The Effects of Tonic Muscle Pain
on the Sympathetic and Somatic
Motor Systems in Humans
Azharuddin Fazalbhoy
A thesis in fulfillment of the requirements for the degree of
Doctor of Philosophy
Prince of Wales Clinical School
Faculty of Medicine
January 2014
ORIGINALITY STATEMENT
‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’
Signed ......
Date ...... 22/01/2014......
ii
PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Fazalbhoy First name: Azharuddin Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Prince of Wales Clinical School Faculty: Medicine Title: The effects of tonic muscle pain on the sympathetic and somatic motor systems in human
Abstract 350 words maximum: (PLEASE TYPE)
Mechanisms underlying the development of chronic muscle pain in humans remain unknown. Our current understanding is based on experimentation in animals, which has resulted in a multitude of theories to postulate the mechanisms in humans. The vicious cycle theory – a popularised but unsupported theory in humans – suggests that muscle metabolites produced by static muscle contractions stimulate group III and IV muscle nociceptors leading to an excitation of gamma-motoneurons through a reflex mechanism. Increased gamma-motor drive causes intrafusal fibres to contract increasing muscle spindle afferent firing. This activity in turn will raise the activation level in the pool of alpha-motorneurones projecting to the primary muscle increasing resting muscle tonus. Muscle spindle afferents and postganglionic sympathetic outflow to skin and muscle was recorded from healthy subjects using the technique of microneurography. Cardiorespiratory function was measured non-invasively. Muscular pain was induced using a solution of hypertonic saline tonically infused via indwelling cannulae delivered by an infusion pump. The rate of infusion was controlled to ensure subjects experienced mild to moderate pain. Muscle spindle afferents fail to show any change in spontaneous discharge rate in response to tonic muscle pain contradicting the observations and conclusions from animal studies. Sympathetic outflow to skin demonstrated a transient increase followed by a sustained decrease during infusion of hypertonic saline. Sympathetic outflow to muscle demonstrated a dichotomy of responses during muscle pain: half of the subjects showing increasing sympathetic activity, BP, and HR; whilst the others showed decreasing sympathetic activity, BP, and HR. Consistent responses were seen in the same individuals when the study was reproduced in a second recording. This investigation clarifies our understanding of the physiological consequences tonic pain has on sympathetic outflow and muscle spindle afferents in the context of developing chronic muscle pain. There is no apparent evidence to suggest that tonic pain increases the discharge rate of spontaneously active muscle spindle afferents to result in chronic muscle pain as proposed by the “vicious cycle” theory. There is, however, evidence of sustained changes in sympathetic outflow to muscle and skin in response to tonic pain that may contribute to the development of chronic pain.
Declaration relating to disposition of project thesis/dissertation
I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).
...... 22/01/2014...... Signature Witness Date
The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.
FOR OFFICE USE ONLY Date of completion of requirements for Award:
THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS
iii ACKNOWLEDGEMENTS
I am in tears as I write this. This PhD thesis marks the end of a long journey I began 11 years ago after graduating from high school. It was a difficult time for me, but only a few days after starting university I promised myself to turn it around and aim to achieve excellence. I’ve had tremendous amounts of support along the way; hence, I would like to dedicate this thesis to my family.
To my wife Farzana, the last 18 months have been a tremendous struggle for both of us. I cannot thank you enough for your love, patience, and support during this time. I know at times I have been very demanding and unpleasant to be around. Thank you so much for understanding and being patient as I spent countless hours at Neura and at home trying to finish this PhD. I would not have finished this if it weren’t for your support and encouragement. No longer will we spend weekends at home so that I can finish my work. From now on it will be just you and I.
To my parents Papa and Mummy, you have by far been my greatest support throughout this journey. You sacrificed everything you had and everything you knew to come to a foreign land and start a new life with only one vision that your children get a good education. I hope I have lived up to your expectations and made you proud by achieving the highest form of tertiary education. There is nothing in this world that I could ever give you that will equate to the amount of love and support you have given me. I make dua that it all comes back to you.
To my brother Faris, you have been my greatest inspiration in completing this journey. From my first day at university you have been extremely pushy about me being as motivated, dedicated, and hard working as you are. The pressure was relentless and there were times when you just simply got on my nerves, but I am so grateful that you did because if it weren’t for that pressure I may not be where I am today. Thank you so much for everything you have done for me. Also, thank you to my sister in-law Aliya and my little nephew Zeyaan, cha cha has a PhD now.
iv To my lab colleagues Elie, Rania, Cheree, and Rachael, thank you so much for all your help running experiments and more importantly for your friendship. We’ve had the most amazing times together in the lab and at conferences, which simply would not have been the same without you all. I wish you all the best with your future and hope that we can continue our friendship long until we are old and miserable.
To my supervisor Ingvars, I am truly grateful for what you have taught me about being a good scientist. Your high standards of scientific research, critical evaluation, and constructive criticism are something to be envious of. Yes, at times they have been over the top and frustrating but I have nothing but respect for you as these qualities have certainly influenced me and have given me a foundation which I believe will give me great success in a career of research. I also thank you for your friendship and support especially in the first 12 months of my PhD. There were times when you literally had to spoon-feed me, but never did you make me feel as if I couldn’t knock on your door and ask for help.
Last but not least I would like to thank my supervisor Vaughan, there are simply no words in the English language that can express my gratitude for what you have done for me. I approached you to do a PhD with nothing except a little enthusiasm and motivation and you have helped turn it into a reality. Thank you so much for your advice, your help, your time, and your friendship. I couldn’t have asked for a better PhD supervisor. You truly have made my time as a PhD student an absolute delight. All I can say is when I grow up I want to be like you.
v ABSTRACT
Mechanisms underlying the development of chronic muscle pain in humans remain unknown. Our current understanding is principally based on experimentation in animals, which has resulted in the formulation of a multitude of theories and physiological models to postulate these mechanisms in humans. The vicious cycle theory – a popularised but unsupported theory in humans – suggests that muscle metabolites produced by static muscle contractions stimulate group III and IV muscle nociceptors leading to an excitation of gamma-motoneurones through a reflex mechanism. Increased gamma-motor drive causes intrafusal fibres to contract increasing muscle spindle afferent firing. This activity in turn will raise the likelihood of activation in the pool of alpha-motorneurones projecting to the primary muscle increasing resting muscle tonus.
Muscle spindle afferents and postganglionic sympathetic outflow to skin and muscle were recorded from healthy subjects using the technique of microneurography.
Cardiorespiratory function (blood pressure, heart rate, and respiration) was measured non-invasively. Muscle pain was induced using a solution of hypertonic saline tonically infused via indwelling cannulae delivered by an infusion pump. The rate of infusion was controlled to ensure subjects experienced mild to moderate pain.
Muscle spindle afferents fail to show any change in spontaneous discharge rate in response to tonic muscle pain contradicting the observations and conclusions from animal studies. Sympathetic outflow to skin demonstrated a transient increase followed by a sustained decrease during infusion of hypertonic saline. Sympathetic outflow to muscle demonstrated a dichotomy of responses during muscle pain: half of
vi the subjects showing increasing sympathetic activity, blood pressure, and heart rate; whilst the others showed decreasing sympathetic activity, blood pressure, and heart rate. Consistent responses were seen in the same individuals when the study was reproduced in a second recording.
This investigation advances our understanding of the physiological consequences tonic pain has on sympathetic outflow and muscle spindle afferents in the context of developing chronic muscle pain. After undertaking experimentation in awake human subjects there is no apparent evidence to suggest that tonic pain increases the discharge rate of spontaneously active muscle spindle afferents to result in increased muscle tone as proposed by the “vicious cycle” theory. There is, however, evidence of sustained changes in sympathetic outflow to muscle and skin in response to tonic pain that may contribute to the development of chronic pain.
vii Journal publication arising from this thesis
Chapter III:
Fazalbhoy, A., Macefield, V. G., & Birznieks, I. (2013). Tonic muscle pain does not increase fusimotor drive to human leg muscles: implications for chronic muscle pain.
Exp Physiol. 2013, 98(6), 1125-32
Chapter IV:
Fazalbhoy, A., Birznieks, I., & Macefield, V. G. (2012). Individual differences in the cardiovascular responses to tonic muscle pain: parallel increases or decreases in muscle sympathetic nerve activity, blood pressure and heart rate. Exp Phys, 97(10),
1084-1092.
Chapter V:
Fazalbhoy, A., Birznieks, I., & Macefield, V. G. (2014). Consistent inter-individual increases or decreases in muscle sympathetic nerve activity during tonic muscle pain: implications for chronic pain. Exp Brain Res (Epub ahead of print)
Chapter VI:
Hall, S. C., Fazalbhoy, A., Birznieks, I., & Macefield, V. G. (2012). Biphasic effects of stimulation of muscle nociceptors on skin sympathetic nerve activity in human subjects. Exp Brain Res, 221(1), 107-14.
viii Conference publications arising from this thesis
31st Annual Meeting of the Australian Neuroscience Society, Auckland, New
Zealand, 2011 Fazalbhoy A., Birznieks I., & Macefield V. G. Effects of sustained muscle pain on spontaneous muscle sympathetic nerve activity in awake human subjects.
Musculoskeletal Disorders and Chronic Pain: Evidence-based approaches for clinical care, disability prevention and claims management, Canadian Institute for the Relief of Pain and Disability, Los Angeles, California, U. S. A. Fazalbhoy A., Birznieks I.,
& Macefield V. G. Effects of tonic muscle pain on spontaneous muscle sympathetic nerve activity in humans.
Brain Sciences Symposium UNSW, Sydney, Australia. Fazalbhoy A., Birznieks I.,
& Macefield V. G. Changes in Muscle Sympathetic Nerve Activity in Response to
Sustained Muscle Pain.
UWS College of Health and Sciences student and post-doctoral conference 2011,
Sydney, Australia. Fazalbhoy A., Birznieks I., & Macefield V. G. Changes in muscle sympathetic nerve activity in response to sustained muscle pain.
32nd Annual Meeting of the Australian Neuroscience Society, Gold Cost, Australia
Fazalbhoy A., Macefield V. G., & Birznieks I. Changes in firing of spontaneously active muscle spindle afferents.
ix 14th World congress on pain, Milan, Italy. Fazalbhoy A., Macefield V. G., &
Birznieks I. Does tonic muscle pain alter the discharge rate of spontaneously active muscle afferents in awake human subjects?
33rd Annual Meeting of the Australian Neuroscience Society, Melbourne, Australia.
Fazalbhoy A., Birznieks I., & Macefield V. G. Invariable inter-individual cardiovascular responses to sustained muscle pain.
24th International Symposium on the Autonomic Nervous System, the Big Island,
Hawaii. Fazalbhoy A., Birznieks I., & Macefield V. G. Tonic muscle pain evokes individually consistent cardiovascular and effector organ responses: implications for hypertension with chronic pain.
Society for Neuroscience 2013, San Diego, California. Fazalbhoy A., Birznieks I., &
Macefield V. G. Consistent individual cardiovascular responses to long-lasting muscle pain in humans: implication for the development of hypertension with chronic pain.
x TABLE OF CONTENTS
Originality Statement...... ii
Acknowledgements...... iv
Abstract...... vi
Table of Contents...... xi
List of Figures...... xv
List of Tables...... xvi
INTRODUCTION
Statement of the problem……………………………………………………………………..……1
Motivation for the study ...... 2
Overview ...... 5
CHAPTER ONE - LITERATURE REVIEW
1.1 Nociception and pain ...... 8
1.2 Peripheral nervous system and its subdivisions...... 11
1.3 Autonomic nervous system ...... 12 1.3.1 Sympathetic nervous system...... 13 1.3.1.1 Sympathetic outflow to muscle ...... 14 1.3.1.2 Sympathetic outflow to skin ...... 15 1.3.2 Parasympathetic nervous system...... 16 1.3.3 Blood pressure control and baroreceptor reflex mechanisms...... 17 1.3.4 Relationships between pain and the cardiovascular system ...... 18 1.3.4.1 Animal studies of pain and the autonomic nervous system ...... 20 1.3.4.2 Human studies of pain and the autonomic nervous system...... 21 1.3.5 Pain and the baroreflex mechanism ...... 24
1.4 The somatic motor system...... 25 1.4.1 Structure of muscle spindles ...... 26
xi 1.4.2 Function of muscle spindles ...... 27 1.4.3 Sympathetic innervations of human muscle spindles ...... 28 1.4.4 Pain and proprioception...... 28 1.4.5 Pain and muscle stretch reflexes ...... 30 1.4.6 Muscle tone and muscle spindle activity: the vicious cycle theory ...... 32 1.4.7 Increase in fusimotor drive as a result of pain...... 33 1.4.7.1 Animal studies ...... 33 1.4.7.2 Human studies...... 40 1.4.8 Fusimotor drive and the activation threshold of the alpha motoneurone pool ...... 41
1.5 Microneurography...... 42
1.6 Experimental muscle pain...... 44 1.6.1 Hypertonic saline ...... 45
1.7 Aims ...... 46
CHAPTER TWO - GENERAL METHODS
2.1 Subjects...... 48
2.2 Noxious stimulation ...... 49
2.3 Recordings...... 50 2.3.1 Microneurography ...... 50 2.3.2 Cardiorespiratory ...... 51 2.3.3 Radial tonometry...... 51 2.3.4 Photoplethysmography...... 52
CHAPTER THREE - TONIC MUSCLE PAIN AND MUSCLE SPINDLE AFFERENT DISCHARGE RATE
3.1 Introduction...... 54
3.2 Methods ...... 56 3.2.1 Muscle spindle recordings ...... 57 3.2.2 Noxious stimulation...... 58 3.2.3 Data analysis ...... 58
xii 3.2.3.1 Statistics...... 59
3.3 Results...... 59
3.4 Discussion...... 64 3.4.1 Limitations...... 67
3.5 Conclusions ...... 69
CHAPTER FOUR - MUSCLE SYMPATHETIC NERVE ACTIVITY RESPONSES TO TO TONIC MUSCLE PAIN
4.1 Introduction...... 71
4.2 Methods ...... 73 4.2.1 Experimental procedures...... 73 4.2.2 Noxious stimulation...... 74 4.2.3 Analysis...... 75
4.3 Results...... 76
4.3.1 Subjective experience of tonic muscle pain ...... 76
4.3.2 Muscle sympathetic nerve activity during tonic muscle pain ...... 77
4.3.3 Blood pressure and heart rate during tonic muscle pain ...... 80
4.3.4 Heart rate variability...... 83
4.4 Discussion...... 84
4.5 Conclusions ...... 90
CHAPTER FIVE - CONSISTENT RESPONSES IN MUSCLE SYMPATHETIC NERVE ACTIVITY DURING TONIC MUSCLE PAIN
5.1 Introduction……………………………………………………………………………………….87
5.2 Methods ...... 94 5.2.1 Subjects...... 94 5.2.2 Experimental procedures...... 95 5.2.3 Noxious stimulation...... 96
xiii 5.2.4 Data analysis ...... 97
5.3 Results...... 97
5.4 Discussion...... 103 5.4.1 Methodological considerations ...... 106
CHAPTER SIX - SKIN SYPATHETIC NERVE ACTIVITY RESPONSES TO TONIC MUSCLE PAIN
6.1 Introduction...... 109
6.2 Methods ...... 111 6.2.1 Recording procedures ...... 111 6.2.2 Noxious stimulation...... 113 6.2.3 Analysis...... 114
6.3 Results...... 115
6.4 Discussion...... 121
6.5 Conclusions ...... 126
CHAPTER 7 - GENERAL DISCUSSION
7.1.1 Group III and IV nociceptors and muscle spindle discharge activity...... 129 7.1.2 Lack of alpha-motoneuron activity ...... 131 7.1.3 The effects of tonic pain on sympathetic outflow to muscle...... 131 7.1.4 The effects of tonic pain on skin sympathetic outflow ...... 134
7.2 Limitations ...... 135
CHAPTER EIGHT - CONCLUSIONS
CONCLUSIONS ...... 138
xiv List of figures
Figure 1.1 Schematic representation of the central nervous system…………………13
Figure 1.2 Diagrammatic representation of the sympathetic nervous system……….16
Figure 1.3 Diagrammatic representation of the parasympathetic nervous system…..21
Figure 1.4 Muscle spindle apparatus highlighting static and dynamic nuclear bag and nuclear chain fibres………………………………………………………….……………….……32
Figure 1.5 Depiction of the technique of microneurography……………...….……...49
Figure 3.1 Experimental record from a single subject – MSNA and pain…….…..…66
Figure 3.2 Individual trends of muscle spindle discharge plotted over time…..….…67
Figure 3.3 Mean discharge rate of muscle spindle afferents and mean pain profiles of all subjects…………………………………………………………………….…...... 68
Figure 4.1 Experimental records from one subject…………………………….....…82
Figure 4.2 Mean changes in MSNA burst frequency and burst amplitude during tonic muscle pain…………………………………………………………………….……83
Figure 4.3 Mean changes in blood pressure and heart rate in the group showing an increase in MSNA during tonic muscle pain………………………………….……85
Figure 4.4 Mean changes in low-frequency and high-frequency power in the heart variability spectrum……………………………………………………..…….……87
Figure 5.1 Experimental records from one subject…………………………...... …103
xv Figure 5.2 Changes in mean MSNA burst amplitude, mean blood pressure and heart rate during tonic muscle pain………………………………………………….……104
Figure 5.3 Data from all subjects, showing the relationships between the peak changes in MSNA burst amplitude during tonic muscle pain………………………..………106
Figure 6.1 Experimental records from one subject………………………………....120
Figure 6.2 Changes in skin sympathetic nerve activity (SSNA) and skin blood flow during tonic muscle pain.……………………………..……………………….……122
Figure 6.3 Changes in heart rate and blood pressure during tonic muscle pain……123
Figure 6.4 Changes in respiratory rate and amplitude during tonic muscle pain…..125
List of tables
Table 4.1: Mean changes in low-frequency (LF) and high-frequency (HF) power in the heart variability spectrum………………………………………………………..89
xvi Introduction
INTRODUCTION
Statement of the problem
Chronic pain research has evolved over the last decade in terms of its importance and lines of investigation. Advancing knowledge in this field has enabled a convergence in the scope of investigation, and further progress is being made. Broadly speaking, most chronic pain research endeavours to address one of three central questions.
Firstly, the aetiology of chronic pain – is it physiological or psychological in origin?
Secondly, the development of chronic pain – what are the elements and factors that begins its transformation from acute (short-lasting) to chronic (long-lasting)? Thirdly, the maintenance of chronic pain – in the absence of continuous noxious input how does it self-perpetuate? Considering that chronic pain is being increasingly recognized as a disease state rather than simply a symptom, a fourth question is emerging – what are the consequences of chronic pain, in terms of their impact on the regulation of other body systems (Siddall & Cousins 2004).
The framework of chronic pain research can be placed along a continuum, consisting of a body with two ends. At one end are investigations into the neurophysiological processes; at the other end the cognitive/psychological processes. This is due to the duality of pain being a physiological and psychological experience. The present thesis is located within the neurophysiological realm of chronic pain, though does not directly study chronic pain. Rather, it looks at long-lasting experimental muscle pain, as a model for examining the physiological changes that ultimately develop into the expression of chronic pain. In particular, the work presented in this thesis considers the impact of long-lasting muscle pain on the cardiovascular system and its regulation
1 Introduction
by the sympathetic nervous system, as well as the effects of muscle pain on the somatic motor system.
Pain is described as a sensation that takes a high degree of priority in the central nervous system. The basic premise of its function is to signal, alarm, and bring to attention a compromise to the integrity of the body. Triggered by the presence of actual tissue damage, or the potential of tissue damage, it sets in motion a number of cascading events to preserve the structure and function of the body.
Motivation for the study
For decades chronic muscle pain has been an enormous challenge for sufferers and clinicians alike. Sufferers find it difficult to function, incapable of completing basic activities of daily living (ADL). It can also lead to time off work costing employers in loss of productivity. Clinicians are limited by a lack of specific treatment options available, which results in ineffective management and frustration for the sufferer.
Furthering our understanding of the aetiology, and more importantly the mechanisms that precipitate chronic muscle pain, will enable us to intervene by stopping the progression from acute to chronic, and provide a foundation for developing better treatment options for chronic pain sufferers. Our current understanding of the development of chronic muscle pain syndromes primarily comes from animal models.
This in it self presents a challenge for developing effective treatments due to its lack of evidence in humans, despite it being a popular explanation of chronic muscle pain.
2 Introduction
A resonating challenge that exists with medical research utilising animal models is the translation of findings into humans, particularly in to a clinical setting. Many research questions are investigated and addressed using models that abide by universally acceptable ethical and moral guidelines. The use of animal models, for example, provides a pathway to studying neurophysiology among other things. However, observations made in animals don’t always necessarily hold true in humans. It is therefore imperative that animal data be tested and proved to be similar in humans in order to treat clinical disorders.
The dominating theory on the aetiology of chronic muscle pain is coined the ‘vicious- cycle theory’ (Johansson & Sojka 1991). It is a physiological model based on particular observations made in animals implicating the increase of muscle spindle afferent firing in the development of chronic muscle pain. The cycle is initiated by activation of group III and IV afferent nociceptors located in muscle tissue by noxious stimulation. This activation creates a reflex-mediated increase in the activity of gamma-motoneurones (fusimotor neurones), which innervate the intrafusal muscle fibres within the muscle spindle apparatus. Fusimotor excitation causes the intrafusal muscle fibres, located at the poles of the spindle, to contract and thereby stretch the central sensory region (Matthews 1972). This increases the background firing of spontaneously active muscle spindle afferents (Group Ia and II afferents), and may recruit silent afferents. Consequently, the excitability of spinal alpha-motoneurones - which innervate the extrafusal (skeletal) muscle fibres - is increased, proximating the activation threshold and thereby increasing the likelihood of contraction (or increasing the activity of already active motonuerones). As a result of this reflexly generated contraction muscle metabolites accumulate, further stimulating group III
3 Introduction
and IV afferents (Johansson & Sojka 1991). This self-perpetuating cycle has been used to form the basis of a physiological explanation for the development of chronic muscle pain.
The vicious cycle theory has been confirmed in the cat (Thunberg et al. 2002a).
Thunberg et al. (2002a) induced experimental muscle pain by bolus injection of hypertonic saline into heteronymous and homonymous muscles of the leg in the anaesthetised cat. Direct recordings from single muscle spindle afferents showed an increase in spontaneous firing rate by ~80%, which was used to support the vicious cycle theory. However, translating this into the clinical setting is unwarranted because there is no evidence that such a model of chronic pain exists in humans. In fact, bolus intramuscular injection of hypertonic saline into human leg muscles – sufficient to cause a strong level of pain - does not increase the firing of spontaneously active muscle spindles, rather it causes a slight decrease (Birznieks et al. 2008). Indeed, the increase in muscle spindle stretch sensitivity during muscle pain may be a feature of the cat, or the result of experimental design such as using anaesthesia, which may not respond the same way in awake humans. So, translatability is certainly an issue with animal research.
Any therapeutic intervention should be carefully tested in a methodical manner until it is proven to be scientifically and clinically effective. Acceptance of the vicious cycle theory in a clinical setting without evidence of the mechanism and pathophysiological cycle existing in humans is misleading and undermines the effectiveness of therapeutic intervention developed on its premise. Furthermore, acceptance of this theory as the aetiology behind chronic muscle pain creates complacency making
4 Introduction
further investigation unwarranted. No doubt the anatomical circuitry exists in humans but it is not known whether long-lasting muscle pain affects the firing rate of human muscle spindle afferents and its functional significance.
In addition to effects on muscle spindles, noxious stimulation of deep tissue such as muscle has been shown to modulate sympathetic outflow to both muscle and skin
(Burton et al. 2009a; 2009b). Considering sympathetic outflow contributes to the regulation of heart rate and blood pressure, withdrawals can dramatically impact on the cardiovascular system. It is expected that any change in sympathetic outflow remains sustained as long as the noxious input continues. Accordingly, a study of the effects of long-lasting muscle pain on sympathetic outflow could provide insights into the underlying basis of the physiological signs associated with pain.
Overview
This thesis comprises a further eight chapters split into three parts. In Part I (Chapters
1 & 2) I situate the research project into the context of chronic pain by identifying the gaps in knowledge and further go on to establish an appropriate research methodology. Chapter 1 explores the current understanding of the differential effects and mechanisms that pain have, identified to date. It details the normal actions of both the sympathetic nervous system and its roles in the regulation of the cardiovascular system, and describes the sensory feedback organs located in skeletal muscle (muscle spindles). It presents evidence obtained from both animal and human experiments carried out to explain the neurophysiological responses to pain. I also present some work that has been conducted in our laboratory by a previous PhD student. Chapter 2 familiarises the reader with the research methodology and presents the rationale for
5 Introduction
invasive microelectrode recordings from peripheral nerves in awake humans
(microneurography) and the use of intramuscular injection of hypertonic saline as a model of experimental muscle pain in healthy subjects. It also outlines the benefits of using healthy humans without pre-existing pain as opposed to actual chronic pain sufferers.
Part II (Chapters 3-6) details the experimental work undertaken to investigate the effects of long-lasting muscle pain on the firing of muscle spindle afferents and on sympathetic nerve activity to muscle and skin. Chapter 3 presents results of the study investigating changes in discharge rate of spontaneously active muscle spindle afferents during infusion of hypertonic saline. It assesses the validity of the “vicious cycle theory” in human subjects. Chapter 4 provides results of a study investigating the effects of tonic muscle pain on muscle sympathetic nerve activity (MSNA). In addition to examining changes in MSNA it includes an examination of the changes in blood pressure, heart rate, and heart rate variability during intramuscular infusion of hypertonic saline. Chapter 5 extends this work by investigating the effects of tonic muscle pain on MSNA in a cohort of subjects in whom recordings were made on two occasions. It assesses the reproducibility of the sympathetic responses to pain.
Chapter 6 investigates the effects of long-lasting muscle pain on skin sympathetic nerve activity (SSNA), as well as on skin blood flow and sweat release.
Part III (Chapters 7 & 8) brings together the most significant findings from my investigations to discuss them in the context of the literature. Chapter 7 in particular explores the principal outcomes of each study and addresses the findings with a view of filling in the gaps of knowledge identified in Part I. Chapter 8 finally is a synthesis
6 Introduction
of my work demonstrating how it fulfills the aims outlined in the Introduction. It summarizes the implications of tonic muscle pain on the sympathetic and somatic motor systems in humans, with respect to chronic muscle pain.
7 Chapter 1 – Literature review
CHAPTER ONE
LITERATURE REVIEW
1.1 Nociception and pain
Pain is a protective mechanism essential for the preservation and survival of an individual. Its function is to warn of bodily harm from trauma and injury, but it can also signal the presence of unhealthy conditions or inflammation in tissues of the body. As a complex somatic distress signal, it is a multidimensional phenomenon that includes sensory-discriminative, affective–motivational, motor, and autonomic components (Maihofner et al. 2010). This sensation triggers a cascade of events that are essential for preventing further damage and promoting healing. The International
Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (Merskey & Bogduk 1994). Alternative definitions of pain are still used, but this definition in particular comprises two central ideas that comprehensively encompass the experience of pain. Firstly, tissue damage doesn’t necessarily need to be present; the perception of potential harm is sufficient to trigger similar physiological and psychological responses to actual pain. Secondly, pain is a sensation that is physiologically sensory but also incorporates a psychological experience, and thus has cognitive elements. This indicates both the presence of an injurious stimulus, but also – and more importantly – the need for behavioural change motivated by distinct unpleasantness. Wall (1979) suggested that pain represents first
8 Chapter 1 – Literature review
and foremost the awareness of a “need state,” such as hunger or thirst, rather than an exteroceptive discriminative sense, such as touch, hearing, or vision. The concept of multidimensional pain and the activation of multiple systems as a result of applying painful stimuli have been shown in several human brain imaging studies (Jones et al.
1991; Coghill et al. 1994; Derbyshire & Jones 1998; Paulson et al. 1998). Presenting painful stimuli activates multiple cortical regions, engaging systemic survival mechanisms at several functional levels: autonomic, homeostatic, motor, sensory, and behavioural.
It is important to understand the difference between nociception and pain.
Nociception defines the neuronal activity generated following noxious stimulus that can be filtered by the sub-conscious mind to pass through to the conscious mind or not. Nociceptors are not supposed to signal the presence of tissue damage but prevent damage by informing the central nervous system that tissues are approaching structural or functional limits (Mense 1997). Pain is the conscious perception and recognition of the stimulus as potentially damaging. The level of nociception that is regarded as painful varies across individuals, contexts and cultures, extending over a range of perceived intensities in all individuals (Madan et al. 2008).
Nearly all tissues of the periphery are lined with receptors that transmit their activity to the central nervous system via axons coursing through sensory nerves. Sir Charles
Sherrington labelled receptors specifically coding for noxious or potentially damaging stimuli as nociceptors, which are high-threshold small-diameter fibres with associated small cell bodies (Sherrington 1900). They include fast conducting – small myelinated fibres (A∂ or group III), - and slow conducting – small unmyelinated
9 Chapter 1 – Literature review
fibres (C-fibres or group IV). Physiological subtypes of nociceptors have been differentiated in skin and other somatic tissues such as muscle, ligament, joint, and viscera thus giving rise to a classification of pain based on location (Messlinger
1997). Superficial pain and deep pain are traditionally separated because of their unique and contrasting characteristics. Pain originating from deep body structures, such as skeletal muscle tissue, evokes a dull, aching sensation that can be very difficult to localize, whereas pain originating in skin evokes a sharp, burning sensation that is well localised (Henderson et al. 2006; Burton et al. 2009a). These sensations differ in part due to the properties of group III and group IV fibres, but also due to the central processing of nociception relaying back to the dorsal horn and activating different parts of the brain.
Nociceptive signals enter the spinal cord through the dorsal rami of spinal nerves and synapse onto neurones in the dorsal horn of the spinal cord. Slow conducting nociceptive fibres, cutaneous C nociceptive fibres, terminate in lamina I, and the outer portions of lamina II. Cutaneous A∂ fibres, fast conducting fibres, also terminate on lamina I but contain additional projections terminating on the deeper lamina V (Iggo et al. 1985). Afferents from deeper structures such as muscle, joints, ligaments, and viscera primarily terminate in lamina I but additionally extend into laminae II, V, and
X (Sugiura et al. 1989). From this point, cells of both lamina I and lamina V encode the intensity of the noxious stimulus, in terms of frequency of discharge and the number of neurones activated, and transmit this information via ascending axons to the brain. Projections of nociceptive neurones from lamina I and V ascend to terminate in several hierarchically organised regions that reflect the various reactions to a painful stimulus (Craig 2002).
