactone only visits. Cardiovascular and psychological responses,

however, were not affected by metyrapone pre-treatment. The likely explanation for

this is that whilst metyrapone reduced cortisol levels by about 50%, there was still

sufficient cortisol present to ensure normal glucocorticoid responsiveness, particularly

within the brain. Alternative possibilities include the effect of cortisol-like molecules

152

formed from other steroid biosynthetic pathways associated with the inhibition of 11β-

hydroxylase [Lamberts et al 1987].

The combination of spironolactone and metyrapone again had no effect on

psychological and cardiovascular responses, although this group did demonstrate a

reduced cortisol response to the CO2 challenge. Given both spironolactone alone and

metyrapone alone were each associated with significant cortisol response to the CO2

challenge, it is difficult to postulate a mechanism whereby the combination would

suppress a cortisol response. The anticipated enhanced cardiovascular response from

increased CRH action on sympathetic brainstem centres was not apparent, suggesting

the principal cardiovascular and psychological responses are driven from a primary

stimulation of brainstem noradrenergic centres and/or limbic centres with HPA

responses occurring as a secondary phenomenon.

153

4.5. Lactation study


Lactation, at least in animal models, represents a time of relative neurohormonal

hyporesponsiveness to a variety of stressors [Lightman et al 1997, Lightman et al 2001,

Heinrichs et al 2002]. Studies in rodents that have included both psychological

stressors such as noise [Windle et al 1997], conditioned footshock [Shanks et al 1997]

and restraint [Shanks et al 1999], as well as physical stressors such as intra-peritoneal

NaCl [Lightman and Young 1989] or lipopolysaccharide [Lightman 1993], have

demonstrated attenuated ACTH, cortisol, catecholamine, oxytocin and prolactin

responses to stress [Higuchi 1989, Lightman 1993, Altemus et al 1995, Neumann et al

1998, Heinrichs et al 2001, Lightman et al 2001, Russell 2001, Heinrichs et al 2002].

Further, behavioural responses to stress in rodents also appeared attenuated during

lactation [Altemus et al 1995, Heinrichs et al 2001, Lightman et al 2001, Russell 2001,

Heinrichs et al 2002]. This hyporesponsiveness was a generalised feature of lactation

and was not limited to selective times such as immediately following suckling [Windle

et al 1997, Heinrichs et al 2002]. It has been felt that neuroendocrine and behavioural

stress hyporesponsiveness during lactation is advantageous both for the mother and the

infant. For the mother, attenuated stress responsiveness is thought to protect against

distracting stimuli during feeding, conserve energy for suckling and rearing, improve

nutritional milk quality and promote protective immune activity [Lightman et al 1997,

Lightman et al 2001, Russell 2001, Heinrichs et al 2002]. For the infant it protects

against the potentially damaging effects of exposure to high glucocorticoid levels at a

time of vulnerability of the developing brain [Lightman et al 1997, Russell 2001,

Heinrichs et al 2002]. The neurobiological pathways mediating this reduced stress

154

reactivity are yet to be fully elucidated. The principal hormones involved in mammary

gland development, growth, milk production and ejection include oxytocin, prolactin,

estrogens, GH, thyroid hormone, ACTH and glucocorticoids, most of which also have

significant roles in behavioural modification [Lightman and Young 1989, Tucker 1994,

Lightman et al 1997, Neumann et al 2000, Lightman et al 2001, Heinrichs et al 2002]

and stress responsiveness. Oxytocin and prolactin, for example, are both increased

during lactation and are both inhibitors of the HPA axis [Heinrichs et al 2002].

Endogenous opiates are also modulators of HPA axis activity, inhibiting ACTH and

cortisol release. Hypothalamic opiate receptors have been shown to change during

lactation and are associated with reduced oxytocin and HPA activity [Carter and

Lightman 1987, Hammer and Bridges 1987]. It is likely that the complex interplay of

these and a number of other neurohormones, neurotransmitters as well as environmental

influences are involved in the plasticity of stress responses seen during lactation.

The effect of lactation on stress-response systems in humans is not well characterised

but appears more complex than the situation in rodents. The first study to be performed

in this area in humans examined neuroendocrine responses to physical stress (treadmill

exercise) in healthy women 7-18 weeks post-partum [Altemus et al 1995]. In that

study, breastfeeding began 60 minutes before the start of the exercise challenge and

basal ACTH and cortisol levels were the same for lactating women and non-lactating

controls. Basal prolactin levels were significantly higher in the lactating group. In

response to the exercise, ACTH and cortisol increased to a lesser degree in the lactation

group than in the control group, and prolactin levels fell steadily. Oxytocin levels were

unchanged. The authors concluded that HPA axis responses to physical stress were

suppressed in lactating women, and this effect was independent of oxytocin. A second

study by the same group [Altemus et al 2001] used a psychological stressor (Trier

155

Social Stress Test) in healthy women 6-24 weeks post-partum administered 60 minutes

after feeding. This study also examined bottlefeeding mothers as well as a non-lactation

control group. Baseline ACTH and cortisol levels were equivalent as were the

increases in ACTH, cortisol, blood pressure, heart rate and anxiety symptoms in all

groups. A similar lack of endocrine hyporesponsiveness to psychosocial stress was seen

in a study by Redwine et al [2001]. In this study breast or bottlefeeding within an hour

of being challenged (TSST) had no effect on adrenocortical responses.

It has been well established that suckling itself will suppress the HPA axis probably as a

result of the inhibitory actions of oxytocin and prolactin [Amico et al 1994]. Heinrichs

et al [2001] undertook a study using a similar psychological stressor (TSST) to

determine the effects of acute suckling on HPA responsiveness. The aim was to see if

there is a sensitive time period during lactation where the HPA axis may be

hyporesponsive as opposed to the apparent general hyporesponsiveness of the HPA axis

throughout lactation in rodents. Healthy women, 6-18 weeks post-partum either

breastfed or just held their infant for a 15 minute period with the onset within 30

minutes of receiving the TSST challenge. Baseline ACTH, cortisol, oxytocin, prolactin

and catecholamine levels were equivalent prior to exposure, however, HPA axis

responses were markedly diminished in the breastfeeding group. Prolactin levels in this

group also fell steadily throughout and following the stress. The authors conclude that

unlike rodents humans demonstrate a time sensitive period immediately following

suckling that is associated with significant blunting of HPA responses to psychological

stress. This blunting is not apparent if the stress exposure occurs about 1 hour after

suckling [Heinrichs et al 2001, Heinrichs et al 2002].

Twenty two healthy women participated in the study after giving written informed

consent. Potential breast and bottle-feeding subjects received information regarding the

156

study antenatally during a routine visit to the Antenatal Day Assessment Unit at St

Michael’s maternity Hospital, Bristol. Subjects were then recruited approximately 4

weeks after delivery. All subjects were paid for their participation. Subjects were asked

to attend once, where they all received a single breath of 35% CO2 according to the

same protocol as described above.

Fourteen subjects were 6-8 weeks post-partum and were in good general health

following uncomplicated term deliveries. Participants were non-smokers who were not

taking any regular medications. All post-partum participants completed an interview,

physical examination and the Edinburgh Post-Natal Depression score (see appendix 6)

[Cox et al 1987] prior to inclusion in the study. In addition to the standard inclusion

and exclusion criteria applied in previous studies, subjects were only included if they

had a low risk of post-natal depression based on the EPDS. Eight of the fourteen post-

partum women (age range 18 – 34) were exclusively breastfeeding (BF) their infants,

whilst the remaining six (age range 22-36) had been exclusively bottlefeeding (BO)

their infants from birth. Eight control (C) women (age range 22-27) were recruited

from within the University of Bristol. None of these subjects were, or had ever

previously been, pregnant.

All subjects attended at 11 am, and an antecubital intravenous line was placed 45

minutes before the CO2 exposure. Following insertion of the cannula, mothers fed

(either breast or bottle according to their standard practice) their infants for

approximately 15 minutes. Thirty minutes after arrival, the first of two baseline blood

samples were taken. Cardiovascular monitoring commenced and the second baseline

sample was taken after a further 10 minutes. The breath of 35% CO2 was then

administered. The time from cessation of feeding to receiving the CO2 was

157

approximately 20 to 30 minutes. Sampling continued for 30 minutes following

exposure. A nurse was present during the procedure to assist with caring for the infants.

Two-way, repeated measures, analysis of variance was used to determine between-

group differences and time effects, with two-tailed t-tests used to compare single time-

point data including baseline differences. Paired t-test analysis was used to determine

differences in maximum response from baseline within each group.

4.5.2. Results

One individual (from the bottlefeeding group) did not take an adequate breath of CO2

(VC < 80% of baseline) and data from this individual has been excluded from the

analysis. All other subjects completed the study without significant adverse effects.

Physical, hormonal and psychometric characteristics of all subjects at baseline are

presented in Table 4.02. There was no difference at baseline between the control and

bottlefeeding groups, however, as shown in Figure’s 4.11 and 4.12, baseline cortisol

levels in the breastfeeding group were significantly reduced compared with both the

control and bottlefeeding groups (p = 0.002 and p = 0.003 respectively). In addition,

baseline prolactin levels in the breastfeeding group were significantly higher compared

with both the control and bottlefeeding groups (p = 0.0003 and p = 0.003, respectively).

For all the groups combined, there was a significant increase in cortisol, SBP, and

subjective feelings of anxiety, fear and breathlessness (p < 0.05 for all). Further, there

was also a significant fall in heart rate (p < 0.05). For cortisol, two-way ANOVA

revealed a significant group effect between control (C) and breastfeeding (BF) (F(1.4) =

40.2, p < 0.0001) and between breastfeeding (BF) and bottlefeeding (BO) (F(1.4) = 26.3,

p < 0.0001). Post-hoc analysis indicated significant differences at all time points for C

158

and BF (p < 0.05) and at –5, 2 and 10 minutes for BF and BO (p < 0.05). Maximum

cortisol change from baseline, however, was equivalent in all three groups (Figure

4.11).

Two-way ANOVA also revealed a significant group effect between control and

breastfeeding (F(1.3) = 80.3, p < 0.0001) and between breastfeeding and bottlefeeding

(F(1.3) = 39.5, p < 0.0001) for prolactin responses. Post-hoc analysis indicated

significant differences at all time points for both C and BF (p < 0.01 for all) and for BF

and BO (p < 0.05 for all). There was no significant increase in prolactin from baseline

in the C or the BO group, however, there was sustained and marked fall in prolactin in

the BF group with a significant maximum decrease from baseline (p = 0.04) (Figure

4.12).

Baseline SBP and HR measures were no different between each group. Within each

group there was a significant increase in SBP and a significant fall in HR following CO2

exposure (p < 0.05 for maximum change from baseline for SBP and HR in all three

groups), as shown in Figure 4.13. Between groups, the change in cardiovascular

parameters over time and the maximum change in SBP and HR were equivalent.

Similarly, anxiety, fear, breathlessness and other somatic symptoms of fear were

equivalent at baseline. Within each group there was a significant increase in these

symptoms (p < 0.05 for all) that was equivalent between each group (Figure 4.14).

159

Table 4.02. Baseline hormonal, cardiovascular and psychological parameters for

control (C), breastfeeding (BF) and bottlefeeding (BO) women.

Control (n=8) Breastfeeding (n=8) Bottlefeeding (n=5)

Age (yr) 24.7+/-1.5 29.2+/-1.9 31.2+/-3.0

Cortisol (nmol/l) 300.8+/-25.9 191.1+/-12.8* 274.0+/-16.7

Prolactin (nmol/l) 138.0+/-17.6 2071+/-432.9† 107.0+/-15.1

SBP (mmHg) 108.8+/-4.9 110.0+/-4.2 114.2+/-4.0

HR (b/m) 67. 8+/-3.7 76.1+/-1.6 73.0+/-2.3

Anxiety (mm) 10.6+/-3.9 11.8+/-5.9 13.0+/-5.8

Fear (mm) 6.8+/-2.6 6.2+/-4.0 11.0+/-5.5

Breathlessness

(mm)

1.2+/-0.8 3.1+/-3.1 1.0+/-1.0

*p=0.002 C v BF and p=0.003 BF v BO

†p=0.0003 C v BF and p=0.003 BF v BO

160

Baseline cortisol

0

100

200

300

400controlbreastfeedingbottlefeeding

nmol

/l

Cortisol response to 35% CO 2

Con

trol

Brea

stfe

edin

g

Bottl

efee

ding

-100

0

100

200

300

nmol

/l

Absolute cortisol response to35%CO2

-10 0 10 20 30 40

100

200

300


Time (mins)

nmol

/l

**

Figure 4.11. Baseline (top panel), absolute (middle panel) and maximum cortisol (lower panel) responses to a single breath of 35% CO2 in control women (C; n=8), breastfeeding women (BF; n=8) and bottlefeeding (BO; n=5) women. *p=0.002: C v BF; †p=0.003: BF v BO; p=ns: C v BO. **p<0.05: C v BF all time-points and BF v BO at 0, 2, 10 mins. ‡p<0.05 ∆max cortisol within each group from baseline. Responses between each group were not significantly different.

161

Baseline prolactin

0

1000

2000


nmol

/l

Prolactin response to 35% CO 2C

ontro

l

Bre

astfe

edin

g

Bot

tlefe

edin

g

-500

-250

0

250

nmol

/l

Percent prolactin response to35% CO2

-20

-10

0

10

20ControlBreastfeedingBottlefeeding

%

162
Figure 4.12. Baseline (top panel) and stimulated prolactin (middle andlower panels) responses to a single breath of 35% CO2 in control women (C; n=8), breastfeeding women (BF; n=8) and bottlefeeding (BO; n=5) women. Middle panel is absolute change, lower panel is percent change. *p=0.0003: C v BF †p=0.003: BF v BO p=ns: C v BO. **p=0.04 maximum absolute change in prolactin; ‡p<0.05 maximum

SBP response to 35% CO 2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

80

100

120

140


time

mm

Hg

SBPresponse to 35% CO2

Con

trol

Bre

astfe

edin

g

Bot

tlefe

edin

g

0

10

20

30

40

mm

HgHR response to 35% CO 2

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

80


Time (min)

b/m

HR response to 35%CO 2

Con

trol

Bre

astfe

edin

g

Bot

tlefe

edin

g

-30

-20

-10

0

10b/

m

Figure 4.13. SBP (top panels) and HR (lower panel) responses to a single breath of 35% CO2 in control (C), breastfeeding (BF) and bottlefeeding (BF) women. *p<0.05 ∆max SBP and ∆max HR. Responses between groups are all equivalent.

163

Psychological responses to

0

25

50

75

100ControlBreastfeedingBottlefeeding

%

Anx

iety

Fear

Bre

athl

essn

ess

Figure 4.14. Anxiety, fear and breathlessness responses to a single breath of35% CO2 in control (C), breastfeeding (BF) and bottlefeeding (BF) women. *p<0.05 ∆max anxiety, fear and breathlessness from baseline. Responses between groups are all equivalent.

35% CO2 * * *

164

4.5.3. Discussion

In this study, the CO2 challenge was timed to occur within 30 minutes of breastfeeding

so as to coincide with the expected HPA hyporesponsive period. Baseline cortisol

levels in the breastfeeding group were significantly lower than in the control or

bottlefeeding group consistent with the expected effect of suckling induced HPA axis

suppression. Baseline prolactin levels in this group were also significantly elevated

again consistent with the recent suckling. This is in contrast to the study by Heinrichs et

al [2001] where sampling after a similar time period from the completion of suckling

was not associated with significant differences in baseline cortisol or prolactin levels. It

is, however, consistent with the findings of Amico et al [1994] that did demonstrate

cortisol suppression 15 minutes after the completion of feeding.