10 Chapter 1 – Literature review
At a spinal level, lamina I (and some lamina V) cells project to the preganglionic sympathetic regions in the thoracolumbar cord, and to parasympathetic regions in the sacral spinal cord. Axons projecting to the lower brain stem from lamina I terminate in several autonomic sites bilaterally, providing a basis for the somatoautonomic reflexes caused by site-specific noxious stimuli (Craig 2002). Furthermore, a large density of projections from lamina I are destined towards the parabrachial nucleus located near the pontomesencephalic junction, which also receives vagal afferent input from the nucleus tractus solitarius, making it a major viscerosensory integration site and linking it with the periaqueductal gray, hypothalamus, and amygdala
(Sikandar & Dickenson 2012). The extensive network of axons cross linking areas of the brain specific with pain processing and homeostatic maintenance provides a neuroanatomical basis for activation of these areas during noxious stimulation.
Autonomic responses are reflexly generated by painful stimuli between distinct areas of the brain and projecting fibres (Janig 1985). Functional imaging using PET and
MRI, and evoked potential studies of the human cerebral cortices have demonstrated activation of the insula and the anterior cingulate regions of the brain in response to painful thermal stimuli (Besson et al. 1995). Other studies utilising similar techniques report activation in the primary and secondary somatosensory regions (Maihofner et al. 2010; Vierck et al. 2013).
1.2 Peripheral nervous system and its subdivisions
The autonomic nervous system and the somatic nervous system are subdivisions of the peripheral nervous system that principally coveys sensory information and motor control to and from several target tissues in the periphery (See fig 1). As a complex network of neurones working synchronously and synergistically, the peripheral
11 Chapter 1 – Literature review
nervous system is the pathway for neural control from the central nervous system to the vast periphery. The autonomic nervous system comprises neural structures and connecting pathways that are functionally responsible for transmitting sensory input from the visceral organs, such as the digestive system and cardiovascular system. In addition, it also provides motor control to smooth and cardiac muscle, and to glands of the viscera. Autonomic pathways coordinate these motor outputs involuntarily and independent of conscious decision. Similarly, the somatic nervous system comprises a series of neural structures responsible for conveying and processing conscious and unconscious sensory information originating in the body wall, skeletal muscles, joints, ligaments and tendons, such as proprioception, touch and pain. Motor pathways of the somatic nervous system innervate striated (skeletal) muscles under voluntary control
(Guyton & Hall 2000)`.
1.3 Autonomic nervous system
The autonomic nervous system comprises a complex network of neurones that provide involuntary control to target tissues and organs, particularly those governing homeostasis. Working with regions of the hypothalamus, brain stem, and spinal cord
(Kandel et al. 2000), the autonomic system regulates smooth muscle, cardiac muscle, glandular (secretory) cells, and mediates a variety of visceral reflexes (Powley 1999).
Classified into three subdivisions, sympathetic (thoracolumbar), parasympathetic
(craniosacral) and enteric (gastrointestinal tract), their functions are not antagonistic to one another, but rather they operate synergistically and differentially under various physiological conditions (Brooks 1981). Both the sympathetic and parasympathetic systems utilise a sequential two-neurone efferent pathway to connect the central nervous system to the effector organ (see Fig 1.2 and 1.3). A preganglionic neurone,
12 Chapter 1 – Literature review
located in the gray matter of the central nervous system, must first synapse onto a postganglionic neuron, which is located in the paravertebral sympathetic chain or other plexuses. The postganglionic neurone will then synapse onto the target organ.
Some structures receive innervation from only one system, such as systemic blood vessels, sweat glands and the arrector pili (hair) muscles, which are innervated solely by sympathetic fibres, whilst other structures such as the heart receive dual innervation by sympathetic and parasympathetic fibres (Brooks 1983). Differentiated responses and functions of the autonomic nervous system are achieved primarily through two classes of postganglionic neurotransmitters: norepinephrine for the sympathetic system and acetylcholine for the parasympathetic (Guyton & Hall 2000).
1.3.1 Sympathetic nervous system
The sympathetic nervous system provides motor innervation to a number of different organs and target tissues (see Fig 1.2). Short preganglionic fibres project from the spinal cord and synapse onto neurones within the mediolateral cell column located in the thoracic and upper lumbar regions. Paravertebral (sympathetic chain) and visceral
(prevertebral) ganglia then project post-ganglionic fibres to target tissues and organs.
Cardiovascular, respiratory, gastrointestinal, and urogenital systems all receive efferent sympathetic innervation. The traditional view of the workings of the sympathetic nervous system has been an all-or-nothing approach, with terms like
“flight or fight” summarising its function (coined by Walter B. Canon in 1915)
(Cannon 1915). Such a basic premise simply doesn’t hold true, as the sympathetic nervous system is a highly organised and differentiated neural network with specific subdivisions, each regulated and governed by its own set of control systems (Janig &
13 Chapter 1 – Literature review
McLachlan 1992; Wallin & Charkoudian 2007). The primary neurotransmitter for all sympathetic preganglionic neurones is acetylcholine, whereas the primary neurotransmitter for postganglionic neurones is noradrenaline (Guyton & Hall 2000).
Of primary importance in the regulation of the cardiovascular system, sympathetic nerve activity controls peripheral vasculature in muscle and skin by modulating resistance of the blood vessels, which is essential for the maintenance of arterial blood pressure. In humans, the greater majority of postganglionic sympathetic neurones are vasoconstrictor in function (McLachlan 2007). Considering that vascular beds of skeletal muscles make up a large majority of the cardiovascular system, neural control of blood vessels through sympathetic activity is a vital part of systemic hemodynamics (Wallin & Charkoudian 2007). In contrast to muscle, skin sympathetic nerve activity is more complex, receiving four different types of neurones: cutaneous vasoconstrictor, vasodilator, sudomotor, and pilomotor (Wallin & Charkoudian 2007).
1.3.1.1 Sympathetic outflow to muscle
There is a large mass of skeletal muscle in the body, which requires a large amount blood flow. It is estimated that at rest blood flow to muscle is approximately 3-
4ml/min/100g of muscle, increasing to 50-80ml/min/100g of muscle during rigorous exercise (Laine et al. 1998). Fluctuation in blood volume to keep up with demand is partly achieved by vasoconstrictor function. Microelectrode recordings from muscle fascicles of human peripheral nerves have revealed that muscle sympathetic nerve activity (MSNA) occur as bursts of pulse-synchronous impulses followed by a period of quiescence (Vallbo & Hagbarth 1967). Muscle vasoconstrictor neurones are tightly coupled to the cardiac rhythm via the arterial baroreceptors (Delius et al. 1972a;
14 Chapter 1 – Literature review
Sundlof & Wallin 1978; Janig 2006). There is considerable inter-individual differences in the frequency of MSNA impulses that is reproducible over weeks, months, and years (Sundlof & Wallin 1977).
Any procedure that leads to changes in arterial blood pressure or the volume of venous capacitance vessels generates a reflex change in MSNA (Janig 2006). Animal studies have shown that stimulation of cutaneous nociceptors, distension of viscera
(Traub et al. 1996), stimulation of cutaneous low-threshold mechanoreceptor afferents, stimulation of arterial chemoreceptors (Boczek-Funcke et al. 1992a), and trigeminal receptors also increase MSNA (Boczek-Funcke et al. 1992b). Noxious stimuli such as mechanical pressure, heat, cold-water immersion (Victor et al. 1987), or apnoea (Fagius & Sundlof 1986) activate muscle vasoconstritor neurons in humans and the cat .
1.3.1.2 Sympathetic outflow to skin
Thermoregulation is among the many important functions of the integumentary system, achieved through excitation or inhibition of vasomotor activity (Guyton &
Hall 2000). Spinal nerves destined for skin carry postganglionic sympathetic axons that are mostly vasoconstrictor in function innervating blood vessels, as well as sudomotor neurones to sweat glands, and piloerector neurones to the hairs (Kellogg
2006). Depending on location there may also be vasodilator neurones (Janig 2006).
In cold conditions an increase in cutaneous vasoconstrictor drive decreases skin blood flow, and increases in pilomotor drive stimulates erection of hairs on non-glabrous skin, resulting in heat conservation. Conversely, in warm conditions a decrease in
15 Chapter 1 – Literature review
cutaneous vasoconstrictor drive increases skin blood flow and an increase in sudomotor drive stimulates sweating, resulting in heat dissipation (Wallin &
Charkoudian 2007). Thus, skin sympathetic nerve activity (SSNA) plays a key role in thermoregulation.
Under thermoneutral conditions, many cutaneous vasoconstrictor neurons are spontaneously active in the anaesthetised cat and rat (Janig 2006). Microneurographic recordings of SSNA from humans via microelectrodes inserted percutaneously into peripheral nerves reveal irregular bursts of activity, the frequency and amplitude of which are lowest at thermoneutral ambient temperatures (Bini et al. 1980a; Macefield
& Wallin 1999). Unlike MSNA, SSNA is weakly associated with the cardiac rhythm
(Hagbarth et al. 1972; Macefield & Wallin 1999), but bursts of activity are caused by brisk inspiratory efforts and various ‘arousal stimuli’, including unexpected sounds, mental arithmetic and emotional stimuli (Delius et al. 1972b). Studies investigating the influence of mental or emotional stress on SSNA are limited (Wallin &
Charkoudian 2007). There is evidence to suggest that stimuli such as isometric handgrip, vibration, and metabolic-hormonal influence SSNA (Wallin et al. 1997)
1.3.2 Parasympathetic nervous system
The primary role of the parasympathetic nervous system is to respond transiently to specific stimuli in localised and discrete regions counteracting the effects of sympathetic activity (Tsuboi et al. 1987; Levenson 2003). Subdivided into two separate regions, the cranial portion is associated with four cranial nerves (III, VII,
IX, and X) that supply the head, neck, thorax, and most of the abdominal viscera (see
Fig 2.2). The sacral portion originates in the spinal cord from S2-4 and innervates the
16 Chapter 1 – Literature review
lower abdominal and pelvic viscera. Preganglionic axons synapse in terminal ganglia located in or near the targets of the very short postganglionic axons with little divergence, making the effects localised rather than widespread. Parasympathetic innervation of the heart and upper gastrointestinal tract (GIT) are through post- ganglionic branches of the vagus nerve. At rest, the heart receives tonic low-level vagal activity, mediated by acetylcholine acting upon muscarinic receptors, activation of which reduces heart rate and cardiac contractility (Carlsten et al. 1957; Campbell et al. 1989).
1.3.3 Blood pressure control and baroreceptor reflex mechanisms
Beat-to-beat (short-term) neural control of blood pressure hinges on a reflex-feedback loop balancing efferent and afferent activity. Regulation of blood pressure by the autonomic nervous system is primarily carried out through the arterial baroreflex mechanism (Cowley et al. 1973; La Rovere et al. 2008). The baroreflex is a mechanism that employs a set of stretch-sensitive sensory afferents within the walls of the heart, the carotid arterial bifurcation, aortic arch and vena cavae (Guyton &
Hall 2000). In recent years Thrasher (Thrasher 2002, 2004, 2005) has established the importance of arterial baroreceptors in the long-term regulation of blood pressure with his experimental observations in dogs. These observations have refuted the traditional understanding of arterial baroreceptors having limited impact on the long-term regulation of mean arterial blood pressure. Evidence suggests that baroreflex sensitivity is lowered in hypertensive patients (Gribbin et al. 1971) and has heritable qualities (Parmer et al. 1992), including potentially altered baroreflex sensitivity in patients with family histories of hypertension (Iwase et al. 1984; Tank et al. 2001).
17 Chapter 1 – Literature review
During increases of blood pressure, specifically systole, the stretch of the aorta and carotid arteries causes baroreceptors located within the aortic arch and carotid sinus to increase their firing rate. These signals are transmitted via the vagus and glossopharyngeal nerves, respectively, synapsing onto neurones within the nucleus tractus solitarius (NTS) located in the medulla. Glutaminergic (excitatory) fibres from
NTS project to the caudal ventrolateral medulla (CVLM), which then sends
GABAergic (inhibitory) projections to the rostral ventrolateral medulla (RVLM)
(Dampney 1981). The RVLM is the primary source of pre-sympathetic vasoconstrictor neurones destined for the intermediolateral column of the spinal cord, and is the primary output nucleus for MSNA. An increase in negative feedback provided by the baroreceptors results in inhibition of the RVLM and, via a direct glutamatergic projection to nucleus ambiguous and the dorsal motor nucleus of the vagus, an increase in cardiac vagal activity. Because acetylcholine (ACh) is inhibitory at the heart, an increase in cardiac vagal outflow results in a decrease in heart rate and contractility (Habermeier-Muth & Muscholl 1988). Together, the decreased heart rate, stroke volume, and – especially – the decrease in total peripheral resistance brought about by withdrawal of sympathetic vasoconstrictor drive causes blood pressure to fall and normalise around the levels set for homeostatic purposes.
1.3.4 Relationships between pain and the cardiovascular system
Sir Thomas Lewis undertook the earliest set of investigations describing autonomic reflexes generated by noxious stimuli. He observed that pain originating from deep structures evoked a vastly different response to pain originating in skin (Lewis 1942).
Deep pain evoked a “slowing of the pulse” and “falling of blood pressure”, whereas skin pain evoked “a rise of pulse rate” and a “sense of invigoration”. Feinstein and
18 Chapter 1 – Literature review
colleagues (1954) confirmed these initial findings when they observed a fall in blood pressure and bradycardia with muscle pain in awake human subjects. It has been hypothesised that these observations are a result of differential activation of the periaqueductal gray (Keay et al. 1994). The periaqueductal gray refers to the gray matter surrounding the cerebral aqueduct of the midbrain, which is functionally involved in pain modulation (Magoun et al. 1937; Nashold et al. 1969; Mantyh &
Peschanski 1982) and cardiovascular control (Kabat et al. 1953; Abrahams et al.
1960). Neurones are organised in a columnar fashion: dorsomedial, dorsolateral, lateral, and ventrolateral. Stimulation of the dorsal column is involved in pressor responses, while the ventrolateral column is involved in depressor responses
(Behbehani 1995). Various functions of the PAG are inter-related and there is significant interaction between different functional components of the PAG. Melzack and colleagues studied cats with lesions in the ventrolateral PAG and demonstrated significant attenuation in pain perception (Melzack et al. 1958). Studies in humans were taken further with stimulation of the PAG evoking a sensation of burning pain, vibration, occular movements, and fear (Nashold et al. 1969). Anatomical experiments in animals using horseradish peroxidase (HRP) tracer, soon after it was developed, revealed the termination of the spinothalamic tract in the PAG (Yezierski
1988). These investigators also showed that spinal afferents projecting to the PAG terminate in a site-specific distribution pattern (Holstege 1988). This is significant because the spinothalamic tract is known to transmit pain, itch, as well as crude touch.
This type of sensory information is known as affective sensation, which means the sensation is accompanied by a compulsion to act. It has been shown in two studies that microinjection of morphine into the PAG in rats resulted in hyperalgesia and explosive motor behaviour (Jacquet & Lajtha 1973, 1974).
19 Chapter 1 – Literature review
Kabat and colleagues carried out the earliest studies examining the reflex between the
PAG and the cardiovascular system when they showed an increase in blood pressure with specific stimulation of the midbrain (Kabat et al. 1953). Further studies showed that stimulation of the dorsolateral PAG, in particular, increases blood pressure, increases blood flow to muscles and decreases blood flow to skin along with piloerection (Abrahams et al. 1960).
1.3.4.1 Animal studies of pain and the autonomic nervous system
Horeyseck and Jänig demonstrated in a series of experiments the effects of noxious and non-noxious skin stimulation on sympathetic outflow to muscle and skin in the cat (Horeyseck & Jänig 1974a; Horeyseck & Jänig 1974b). Cutaneous nociceptors were stimulated using mechanical pressure or heat: sympathetic outflow to skin decreased in the superficial (cutaneous) peroneal nerve, but sympathetic outflow to the gastrocnemius muscles increased. They found similar results with both the anaesthetised (Horeyseck & Jänig 1974b) and spinalised (Horeyseck & Jänig 1974a) cats, implicating a spinal-reflex mechanism.
Noxious stimulation of deep tissues shows differential effects on autonomic activity in comparison to superficial tissues. Sato and colleagues (Sato et al. 1984) observed that normal rotation of the knee within the physiological range of motion in anaesthetised cats produced no change in autonomic activity. When the experiment was repeated in cats with knee joints sensitised by inflammation rotation of the knee through the same physiological range of motion resulted in an increase in blood pressure and heart rate (Sato et al. 1984). In addition to this they demonstrated that
20 Chapter 1 – Literature review
noxious rotation outside the physiological range of motion in both normal and inflamed knee joints yielded greater increases in blood pressure (BP) and heart rate
(HR). Considering sympathetic nerve activity plays a role in the regulation of blood pressure, it can be inferred that in this study the increases in blood pressure observed also resulted in increases of MSNA, although Sato and colleagues never directly measured it.
Contrasting with these findings, Keay and colleagues (1994) reported that pain originating in deep structures - such as joints, muscle and viscera - evokes profound decreases in blood pressure and heart rate in rats. Bilateral injections of formalin, an algesic substance, injected into the deep dorsal neck muscles evoked a significant increase in the duration of quiescence and hyporeactivity (Keay et al. 1994). This observation was similar to that evoked by direct injection of an amino acid in to the ventrolateral PAG (Keay et al. 1994), a region known to result in hypotension and bradycardia with electrical stimulation (Behbehani 1995). Again, although MSNA was not explicitly recorded, a decrease in blood pressure and heart rate is achieved through withdrawal of muscle (and splanchnic) vasoconstrictor and cardiac sympathetic drive. These studies together suggest that nociceptive pain from deep tissues affects the ouflow of MSNA in animals.
1.3.4.2 Human studies of pain and the autonomic nervous system
An array of different stimuli has been observed to increase MSNA in human subjects.
Noxious stimuli such as instillation of soapy solution in the eyes, strong mechanical pressure on the nail (Nordin & Fagius 1995), immersing the hand in ice-water (Fagius et al. 1989), and mechanical pressure on the skin (Schobel et al. 1996) all cause
21 Chapter 1 – Literature review
increases in MSNA and blood pressure. Perhaps the most relevant studies using microneurography and hypertonic saline as a noxious agent to induce deep pain in awake human subjects were those carried out by Burton and colleagues (Burton et al.
2009a; Burton et al. 2009b). They showed that selective stimulation of muscle nociceptors, induced by bolus (0.5 ml) intramuscular injection of hypertonic saline into the leg of awake human subjects caused deep pain that had disappeared after 10 minutes, and an increase in MSNA, blood pressure and heart rate that essentially followed the pain profile (Burton et al. 2009a). The same was true for skin pain induced by subcutaneous injection of hypertonic saline (Burton et al., 2009b). It is not known whether tonic muscle pain, induced by intramuscular infusion of hypertonic saline, will cause long-lasting increases in MSNA, blood pressure and heart rate. The present project aims to observe whether the increase in MSNA is sustained with tonic muscle pain lasting for ~1 hour. The working hypothesis is that muscle vasoconstrictor drive will show a sustained increase in MSNA and blood pressure.
Few studies in humans have investigated the effects of painful stimuli on skin sympathetic nerve activity. The large majority of investigations with noxious stimulation have been used to identify SSNA and its characteristics (Iwase et al.
2000; Mano et al. 2006). Acute noxious stimulation of muscle or skin—induced by bolus intramuscular or subcutaneous injection of hypertonic saline— whilst recording
SSNA from awake human subjects revealed transient increases of SSNA during both types of acute pain (Burton et al. 2009b). This increase was interpreted as an arousal response, rather than a spinal reflex response, evidence supported by the absence of any increases in blood pressure, heart rate, sweat release or cutaneous vasoconstriction when hypertonic saline was injected into a leg muscle in patients
22 Chapter 1 – Literature review
with spinal cord injury (Burton et al., 2008). The current project aims to extend this work in awake human subjects and observe whether the increase in SSNA to tonic muscle pain is sustained during the period of pain. The working hypothesis is that tonic muscle pain causes a sustained increase in SSNA, related to the affective component of pain.
Given that both muscle and skin pain can increase MSNA, and that an increase in
MSNA can increase blood pressure, it is likely that ongoing pain- even before it becomes chronic – will result in a sustained elevation of blood pressure. Indeed, it is known that elevated levels of MSNA are associated with essential hypertension (Esler
& Kaye 2000), renovascular hypertension (Johansson et al. 1999) and the high blood pressure that results from untreated obstructive sleep apnoea (Takeuchi et al. 1994;
Trombetta et al. 2010). Moreover, an increase in sympathetic outflow to the muscle vascular bed is often paralleled by an increase in cardiac sympathetic drive, leading to cardiac function and ultimately resulting in heart failure. Studies in rats have shown that chronically raised sympathetic tone results in abnormal cardiac function (Brum et al. 2002). Experimental evidence from mice lacking genes to regulate sympathetic activity in the midbrain and presynaptic autoinhibition shows that elevated sympathetic tone including a higher baseline heart rate and a modest elevation in systolic blood pressure resulted in cardiac dysfunction: decreased maximal exercise capacity and contractility (Brum et al. 2002). Furthermore, Brum and colleagues
(2002) examined myocardial tissue via electron microscopy and saw significant abnormalities in the ultra structure of cardiac myocytes. This indicates that a sustained increase in sympathetic activity can cause end-organ damage.
23 Chapter 1 – Literature review
1.3.5 Pain and the baroreflex mechanism
The functional interaction between pain and the autonomic nervous system is further highlighted by observations of blood pressure related hypoalgesia. Experiments involving baroreceptor stimulation have identified that pain sensitivity is lower in hypertensive compared to normotensive states (Schobel et al. 1996; D'Antono et al.
2000; Bruehl & Chung 2004; Ditto et al. 2009). The effects of pain on the baroreflex mechanism has been investigated by delivering a noxious stimulus and observing the alteration of blood pressure and heart rate; the resultant changes in cardiovascular parameters infers modulation of baroreceptor activity (Reyes del Paso et al. 2011).
D’Antono and colleagues (2000) applied noxious mechanical pressure to the finger in healthy male subjects following either a period of supine rest, rest with periodic valsalva manouevres to increase MSNA and blood pressure, or passive leg elevation to stimulate baroreceptors. Investigators inferred modulation of baroreceptor activity by monitoring the change in blood flow to the arm and the thorax via plethysmography - impedance cardiography - in response to the experimental manoeuvres mentioned above. Results showed that for the same amount of noxious pressure pain ratings were significantly lower following passive leg elevation in subjects with relatively elevated systolic blood pressure for the same intensity of nociceptive stimulation (D'Antono et al. 2000). This suggests a reflex interaction between pain and the baroreflex mechanism in the ability to modulate one another.
In a related study cutaneous nociceptors were noxiously stimulated acutely using a thermal probe in borderline hypertensive and normotensive male subjects (Ditto et al.
2009). The use of a thermode heated to a temperature of 46.5°C has previously been shown to be painful yet evidently tolerable in humans (Hofbauer et al. 2001). Painful
24 Chapter 1 – Literature review
stimuli were delivered at 30-sec intervals with alternating experimental manoeuvres, legs lying flat or with the participant’s legs elevated to 60° by an inclined ramp. After delivering each painful stimulus, the participant provided ratings of pain intensity (no pain—extremely intense pain) and unpleasantness (not unpleasant—extremely unpleasant) on two visual analogue scales (Ditto et al. 2009). Interestingly, pain ratings reported by borderline hypertensive subjects were lower when the legs were elevated compared to the legs laying flat for the same intensity of stimulus.
Stimulating the low-pressure (cardiopulmonary) baroreceptors during leg elevation distinctly shows hypoalgesia in hypertensive subjects, highlighting the existential relationship between blood pressure changes and pain sensitivity.
1.4 The somatic motor system
The somatic nervous system comprises an integrated network of somato-sensory neurons, located mostly in the dorsal root ganglia, which perceive external stimuli
(primarily mechanical and olfactory) and govern an appropriate locomotor response
(Blessing 1998). Some of the earliest work attempting to classify different afferent fibres coming from muscle was done by Lloyd and Chang (Lloyd & Chang 1948).
Four groups of fibres were identified based on the size of the fibre in diameters: group
I, large myelinated afferents; group II, medium-sized; group III, small; and group IV, unmyelinated. Efferent fibres in nerves destined for muscle are classified into three groups based on their size and function: large myelinated α-efferents from the α motoneurons in the anterior horn of the spinal cord, innervating the contractile bulk of the muscle; small myelinated γ-efferents from γ motoneurons (fusimotor), innervating the modified muscle fibers inside the muscle spindle; and unmyelinated sympathetic efferents innervating intramuscular blood vessels.
25 Chapter 1 – Literature review
1.4.1 Structure of muscle spindles
Nearly all human skeletal muscles contain spindles except for facial muscles and the digastric, a jaw-opening muscle (Kubota and Masegi, 1977). Muscle spindles are fusiform sensory receptors (see Fig 1.4) located in the belly of skeletal muscles, encapsulating 2–14 modified striated muscle fibres referred to as intrafusal fibres
(Cooper and Daniel, 1963; Kennedy, 1970; Kucera and Dorovini-Zis, 1979; Swash and Fox, 1972). A fibrous capsule surrounds the middle third of the spindle and is dilated to enclose a fusiform fluid-filled space. They are oriented in parallel with the extrinsic muscle fibres and transmit information about the length and rate of change in length of the muscle. Intrafusal fibres are of three different types that can be differentiated by the intensity of their staining for myosin ATPase, under acidic and alkaline conditions (Barker, 1974; Harriman et al., 1974; Kucera and Dorovini-Zis,
1979; Ovalle and Smith, 1972). Dynamic nuclear bag fibres and static nuclear bag fibres are long and large containing a central enlargement or bag shape that contains numerous nuclei. Nuclear chain fibres differ in formation and tend to be thinner and shorter as the central region contains a row of some 20-50 nuclei resembling a chain
(Kucera and Dorovini-Zis, 1979). Dynamic responses represent sensitivity of the primary sensory ending of nuclear bag fibres, which react to the size of a muscle length change and its speed. Static responses represent sensitivity of both primary and secondary sensory endings of nuclear chain fibres, which react to only the length change (Matthews, 1972; Proske et al., 1991).
Each muscle spindle receives both sensory and motor innervations. Primary sensory endings receive one large myelinated (group Ia) afferent and secondary endings receive 1-5 smaller myelinated (group II) afferents forming a single nerve bundle
26 Chapter 1 – Literature review
wrapping their sensory terminals about the central portion of the spindle (Kennedy et al., 1975; Swash and Fox, 1972). Polar ends of the muscle spindle are innervated by gamma motoneurones (fusimotor neurones) making them exclusive in terms of it innervation. Fusimotor neurones innervating dynamic nuclear bag fibres rarely innervate any other interfusal fiber hence have a predominantly dynamic effect on the sensitivity of the primary ending (Boyd and Gladden, 1985).
1.4.2 Function of muscle spindles
Proprioception is the awareness of limb position and movement, which is a prerequisite for muscle contraction, body balance, coordinated movement, and body orientation (Sherrington 1900). Muscle spindles are a kinesthetic sensor and major contributor enabling the sense of position and movement of our limbs and trunk
(Proske & Gandevia, 2012). This is achieved by providing information about length changes in muscles and derives a positional cue from the effort required to hold a limb against the force of gravity (Proske 2005). The proximity of skin receptors adjacent to each joint allows them to provide joint-specific information (Collins et al.
2005). Initially it had been thought that joint receptors were all-important in proprioception but their contribution of limb position at most joints is likely to be minor (Proske & Gandevia, 2009).
Goodwin and colleagues (1972) carried out an interesting study implicating the muscle spindle apparatus in position sense. They showed that during vibration of muscles surrounding the elbow subjects experienced illusions of forearm movement and displacement. When the vibration stimulus was moved from the muscle belly to the elbow joint itself, illusions disappeared. This supports the view of a significant
27 Chapter 1 – Literature review
contribution from muscle spindles.
1.4.3 Sympathetic innervations of human muscle spindles
The suggestion of sympathetic branches innervating muscle spindles has given rise to the idea that strong excitatory “flight-fight” responses alter discharge activity of spindle afferents. Histological studies have shown that muscle spindles do receive sympathetic innervation in the cat (Barker & Saito 1981). It has also been shown in cats that electrical stimulation of the cervical sympathetic trunk affects stretch sensitivity and background mean discharge rate of Ia muscle spindle afferents innervating the trapezius and splenius muscles (Hellström et al. 2005). Investigations in human subjects, however, have thus far been unable to establish sympathetic innervations of muscle spindles. A physiological and sustained increase in muscle sympathetic nerve activity failed to influence the firing of spontaneously active muscle spindles in human subjects (Macefield et al. 2003). This observation suggests that human muscle spindles may not receive sympathetic innervations.
1.4.4 Pain and proprioception
Studies investigating movement and posture in human subjects suffering from musculoskeletal pain syndromes have reported alterations in the control and awareness of movement and position (Sainburg et al. 1993). Similarly in healthy subjects experimental muscle pain induced in muscles of mastication showed changes in positioning and movement of the jaw during mastication, suggesting that proprioceptive control is altered in the presence of pain (Stohler et al. 1996; Svensson et al. 1997).
28 Chapter 1 – Literature review
Ankle joint proprioception in healthy human subjects, examined in the presence of experimentally induced muscle pain, contrasted previous findings that pain alters proprioception. The investigators reported that, despite a large spread of muscle pain, the subjects’ ability to recognise a reference position and detect movement was not disturbed as a result of pain (Matre et al. 2002). However, in the same study, a moderate intensity of pain in two muscles disturbed the ability to detect movement changes. This is believed to be the result of the large number of muscle tendons crossing the ankle joint providing a rich source of muscle spindle afferent information from several muscles, which is unlikely to be dampened by small areas of pain
(Bergenheim et al. 2000). The central nervous system still receives an abundance of information from agonist and antagonist muscles. Proprioceptive acuity is only disturbed when the distribution of pain is large enough to affect a large number of muscle spindles and their afferent signals.