In response to the CO2 challenge, the absolute (and percent) maximum increase in

cortisol from baseline was equivalent in all three groups. This suggests the ability of

the HPA axis to respond to this challenge is not blunted during the lactation phase and

is not affected by a suppressed baseline cortisol level. This would also be consistent

with the notion that acute CO2 exposure (single breath of 35% CO2) represents an

immediate threat to respiratory homeostasis and a diminished response to this stressor

would be inappropriate particularly for a nursing mother.

Consistent with previously mentioned studies in humans, prolactin levels fell steadily

following CO2 exposure in the breastfeeding group. Of note, however, there was only a

small increase in prolactin following CO2 in the control group (as opposed to prolactin

responses in earlier studies) and no significant change in prolactin from baseline in the

bottlefeeding group. The reason for this is yet to be determined.

165

As in the study by Altemus et al [2001] baseline SBP was equivalent in all three groups.

However, unlike the Altemus study that indicated bottlefeeders have a reduced cardiac

vagal tone associated with significantly increased basal heart rates, there was no

difference in HR in all three groups in this study. A single breath of 35% CO2 increased

SBP and reduced HR to a similar degree in all groups indicating that the SAM response

to 35% CO2 is not attenuated in lactation and is not influenced by background HPA axis

activity. Similarly, as was the case in the Altemus study [2001], baseline anxiety and

somatic symptoms of fear were no different between the groups and 35% CO2 caused

significant anxiety responses that were equivalent between the three groups. This

would again suggest that lactation has no effect on anxiety responses to 35% CO2 and

that these responses are also not dependent on basal cortisol levels. Similar findings

have been found with various other stressors applied during lactation in both human and

animal studies [Heinrichs et al 2001]. Indeed even in those studies where HPA axis

responses were suppressed, anxiety and emotional arousal responses were no different

in the breastfeeding groups compared with either control or bottle-feeding mothers

[Heinrichs et al 2001].

Overall, this study confirms that within 20-30 minutes of suckling, baseline cortisol

levels in breastfeeding mothers are suppressed, an effect that is probably mediated

through the inhibitory effects of prolactin and oxytocin on the PVN. However, unlike

the response to a psychological stressor, exposure to acute hypercapnia is not associated

with diminution of HPA, autonomic or psychological stress responses. This would be

compatible with the notion that suppression of stress responses may be advantageous to

a nursing mother faced with stressors that are not pertinent to survival, but when

physiological homeostasis is threatened survival demands a full neuroendocrine and

behavioural response.

166

4.6. Peripheral versus central autonomic nervous system effects


The role of the autonomic nervous system in the CO2 response was examined when the

opportunity to study an adult with the congenital central hypoventilation syndrome

(CCHS) presented itself. This syndrome (as described below) is associated with central

CO2 insensitivity and was compared to an individual who had peripheral pulmonary

denervation following dual lung-heart transplant for cystic fibrosis. Because of the

extreme rarity of the CCHS syndrome (approximately 200-300 living children and

young adults worldwide [American Thoracic Society 1999]) and the limited number of

heart-lung transplant patients well enough to undergo evaluation in Bristol, only one

individual with each condition was studied. As a result, formal statistical evaluation of

their responses has not been performed and the significance of their results should be

considered in this context.

One of the individuals studied was a 27 year old male who had had a dual lung-heart

transplant 3 years previously for cystic fibrosis and at the time of this study was well,

being maintained on anti-rejection therapy that included prednisolone 5 mg daily,

cyclosporin and azathioprine. His cystic fibrosis had been complicated by both

exocrine and endocrine pancreatic failure and he was on pancreatic enzyme

supplements and insulin, but had no evidence of diabetic complications and was

normotensive at the time of the study. The other participant was a 43 year old male

with CCHS. He was on non-invasive night-time ventilation and his condition had been

complicated by secondary polycythaemia that had required previous venesection. He

was on no regular medications and was normotensive at the time of the study.

167

4.6.2. Results

As shown in Figure 4.15, the lung transplant individual, as a result of his exogenous

glucocorticoid use, had a suppressed baseline cortisol with no change in his cortisol

level seen following CO2 exposure. He did, however, demonstrate a rise in prolactin to

a similar degree as that seen in healthy volunteers. The individual with CCHS showed

no change in his cortisol and only a small (5 nmol/l or 2.5%) increase in prolactin

following CO2 exposure.

The cardiovascular responses between the two individuals were markedly different.

Pulmonary denervation was associated with a slightly higher baseline heart rate, a

significant rise in SBP (+11 mmHg) in response to CO2 but no bradycardia (+2 b/m).

The cardiovascular responses over time and the peak change following CO2 exposure

are shown in Figure 4.16. In contrast, the individual with CCHS demonstrated no rise

in SBP (+0 mmHg) but he did show a marked bradycardic response (-12 b/m). Figure

4.17 illustrates the psychological response to CO2. Both individuals demonstrated mild

anticipatory anxiety, but whilst the lung transplant subject had a significant anxiety

response to the CO2 challenge equivalent to that seen previously in healthy volunteers,

the CCHS individual experienced no significant anxiety. Similarly, the lung transplant

individual also experienced a significant increase in the symptoms of fear, feeling hot,

blurred vision and dizziness to a degree that was very similar to the response of normal

individuals. The CCHS subject experienced none of these symptoms apart from some

very mild dizziness and breathlessness. The lung transplant subject experienced no

breathlessness.

168


Lung Tx CCHS0

100

200

300

400PrePost

nmol

/l

Prolactin response to 35% CO2

Lung Tx CCHS

150

175

200

225PrePost

nmol

/l

Figure 4.15. Cortisol (upper panel) and prolactin (lower panel) response to a single breath of 35% CO2 in an individual with cardiopulmonary denervation (lung Tx) and in an individual with central CO2 insensitivity (CCHS). Note the subject post heart-lung transplant was on exogenous glucocorticoids.

169

SBP

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

125

135

145

155Lung TxCCHS

Time (mins)

mm

Hg

SBP response to 35% CO2

Lung Tx CCHS

140

145

150

155

160PrePost

mm

Hg

HR

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

85

90

95

100

105Lung TxCCHS

Time (mins)

b/m

HR response to 35% CO 2

Lung Tx CCHS

85

90

95

100

105prepost

b/m

Figure 4.16. SBP (upper panel) and HR (lower panel) response to a single breath of 35% CO2 in an individual with cardiopulmonary denervation (lung Tx) and in an individual with central CO2 insensitivity (CCHS).

170

Anxiety

Lung Tx CCHS

0

25

50

75PrePost

mm

Fear

Lung Tx CCHS

0

25

50

75

mm

Breathlessness

Lung Tx CCHS

0

25

50

75

mm

Feeling hot

Lung Tx CCHS

0

25

50

75

mm

Blurred vision

Lung Tx CCHS

0

25

50

75

mm

Dizziness

Lung Tx CCHS

0

25

50

75

mm

Figure 4.17. Psychological (anxiety, fear, breathlessness and the common somatic symptoms of fear associated with CO2 exposure) response to a single breath of 35% CO2 in an individual with cardiopulmonary denervation (lung Tx) and in an individual with central CO2 insensitivity (CCHS).

171

4.6.3. Discussion

Idiopathic congenital central hypoventilation is a rare syndrome characterised by a

failure of the automatic control of breathing [Gozal 1998, American Thoracic Society

1999]. Typically, ventilation is most severely affected during non-rapid eye movement

(NREM) sleep, a time where automatic neural control of breathing predominates.

Severe hypoventilation usually requiring assisted ventilation occurs during sleep, with

lesser, albeit variable, degrees of hypoventilation during wakefulness. When awake

many patients can sustain normal ventilation without assistance, although some will

require continuous ventilatory assistance [Gozal 1998]. A similar condition has been

described following high cervical trauma or in people with cerebrovascular

malformations, infections or tumours in and around the brainstem [Severinhaus and

Mitchell 1962, Bower and Adkins 1980, Jensen et al 1988, Mukhopadhyay and

Wilkinson 1990, Weese-Mayer et al 1992, Gozal 1998, Weese-Mayer et al 2001] and a

related animal model has been developed by inducing lesions of the intermediate area of

the ventral medullary surface [Schlaefke et al 1979]. Despite this, the majority of

patients with idiopathic CCHS do not have discrete anatomical or neuropathological

lesions. There have only been two case reports of discrete anatomical lesions identified

in children with idiopathic CCHS, one with arcuate nucleus agenesis [Folgering et al

1979] and another with neuronal loss in the region of the reticular nuclei and the lower

cranial nerve nuclei [Weese-Mayer et al 2001]. More likely, the origin of the condition

is genetic, with about 20% of sufferers having associated Hirschbrung’s disease (a

neurocristopathy characterised by loss of parasympathetic intrinsic ganglion cells of the

hindgut and associated with mutation of the RET proto-oncogene) [Haddad et al 1978,

American Thoracic Society 1999]. There are a few case reports of families with the

condition either having affected siblings or having a common RET or other gene

172

mutation, although these are only the minority [Gozal 1998, American Thoracic Society

1999].

Associated autonomic dysfunction occurs relatively commonly, although to a variable

degree. Commonly reported features include; constipation, oesophageal dysmotility,

pupillary abnormalities, abnormalities of cardiac rhythm, decreased basal body

temperature and altered sweating [Severinhaus and Mitchell 1962, Weese-Mayer et al

1992, Commare et al 1993, Weese-Mayer et al 1993, Silvestri et al 2000]. Further,

neuropsychological studies have reported reduced pain perception [American Thoracic

Society 1999], reduced anxiety perception [Pine et al 1994] and a reduced sensation of

breathlessness with prolonged breathholding [American Thoracic Society 1999,

Spengler et al 2001]. Physiological evidence of intact peripheral chemoreceptor activity

with impaired central chemoreceptor function [Gozal et al 1993, Spengler et al 2001]

has led to the current hypothesis that the condition is principally related to impaired

central CO2 chemosensation along with abnormalities of central autonomic integration.

The origin of this abnormality is most likely within the brainstem resulting

predominantly in ventilatory but also other autonomic abnormalities [Gozal 1998,

American Thoracic Society 1999, Spengler et al 2001, Weese-Mayer et al 2001].

In contrast to the situation of central chemoreceptor and autonomic activity described in

CCHS, pulmonary denervation, as occurs following heart-lung transplant, represents a

model of peripheral autonomic denervation affecting both afferent and efferent

pulmonary autonomic pathways. Amphibians and reptiles possess CO2 sensitive

olfactory receptors that cause a dose-dependent decrease in breathing when stimulated

by CO2 [Coates 2001] and there is some evidence that mammals may possess similar

chemoreceptors that play a role in olfaction [Coates 2001]. Pulmonary CO2 sensitive

173

chemoreceptors have not been identified in humans [Lahiri and Forster 2003] and

whether chemoreceptors located within the laryngopharynx exist, or have a role in

mediating rapid responses to acute CO2 exposure in humans is not known.

Pulmonary stretch receptors and their vagal afferents are known to mediate important

ventilatory [Trachiotis et al 1994] and cardiovascular responses to breathing [Taha et al

1995]. Taha et al [1995], for example, have shown that in pulmonary denervated

individuals, respiratory sinus arrhythmia is significantly reduced compared to normal

individuals as a result of altered vagal feedback pathways.

It is important to remember that whilst these individuals represent interesting

physiological models of impaired autonomic control of hypercapnia, only single

individuals with each condition were tested because of the rarity of their respective

conditions. As such their responses are purely descriptive and formal statistical analysis

has not been used to examine differences in responses. Similarly, given the intra-

individual variability in responses in normal individuals, the responses seen in these two

individuals should not be extrapolated to include everyone with similar conditions.

The individual following lung transplantation was taking exogenous glucocorticoids

(prednisolone 5 mg daily) as part of his anti-rejection regime and this would explain

both the suppressed basal cortisol level and the absent change in cortisol following CO2

exposure. His prolactin response was similar to that seen for normal individuals. In

contrast the CCHS subject had normal baseline cortisol and prolactin levels, but failed

to show a response of either to CO2.

174

Consistent with the vagal denervation following lung transplantation, the post-transplant

subject had a higher resting heart rate, and failed to show any drop in heart rate

following CO2, although his SBP response was normal. In contrast, the CCHS subject

demonstrated a normal bradycardic response, but failed to show a rise in SBP. As

expected neither subject described much in the way of breathlessness following the

CO2, however, anxiety, fear and their associated somatic symptoms were experienced

normally by the lung transplant individual, but not at all by the CCHS individual.

Taken together, these results suggest that central chemoreceptor sensation and

integration is essential for generating a neurohormonal (cortisol and prolactin) and

psychological response to CO2. Further, central brainstem stimulation is necessary for a

SBP response. The bradycardia, on the other hand, is likely to be a direct vagal

response as a result of direct stimulation of either the nerve or its central nuclei. It had

been suggested by Tenney [1956] in early experiments of CO2 on isolated heart muscle

preparations that there is a direct effect of CO2/pH causing a decrease in the

spontaneous firing rate of cardiac myocytes and that this may have been an explanation

for the observed bradycardia. If this were the case, it would be expected that heart rate

slowing would still occur in the subject post heart-lung transplant. Vagal denervation

did, however, result in loss of the bradycardic response. In addition, at least in this one

individual with CCHS, stimulation of the vagus was preserved and thus appears to be

separate from other centres involved in mediating the integration and/or response to

CO2 exposure.

175

4.7. Conclusion

The results of the above experiments provide further evidence to suggest that vagally

mediated bradycardia is the first response to CO2 and this is mediated by direct

stimulation of the vagus nerve itself or of its nuclei located within the brainstem. In

addition, it appears that stimulation of the vagus occurs independently of the stimulation

of other brainstem sites responsible for the integration and output of other autonomic

responses to CO2. Stimulation of brainstem noradrenergic centres, particularly the

VLM and the LC amongst others, are likely to be responsible for activation of

descending sympathetic nervous system pathways generating the acute pressor

response. The origins of both the vagal and sympathetic responses are discussed more

fully in the next chapter.

The absence of CO2 chemoreceptors within the hypothalamus and pituitary, coupled

with experimental evidence showing a close correlation between the SAM response and

the cortisol response and the absence of a cortisol response in the subject with central

CO2 insensitivity, suggests HPA axis stimulation occurs as a result of projections from

brainstem noradrenergic centres to the PVN and/or other hypothalamic sites. Further,

the absence of a significant effect on SAM responses in the setting of a

pharmacologically (metyrapone) or physiologically (lactation) suppressed HPA axis

again suggests HPA axis stimulation is driven by brainstem sympathetic centres.

Similarly, SAM responses were not increased in the setting of a disinhibited HPA axis

(naltrexone), although this does not necessarily exclude a role for CRH projections to

the brainstem enhancing or modifying SAM responses to CO2 exposure.

176

Psychological responses also appear to require intact central chemoreceptor

mechanisms and are not dependent on baseline or subsequent cortisol responses. Indeed

this was also evident from studies of panic whereby some panicogens produce panic in

association with cortisol release whilst others produce the same emotional response

without any evidence of HPA axis activation [Sinha 1999]. This suggests the pathways

mediating the psychological response to CO2 operate separately to those that may

mediate the cortisol response. Whether the psychological response occurs as a result of

direct stimulation of limbic centres by CO2 or from indirect activation via the VLM and

LC is not certain. Evidence for a separate pathway, or at least a pathway with different

thresholds of activation, comes from the dose response study that indicated the

significantly lower doses required to produce a psychological response as compared to a

sympathetic (or cortisol) response. As discussed in Chapter 3, acute CO2 exposure may

well represent a ‘limbic insensitive’ threat [Herman and Cullinan 1997] with initial

brainstem activation whilst prolonged exposure to lower doses of CO2 may represent a

‘limbic sensitive’ threat requiring cortical processing first, with subsequent autonomic

and HPA activation.

Manipulating central serotonin, noradrenaline or opiate pathways in normal individuals

had no effect on either psychological or sympathetic responses although there is

considerable evidence that in susceptible individuals serotonin as well as other

neurotransmitters do have a modifying effect on psychological responses to CO2

exposure [Bertani et al 1997, Klaasen et al 1998, Ben Zion et al 1999, Miller et al 2000,

Schruers et al 2000, Perna et al 2002, Schruers et al 2002, Bertani et al 2003].

177

Figure 4.18 indicates schematically a hypothetical pathway that may mediate the

respective hormonal, cardiovascular and psychological response to a single breath of

35% CO2.

In the following chapter, another series of experiments has been performed that attempt

to further examine and characterise the potential mechanisms and pathways that may

mediate the response to acute CO2 exposure. In addition, these studies will also

examine some of the potential clinical implications an altered CO2 response system may

have for particular individuals.

178

Single breath of

35% CO2

Noradrenergic brainstem

Limbic centres

(CnA)

?

FmVrPsbHap(

Vagal centres/vagus nerve
centres
(VLM, LC) ?

PVN Bradycardia

NA ACTH

Emotional arousal ↑SBP Cortisol

igure 4.18. Schematic diagram suggesting the principal pathways of CO2ediated HPA, cardiovascular and psychological responses (solid arrows). agal and noradrenergic brainstem centres are directly stimulated by CO2

esulting in bradycardia and an acute pressor response respectively. sychological responses are probably due to a combination of direct CO2timulation of a central fear circuit as well as indirect stimulation via rainstem NA centres. PA responses occur indirectly in response to brainstem NA stimulation and

lso possibly from the CnA. CRH neurones from the PVN also have the otential for augmenting sympathetic and psychological responses represented by hashed arrows), although this role is less clear.

179

CHAPTER 5

THE 35% CO2 MODEL: RESPONSES IN SPECIFIC

SUBPOPULATIONS – FURTHER MECHANISMS

AND POTENTIAL CLINICAL RELEVANCE

180

5.1. The role of the HPA axis

Earlier experiments had identified emotional arousal and autonomic activation as the

principal responses to CO2 inhalation, with HPA axis activation probably occurring in

response to stimulation of either or both of the former two centres. Autonomic

activation appeared to involve direct parasympathetic stimulation as well as central

sympathetic stimulation with the pressor response occurring as a result of noradrenaline

mediated peripheral vasoconstriction. The pressor response to stress is, however,

complex and the reported role cortisol plays in regulating this response [Harbuz and

Lightman 1992, Chrousos 1998, Sapolsky et al 2000, Habib et al 2001, Tsigos and

Chrousos 2002] varies in the literature depending in the nature of the stressor, the

individual within whom it is occurring and the methods used to measure the response

[Kvetnansky et al 1995, Sapolsky et al 2000]. Traditionally, glucocorticoids enhance

the cardiovascular response to stress by increasing blood pressure and cardiac output

[Sapolsky et al 2000]. Similarly, glucocorticoid deficiency is associated with

hypotension and impaired cardiac function, particularly under conditions of stress

where, if the increased cardiovascular demands are not met, an acute adrenocortical

crisis may occur that could be rapidly fatal [Stewart 2003].

The interaction between the HPA and SAM axes, as detailed previously, occurs on

several levels within the brain and the periphery. CRH neurones are known to project

to brainstem noradrenergic centres where they increase activity of sympathetic

autonomic centres [Jezova et al 1999, Habib et al 2001, Gammatopoulos and Chrousos

2002]. Cortisol itself has direct actions on cardiac and vascular tissues [Habib et al

2001], although, its most important function in enhancing the cardiovascular responses

181

to stress stems from its role in augmenting the activity of catecholamines and other

vasoconstrictors [Habib et al 2001, Stewart 2003].

Glucocorticoids produced in the adrenal cortex are transported via a portal capillary

route to the adrenal medulla where they are required for the survival and maintenance of

adrenomedullary chromaffin cells [Kvetnansky et al 1995, Habib et al 2001,

Zuckerman-Levin et al 2001, Stewart 2003]. In addition, expression of the enzyme

phenylethanolamine N-methyltransferase (PNMT), required for the conversion of

adrenomedullary noradrenaline to adrenaline, depends on glucocorticoids at the

transcriptional level [Kvetnansky et al 1995, Sapolsky et al 2000, Zuckerman-Levin et

al 2001, Stewart 2003]. Glucocorticoids also enhance catecholamine action by

increasing α-adrenergic receptor binding sensitivity and catecholamine-induced cyclic

AMP synthesis [Sapolsky et al 2000]. Finally, glucocorticoids also inhibit the

vasodilatory effects of prostaglandins [Sapolsky et al 2000].

On the other hand others have shown that adrenocortical deficiency (for example

Addison’s disease) is associated with compensatory noradrenaline overproduction with

an increase in the noradrenaline to adrenaline ratio and increased cardiovascular

responses to stress [Bornstein et al 1995, Kvetnansky et al 1995]. Further, using a

model of immobilisation stress Kvetnansky et al [1995] describe the predominant acute

effects of glucocorticoids on the SAM and sympathoneural response to stress as reduced

catecholamine release and turnover with suppression of sympathoadrenal activity. In

accordance with the theory of Munck et al [Munck and Naray-Fejes-Toth 1994,

Sapolsky et al 2000], these authors imply that glucocorticoids act to restrain and modify

the stress response to prevent the damaging effects of excessive exposure to stress

mediators. Further, they ensure the response is kept appropriate to the intensity and

182

duration of the stress being faced [Munck and Naray-Fejes-Toth 1994, Sapolsky et al

2000]. More than likely, the effect of glucocorticoids and the HPA axis on SAM and

sympathoneural responses to stress is likely to vary (enhanced in some cases, inhibited

in others) depending several factors. These may include: the nature of the

glucocorticoid abnormality (central vs peripheral deficiency or excess, or exogenous

exposure); the duration and severity of the hormone alteration; associated changes such

as mineralocorticoid deficiency; the specific characteristics of the stressors involved;

and the specific characteristics of the individual in whom they are occurring.

A study of isolated glucocorticoid deficiency due to inherited ACTH resistance in a

family of six individuals was studied by Zuckerman-Levin et al [2001] with the

hypothesis that this condition will be associated with impaired adrenaline production,

compensatory noradrenaline release and physiological changes reflecting these

alterations. Subjects were examined at rest and following three stressors – upright

posture, the cold pressor test and exercise. Results showed significantly reduced

adrenaline levels compared with matched controls with slightly higher noradrenaline

levels and a marked increase in the noradrenaline:adrenaline ratio under all conditions.

With this, the pulse rate response to upright posture in patients was increased (an effect

of noradrenaline), whilst the systolic blood pressure response to cold was impaired

(probably due to insufficient adrenaline). Finally the diastolic blood pressure response

to exercise was increased in the patients, also thought to be secondary to the effect of

the increased noradrenaline:adrenaline ratio [Zuckerman-Levin et al 2001].

Experiments performed in this thesis examining the effect of changes to the HPA axis

have not identified a specific effect of cortisol on the cardiovascular response to CO2.

Disinhibiting the axis with naltrexone raised baseline cortisol levels but did not affect

183

SBP or HR responses. Similarly lactating women who have lower baseline cortisol

levels also had equivalent responses to controls. In addition the administration of

metyrapone (to decrease cortisol synthesis) with or without mineralocorticoid inhibition

(with spironolactone) had no effect on the cardiovascular response to CO2. It is

possible, however, that in all these conditions, particularly those of glucocorticoid

deficiency, there was still enough circulating cortisol to facilitate the necessary SAM

and sympathoneural responses associated with this challenge. To further elucidate the

importance of glucocorticoids in the CO2 response, and to assess any clinical role

cortisol deficiency may have in modulating the SAM and sympathoneural response to

stress, the CO2 challenge was performed in patients with Addison’s disease (primary

adrenal failure, see below). The hypothesis being that combined glucocorticoid and

mineralocorticoid deficiency will result in impaired cardiovascular responses to the

challenge and potentially increased emotional arousal through impaired feedback and

increased CRH.

184

5.2. Addison’s disease


In western countries, primary adrenocortical failure, or Addison’s disease, is most

commonly due to autoimmune adrenalitis [Stewart 2003]. In this condition, adrenal

antibodies cause destruction of the adrenal cortex but leave the adrenal medulla

unaffected. In contrast to secondary (pituitary) adrenocortical insufficiency (or ACTH

resistance as in the study by Zuckerman-Levin et al [2001]), Addison’s disease is

associated with both glucocorticoid and mineralocorticoid deficiency and clinical

symptoms are due to their combined lack. Adrenal androgens are also deficient and

some clinical symptoms may be due to their deficiency [Stewart 2003]. The clinical

presentation is usually of an insidious onset with malaise, fatigue, anorexia, nausea,

vomiting, abdominal pain and symptoms of postural hypotension. Skin pigmentation

due to ACTH stimulation of the melanocortin-2 receptor distinguishes primary from

secondary adrenocortical failure. Less commonly, long standing Addison’s disease may

be associated with overt psychiatric symptoms including memory impairment,

depression or anorexia nervosa [Stewart 2003]. Acute adrenal insufficiency or

addisonian crisis is an important, life threatening state that usually occurs during times

of intercurrent illness or stress. It is characterised by hypotension and shock out of

proportion to the current illness and is often associated with nausea, vomiting, acute

abdominal pain, hypoglycaemia, fever and electrolyte disturbances [Stewart 2003].

Addison’s disease is treated with the replacement of both glucocorticoids and

mineralocorticoids. Glucocorticoids are given in divided doses (less in the afternoon) to

try and mimic the normal diurnal rhythm of cortisol. Additional glucocorticoids need to

185

be given during times of illness, accident, surgery or other significant (including

mental) stress. Mineralocorticoids are given once daily to prevent postural hypotension

and electrolyte imbalance. These traditional replacement strategies have been

considered sufficient to allow patients with Addison’s disease to live normally,

however, many patients with this condition continue to complain of persistent fatigue,

malaise, poor exercise tolerance, a reduced ability to perform normally at work and

reduced stress tolerance [Riedel et al 1993, Arlt et al 1999, Lovas et al 2002]. Lovas et

al [2002] used several fatigue, general health and vitality perception scales to compare a

population of patients with Addison’s disease to the general population and identified

the Addison’s group as being quite heterogenous in their responses. Whilst many

patients had normal responses, there was a substantial group that described markedly

reduced subjective feelings of general health and working ability.

Adrenal androgens are not commonly replaced in clinical practice, although there is

some evidence that their administration may improve subjective health status and

sexuality, particularly in females [Riedel et al 1993, Arlt et al 1999, Stewart 2003]. On

the whole, however, they have failed to significantly improve the feelings of fatigue and

impaired health held by many patients [Riedel et al 1993, Hunt et al 2000, Lovas et al

2002]. It is not clear why these subjective feelings of fatigue and ill-health are so

prominent in many patients, or why they often feel less able to manage mild day-to-day

stress. Refinements and modifications of glucocorticoid replacement schedules

(including both the type of glucocorticoid used and the timing of its administration) are

used extensively to try and improve feelings of fatigue and well-being in Addison’s

patients. This is based on the premise that fixed twice or three times a day dosing is

insufficient to account for the normal day-to-day fluctuations caused by mild to

moderate stress normally experienced by all individuals. One principal objective of

186

studying patients with Addison’s disease was to assess whether their response to a mild

stressor (CO2 exposure) varied according to the state of hormone replacement and

whether this could provide any insights into the potential reasons for their general

feelings of ill-health.

It has also been suggested that Addison’s disease is associated with an increased

prevalence of affective and anxiety symptoms [Musselman and Nemeroff 1996],

although in normally replaced individuals the literature supporting an increased

prevalence of anxiety disorders is scant.

Five (2 male) subjects with known Addison’s disease of at least 5 years duration were

recruited from a pool of Addison’s disease sufferers who attend the Endocrinology

clinic at the Bristol Royal Infirmary. The subjects had a mean age of 49.8 years (range

44–55 years) and were currently well controlled on glucocorticoid and

mineralocorticoid replacement. Three subjects also had autoimmune primary

hypothyroidism and all were clinically and biochemically euthyroid on thyroxine

replacement at the time of testing. Subjects were free of all other new medications in

the two weeks prior to testing as well as during the testing weeks. During the testing

weeks, all subjects remained well with no evidence of intercurrent illnesses. In a

randomised, placebo controlled, double blind fashion, subjects attended for testing on

two occasions one to three weeks apart. For 48 hours prior to one visit subjects

received hydrocortisone 20 mg and fludrocortisone 100 mcg in the morning with a

further 10 mg hydrocortisone in the afternoon instead of their usual glucocorticoid and

mineralocorticoid replacement. For the other visit, subjects received an equal number

of matched placebo containing capsules taken at the same times.

187

All the tests were conducted between 1 and 3 pm and all subjects completed all the

tests. The tests were performed using the same protocol as described for previous

experiments and included the recording of baseline and test VC. Cardiovascular

measures were recorded with the Dynamap monitor recording pulse rate and blood

pressure every minute for 5 minutes before and 5 minutes after CO2 exposure.

Psychological responses were recorded using the same visual analogue scales. An

intravenous line for blood sampling was placed 30 minutes before testing commenced

and samples for prolactin levels were taken at baseline, 10, 20 and 30 minutes after

exposure. Cortisol levels are not reported because of the use of exogenous

glucocorticoids as part of their replacement therapy.


exposure with paired t-test analysis used to determine differences between single time

points including baseline measures for the placebo and active replacement visits for

each subject.

5.2.2. Results

Baseline prolactin levels were not significantly different between replacement and

placebo visits. CO2 stimulated increases in prolactin were small and did not reach

statistical significance in either condition. As shown in Table 5.1, compared with the

healthy volunteers who participated in the mineralocorticoid/glucocorticoid study,

baseline SBP and HR were non-significantly higher in the subjects with Addison’s

disease, although these subjects were also older (49.8 compared with 22.0 years). The

bradycardic response under both Addison’s replacement conditions was significant (p <

188

0.05), however, the fall was the same on both occasions (-9.0 +/- 3.4 bpm on

replacement; -10.6 +/- 3.3 bpm off replacement). There was a significant (p < 0.05)

pressor response for the visit when subjects were taking replacement hormones (+15.6

+/- 5.0 mmHg), but no significant response off replacement (+4.2 +/- 3.3 mmHg; p =

0.02) (Figure 5.01). As a whole, anxiety, fear, breathlessness and selected somatic

symptom increases were significant (p <0 .05), however, baseline and stimulated

anxiety, fear and breathlessness responses were no different between each visit.

Similarly, somatic symptom responses between each visit were equivalent (Figure

5.02).

189

Table 5.01. Baseline and stimulated SBP and HR responses in patients with Addison’s Disease both off and on standard replacement therapy (n=5). For comparison, responses in young healthy volunteers taking placebo, spironolactone, metyrapone or both spironolactone and metyrapone are presented (n=9).