Chronic low back pain sufferers were investigated to assess whether pain modulates motor performance, particularly whilst attempting to carry out standardised activities of daily living such as gait or mastication. In this study the activity of lumbar paraspinal muscles were monitored through the use of electromyography (EMG) during gait analysis. Chronic low back pain sufferers were tested against control subjects with experimental muscle pain induced with a bolus injection of hypertonic saline. During the swing phase of the gait cycle, a phase in which lumbar muscles are normally inactive, lumbar paraspinal EMG activity of back pain sufferers was significantly higher compared to control subjects. During the double stance phase of the gait cycle, in which lumbar spinal muscle are active, similar EMG activity was observed from both groups (Arendt-Nielsen et al. 1996). Although the study
29 Chapter 1 – Literature review
concluded that the motor control involved in gait is altered significantly in chronic low back pain sufferers it lacks a bridge between methods of investigation and the conclusions being made. It is rather misleading to suggest that elevated EMG activity in paraspinal muscles during walking is suggestive of proprioceptive changes and altered muscular control. It is unclear and ambiguous that the overactivity is a result of nociceptive activity on muscle spindle firing.
1.4.5 Pain and muscle stretch reflexes
Muscle stretch reflexes are the simplest manifestation of muscle spindle function, where a rapid stretch of muscle spindles reflexly excites spinal motoneurones and evokes a myotatic stretch reflex. Muscle stretch reflexes are regularly tested in clinical settings to ascertain integrity of the neuronal circuitry of skeletal muscles and test the excitability of the spinal neurones. Striking the patellar tendon with a tendon reflex hammer, for example, evokes a relatively synchronous volley in the fastest conducting afferent fibres from the muscle spindles (Burke et al. 1983). The afferent volley evokes excitatory postsynaptic potential in a variety of spinal neurones, predominantly in the interneurons at the relevant segmental level, but also in the motor neurones (Dick 2003). Type Ia afferents enter the dorsal root of the spinal cord, projecting towards the brain and sending collateral axons to the anterior horn of the gray matter to synapse onto anterior motor neurons. The type Ia afferent fibres synapse directly onto proximal dendrites and soma of the motor neurone making it extremely likely that the tendon reflex is monosynaptic (Rothwell 1994). Formally the stimulus causes a phasic stretch of the muscle indicating the integrity of the neuronal circuitry.
30 Chapter 1 – Literature review
Experimental muscle pain in humans has been observed to enhance the stretch reflex of the muscle infused with hypertonic saline (Matre et al. 1998). Investigators infused hypertonic saline into the soleus muscle (SOL) and tibialis anterior (TA) muscle in healthy male subjects and tested the stretch reflex and H-reflex. The H reflex, which is the electrical equivalent of the tendon jerk, bypasses fusimotor drive and the potentially variable intrafusal muscle tension of the muscle spindle (Matthews 1970).
EMG and torque of the reflex elicited were recorded and analysed. Interestingly, the torque response, and the short and medium latency of EMG response in both soleus and TA increased during experimental muscle pain were greater when compared to baseline. Enhanced stretch reflexes in the SOL as a result of pain in the antagonist muscle is indicative of the pain-adaptation model (Lund et al. 1991) which predicts that increased antagonist EMG when pain is present in the agonist muscle. The increases in muscle stretch reflexes of the agonist muscle during pain suggests a possible change in spinal excitability and muscle spindle sensitivity (Matre et al.
1998).
Considering they had observed enhanced muscle stretch reflexes in painful agonist and antagonist muscle, in a separate study Matre and colleagues further investigated the effects of experimental muscle pain on the stretch reflex while at rest and during the performance of motor tasks (Matre et al. 1999). Unsurprisingly, similar results were observed for muscle stretch reflexes during pain, confirming their previous observations. However, experimental muscle pain whilst performing a motor task did not show any change in the amplitude of the muscle stretch reflex. It was hypothesised that the amplitude of the muscle stretch reflex elicited in the presence of muscle pain whilst performing a motor task, such as sitting and walking, would
31 Chapter 1 – Literature review
demonstrate a greater increase than at rest due to the higher “functional” background
EMG levels (Matre et al. 1999). These results confirmed earlier reports by Sinkjaer and colleagues (1996) that the amplitude of a muscle stretch reflex from the soleus muscle remained unchanged during motor active tasks (Sinkjaer et al. 1996). Matre and collegues concluded that as the motor tasks evolved to become more consciously involved and more actively controlled, the effect of pain on the stretch reflex became smaller, to the extent where there was no effect in stance phase when the soleus – agonist – muscle is most active (Matre et al. 1999).
1.4.6 Muscle tone and muscle spindle activity: the vicious cycle theory
As we have explored above, changes in muscle spindle sensitivity has the potential to affect sensorimotor control of skeletal muscle. It has also been implicated as the reason for perpetuation of muscle pain establishing a vicious cycle of muscle pain and stiffness developing in to a chronic problem. The popular ‘vicious cycle’ theory, yet to be proven in humans, was developed by Johansson and Sojka (1991) based on experiments in anaesthetized animals. They suggest that stimulation of group III and
IV muscle afferents by metabolic by-products of muscle contraction reflexly increases gamma-motoneuron drive to intrafusal fibers. Increased firing in the primary muscle spindle afferents raises the probability of action potential firing of alpha-motoneuron pool of the particular muscle, increasing muscle tone and facilitating the release of more muscle metabolites thereby kick starting a vicious cycle.
It is known that thinly myelinated and unmyelinated nociceptive afferents in muscle
(group III and IV afferents) respond to the accumulation of metabolites produced by muscular contraction (Kaufman & Rybicki 1987; Rotto & Kaufman 1988; Andreani et al. 1997). These metabolites include potassium in concentrations that are equivalent
32 Chapter 1 – Literature review
to those found to be produced by static muscle contractions (Hnik et al. 1976; Hirche et al. 1980; Vyskocil et al. 1983; Rybicki et al. 1984; Rybicki et al. 1985; Kaufman &
Rybicki 1987). Lactic acid and arachidonic acid are also known to increase in concentration during muscle contraction and, like potassium, have been shown to enhance the discharge of group III and IV muscle afferents (Rotto & Kaufman 1988).
In addition to normal contraction (Andreani et al. 1997), static (Kaufman et al. 1983;
Kaufman et al. 1984) and ischemic contractions had been shown to activate group III and IV muscle afferents before Johansson and Sojka formalised their theory. Such nociceptive reflexes have been suggested to lead to chronic musculoskeletal pain syndromes characterised by increased muscle tone (stiffness or resistance to stretch) and an increased discharge of spindle Ia and II afferents (Johansson & Sojka, 1991).
1.4.7 Increase in fusimotor drive as a result of pain
The basis of the vicious cycle theory is the increase in fusimotor drive to intrafusal fibres of muscle spindles as a result of noxious stimulation of group III and IV nociceptors. Studies aiming to establish this link have been somewhat contradictory, in particular, between animal and human studies.
1.4.7.1 Animal studies
The larger majority of experimental data supporting the Johansson/Sojka hypothesis comes from observations in decerebrate and/or anaesthetised cats (Johansson 1988;
Mense & Skeppar 1991; Capra & Ro 2000; Thunberg et al. 2002b). Matthews and colleagues have investigated descending control and neuronal pathways to fusimotor
33 Chapter 1 – Literature review
neurones extensively but very few have studied the reflex control of fusimotor neurones (Matthews 1972). Appleberg and colleagues (Appelberg et al. 1983a, b) carried out some of the earliest work aiming to investigate the reflex link between group III afferent fibres and gamma-motoneurones (Appelberg et al. 1983b).
Microelectrode recordings of gamma-motoneurones projecting to the hind-limb muscles of anaesthetised cats whilst electrically stimulating group III muscle afferent fibres resulted predominantly in excitatory effects, though some cells exhibited inhibitory effects. As a result, they suggested that static gamma-motoneurone cells stand out as the predominant recipients of group III action, establishing the reflex neuronal link between afferent fibres and gamma-motor drive.
Mense and Skeppar (1991) were interested in testing whether muscle tone was increased and maintained by activating the fusimotor system. Increased resting muscle tone is effectively a sustained involuntary contraction and is likely to cause pain by either activating muscle nociceptors directly or by impairing the blood supply of the affected muscle through ischemia (Mense & Skeppar 1991). In a study using anaesthetised adult cats (Mense & Skeppar 1991) the nerves innervating the hind limb muscles were exposed. Impulse activity was recorded from nerves innervating gastrocnemius, soleus, and tibialis anterior muscles along with the sciatic nerve.
Myosistis was induced using carrageenan, an inflammation-promoting agent in muscles of the hind limb and the resultant changes in impulse activity was observed.
Carrageenan has been validated as a noxious agent shown to elicit increased afferent activity of group III and IV muscle afferents (Berberich et al. 1988). Results of the study showed that gamma-motoneuron activity to muscles of the hind limb were inhibited when the receptor bearing muscle was inflamed. Interestingly they also
34 Chapter 1 – Literature review
reported that gamma-motorneuron activity to the receptor bearing muscle – medial gastrocnemius – when inflammation was induced in the synergist muscle – lateral gastrocnemius (Mense & Skeppar 1991).
Along similar lines of inducing pain through an inflammatory substance,
Djupsjobacka and colleagues used arachidonic acid (Djupsjobacka et al. 1994 ), and concentrations of bradykinin and 5-HT in two respective studies to investigate the potential of these substances to modulate primary and secondary muscle spindle afferents in the injected and surrounding muscles. Their approach was to use substances native to muscle tissue released by static contractions in the working muscle and during injury as part of the inflammatory “soup” (ie. KCI, lactic acid, arachidonic acid, histamine, and 5-HT). These have been demonstrated to activate chemosensitive group III and group IV muscle afferents (Rybicki et al. 1985; Rotto &
Kaufman 1988). Extensor muscles of anaesthetised cats were injected with the algesic substance and subjected to sinusoidal stretching whilst recording from muscle spindle afferents. The responsiveness of muscle spindle afferents to algesic substances was significant, with 83-89% of recorded afferents increasing their sensitivity to sinusoidal stretch. However, the effects were short lived, with an average of 1-4 mins and some elevations lasting longer. Raising the concentration of arachidonic acid or bradykinin and 5-HT gave rise to fusimotor reflexes, which potently influence both primary and secondary muscle spindle afferents from homonymous as well as heteronymous muscles. A separate study using bradykinin injected into the neck muscles of anesthetised cats observed similar results to the previous study using muscle of the hind-limb. The effects of bradykinin on neck muscle spindle afferents lasted on average between 3.5-4mins with the longest effect lasting 15mins (Pedersen
35 Chapter 1 – Literature review
et al. 1997). Interestingly, as with the previous study, there was a dominance of activation of static fusimotor neurones induced by bradykinin. This work presents the reflex increase in gamma-motor drive through group III and IV afferent stimulation with bradykinin.
Noxious agents stimulating group III afferents has been utilised to extend this work in order to test the vicious cycle theory. Muscles that are particularly rich in muscle spindles, such as the jaw and small muscles of the neck, are mostly used in experimental models (Matthews 1972). Small diameter fibres of the trigeminal mesencephalic nucleus innervate intrafusal fibres of jaw muscle spindles (Capra et al.
2007). Extracellular recordings from the trigeminal mesencephalic nucleus in anaesthetised rats during a jaw-opening ramp and hold activity and nociceptive stimulation, with hypertonic saline preferentially enhancing the amplitude sensitivity of jaw muscle spindle afferents. Some rats showed reduced spontaneous discharge and a few had little or no change. This successful demonstration of an algesic chemical substance producing enhanced responses in fusimotor neurons and spindle afferents predicts that the accumulation of metabolites invariably leads to increased spindle sensitivity as per the vicious cycle theory (Capra et al. 2007). However, the pathophysiological model by Johansson and Sojka predicts increased muscle tone at rest in addition to the increased spindle sensitivity, but these findings suggest increased muscle activity during a voluntary motor activity not at rest.
Acidic saline injected into the massetter muscle in rats was used to produce a constant myalgia in order to mimic pain associated with tissue acidosis and Delayed Onset
Muscle Soreness (DOMS) pain in humans (Lund et al. 2010). The study observed a
36 Chapter 1 – Literature review
long-lasting allodynia-like response developing bilaterally in the massestter muscles, both with unilateral and bilateral injection, causing increased immediate early gene c-
Fos expression in the cell bodies of the related primary muscle spindle mechanoreceptor afferents. Considering c-Fos is a well-established marker of neuronal activity (Hunt et al. 1987), its expression is a sure indicator of nociceptive input, in this case muscle nociception. Changes in electrical properties of the neurones resulted in increased excitability and a caused small but significant fraction to fire spontaneously in vitro (Lund et al. 2010).
Capra and colleagues (Capra et al. 2007) carried out significant work on the effects of pain on muscle spindle afferents. Choosing to mimic clinical muscle pain, they used hypertonic saline to induce experimental pain in the massetter muscle of anaesthetised cats whilst recording from neurons of caudal brain-stem neurons. Afferent activity and proprioception was assessed in two separate studies to observe changes in discharge rates of muscle spindle afferents, recorded from the anaesthetized cat.
Results showed that during various phases of ramp-and-hold jaw movements a dichotomy of responses were observed in the presence of pain: half showing an increase in discharge activity and the other half showing a decrease. Investigators concluded that experimental muscle pain could inhibit or facilitate discharge activity of muscle spindle afferents and explains the duality of responses reported in the literature.
Another study by Capra and Ro aimed to understand the anatomical organisation and functional properties of brain stem neurons that receive afferent input from jaw muscles (Capra & Ro 2000). Results unequivocally show that intramuscular injections
37 Chapter 1 – Literature review
of algesic substances produce significant changes in the proprioceptive properties of these neurones in the cat. Large proportions of dynamic-static units (64%) and static units (89%) were activated in response to passive jaw movements following hypertonic saline injections into the ipsilateral, and in most cases, the contra-lateral massetter muscle (Capra & Ro 2000). A reduction in average firing rate during imposed movements was most commonly observed. This suggests that the mechanism of action is in fact a central mechanism involving small diameter input onto central neurons that have bilateral connections similar to those seen in neck and limb muscles. In light of the vicious cycle theory this observation supports the idea that gamma-motoneurones are modulated to change the stretch sensitivity of muscle spindles. It is not a direct mechanism of pain affecting the muscle spindles rather it involves stimulation of small diameter fibres. As seen in previous studies the effects of hypertonic saline was predominantly seen on static neurons. Given that the dynamic-static neurons exhibited a change in static response without concomitant changes in dynamic response during movement supports the notion that dynamic and static fusimotor activity is independently controlled (Appelberg et al., 1983).
Experimental evidence supporting the functional role of the fusimotor system and its reciprocal relationship between muscle tone and chronic muscle pain is lacking.
Muscle pain may increase the dynamic sensitivity of muscle spindles, variably seen in animal studies, but it doesn’t necessarily increase the excitability of the alpha motoneurones in the relaxed muscle. This is in part due to the mechanism by which muscle contraction is driven. Contraction of the poles of the spindles reflexly or voluntarily induced via the gamma motor efferents causes an accelerated discharge from the primary spindle endings (Marsden et al. 1976a). This activity in turn causes the extrafusal muscle fibres to contract through a spinal stretch reflex arc. Marsden
38 Chapter 1 – Literature review
and colleagues (Marsden et al. 1976b) hypothesise that muscle stretch reflexes potentially employ a cortical arc rather than a spinal reflex arc resulting in a greater latency of the muscle stretch reflex. Previous studies have demonstrated that reflexly- mediated muscle spindle drive is not enough to cause substantial increase in motoneuron excitability during voluntary isometric contractions (Vallbo 1970, 1971).
Indeed, this suggests that increased fusimotor activity is not directly related to increased muscle tension.
The most significant study to support the vicious cycle was carried out by Thunberg and colleagues (2002). The investigators used anaesthetised cats whilst injecting hypertonic saline intramuscularly. Muscle spindle afferents were recorded from gastrocnemius medialis and/or gastrocnemius lateralis whilst hypertonic saline (5%) was injected into either the receptor-bearing muscle (homonymous muscle) or into a close synergistic muscle such as posterior biceps (heteronymous muscle). Muscle spindle afferent responses to sinusoidal stretch were recorded, 90% of which exhibited statistically significant responses to hypertonic saline into homonymous and/or heteronymous muscles. The average maximal increase in mean discharge rate was 74%, demonstrating a positive correlation between noxious input and fusimotor drive. The majority of the responses (72%) were compatible with reflex action on static fusimotor neurones, whereas 20% of the responses could be attributed to mixed static and dynamic fusimotor action (Thunberg et al. 2002b). That static fusimotor neurones were actvated is in agreement with previous studies highlighting the different responses between static and dynamic-static neurons. No statistically significant difference in response was observed between injections into heteronymous and homonymous muscles. A control injection of Tyrode – an aqueous solution of
39 Chapter 1 – Literature review
salts and glucose whose ion concentration and osmotic pressure are similar to blood plasma – did not induce any change in muscle spindle afferent responses confirming that hypertonic saline causes changes via fusimotor reflexes.
1.4.7.2 Human studies
In comparison to the depth of experimental studies in animals demonstrating support for the vicious cycle theory, strong evidence is lacking when it comes to human studies. A study that sought direct evidence for the applicability of the vicious cycle theory in humans used microneurographic recordings of spontaneously active muscle spindle afferents during bolus intramuscular injections of hypertonic salines. Short- lasting acute pain was induced using a bolus injection of hypertonic saline into the belly of tibialis anterior and the overlying skin whilst the discharge rate of muscle spindle afferents was observed. Mean discharge rate of afferents demonstrated no increase in firing rate of muscle spindles in relaxed human leg muscles, rather a small decrease was observed (Birznieks et al. 2008). This observation was in stark contrast to the experimental work in anaesthetized cats, where muscle spindle firing rate increased by ∼80% in response to the same noxious stimulus (Thunberg et al. 2002).
Experimentally induced muscle and skin pain in healthy human subjects failed to demonstrate a reflex-driven increase in muscle spindle afferent discharge, nor was there any recruitment of silent muscle spindles. A significant increase in fusimotor drive would invariably recruit surrounding silent muscle spindles (Birznieks et al.
2008). As noted above, a high percentage of animal studies indicate the modulation of muscle spindle afferents are attributed to the action of static neurones, and not mixed
40 Chapter 1 – Literature review
static and dynamic neurones (Capra & Ro 2000; Ro & Capra 2001; Hellström et al.
2005). Relaxed muscles have very little fusimotor activity therefore it is unclear whether the decrease in discharge rate can be attributed to nociceptive modulation of gamma-drive (Gandevia et al. 1986). Furthermore, the decrease in discharge rate failed to align with the pain profiles, suggesting a lack of correlation between nociception and gamma-drive (Birznieks et al. 2008). It is plausible that nociceptor– fusimotor reflexes require a longer time to be activated in humans and thus cannot be achieved with acute pain – given that the pain profile following a bolus intramuscular injection of 0.5 ml hypertonic saline reaches a peak within 2 min and has all but disappeared after 8 min. The aim of the current project is to induce longer-lasting pain and observe whether this will activate nociceptor-fusimotor reflexes in awake human subjects. The working hypothesis for the current project is that tonic muscle pain will cause an overall increase in static and dynamic stretch sensitivity of human muscle spindles via increases in fusimotor drive.
1.4.8 Fusimotor drive and the activation threshold of the alpha motoneurone pool
Muscle pain and myalgia are often associated with taut muscle bands – fibres that are rigid and painful on palpation. Within these taut bands are areas that are most painful and sensitive referring pain to a distant region spontaneously or upon compression known as trigger points (Partanen et al. 2010). Facilitation of these segments is brought about by release of acetylcholine, at the nerve-ending terminals of an abnormal motor end plate under resting conditions (Yap 2007; Jiang et al. 2011).
Surface EMG is a common non-invasive tool used to measure muscle contracture and has shown increased electrical activity in affected muscles with trigger points
41 Chapter 1 – Literature review
(Couppe C et al. 2001). Hagberge and Kvarnström found that the characteristics of surface EMG signals from myofascial pain patients were identical to those from fatigued muscle, in terms of reduced mean power frequency (Hagberg & Kvarnstrom
1984). The vicious cycle theory explains sustained muscle contraction as an increase in fusimotor drive, raising the background firing of muscle spindle afferents and resulting in active contraction of extrafusal muscle - expressed as taut bands.
However, physiologically, muscle tone (ie. taut bands) depends on two factors: viscoelastic properties of the soft tissues and/or the degree of activation of the contractile apparatus (Simons & Mense 1998). EMG sufficiently identifies an electrogenic contraction generated by neuromuscular activation, but does not detect endogenous contracture of the contractile apparatus of skeletal muscle (Simons &
Mense 1998). In healthy human subjects, that aren’t completely relaxed, the alpha- motor neurones and the innervated muscle fibres dictate muscle tone and stiffness, observable as EMG activity. Failure to observe any activation of surface EMG by
Birznieks and colleagues in their study (Birznieks et al. 2008) may be a significant finding, as it could suggest a lack of alpha-motorneurones activating in response to pain. There is a possibility that recruitment of gamma-motor neurones might require plastic changes in nociceptive circuits that might develop over a longer time, hence, exciting alpha-motorneurones. The current project aims to observe these changes in a tonic pain state.
1.5 Microneurography
A refinement in the assessment of sympathetic and muscle spindle activity has enabled a better understanding of their function. Contemporary methodological approaches allow direct assessment of systemic and regional sympathetic
42 Chapter 1 – Literature review
cardiovascular drive in humans. Microneurography is an electrophysiological investigative tool that permits the study of the somatosensory, motor, and cardiovascular systems through direct recording of nerve traffic. The technique was developed in Sweden in the late 1960s by Hagbarth and Vallbo (Vallbo & Hagbarth
1968) , and has since been applied in both human (Sundlof & Wallin 1977; Wallin et al. 1992; Macefield 2005) and animal studies (Horeyseck & Jänig 1974b; Thunberg et al. 2002b). In human studies, the technique utilises two tungsten microelectrodes percutaneously inserted into an accessible peripheral nerve (Macefield et al. 1999). A recording electrode inserted into the nerve records action potentials directly from myelinated and unmyelinated axons within the nerve fascicles (see fig 1.5). The recording electrode registers the voltage difference between itself and the reference electrode inserted subcutaneously. The use of this technique in awake human subjects has enabled nerve discharge data to be matched with the sensory experience particularly with pain studies (Birznieks et al. 2008; Burton et al. 2009a; Burton et al.
2009b). Furthermore, it is the only technique that provides direct insight into the changes in neural discharge in experimental physiology and pathophysiology.
Recording from muscle spindles can be used to analyze mechanisms of fusimotor control of spindles in movement and muscle tone, for example. Recordings of sympathetic efferent activity are essential to understand the neural mechanisms of autonomic functions (Mano et al. 2006).
43 !"#$%&'()(()%'%(#(%&'#%'&(&& Chapter 1 – Literature review
Figure 1.5 Depiction of the technique of microneurography. A crossection of the common peroneal nerve
shows the bundles on skin and muscle fascicles with a tungsten microelectrode impaling a muscle fascicle, in this !C("#($&$%7(%"&(%&"I&(9(A'&'C'#$"@6()(%"7(&P#A$4&(#('&%('&'C(9(A74&(7@A$#%"&%( &'&(#%%@(Gillustration. The$% !&H(#(#(%C7%&(A'&4&%'&(7&'%&(%(#(A74&(9#74&(9(%"&(AA($&'(top right hand panel shows the bursts of MSNA in the raw neurogram with a root mean square
&'&((#(#calculated integrated#&&("A#(78 nerve signal'&%(7(A#&6(""&(%$('C"%(7" directly below. EGC signal is 7(%"&(8'7%7(9(#%%@((%"&('#illustrated showing the time-locked (&'C'#A(relationship ""(#'&(A$'7&(9(A4%$4&(#%($%&%#473(8&4 (%"7(7(%"&(%&C'#%&(&'&( ""(&$%7(%"&( between ECG and the integrated nerve signal due to the baroreceptor reflex mechanism. Blood pressure and A4#%&(#%%@(&'&(9'A(%"&('# (&'C'#A((8&4 (%"7(7(%"&('!)D(%&(%"&(%A&('&4#%7"$( respiration are illustrated in the last two panels. Adapted from Macefield et al., (1999) 8&% &&(%"&('!)(#(%&C'#%&(&'&(&(%(%"&(8#''&&$%'('&94&P6(""&(8%%A(% (%'#&7(&$%(84( $'&77'&(#('&7$'#%6(&#$%&(9'A( $#&9&4(&%(#463(G0111H
1.6 Experimental muscle pain )($#'%4#'3(%"&(7&(9(A'&'C'#$"@((%"&(7%@(9(%"&(7@A$#%"&%(&'7(7@7%&A( "#7($'&(%(8&(&'@(9'%946(""&(%&"I&("#7(A#&($7784&(%"&(##4@77(9(#(#'&%@(9(Experimental pain models offer the ability to explore and understand pain &'#4('&C4#%'@(A&"#7A7(%"#%('A#44@(%mechanisms under controlled settings (Mano'4(%"&(%94 et al. 2006) (9(7@A$#%"&%(A$47&7(%(. Every aspect can be 99&'&%(%@$&7(9(#%A(&99&%'('C#7(("A#7(!'(&P#A$4&3(%"&(%&"I&("#7( carefully controlled including the nature, localisation, intensity, frequency, and "C"4C"%&(%"&(99&'&&7(8&% &&(7@A$#%"&%(%94 (%(A74&7(&'77(7&(G*#C8#'%"3( "++"durationH6 of the stimulus all the while providing quantitative measures of the
psychological, behavioural, and neurophysiological responses (Arendt-Nielsen et al. D(1(D 2007). It also bridges the gap between animal studies and clinical observations,
permitting the testing of mechanisms demonstrated in animals to be evaluated in
healthy humans before entering a clinical setting.
44 Chapter 1 – Literature review
1.6.1 Hypertonic saline
In experimental pain, the use of hypertonic saline as a noxious agent has gained acceptance for many reasons. Its first use in a study was experimental muscle pain in humans documented in 1938 when Kellgren injected his patients with hypertonic saline in order to understand referred pain (Kellgren 1938). Most importantly, the sensation of pain experienced by the subject in experimental muscle pain closely resembles that which is described by clinical patients. In a clinical setting patients describe chronic muscle pain as a deep dull ache, which is diffuse and difficult to localise or pin point the origin and epicentre of the pain (Arendt-Nielsen et al. 2007).
It also has the ability to refer to distant structures making diagnosis and treatment a challenging task for clinicians. The intensity of the pain varies from strong pain to mild pain but the sensation is constant and at times overpowering affecting activities of daily living. Often it is difficult to find a comfortable position in order to decrease the pain intensity.
Bolus injection or infusion of hypertonic saline intramuscularly manifests a pain most similar to clinical muscle pain, experienced by subjects as a cramp-like, diffuse, aching pain in the muscle accompanied by pain referred to distant somatic structures
(Kellgren 1938). It has also been observed to modify sensibility both superficially and deep, motor control, proprioception, and evoke autonomic reactions (Graven-Nielsen et al. 1997; Thunberg et al. 2005). In clinical studies hypertonic saline induced muscle pain has been used to quantify the cortical changes in subjects with whiplash associated disorders (Koelbaek Johansen et al. 1999), and fibromyalgia (Sorensen et al. 1998).
45 Chapter 1 – Literature review
1.7 Aims
The aim of this research is to observe the physiological changes in the regulation of sympathetic outflow, and muscle spindle afferent activity in response to long-lasting
(tonic) experimental muscle pain in human subjects. An alteration in these systems has been implicated in the development of chronic muscle pain in animals but is yet to be confirmed in human subjects. Through this research we can observe the early onset changes in the regulation of these systems in response to tonic pain and predict the implications of such physiological alterations in the establishment of chronic pain.
Limitations of this research are noted from the beginning. Firstly, this series of investigations does not draw conclusions about the actual development of chronic pain. Rather, it explores and challenges the validity of the current model and framework of understanding adapted from animal experimentation by investigating it in awake human subjects. Thereby, we can confirm or refute the current understanding of the physiological effects of pain, with respect to its applicability to awake humans. Secondly, all of the experiments to be described in this thesis were undertaken in healthy human subjects, not on subjects suffering from chronic pain.
This may, of course, confound the generalisation of our findings to a population of chronic pain sufferers. However, utilising subjects with chronic pain in a study of this nature would be misleading due to the complex changes in physiology and psychology chronic pain sufferers already have undergone. Studying healthy subjects provides a standardised baseline from which observations can be contextualised and understood. Thirdly, it is difficult to obtain resting physiological data in patients with chronic pain, given that – by definition – chronic pain would have been established
46 Chapter 1 – Literature review
for three months or more and we would need to have obtained baseline data prior to the event that precipitated chronic pain.
47 Chapter 2 – Methods
CHAPTER TWO
GENERAL METHODS
A range of tools and experimental procedures were common among the individual experiments in the research work undertaken. This chapter describes these procedures and tools, elaborating on their use in data acquisition. Reliability and validity have been discussed in previous chapters and are also further discussed within each results chapter.
2.1 Subjects
Subjects included in the studies were aged between 18-65 years old. Current or previous medical history of chronic musculoskeletal pain was a significant exclusion criterion as it may have proven to be confounding and undermining to the results of the study. The essence of the research work undertaken is to observe early physiological changes occurring in the sympathetic and somatic motor system, if a person has already suffered chronic pain or is currently suffering chronic pain this would be illogical as it goes against the whole premise of the study. To add to this rationale, considering that the sensation of pain involves both physiological and psychological mechanisms, chronic pain could be an expression of the alteration in these systems. Hence, using subjects that already suffer from chronic pain for these studies may confound our results. This is undetermined, however, one can expect that an alteration has taken place in both systems. Subjects were asked to disclose any
48 Chapter 2 – Methods
known issues about their cardiovascular health, and were excluded from the study if their blood pressure demonstrated irregular fluctuations at rest.
Female subjects were additionally asked about their menstrual cycle and the phase they were in at the time of the experiment. Experimental evidence suggests heightened sensitivity to pain and autonomic arousal in the premenstrual phase
(Kuczmierczyk et al. 1986).
Ethics approval was received from the University of Western Sydney Human
Research Ethics Committee for the design and methodology of the study. The research also conformed to the guidelines set out in the Declaration of Helsinki.
Subjects gave written informed consent and were given the option to withdraw from the study without repercussion or consequence. During the experiment subjects were in a comfortable, semi-reclined position, in a room with a neutral ambient temperature. Particular care was taken to ensure that the environment was quiet and calm minimizing the amount of spontaneous arousal reaction due to external noise interfering with the experimental procedure.
2.2 Noxious stimulation
Experimental muscular pain was induced in healthy human subjects by infusing a noxious stimulant in to tibialis anterior muscle. To obtain a sustained long-lasting effect of muscular pain a sterile solution of hypertonic saline was prepared for intramuscular infusion. Hypertonic saline (20%) was diluted with water for injections to form a solution of hypertonic saline (7%). The solution was drawn up into two
10mL syringes and connected to a three-way tap using 25cm extension tubing. A 23-
49 Chapter 2 – Methods
gauge butterfly needle attached to a cannula is also connected via 25cm extension tubing to the three-way tap. The entire apparatus was primed with the solution of hypertonic saline. Locating the muscle belly of the ipsilateral tibialis anterior was done with palpation and a spot was identified for insertion, generally 1.5 cm deep, and
7-9cm inferolateral to the tibial tuberosity. An infusion pump (Harvard Instruments,
USA) beginning with a rate of 250 µL per minute was used to continuously infuse the noxious solution. The rate of infusion was adjusted spontaneously according to subjects’ feedback of the pain rating in order to maintain a constant level of moderate pain. Feedback on pain was instantaneously updated using a potentiometer and a visual analogue scale (VAS) where “0” was no pain and “10” was considered worst pain imaginable. The pain experience lasted ~60mins.
2.3 Recordings
2.3.1 Microneurography
Neural recordings were acquired from muscle and skin fascicles of the common peroneal nerve. The common peroneal nerve was identified using anatomical landmarks and palpation on the skin surface. Electrical stimulation using a surface probe between ranges of 1 to 10mA were applied to the skin to identify the course of the nerve. A location resulting in the strongest twitch at a low current was chosen for insertion of the recording electrode. Two Tungsten microelectrodes were inserted percutaneously, one just under the skin serving as a reference electrode; while the other into the common peroneal nerve serving as a recording electrode. The recording electrode was further guided into a muscle or skin fascicle using internal electrical stimulation until a twitch was elicited at a current as low as 0.2mA. Further manipulation of the recording electrode was done until spontaneous sympathetic
50 Chapter 2 – Methods
activity or spindle activity was audible through sound and visually seen on the data acquisition program. Specific detail of how afferent activity identified and confirmed is outlined in respective results chapters.
2.3.2 Cardiorespiratory
Heart rate (HR) was recorded by using standard Ag-AgCl ECG chest electrodes. HR
Variability was calculated from the R-R interval. For skin sympathetic activity sweat release was monitored and inferred from a change in electrical conductance of the glabrous skin on the second digit of the ipsilateral foot (GSR Amplifier,
ADInstruments, Sydney, Australia). Respiration was recorded with a strain gauge transducer attached to a strap fastened around the subjects’ chest (Pneumotrace,
Morro Bay, CA, USA).
2.3.3 Radial tonometry
Continuous blood pressure (BP) was monitored and recoded non-invasively via the use of radial tonometry (CBM-7000, Colin Corp, Japan). A pulse-sensing transducer is placed directly over the radial artery and deforms the artery. Blood flowing through the artery resonate an impulse detected by the transducer and converted into an electrical signal providing continuous pulse recording. For the purpose of calibrating the tonometric sensor, a blood pressure cuff is strapped on the contralateral brachial artery. Utilization of the transducer on large superficial arteries has been shown to be as reliable as sphygmomanometric and intra-arterial blood pressure measurements
(Drzewiecki et al. 1983; Sato et al. 1993).
51 Chapter 2 – Methods
2.3.4 Photoplethysmography
The photoplethysmogram (PPG) waveform estimates arterial oxygen saturation based on the absorption of red/infrared light by circulating blood in the underlying tissues.
Given that infrared light can penetrate deep into the skin and subcutaneous tissue and is relatively sensitive to blood oxygenation (Lindberg & Oberg 1991; Hales et al.
1993; Allen 2007), the resultant PPG waveform can reflect intravascular blood volume change at a peripheral site (typically the fingertip, ear or forehead), under the combined influences of central perfusion pressure and local vascular control mechanism. PPG were measured from the tip of the second digit of the ipsilateral leg by reflection mode infrared probe utilizing light at 940 nm (MLT1020PPG,
ADInstruments, Sydney, Australia).
52 Chapter 3 – Tonic muscle pain and spindles
CHAPTER THREE
TONIC MUSCLE PAIN AND MUSCLE SPINDLE
AFFERENT DISCHARGE RATE
Chapter abstract
Experimental pain induced in animals has shown that noxious stimulation of group III and IV afferents increases the firing of muscle spindles via a reflex excitation of fusimotor (γ) motoneurones. Chronic muscle pain has been hypothesized to develop as a result of a vicious cycle involving this mechanism. In order to explore the effects of long-lasting muscle pain on the fusimotor system, single unit muscle spindle afferents were recorded from 15 subjects. Afferent activity was recorded from foot and ankle extensor muscles whilst infusing hypertonic saline into the tibialis anterior muscle of the ipsilateral leg, producing moderate–strong pain lasting for ~60 min. A change in fusimotor drive was inferred by observing changes in the mean discharge rate of spontaneously active muscle spindle afferents. Homonymous and heteronymous muscles remained relaxed and showed no increase in activity, arguing against any fusimotor-driven increase in motor activity, and there was no net change in the firing of muscle spindle afferents. We conclude that long-lasting stimulation of group III and IV afferents fails to excite fusimotor neurones and increase muscle spindle discharge. Accordingly, the vicious cycle theory has no functional basis for the development of myalgia in human subjects.
53 Chapter 3 – Tonic muscle pain and spindles
3.1 Introduction
In addition to being caused by overt muscle damage, muscle pain (myalgia) more often develops from the demand for recurrent isometric muscular contractions involved in repetitive activity. Characteristically, there is increased ‘muscle tone’, mild to severe pain, tender areas of hypersensitivity to mechanical stimulation in the muscle and spread of this hypersensitivity to surrounding muscles, each of which can affect the performance of synergist muscles (Johansson & Sojka 1991; Hodges et al.
2008). However, the mechanisms by which acute muscle pain becomes pathological are yet to be understood (Arendt-Nielsen et al. 2011). One idea relies on changes in muscle spindles, which are highly sensitive stretch receptors located in skeletal muscles throughout the body. Muscle spindles are unusual sensory organs in that they have their own motor innervation – fusimotor motoneurones (γ-motoneurones) – which can modulate the sensitivity of muscle spindles to static and dynamic muscle stretch and thereby influence their capacity to encode changes in muscle length and hence the sensory feedback they provide (Matthews 1988).
It is known that thinly myelinated and unmyelinated nociceptive afferents in muscle
(group III and IV afferents) respond to the accumulation of metabolites produced by muscular contraction (Kaufman & Rybicki 1987; Rotto & Kaufman 1988; Andreani et al. 1997; Andreani & Kaufman 1998; Kaufman et al. 2002), and that these afferents reflexly excite fusimotor neurones and thereby change the sensitivity of muscle spindles to stretch (Appelberg B 1983b; Johansson et al. 1993; Djupsjobacka et al.
1995a). Such nociceptive reflexes have been suggested to lead to chronic musculoskeletal pain syndromes (Johansson & Sojka 1991). According to this model, the metabolic products of muscle contractions evoke a ‘vicious cycle’ that sustains
54 Chapter 3 – Tonic muscle pain and spindles pain, as follows: (i) activation of nociceptors by muscle metabolites reflexly excites fusimotor neurones and thereby increases the background firing and stretch sensitivity of muscle spindles; (ii) these then reflexly excite α-motoneurones and increase muscle tone, which in turn (iii) leads to accumulation of metabolites, exciting muscle nociceptors and so on and so on (Johansson & Sojka 1991). This model was established from studies in anaesthetized experimental animals, but it is not known whether this applies to awake human subjects. We recently sought direct evidence for the applicability of this model to humans. We showed that bolus intramuscular injections of hypertonic saline, which excite group III and IV muscle afferents, in fact caused a small decrease in the background discharge of muscle spindles in relaxed human leg muscles (Birznieks et al. 2008). By contrast, in the anaesthetized cat, muscle spindle firing rate increased by ∼80% in response to the same noxious stimulus (Thunberg et al. 2002a). We posit that nociceptor–fusimotor reflexes require a longer time to be activated in humans and thus cannot be seen with pain that lasts only a few minutes, particularly given that the pain profile following a bolus intramuscular injection of 0.5 ml hypertonic saline reaches a peak within 2 min and has all but disappeared after 8 min.
In the present study, we used intramuscular infusion, rather than bolus injection, of hypertonic saline into the tibialis anterior muscle to produce strong yet tolerable pain for an hour. Unlike the short-lasting pain we had previously induced by bolus intramuscular injections of hypertonic saline (Birznieks et al.
2008), we believe the physiological responses to tonic muscle pain will more closely align with those one might expect to see in patients with chronic pain.
However, because one cannot obtain baseline data in such patients our aim was
55 Chapter 3 – Tonic muscle pain and spindles to use an experimental model of chronic pain (Capra & Ro 2004), allowing us to assess the potential effects of long-lasting activation of intramuscular nociceptors on the firing rates of muscle spindles. We hypothesized that tonic noxious input would cause an increase in muscle spindle firing rates via an increase in fusimotor drive. Of course, we recognize that chronic pain does not necessarily depend on continuous activity of peripheral nociceptors and can be sustained by plastic changes within the central nervous system, but it is generally acknowledged that chronic pain is established from a protracted episode of nociceptive pain, usually involving damage to deep tissues, such as muscles (Arendt-Nielsen et al. 2011).
3.2 Methods
Successful experiments were performed on 15 healthy subjects (10 men and five women) aged between 18 and 37 years. Prior to participation, subjects gave informed written consent for the experimental procedures. Ethical approval for the study was obtained from the Human Research Ethics Committee at the
University of Western Sydney and conformed to the principles established in the
Declaration of Helsinki. Subjects were seated in a comfortable chair with their legs supported horizontally in front of them; their knees were flexed ∼15 deg from full extension and the feet were planted on a rigid footplate at ∼30 deg plantar flexion.
56 Chapter 3 – Tonic muscle pain and spindles
3.2.1 Muscle spindle recordings
The common peroneal nerve at the fibular head was located by external stimulation
(0.2 ms pulses, 1 Hz, 3– 10 mA; Stimulus Isolator; ADInstruments, Sydney, NSW,
Australia). Intraneural recordings were made from muscle fascicles of the common peroneal nerve via tungsten microelectrodes (FHC, Bowdoinham, ME, USA) inserted percutaneously at the level of the fibular head. Muscle fascicles were identified as supplying the peronei muscles, extensor digitorum longus, extensor hallucis longus or tibialis anterior muscles according to the muscle twitches produced by intraneural stimulation. Neural activity was amplified (gain 10,000, bandpass 0.3–5.0 kHz) using an isolated amplifier (NeuroAmpEX; ADInstruments) and stored on computer (10kHz sampling) using a computer-based data acquisition and analysis system (PowerLab
16SP hardware and LabChart 7 software; ADInstruments). Surface EMG over the pretibial flexors was recorded with Ag–AgCl electrodes located over tibialis anterior on the same side as the spindle recording (10 Hz to 1 kHz bandpass, 2 kHz sampling;
BioAmplifier; ADInstruments).
Intraneural recording confirmed that the micro- electrode tip was located within a muscle fascicle; there were no afferent responses to light stroking of the skin, but afferent activity could be evoked by passive stretch or palpation of the muscle belly, or percussion over the tendon. Muscle spindles were identified as such if they presented a tonic discharge, the mean frequency of which could be increased by stretching the receptor-bearing muscle and decreased by unloading the muscle. These spontaneously active spindles were further subdivided into presumed primary and secondary endings. Primary endings showed a characteristic silent period following release of the stretch (passive shortening) and typically expressed a high dynamic
57 Chapter 3 – Tonic muscle pain and spindles sensitivity to passive stretch. Secondary endings typically decelerated their discharge, without ceasing completely, when the stretched muscle was returned to its resting length (Edin & Vallbo 1990).
3.2.2 Noxious stimulation
Sterile hypertonic saline (20%) was diluted with sterile water for injection to create a solution of 7% hypertonic saline, and drawn up into two 10ml syringes. The syringes were connected in parallel to a three-way stopcock via extension tubing, primed with hypertonic saline, and placed in an infusion pump (Harvard Instruments, Holliston,
MA, USA). A single 23 gauge butterfly cannula attached to the stopcock was inserted approximately 1.5 cm deep into the belly of the tibialis anterior muscle, approximately 4 cm lateral and 8 cm inferior to the tibial tuberosity. Baseline activity was recorded for 5 min prior to commencing the infusion, which began at a time unknown to the subject and lasted for 45 min. The infusion began at a rate of 250µl min−1 and was constantly adjusted by the experimenter to maintain the subjective pain rating at a level of 5–6 out of 10. During the infusion, subjects provided feedback about the intensity of pain they were experiencing using a potentiometer on a visual analog scale, where ‘0’ indicated ‘no pain’ and ‘10’ the ‘worst pain imaginable’. At the end of the experiment, subjects completed a McGill Pain Questionnaire and drew the area(s) of pain on an image of a leg.
3.2.3 Data analysis
A 30 s sample of muscle spindle discharge activity was selected at sequential 5 min intervals. The data were then exported to custom-designed spike recognition software written in IGOR Pro 5 (WaveMetrics Inc., Portland, OR, USA). Single afferent spikes
58 Chapter 3 – Tonic muscle pain and spindles were detected by setting a minimal discharge threshold. Spike superimposition was used to confirm the uniform spike morphology of each spike train, and the mean frequency was calculated. Mean EMG activity was measured from the root mean square- processed (RMS) EMG signal (100 ms moving average), calculated as a mean of the absolute value in 5 min blocks over the course of the experiment and expressed relative to the baseline prior to the infusion.
3.2.3.1 Statistics
Repeated measures, one-way ANOVA with Dunnett’s post hoc test was performed using GraphpPad Prism v5.00 for Mac OS X (GraphPad software, San Diego, CA,
USA). Given that the absolute duration of recording over which a given muscle spindle afferent could be held varied across afferents, repeated measures ANOVA was computed over different epochs with differing numbers of afferents comprising the sample. Changes in mean firing rate of each spontaneously active muscle spindle afferent during the infusion are presented relative to the baseline firing rate. For all statistical tests, a probability level of <0.05 was regarded as significant. All values are expressed as means and SEM.
3.3 Results
Tonic muscle pain was induced by intramuscular infusion of hypertonic saline into the tibialis anterior muscle. The rate of infusion was continually adjusted to maintain a constant, moderately strong, yet tolerable deep level of pain that lasted for ∼60min.
No subject reported being unable to cope with the pain, and none requested termination of the infusion. The mean steady-state pain rating across subjects was
5.1±0.4 out of 10 (a.u). On average, the total volume of hypertonic saline infused into
59 Chapter 3 – Tonic muscle pain and spindles the muscle was 13.6 ± 1.2 ml. Pain was experienced solely in the muscle belly of tibialis anterior in 11 of 15 subjects, and in four cases referred pain was also experienced in the ipsilateral ankle or foot. A McGill Pain Questionnaire administered after the experiment showed that the most common terms used to describe the pain were ‘aching’ (nine of 15), ‘hurting’ (eight of 15) and ‘heavy’ (eight of 15). Thirteen subjects also used the terms ‘continuous’, ‘steady’ and ‘constant.’ Throughout the infusion, EMG recordings confirmed that the muscles remained completely relaxed; there was no evidence of recruitment of motor units during the infusion.
Unitary recordings were obtained from 15 spontaneously active muscle spindle afferents, seven supplying the peronei muscles, five extensor digitorum longus, two extensor hallucis longus and one the tibialis anterior muscle. Nine afferents were identified as type I (primary spindle endings) and six were identified as type II afferents (secondary endings), based on criteria described in the Methods. While our aim was to record the ongoing activity of a single muscle spindle continuously for the entire duration of the infusion, in addition to the 5min pre-infusion baseline period, this proved problematic; some recordings deteriorated within a few minutes of the infusion, resulting in termination of the experiment. Moreover, in a given experiment we could infuse hypertonic saline only once, to avoid potential history-dependent changes. While one spindle afferent recording was lost after 10 min into the infusion, for the remaining 14 spindle afferents stable recordings were held for at least 20 min of the infusion, and 10 were followed for at least 30 min; continuous recordings up to
50 min were obtained from eight of these afferents before the site was lost.
60 Chapter 3 – Tonic muscle pain and spindles Background and Research Plan 8 APP1029782 Figure 4: Changes in spontaneous firing of a muscle spindle secondary ending, located in extensor digitorum longus, during a 45-minute infusion of hypertonic saline into tibialis anterior. Baseline activity immediately prior to the infusion is shown in the left panel; activity 40 minutes following the onset of pain is shown in the right panel. Note the deceleration in firing of the spindle during pain. Figure 3.1 Changes in spontaneous firing of a muscle spindle secondary ending, located in extensor digitorum longus,Relevance: during a 45- minuteIf theseinfusion of experiments hypertonic saline into demonstrate tibialis anterior. Baseline that activity muscle immediately spindle prior stretch sensitivity increases during a period of tonic pain, this will be the first study to reveal the existence of excitatory to the infusion is shown in the left panel; activity 40 minutes following the onset of pain is shown in the right nociceptive inputs onto fusimotor neurones in humans, as predicted by corresponding animal panel.experiments. Note the deceleration Such in firing results of the spindle would during corroborate the infusion. a candidate mechanism (“vicious cycle” hypothesis) that explains how clinical muscle pain is being sustained and even transformed into chronic state in some patients. Our findings would substantiate development of preventive and clinical treatment Experimentalstrategies based records on from this one fundamental subject are shown physiological in Fig 3.1. This mechanism. afferent, located In incontr ast, if a decrease in muscle extensorspindle digitorum activity longus, is seen showed (as seena decrease in Fig. in mean 4), thenfiring rateour ofstudy 3.2 Hz will following provide the strongest experimental support to date for the alternative “pain adaptation” model, which until now has been supported 40only min by of tonic EMG pain; data. this was Finally, the maximal any nociceptive fall observed. Otherreflex afferents-induced showed changes in how muscle spindles slightrespond increases to stretch or no changestimuli in will spontaneous affect proprioception discharge; the maximal and sensorimotor increase, control, which may have implications for sports-related injuries and falls in the elderly. recorded from an afferent located in tibialis anterior, was 6.5 Hz. It is possible that STUDY III: To examine the effects of tonic muscle pain on fusimotor control during thisvoluntary increase was contractions related, in part or in whole, to the increase in volume of the muscle andOne the well associated-known radial difference and/or longitudinal between stretch human of muscle and in animal the vi cinity muscle of the spindles is that firing rates are generally much lower in humans than in animals. For example, the fusimotor-induced increase in spindlehuman ending; muscle it isspindle worth pointing firing rate out that during this wasisometric the only contractions spindle recording (20 in imp/s) is considerably less than whichthat inhypertonic awake catssaline ( abovewas infused 100 into imp/s the )homonymous (for discussion muscle see (tibialis Ribot anterior).-Ciscar et al, 2000). It is possible that some of the discrepancies between human experimental data and those obtained in anaesthetised Nevertheless,animals could another be spindleexplained afferent, by locateddifferences in extensor in background digitorum lon gusfusimotor and held drive when the leg muscles are relaxed. Therefore in study III we will activate the fusimotor system by asking subjects to generate forvoluntary 20 min during con tractionsthe infusion, to also target showed forces. an increase Using in mean the firing same rate experimental (from 7.9 methodology employed in toStudy 10.6 Hz). II we shall again record from muscle spindles in the pretibial flexors. However, these will be fusimotor driven during a voluntary contraction, as determined by standard procedures – only fusimotor driven afferents can increase their firing rate during the voluntary contraction of the same muscle where muscle spindle is located. Thus in study III we will assess whether, during a voluntary contraction, the descending excitatory drive to the spinal cord increases the synaptic strength of the oligosynaptic connections between nociceptors in61 the muscle and the fusimotor neurones supplying that muscle and its synergists.
Unitary recordings will be obtained from ~40 fusimotor-driven muscle spindle afferents that either are (i) spontaneously active at rest yet increase (or maintain) their firing during an isometric contraction) or (ii) silent at rest yet can be recruited during voluntary effort. After characterising the spindle afferent, subjects will be asked to isometrically contract the receptor-bearing muscle to the target force required to recruit the muscle spindle or to increase its discharge and to hold that for 60 s. During the first half of the contraction period (~ 30s) the subject will be given visual feedback, after which the subject will be deprived of visual feedback and asked to maintain the same force Chapter 3 – Tonic muscle pain and spindles
Figure 3.2 Individual (n=15) trends of muscle spindle discharge plotted over time. Time “0” indicates the baseline activity prior to commencement of infusion. Data for two afferents that showed an overt increase in baseline firing during the infusion are shown with dotted lines. Note that individual spindle afferents were recorded for different durations.
Mean visual analog scale scores associated with the steady-state pain level for these two subjects were 5.5 and 2.6, respectively, so we do not believe that differences in perceived pain can account for the excitatory responses of these two muscle spindles to infusion of hypertonic saline.
Data from all recordings are shown in Fig 3.2. With the exception of the two afferents noted above, on the whole there was no net change in firing. Repeated measures
ANOVA, conducted on the afferents held for 20min (n=14), 30min (n=10) and 50min
(n=8), revealed no significant change in mean firing rate during tonic pain (P = 0.874,
0.830 and 0.971, respectively).
62 Chapter 3 – Tonic muscle pain and spindles
Mean firing rates are shown in Fig. 3.3, together with mean pain ratings.
Figure 3.3 A, mean discharge rate of muscle spindle afferents plotted with standard errors of the mean.
The numbers in the columns refer to the number of spindles that contributed to the data, owing to loss of recording sites at different times throughout. B, mean pain profile, calculated from all subjects.
63 Chapter 3 – Tonic muscle pain and spindles
3.4 Discussion
We have studied, for the first time, the effects of long- lasting muscle pain on the spontaneous discharge of muscle spindle afferents in the relaxed leg muscles of awake human subjects. According to the animal literature, we should have seen a marked increase in spindle firing rate during the infusion of hypertonic saline, but saw no consistent change. Despite moderately strong pain that lasted for about an hour, we failed to show any evidence of a fusimotor-driven increase in muscle spindle activity.
We also failed to see any recruitment of silent muscle spindles, which would be expected if fusimotor neurones had been recruited; it is generally accepted that there is essentially no resting static fusimotor drive to muscle spindles in relaxed human muscles (Burke et al. 1979; Nordh et al. 1983; Macefield 2013). Moreover, the receptor-bearing muscle and adjacent muscles remained silent during the infusion; there was no evidence of activation of α-motoneurones, expected if the facilitation of the motoneurone pool by muscle spindle afferents was sufficiently strong to produce a sustained contraction. On the basis of the present results, as well as the results of our earlier study (Birznieks et al. 2008), we reject the hypothesis we set out to test.
It is important to recognize that what is found in anaesthetized and often surgically reduced animal preparations may not hold true in awake human subjects. Indeed, our observations are in contrast to those of several animal studies that have found evidence of a significant relationship between group III and IV afferents and the fusimotor system (Appelberg B 1983b; Mense & Skeppar 1991; Johansson et al.
1993; Djupsjobacka et al. 1995a; Capra & Ro 2000). Noxious afferent input is known to be one of many factors that alter muscle spindle sensitivity in experimental animals
(Matthews 1988), and the ‘vicious cycle’ theory was proposed as the underlying mechanism for the development of chronic muscular pain syndromes (Johansson &
64 Chapter 3 – Tonic muscle pain and spindles
Sojka 1991). The hypothesis posits that noxious stimulation of group III and IV afferents reflexly excites fusimotor neurones, with the autogenic facilitation of the α- motoneurone pool via the muscle spindles in turn causing an increase in muscle activity and accumulation of metabolites, particularly if the contraction is sustained and local muscle ischaemia develops (Johansson & Sojka 1991) . It is known that electrical stimulation of group III afferents reflexly excites γ-motoneurones (Ellaway et al. 1982; Appelberg B 1983b). It is also known that group IV (and group III) afferents respond to certain products of muscle metabolism (Kaufman & Rybicki
1987; Rotto & Kaufman 1988; Andreani et al. 1997; Andreani & Kaufman 1998;
Kaufman et al. 2002) and increase static fusimotor drive (Johansson et al. 1993;
Djupsjobacka et al. 1995a).
Accordingly, if the production of metabolites is high enough to excite intramuscular nociceptors or if the nociceptive input from joints (Johansson 1988) and ligaments
(Johansson et al. 1989) also contributes, a process sustaining a vicious cycle might be initiated, resulting in chronic muscle pain. In addition, experimental evidence obtained with electrical stimulation suggests that there might be a second positive feedback loop sustained by secondary muscle spindle afferents acting on γ- motoneurones directly, thereby bypassing nociceptive afferents (Appelberg B 1983a) .
Together with other feedback loops, this mechanism may strengthen a self- perpetuating vicious cycle reflex.
Intramuscular injection of hypertonic (5–7%) saline, either as a bolus or as a continuous infusion, has become a standard model for inducing muscle pain – by depolarizing group IV (and group III) afferents – in human subjects (Graven-Nielsen
65 Chapter 3 – Tonic muscle pain and spindles et al. 1997; Svensson et al. 1998; 2000; Hodges et al. 2008; Tucker et al. 2009;
Fazalbhoy et al. 2012). We have previously shown that bolus injection into tibialis anterior had little effect on the resting discharge of 19 spontaneously active muscle spindle afferents, both primary and secondary endings, recorded from relaxed leg muscles in human subjects; rather, what was observed was a decrease in firing in some afferents, or no effect whatsoever (Birznieks et al. 2008). Similar results were obtained with infusion of hypertonic saline in the present study, with some afferents showing an overt fall in firing rate and some an increase, but the majority showing no change over time. Conversely, intramuscular injection of hypertonic saline into leg muscles of anaesthetized cats caused large increases in the firing of 38 of 42 muscle spindles, with mean firing rate increasing by 74% when the injection was made into the receptor-bearing muscle and by 87% when injected into heteronymous muscles, irrespective of their identity as primary or secondary endings (Thunberg et al. 2002a).
Although our observations in awake human subjects contradict those obtained in the cat, our data are supported by indirect evidence suggesting that muscle pain causes an inhibition of muscle spindle activity. Stretch-evoked reflexes in single motor units from the masseter muscle were reduced by injection of hypertonic saline into the ipsilateral muscle (Svensson et al. 2000), and motor unit firing rates in the upper and lower limb muscles were reduced by intramuscular injection of hypertonic saline
(Farina et al. 2005; Hodges et al. 2008; Tucker et al. 2009). Given that the firing rates of individual human motoneurones are facilitated by approximately 30% at all levels of voluntary drive by muscle spindle input (Macefield et al. 1993), the fall in firing rates of some motor units during muscle pain could, in part, be explained by a decrease in excitatory drive to the motoneurone pool by some muscle spindles.
66 Chapter 3 – Tonic muscle pain and spindles
Importantly, hypertonic saline has never caused recruitment of motor units in relaxed muscles, and it needs to be pointed out that the validity of the vicious cycle hypothesis in humans is controversial (Matre et al. 1998; Matre et al. 2002; Birznieks et al. 2008); for review see (Knutson 2000). The limitation of most human studies is that they are based on indirect evidence, rather than direct assessment of the effects of muscle pain on the activity of muscle spindles. Lund et al. (1991) reviewed a wide range of clinical literature and experimental studies and came to the conclusion that chronic pain tends to inhibit, not facilitate, voluntary and reflex contractile activity of a painful muscle or its agonists. These authors suggest that those effects are beneficial and provide protective adaptation (i.e. the ‘pain adaptation’ model), which fits well with our own data obtained with short-lasting (Birznieks et al. 2008) and long-lasting pain (present study), as well as with data obtained from animal experiments that used an inflammatory agent to induce pain (Mense & Skeppar 1991). Interestingly, Simons
& Mense (1998) provided electromyographic evidence showing that painful muscles in humans are mostly silent and, if EMG activity is present, it does not correlate with pain in either the time or the intensity domain. Likewise, Svensson et al. (1998) obtained no evidence that pain could induce long-lasting muscle hyperactivity.
3.4.1 Limitations
As noted above, our aim was to follow the spontaneous discharge of identified muscle spindle afferents for the entire duration of the infusion, but given that we also needed to record an adequate baseline period prior to the infusion and lost recordings at various times during the infusion, we do not have complete data sets for all 15 afferents studied. Moreover, we could induce muscle pain only once in each subject,
67 Chapter 3 – Tonic muscle pain and spindles so if we lost the recording site we had to terminate the experiment; we wanted to ensure that each recording of a muscle spindle response to muscle pain was not preceded by a previous episode of pain. Nevertheless, if there was a significant effect of muscle pain on the fusimotor system we might have expected to see a change in the spontaneous discharge of muscle spindle afferents within 10 min of a steady-state level of pain (all 15 afferents were held for at least 10 min of the infusion). The fact that we did not see consistent changes in mean firing rate either early or late in the infusion leads us to conclude that there are no reflex effects of muscle pain on human fusomotor neurones, at least in the relaxed state.
However, two spindle afferents did show an increase in mean firing rate during the course of the infusion. The largest increase (40%) occurred in the spindle afferent that was located in the same muscle in which the hypertonic saline was infused. It is possible that this is a real response to noxious stimulation of the homonymous muscle but, as noted above, Thunberg and colleagues (2002a) found no differences in the magnitude of the excitatory responses of muscles spindles located in homonymous and heteronymous muscles during intramuscular injection of hypertonic saline into the tibialis anterior muscle of the cat. Indeed, the authors noted that, following injection of hypertonic saline into ‘homonymous, close and/or remote heteronymous muscle the fusimotor drive to a particular spindle was of the same character no matter what muscle was stimulated. Moreover, the authors specifically chose intramuscular injections rather than some other form of noxious stimulation in order to make the study comparable to corresponding studies in humans. Accordingly, we feel it unlikely that recording from more muscle spindle afferents located in the homonymous muscle will reveal further excitatory responses to infusion of hypertonic
68 Chapter 3 – Tonic muscle pain and spindles saline, but this may well need to be explored in future work. It is also possible that the observations in the cat were a consequence of anaesthesia, which uncovered strong nociceptor–fusimotor spinal reflexes that are normally suppressed in the awake state.