Addison’s

On

Addison’s

Off

Control Spironolact-

one

Metyrapone Spironolact-

one /

Metyrapone

Baseline SBP

118.2+/-6.9

114.6+/4.5

110.3+/2.6

110.7+/2.9

108.4+/-2.8

109.6+/-2.1

∆max SBP 15.6+/-5.0 1.0+/-4.4* 10.2+/-3.4 13.8+/-2.7 11.8+/-2.9 11.5+/-3.1

HR 72.0+/-3.2 71.0+/-4.3 68.0+/-3.5 65.7+/-3.5 68+/-3.7 65.8+/-2.9

∆max HR -9.0+/-3.4 -10.6+/-3.3 -14.7+/-2.4 -9.1+/-3.2 -18.1+/-4.2 -16.0+/-3.5

190

SBP

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

105110115120125130135140145

On replacementOff replacement

Time (mins)

mm

Hg

SBP response to 35% CO2

On replacement Off replacement

-10

0

10

20

30

40

*

mm

Hg

PR

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

55

60

65

70

75

80On replacementOff replacement

Time (mins)

b/m

PR response to 35% CO2

On replacement Off replacement

-30

-20

-10

0

b/m

Figure 5.01. SBP (upper panel) and HR (lower panel) in response to a single breath of 35% CO2 in patients with Addison’s disease on and off standard gluciocorticoid and mineralocorticoid replacement. Left hand panels show responses over time and right hand panels show peak stimulated response. n=5. *p=0.02

191

B a s e lin e fe e lin g s o f a n x ie ty ,fe a r a n d b re a th le s s

Anxi

ety

Fear

Bre

athl

essn

ess

0

2 5

5 0

7 5On rep lac em entOff rep lac em ent

mm

An x ie ty , fe a r a n db re a th le s s n e s s re s p o n s e to

3 5 % C O 2

Anx

iety

Fear

Bre

athl

essn

ess

0

2 5

5 0

7 5

1 0 0On rep lac em entOff rep lac em ent

mm

S o m a tic s y m p to m re s p o n s e to3 5 % C O 2

Hea

rt ra

cing Hot

Blu

rred

vis

ion

Diz

zine

ss

0

2 5

5 0

7 5On rep lac em entOff rep lac em en t

mm

192

Figure 5.02. Baseline (upper panel) and stimulated (middle panel) anxiety, fear and breathlessness responses to a single breath of 35% CO2 in patients with Addison’s disease on and off standard gluciocorticoid and mineralocorticoid replacement. Lower panel indicates somatic symptom responses. n=5. Baseline and stimulated responses between groups are all equivalent.

5.2.3. Discussion

In this study using a single breath of 35% CO2 as a challenge, no difference in baseline

subjective anxiety or fear was identified in subjects on full replacement compared with

no replacement for 48 hours. Anxiety and fear scores were also equivalent to normal

healthy volunteers and to volunteers treated with either or both spironolactone and

metyrapone. Similarly, subjective anxiety and fear responses were equivalent in the

replaced and placebo conditions and was also no different to healthy controls or the

spironolactone and metyrapone conditions. This would suggest that baseline HPA axis

function does not affect psychological responding to the 35% CO2 challenge.

Patients with Addison’s disease on full glucocorticoid and mineralocorticoid

replacement were able to mount the typical pressor response to the 35% CO2 challenge

that was similar in magnitude to young healthy controls. They also showed the typical

bradycardic response. Without replacement, the pressor response was significantly

impaired, although the bradycardia was not significantly different to the replacement

condition or to controls. There are several potential mechanisms that would explain this

observation. Firstly, the renin-angiotensin system is an important mediator of the

response to cardiovascular stressors such as postural change and volume loss [Van de

Kar and Blair 1999]. Angiotensin II is an important vasoconstrictor and also acts to

augment SAM activity both centrally and peripherally [Reid 1992, Van de Kar and

Blair 1999]. Deficiency of angiotensin II or inhibition of its receptor centrally is

associated with reduced SAM and hormonal stress responses and a reduction in blood

pressure [Armando et al 2003]. Aldosterone, the principal mineralocorticoid secreted

by the adrenal cortex in response to renin-angiotensin stimulation, acts to increase

sodium and water reabsorption in the kidney, thereby increasing plasma volume and

193

blood pressure [Stewart 2003]. Deficiency of aldosterone is the principal reason for

symptoms of postural hypotension in untreated Addison’s disease and may explain the

reduced pressor response observed. Of note, in isolated glucocorticoid deficiency

[Zuckerman-Levin et al 2001] with an intact renin-aldosterone system, the

haemodynamic challenge of postural change was associated with a normal blood

pressure response suggesting glucocorticoids are not involved in this response. In this

same study [Zuckerman-Levin et al 2001], the pressor response to cold was impaired

suggesting an impaired SAM response to this challenge as a consequence of

glucocorticoid deficiency. This may represent an alternative explanation for the

impaired pressor response to CO2. As previously discussed, glucocorticoids play an

important part in enhancing the SAM response to stress both at a central (brainstem)

and peripheral level including maintaining adrenal medullary chromaffin cells and the

production of adrenal catecholamines [Bornstein et al 1995, Sapolsky et al 2000,

Laborie et al 2003]. Glucocorticoid deficiency may then result in impaired adrenaline

mediated responses to this challenge. CO2 challenge studies described earlier indicated

the pressor response is predominantly due to a noradrenaline mediated increase in total

peripheral resistance, however, these studies have not excluded the potential

contribution of adrenal catecholamine to this response. Importantly, whilst samples for

adrenaline and noradrenaline were taken and measured, inconsistencies in and problems

with the HPLC assay made their interpretation unreliable and results have not been

reported.

Earlier studies of this challenge had suggested the bradycardic response was mediated

independently of the pressor response most likely through direct stimulation of vagal

centres by CO2 itself. The bradycardia observed in patients with Addison’s was the

same regardless of replacement status and was similarly no different to healthy

194

volunteer controls and to those taking either spironolactone, metyrapone or both. This

would again reinforce the likelihood that the bradycardia is occurring independently of

the HPA axis and also the pathways involved in mediating the pressor response.

Prolactin release is also seen following stress exposure including haemodynamic

challenges [Van de Kar and Blair 1999], although its role is uncertain. In patients with

Addison’s disease, prolactin responses to 35% CO2 were not affected by hormone

replacement status, suggesting prolactin release occurs independently of HPA axis

activity.

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5.3. The role of the autonomic nervous system

The principal effector arms of the stress response (the HPA and SAM axes) are

interconnected at several levels [Chrousos 1998, Bornstein and Chrousos 1999, Habib et

al 2001, Wurtman 2002]. In addition to components of the HPA axis being important in

the maintenance, regulation and enhancement of the SAM response to stress (as detailed

above), the SAM system plays an essential role in the regulation of the HPA response to

specific stressors. Projections of noradrenergic neurones from brainstem autonomic

centres to the hypothalamic PVN significantly influence CRH and HPA activity

[Kvetnansky et al 1995, Jezova et al 1999, Koob 1999]. Stimulation of these neurones

or administration of noradrenaline directly into the area of the PVN significantly

increases CRH mRNA production, CRH synthesis and release with a dose dependent

increase in ACTH and glucocorticoids [Itoi et al 1994, Kvetnansky et al 1995, Pacak et

al 1995]. Inhibition of these neurones results in the opposite effect [Kvetnansky et al

1995]. Importantly, however, significant stressor specificity exists such that the

presence of an HPA and SAM response as well as the intensity and duration of the

response will vary according to the nature of the stressor applied [Kvetnansky et al

1995, Pacak et al 1995]. Further, there is evidence to suggest that stressor-specific

activation of noradrenergic brainstem systems is an important contributor to the

differential regulation of HPA and SAM responses to specific stressors [Kvetnansky et

al 1995, Pacak et al 1995, Pacak et al 1998].

Activation of the SAM/sympathoneural system in response to stress exposure is

responsible for the early ‘fight or flight’ response. This response comprises immediate

stereotypical behaviour (such as freezing or fleeing) and activation of multiple

physiological processes designed to release energy stores, heighten alertness, increase

196

motor activity, protect against injury and inhibit non-essential functions [McCarty 1994,

Young and Landsberg 1998]. The peripheral components of this system, the

sympathetic and parasympathetic nervous systems, are essentially antagonistic, but act

in a functionally synergistic fashion to enhance their control. Activation of the

sympathetic system, for example, is usually accompanied by withdrawal of the

opposing parasympathetic inputs [Gilbey and Spyer 1993, Janig 1983, Elenkov et al

2000].

Studies of cardiorespiratory regulation in reptiles and mammals has led to the

identification of opposing vagally mediated responses and the subsequent description of

the likely way in which vagal pathways mediate heart rate responses to novelty and

various other stressors [Porges 1995]. This ‘Polyvagal Theory’ identifies two

anatomically and functionally discrete vagal systems [Porges 1995, Porges et al 2003].

The first, a phylogenetically more primitive system, originates from the Dorsal Motor

Nucleus of the vagus (DMNX) and is characterised by unmyelinated fibres that produce

heart rate slowing and neurogenic bradycardia. The second, an evolutionarily more

recent system, comprises myelinated fibres originating from the Nucleus Ambiguus.

This system, is responsible for producing respiratory sinus variation through tonic

inhibition of the sympathetic nervous system and therefore acting as a ‘brake’ on the

energy expensive and metabolically demanding ‘fight-or-flight’ sympathetic system

[Porges 1995, Porges et al 2003]. In addition to mediating heart rate responses, these

systems are also intimately involved with regulating the stereotypical emotional and

behavioural responses to stress through their relationship with other cranial nerves (for

example those involved with facial expression and vocalisation [Porges et al 2003]), as

well as other brainstem and higher centres [Porges 1995]. The theory postulates that

during times of stress, control of autonomic function shifts from the phylogenetically

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newer to the older and more primitive systems [Porges et al 2003]. In other words,

when faced with a challenge, withdrawal of the nucleus ambiguus system would allow

increased activity of the sympathetic system necessary for meeting the metabolic

demands of that challenge, but would make the individual more susceptible to

stimulation of the DMNX system with significant bradycardia and even syncope. This

theory has thus been used to explain vasovagal syncope associated with fright [Porges

1995, Porges et al 2003], and with the heart rate changes observed in response to

hypoxia during foetal heart rate monitoring [Reed et al 1999].

The brainstem cardiorespiratory centres, particularly the more primitive centres, are

much more sensitive to CO2 than O2 [West 1974, Porges 1995], and it is possible that

the 35% CO2 challenge is directly stimulating the DMNX system thereby producing the

significant bradycardia. Syncope is prevented by sympathoneural activation with

peripheral vasoconstriction, increased total peripheral resistance and the subsequent

pressor response.

198


Three clinical models of autonomic dysfunction have been studied in order to further

clarify some of the mechanisms that may regulate the cardiovascular and HPA

responses to 35% CO2, and to also evaluate the potential clinical relevance of an altered

response in these individuals. The groups chosen included: patients with diabetic

autonomic neuropathy (DAN) (a peripheral neuropathy with predominant

parasympathetic dysfunction); patients with pure autonomic failure (PAF) (a model of a

purely peripheral mixed sympathetic and parasympathetic neuropathy); and patients

with multiple systems atrophy (MSA) (representing a model of central autonomic

failure). Based on earlier experiments, it was hypothesised that peripheral

parasympathetic failure (DAN and PAF) would result in loss of the bradycardic

response but the pressor response would be preserved if sympathetic function was not

affected (that is, preserved in early DAN but not in PAF). Central autonomic failure

(MSA) would be associated with a loss of the pressor response but not of the

bradycardia. HPA and psychological responses would be preserved in peripheral (DAN

and PAF) but not central (MSA) autonomic failure syndromes. Finally an attempt was

made to determine whether the responses were sufficiently discrete and predictable

enough (based on the pathophysiology of the underlying condition), to use the CO2 test

clinically when investigating these conditions.

199

5.5. Diabetic autonomic neuropathy


Autonomic neuropathy is an important chronic complication of diabetes producing a

variety of clinical features and physiological changes related to the widespread

involvement of both the sympathetic and parasympathetic divisions of the autonomic

nervous system [Ward 1992, Forst et al 1996]. Damage to peripheral nerves is related

to glycaemic control and the frequency of clinically apparent neuropathy is related to

both the duration of diabetes and the adequacy of control [Ward 1992]. The exact

pathological mechanism is, however, unknown although popular theories include nerve

damage secondary to sorbitol accumulation or from ischaemia following microvascular

occlusion [Ward 1992]. The prevalence of autonomic neuropathy is highly variable

ranging from 0% - 93% depending on the diagnostic criteria used and the population

studied [Kempler et al 2002]. More recently, studies in type 1 diabetics have suggested

prevalence rates of between 20 and 36% [Stella et al 2000, Kempler et al 2002]. Many

patients with diabetes will demonstrate abnormalities of autonomic function on specific

testing without having any clinical symptoms [Ward 1992], however, once established

autonomic neuropathy is associated with a significant deterioration in quality of life and

increased morbidity and mortality [O'Brien et al 1991, Ward 1992, Ziegler 2001,

Wheeler et al 2002, Ziegler 2002]. The manifestations of autonomic neuropathy are

diverse involving numerous organ systems [Ziegler 2001]. Most commonly this

involves:

i. The cardiovascular system with resting tachycardia, reduced heart rate

variability, impaired heart rate and blood pressure responses to exercise and

200

postural changes, silent myocardial ischaemia and possibly an increased risk

of increased arrhythmias and sudden death.

ii. The respiratory system with impaired ventilatory responses to hypoxia and

hypercapnia [Tantucci et al 2001], and sleep apnoea.

iii. The gastrointestinal system with oesophageal dysmotility, gastroparesis,

diarrhoea and anorectal dysfunction.

iv. The genitourinary tract with bladder and erectile dysfunction.

Several studies have demonstrated an increased risk of premature death associated with

the presence of diabetic autonomic neuropathy [O'Brien et al 1991, Wheeler et al 2002,

Ziegler 2002]. O’Brien et al [1991], for example, demonstrated a 5-year mortality rate

that was more than 5-fold greater in type 1 diabetic patients with autonomic neuropathy

compared with those without neuropathy. The mechanism behind this increased

mortality is unclear, however, most hypotheses have implicated the increased

arrhythmogenic potential of cardiac autonomic neuropathy [Wheeler et al 2002]. The

parasympathetic nervous system (PNS) is typically involved earlier in diabetic

autonomic neuropathy [Edmonds and Watkins 1992]. This system provides tonic

inhibitory control over the heart, and the bradycardic response seen in the normal

response to the 35% CO2 challenge may be mediated by direct stimulation of the region

of the DMNX [Porges 1995]. In addition, withdrawal of the nucleus ambiguus

component of the vagal response allows sympathetic stimulation to predominate. It has

been postulated the withdrawal of the nucleus ambiguus component leaves the

individual susceptible to the effects of excessive neurogenic bradycardia if sympathetic

compensation is inadequate. However, it could also be postulated that parasympathetic

neuropathy removes both vagal components that would normally act to protect the

individual from excessive sympathetic stimulation increasing the risk of cardiac

201

tachyarrhythmias and resulting in an increased mortality associated with diabetic

autonomic neuropathy [Porges 1995, Reed et al 1999, Wheeler et al 2002, Porges et al

2003]. However, not all studies have shown an increase in cardiovascular (as opposed

to all-cause) mortality or an increased prevalence of non-fatal arrhythmias [Wheeler et

al 2002]. It has also been noted that most deaths appear to occur during times of acute

stress or during sleep [Tantucci et al 2001] and several studies have examined the

integrity of components of the stress response following various challenges.

In view of its immediate clinical relevance, the most commonly evaluated challenge has

been that of hypoglycaemia. A recent study by Meyer et al [1998] in longstanding

diabetics demonstrated impaired counter regulatory hormone production including

noradrenaline, adrenaline, cortisol and GH in all subjects, but catecholamine responses

were more severely impaired in those with autonomic neuropathy compared with those

without. Other studies that have incorporated such challenges as the valsalva

manoeuvre, postural changes, the cold pressor test and exercise [Forst et al 1996,

Granados et al 2000] have all indicated autonomic neuropathy is associated with low

baseline noradrenaline levels and impaired noradrenaline responses to stress. Both

baseline and stimulated adrenaline levels on the other hand, were more variable. Forst

et al [1996] examined catecholamine responses to a psychological stressor and

identified normal stimulated noradrenaline responses, but impaired adrenaline responses

in those with diabetic autonomic neuropathy compared with those without.

Haemodynamic differences, however, were not were not observed. Most studies

conclude that there is evidence of impaired adaptive responses to stress particularly

involving the SAM and sympathoneural systems in patients with diabetic autonomic

neuropathy, and that this may be important in the observed increased mortality

202

associated with this particular diabetic complication [Forst et al 1996, Meyer et al 1998,

Granados et al 2000, Wheeler et al 2002].

Another facet that has been investigated in subjects with diabetic autonomic neuropathy

in order to further understand their increased mortality has been the regulation of

breathing in these subjects. Several studies have indicated impaired peripheral

chemosensitivity and reduced hypoxic drive [Nishimura et al 1989, Tantucci et al

2001], although central chemosensitivity and responses to CO2 have been conflicting.

Whilst some studies have shown normal or increased ventilatory and haemodynamic

responses to hypercapnia [Nishimura et al 1989, Tantucci et al 2001], others have

shown impaired responses [Williams et al 1984, Tantucci et al 2001]. More recently,

Tantucci et al [2001] have demonstrated altered chemosensitivity to hypercapnia

depending on the extent and severity of the underlying autonomic dysfunction. Subjects

with autonomic neuropathy that included postural hypotension (reflecting both

sympathetic and parasympathetic dysfunction) were shown to have an increased

ventilatory and cerebrovascular reactivity responses to progressive hypercapnia, as

compared with those with autonomic neuropathy without postural hypotension

(parasympathetic dysfunction alone). This suggested central autonomic function was

impaired as a result of peripheral dysfunction of the autonomic nervous system and the

extent of this impairment reflected the balance between the activities of the sympathetic

and parasympathetic systems [Tantucci et al 2001].

Male subjects aged between 18 and 70 years with diabetes of at least 3 years duration

were recruited from the Bristol Royal Infirmary’s diabetes outpatient clinic. The first

12 subjects were recruited on the basis of a high clinical likelihood of the presence of

autonomic neuropathy (including the presence of peripheral neuropathy and/or

203

suggestive symptoms). No subject had previously had formal autonomic function

studies performed. These first subjects underwent detailed autonomic functioning

testing (as described below) followed immediately by a 35% CO2 challenge study using

the same protocol as already described. Autonomic function tests were not analysed

until after the completion of the CO2 test. Of the twelve, ten were identified as having

autonomic neuropathy (AN), whilst the remaining two were not and were included in

the control group (C). Subsequently, a further eight consecutive subjects were

recruited. Of these, one was identified with autonomic neuropathy and included in that

group and data from one was excluded because of an inadequate intake of CO2 during

the challenge (VC < 80% of baseline). As a result, there were 11 subjects with

autonomic neuropathy and 8 control subjects. As shown in Table 5.02, those with

autonomic neuropathy were significantly older (60.0 +/- 2.4 compared with 40.6 +/-

5.23 years, p = 0.002) and had had diabetes for longer (25.9 +/-4.0 compared with 12.4

+/- 3.6 years, p = 0.03). In addition, those with autonomic neuropathy were more likely

to have other microvascular complications of diabetes, and were more likely to be on

anti-hypertensive medication. The number of subjects with type 1 compared with type

2 diabetes within each group was not significantly different.

Standard exclusion criteria for the CO2 test as previously described were applied in this

study. Specifically, subjects were excluded if they had uncontrolled diabetes (defined

as an HbA1c ≥ 10%), uncontrolled hypertension, a history of recent angina or ischaemic

heart disease or any history of cerebrovascular disease. Other exclusion criteria

included age over 70 years, a history of asthma or a history of panic disorder or severe

anxiety. Patients were allowed to remain on their usual medications as long as there

had been no recent (within 8 weeks) change and as long as they were not taking

exogenous glucocorticoids, anti-depressants, anti-psychotics or anti-anxiety agents, β-2

204

agonists or centrally acting anti-hypertensives. Other anti-hypertensives were allowed,

and one subject (in the autonomic neuropathy group) was taking a β-blocker. Subjects

were asked to avoid consuming cigarettes, alcohol and caffeine for 24 hours prior to

testing.

CO2 testing was performed according to the same protocol as described for the

experiment involving subjects with Addison's disease and used the same cardiovascular

monitoring techniques, visual analogue scales, and blood sampling procedures.

Methods of statistical analysis were also the similar using repeated measures ANOVA

and unpaired t-test analysis.

On the day of testing, all subjects were asked to attend between 11 am and midday and

had their blood glucose level measured on arrival. Testing was only undertaken if blood

glucose levels were between 5 to 12 mmol/l. An intravenous line was placed in an

antecubital vein as before and subjects where asked to rest quietly on a bed prior to

autonomic function studies being performed. A 12-lead ECG was recorded and

autonomic function studies performed according to the method of Ewing and Clark

[1982]. This procedure involved determining the R-R interval from lead II of the ECG

and classifying subjects according to the following [Granados et al 2000]:

1. The supine position test determines the variation in sequential R-R intervals

after a one-minute rest (normal, 1.4 or more; borderline, 1.1 –1.3; abnormal,

1.0 or less).

2. The expiration-to-inspiration ratio (E/I) with the subject breathing at

maximal vital capacity with a regular rate of six breaths per minute. During

each expiration-inspiration cycle, the ratio of the longest to the shortest R-R

interval is calculated and the mean of six ratios taken as the E/I ratio

205

(normal, 15 beats/min or more; borderline, 11-14 beats/min; abnormal, 10

beats/min or less).

3. The Valsalva index is calculated from the longest R-R interval following a

Valsalva manoeuvre divided by the shortest R-R interval during the

manoeuvre (normal, 1.21 or more; abnormal, 1.20 or less).

4. Postural hypotension was determined by comparing supine SBP after a 5

minute rest with SBP after 1 minute in the standing position (normal, 10

mmHg or less; borderline, 11-29 mmHg; abnormal 30 mmHg or more).

Using these criteria, subjects were classified as having no autonomic neuropathy (all

four tests normal) or as definite autonomic neuropathy (first 3 tests all abnormal) with

or without postural hypotension (depending on the result of the fourth test). As shown

in Table 5.02, 3 (27%) of the 11 subjects with autonomic neuropathy also had postural

hypotension.

5.5.2. Results

Baseline cortisol and prolactin levels were equivalent in both groups (Figure 5.03). In

response to a single breath of 35% CO2 there was a significant increase in cortisol in

both groups (p < 0.05) with no difference in the maximum cortisol change between the

control (C) and autonomic neuropathy (AN) groups (Figure 5.03). The prolactin

response to 35% CO2 did not reach statistical significance in either group.

As shown in Table 5.02 and Figure 5.04, baseline blood pressure in the AN and C

groups was similar (135/74 and 137/70 mmHg, respectively) and whilst heart rates were

206

consistently higher in the AN group compared with the C group at baseline (76 +/- 3.5

and 69 +/- 3.2 b/m, respectively) and at all time points during the study, these

differences did not reach statistical significance. In response to 35% CO2, there was a

significant pressor response in both groups (+13.2 +/- 1.7 mmHg in the C and +12.3 +/-

3.7 mmHg in the groups respectively). However, whilst the C group demonstrated a

significant bradycardic response similar to that seen in normal healthy volunteers (-10.5

+/- 1.9 bpm), no subject with AN demonstrated a bradycardic response. The mean

maximum change in heart rate in the AN group was +2.7 +/- 1.3 bpm (p < 0.0001

compared with the C group) (Figure 5.04).

Baseline anxiety, fear and somatic symptoms of anxiety were minimal in all subjects

and did not differ between groups. In response to 35% CO2 there was a significant

increase in subjective feelings of anxiety, fear, breathlessness and in some specific

somatic sensations (p < 0.05). There was no difference in the change in these feelings

between the two groups (Figure 5.05) apart from a trend towards less breathlessness in

the AN group.

207

Table 5.02. Baseline characteristics of diabetic subjects with (n=11) and without (n=8) autonomic neuropathy

Control Autonomic

Neuropathy

p

Age (years)

40.6 +/- 5.3

60.0 +/- 2.4

0.002

Age range 24 – 36 40-69

Diabetes type

Type 1 75% 55%

Type 2 25% 45%

Duration of diabetes (years) 12.4 +/- 3.6 25.9 +/- 4.0 0.03

Range of duration 3 – 36 3 – 43

Complications

Diabetic retinopathy 38% 63% <0.05

Nephropathy (including

microalbuminuria)

0% 45% <0.05

Peripheral neuropathy 25% 91% <0.05

Cardiovascular parameters

Resting blood pressure (mmHg) 137 / 70 135 / 74 ns

Postural hypotension - 27%

Resting heart rate 69.3 +/- 3.2 76.4 +/- 3.5 ns

Antihypertensive medications <0.05

Nil 87.5% 45%

ACEI alone 12.5% 18%

ACEI plus other - 27%

AngIIAntagonist - 9%

β-blocker - 9% (1 subject)

208

Baseline cortisol

Control AN

0

100

200

300

400

500

nmol

/l


Control AN

-100

0

100

200

300

400

nmol

/lBaseline prolactin

Control AN

0

100

200

300

nmol

/l

Prolactin response to 35% CO 2

Control AN

-100

0

100

200

nmol

/l

Figure 5.03. Baseline and stimulated cortisol (upper panel) and prolactin (lower panel) responses to a single breath of 35% CO2 in diabetic subjects with (n=11) and without (n=8) autonomic neuropathy. Differences between groups are all non-significant.

209

SBP

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

125

150

175ControlAN

Time (mins)

mm

Hg

SBP

Control AN

-10

0

10

20

30

mm

Hg

HR

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

50

60

70

80

90ControlAN

Time (mins)

bpm

HR

Control AN

-30

-20

-10

0

10

20

bpm

*

Figure 5.04. SBP (upper panel) and HR (lower panel) responses to a single breath of 35% CO2 in diabetic subjects with (n=11) and without (n=8) autonomic neuropathy. *p<0.0001

210

Psychological responses to35% CO2

Anxiety Fear Breathlessness

0

25

50

75ControlAN

mm

Somatic symptom responsesto 35% CO2

Hea

rt B

eat

Feel

ing

Hot

Blu

rred

Vis

ion

Diz

zine

ss

0

25

50

75ControlAN

mm

Figure 5.05. Subjective anxiety, fear and breathlessness (upper panel) and somatic symptom (lower panel) responses to a single breath of 35% CO2 in diabetic subjects with (n=11) and without (n=8) autonomic neuropathy. Differences between groups are all non-significant.

211

5.5.3. Discussion

The most obvious abnormality between those with autonomic neuropathy compared to

those without was the failure to observe a bradycardic response in any of the AN +

subjects. In an earlier experiment involving a subject with pulmonary denervation, the

absence of bradycardia (with a normal pressor response) implied an intact vagus was

necessary for this response, whilst earlier studies showing the bradycardia occurring

prior to the pressor response implied this is not reflex bradycardia. The observation in

the diabetic subjects with autonomic neuropathy supports the notion that this is a direct

vagally mediated response that is lost in the presence of parasympathetic dysfunction.

A maintained bradycardia with no change in blood pressure in the Addison’s patients

off treatment also supports this concept.

Furthermore, all of these subjects demonstrated a pressor response that was no different

to the control group. As mentioned, it has been consistently shown that diabetic

autonomic neuropathy is associated with reduced basal and impaired stimulated

noradrenaline responses [Forst et al 1996, Granados et al 2000]. Since the pressor

response to 35% CO2 is thought to be predominantly mediated through noradrenaline

mediated vasoconstriction, it had been postulated that the pressor response might be

impaired in this group as well. Indeed, Forst et al [1996] had indicated declining

catecholamine responses to stress with increasing severity of autonomic dysfunction

(reflecting the increasing significance of sympathetic nervous system, involvement),

although no difference in haemodynamic responses were noted even in the severe group

with the most marked catecholamine difference. As with this study, Tantucci et al

[2001] demonstrated significantly higher resting heart rates in subjects with autonomic

neuropathy, although resting blood pressure was equivalent. These authors also showed

heart rate and blood pressure responses to progressive hypercapnia (breathing 7% CO2

212

according to the Read re-breathing method) were no different in control subjects as

compared with diabetic subjects with autonomic neuropathy, including when these were

sub-divided into those with or without postural hypotension. They did, however, show

differences in cerebrovascular reactivity in controls compared with autonomic

neuropathy subjects both with and without postural hypotension. In this study, 3 of the

11 AN+ subjects had postural hypotension >30mmHg. There was no difference in the

pressor response in these three individuals as compared with either the other AN+

subjects or the control group. However, it may be that this number was too small to

identify a real difference.

Baseline and stimulated cortisol responses were equivalent in those with and without

diabetic autonomic neuropathy. This would suggest the HPA axis to this challenge

remains intact and is independent of peripheral autonomic dysfunction. Studies of

uncontrolled diabetes in rats have indicated HPA axis dysregulation with increased

baseline CRH, ACTH and cortisol levels and impaired cortisol responses to applied

stress [Williams et al 1984, Chan et al 2002]. Interestingly, this dysregulation was

normalised with insulin treatment, although similarly impaired adrenaline responses

were not affected by the same treatment [Williams et al 1984]. It is likely that subjects

with acute hyper or hypoglycaemia will have alterations in HPA axis output as a

reflection of the glucose levels at the time and that subjects with persistent poorly

controlled blood glucose levels or frequent hypoglycaemia will have altered HPA axis

activity. In this study with treated control and autonomic neuropathy subjects matched

for glycaemic control and having relatively normal glucose levels at the time of testing

the effect of glucose levels on the HPA axis was minimised.

213

Cortisol responses to graded exercise have been shown to be impaired in diabetic

subjects with autonomic neuropathy compared with those without, although only at

maximum workload [Hilsted et al 1980 , Chan et al 2003]. The mechanism of this is

unclear, although it has been suggested that this is a function of impaired sympathetic

afferent impulses [Chan et al 2003]. Exercise at maximal workload is a complex

stressor that involves other neurohormonal pathways such as GH (which is also blunted

in those with diabetic autonomic neuropathy) and the HPA axis response that is almost

certainly mediated through different pathways to the CO2 challenge [Iranmanesh et al

1990].

Reduced circulating and stimulated prolactin levels have previously been demonstrated

in poorly controlled diabetic patients and laboratory animals with induced diabetes

[Iranmanesh et al 1990, Arroba et al 2003]. The mechanism producing this is not fully

understood, however, recent evidence suggests a reduction in lactotroph number within

the pituitary is in part responsible [Arroba et al 2003]. The consequences of this change

in prolactin secretion is also not fully understood, although one group [Hartmann and

Cregan 2001] has suggested diabetic women could have delayed or reduced breast milk

production as a result. Compared with results from non-diabetic healthy volunteers,

baseline prolactin levels were no different in those with diabetes regardless of their

neuropathy state. Apart from two diabetic control subjects, none of the other diabetic

subjects demonstrated a prolactin response to the CO2 challenge. This is in contrast to

the significant prolactin secretion seen in other experiments involving healthy non-

diabetic volunteers. This would be consistent with the already mentioned literature

regarding prolactin release in diabetics and, although at the time of testing blood sugar

control was not poor, most of the subjects tested have had diabetes for many years and

may have had periods of poor control in the past.

214

Finally, emotional arousal, as measured by subjective anxiety, fear, breathlessness and

somatic symptoms of fear where no different to those seen in healthy volunteers with

autonomic neuropathy having no additional effect on anxiety or other psychological

responses. This would appear to reinforce the notion that psychological responses to

35% CO2 appear to be mediated through pathways involving central chemoreceptors

that are probably stimulated independently, but that may subsequently influence

hypothalamic (HPA and prolactin) and brainstem (SAM) centres through reciprocal

connections.

215

5.6. Chronic autonomic failure syndromes


The autonomic failure syndromes are numerous in origin, most commonly occurring in

association with other known pathologies that also involve the autonomic nerves. These

‘secondary’ forms of autonomic neuropathy most commonly occur in association with

diabetes and other metabolic insults, amyloidosis, inflammatory disease, malignancy

and exposure to various medications or poisons. Other associations include a number of

inherited diseases, chronic infection and the aging process [Bannister and Mathias

1992a]. Chronic ‘primary’ autonomic neuropathy (or that not occurring in association

with other known pathologies) has traditionally been subdivided into three forms:

i. PAF,

ii. MSA,

iii. Autonomic failure with Parkinson’s disease (PD) [Bannister and Mathias

1992a].