An alternative interpretation is that the increase in discharge rate for this afferent was not related to the noxious stimulus per se, but rather to the increase in muscle volume produced by the infusion. Indeed, the time lag of reabsorption of the saline means that subjects often have a more prominent muscle belly on the ipsilateral leg compared with the contralateral leg. Given that muscle spindles are stretch-sensitive sensory organs, it is plausible that an increase in intramuscular volume would cause stretch of the muscle in the vicinity of the spindle afferent and that this response is, essentially, an artifact of the experimental model rather than of any fusimotor-driven response to muscle pain. However, to test this we would need to infuse an equivalent volume of isotonic saline into the receptor-bearing muscle.
3.5 Conclusions
We conclude that, despite the presence of a constant level of moderately strong muscle pain for about an hour, there was no reflexly generated recruitment of fusimotor neurones by muscle nociceptors. This is in marked contrast to the ∼80% increase in muscle spindle firing rates in anaesthetized cats following bolus injection of hypertonic saline into the homonymous or heteronymous muscles. Together with our previous study (Birznieks et al. 2008), the present investigation does not support the concept that nociceptor-driven increases in fusimotor drive are responsible for the development of myalgia and chronic muscle pain.
69 Chapter 4 – MSNA responses to tonic muscle pain
CHAPTER FOUR
MUSCLE SYMPATHETIC NERVE ACTIVITY RESPONSES TO
TONIC MUSCLE PAIN
Chapter abstract
It has been recently shown that acute muscle pain, induced by bolus intramuscular injection of hypertonic saline, causes a sustained increase in muscle sympathetic nerve activity (MSNA) and a modest increase in blood pressure and heart rate.
However, it is not known whether long- lasting (tonic) pain, which more closely resembles chronic pain, causes a sustained increase in MSNA and blood pressure. We tested this hypothesis by recording MSNA in 12 healthy subjects. Tonic pain was induced for ∼60 min by slow intramuscular infusion of hypertonic saline (7%) into the ipsilateral tibialis anterior muscle. Pain was sustained at a tolerable level (5/10 to
6/10 on a visual analog scale). Seven subjects showed progressive increases in mean
MSNA amplitude during tonic pain, increasing to 154 ± 17% (SEM) at 45 min and remaining essentially constant for the duration of the infusion. In these subjects, blood pressure and heart rate also increased. Conversely, for the other five subjects MSNA showed a progressive decline, with a peak fall of 67 ± 11% at 40 min; blood pressure and heart rate also fell in these subjects. In conclusion tonic muscle pain has long- lasting effects on the sympathetic control of blood pressure, causing a sustained increase in some subjects yet a sustained decrease in others. This may have implications for individual differences in the cardiovascular consequences of chronic pain.
70 Chapter 4 – MSNA responses to tonic muscle pain
4.1 Introduction
Pain originating from deep body structures, such as skeletal muscle tissue, evokes a dull, aching sensation that can be very difficult to localize (Henderson et al. 2006;
Burton et al. 2009a). Increasing evidence from animal and human research shows that, in addition to the painful experience, the presence of pain affects the regulation of other physiological processes in the body (Schobel et al. 1996). A number of sensory, motor and behavioural changes have been reported in response to painful stimuli, such as enhanced cardiovascular activity, central and peripheral sensitization, suppression of immune function and secretion of cortisol (Hervey 1983; Edwards &
Fillingim 2005). Noxious stimulation of deep structures, in particular, evokes a very different behavioural and cardiovascular response compared with pain originating in superficial structures (Lewis 1942; Horeyseck & Jänig 1974b; Sato et al. 1984).
Early experiments on the cardiovascular responses to experimental pain in human subjects reported that noxious inputs originating in deep structures evoked a ‘slowing of the pulse’ and ‘falling of the blood pressure’ (Lewis 1942; Feinstein et al. 1954).
These early observations have formed the basis of other studies looking at the effects of pain on the cardiovascular system in awake human subjects.
Unlike the discrete, well-localized pain induced by noxious stimulation of the skin, that induced by noxious stimulation of deep tissues, such as muscle, is typically diffuse and often refers to distal structures. Indeed, we have shown in a longitudinal study that repeated noxious stimulation of muscle may result in an increase in the area of the referred pain and a reduction in the area of pain localized to the muscle belly
(Rubin et al. 2010). This emphasizes the difficulty in treating deep pain; over time, the apparent site of pain may change, which may leave patients suffering from
71 Chapter 4 – MSNA responses to tonic muscle pain
protracted pain for a significant part of their lives. Chronic pain is frequently established from activation of nociceptors located in deep tissues, such as muscle, ligaments or joints.
The sympathetic nervous system plays an important role in the control of blood pressure and blood flow, and noxious stimulation could affect either of these parameters. A sustained increase in muscle vasoconstrictor activity can lead to high blood pressure (hypertension), and an increase in cutaneous vasoconstrictor activity will reduce skin blood flow, potentially contributing to poor wound healing. In a retrospective study, Bruehl and colleagues (2005) showed that patients with postsurgical chronic pain have nearly twice the prevalence of clinical hypertension
(39%) as medical patients without pain (21%). We recently showed, for the first time, that selective stimulation of muscle nociceptors, induced by bolus (0.5 ml) intramuscular injection of hypertonic saline into the leg of awake human subjects, caused a sustained increase in muscle sympathetic nerve activity (MSNA), blood pressure and heart rate (Burton et al. 2009a). Conversely, skin sympathetic nerve activity (SSNA) showed a transient increase, which we believe is simply a reflection of the alerting (arousal) response to the pain (Burton et al. 2009b). The finding that
MSNA increased with muscle pain is contrary to the decreases in sympathetic outflow, blood pressure and heart rate observed in anaesthetized animals during deep pain (Keay et al. 1994).
In the present study, we use infusion, rather than bolus injection, of hypertonic saline into the tibialis anterior muscle to cause a sustained, steady-state level of pain lasting for 1h. Unlike the short-lasting pain we had previously induced by bolus
72 Chapter 4 – MSNA responses to tonic muscle pain
intramuscular injections of hypertonic saline (Burton et al. 2009a; 2009b), the responses to which could be seen simply as reflections of the ‘fight-or-flight’ alerting response pattern, we believe the physiological responses to tonic pain will more closely replicate episodes during which chronic pain patients are suffering and coping with their pain. Indeed, persistent deep pain has been shown to provoke a passive coping response, i.e. conservation/withdrawal (Keay & Bandler 2002). The aim of this study was thus to examine the cardiovascular responses to tonic muscle pain by inducing long-lasting pain, and to observe whether the changes in MSNA, blood pressure and heart rate are transient or sustained during tonic pain. This may provide us with fundamentally different results from those obtained in our earlier studies of short-lasting (acute) pain and reveal novel physiological processes that cannot be identified otherwise.
4.2 Methods
The study was conducted with the approval of the Human Research Ethics
Committee, University of Western Sydney, and satisfied the Declaration of Helsinki.
Prior written informed consent was obtained from all subjects, and they were given the option to withdraw consent at any time. Twelve subjects (11 male and one female), ranging in age from 18 to 48 years, with no history of chronic muscle pain, were recruited for this study. Subjects were asked to refrain from caffeine and excessive physical activity for 24 h prior to the experiment.
4.2.1 Experimental procedures
Subjects were seated in a semi-reclined posture in a comfortable chair, with the legs supported in the extended position. Muscle sympathetic nerve activity was recorded
73 Chapter 4 – MSNA responses to tonic muscle pain
from muscle fascicles of the common peroneal nerve supplying the ankle or toe extensor or foot everter muscles via tungsten microelectrodes (FHC, Bowdoinham,
ME, USA) inserted percutaneously at the level of the fibular head. Multiunit neural activity was amplified (gain 20,000; bandpass 0.3–5.0 kHz) using an isolated amplifier (NeuroAmp EX; ADInstruments, Sydney, NSW, Australia) and stored on a computer (10kHz sampling) using a computer-based data acquisition and analysis system (PowerLab 16SP hardware and LabChart 7 software; ADInstruments). A root- mean-square (RMS) processed version of this signal was also computed, with a moving average of 200 ms. The ECG (0.3–1.0 kHz) was recorded with Ag–AgCl surface electrodes on the chest and sampled at 2kHz. Blood pressure was recorded continuously using radial arterial tonometry (NIBP 7000; Colin Corp., Kamaki,
Japan) and sampled at 400 Hz. Respiration (DC to 100 Hz) was recorded using a strain-gauge transducer (Pneumotrace; UFI, Morro Bay, CA, USA) wrapped around the chest. Surface EMG was recorded with standard Ag–AgCl electrodes placed over the ipsilateral tibialis anterior muscles to ensure that the subject was fully relaxed.
4.2.2 Noxious stimulation
Sterile 20% saline was diluted with sterile water to create a 7% hypertonic solution.
Two 10ml syringes were connected via extension tubing to a three-way tap and primed with hypertonic saline. A single 23gauge butterfly cannula attached to the three-way tap was inserted approximately 1.5 cm deep into the belly of the ipsilateral tibialis anterior muscle, approximately 5 cm lateral and 10 cm inferior to the tibial tuberosity. Baseline MSNA was recorded for 5 min prior to commencing the infusion of hypertonic saline, which began at a time unknown to the subject and lasted for 45 min. The rate of infusion began at 250µl min−1, but this was adjusted to maintain the
74 Chapter 4 – MSNA responses to tonic muscle pain
pain at a moderate level [5/10 to 6/10 on visual analog scale (VAS)]. During infusion, subjects provided instantaneous feedback about the intensity of pain they were experiencing using a potentiometer that was calibrated to a VAS that ranged from 0 to
10, where ‘0’ was considered ‘no pain’ and ‘10’ as ‘the worst pain imaginable’.
Subjects were informed that they could stop the pain at any time by asking the experimenter to stop the infusion pump; no subject requested early termination of the experiment. At the end of the experiment, subjects completed a McGill Pain
Questionnaire and drew the area(s) of pain on an image of a leg.
4.2.3 Analysis
The neurogram was critically reviewed to ensure the recording was stable and that no shifting of the recording electrode was apparent. Individual bursts of MSNA were analysed from the RMS-processed signal using the Peak Analysis module, part of
LabChart v7.2.2 software (ADInstruments). The commencement of infusion was denoted time zero; data recorded 5 min prior to the infusion were termed ‘baseline’.
The mean number of bursts per minute and the total burst amplitude were calculated during the baseline period and during infusion of hypertonic saline in 5 min blocks.
Values of MSNA were normalized to individual baseline values and expressed as a percentage change from baseline. Heart rate variability was computed over 5 min periods of stationary data using the heart rate variability (HRV) software module; low-frequency (LF) and high- frequency (HF) power and the LF/HF ratio were calculated (LabChart v7.2.2; ADInstruments). All statistical analysis was performed using Prism v5.00 for Mac OS X (GraphPad Software, San Diego, CA, USA).
Repeated-measures, one- way ANOVA with Dunnett’s post hoc test were used to
75 Chapter 4 – MSNA responses to tonic muscle pain
compare changes in MSNA, mean blood pressure and heart rate relative to baseline.
For all statistical tests, a probability level of P < 0.05 was regarded as significant. All values are expressed as means and standard errors.
4.3 Results
4.3.1 Subjective experience of tonic muscle pain
Infusion of hypertonic saline into the tibialis anterior muscle for 45 min induced pain that lasted approximately 60 min in all subjects, but no subject reported not being able to cope with the pain. By adjustment of the infusion rate during the course of the experiment, we were able to titrate the pain and keep it at a constant level, around
5/10 to 6/10 on a VAS. On average, 14.2 ml of hypertonic saline was infused into the muscle, with a starting rate of 250µl min−1. The mean VAS rating across subjects was 5.5 ± 0.3/10. After the pain had subsided, subjects identified on an anatomical map of their leg where they experienced the pain; while all subjects reported pain in the muscle belly of tibialis anterior, nine of 12 subjects also reported pain that referred into the foot. Completion of the McGill Pain Questionnaire indicated that the overall severity of pain was 2.17 ± 0.15 (on a scale of 5), with most subjects rating it as
‘discomforting’ (eight of 12). The most commonly used pain descriptors were
‘continuous’ (eight of 12), ‘constant’ (six of 12) and ‘tight’ (five of 12), with ‘dull’
(seven of 12) and ‘aching’ (four of 12) also being common.
76 Chapter 4 – MSNA responses to tonic muscle pain
Figure 4.1 Experimental records from one subject. Neural activity was recorded from the motor fascicle supplying extensor digitorum longus; the root mean squared (RMS)-processed signal is shown in the lowest trace. This subject generated little spontaneous muscle sympathetic nerve activity (MSNA) at rest, shown immediately prior to the commencement of the infusion of hypertonic saline into the ipsilateral tibialis anterior muscle (A), but both burst frequency and amplitude increased during tonic muscle pain, shown 50 minutes after the start of the infusion
(B). It can be seen that blood pressure also increased. Pain was maintained at a visual analog scale (VAS) level of
5.5 throughout the infusion period.
4.3.2 Muscle sympathetic nerve activity during tonic muscle pain
Experimental records from one subject are shown in Fig. 4.1. Muscle sympathetic nerve activity occurred as spontaneous bursts that were time locked to the R wave of the ECG signal. During infusion of hypertonic saline into the tibialis anterior muscle, the mean frequency of MSNA (in bursts per minute) increased [F(10,11) = 2.38, P =
0.01] in comparison to the control period measured over the 5min immediately preceding the start of the infusion. In individual subjects, the relative burst frequency averaged over the period of pain ranged from 104.4 to 119.0%. This is illustrated graphically in Fig. 4.2A.
77 Chapter 4 – MSNA responses to tonic muscle pain
A
130 ) %
( 120
y c n e
u 110 q e r f
t s r
u 100 b
90 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 B
180
160 ) % ( 140 e d u
t 120 i l p
m 100 a
t s
r 80 u b 60
40 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 time (mins)
Figure 4.2 Mean (+/- SE) changes in MSNA burst frequency (A) and burst amplitude (B) during tonic muscle pain. Whilst burst frequency showed an increase, two groups were apparent in the changes in MSNA burst amplitude: a group in whom amplitude increased (closed symbols; n=7) and a group in whom amplitude decreased
(open symbols; n=5). The dashed line (half-filled symbols; n=12) shows burst amplitude when subjects are pooled together.
Interestingly, in terms of burst amplitude there was no net change [F(10,11) = 0.56, P
= 0.85], as demonstrated by the dotted line in Fig. 4.2B. However, close examination of individual data revealed that there were in fact two types of responses, an increase or a decrease in burst amplitude, that, when the data were pooled, essentially cancelled each other out (Fig. 2B). Based on this post hoc assessment, seven subjects
78 Chapter 4 – MSNA responses to tonic muscle pain
showed progressive increases in mean burst amplitude during tonic pain [F(10,6) =
2.56, P = 0.01], with a peak increase of 154 ± 17% at 45 min. Conversely, for the other five subjects burst amplitudes showed a progressive fall [F(10,4) = 2.39, P =
0.03], with a nadir of 67 ± 11% at 40 min. There were no significant differences in
VAS ratings between the two groups.
After discovering the two separate responses with regard to mean burst amplitude, mean burst frequency was retrospectively analysed again, to establish the presence of two separate groups. Subjects were divided with respect to the direction of changes in mean burst amplitude. The increasing group (n = 7) demonstrated minimal change in mean burst frequency [F(10,6) = 0.86, P = 0.58]. On the contrary, the decreasing group (n = 5) showed increases in mean burst frequency [F(10,4) = 3.17, P = 0.004].
Furthermore, we compared absolute values of resting MSNA in the 5 min immediately preceding the infusion in the two groups. Interestingly, the seven subjects who demonstrated increases in MSNA amplitude during tonic pain showed a higher resting mean burst frequency (25.1 ± 3.4 bursts min−1) than the five subjects who demonstrated a decline in burst amplitude during long-lasting pain (19.6 ± 3.6 bursts min−1); however, this difference failed to reach statistical significance.
79 Chapter 4 – MSNA responses to tonic muscle pain
Figure 4.3 Mean (+/- SE) changes in blood pressure (A) and heart rate (B) in the group showing an increase in
MSNA amplitude during tonic muscle pain (closed symbols; n=7) and the group showing a decrease in MSNA amplitude (open symbols; n=5). The changes in blood pressure and heart rate paralleled the changes in MSNA amplitude in the two groups.
4.3.3 Blood pressure and heart rate during tonic muscle pain
Pooling the results of the changes in mean blood pressure from all subjects during tonic muscle pain revealed a similar profile to that observed with MSNA amplitude.
In order to determine whether two divergent responses were present, data were analysed from each individual subject, independent of any grouping. The results
80 Chapter 4 – MSNA responses to tonic muscle pain
showed that the same two divergent responses seen with burst amplitude were apparent with respect to mean blood pressure; the same subjects in whom increases in
MSNA were apparent showed increases in blood pressure, while falls in blood pressure occurred in the group showing a decline in burst amplitude during tonic pain.
Mean changes in blood pressure and heart rate were analysed with respect to whether
MSNA amplitude increased or decreased during tonic muscle pain; data are shown graphically in Fig. 4.3. The group that exhibited an increase in burst amplitude also showed an increase in mean blood pressure [F(10,6) = 2.65, P = 0.01] to 115 ± 0.8% at its peak following the onset of the infusion (Fig. 4.3A). Conversely, in the decreasing group the mean blood pressure dropped [F(10,4) = 3.40, P = 0.003], with a peak fall at 30 min of 87 ± 4% compared with control values. Likewise, heart rate in the increasing group showed an overall increase [F(10,6) = 3.85 (P < 0.001)], peaking at 15 min to 106 ± 3%, whereas for the decreasing group an overall fall [F(10,4) =
6.63, (P < 0.001)] in heart rate occurred, with a peak fall at 40 min of 93 ± 2% (Fig.
4.3B). Given these divergent changes in blood pressure and heart rate, it is not surprising that pooling the data from both groups revealed overall changes in blood pressure (104±5%) and heart rate (99±2%) that were not significantly different from baseline.
81 Chapter 4 – MSNA responses to tonic muscle pain
Figure 4.4 Mean (+/- SE) changes in low-frequency (LF; A) and high-frequency (HF; B) power in the heart variability spectrum and RMSSD (the square root of the mean of the squared differences between adjacent NN intervals), each calculated over 5 minutes, in the group showing an increase in MSNA amplitude during tonic muscle pain (closed symbols; n=7) and the group showing a decrease in MSNA amplitude (open symbols; n=5).
Low-frequency power was higher, and high-frequency power lower, in the group showing an increase in MSNA amplitude during tonic pain. RMSSD values were lower in this group.
82 Chapter 4 – MSNA responses to tonic muscle pain
4.3.4 Heart rate variability
Heart rate variability was assessed over a 5 min steady- state period at 10, 30, 40 and
60 min following the onset of the infusion, and compared with the 5 min baseline period immediately prior to the infusion. Heart rate variability parameters were analysed separately for the two groups, defined according to whether MSNA increased or decreased during tonic muscle pain. Interestingly, as shown in Fig. 4.4 and Table 4.1, the increasing group showed significantly higher LF power and lower
HF power, with a higher LF/HF ratio than the decreasing group, both at rest and during tonic pain. Conversely, RMSSD (root mean square successive difference of cardiac intervals), another measure of variability, was lower in the increasing group.
For both groups, there were no significant changes in any of these parameters during tonic muscle pain.
83 Chapter 4 – MSNA responses to tonic muscle pain
Baseline 10 min 30 min 40 min 60 min
LF Power 65.1 +/- 5.1 58.6 +/- 4.2 69.3 +/- 2.9 66.2 +/- 4.2 64.8 +/- 5.4
46.0 +/- 5.0 43.8 +/- 10.1 45.1 +/- 8.8 39.3 +/- 10.3 43.9 +/- 8.8
HF Power 30.1 +/- 4.4 36.3 +/- 3.7 26.3 +/- 1.8 29.7 +/- 3.6 31.2 +/- 4.9
47.6 +/- 4.1 48.7 +/- 8.5 48.1 +/- 7.1 54.2 +/- 9.1 50.0 +/- 6.9
LF/HF 2.63 +/- 0.55 1.79 +/- 0.30 2.76 +/- 0.30 2.56 +/- 0.48 2.49 +/- 0.54
1.04 +/- 0.20 1.60 +/- 1.00 1.33 +/- 0.65 1.22 +/- 0.75 1.12 +/- 0.47
RMSSD 40.3 +/- 5.3 36.9 +/- 5.6 38.9 +/- 5.1 41.2 +/- 5.0 37.8 +/- 4.8
59.5 +/-5.7 68.5 +/- 7.4 72.1 +/- 8.3 65.4 +/- 5.8 64.2 +/- 5.4
Table 4.1: Mean (+/- SE) changes in low-frequency (LF) and high-frequency (HF) power in the heart variability
spectrum (calculated over 5 minutes), LF/HF ratio and RMSSD (the square root of the mean of the squared
differences between adjacent NN intervals) in the groups showing an increase in MSNA during tonic muscle pain
(bold text; n=7) and the group showing a decrease in MSNA (light text; n=5). Low-frequency power was
significantly higher, and high-frequency power significantly lower, in the group showing an increase in MSNA
during tonic pain. Accordingly, LF/HF ratio was significantly higher in this group.
4.4 Discussion
The results of this study have shown, for the first time, that tonic muscle pain,
induced by intramuscular infusion of hypertonic saline, has long-lasting effects on
MSNA, blood pressure and heart rate in awake human subjects. Interestingly,
84 Chapter 4 – MSNA responses to tonic muscle pain
approximately half of the subjects showed increases in MSNA, with the other half showing decreases. Independent analysis of mean blood pressure from individual subjects also revealed two distinct responses, asserting the existence of two groups.
Given that MSNA is vasoconstrictor in function and is a primary determinant of blood pressure, it is perhaps not surprising that we saw essentially parallel changes in
MSNA and blood pressure. Heart rate also increased or decreased in parallel with
MSNA and blood pressure. Within the first few minutes of commencement of the infusion a change in MSNA burst amplitude was apparent. The changes in blood pressure and heart rate followed the same trend, either an increase or decrease, but were delayed with respect to the changes in MSNA. All subjects showed an increase in mean blood pressure within the first 10 min; thereafter, in the increasing group blood pressure continued to rise, whereas in the decreasing group it started to fall.
These same changes were also observed with heart rate. Although cardiac sympathetic nerve outflow cannot be measured directly in human subjects, it is known that, at rest, cardiac noradrenaline spillover essentially parallels resting MSNA
(Wallin et al. 1992); it is plausible that parallel changes in cardiac and muscle sympathetic outflow also occurred during tonic muscle pain.
It has been established that noxious stimulation alters the regulation of sympathetic activity in animals and humans (Nordin & Fagius 1995; Schobel et al. 1996).
Likewise, in the present study, it is apparent that the changes in MSNA, blood pressure and heart rate are the result of the intramuscular infusion of hypertonic saline. It may be argued that anticipation and anxiety associated with the experimental procedures may influence the results, which to an extent are a hardwired cognitive process integral to pain perception (Burgmer et al. 2011), but the subjects were
85 Chapter 4 – MSNA responses to tonic muscle pain
unaware of when the infusion was going to commence. Moreover, the changes observed during infusion compared with the control period indicate that these are responses to the noxious stimulation. Had anticipation and anxiety exercised a profound effect on MSNA, these effects would have been observed during the control period, and once infusion commenced little or no change would have occurred. Also, if anticipation of pain was critical, we would expect that subjects would habituate during the course of the infusion, with MSNA, blood pressure and heart rate returning to control levels. Clearly, this was not the case; rather, increases or decreases in
MSNA, blood pressure and heart rate commenced shortly after the infusion commenced and were sustained through the experiment.
Several studies that have examined the effects of mental stress have shown that it has a minor impact on MSNA, although a significant impact on the fluctuation of sympathetic activity (Anderson et al. 1987; Freyschuss et al. 1990). Repeat measurements of MSNA in resting awake human subjects made 3 weeks to 21 months apart have shown that intra-individual resting activity was relatively unchanged between recordings, suggesting an individually constant level of sympathetic activity in muscle nerves (Sundlof & Wallin 1977); interindividual reproducibility is maintained even after 10–14 years (Fagius & Wallin 1993). Anxiety associated with the different experimental procedures in which microneurography has been utilized would be likely to influence the first recording and thereby make subsequent recordings incomparable, which is evidently not the case. Accordingly, we do not believe the observations we have made in the present study can be explained by anticipation or anxiety.
86 Chapter 4 – MSNA responses to tonic muscle pain
Heart rate variability, which purportedly reflects the degree of sympathetic and parasympathetic control over the heart, indicates that the group in whom increases in
MSNA occurred showed higher power in the LF band and lower power in the HF band at rest and during tonic pain. The HF band is believed to reflect vagal cardiac control (Bernston et al. 1997), while the LF band has been suggested to represent
(primarily) sympathetic cardiac activation (Malliani et al. 1991); the LF/HF ratio has been proposed as an index of the sympathovagal balance, i.e. the relative sympathetic contribution to cardiac regulation (Cohen et al. 2000; Martínez-Lavín 2004; Staud
2008; Reyes del Paso et al. 2011). While the utility of HRV in differentiating between cardiac sympathetic and parasympathetic outflow is questionable (Goldstein et al.
2011), it is interesting to note that the resting LF power and LF/HF ratio values were significantly higher for the group showing an increase in MSNA during tonic pain, while the RMSSD values were lower. Based on the standard (simplistic) interpretation of HRV, these data could be interpreted to suggest that resting cardiac sympathetic outflow is higher, and cardiac vagal outflow lower, in individuals who respond to tonic muscle pain by increasing their MSNA, blood pressure and heart rate. However, in both groups there were no significant changes in any of these parameters during long-lasting muscle pain.
In our previous study, in which we recorded MSNA during a bolus injection of hypertonic saline (0.5ml) into the tibialis anterior muscle, we showed that MSNA amplitude increased during pain, with a moderate increase in blood pressure and heart rate (Burton et al. 2009a). Interestingly, although we did not think much of it at the time, we noted that three of the 15 subjects exhibited a decrease, rather than an
87 Chapter 4 – MSNA responses to tonic muscle pain
increase, in blood pressure or heart rate. This fits with what we observed with long- lasting muscle pain in the present study; some subjects showed an increase in blood pressure and heart rate, while some showed a decrease. It is important to note that in that earlier study the pain lasted on average for only 6 min, whereas it lasted 10 times as long in the present study. Clearly, the changes in cardiovascular expression can be observed with short-lasting as well as long-lasting muscle pain.
In the present study, we observed an increase in mean MSNA burst frequency, yet
MSNA burst amplitude showed two distinct patterns, increasing in some subjects and decreasing in others. Although this may seem contradictory, exclusive changes in one parameter, either burst amplitude or burst count, have been known to occur. For example, mental stress causes an increase in sympathetic burst amplitude yet no change in burst count (Hjemdahl et al. 1989). In a separate study using rats, it was observed that heat stimulation of the tail increased, and acute volume loading decreased, renal sympathetic nerve activity through changes in burst amplitude exclusively, whilst burst frequency remained unchanged (DiBona & Jones 1998).
This is thought to be the result of two independent mechanisms governing burst threshold and burst strength (Kienbaum P et al. 2001; Keller et al. 2006). Although the precise mechanism that governs these two aspects of MSNA burst activity are unknown, it has been suggested that the central nervous system utilizes two separate sites as part of a feedback loop interacting with the arterial baroreceptors, with one determining the incidence of bursts and the other the strength of the bursts (Kienbaum et al. 2001).
88 Chapter 4 – MSNA responses to tonic muscle pain
Supraspinal centres regulating cardiovascular activity are known to overlap substantially with those receiving nociceptive input (Bruehl & Chung 2004). Of particular interest are two structures, the nucleus tractus solitarii (NTS) and the periaqueductal grey (PAG), which share direct and indirect neuronal projections. The
NTS provides a platform of interaction between the autonomic and sensory systems by acting as the first synapse in the baroreflex pathway, whilst concurrently playing an important role in the processing of visceral information, receiving baroreceptor input from the glossopharyngeal and vagus nerves and projection neurones from spinal laminae involved in nociceptive processing (Bruehl & Chung 2004). Based on this anatomical relationship, it would seem that nociceptive input may, via NTS, influence the gain of the baroreflex and bring about changes in MSNA, blood pressure and heart rate. The PAG has also been shown to be involved in the processing of noxious stimuli. Stimulation of the lateral columns of the PAG preferentially evokes a flight/fight response coupled with increased arousal, blood pressure and heart rate, similar to that evoked by cutaneous pain (Depaulis et al.
1992). Conversely, stimulation of the ventrolateral columns of the PAG preferentially evokes a rest/digest response coupled with decrease in blood pressure and heart rate, similar to that evoked by deep pain in experimental animals (Depaulis et al. 1992).
We suspect, however, that individual traits govern the responses evoked by noxious stimulation, despite the predicted response a given noxious stimulus would evoke.
Another important player in the relationship between pain and blood pressure is the baroreflex. Through its buffering effect on blood pressure oscillations, the baroreflex contributes to short-term regulation of blood pressure (Eckberg & Sleight 1992;
Reyes del Paso et al. 2011). If we consider the two distinct responses, one group increasing and the other decreasing, as the coping strategy to painful stimuli, it is
89 Chapter 4 – MSNA responses to tonic muscle pain
possible that the initial effects, where all subjects show the same response, is only an arousal response, similar to that seen in the acute pain model we had previously reported (Burton et al. 2009a). Wall (1979) suggested that a noxious event signals first and foremost the necessity to respond; it follows that the behavioural significance of the noxious event (its escapability or inescapability; (Keay & Bandler 2002) may determine the nature of the response. Initially, therefore, the cardiovascular response is a reaction to the noxious stimulus, reflected by an increase in blood pressure and decrease in heart rate. Subsequently, the behavioural significance of the noxious input is projected, which is where we observe different responses across subjects, perhaps relating to their different coping strategies. While we must acknowledge that the pain induced in this experimental environment was not strictly inescapable, in that the research participants knew that they could ask the infusion pump to be stopped at any time, it is worth emphasizing that the long-term cardiovascular responses to pain may exhibit interindividual differences, with some showing increases in blood pressure and others decreases. There were no obvious differences in the subjects between the two groups; the increasing and decreasing groups were not different in either age or sex (as pointed out in Methods, only one subject in the cohort was female; she was in the increasing group).
4.5 Conclusions
Using intramuscular infusion of hypertonic saline as a model of tonic muscle pain, we have shown, for the first time, that different people react to long-lasting muscle pain differently. Despite similar ratings of pain, some subjects showed increases in MSNA, blood pressure and heart rate, while others showed decreases. This study emphasizes the diverse physiological consequences of pain; consequences that we believe may
90 Chapter 4 – MSNA responses to tonic muscle pain
contribute to the cardiovascular disturbances associated with chronic pain. Given that some patients with postsurgical chronic pain go on to develop chronic pain, whereas others do not (Bruehl et al. 2005), we believe that our results may go towards explaining why this occurs.
91 Chapter 5 – Consistent MSNA responses to tonic muscle pain
CHAPTER FIVE
CONSISTENT RESPONSES IN MUSCLE
SYMPATHETIC NERVE ACTIVITY DURING TONIC
MUSCLE PAIN
Chapter Abstract
In the previous chapter we observed two patterns of cardiovascular responses across subjects: one group showed parallel increases in muscle sympathetic nerve activity
(MSNA), blood pressure and heart rate, while the other group showed parallel decreases. Given that MSNA is consistent day-to-day, we tested the hypothesis that individuals who show increases in MSNA during experimental muscle pain will show consistent responses over time. MSNA was recorded from the peroneal nerve, together with blood pressure and heart rate, during an intramuscular infusion of hypertonic saline causing pain for ~an hour in 15 subjects on two occasions, 2-27 weeks apart. Pain intensity ratings were not significantly different between the first
(5.8 ± 0.4/10) and second (6.1 ± 0.2) recording sessions. While 4 subjects showed significant decreases in the first session (46.6 ± 9.2 % of baseline) and significant increases in the second (159.6 ± 8.9 %), in 11 subjects there was consistency in the changes in MSNA over time: either a sustained decrease (55.6 ± 6.8%, n=6) or a sustained increase (143.5 ± 6.1 %, n=5) occurred in both recording sessions. There were no differences in pain ratings between sessions for any subjects. In conclusion the changes in MSNA during long-lasting muscle pain are consistent over time in the majority of individuals, reflecting the importance of studying inter-individual differences in physiology.
92 Chapter 5 – Consistent MSNA responses to tonic muscle pain
5.1 Introduction
Dysregulation of the autonomic nervous system has been implicated in many chronic muscle pain syndromes such as fibromyalgia, chronic fatigue syndrome, and complex regional pain syndrome (CRPS). In addition to muscular pain some of the clinical manifestations presented to clinicians are Raynaud’s-like phenomena, anxiety, nociceptive sensitization, and vasomotor dysfunction. Investigations exploring the role of the sympathetic nervous system in chronic muscular pain syndromes are sporadic and contradictory, with some studies showing evidence of increases in sympathetic outflow (Hallman et al. 2011; Oliveira et al. 2012) and others decreases
(Reyes del Paso et al. 2011). Campero and colleagues (2010) recently showed, in a large sample of patients with CRPS, that physiological manoeuvres known to increase sympathetic outflow to the skin had no effect on spontaneous or evoked activity in cutaneous nociceptors. Indeed, we believe that the sympathetic nervous system has very little role to play in the maintenance of pain; rather it provides an appropriate physiological response to noxious inputs (Macefield 2010).
The nature of these physiological responses is uncertain, with some differences being observed between studies on awake human subjects and anaesthetized or decerebrate animals. Increases in muscle sympathetic nerve activity (MSNA) and blood pressure during acute pain have been reported following instillation of soap solution in the eye
(Nordin & Fagius 1995), strong pressure on the nail (Nordin & Fagius 1995), immersion of a hand in ice-water (Fagius & Karhuvaara 1989) and mechanical pressure on the skin (Schobel et al. 1996). Our own work on the sympathetic effects of acute pain in awake human subjects revealed transient increases in skin sympathetic nerve activity (SSNA) yet sustained increases in muscle sympathetic
93 Chapter 5 – Consistent MSNA responses to tonic muscle pain nerve activity (MSNA) following bolus intramuscular or subcutaneous injection of hypertonic saline (Burton et al. 2009a; 2009b). Moreover, we recently showed that sustained intramuscular infusion of hypertonic saline, producing tonic pain over a 60 minute period, evoked an initial increase and then a sustained decrease in SSNA in all subjects (Hall et al. 2012), yet sustained increases in MSNA in some individuals and sustained decreases in others (Fazalbhoy et al. 2012). Importantly, for those subjects in whom increases in MSNA were evoked, blood pressure and heart rate also increased, while parallel decreases in blood pressure and heart rate were produced in those subjects in whom decreases in MSNA occurred (Fazalbhoy et al. 2012). Why there would be such divergent responses we do not know, but the purpose of the present study was to test the hypothesis that an individual subject’s pattern of response is consistent over time. This is based on the fact that, while resting levels of
MSNA differ across individuals, they are internally consistent over time (Sundlof &
Wallin 1977; Fagius & Wallin 1993); it is not unreasonable to speculate that the pattern of response to tonic pain will also show internal consistency.
5.2 Methods
5.2.1 Subjects
Fifteen healthy subjects (11 males, 4 females) with a mean age of 24 years (18-37 years) were recruited for the study. Subjects were screened for history of musculoskeletal pain, and were asked to refrain from excessive physical activity and consumption of caffeine for a minimum of twenty-four hours prior to the experiment.
Written informed consent was obtained from all participating subjects, who were informed that they could withdraw from the experiment at any time with no consequences. The study was approved by the Human Research Ethics Committee,
94 Chapter 5 – Consistent MSNA responses to tonic muscle pain
University of Western Sydney, and conformed to the criteria outlined in the
Declaration of Helsinki. A controlled environment with an ambient room temperature of 22 oC was used for the study. Subjects were seated in a semi-reclined posture in a comfortable chair with the legs supported in the extended position.
5.2.2 Experimental procedures
Nerve recordings were obtained from all subjects on two separate occasions with an interval between recordings ranging from two weeks to twenty-seven weeks. Muscle sympathetic nerve activity was recorded from muscle fascicles of the common peroneal nerve supplying the ankle, toe extensor, and foot everter muscles through tungsten microelectrodes (FHC, Bowdoinham, ME, USA) inserted percutaneously at the level of the fibular head. A muscle fascicle was defined as such if discrete twitches of the innervated muscle could be evoked by intraneural stimulation at currents of ≤20 µA and muscle spindle afferent activity could be recorded either as a spontaneous discharge that increased during stretch of the receptor-bearing muscle, or was evoked by muscle stretch or palpation over the muscle belly, and there were no afferent responses to light stroking of the skin. Muscle sympathetic nerve activity
(MSNA) was defined as such if it occurred as spontaneous bursts with a clear cardiac rhythmicity, the activity increasing during a maximal inspiratory breath-hold but not during delivery of unexpected arousal stimuli. Multi-unit neural activity was amplified (gain 20 000, bandpass 0.3–5.0 kHz) using an isolated amplifier
(NeuroAmp EX, ADInstruments, Sydney, Australia), and stored on computer (10-kHz sampling) using a computer-based data acquisition and analysis system (PowerLab
16SP hardware and LabChart 7 software; ADInstruments, Sydney, Australia). A root- mean-square (RMS) processed version of this signal was also computed, with a
95 Chapter 5 – Consistent MSNA responses to tonic muscle pain moving average of 200 ms. ECG (0.3–1.0 kHz) was recorded with Ag–AgCl surface electrodes on the chest and sampled at 2 kHz. Blood pressure was recorded continuously using radial arterial tonometry (NIBP 7000, Colin Corp., Japan) and sampled at 400 Hz. Respiration (DC-100 Hz) was recorded using a strain-gauge transducer (Pneumotrace, UFI, Morro Bay CA, USA) wrapped around the chest.
Surface EMG was recorded with standard Ag–AgCl electrodes placed over the ipsilateral tibialis anterior muscles to ensure that the subject was fully relaxed.
5.2.3 Noxious stimulation
Sterile hypertonic saline (20 %) was diluted with sterile water to create a 7 % hypertonic solution drawn up into two 10 ml syringes. The syringes were connected to a three-way tap via 30 cm extension tubing primed with hypertonic saline. A single butterfly cannula (23 G) attached to the three-way tap was inserted approximately 1.5 cm deep into the belly of the ipsilateral tibialis anterior muscle, approximately 5 cm lateral and 10 cm inferior to the tibial tuberosity. A baseline recording of MSNA and all other parameters was recorded for five minutes prior to commencing the infusion of hypertonic saline, which began at a time unknown to the subject and lasted for forty-five minutes. The rate of infusion began at 250 µl per minute, but this was constantly adjusted to maintain the pain at a moderate level (5-6/10 on a Numerical
Rating Scale, NRS). During infusion, subjects provided instantaneous feedback about the intensity of pain they were experiencing using a linear potentiometer that was calibrated to the NRS, where “0” was considered “no pain” and “10” as “the worst pain imaginable”. Subjects were informed that they could stop the pain at any time by asking the experimenter to stop the infusion pump; no subject requested early
96 Chapter 5 – Consistent MSNA responses to tonic muscle pain termination of the infusion. At the end of the experiment subjects completed a McGill
Pain Questionnaire and drew the area(s) of pain on an image of a leg.
5.2.4 Data analysis
The commencement of infusion was denoted time zero; data recorded five minutes prior to the infusion were termed ‘baseline’. Individual bursts of MSNA were analysed from the RMS-processed signal using Peak Analysis module, part of
LabChart 7 software (ADInstruments, Sydney, Australia) and the amplitude of each burst computed. The mean number of bursts per minute and total burst amplitude was calculated during the baseline period and during infusion of hypertonic saline in five- minute blocks. MSNA values were normalized to individual baseline values and expressed as a percentage of change compared with baseline. Blood pressure (BP) and heart rate (HR) were calculated using the Blood Pressure and ECG modules respectively part of LabChart v7.3.1 software (ADInstruments, Sydney, Australia) as absolute values and averaged into five-minute blocks. All statistical analysis was performed using Prism v6.00 for Mac OS X (GraphPad software, San Diego,
California, USA). For all statistical tests, a probability level of <0.05 was regarded as significant. All values are expressed as means and standard error.
5.3 Results
Intramuscular infusions of hypertonic saline were made into the tibialis anterior muscle of 15 subjects on two occasions, 2 to 27 weeks apart. By controlling the infusion rate we were able to produce steady-state levels of pain in the muscle belly for 60 minutes; in 8 subjects the pain also referred to the ankle. At the completion of the recording subjects were asked to describe the pain according to the McGill Pain
97 Chapter 5 – Consistent MSNA responses to tonic muscle pain
Questionnaire: the most common descriptors were “sore” (80%), “dull” (75%),
“aching” (75%) and “hurting” (68%). Subjects were also asked to draw the area of pain on a picture of the leg: there were no differences in the regions of muscle pain between the two recording sessions. Moreover, pain intensities, as measured on a visual analog scale (0-10), were not significantly different between the first (5.8 ±
0.4) and second (6.1 ± 0.2) recording sessions.
There were no differences in resting MSNA, measured as burst frequency, between the first (21.5 ± 1.4 bursts/min) and second (23.0 ± 2.6 bursts/min, p=0.83; paired t- test) sessions. Between the two recording sessions there were no differences in resting mean blood pressure (90.7 ± 3.9 vs 89.4 ± 4.4 %, p=0.93; paired t-test) or heart rate
(71.1 ± 2.8 vs 74.1 ± 4.1 %, p=0.46; paired t-test). In addition, there were no differences in the peak changes in blood pressure or heart rate in the two recording sessions during the infusion of hypertonic saline.
As described previously (Fazalbhoy et al. 2012), we could define two groups of subjects: (i) those in whom muscle sympathetic nerve activity (MSNA) mean burst amplitude increased during long-lasting muscle pain, and (ii) those in whom amplitude decreased. In the current study 10 of the 15 subjects showed a significant decrease in burst amplitude (52.0 ± 5.4 %, p<0.0001, t-test) and five an increase
(143.5 ± 6.1 %, p=0.002, t-test) in the first recording session. Experimental records from one subject who exhibited an increase in MSNA during the infusion of hypertonic saline are shown in Fig. 5.1.
98 Chapter 5 – Consistent MSNA responses to tonic muscle pain
Figure 5.1 Experimental records from one subject. Neural activity was recorded from the motor fascicle supplying extensor digitorum longus; the RMS-processed signal is shown in the lowest trace.
This subject generated little spontaneous muscle sympathetic nerve activity (MSNA) at rest, shown immediately prior to the commencement of the infusion of hypertonic saline into the ipsilateral tibialis anterior muscle (A), but both burst frequency and amplitude increased during tonic muscle pain, shown
27 minutes after the start of the infusion (B). Blood pressure also increased. Pain was maintained at a level of 5/10 throughout the infusion period.
99 Chapter 5 – Consistent MSNA responses to tonic muscle pain
Figure 5.2 Changes in mean MSNA burst amplitude (A, B), mean blood pressure (C, D) and heart rate
(E, F) during tonic muscle pain induced by intramuscular infusion of hypertonic saline for two subjects
(A, C, E and B, D, F) during two recording sessions. The subject illustrated on the left exhibited a sustained increase in MSNA, BP, and HR during the course of infusion, while the subject illustrated on the right showed a decrease. For both subjects the direction of the changes was the same in both recording sessions. Mean burst amplitudes were calculated over 5 min epochs, normalised to the 5 min period immediately preceding infusion.
100 Chapter 5 – Consistent MSNA responses to tonic muscle pain
Eleven (73%) subjects showed a consistent pattern in both recording sessions: six of the 10 subjects in whom MSNA decreased in the first session showed the same pattern in the second session, while all five subjects who exhibited an increase in the first session also showed an increase in the second. Changes in mean burst amplitudes, mean blood pressure and heart rate are shown for two subjects in Fig. 5.2.
It can be seen that the overall magnitude of the change in burst amplitude, blood pressure and heart rate are similar in both recording sessions, although the time at which the peak change occurred were not necessarily the same. Nevertheless, on average, there were no significant differences in the magnitudes of the changes in burst amplitude across sessions for the group showing consistent decreases in MSNA
(55.6 ± 6.8 vs 52.1 ± 8.1 %, p=0.83, n=6; Wilcoxon) or the group showing consistent increases (143.5 ± 6.1 vs 149.9 ± 15.7 %, p=0.81, n=5; Wilcoxon); the same was true for the times at which the peak decrease (34.2 ± 4.7 vs 31.7 ± 5.6 min, p>0.99;
Wilcoxon) or peak increase (28.0 ± 7.5 vs 31.0 ± 7.0 min, p=0.25; Wilcoxon) occurred. Furthermore, there was no significant difference in baseline MSNA for the group that showed a consistent increase and the group that showed a consistent decrease in MSNA during tonic muscle pain (23.6 ± 1.5 vs 18.3 ± 1.5 bursts/min, p=0.07; Mann-Whitney). Finally, there was no apparent effect of gender that could account for the divergent changes in MSNA: of the subjects who showed a consistent decrease in MSNA, five were male and one was female, and of the group that showed a consistent increase in MSNA, three were male and two female.
101 Chapter 5 – Consistent MSNA responses to tonic muscle pain
Figure 5.3 Data from all subjects, showing the relationships between the peak changes in MSNA burst amplitude during tonic muscle pain recorded in session 1 (X axis) and session 2 (Y axis). Six subjects
(open circles) showed consistent decreases in burst amplitude while 5 (filled circles) showed consistent increases. The asterisks represent data from 4 subjects who did not demonstrate consistency across sessions: they each showed decreases in MSNA during the first session but increases in the second.
Figure 5.3 shows the relationships between the changes in burst amplitude during tonic muscle pain in session 1 and session 2. For the 11 subjects who showed consistent changes in MSNA burst amplitude there was a very good linear relationship between the changes observed in one recording session and the next
(r2=0.82, p<0.0001). While the four remaining subjects (three males, one female)
102 Chapter 5 – Consistent MSNA responses to tonic muscle pain indicated by asterisks in Fig. 5.3 did not exhibit reproducibility in response pattern over time, they nevertheless shared certain features: they all showed a marked decrease in burst amplitude with the first exposure to tonic muscle pain (46.6 ± 9.2 % of baseline), and a marked increase on the second exposure (159.6 ± 8.9 %). There were no differences in the magnitudes of these changes compared to those seen in the subjects who exhibited consistent responses. Moreover, there were no differences in pain ratings between the first and second sessions (5.4 ± 0.5 vs 5.8 ± 0.7; paired t-test) that could potentially account for these disparate responses, and no differences in pain ratings between these subjects and the 11 who did show consistent patterns over time
(5.9 ± 0.5 and 6.2 ± 0.2 for sessions 1 and 2, respectively).
5.4 Discussion
The current study extends our recent work on the effects of selective stimulation of nociceptors in muscle or skin on sympathetic outflow to muscle or skin (Burton et al.
2009a; Burton et al. 2009b; Fazalbhoy et al. 2012; Hall et al. 2012), and on fusimotor drive to relaxed leg muscles (Birznieks et al. 2008; Fazalbhoy et al. 2013). In each of the studies in which we used intramuscular infusions of hypertonic saline (Fazalbhoy et al. 2012; Hall et al. 2012; Fazalbhoy et al. 2013), we titrated the rate of infusion to produce a steady-state level of pain and interpreted our results in terms of changes in the output in the particular system we were studying - sympathetic outflow to skin, sympathetic outflow to muscle, or fusimotor drive to muscle – and did not consider the potential effects of any changes in blood pressure on the output. This is not unreasonable, given that (i) changes in skin sympathetic nerve activity are independent of any changes in blood pressure (Hall et al. 2012), that (ii) the increases or decreases in MSNA preceded the changes in blood pressure (Fazalbhoy et al.
103 Chapter 5 – Consistent MSNA responses to tonic muscle pain
2012) and that (iii) decreases in blood pressure have no effects on the firing of human muscle spindle afferents (Macefield et al. 2003). In the current study we investigated the effects of tonic muscle pain on muscle sympathetic nerve activity over two sessions on two separate occasions, in order to determine whether the pattern of response is reproducible over time. This was based on the premise that resting MSNA is consistent day to day, month to month, year to year (Sundlof & Wallin 1977;
Fagius & Wallin 1993). Previously we have shown, in 12 subjects, that there are two distinct responses to tonic muscle pain – an increase in MSNA, and a parallel increase in BP and HR, or a decrease in MSNA and parallel decrease in BP and HR
(Fazalbhoy et al. 2012). Here we have shown that the changes in MSNA during tonic muscle pain are consistent over time, with a tight linear relationship. And while we only studied two time points, the consistency in the direction of changes in MSNA in the first and second recording sessions in the majority of subjects, despite the two sessions being weeks to months apart, suggests that subsequent recordings would show the same patterns.
What is perplexing is why there were two patterns of vasomotor response to long- lasting muscle pain – an increase or a decrease in MSNA – despite identical pain ratings. A decrease in MSNA fits with the bradycardia and hypotension reported during early studies of deep pain in human subjects (Lewis 1942; Feinstein et al.
1954). We had shown that intramuscular injection of hypertonic saline into a leg muscle failed to induce any changes in blood pressure or heart rate in people with spinal cord injury, leading us to conclude that nociceptor-driven changes in sympathetic outflow depend on supraspinal, rather than spinal, mechanisms (Burton et al. 2008). Indeed, we know that muscle pain engages many areas of the brain
104 Chapter 5 – Consistent MSNA responses to tonic muscle pain involved in autonomic control, including the insula and anterior cingulate cortex, that there are differences in the central processing of noxious inputs originating in muscle and skin (Henderson et al. 2006; Henderson et al. 2007; Macefield et al. 2008;
Henderson et al. 2011), and that there are multiple sites at which interaction between noxious inputs and cardiovascular control can occur (Bruehl & Chung 2004). What we do not know is whether there are differences in central processing of noxious inputs in subjects with divergent sympathetic responses.
It is likely that one site in particular – the midbrain periaqueductal gray (PAG) – is involved in generating these opposing sympathetic responses. Studies in experimental animals have shown that the PAG plays an important role in furnishing the autonomic and behavioural responses to pain (Carrive & Bandler 1991; Depaulis et al. 1992).
Organised in a columnar fashion, the lateral columns of the PAG receive noxious inputs from the skin, while noxious inputs from muscle project to the ventrolateral columns (Keay & Bandler 1993; Keay et al. 1994; Keay & Bandler 2002).
Importantly, stimulation of the lateral columns increases blood pressure, heart rate and arousal - typical components of a fight or flight response - whereas activation of the ventrolateral columns evokes resting behavior, coupled with hypotension and bradycardia (Depaulis et al. 1992). According to these input-output relationships of the ventral and ventrolateral columns of the PAG, in which the behavioural and autonomic responses can be interpreted as appropriate physiological reactions to escapable and inescapable pain (Keay et al. 1994), one would attribute the falls in
MSNA, blood pressure and heart rate seen in our previous study (Fazalbhoy et al.
2012) and the current investigation to activation of the ventrolateral columns of the
PAG. Conversely, the increases in MSNA, blood pressure and heart rate could be
105 Chapter 5 – Consistent MSNA responses to tonic muscle pain ascribed to activation of the lateral columns, i.e. the areas activated by cutaneous pain in experimental animals. Nevertheless, we must reiterate that the cardiovascular responses to pain in anaesthetised animals are not necessarily the same as those seen in awake human subjects. Indeed, based on the animal literature, we would not have predicted that tonic muscle pain would cause a sustained decrease in skin sympathetic nerve activity (SSNA), accompanied by a sustained increase in skin blood flow, after the initial transient increase in SSNA and fall in skin blood flow (Hall et al. 2012).
5.4.1 Methodological considerations
Intramuscular injection or infusion of hypertonic saline is a well-validated model of muscle pain, with infusion for 20 minutes having been accepted as a model of chronic pain (Capra & Ro 2004). In our laboratory we have used this model to show that muscle pain does not affect the firing of spontaneously active muscle spindle afferents in relaxed leg muscles via changes in fusimotor drive (Birznieks et al., 2008a,b;
Fazalbhoy et al., 2013), yet does change sympathetic nerve activity to both muscle
(Burton et al., 2009a; Fazalbhoy et al., 2012) and skin (Burton et al., 2009b; Hall et al., 2012). The effects of long-lasting muscle pain on muscle sympathetic nerve activity have been further validated in the current study, in which the same subjects were studied on two occasions. Obviously, subjects knew what to expect in the second experimental session, conducted weeks to months after the first, but were still not aware of when the infusion of hypertonic saline would commence. It should be emphasized that we are looking at experimental muscle pain: the subjects are reporting pain from a baseline of zero. This is not the same as a chronic pain condition in which the pain has persisted for three months or more.
106 Chapter 5 – Consistent MSNA responses to tonic muscle pain
While we observed consistency in the sympathetic responses to tonic muscle pain in the majority of subjects examined in the current study, it was interesting that four subjects showed marked falls in MSNA during their initial exposure to experimental muscle pain but marked increases on their second exposure. The magnitudes of these changes were similar to those seen in the subjects that showed consistent decreases or increases, but why they showed divergent responses on the two occasions we are at a loss to explain. Certainly, there were no differences in resting blood pressure, heart rate or resting MSNA, and no differences in subjective pain ratings. Could it be that these subjects were more anxious the second time, knowing that they were going to be experiencing strong pain or an hour? If this was the case, we might have expected their resting levels of MSNA, as well as blood pressure and heart rate, to be elevated, but this was not the case. We do not believe there were any differences in state of mind in these four subjects on the two occasions, but we did not administer any psychological assessments to any of the subjects. Of course, it is possible that the increases in MSNA seen during the second exposure in these subjects may reflect central neuronal changes, perhaps related to central sensitization; future experiments will need to address this, but in other experiments we had shown that the central representation of pain could change over time (Rubin et al. 2010).
Acknowledgements
This work was supported by the National Health & Medical Research Council of
Australia. We are grateful to Dr Rachael Brown for her assistance in the experiments.
107 Chapter 6 – SSNA responses to tonic muscle pain
CHAPTER SIX
SKIN SYMPATHETIC NERVE ACTIVITY RESPONSES
TO TONIC MUSCLE PAIN
Chapter abstract
Skin sympathetic nerve activity (SSNA) controls skin blood flow and sweat release, and acute noxious stimulation of skin has been shown to cause a decrease in SSNA in the anaesthetised or spinal cat. In awake human subjects, acute muscle pain causes a transient rise in SSNA, but the impact of long-lasting stimulation of muscle nociceptors on skin sympathetic outflow, blood flow and sweat release is unknown.
We tested the hypothesis that tonic stimulation of muscle nociceptors causes a sustained increase in sympathetic outflow to the skin. SSNA was recorded from the common peroneal nerve of 10 awake human subjects. Tonic muscle pain was induced by infusing hypertonic saline (7 %) into the tibialis anterior muscle over ~40 min, titrated to achieve a constant level of muscle pain. SSNA initially increased following the onset of the infusion, reaching a peak of 164 % of baseline within 5 min, but then showed a prolonged and sustained decrease, reaching a nadir of 77 % in 20 min.
Conversely, skin blood flow (and vascular conductance) initially decreased, followed by a progressive increase; there were no consistent changes in sweat release. We conclude that sympathetic outflow to skin exhibits a biphasic response to long-lasting stimulation of muscle nociceptors: an initial increase presumably related to the
‘arousal’ component of pain, characterised by increased SSNA and decreased skin blood flow, followed by a prolonged decrease in SSNA and increased skin blood flow. The latter may be a purposeful response that contributes to wound healing.
108 Chapter 6 – SSNA responses to tonic muscle pain
6.1 Introduction
The sympathetic nervous system exercises a number of functions essential to the maintenance of a constant internal environment. In the skin, the sympathetic nervous system controls blood flow through cutaneous vasoconstrictor (and vasodilator) neurones and sweat release through sudomotor neurones; these actions are critically important in thermoregulation (Kellogg 2006). Direct recordings of skin sympathetic nerve activity (SSNA) via microelectrodes inserted percutaneously into peripheral nerves (microneurography) reveal irregular bursts of activity, the frequency and amplitude of which are lowest at thermoneutral ambient temperatures yet increase as temperature increases, thereby bringing about sweat release and cutaneous vasodilatation, or as temperature decreases, bringing about cutaneous vasoconstriction
(Bini et al. 1980a, b; Macefield & Wallin 1995, 1999). SSNA is only weakly associated with the cardiac rhythm (Hagbarth et al. 1972; Macefield & Wallin 1999), but bursts of activity are caused by brisk inspiratory efforts and various ‘arousal stimuli’, including unexpected sounds, mental arithmetic and emotional stimuli
(Delius et al. 1972b).
Nociceptive pain, originating from stimulation of nociceptors in the periphery, has a complex interaction with the sympathetic nervous system, as sympathetic outflow is dependent upon both the target organs and the tissue from which the noxious stimulus originates. In the rat, stimulation of nociceptors in muscle or the viscera (deep pain) preferentially activates the ventrolateral column of the periaqueductal grey matter
(vlPAG) (Keay & Bandler 1993), with a subsequent fall in blood pressure and heart rate (Keay et al. 1994). Conversely, stimulation of cutaneous nociceptors (superficial pain) activates the lateral column (lPAG) (Keay & Bandler 1993), which causes blood
109 Chapter 6 – SSNA responses to tonic muscle pain pressure and heart rate to increase (Depaulis et al. 1992). Horeyseck and Ja ̈nig
(1974a; 1974b) demonstrated that, in the anaesthetised cat, mechanical and heat- induced noxious stimulation of the skin decreases sympathetic nerve activity in the superficial (cutaneous) peroneal nerve, but increases sympathetic nerve activity to the gastrocnemius muscles. In awake human subjects, however, acute noxious stimulation of muscle or skin—induced by bolus intramuscular or subcutaneous injection of hypertonic saline—increases both SSNA (Burton et al. 2009b) and MSNA (Burton et al. 2009a). However, unlike the sustained increase in MSNA, which essentially paralleled the pain profile, the rise in SSNA was only transient and did not follow the pain profile; this transient increase in SSNA may simply be a reflection of the well- known ‘arousal response’ of SSNA to alerting stimuli. The mechanisms that underlie the sympathetic responses to pain are unclear. Horeyseck and Ja ̈nig found similar results with both the anaesthetised (Horeyseck & Jänig 1974b) and spinalised
(Horeyseck & Jänig 1974a) cats, implicating a spinal- reflex mechanism. However, in persons with spinal cord injury, both deep (muscular) and superficial (skin) pain failed to cause any significant change in skin blood flow or sweat release, implicating supraspinal mechanisms in mediating the responses to noxious stimulation in humans
(Burton et al. 2008). In other words, we believe that—in awake human subjects—the sympathetic responses to noxious stimulation reflect the higher-order processing of pain, specifically the affective components, rather than any spinal reflex.
Currently, the effects of long-lasting muscular pain on skin sympathetic nerve activity are unknown. Tonic muscle pain has been used as a model for chronic musculoskeletal pain (Capra & Ro 2004) and has the advantage of allowing a controlled investigation into how such pain may modulate SSNA, skin blood flow and
110 Chapter 6 – SSNA responses to tonic muscle pain sweat release. Conversely, assuming everything else is equal, one would need to know the level of SSNA in a person prior to the development of chronic pain in order to interpret any changes in sympathetic outflow. Indeed, microelectrode recordings of sympathetic nerve traffic in human subjects have found no differences in sympathetic outflow to a painful limb compared with the contralateral non-painful limb in patients with chronic regional pain syndrome that was suspected to be sympathetically maintained because of the marked cutaneous vasoconstriction (Casale & Elam 1992).
We recently showed that prolonged intramuscular infusion of hypertonic saline into the tibialis anterior muscle, which causes a deep pain that lasts up to an hour, causes sustained increases in muscle sympathetic nerve activity (MSNA), blood pressure and heart rate in some individuals yet sustained decreases in each of these parameters in others (Fazalbhoy et al. 2012). The purpose of the present study is to use this same model to test the hypothesis that tonic muscle pain causes a sustained increase in
SSNA, related to the affective component of pain.
6.2 Methods
6.2.1 Recording procedures
Experiments were performed on 10 healthy volunteers (9 male, 1 female; age range
19–31). All subjects were seated in a semi-reclined position at a comfortable ambient temperature in a temperature-controlled room (22 °C). Sympathetic nerve activity was recorded from cutaneous fascicles of the common peroneal nerve via a tungsten microelectrode (FHC, Bowdoinham, ME, USA) inserted percutaneously at the fibular head; an uninsulated reference microelectrode was inserted subdermally ~1 cm away.