Since the autonomic nervous system innervates all organ systems in the body, the

clinical manifestations of autonomic failure are widespread and varied. Early changes

are often subtle with few clues as to the underlying disease process due to the existence

and activation of multiple compensatory reflexes [Bannister and Mathias 1992b]. As

will be detailed further, the pathophysiology of PAF involves post-ganglionic fibres and

thus represents model of failure of the peripheral autonomic nervous system. In

contrast, MSA involves pre-ganglionic systems and therefore represents central

autonomic failure.

216

Postural hypotension is the most common and most dramatic clinical feature of all three

forms of chronic autonomic failure [Bannister and Mathias 1992b]. Other clinical

features include visual disturbance, defective sweating, cardiorespiratory abnormalities

and problems with urogenital function including bowel, bladder and sexual dysfunction

[Bannister and Oppenheimer 1972, Bannister and Mathias 1992b]. Movement disorders

are also apparent in MSA and PD forms [Bannister and Mathias 1992b, Parikh et al

2002]. Patients vary considerably in the degree of involvement of various components

of the autonomic nervous system. However, early in their presentation the clinical

features are strikingly similar and it can often take several years before it is clinically

apparent which form of the disease a particular patient may have. This distinction is

critical, as the prognosis of the various forms is very different [Bannister and Mathias

1992b]. PAF is typically characterised by a relatively benign course with predominant

postural hypotension, no movement disorder and an essentially normal life expectancy

[Bannister and Mathias 1992a, Bannister and Mathias 1992b]. MSA on the other hand

runs a debilitating, progressive and distressing course with increasing involvement of

autonomic systems, progressive motor weakness and rigidity, and is invariably fatal

with a life expectancy of less than 10 years [Bannister and Mathias 1992b, Parikh et al

2002].

In recent years, much has been learnt about the pathophysiology of these forms of

autonomic failure and several new tests have been developed to aid in their diagnosis.

As yet, however, no single test exists that can accurately distinguish peripheral from

central autonomic failure and the diagnosis remains a clinical one. MSA and PAF have

a distinct pathophysiological basis, and their detailed evaluation has provided an

enormous amount of valuable information on the mechanisms of autonomic function

217

and has led to the considered introduction of new and more effective therapies

[Bannister and Mathias 1992b].

The primary site of pathological involvement in PAF is the post-ganglionic sympathetic

neuron. This condition is characterised by reduced intra-neuronal noradrenaline

synthesis as well as reduced noradrenaline re-uptake from the synaptic cleft [Ziegler et

al 1977, Parikh et al 2002] and is reflected by lower plasma noradrenaline levels

compared to both normal individuals and those with MSA or PD [Bannister and

Mathias 1992b, Parikh et al 2002]. Consistent with this, histological studies of PAF

subjects show markedly reduced post-ganglionic nerve fibres with remaining nerve

endings showing abnormalities including Lewy bodies, distorted neurites and high

concentrations of lysosomes [Bannister and Mathias 1992b, Parikh et al 2002].

Functionally they are characterised by post-synaptic receptor hypersensitivity to

noradrenaline with associated baseline vasoconstriction and an increased TPR. Whilst

sympathetic failure and reduced baroreflex buffering capacity with postural hypotension

is the most significant clinical symptom [Bannister and Mathias 1992b, Shannon et al

2000, Parikh et al 2002], a large number of patients have associated supine hypertension

that is not mediated by increases in cardiac output and only partly due to residual

sympathetic tone mediated by post-synaptic hypersensitivity [Shannon et al 2000,

Parikh et al 2002].

Parasympathetic dysfunction in PAF has also been described [Parikh et al 2002], again

demonstrating a post-ganglionic defect with post-synaptic receptor hypersensitivity to a

variety of stimuli.

In contrast to the peripheral post-ganglionic defect seen in PAF, MSA is characterised

by features suggesting a central or preganglionic abnormality. Noradrenaline levels are

218

normal but they fail to respond to stimuli that require centrally mediated interpretation

or integration [Bannister and Mathias 1992b, Shannon et al 2000, Parikh et al 2002].

Histologically, the most striking abnormality is loss of cells from the intermediolateral

column of the spinal cord as well as other sites within the brainstem [Daniel 1992,

Parikh et al 2002]. Supine hypertension is seen more often in MSA subjects and is

more clearly related to residual post-ganglionic sympathetic tone [Polinsky et al 1991,

Parikh et al 2002], and is also characterised by orthostatic tachycardia [Parikh et al

2002]. Parasympathetic dysfunction has similarly been described in MSA, although

patients with MSA are difficult to distinguish from those with PAF based on

parasympathetic changes [Parikh et al 2002].

A study of the 35% CO2 challenge was therefore undertaken in patients with either

MSA or PAF to examine the role of the autonomic nervous system in regulating the

specific response to acute CO2 exposure, and to assess its potential to distinguish

between these two forms of autonomic failure.

Male and female patients with clinically established MSA or PAF were recruited from

the neurology outpatient clinics of the National Hospital for Neurology and

Neurosurgery, Queen Square, London and St Mary’s Hospital, Praed Street, London.

Control subjects were recruited from within the Autonomic Unit or were a well family

member of recruited patients. Nine MSA, 9 PAF and 5 control subjects were recruited.

MSA and PAF were defined using established diagnostic criteria [Gilman et al 1998,

Mathias and Bannister 2002]. All patients had documented sympathetic and

parasympathetic dysfunction with severe orthostatic hypotension. No subjects were on

anti-parkinsonian medication and all vasoactive medications were withdrawn the night

prior to the study. Control subjects were healthy with no active or chronic medical

219

illnesses and had not been on any medications apart from simple anaelgesia for the 2

weeks prior to the study. Exclusion criteria for this study included age over 70 years,

uncontrolled hypertension, a history of recent angina or ischaemic heart disease or any

history of cerebrovascular disease, asthma or a history of panic disorder or severe

anxiety. Subjects were asked to avoid consuming cigarettes, alcohol and caffeine for 24

hours prior to testing.

Subjects were asked to attend once, where they all received a single breath of 35% CO2

according to the protocol described previously. The ethics committees of the United

Bristol Healthcare Trust, the National Hospital for Neurology and Neurosurgery, and St

Mary’s Hospital all approved the study and all subjects provided written informed

consent prior to participation.

The study took place in a dedicated autonomic laboratory between 10 am and 1 pm.

After emptying the urinary bladder, the subject was seated and an intravenous line

(Venflon, Viggo Spectramed, Helingsborg, Sweden) was placed in an antecubital vein.

After a 10 minute rest, cardiovascular monitoring commenced. Continuous

measurement of beat-to-beat heart rate and blood pressure was made using a Portapres

II (TNO-TPD Biomedical Instruments, Amsterdam) device on the middle finger of the

right hand. Subsequent calculation of cardiac output (CO), stroke volume (SV) and

total peripheral resistance (TPR) using Modelflow analysis (Beatscope Software)

according to previously validated methods. Modelflow method uses a three-element

model of the aortic input impedance to compute flow from the pulsation of the arterial

pressure [Wesseling et al 1993, Langewouters et al 1998].

On the left side one Laser Doppler (Perimed) probe was taped over the pulp of the index

finger and one probe over the anterior forearm. Skin blood flow was measured by laser

220

Doppler flowmetry [Perimed Periflux 5000/5010 solid state diode laser set at 780 nm

with a 1 mW maximal power output at the probe tip]. Fibre optics permit the Laser

Doppler probe to shine a laser light directly at the skin surface. Light is reflected back to

the recording element of the probe from red blood cells in the skin capillaries. Light

reflected back to the probe has its wavelength altered by the Doppler shift caused by the

movement of the red blood cells relative to the probe. This altered wavelength is then

used to calculate an arbitrary perfusion rate (“Perfusion units”; PU), rather than absolute

flow, which is then visualised as a real-time trace. Cutaneous vasoconstriction will

therefore be shown as a reduction in perfusion units relative to baseline perfusion. The

forearm probe was used as a control to exclude movement or temperature artefact, as

forearm skin blood flow is under thermoregulatory rather than the “emotional”

sympathetic regulation present in the fingertip skin [Johnson et al 1995, Saad et al

2001].

One way analysis of variance was used to determine between-group differences with

two-tailed t-tests used to compare single time-point data including baseline differences.

Cardiovascular responses for a particular time point were calculated as the mean of +/-3

beats from that point.

For skin blood flow, from the individual perfusion unit data the calculation of %

reduction in skin blood flow following inhalation of air or CO2 was calculated as shown

in Figure 5.06. This allowed intra- and inter-groups comparisons. Results (both for skin

blood flow at baseline and reduction ratios after gasp of air or CO2) were compared by

ANOVA with Newman-Keuls post-hoc testing.

221

Figure 5.06. Calculation of % reduction in Skin Blood Flow (SBF) following inhalation of air or 35% CO2

222

5.6.2. Results

Anxiety symptoms were all mild and transient and the CO2 challenge was well tolerated

by all participants with no significant adverse effects recorded. In all cases, the test

breath was considered adequate being > 80% of the measured baseline VC.

A total of 18 patients (9 with PAF and 9 with MSA) and 5 control subjects were

studied. Baseline characteristics are given in Table 5.03. The PAF group comprised 5

males and 4 females with a mean age of 66.4 +/- 2.1 years. The MSA group comprised

4 males and 5 females with a mean age of 57.1 +/- 3.1 years whilst the control group

comprised 4 males and 1 female with a mean age of 47.0 +/- 6.8 years.

Cortisol, prolactin and salivary amylase were not significantly different between the

three groups at rest. Compared with both control and MSA subjects, PAF subjects had

significantly lower baseline noradrenaline levels (126.8 +/- 19.7 pg/ml compared with

288.4 +/- 31.3 and 246.9 +/- 13.1 pg/ml respectively, p < 0.001 for both). Baseline

subjective anxiety and somatic symptom VAS ratings were also not significantly

different between the three groups.

As demonstrated in Table 5.04 and Figure 5.07, in response to a single breath of 35%

CO2, systolic blood pressure increased significantly from baseline in control (+60.2 +/-

13.9 mmHg, p = 0.01), PAF (+26.8 +/- 3.2 mmHg, p < 0.001) and MSA (+18.3 +/- 2.7

mmHg, p = 0.002) subjects. The rise in SBP was, however, significantly greater in

control subjects compared with either PAF or MSA subjects (p < 0.01 and p < 0.001,

respectively). This peak pressor response occurred 32.4 +/- 2.1 seconds following the

CO2 breath in control. In contrast, the peak pressor response was significantly delayed

223

in both the PAF (152.4 +/- 23.9 secs) and MSA subjects (140.2 +/- 35.5 secs) (p = 0.03

and p = 0.04 compared with controls respectively). Diastolic blood pressure also

increased significantly in control (+21.8 +/- 5.0 mmHg, p = 0.01), PAF (+13.7 +/- 2.0

mmHg, p = 0.002) and MSA subjects (+10.5 +/- 2.7 mmHg, p = 0.004). There was a

similar, although non-significant trend towards a smaller rise in DBP in the PAF and

MSA groups compared with controls. Bradycardia was observed following CO2

exposure in 4 of the 5 controls, although peak change from baseline was not significant

(–9.1 +/- 3.5 bpm, p = 0.06). Bradycardia was only observed in 3 of 9 PAF and 2 of 9

MSA subjects.

Corresponding to the pressor response in control subjects, there was a non-significant

trend toward a rise in both SV and TPR with no change in cardiac output (Table 5.04).

MSA subjects, similarly showed no change in CO, SV or TPR whereas PAF subjects

demonstrated a significant increase in TPR from baseline (p = 0.04) that was

significantly greater than the peak response seen in control subjects (p = 0.03). As seen

in Table 5.04 and Figure 5.08, there was a marked increase in noradrenaline levels from

baseline in control subjects (+41.7 +/- 7.1 %, p < 0.0001). This increase was

significantly greater than the noradrenaline response seen in either PAF (+4.2 +/- 2.2 %)

or MSA (+8.0 +/- 2.4 %) subjects (p < 0.0001 for both). No significant change from

baseline was seen in salivary amylase or prolactin in any of the groups. There was a

trend towards a greater increase in cortisol in control (+35.2 +/- 3 0.6 %) and PAF

(+23.7 +/- 14.3 %) subjects compared with MSA subjects (+8.8 +/- 8.9 %) (Table 5.04

and Figure 5.08).

Figure 5.09 demonstrates the subjective psychological responses to the CO2 challenge.

Baseline psychological symptoms were similar in all three groups. Changes in anxiety,

224

fear and breathlessness VAS responses were not significantly different. Overall, MSA

subjects noted fewer somatic symptoms than either the control or PAF subjects. Of the

4 most commonly experienced symptoms (awareness of one’s heart beat; feeling hot;

having blurred vision; and dizziness), only 2 of the 9 MSA subjects experienced more

than one of these symptoms compared with all of the controls and 7 of the 9 PAF

subjects. Considered individually, there was only a non-significant trend towards lower

mean changes in VAS scores in the MSA group however.

Baseline systolic and diastolic blood pressures were the same in all three groups,

although MSA subjects had a higher resting heart rate (77 +/- 4.1 bpm compared with

60.9 +/- 3.9 bpm in controls, p = 0.02). PAF subjects had a lower resting cardiac output

(2.8 +/- 0.4 L/min) compared with both control (4.9 +/- 0.9 L/min, p = 0.02) and MSA

subjects (4.7 +/- 0.6 L/min, p = 0.02). In association with their lower CO, PAF subjects

also had a lower resting stroke volume and higher resting total peripheral resistance (p =

0.02 and p = 0.03 respectively) compared with controls.

Baseline skin blood flow was significantly lower in PAF compared to either controls (p

= 0.000006) or MSA (p = 0.026) (Table 5.05); MSA baseline skin blood flow was lower

than in controls (p = 0.002). Whilst all 3 groups showed reduction in skin blood flow

with inhalation of air, the reduction relative to baseline was less in PAF than controls (p

< 0.0006) or MSA (p < 0.04); (Table 5.05, Figure 5.10). MSA % reduction of skin

blood flow with air was less than seen in controls (p < 0.012). The time in seconds from

inspiratory gasp to onset of the skin vasomotor response was 3.1 +/- 0.3 for controls, 2.9

+/- 0.3 for MSA and 4.1 +/- 0.6 for PAF without significant differences between the

groups. The mean length of vasoconstriction following inhalation of air was 87.6 +/-

225

28.9 seconds for controls, 62.2 +/- 30.6 seconds for MSA without significant difference

between the groups. PAF showed vasodilatation rather than vasoconstriction.

After CO2 healthy controls showed a similar skin blood flow response to that seen with

inhalation of air over a similar time course (onset: 2.9 +/- 0.4 seconds after CO2;

duration: 91 seconds +/- 41.3 seconds). In PAF there was no reduction in skin blood

flow following CO2, but a striking transient increase in skin blood flow was observed in

PAF within 45 seconds (+/- 1.1 seconds) of CO2 inhalation (Figures 5.10 and 5.11).

This increase in skin blood flow was significantly greater than in controls (p < 0.00003)

or MSA (p < 0.00013). No increase in skin blood flow after CO2 inhalation was seen in

the controls, but a small increase was noted in 5/9 MSA subjects (+28.5% +/- 12.4),

which was less than that seen in PAF (p < 0.06).

226

Table 5.03. Baseline characteristics of subjects.