Intraneural stimulation (0.01–1.0 mA, 1 Hz, 0.2 ms pulses), delivered to the
111 Chapter 6 – SSNA responses to tonic muscle pain microelectrode via an isolated stimulator (Stimulus Isolator, ADInstruments, Sydney,
Australia), was used to guide the microelectrode tip into a cutaneous fascicle of the nerve, as judged by the generation of radiating paraesthesiae—either down the lateral aspect of the leg or on the dorsum of the foot—and the absence of muscle twitches at currents B0.02 mA. Neural activity was amplified (gain 2 9 104) and filtered at 0.3–
5.0 kHz using a low-noise amplifier (NeuroAmpEX, ADInstruments, Sydney,
Australia) and the identity of the cutaneous fascicle confirmed by the afferent responses to light stroking of the fascicular innervation zone. The microelectrode was manually advanced until spontaneous bursts of skin sympathetic nerve activity
(SSNA) were encountered, identified by the following features: (1) a burst could be evoked by a brisk sniff, and, with the subject’s eyes closed, an arousal burst could be evoked by an unexpected tap on the nose or a loud shout (Delius et al. 1972b); (2) the bursts were typically longer than those comprising muscle sympathetic nerve activity
(MSNA); and (3) unlike MSNA, there was no sustained increase in burst amplitude and frequency during an inspiratory-capacity apnoea (Macefield & Wallin 1995).
Subjects were asked not to talk during the recording and the experimenters also refrained from talking; extraneous noise or distractions in the laboratory were kept to a minimum.
Heart rate was monitored via standard surface Ag/AgCl ECG chest electrodes (0.3–
1.0 kHz) and sampled at 2 kHz. Continuous blood pressure was measured non- invasively using finger pulse plethysmography (Finometer Pro, Finapres Medical
Systems, The Netherlands) and sampled at 400 Hz. Respiration (DC-10 Hz) was recorded via a strain gauge transducer (Pneumotrace, UFI, USA) strapped around the chest and sampled at 100 Hz. Changes in pulsatile skin blood volume (0.3–100 Hz),
112 Chapter 6 – SSNA responses to tonic muscle pain reflecting changes in skin perfusion (blood flow), were monitored via infrared photoelectric plethysmography probes attached to the pad of the ipsilateral big toe
(MLT1020, ADInstruments, Sydney, Australia) and sampled at 400 Hz. Changes in electrical skin potential (0.1–10 Hz) between the plantar and dorsal surfaces of the foot (BioAmplifier, ADInstru- ments, Australia) were used as a measure of sweat release. Because of technical reasons, skin blood flow could not be recorded in one subject and sweat release could not be recorded in three subjects. All data were acquired on a computer-based data acquisition and analysis system (PowerLab 16SP,
ADInstruments, Sydney, Australia).
6.2.2 Noxious stimulation
A 23G butterfly cannula, connected to an infusion pump (Harvard Instruments, USA), was inserted 2 cm into the belly of the tibialis anterior muscle. Resting SSNA, ECG, blood pressure, respiration, skin blood flow and sweat release were recorded for a minimum of 10 min to establish a baseline (control period). After the control period, the intramuscular infusion of 7 % hypertonic saline was commenced at 0.25 ml/min at a time unknown to the subject. The subjective pain level was monitored on a visual analogue scale (VAS), where 0 is described as ‘no pain’ and 10 as ‘the worst pain ever experienced’. The instantaneous pain level was recorded by asking the subject to turn a calibrated, labelled potentiometer and the infusion adjusted to maintain a constant level around 6/10. The infusion was continued for up to 50 min, with the pain gradually abating over ~10 min following cessation of the infusion. At the conclusion of the experiment, subjects were asked to describe the quality of the sensations using the McGill Pain Questionnaire.
113 Chapter 6 – SSNA responses to tonic muscle pain
6.2.3 Analysis
The time between cannula insertion and the commencement of the infusion was used to calculate baseline values for all parameters. The infusion period was separated into
5-min intervals. This included the first 30 min (6 consecutive blocks), a period of variable duration (10.1 ± 1.5 min; range 2.1–14.7), and the final 5 min of the infusion.
This allowed analysis to accommodate for variation in infusion duration, which depended on the infusion rate required to maintain a constant level of pain. The raw
SSNA signal was converted into a root mean square (RMS)-processed signal with a moving time average calculated over 0.1 s. Bursts of sympathetic nerve activity were identified using Peak Analysis (ADInstruments, Sydney, Australia). SSNA burst area was calculated using the area under the curve minus baseline neural activity. The mean SSNA burst area for each 5-min period was normalised to the control period and expressed as a percentage change. Skin perfusion was measured as the mean amplitude of the pulse plethysmogram over each 5-min epoch, normalised to the mean amplitude in the baseline period; respiratory amplitude was measured in a similar fashion. Skin potential was measured as the absolute value over each 5-min period, again normalised to the baseline period. Blood pressure, heart rate and respiratory rate were measured as absolute values, calculated over 5-min epochs.
Changes in cutaneous vascular conductance were calculated as the ratio between normalised skin perfusion and normalised mean blood pressure. Data are presented as mean ± standard error of the mean (SEM). Repeated-measures analysis of variance
(ANOVA), coupled with Tukey’s multiple comparison tests, was performed using
Prism 5.0 for Macintosh (GraphPad Software, USA).
114 Chapter 6 – SSNA responses to tonic muscle pain
6.3 Results
Intramuscular infusion of hypertonic saline caused a constant pain, which was typically described as dull and aching. On average, the onset of pain occurred after 81
± 17 s (range 10–152 s). Pain VAS scores averaged 5.02 ± 0.31 (range 3.58–6.42/10) over the infusion period. The total duration of infusion averaged 41.1 ± 3.2 min.
Spontaneous skin sym- pathetic nerve activity (SSNA) occurred as apparently random bursts of activity, at a baseline rate of 25.7 ± 1.5 bursts/min (range 18–33 bursts/min).
Burst area and amplitude were directly proportional (R2 = 0.6879; two-tailed p \
0.0001); accordingly, only burst area was used in the analysis.
Experimental records from one subject are shown in Fig. 6.1. Skin sympathetic nerve activity (SSNA) increased above the baseline values during the first few minutes of the infusion, but then decreased, despite the persistent pain, which this subject rated at
6/10. These increases in SSNA were observed in 8 of 10 subjects. The period of highest SSNA occurred within the first 5 min of the infusion. Across subjects, there was no relationship between the magnitude of the peak increase in SSNA and the pain rating (p = 0.96). All subjects exhibited a sustained decrease in SSNA after the initial increase.
115 Chapter 6 – SSNA responses to tonic muscle pain 110 Exp Brain Res (2012) 221:107–114
Fig. 1 Experimental records from one subject. Spontaneous SSNA and cutaneous vasoconstriction were present in the control period, recorded 1 min prior to the start of the infusion of hypertonic saline (a). This increased within the first few minutes of the infusion (b) but then showed a subsequent decrease, coupled with an increase in skin blood flow, later in the infusion (c) despite a constant level of pain. SSNA started to increase once the infusion had stopped during the recovery period (d); a technical issue meant the blood pressure recording was not available during this period
Figure 6.1 Experimental records from one subject. Spontaneous SSNA and cutaneous vasoconstriction were magnitude of the peak increase in SSNA and the pain lowest level in 20 min (76.8 ± 2.9 %; p \ 0.001) and rating (p = 0.96). All subjectspresent inexhibited the control a period, sustained recorded 1 remained min prior to low the for start the of the remainder infusion of of hypertonic the infusion—with saline (a). This the decrease in SSNA after theincreased initial increase. within the first In mostfew minutes sub- of theexception infusion (b) of but the then final showed 5 min a subsequent of infusion, decrease, when coupled it returned with an to jects (8/10), the SSNA dropped to levels below those of the control levels (100.7 ± 4.3 %). Mean data from all 10 control period, although in twoincrease subjects in skin blood SSNA flow, remained later in the infusionsubjects (c) despite areshown a constant graphically level of pain. in SSNA Fig. started2a. to increase once elevated throughout the infusion. It is worth pointing out All subjects showed an initial decrease in skin blood the infusion had stopped during the recovery period (d); a technical issue meant the blood pressure recording was that, although the indwelling cannula had been inserted flow and vascular conductance, as measured from the pad prior to recording the baselinenot available period, during subjects this period did know of a toe. However, after the initial vasoconstriction, the that—at some point in time unknown to them—the infu- effect reversed its direction and skin blood flow started to sion was going to commence and that they were going to increase; in 6 subjects, this increase exceeded the baseline experience deep pain in the leg that would last up to an skin blood flow. Mean data from all subjects are shown in hour. Because of this, one would expect a certain degree of Fig. 2b. Skin blood flow was lowest during the first 5 min anxiety during this baseline period which would evidence (75.3 ± 1.2 %; p \ 0.001), after which it increased itself as an overall elevation of resting SSNA, even though beyond the control levels, reaching a maximum in the final they knew that, should the pain become intolerable, they 5 min of the infusion (163.2 ± 3.4 %; p \ 0.001). Skin could ask the infusion of hypertonic saline to be stopped at blood flow remained high during the recovery period. any time. On average, SSNA was highest during the first Although the same trends were seen in vascular conduc-
5 min of infusion (163.7 ± 6.4 %; p \ 0.001), reached its tance, these failed to reach statistical significance (Fig. 2c).
123 116 Chapter 6 – SSNA responses to tonic muscle pain
In most subjects (8/10), the SSNA dropped to levels below those of the control period, although in two subjects SSNA remained elevated throughout the infusion. It is worth pointing out that, although the indwelling cannula had been inserted prior to recording the baseline period, subjects did know that—at some point in time unknown to them—the infusion was going to commence and that they were going to experience deep pain in the leg that would last up to an hour. Because of this, one would expect a certain degree of anxiety during this baseline period which would evidence itself as an overall elevation of resting SSNA, even though they knew that, should the pain become intolerable, they could ask the infusion of hypertonic saline to be stopped at any time. On average, SSNA was highest during the first 5 min of infusion (163.7 ±
6.4 %; p < 0.001), reached its lowest level in 20 min (76.8 ± 2.9 %; p < 0.001) and remained low for the remainder of the infusion—with the exception of the final 5 min of infusion, when it returned to control levels (100.7 ± 4.3 %). Mean data from all 10 subjects are shown graphically in Fig. 6.2a.
117 Chapter 6 – SSNA responses to tonic muscle pain Exp Brain Res (2012) 221:107–114 111
Fig. 3 Changes in heart rate and blood pressure during tonic muscle pain. Tonic muscle pain caused significant increases in heart rate (a) but no significant change in mean blood pressure (b). The period of infusion is represented by the black bars. **p \ 0.02, ***p \ 0.001
Skin potential (sweat release) fluctuated throughout both control and infusion periods, but there was no consistent change at any stage (p = 0.43). Baseline values were obtained for heart rate (61.4 ± 2.4 beats/min; range 52.1–75.8 bpm), mean blood pressure (81.1 ± 3.3; range 63.6–96.9 mmHg) and respiratory rate (16.8 ± 0.65 breaths/min; range 12.9–19.0 breaths/min). Heart rate showed a small but significant increase throughout the entire infusion (Fig. 3a). Mean blood Fig. 2 Changes in skin sympathetic nerve activity (SSNA) and skin pressure increased in five of the nine subjects in whom it Figure 6.2 Changes in blood skin sympathetic flow during nerve tonic muscle activity pain. (SSNA) Tonic and muscle skin pain blood caused flow an during was tonicrecorded muscle pain. and showed a slight decrease in four; on initial increase in SSNA (a) and a decrease in skin blood flow (b) and average, there was a tendency for blood pressure to Tonic muscle pain causedvascular an initial conductance increase (c ), in followed SSNA (a) by a and subsequent a decrease decrease in skin in SSNA blood flowincrease, (b) and butvascular this failed to reach statistical significance and increase in skin blood flow and vascular conductance. The period conductance (c), followedof by infusion a subsequent is represented decrease byin SSNA the black and increase bars. ** inp \ skin0.02, blood (Fig. flow 3 andb). vascular Respiratory rate and depth remained stable ***p \ 0.001 throughout the entire infusion, with the exception of the conductance. The period of infusion is represented by the black bars. **p < 0.02, ***p < 0.001
123
118 Chapter 6 – SSNA responses to tonic muscle pain
All subjects showed an initial decrease in skin blood flow and vascular conductance,
as measured from the pad of a toe. However, after the initial vasoconstriction, the
effect reversed its direction and skin blood flow started to increase; in 6 subjects, this
increase exceeded the baseline skin blood flow. Mean data from all subjects are
shown in Fig. 6.2b. Skin blood flow was lowest during the first 5 min (75.3 ± 1.2 %; p
< 0.001), after which it increased beyond the control levels, reaching a maximum in
the final 5 min of the infusion (163.2 ± 3.4 %; p < 0.001). Skin blood flow remained
high during the recovery period. Although the same trends were seen in vascular
Exp Brain Res (2012) 221:107–114 conductance, these failed to reach statistical significance (Fig. 6.2c).111
Figure 6.3 Changes in Fig. heart 3 Changes rate and in blood heart rate pressure andblood during pressure tonic during muscle tonic pain. muscle Tonic muscle pain caused pain. Tonic muscle pain caused significant increases in heart rate (a) but no significant change in mean blood pressure (b). The period significant increases in heart rate (a) but no significant change in mean blood pressure (b). The period of infusion of infusion is represented by the black bars. **p \ 0.02, ***p \ 0.001 is represented by the black bars.**p < 0.02, ***p < 0.001
Skin potential (sweat release) fluctuated throughout both control and infusion periods, but there was no consistent change at any stage (p = 0.43). 119 Baseline values were obtained for heart rate (61.4 ± 2.4 beats/min; range 52.1–75.8 bpm), mean blood pressure (81.1 ± 3.3; range 63.6–96.9 mmHg) and respiratory rate (16.8 ± 0.65 breaths/min; range 12.9–19.0 breaths/min). Heart rate showed a small but significant increase throughout the entire infusion (Fig. 3a). Mean blood Fig. 2 Changes in skin sympathetic nerve activity (SSNA) and skin pressure increased in five of the nine subjects in whom it blood flow during tonic muscle pain. Tonic muscle pain caused an was recorded and showed a slight decrease in four; on initial increase in SSNA (a) and a decrease in skin blood flow (b) and average, there was a tendency for blood pressure to vascular conductance (c), followed by a subsequent decrease in SSNA increase, but this failed to reach statistical significance and increase in skin blood flow and vascular conductance. The period of infusion is represented by the black bars. **p \ 0.02, (Fig. 3b). Respiratory rate and depth remained stable ***p \ 0.001 throughout the entire infusion, with the exception of the
123 Chapter 6 – SSNA responses to tonic muscle pain
Skin potential (sweat release) fluctuated throughout both control and infusion periods, but there was no consistent change at any stage (p = 0.43).
Baseline values were obtained for heart rate (61.4 ± 2.4 beats/min; range 52.1–75.8 bpm), mean blood pressure (81.1 ± 3.3; range 63.6–96.9 mmHg) and respiratory rate
(16.8 ± 0.65 breaths/min; range 12.9–19.0 breaths/min). Heart rate showed a small but significant increase throughout the entire infusion (Fig. 6.3a). Mean blood pressure increased in five of the nine subjects in whom it was recorded and showed a slight decrease in four; on average, there was a tendency for blood pressure to increase, but this failed to reach statistical significance (Fig. 6.3b). Respiratory rate and depth remained stable throughout the entire infusion, with the exception of the final 5 min, where there was a significant increase in depth (p <0.01), but not rate. Mean data are shown in Fig. 6.4.
120 Chapter 6 – SSNA responses to tonic muscle pain 112 Exp Brain Res (2012) 221:107–114
(Burton et al. 2009b). We believed this was due to the emotional valency of the stimulus—an ‘arousal response’ to the perception of deep muscle pain and interpretation of its emotional valency, rather than to a nociceptor-driven reflex. Interestingly, unlike conscious humans, studies using anaesthetised and spinalised cats have shown that noxious stimulation of skin actually causes a decrease in SSNA (Horeyseck and Ja¨nig 1974a, b). Moreover, we saw no evidence for divergent responses across subjects: we recently showed that, in some subjects, intramuscular infusion of hypertonic saline causes a sustained increase in sympathetic nerve activity to muscle, an increase in heart rate and an increase in blood pressure, whereas in other subjects sustained decreases in each of these parameters were observed (Fazalbhoy et al. 2012). The biphasic response of SSNA to tonic muscle pain seen in our study may provide an explanation for the dis- parity between human and animal data. Nociceptive pain may cause a reduction in SSNA in both cats and humans, but the onset of pain is associated with an initial, overriding ‘arousal response’ in conscious subjects; we do not know whether pain causes decreases in sympathetic outflow to skin in awake cats, and strongly suspect that cutaneous vasoconstriction would occur in the awake cat as in the human. The tonic pain model used in the current study allowed us to monitor changes in SSNA beyond the initial responses to the onset of pain. After the initial increase, we found that SSNA fell below baseline levels, which is more consistent with the results found in the anaesthetised or spinal cat. In the majority of subjects, SSNA and skin Fig. 4 Changes in respiratory rate and amplitude during tonic muscle blood flow were inversely related, suggesting that changes Figure 6.4 Changes in pain.respiratory Tonic muscle rate and pain amplitude caused significant during changestonic muscle in respiratory pain. rate Tonic musclein SSNA pain reflected caused the activity of noradrenergic vasocon- (a) but a significant increase in respiratory depth during the final strictor neurons, rather than the cholinergic vasodilator 5 min of the infusion and during the recovery period (b). The period significant changes in respiratory rate (a) but a significant increase in respiratory depth duringneurons—where the final 5 min of a direct relationship would be expected. of infusion is represented by the black bars.*p \ 0.05, **p \ 0.02 the infusion and during the recovery period (b). The period of infusion is represented by the blackOf bars. course, *p \ 0.05, one must recognise that we were measuring SSNA to the hairy skin, but blood flow was measured from **p \ 0.02 final 5 min, where there was a significant increase in depth the pad of a toe—glabrous skin. This is a clear limitation of (p \ 0.01), but not rate. Mean data are shown in Fig. 4. this study, though the most parsimonious interpretation is that reductions in blood flow occurred also in the hairy skin to which the recorded SSNA was directed. It is known that 6.4 Discussion Discussion the glabrous skin of the fingers and toes contains many arterioles and arteriovenous anastomoses, through which We have documented,We have for documented, the first fortime, the first the time, effects the effects of long of long-- lastingthe experimental flow is determined exclusively by cutaneous vasocon- lasting experimental muscle pain on skin sympathetic strictor neurones (Roddie 1983). Conversely, blood flow muscle pain on skinnerve sympathetic activity in awake nerve human activity subjects. in Our awake findings human do in subjects. hairy skin Our is governed also by active vasodilatation not lend support to our original hypothesis that tonic (Kellogg 2006). While there is strong evidence for the muscle pain causes a sustained increase in SSNA. Rather, findings do not lend support to our original hypothesis that tonic musclepresence pain causes of sympathetic a cholinergic neurones that mediate there was an initial increase followed by a prolonged vasodilation (Kellogg et al. 1995; Sugenoya et al. 1998), decrease in SSNA. We had previously shown that acute there is little evidence to show that sympathetic vasodilator sustained increasemuscle in SSNA. and skin Rather, pain, induced there by was bolus an intramuscular initial increase or followedand sudomotor by aneurones are separate entities. Sugenoya subcutaneous injections of hypertonic saline, was found to et al. (1998) showed that in a mildly heated environment, prolonged decreasecause in SSNA. increases We in SSNA;had previously however, the shown rise peaked that acute soon muscleisolated and vasodilation skin followed just 1 % of SSNA bursts, after the onset of pain without following the pain profile compared with 10 % for ‘sweat only’ and 70 % for pain, induced by bolus intramuscular or subcutaneous injections of hypertonic saline, 123
121 Chapter 6 – SSNA responses to tonic muscle pain was found to cause increases in SSNA; however, the rise peaked soon after the onset of pain without following the pain profile (Burton et al. 2009b). We believed this was due to the emotional valency of the stimulus—an ‘arousal response’ to the perception of deep muscle pain and interpretation of its emotional valency, rather than to a nociceptor-driven reflex. Interestingly, unlike conscious humans, studies using anaesthetised and spinalised cats have shown that noxious stimulation of skin actually causes a decrease in SSNA (Horeyseck & Jänig 1974a; 1974b). Moreover, we saw no evidence for divergent responses across subjects: we recently showed that, in some subjects, intramuscular infusion of hypertonic saline causes a sustained increase in sympathetic nerve activity to muscle, an increase in heart rate and an increase in blood pressure, whereas in other subjects sustained decreases in each of these parameters were observed (Fazalbhoy et al. 2012).
The biphasic response of SSNA to tonic muscle pain seen in our study may provide an explanation for the disparity between human and animal data. Nociceptive pain may cause a reduction in SSNA in both cats and humans, but the onset of pain is associated with an initial, overriding ‘arousal response’ in conscious subjects; we do not know whether pain causes decreases in sympathetic outflow to skin in awake cats, and strongly suspect that cutaneous vasoconstriction would occur in the awake cat as in the human. The tonic pain model used in the current study allowed us to monitor changes in SSNA beyond the initial responses to the onset of pain. After the initial increase, we found that SSNA fell below baseline levels, which is more consistent with the results found in the anaesthetised or spinal cat. In the majority of subjects,
SSNA and skin blood flow were inversely related, suggesting that changes in SSNA reflected the activity of noradrenergic vasoconstrictor neurons, rather than the
122 Chapter 6 – SSNA responses to tonic muscle pain cholinergic vasodilator neurons—where a direct relationship would be expected.
Of course, one must recognise that we were measuring SSNA to the hairy skin, but blood flow was measured from the pad of a toe—glabrous skin. This is a clear limitation of this study, though the most parsimonious interpretation is that reductions in blood flow occurred also in the hairy skin to which the recorded SSNA was directed. It is known that the glabrous skin of the fingers and toes contains many arterioles and arteriovenous anastomoses, through which the flow is determined exclusively by cutaneous vasoconstrictor neurones (Roddie 1983). Conversely, blood flow in hairy skin is governed also by active vasodilatation (Kellogg 2006). While there is strong evidence for the presence of sympathetic cholinergic neurones that mediate vasodilation (Kellogg et al. 1995); (Sugenoya et al. 1998), there is little evidence to show that sympathetic vasodilator and sudomotor neurones are separate entities. Sugenoya et al. (1998) showed that in a mildly heated environment, isolated vasodilation followed just 1 % of SSNA bursts, compared with 10 % for ‘sweat only’ and 70 % for simultaneous ‘vasodilation and sweat release’. It is thus more likely that one body of cholinergic sympathetic neurones mediate both vasodilation and sweat release, making it reasonable to conclude that any SSNA-induced vasodilation should be associated with changes in sweat release. It is known that there is little spontaneous sudo- motor activity to the hairy skin of the foot in thermoneutral conditions (Bini et al. 1980a, b), and that there was no significant change in sweat release in the current study provides further support to the conclusion that the observed changes in SSNA represent changes in activity of the vasoconstrictor population—at least to the hairy skin. However, we do need to exhibit caution here: it should be recognised that sweat release was measured indirectly as a change in skin potential, measured across the
123 Chapter 6 – SSNA responses to tonic muscle pain sole of the foot (glabrous skin) and the dorsum of the foot (hairy skin). Nevertheless, if there was significant sweat release at either site, we believe this would have been reflected in shifts in skin potential away from zero.
After falling below baseline levels during tonic muscle pain, SSNA remained relatively stable for much of the remainder of the infusion. By contrast, after increasing above the baseline levels, skin blood flow showed a progressive increase for the remainder of the infusion. Although primarily controlled by SSNA, local factors are also known to influence skin blood flow (Kellogg 2006). However, given that the infusion was made into the muscle, local cutaneous factors seem unlikely to play a prominent role. Therefore, why skin blood flow continued to increase despite a steady reduction in SSNA remains unexplained. Nevertheless, it is worth pointing out that prior to the commencement of the infusion, subjects would have been somewhat anxious: this would cause an increase in skin sympathetic outflow and consequent reduction in skin blood flow. This is evident in the skin blood flow records of Fig.
6.1. Accordingly, the reduction in SSNA and increases in skin blood flow during the course of the infusion, no doubt, can be explained by the subjects realising that the pain was not going to increase, but was going to remain at this constant and tolerable level.
It is known that the ‘arousal response’ is strongly influenced by conscious cognitive processes (Delius et al. 1972b; Hagbarth et al. 1972; Ito et al. 1996) and is thus likely to originate in higher cortical areas. The level of integration at which pain mediates the reduction in SSNA is less clear, though that a reduction in sympathetic outflow to the skin has been observed in the spinalised cat implicates a spinal reflex (Horeyseck
124 Chapter 6 – SSNA responses to tonic muscle pain
& Jänig 1974b). Whether the fall in SSNA we observed after the initial increase reflects a spinal mechanism is unknown, but we certainly had not seen such a decrease in patients with spinal cord injury following injection of hypertonic saline into muscle or skin of the leg (Burton et al. 2008); obviously, noxious stimulation below the lesion generated no perceptions of pain.
Deep breaths are known to reliably cause large bursts of SSNA (Delius et al. 1972b), a feature which assists in confirming the identity of SSNA during the search procedures. It is therefore important to identify any changes in respiration throughout the experiments. Our continuous recording of respiration allowed detection of even minor changes in respiratory rate or depth. While respiratory rate remained stable at all stages, the depth of respiration was significantly higher in the final 5 min of infusion: this increase in respiratory depth would reasonably be expected to increase
SSNA. We believe this increase in respiratory depth and SSNA represents the relief that subjects feel as the infusion is stopped: indeed, after the experiment, subjects reported relief that the infusion had stopped, even though they could tolerate the pain and were informed at the outset that they could stop the pain whenever they wanted.
It would appear that nociceptor-induced increases in skin blood flow may serve a valuable physiological role in the promotion of wound healing, consistent with the passive coping strategy described by Lewis (1942) and Feinstein et al. (1954). It is worth noting that this response is only evident when the onset of pain (e.g. the time of injury) has subsided and if there are no other alerting stimuli in the environment.
Thus, pain in an alerting environment reduces skin blood flow, but prolonged pain increases blood flow. Pain usually accompanies a healing wound, and there are likely
125 Chapter 6 – SSNA responses to tonic muscle pain to be important physiological reasons for this. Deep pain promotes healing through a passive, protective strategy aimed at minimising further damage (Keay & Bandler
1993). However, pain may also play a more active role in wound healing through interactions with SSNA and skin blood flow. Thus, knowledge of how pain influences
SSNA is important in gaining a better understanding of the role of nociceptive pain in tissue injury and repair. Indeed, persistent, pain-induced alterations in cutaneous vascular tone could have significant clinical implications. Hypothetically, nociceptor- mediated vasoconstriction would lead to a reduction in skin blood flow, which has the potential to impair effective wound healing. Conversely, nociceptor- mediated vasodilation may serve an important but unrecognised role in wound healing, such as at the site of a deep penetrating wound. Knowledge of these mechanisms may have secondary implications for wound healing in persons with chronic pain and for the use of analgesics in the context of open wounds, operations or chronic ulcers.
6.5 Conclusions
Prolonged intramuscular infusion of hypertonic saline provides an established model for chronic musculoskeletal pain (Capra & Ro 2004), allowing controlled investigation into the physiological responses involved. Here, we demonstrated that tonic muscle pain induces a persistent decrease in skin sympathetic outflow, following an initial increase in SSNA that we attribute to the initial arousal.
126 Chapter 7 – General discussion
CHAPTER SEVEN
GENERAL DISCUSSION
The body of experimental work comprising this thesis has provided new information towards understanding the physiological changes that occur during long-lasting muscle pain. Experiments were carried out in healthy subjects using microneurography, an invasive research technique that allows one to directly record neural activity through the use of tungsten microelectrodes inserted percutaneously into a peripheral nerve of an awake human volunteer. In each experiment we infused hypertonic saline into the tibialis anterior muscle to induce muscle pain for ~1 hour,
Intramuscular infusion of hypertonic saline for 40 minutes has been validated as a suitable model for experimental chronic muscle pain in animal and human studies
(Capra & Ro 2004), though I reiterate that the research reported herein was not looking directly at chronic pain. My research sought direct evidence from awake human subjects of the effects of long-lasting muscle pain on the sympathetic and somatic motor systems, areas that have been the study of little investigation in human subjects yet have generated theories on chronic pain on the basis of studies performed in anaesthetized experimental animals. In particular, this investigation has also attempted to uncover the functional existence of the vicious cycle theory (Johansson
& Sojka 1991) in human subjects.
127 Chapter 7 – General discussion
Patients suffering from chronic muscular pain syndromes experience tenderness upon pressure applied to a painful muscle, as well as pain on movement and exercise
(Mense 2008). Functionally, they experience decreased work tolerance, fatigue, and weakness (Borg-Stein & Simons 2002). Clinicians assessing patients for muscular pain syndromes routinely identify increased muscle tone when they palpate both the affected and surrounding muscles. When applying force or pressure upon muscles with increased tonicity the muscles show resistance to movement, stiffness, and tenderness, elements that can each be assessed objectively as well as subjectively. The vicious cycle theory proposes that muscle tone are determined by an increase in excitability of the alpha-motoneuron pool due to an increase in activity of primary (Ia) muscle spindle afferents, which are regulated by the dynamic and static fusimotor neurones (Matthews 1972; Hulliger 1984; Johansson 1988). While fusimotor neurones are typically driven by supraspinal outflow, they are also influenced by reflexes from receptor afferents in the skin, joints, and muscles (Johansson & Sojka
1985; Johansson et al. 1986; Johansson & Sojka 1991). Hence, reflex increases in fusimotor neurone activity will increase the sensitivity of the muscle spindle afferents, which – according to the theory – will reflexly excite alpha motoneurones and hence increase muscle tension, stiffness, and tone (Johansson & Sojka 1991). As a result of the above nociceptive-fusimotor reflexes, muscles release an increased amount of chemical metabolites, which are normally removed by blood flowing through the muscles. However, according to the theory, due to the increased muscle contraction flow within the muscle is reduced, resulting in accumulation of the metabolites of contraction within the muscle. Furthermore, the reduction in blood flow deprives the muscle of oxygen creating a state of hypoxia in the muscle. Ischemic muscle pain can ensue adding to the nociceptor stimulation caused by increasing muscle metabolite
128 Chapter 7 – General discussion accumulation. Thus, in addition to examining the effects of muscle pain on muscle spindles, it is important to investigate the nature of the effects of muscle pain on muscle vasoconstrictor activity, given that a sustained increase in MSNA may be expected to reduce flow to the contracted muscle.