Control PAF MSA p

n

5 9 9

Age (years)

47 +/- 6.8 57.1 +/- 3.1 66.4 +/- 2.1

Cardiovascular parameters

SBP (mmHg)

122.5 +/- 12.3 128.8 +/- 13.6 132.7 +/- 8.7 -

DBP (mmHg)

66.9 +/- 3.8 75.7 +/- 4.3 76.1 +/- 7.2 -

HR (b/m)

60.92 +/- 3.925 66.8 +/- 2.6 77.0 +/- 4.1 0.02 (CvPAF)

CO (L/min)

4.9 +/- 0.9

2.8 +/- 0.4 4.7 +/- 0.6 0.04 (CvPAF) 0.02 (PAFvMSA)

SV (ml)

82.6 +/- 6.1

43.5 +/- 7.6 65.9 +/- 0.9 0.02 (CvPAF)

Skin Blood Flow (perfusion Units)

278.8 +/- 18.2 59.8 +/- 14.6 143.7 +/- 29.5 0.003 (CvPAF) 0.03 (PAFvMSA)

0.02 (CvMSA)

TPR (PRU)

1.3 +/- 0.2

2.2 +/- 0.2 1.5 +/- 0.3 0.03 (CvPAF)

Endocrine parameters

Cortisol (nmol/l)

439.8 +/- 75.8

347.1 +/- 52.0 403.7 +/- 53.0 -

Prolactin (mIU/l)

109.8 +/- 14.1 119.1 +/- 12.8 148.3 +/- 26.7 -

Salivary amylase (IU/l)

127643 +/- 20070

126686 +/- 35460

254867 +/- 92630

-

Noradrenaline (pg/ml)

288.4 +/- 31.3 126.8 +/- 19.7 246.9 +/- 13.1 <0.001 (CvPAF) <0.001 (PAF

vMSA)

227

Peak SBP response

control PAF MSA

0

25

50

75

mm

Hg

Time to peak SBP

control PAF MSA

0

100

200

secs

Peak DBP response

control PAF MSA

0

10

20

30

mm

Hg

Peak HR response

control PAF MSA

-15

-10

-5

0

b/m

Figure 5.07. Peak (A) and time to peak (B) systolic blood pressure; diastolic blood pressure (C) and heart rate (D) responses to a single breath of 35% CO2 in control, PAF and MSA subjects. The peak increase in SBP was significantly greater in controls compared to both PAF (p<0.01) and MSA (p<0.001) patients and the time to this peak was significantly shorter in the control subjects (p=0.03 and p=0.04 compared with PAF and MSA patients respectively).

228

Table 5.04. Peak cardiovascular and endocrine responses to CO2 exposure.

Control PAF MSA p Cardiovascular parameters

SBP (mmHg) ∆max 60.2+/-13.9 26.8+/-3.2 18.3+/-2.7 <0.01 (CvPAF)

%∆max 50.3+/-11.8 23.2+/-3.9 15.1+/-2.5 <0.001 (CvMSA) DBP (mmHg)

∆max 21.8+/-5.0 13.7+/-2.0 10.5+/-2.7 - %∆max 32.3+/-7.4 14.7+/-3.1 18.7+/-2.9

HR (b/min) ∆max -9.1+/-3.5 -4.3+/-2.3 -4.1+/-2.1 -

%∆max -16.1+/-6.9 -7.1+/-3.7 -6.4+/-3.2 CO (L/min)

∆max -0.6+/-0.5 -0.1+/-0.1 -0.1+/-0.1 - %∆max -6.2+/-9.5 -4.5+/-3.7 -3.3+/-4.0

SV (ml) ∆max 19.5+/-34.3 -1.7+/-1.4 0.6+/-2.4 -

%∆max 18.4+/-14.5 -0.5+/-5.7 2.0+/-4.8 TPR (PRU)

∆max 1.7+/-0.2 2.8+/-0.3 1.8+/-0.4 0.03 (CvPAF) %∆max 14.3+/-9.0 24.6+/-5.5 17.8+/-7.5

Endocrine parameters

Cortisol (nmol/l) ∆max 105.2+/-79.9 72.8+/-45.6 38.2+/-32.2 -

%∆max 35.2+/-30.6 23.7+/-14.3 8.8+/-8.9 Prolactin (mIU/l)

∆max 1.8+/-4.7 15.2+/-11.4 -5.4+/-9.3 - %∆max 1.7+/-4.0 7.0+/-5.4 -2.0+/-4.3

Salivary amylase (IU/l)

∆max 5171+/-12411 16651+/-13958

15551+/-5065 -

%∆max 3.7+/-9.1 15.2+/-12.5 15.2+/-13.8 Noradrenaline

(pg/ml)

∆max 124.2+/-19.5 5.2+/-2.8 20.0+/-6.4 <0.001 (CvPAF) %∆max 41.7+/-7.1 4.2+/-2.2 8.0+/-2.4 <0.001 (CvMSA)

229

Peak % cortisol response

control PAF MSA

0

25

50

75

%

Peak % prolactin response

control PAF MSA

-10

0

10

20

%Peak % salivary amylase

response

control PAF MSA

0

10

20

30

%

Peak % noradrenalineresponse

control PAF MSA

0

25

50

%

Figure 5.08. Peak percent cortisol, prolactin,

salivary amylase and noradrenaline levels following a single breath of 35% CO2.

230

Change in anxiety, fear andbreathlessness responses

Anx

iety

Fear

Bre

athl

essn

ess

0

25

50

75ControlPAFMSA

%

Somatic symptom responses

Hea

rt be

at

Hot

Blu

rred

vis

ion

Diz

zine

ss

-25

0

25

50

75

%

Figure 5.09. Percent change in anxiety, fear, breathlessness (top panel) and somatic symptom (lower panel) responses to 35% CO2 exposure.

231

Table 5.05. Changes in Skin Blood Flow in perfusion Units (+/- SE) after single breath of air or CO2

Baseline % Change in flow with

Room air

% Change in flow with

CO2

Controls 278.8 -83.8 ( 3.9) - 79.0 ( 5.6)

MSA 143.7 -52.7 ( 8.6) - 4.8 (15.9)

PAF 59.8 -31.7 (14.6) + 151.2 (40.2)

232

0

100

200

300

400

CONTROL R

0

100

200

300

400

8

0

100

200

300

400

Figure ControlPerfusio

AI

MSA

R
AI
5 2 9 6 3 0 7 4 8 5 2 9 6 3 0 7 4 8 5 2 9 6 3 0 7 4 8 5 2 9 6

PAF

AIR

0 45 90 135 Time (in seconds) po

5.10. Skin Blood Flow changes follo Subject (top), PAF (middle) and Mn Units.

CO2

st

wSA

CO2

CO2

0 45 90 135 inhalation of air or CO2

ing inhalation of air or 35%CO2 in (lower). Y-axis shows skin blood flow in

233

-200

-100

0

100

200

300

Mea

n %

Cha

nge

in S

BF

follo

win

g in

hala

tion

AIR CO2 AIR CO2 AIR CO2

MSA (n = 9) PAF (n = 9) CONTROLS (n = 5)

Figure 5.11. Skin Blood Flow changes following inhalation of air or 35%CO2 in Control Subjects (gray), MSA (white) and PAF (black). Error bars = SE. Y-axis shows % change in SBF relative to baseline.

234

5.6.3. Discussion

In keeping with the known pathophysiology of MSA and PAF, we observed a

significant reduction in baseline noradrenaline levels in PAF subjects that was

associated with evidence of post-ganglionic hypersensitivity with vasoconstriction

(reduced skin blood flow and increased TPR) with a slightly reduced CO and SV

[Bannister and Mathias 1992a, Bannister and Mathias 1992b, Shannon et al 2000].

Also, as expected, MSA subjects had higher baseline heart rates although their other

cardiovascular parameters were no different to controls [Shannon et al 2000]. Apart

from noradrenaline, other hormone levels were no different between the three groups.

In control subjects, following a single breath of 35% CO2 there was the expected

increased in systolic and diastolic blood pressure associated with a marked increase in

plasma noradrenaline. Cardiac output remained unchanged and there was a trend

toward increased TPR. Both MSA and PAF subjects demonstrated a significant systolic

and diastolic pressor response following 35% CO2 exposure. However, these responses

were both smaller in magnitude, and occurred later than the responses observed in

normal controls. Whilst the magnitude of the responses were similar in MSA and PAF,

as is the case with supine hypertension in these subjects, it is likely they occurred

through different mechanisms.

Both MSA and PAF subjects showed a markedly reduced noradrenaline response

compared with controls, however, PAF subjects did increase their TPR compared to

both MSA and controls. It has been shown previously that resting noradrenaline levels

are reduced in PAF as are stimulated noradrenaline levels following various

haemodynamic and pharmacological challenges [Bannister and Mathias 1992a,

235

Bannister and Mathias 1992b, Shannon et al 2000]. Despite the histopathological

damage to peripheral autonomic nerves with the resultant impairment in noradrenaline

synthesis and release, sympathetically-mediated increases in blood pressure are seen in

PAF subjects, for example following the administration of the α-adrenoreceptor agonist

yohimbine [Shannon et al 2000]. This is most likely the result of post-ganglionic

receptor hypersensitivity to the small changes in noradrenaline seen in PAF subjects

[Bannister and Mathias 1992b, Shannon et al 2000], partly due to adrenoreceptor up-

regulation. This receptor hypersensitivity that increases the sensitivity of PAF patients

to the action of other pressor agents and vasodilators [Shannon et al 2000], is also

responsible for preserving, at least in part, the pressor response to CO2.

MSA, on the other hand, is characterised by features suggesting a central or

preganglionic abnormality. Noradrenaline levels are normal but they fail to respond to

stimuli that require centrally mediated interpretation or integration [Bannister and

Mathias 1992b, Shannon et al 2000, Parikh et al 2002]. The adreno-medullary response

to hypoglycaemia, for example, requires activation of central glucose receptors and an

efferent pre-ganglionic sympathetic arc [Shannon et al 2000]. This response is blunted

in both PAF and MSA, but can be restored in MSA subjects through the application of

post-ganglionic stimuli [Shannon et al 2000]. As mentioned, the pressor response to a

low dose CO2 challenge was lost in MSA subjects compared to controls presumably due

to impaired or absent responses of sympathetic autonomic nervous system structures in

the brainstem or cervical trunk [Braune et al 1997]. Supine hypertension, however, is

often seen in MSA subjects related to residual post-ganglionic sympathetic tone

[Shannon et al 2000, Parikh et al 2002] that occurs due to involvement of the

baroreceptor reflex arc with subsequent loss of baroreceptor-mediated buffering

[Shannon et al 2000] as well as post-ganglionic receptor hypersensitivity. This residual

236

post-ganglionic sympathetic tone is the likely mechanism producing the pressor

response observed in this study. In addition, it also likely contributes to the resting and

orthostatic tachycardia seen in MSA [Shannon et al 2000].

Only one MSA and 2 PAF subjects demonstrated a bradycardic response to the CO2 in

contrast to the control response. Again this is consistent with the parasympathetic

dysfunction previously described in both these conditions [Parikh et al 2002]. PAF

subjects behave in a manner similar to vagotomised subjects (such as the individual post

heart-lung transplant described in Chapter 4). MSA subjects differ from the individual

with CCHS described previously in that whilst both have central lesions, the process in

MSA is more diffuse with more severe autonomic involvement that is thus more likely

to involve parasympathetic centres that the more focal deficit that characterises CCHS

[Bannister and Mathias 1992b, Shannon et al 2000].

Along these lines, study by Braune et al [1997] evaluated the response of MSA patients

to a low dose (7%) CO2 challenge. MSA subjects failed to demonstrate the rise in

systolic blood pressure or heart rate seen in a comparison group of normal control

subjects again indicating the sympathetic response to CO2 is dependent on the presence

of intact preganglionic brainstem and cervical trunk centres. Based on this, our a priori

hypothesis was that PAF subjects would be expected to show impaired sympathetic and

parasympathetically mediated cardiovascular responses (as detailed above), but would

have preserved neurohormonal and psychological responses. Further, MSA subjects

would showed impaired autonomic cardiovascular responses, but would also show

impaired hormonal and psychological responses as these are centrally mediated.

Consistent with this hypothesis, endocrine and psychological responses in PAF subjects

were no different than in controls. MSA subjects on the other hand, showed a trend

towards lower cortisol responses compared with controls (8.8 +/- 8.9 % vs 35.2 +/- 30.6

237

% respectively). Similarly, there was an overall trend toward fewer somatic symptom

responses in MSA subjects compared with both control and PAF subjects.

Acute sympathetic denervation results in increased skin blood flow, however, chronic

denervation results in reduced skin blood flow [Barcroft and Walker 1949]. In this

study, subjects with PAF (which is a condition of chronic denervation), had

significantly lower skin blood flow readings compared with either MSA or control

subjects. Previous studies have shown that the inspiratory gasp reflex is associated with

a significant reduction in skin blood flow and that this effect is preserved in subjects

with MSA but impaired in those with PAF [Asahina et al 2003]. It is also known that

CO2 can cause cutaneous vasodilation (and an increase in skin blood flow) by non-

neural mechanisms [Bullard 1964, Ito et al 1989]. Following 35% CO2 control subjects

showed the expected reduction in skin blood flow following both an inspiratory gasp

(air breath) and a gasp plus CO2 (the 35% CO2 breath). MSA subjects showed a

blunting of the skin blood flow fall in response to both gasp and CO2 exposure, whilst

PAF subjects showed a blunted gasp response and a striking increase in skin blood flow

(or vasodilatory response) following 35% CO2 exposure. It seems likely that in normal

controls, the non-neurally mediated vasodilation produced by CO2 is masked by the

vasoconstriction produced by both the gasp reflex and the neurally mediated CO2-

induced noradrenaline release. In MSA subjects, the combination of a preserved gasp

reflex with a blunted neurally mediated CO2-induced vasoconstriction is still sufficient

to produce a vasoconstrictor response, albeit a smaller one as compared with controls.

PAF subjects on the other hand, have both blunted gasp reflex and an impaired neurally

mediated CO2 response that unmasks the usually hidden non-neurally mediated CO2-

induced vasodilatory response. This difference in skin blood flow following 35% CO2

exposure may proved a useful adjunct to the tests used to distinguish MSA and PAF.

238

With respect to the 35% CO2 model, these studies in patients with PAF (peripheral

autonomic failure) and MSA (central autonomic failure) add support to the proposed

underlying mechanisms that appear to drive the response to CO2. Overall, peripheral

autonomic failure is associated with an impaired parasympathetic response and

preserved neurohormonal and psychological responses similar to that observed in other

studies of peripheral denervation (heart-lung transplant and diabetic peripheral

neuropathy). This highlights the importance of central chemoreception in generating

these responses. However, the sympathetic involvement in PAF is also associated with

a blunted and delayed pressor response and an un-masking of the cutaneous

vasodilatory effect of CO2 that was previously hidden by the sympathetically-mediated

vasoconstrictor effect. MSA, on the other hand, is associated with impairments in both

the sympathetic and parasympathetic response, as well some in the neurohormonal and

psychological responses. The intact anxiety and fear responses seen in MSA subjects

suggests these occur directly from CO2 stimulation of limbic centres and independently

of brainstem sympathetic activity. However, the impaired somatic symptom responses

seen in both MSA and CCHS subjects suggests these may have more to do with

brainstem activation or at least communication pathways between limbic and brainstem

centres. Further, this also suggests the HPA response is likely secondary to activation

of brainstem sympathetic centres, rather then in response to projects from limbic

centres.

As detailed in Chapter 2, it had been reported elsewhere [Chatterton et al 1996] that

changes in salivary amylase reflects plasma catecholamine (particularly noradrenaline)

levels. In this study, however, there was no association between plasma noradrenaline

and salivary amylase either at rest or in response to the 35% CO2 challenge. At

baseline, PAF subjects had significantly lower noradrenaline levels but amylase levels

239

were no different to either controls or MSA subjects. Following 35% CO2 exposure,

noradrenaline levels increased significantly in controls (+41.7 +/- 7.1 %), but no such

increase in salivary amylase was observed (+3.7 +/- 9.1 %). Based on these results, and

until further studies are performed, salivary amylase does not seem to accurately predict

either resting or stimulated plasma noradrenaline.

240

5.7. Conclusion

The conditions studied in the above series of experiments were chosen as they represent

clinical models of basic pathophysiology. Their study provides useful information

regarding not only some of the potential mechanisms by which the response to CO2

occurs, but also about the disease itself – its pathophysiology, reasons for clinical

symptoms, potential for treatment and their efficacy as well as potential risks and

complications.

In Chapter 4 it was noted that a physiologically or pharmacologically suppressed HPA

axis had no influence over an individuals ability to mount a normal pressor response to

CO2, nor did it influence the bradycardic response or the psychological symptoms

experienced with 35% CO2 inhalation. However, in these situations baseline cortisol

levels were not completely suppressed and it was still possible that low levels of

circulating (and more particularly brain) glucocorticoid was sufficient to mediate the

central (brainstem and limbic) responses to the CO2 challenge. Subjects with Addison’s

disease, without glucocorticoid replacement, still demonstrated the same bradycardic

and psychological response to CO2 than when they were adequately replaced with

glucocorticoid. This would again suggest that the psychological response is

independent of HPA activity as is the bradycardia, which is most likely due to direct

vagal stimulation by CO2. Unreplaced subjects with Addison’s disease showed a

smaller pressor response than when replaced with both glucocorticoid and

mineralocorticoid. The difference was small and may be due to a reduced positive re-

enforcement effect of the HPA axis on brainstem sympathetic activity. However, it is

probably more likely that the lack of mineralocorticoid which, through the renin-

angiotensin system, plays an important role in maintenance of blood pressure in patients

241

with adrenocortical failure was responsible for this difference. The striking differences

in the pressor response seen in the later studies in people with autonomic failure

suggests the sympathetic system per se has a much greater role in determining the

pressor response to CO2 than does the HPA axis.

The study of patients with diabetic autonomic neuropathy has shed more light on the

origin of the bradycardia. The heart rate slowing in response to 35% CO2 exposure fits

well with the Polyvagal theory of Porges [Porges 1995]. The normal slowing following

a single breath of 35% CO2 is consistent with withdrawal of the NA component of the

vagal response. A maintained bradycardia with no change in blood pressure in the

Addison’s patients off treatment also supports this concept.

A similar response has been demonstrated in other situations of acute stress [Porges

1995]. Parasympathetic failure (as occurs in diabetic autonomic neuropathy) may result

in impaired activity of both components of the vagal system thereby allowing

sympathetic activity to predominate. This would be expected to result in higher resting

heart rates and a loss of the bradycardia with 35% CO2 exposure, as was seen in this

group. We would also postulate that this is a potential mechanism to explain the

increased cardiovascular mortality associated with diabetic autonomic neuropathy and

further studies are planned to evaluate this further (see below).

As mentioned, the sympathetic autonomic system appears much more important in

determining the overall response to CO2. Central autonomic failure (MSA) resulted in

a reduced pressor response, less frequently observed bradycardia, fewer somatic

symptoms of fear and a smaller cortisol response. As was also evident from the

individual with CCHS, brainstem sympathetic systems appear to be the principal site

242

regulating the response to 35% CO2. In PAF where peripheral autonomic systems are

involved, both the pressor and bradycardia were affected, but the HPA and

psychological systems were not. Given the previous evidence that acute fear (in the

form of panic attacks) can be precipitated by CO2 exposure without necessarily

producing an HPA response, it is again likely that the HPA response occurs

predominantly following activation of brainstem sympathetic systems. The vagal

response, on the other hand, occurs independently whilst the psychological response

occurs probably as a result of both direct activation as well as with some influence from

brainstem sympathetic systems.

Figure 4.18 can then be updated to include this current evidence to provide an

illustration of the likely pathways mediating the response to a single breath of 35% CO2,

as shown in Figure 5.12.

243

Single breath of

35% CO2

Noradrenergic brainstem

Limbic centres

FoabPsbH

Vagal centres/vagus nerve
centres
(VLM, LC) (CnA)

PVN Bradycardia

NA ACTH

Emotional arousal ↑SBP Cortisol

igure 5.12. Revised schematic diagram suggesting the principal pathways f CO2 mediated HPA, cardiovascular and psychological responses (solid rrows). Vagal and noradrenergic brainstem centres are directly stimulated y CO2 resulting in bradycardia and an acute pressor response respectively. sychological responses occur due to a combination of direct CO2timulation of a central fear circuit as well as indirect stimulation via rainstem NA centres. PA responses occur indirectly in response to brainstem NA stimulation.

244

CHAPTER 6

SUMMARY, CONCLUSIONS AND FUTURE

DIRECTIONS

245

6.1. Summary

The three main objectives of this thesis were to:

i. Design, develop and evaluate the single breath 35% CO2 test as a potential model

of the stress response in humans,

ii. Understand the mechanisms that are involved in mediating the observed

physiological, endocrine and psychological responses to the test, and

iii. Investigate its potential for use in clinically important disease states.

The initial design of the test was based on an existing model from the psychiatric

literature that had been used and evaluated as a means of studying acute anxiety and

panic in susceptible patients. Detailed physiological and endocrine studies had not been

previously performed. The first studies carried out in this thesis evaluated different CO2

doses and confirmed the linear dose related increase in anxiety responses. The

sympathetic and HPA responses, on the other hand, appeared to be threshold dependent

with 35% CO2 producing the most consistent psychological, endocrine and

physiological response whilst being well tolerated with no significant adverse effect.

The neurohormonal response was limited to the HPA axis (ACTH and cortisol) and

prolactin with no release of other anterior pituitary hormones, vasopressin or renin seen.

The physiological cardiovascular response differed from that seen in studies of low dose

CO2 exposure. Low dose and steady state protocols of CO2 exposure, principally used

in the study of anxiety and ventilatory physiology, are associated with tachycardia and

systolic hypertension. A single breath of 35% CO2 is associated with a systolic pressor

response and bradycardia. Initial thoughts that the bradycardia was due to reflex

baroreceptor activity were later proved to be incorrect (see below). The psychological

246

response included general feelings of anxiety, fear and breathlessness and also invoked

a few very specific somatic symptoms.

Subsequent experiments were undertaken to assess the reliability and reproducibility of

the test and also to refine the technique of administration of the CO2. It was clear that

certain operator dependent factors were important particularly the specific instructions

for performing the test and the nature and detail of the symptoms they might expect. In

addition, refinements to the equipment used were made in reverting back to a mouth-

piece and nose clip arrangement rather than a nasal-oral face mask that was associated

with poor inspiratory efforts and limited responses.

With these refinements, the psychological and physiological responses were shown to

be robust and easily reproducible. HPA responses were modest and somewhat more

variable. They appear more easily influenced by a number of potentially confounding

factors such as the individuals level of anticipatory anxiety, the specifics of the

instructions received, their expectations based on details received before taking the

breath, and the performance of the test especially the adequacy of the inspiration.

As mentioned, the HPA axis response was modest and does not appear to be the

primary determinant of either the psychological or autonomic responses.

Administration of naltrexone to remove negative feedback inhibition of the HPA axis

and raise baseline cortisol levels had no effect on either psychological or autonomic

responses. Similarly, the administration of other neurotransmitters that may be

expected to acutely enhance the HPA response to stress (for example paroxetine), also

had no effect on other responses. Further, there was no effect from decreasing the

activity of the HPA axis. This was done by either evaluating subjects with

247

physiologically suppressed axes (lactating women), pathologically suppressed axes

(Addison’s disease) or through pharmacological manipulation with the administration of

metyrapone (decrease cortisol synthesis) or spironolactone (decrease activity of central

mineralocorticoid receptors). In contrast, subjects with deficits of central autonomic

function (MSA) demonstrated a reduced ability to mount a cortisol response to the CO2

challenge.

Clearly distinguishable pattern of sympathetic and parasympathetic responses emerged.

Initial impressions that the bradycardia was as a baroreceptor response to the increased

systolic blood pressure were discounted on the grounds that continuous cardiovascular

monitoring identified the bradycardia as occurring prior to the increase in blood

pressure. Further, vagotomy (heart-lung) transplantation and parasympathetic failure,

both central (MSA) and peripheral (PAF and DAN), abolished the bradycardia

regardless of whether the pressor response was preserved (vagotomy and diabetes) or

impaired (MSA and PAF). The sympathetic response was clearly centrally mediated,

being preserved in peripheral neuropathy (diabetes) and HPA axis impairment (as

above). The response, characterised by systolic and diastolic hypertension was

multifactorial in origin, however, in normal individuals appeared principally to be

associated with significant increases in noradrenaline and peripheral vasoconstriction.

Abnormalities of central CO2 chemoreception and/or integration (CCHS and MSA)

caused significant blunting of the pressor response. The response, however, was not

abolished in MSA subjects but was also not associated with an increase in

noradrenaline. Residual post-ganglionic sympathetic activity (also responsible for

supine hypertension in these subjects) is the likely mechanism by which this response

occurred. The response was also not abolished in subjects with peripheral autonomic

failure (PAF and diabetic peripheral neuropathy with orthostatic hypotension). In this

248

case, post-synaptic receptor hypersensitivity to small changes in noradrenaline is

presumed to be the likely mechanism producing this response.

Subjective anxiety and fear responses were not affected by changes in HPA axis

activity, peripheral neuropathy (diabetes and PAF) or by central autonomic dysfunction

(MSA). This suggests the anxiety component is mediated by direct CO2 stimulation of

limbic fear centres such as the central nucleus of the amygdala. The specific somatic

symptoms of fear typically associated with 35% CO2 exposure were blunted in patients

with abnormalities of brainstem CO2 chemosensation (CCHS) and/or autonomic

integration (MSA). This suggests that projections from brainstem autonomic centres to

limbic fear centres have at least some role to play in generating the overall response to

CO2.

From a clinical perspective, a study of women during the lactation phase has reinforced

the notion that maternal brain plasticity during pregnancy and post-partum alters stress

hormone responsiveness to provide a more advantageous environment for the well-

being of the infant. Previous studies of rodents had demonstrated reduced maternal

HPA responsiveness to stress during lactation [Lightman et al 1997], but in humans

similar suppression following psychological and exercise stress was limited to a specific

time following suckling [Altemus et al 1995, Heinrichs et al 2001]. Following 35%

CO2 exposure, on the other hand, no significant suppression of stress responses was

seen. This would indicate that suppression of stress responses may be advantageous to

a nursing mother faced with stressors that are not pertinent to survival, but when

physiological homeostasis is threatened survival demands a full neuroendocrine and

behavioural response. Failing to appropriately alter stress response patterns during this

time (for example by continuing to respond excessively to non-pertinent survival

249

threats, or alternatively, not responding sufficiently to pertinent threats) may predispose

these individuals to an increased risk of stress related illnesses (such as post partum

depression) during this period.

Another study demonstrated dissociative sympatho-vagal dysfunction in diabetic

autonomic neuropathy. This included preservation of the systolic pressor response

whilst the normally observed bradycardia was abolished. The initial bradycardia seen in

healthy controls could be explained by the Polyvagal theory of Porges [Porges 1995]

which distinguishes between the rhythmic beat-to-beat variations in heart rate (mediated

by phylogenetically newer myelinated fibres from the nucleus ambiguus) and the

marked bradycardia (mediated by more primitive unmyelinated fibres from the DMNX)

that is occasionally observed during acute stress. Active withdrawal of the nucleus

ambiguus component leaves the individual susceptible to the effects of excessive

neurogenic bradycardia if sympathetic compensation is inadequate and this is the likely

explanation for neurogenic syncope [Porges 1995, Reed et al 1999]. In those with

diabetic autonomic neuropathy the loss of both components of the vagal response could

result in unprotected excessive sympathetic activity and this could serve as a potential

explanation for the increased risk of cardiac tachyarrhythmias seen in these patients.

Finally, a study of individuals with autonomic failure syndromes has shed further light

on the presence of residual mechanisms whereby sympathetic tone is maintained despite

severe pathological involvement of either central (MSA) or peripheral (PAF) autonomic

systems. This residual sympathetic tone is an important contributing factor to the

observed supine hypertension many of these subjects display (particularly those with

MSA) and is also an important limiting factor in the treatment of their orthostatic

hypertension. Moreover, this study demonstrated a marked difference in the pattern of

250

skin blood flow changes following 35% CO2 exposure in PAF subjects compared with

MSA and control subjects. Control subjects normally display a significant reduction in

skin blood flow following CO2 that is likely due to the combination of the inspiratory

gasp reflex and the CO2 stimulated increase in noradrenaline that increases TPR. MSA

subjects show a reduction in skin blood flow that is significantly reduced in magnitude

compared with controls most likely from a loss of the CO2-induced sympathetic

stimulation. PAF subjects on the other hand show a completely different skin blood

flow pattern with a striking increase in flow. This probably results from the loss of the

vasoconstrictor effects of both the gasp reflex and the noradrenaline-mediated CO2

effect with an unmasking of a local, non-neural vasodilatory effect of CO2. This

difference could potentially serve as an important adjunct in the clinical differentiation

of these two disorders.

251

6.2. Conclusions and future directions

The 35% CO2 single breath test has shown itself to be a safe, reliable and reproducible

means of generating a general stress response in humans. With a few carefully

considered limitations, the test could be applied to a wide range of individuals without a

significant risk of harm. It is convenient, quick and simple to administer and doesn’t

require sophisticated monitoring. Further work is planned to further define the

limitations of the HPA axis response particularly with regard to potential confounding

variables such as anticipatory anxiety, age and gender differences and menstrual cycle

changes. Further reproducibility studies with larger subject numbers are also planned,

as are studies to compare this model to other established models of both psychological

(such as the TSST) and physical (such as exercise) stress.

Studies to determine whether the test can be performed entirely non-invasively with the

use of non-invasive cardiovascular monitors and salivary biochemical measures

(including salivary cortisol and amylase as surrogates for plasma cortisol and plasma

noradrenaline respectively) are also due to begin shortly.

The model has a large number of potential clinical applications both at an

epidemiological level and at an individual level. There is already the potential to use it

in the diagnosis and assessment of diabetic autonomic neuropathy and in the assessment

of autonomic failure syndromes including the syndrome of unexplained syncope, where

it may be more useful than the conventional tilt test. The model has potential for use in

a multitude of conditions that are characterised by or associated with abnormalities of

HPA or autonomic system dysfunction. These include depression, anxiety and affective

disorders, irritable bowel syndrome, obesity and the metabolic syndrome, chronic

252

fatigue, coronary artery disease, immune and inflammatory conditions and so on. It

could potentially server as a predictor of disease susceptibility, a marker of disease

occurrence or as a determinant of treatment success or outcome. A number of studies

using the model in some the above conditions have commenced or are being planned.

The stress response system has evolved as both an early warning system capable of

recognising potential or existing threats, and as a response system that can initiate and

drive the necessary processes required to escape or confront the threat. By its very

nature, the response is dynamic, beginning rapidly with brain and behavioural activation

followed quickly by physiological activation. These processes are characterised by

positive-feedback and feed forward loops that enhance and reinforce themselves as well

as recruiting other arms of the stress response. Slower acting hormone systems provide

checks and balances to the already active, but energy expensive systems, putting a brake

on the whole response to ensure it is kept appropriate to the type of stress faced, to its

intensity and duration, and to ensure the response is switched off when the threat has

been adequately dealt with. It is well established that impaired functioning of these

stress response systems is central to the pathophysiological pathways that underlie many

psychiatric, immune and physical disorders.

The challenge for the future is a need to develop tools that can accurately and

consistently measure the reactivity of these systems and the consequences of their

activation. It is essential to be able to do this in individuals, in order to predict their

vulnerability to illness, and in large populations, in order to measure the impact of stress

and the efficacy of any therapeutic intervention. Further, there is a need to expand our

current understanding of the mechanisms that produce stress-related pathophysiology in

order to design more effective therapies.

253

CHAPTER 7

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