7.1.1 Group III and IV nociceptors and muscle spindle discharge activity
In Chapter 1, a critical analysis of the literature outlined the experimental evidence for the hypothesis of a “vicious cycle” reflex as the underlying cause for the development of chronic muscle pain. One of the important stages in the vicious cycle theory is the excitation of group III and IV nociceptors increasing fusimotor drive and hence the reflexogenic excitation of alpha motoneurones via the muscle spindles. Many studies were critically reviewed to show the link between noxious stimulation of group III and IV nociceptors and increased activity in directly recorded gamma motoneurones
(Appelberg et al. 1983b; Mense & Skeppar 1991), or increased muscle spindle discharge activity (Djupsjobacka et al. 1994 ; Thunberg et al. 2002b; Djupsjobacka et al. 1995b). These experiments, undertaken in animals, establish the anatomical and functional existence of a fusimotor reflex that responds to group III and IV nociceptor stimulation. However, the conclusions from these studies were conflicting, some showing increasing fusimotor and spindle activity (Djupsjobacka et al. 1995a;
Thunberg et al. 2002b), others reporting decreasing activity (Capra et al. 2007).
Earlier work form our laboratory showed that, in awake human subjects, bolus injection of hypertonic saline did not cause the increase in muscle spindle afferent activity seen in the cat – rather it resulted in a small decrease in the background firing rate of spontaneously active muscle spindle afferents (Birznieks et al. 2008). In the current work I have shown that long-lasting muscle pain also fails to generate a
129 Chapter 7 – General discussion significant increase in spindle firing; again, a small decrease was observed. These results clearly contrast those obtained in the anaesthetized and decerebrated cats that demonstrated a ~80% increase in discharge activity of muscle spindle afferents
(Thunberg et al. 2002b). On the basis of the current data, as well as earlier observations from our laboratory (Birznieks et al. 2008) it can be concluded that an increase in muscle spindle afferent activity cannot be the underlying cause responsible for the development of chronic muscle pain in humans.
Considering the effect on muscle spindle afferents was very limited, it can be concluded that noxious stimulation may not influence fusimotor activity either.
Excitation of the gamma-motor system would lead to increased muscle spindle afferent activity: in the absence of such an observation we can infer that stimulation of group III and IV nociceptors by hypertonic saline does not cause an increase in fusimotor drive in human subjects.
Evidence for fusimotor involvement in muscle hypertonicity of conditions related to muscular spasticity and parkinsonian rigidity is indirect, inconclusive, and circumstantial (Burke 1983). Among the criticisms outlined by Burke, he cites the use of anaesthesia for changing firing rates of muscle spindle afferents and heightening reflex responses in general. This could be a plausible explanation for the large increase in spindle discharge rate (~80%) observed by Thunberg and colleagues in the anaesthetized cat.
130 Chapter 7 – General discussion
7.1.2 Lack of alpha-motoneuron activity
According to the Johansson/Sojka hypothesis, increased muscle spindle afferent activity increases the likelihood of activation of the alpha-motoneuron pool. The increased spindle output caused by excitation of the gamma motoneurones would lead to increased resistance to stretch of an already contracted muscle — hypertonicity.
Reflex gain in alpha-motoneurons by altering fusimotor drive has yet to be demonstrated through conclusive experimental evidence (Burke 1983). Studies have shown intensive increases in spindle afferent discharge activity through stimuli such as muscle vibration resulting in excessive increases in fusimotor activity (Trott 1976).
However, the increased discharge activity does not result in muscles becoming spastic or rigid, contradicting the vicious cycle theory. In the studies undertaken in the current research, surface EMG electrodes were attached to the receptor-bearing muscle for the purpose of ensuring subjects were completely relaxed, but more importantly to monitor the excitation of the alpha-motoneuron pool manifesting in increasing muscle tonicity. This indicates that spindle excitation and increased discharge rate was insufficient to excite alpha-motoneurones and hence cause reflexogenic contraction. Furthermore, increasing fusimotor drive in response to pain would result in the recruitment of silent muscle spindles: we found no such evidence of recruitment during tonic muscle pain.
7.1.3 The effects of tonic pain on sympathetic outflow to muscle
Acute noxious stimuli have been shown to modulate sympathetic outflow to muscle
(Burton et al. 2009a). Previous studies have reported mainly increases in MSNA in response to noxious stimuli (Nordin & Fagius 1995). The current study has shown that long-lasting muscle pain can also increase muscle sympathetic nerve activity, as
131 Chapter 7 – General discussion well as blood pressure and heart rate. However, in some subjects tonic pain caused a decrease in MSNA, together with decreases in blood pressure and heart rate.
Importantly, for the majority of subjects this pattern was reproducible when the study was repeated weeks to months later. The existence of two groups gives us an insight into the possible cognitive influence on pain, resulting in a differing physiological response for a common noxious input. Higher centers of the brain may dictate the cardiovascular response to tonic muscle pain. Predictive factors in determining subjects that may increase MSNA or decrease MSNA are unknown. From the current results it can be said that the difference in response is not related to gender, age, or a difference in the objective measures related to the pain experience. Subjects from both groups selected similar pain descriptors from the McGill Pain questionnaire, strongly suggesting the characteristics and intensity of pain were consistent across all subjects.
During a sustained voluntary muscle contraction blood vessels in the muscle are compressed, such that blood flow may be compromised; this has been observed even at 30-40% of maximal contraction (Bonde-Petersen et al. 1975). Muscle metabolites released as a result of a contraction rely on the flow of blood to flush them out of the muscle. Muscle damage and/or inflammation result in a mixture of chemicals pooling at the site of injury, increasing sensitivity and pain. Findings of increased MSNA resulting in increased vasoconstriction and decreased vascular conductance during noxious stimulation such as muscle pain suggest that accumulation of metabolites sensitize chemonociceptors. So, the reduction in blood flow may perpetuate the pain by limiting reabsorption of metabolites. Prostaglandins and bradykinin are released together in damaged tissue and have been shown to cause a strong sensitizing action on free nerve endings (Mense 1997). Experimental evidence shows that the
132 Chapter 7 – General discussion combination of inflammatory mediators with low doses of 5-HT (serotonin) produces a synergistic increase in the pain response (Hong & Abbott 1994). It has been shown that after only 30 minutes of ischemia cell injury can begin, with irreversible cellular changes occurring after 4 to 6 hours of skeletal muscle ischemia (Sabido et al. 1994).
The current data also show that the reflex increase or decrease in MSNA during tonic muscle pain is maintained as long as the noxious stimulus continues. And, if MSNA remains high so too does blood pressure and heart rate; parallel changes in each of these parameters are also seen in those subjects in whom MSNA decreases during tonic muscle pain. It is tempting to speculate that those subjects who consistently show increases in MSNA, blood pressure and heart rate may, should they develop chronic pain in the future, are those who would also develop hypertension. The increase in MSNA may also be paralleled by an increase in cardiac sympathetic drive, contributing to left ventricular hypertrophy (Esler & Kaye 2000) and contributing to the genesis of ventricular arrhythmias and sudden death (Esler 2000).
In a study conducted by Noll and colleagues, they investigated the role of the sympathetic nervous system in cardiovascular diseases such as hypertension. They concluded that in some cardiovascular disease states such as acute myocardial infarction and heart failure activity of the sympathetic nervous system may determine prognosis significantly (Noll et al. 1998). Experimental studies conducted in humans in order to compare the reactivity of MSNA in response to mental stress in children of normotensive and hypertensive adults. The study used a combination of microneurography and plasma samples to determine the level of norepinephrine and endothelin, providing quantification of noradrenaline spillover to plasma from
133 Chapter 7 – General discussion sympathetic nerves (Esler & Kaye 2000). Results showed that activity of the sympathetic nervous system and plasma norepinephrine and endothelin levels increased during mental stress only in the offspring of hypertensive parents. However, the response to hypoxia in offspring of both hypertensive and normotensive parents was similar, suggesting a genetically determined abnormal regulation of the sympathetic nervous system to certain stressful stimuli in offspring of hypertensive parents. The investigators suggest that this may play a role in the pathogenesis of essential hypertension (Noll et al. 1996).
7.1.4 The effects of tonic pain on skin sympathetic outflow
As noted above, sympathetic outflow to skin comprises cutaneous vasoconstrictor and sudomotor neurones, as well as some vasodilator neurones, mediating thermoregulatory responses (Iwase 2009). Compared to muscle sympathetic outflow, bursts of sympathetic outflow to skin are longer, irregular, and are out of sync with the cardiac cycle. Skin sympathetic nerve activity increases following sudden inspiratory efforts and increases during mental or emotional stress and arousal stimuli such as sound, pain, and electric stimuli (Iwase 2009). In response to tonic muscle pain there was an initial arousal reaction that increases SSNA and reduces blood flow through the skin. After this initial reaction there is a withdrawal of SSNA and resultant increase in skin blood flow. These changes in SSNA are sustained during the period of pain.
In the context of pain, increases in skin blood flow as a result of nociceptor stimulation demonstrate what is conventionally known to be the microvascular
134 Chapter 7 – General discussion response to superficial wounds. Skin sites with nutritive capillary perfusion increase the amount of blood present in the skin through the withdrawal of vasomotor activity and at sites with high arteriovenous perfusion there is vasoconstriction (Rendell et al.
2002). This physiological response promotes wound healing by allowing inflammatory chemicals to pool at the site of injury, consistent with the passive coping strategy described by Lewis (1942) and Feinstein et al. (1954) aimed at minimising further damage (Keay & Bandler 1993). Pain-induced alterations in cutaneous vascular tone could have significant clinical implications particularly in patients with hypertension. Experiments have shown a substantial difference in blood flow to a healing wound between hypertensive and the non-hypertensive rats (Rendell et al. 2000). In the early phases of wound healing a dramatic transient increase in blood flow is observed and there is no apparent capacity to upregulate microvascular resistance in response to increased pressure. However, within several days, microvasculature in the granulation tissue becomes capable of controlling the effects of raised pressure in the hypertensive rat (Rendell et al. 2000).
7.2 Limitations
One of the limitations identified in the study of muscle spindle afferents and tonic muscle pain is that subjects were in an idle position with their legs and muscles completely relaxed with no movement for 30 mins to an hour before recording. This time was utilized to explore the nerve for single muscle spindle afferents. Considering that the larger majority of animal studies reported mostly static and a few dynamic fusimotor neurons responding to noxious stimulation it would seem appropriate to experiment on humans with relaxed muscles. However, it has been reported that relaxed muscles have very little fusimotor activity (Gandevia et al. 1986) and thus, it
135 Chapter 7 – General discussion is plausible that the reduced afferent discharge activity observed in both acute and tonic pain studies may be confounded by the limited fusimotor activity to begin with.
Hence, it is suggested that the response of muscle spindle afferents to noxious stimulation be studied with subjects voluntarily contracting their leg muscles in order to activate dynamic fusimotor neurons.
Infusion of hypertonic saline greater than 20 minutes is an accepted model for chronic pain (Capra & Ro 2004). Sufferers of chronic pain survive with little hope of their pain being completely alleviated, which is different to our experimental subjects. Our research volunteers knew that they could ask the infusion of hypertonic saline to be stopped at any time, yet none did. This indicates that the protocol was tolerable: once a suitable level of pain was obtained subjects could cope with it for an hour, which is quite different to having to deal with months of chronic pain. So, although during the hour of pain the research subjects may be suffering psychological stress, it is probably not the same as that experienced by chronic pain sufferers.
“Radial tonometry was used to monitor arterial blood pressure non-invasively as the device offers the use of a blood pressure cuff wrapped around the brachial artery to calibrate the tonometric sensor. Unlike finger plethysmography, which utilizes micro- vascular arterioles in the finger for measurement, it doesn’t use a cuff for oscillometric calibration. The tonometer measures from the larger, radial artery, which can be calibrated with oscillometric calibration from the brachial artery offering a more accurate reading of arterial blood pressure. Experimental studies suggest that radial tonometry is a validated and reproducible method of blood pressure measurement and under careful controlled operation is comparable in
136 Chapter 7 – General discussion accuracy to sphygmomanometric and intra-arterial blood pressure measurements
(Drzewiecki et al., 1983; Sato et al., 1993).
An accurate reading does rely on appropriate placement of the sensor directly onto the radial artery, which the sensor can sometimes have difficulty detecting a pulse. The subject also needs to be reasonably still as the sensor is sensitive to any movement and can present artefacts in the recordings. These are some limitations of radial tonometry.”
137 Chapter 8 – Conclusions
CHAPTER EIGHT
CONCLUSIONS
The onset of pain triggers a cascade of physiological and behavioral responses from multiple body systems predominantly as a strategy to minimise harm and begin a healing process for the damage sustained. When these responses are short term and the duration of time away from homeostasis is short lived it may have little physiological consequence and significance. It seems, however, many more people are suffering pain for longer periods of time, thus in chronic pain the reflexes may be in overdrive and the long -lasting shift away from homeostasis may impact on the functioning of these systems. They may also be the reason for the development of chronic pain and the transition of pain from acute to chronic.
It has been hypothesized by a popularized theory adapted from animal experimentation that chronic muscle pain may develop as a consequence of a vicious cycle that is self-perpetuating, established through nociceptor stimulation. After carrying out experimentation in awake human subjects it seems unlikely that the development of chronic muscle pain is the result of a vicious cycle that begins with reflex increase in static muscle spindle afferent discharge from group III and IV nociceptor stimulation. Experimental evidence suggests that nociceptor stimulation is insufficient to cause a large enough increase in static muscle spindle discharge activity to increase the excitability of, and cause overt recruitment of, alpha motoneurones. It appears that there may be no evidence, in humans, of a physiologic
138 Chapter 8 – Conclusions reflex of the somatic motor system in response to pain that would contribute to the development of chronic muscle pain. However, there may be another pathway through which the somatic system may contribute to the development of chronic muscle pain.
The sympathetic nervous system appropriately responds to pain with reflex changes to muscle and skin sympathetic outflow. Implications for individuals that specifically respond with an increase in sympathetic outflow to muscle if prolonged can contribute to the development of chronic muscle pain. Vasoconstriction in the muscle increases ischemia and promotes accumulation of muscle metabolites as a result of muscular contraction. In certain individuals prolonged increases in MSNA can result in cardiovascular diseases such as hypertension. This finding suggests that long term suffering of pain can impact on the physiological regulation of multiple systems that contribute to the establishment of chronic pain, in addition to other diseases and health consequences. It also provides an insight into the complexity of pain processing and coping strategies, pointing to the importance of prioritizing appropriate pain management.
Further investigation into this topic would see experimentation into fusimotor involvement in muscle tone and chronic muscle pain. A set of experiments would be designed to increase fusimotor drive through voluntary contractions of the same muscle in which the muscle spindle is located. This way we can assess whether, during a voluntary contraction, the descending excitatory drive to the spinal cord increases the synaptic strength of the oligosynaptic connections between nociceptors in the muscle and the fusimotor neurones supplying that muscle and its synergists. It
139 Chapter 8 – Conclusions would also be of interest to explore the reason we observe individual variations in the cardiovascular responses to muscle pain. We have certainly demonstrated that the observations are reproducible, however, it still remains vague as to the exact reason for diverging responses in MSNA to tonic muscle pain.
140 Bibliography
BIBLIOGRAPHY
Abrahams VC, Hilton SM, Zbrozyna A (1960) Active muscle vasodilatation produced by stimulation of the brain stem: its significance in the defence reaction. The Journal of Physiology 154:491-513.
Allen J (2007) Photoplethysmography and its application in clinical physiological measurement. Physiol Meas 28:R1-39.
Anderson EA, Wallin BG, Mark AL (1987) Dissociation of Sympathetic Nerve Activity in Arm and Leg Muscle During Mental Stress. . Hypertension 9:114-119.
Andreani CM, Hill JM, Kaufman MP (1997) Response of group III and IV muscle afferents to dynamic exercise. J Appl Physiol 82:1811-1817.
Andreani CM, Kaufman MP (1998) Effect of arterial occlusion on responses of group III and IV afferents to dynamic exercise. J Appl Physiol 84:1827-1833.
Appelberg B HM, Johansson H, Sojka P. (1983a) Actions on gamma- motoneurones elicited by electrical stimulation of group II muscle afferent fibres in the hind limb of the cat. J Physiol 255-273.
Appelberg B HM, Johansson H, Sojka P. (1983b) Actions on gamma- motoneurones elicited by electrical stimulation of group III muscle afferent fibres in the hind limb of the cat. J Physiol 275-292.
Appelberg B, Hulliger M, Johansson H, Sojka P (1983a) Actions on gamma- motoneurones elicited by electrical stimulation of group II muscle afferent fibres in the hind limb of the cat. J Physiol 255-273.
Appelberg B, Hulliger M, Johansson H, Sojka P (1983b) Actions on gamma- motoneurones elicited by electrical stimulation of group III muscle afferent fibres in the hind limb of the cat. J Physiol 275-292.
141 Bibliography
Arendt-Nielsen L, Curatolo M, Drewes A (2007) Human experimental pain models in drug development: translational pain research. Curr Opin Investig Drugs 8:41-53.
Arendt-Nielsen L, Fernandez-de-Las-Penas C, Graven-Nielsen T (2011) Basic aspects of musculoskeletal pain: from acute to chronic pain. J Man Manip Ther 19:186-193.
Arendt-Nielsen L, Graven-Nielsen T, Svarrer H, Svensson P (1996) The influence of low back pain on muscle activity and coordination during gait: a clinical and experimental study. Pain 64:231-240.
Barker D, Saito M (1981) Autonomic innervation of receptors and muscle fibres in cat skeletal muscle. Proc R Soc Lond B Biol Sci 212:317-332.
Behbehani MM (1995) Functional characteristics of the midbrain periaqueductal gray. Progress in Neurobiology 46:575-605.
Berberich P, Hoheisel U, Mense S (1988) Effects of a carrageenan-induced myositis on the discharge properties of group III and IV muscle receptors in the cat. J Neurophysiol 59:1395-1409.
Bergenheim M, Ribot-Ciscar E, Roll JP (2000) Proprioceptive population coding of two-dimensional limb movements in humans: I. Muscle spindle feedback during spatially oriented movements. Exp Brain Res 134:301-310.
Bernston G, Bigger J, Eckberg D, Grossman P, Kaufmann P, Malik M (1997) Heart rate variability: origins, methods, and interpretive caveats. Psychophysiol 34:623-648.
Besson JM, Guilbaud G, Ollat H (1995) Forebrain Areas Involved in Pain Processing. Paris: John Libbey Eurotext.
Bini G, Hagbarth KE, Hynninen P, Wallin BG (1980a) Regional similarities and differences in thermoregulatory vaso- and sudomotor tone. J Physiol 306:553- 565.
142 Bibliography
Bini G, Hagbarth KE, Hynninen P, Wallin BG (1980b) Thermoregulatory and rhythm-generating mechanisms governing the sudomotor and vasoconstrictor outflow in human cutaneous nerves. J Physiol 306:537-552.
Birznieks I, Burton AR, Macefield VG (2008) The effects of experimental muscle and skin pain on the static stretch sensitivity of human muscle spindles in relaxed leg muscles. The Journal of Physiology 586:2713-2723.
Blessing W (1998) The lower brainstem and bodily homeostasis. Ann Neurol 43:839.
Boczek-Funcke A, Dembowsky K, Habler HJ, Janig W, McAllen RM, Michaelis M (1992a) Classification of preganglionic neurones projecting into the cat cervical sympathetic trunk. J Physiol 453:319-339.
Boczek-Funcke A, Dembowsky K, Habler HJ, Janig W, Michaelis M (1992b) Respiratory-related activity patterns in preganglionic neurones projecting into the cat cervical sympathetic trunk. J Physiol 457:277-296.
Bonde-Petersen F, Mork AL, Nielsen E (1975) Local muscle blood flow and sustained contractions of human arm and back muscles. Eur J Appl Physiol Occup Physiol 34:43-50.
Borg-Stein J, Simons DG (2002) Focused review: myofascial pain. Arch Phys Med Rehabil 83:S40-47, S48-49.
Brooks CM (1981) The autonomic nervous system, molder and integrator of function. Review of a concept. Braz J Med Biol Res 14:151-160.
Brooks CM (1983) Newer concepts of the autonomic system's role derived from reductionist and behavioral studies of various animal species. J Auton Nerv Syst 7:199-212.
Bruehl S, Chung O, Jirjis J, Biridepalli S (2005) Prevalence of clinical hypertension in patients with chronic pain compared to nonpain general medical patients. Clin J Pain 21:147-153.
143 Bibliography
Bruehl S, Chung OY (2004) Interactions between the cardiovascular and pain regulatory systems: an updated review of mechanisms and possible alterations in chronic pain. Neuroscience & Biobehavioral Reviews 28:395-414.
Brum PC, Kosek J, Patterson A, Bernstein D, Kobilka B (2002) Abnormal cardiac function associated with sympathetic nervous system hyperactivity in mice. American Journal of Physiology - Heart and Circulatory Physiology 283:H1838- H1845.
Burgmer M, Petzke F, Giesecke T, Gaubitz M, Heuft G, Pfleiderer B (2011) Cerebral Activation and Catastrophizing During Pain Anticipation in Patients With Fibromyalgia. Psychosomatic Medicine 73:751-759.
Burke D (1983) Critical examination of the case for or against fusimotor involvement in disorders of muscle tone. In: Advances in neurology: motor control mechanisms in health and disease, vol. 39 (Desmedt, J. E., ed), pp 133- 150 New York: Raven Press.
Burke D, Gandevia SC, McKeon B (1983) The afferent volleys responsible for spinal proprioceptive reflexes in man. J Physiol 339:535-552.
Burke D, Skuse NF, Stuart DG (1979) The regularity of muscle spindle discharge in man. J Physiol 291:277-290.
Burton A, Birznieks I, Bolton P, Henderson L, Macefield V (2009a) Effects of deep and superficial experimentally induced acute pain on muscle sympathetic nerve activity in human subjects. Journal of Physiology 587:183 - 193.
Burton A, Birznieks I, Spaak J, Henderson L, Macefield V (2009b) Effects of deep and superficial experimentally induced acute pain on skin sympathetic nerve activity in human subjects. Experimental Brain Research 195:317 - 324.
Burton AR, Brown R, Macefield VG (2008) Selective activation of muscle and skin nociceptors does not trigger exaggerated sympathetic responses in spinal- injured subjects. Spinal Cord 46:660-665.
144 Bibliography
Campbell GD, Edwards FR, Hirst GD, O'Shea JE (1989) Effects of vagal stimulation and applied acetylcholine on pacemaker potentials in the guinea-pig heart. J Physiol 415:57-68.
Campero M, Bostock H, Baumann TK, Ochoa JL (2010) A search for activation of C nociceptors by sympathetic fibers in complex regional pain syndrome. Clinical Neurophysiology 121:1072-1079.
Cannon WB (1915) Bodily changes in pain, hunger, fear and rage: An account of recent researches into the function of emotional excitement D. Appleton and Company
Capra NF, Hisley CK, Masri RM (2007) The influence of pain on masseter spindle afferent discharge. Archives of Oral Biology 52:387-390.
Capra NF, Ro JY (2000) Experimental muscle pain produces central modulation of proprioceptive signals arising from jaw muscle spindles. Pain 86:151-162.
Capra NF, Ro JY (2004) Human and animal experimental models of acute and chronic muscle pain: intramuscular algesic injection. Pain 110:3-7.
Carlsten A, Folkow B, Hamberger CA (1957) Cardiovascular effects of direct vagal stimulation in man. Acta Physiol Scand 41:68-76.
Carrive P, Bandler R (1991) Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study. Brain Res 541:206-215.
Casale R, Elam M (1992) Normal sympathetic nerve activity in a reflex sympathetic dystrophy with marked skin vasoconstriction. J Auton Nerv Syst 41:215-219.
Coghill R, Talbot J, Evans A, Meyer E, Gjedde A, Bushnell M, Duncan G (1994) Distributed processing of pain and vibration by the human brain. The Journal of Neuroscience 14:4095-4108.
145 Bibliography
Cohen H, Neumann L, Shore M, Amir M, Cassuto Y, Buskila D (2000) Autonomic dysfunction in patients with fibromyalgia: application of power spectral analysis of heart rate variability. Semin Arthritis Rheum 29 217-227.
Couppe C, Midttun A, Hilden J, Jorgensen U, Oxholm P, A. F-F (2001) pontaneous needle electromyographic activity in myofascial trigger points in the infraspinatus muscle: a blinded assessment. J Musculoskel Pain 9:7-17.
Cowley AW, Jr., Liard JF, Guyton AC (1973) Role of baroreceptor reflex in daily control of arterial blood pressure and other variables in dogs. Circ Res 32:564- 576.
Craig AD (2002) Pain. In: Encyclopedia of the human brain(Ramachandran, V. S., ed) London: Acadmeic Press
D'Antono B, Ditto B, Sita A, Miller SB (2000) Cardiopulmonary baroreflex stimulation and blood pressure-related hypoalgesia. Biological Psychology 53:217-231.
Dampney RA (1981) Functional organization of central cardiovascular pathways. Clin Exp Pharmacol Physiol 8:241-259.
Delius W, Hagbarth KE, Hongell A, Wallin BG (1972a) General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand 84:65-81.
Delius W, Hagbarth KE, Hongell A, Wallin BG (1972b) Manoeuvres affecting sympathetic outflow in human muscle nerves. Acta Physiol Scand 84:82-94.
Depaulis A, Keay K, Bandler R (1992) Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat. Exp Brain Res 90:1983-1989.
Derbyshire SWG, Jones AKP (1998) Cerebral responses to a continual tonic pain stimulus measured using positron emission tomography. Pain 76:127-135.
146 Bibliography
DiBona GF, Jones SY (1998) Reflex effects on components of synchronized renal sympathetic nerve activity. American Journal of Physiology - Renal Physiology 275:F441-F446.
Ditto B, Lewkowski MD, Rainville P, Duncan GH (2009) Effects of cardiopulmonary baroreceptor activation on pain may be moderated by risk for hypertension. Biological Psychology 82:195-197.
Djupsjobacka M, Johansson H, Bergenheim M (1994 ) Influences on the gamma- muscle-spindle system from muscle afferents stimulated by increased intramuscular concentrations of arachidonic acid. Brain Research 663:293 - 302.
Djupsjobacka M, Johansson H, Bergenheim M, Wenngren B (1995a) Influences on the gamma-muscle spindle system from muscle afferents stimulated by increased intramuscular concentrations of bradykinin and 5-HT. Neurascience Research 22:325 - 333.
Djupsjobacka M, Johansson H, Bergenheim M, Wenngren B ( 1995b) Influences on the gamma-muscle spindle system from muscle afferents stimulated by increased intramuscular concentrations of bradykinin and 5-HT. Neurascience Research 22:325 - 333.
Drzewiecki GM, Melbin J, Noordergraaf A (1983) Arterial tonometry: review and analysis. J Biomech 16:141-152.
Eckberg D, Sleight P (1992) Human baroreflexes in health and disease. Oxford: Oxford University Press.
Edin BB, Vallbo AB (1990) Classification of human muscle stretch receptor afferents: a Bayesian approach. Journal of Neurophysiology 63:1314-1322.
Edwards RR, Fillingim RB (2005) Styles of pain coping predict cardiovascular function following a cold pressor test. Pain Res Manag 10:219-222.
Ellaway PH, Murphy PR, Tripathi A (1982) Closely coupled excitation of gamma- motoneurones by group III Muscle afferents with low mechanical threshold in the cat. J Physiol 331:481-498.
147 Bibliography
Esler M (2000) The sympathetic system and hypertension. Am J Hypertens 13:99S-105S.
Esler M, Kaye D (2000) Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease. J Cardiovasc Pharmacol 35:S1-7.
Fagius J, Karhuvaara S (1989) Sympathetic activity and blood pressure increases with bladder distension in humans. Hypertension 14:511-517.
Fagius J, Karhuvaara S, Sundlof G (1989) The cold pressor test: effects on sympathetic nerve activity in human muscle and skin nerve fascicles. Acta Physiol Scand 137:325-334.
Fagius J, Sundlof G (1986) The diving response in man: effects on sympathetic activity in muscle and skin nerve fascicles. J Physiol 377:429-443.
Fagius J, Wallin BG (1993) Long-term variability and reproducibility of resting human muscle nerve sympathetic activity at rest, as reassessed after a decade. Clin Auton Res 3:201-205.
Farina D, Arendt-Nielsen L, Graven-Nielsen T (2005) Experimental muscle pain reduces initial motor unit discharge rates during sustained submaximal contractions. J Appl Physiol (1985) 98:999-1005.
Fazalbhoy A, Birznieks I, Macefield VG (2012) Individual differences in the cardiovascular responses to tonic muscle pain: parallel increases or decreases in muscle sympathetic nerve activity, blood pressure and heart rate. Experimental Physiology 97:1084-1092.
Fazalbhoy A, Macefield VG, Birznieks I (2013) Tonic muscle pain does not increase fusimotor drive to human leg muscles: implications for chronic muscle pain. Exp Physiol 98:1125-1132.
Feinstein B, Langton J, Jameson R, Schiller F (1954) Experiments on pain referred from deep somatic tissues. J Bone Joint Surg 36:981-997.
148 Bibliography
Freyschuss U, Fagius J, Wallin B, Bohlin G, Perski A, Hjemdahl P (1990) Cardiovascular and sympathoadrenal responses to mental stress: a study of sensory intake and rejection reactions. Acta Physiol Scand 139:173-183.
Gandevia SC, Miller S, Aniss AM, Burke D (1986) Reflex influences on muscle spindle activity in relaxed human leg muscles. J Neurophysiol 56:159-170.
Goldstein DS, Bentho O, Park MY, Sharabi Y (2011) Low-frequency power of heart rate variability is not a measure of cardiac sympathetic tone but may be a measure of modulation of cardiac autonomic outflows by baroreflexes. Exp Physiol 96:1255-1261.
Graven-Nielsen T, McArdle A, Phoenix J, Arendt-Nielsen L, Jensen TS, Jackson MJ, Edwards RH (1997) In vivo model of muscle pain: quantification of intramuscular chemical, electrical, and pressure changes associated with saline- induced muscle pain in humans. Pain 69:137-143.
Gribbin B, Pickering TG, Sleight P, Peto R (1971) Effect of Age and High Blood Pressure on Barorefiex Sensitivity in Man. Circulation Research 29:424-431.
Guyton AC, Hall JE (2000) Textbook of medical physiology. Philidelphia: W.B. Saunders Company.
Habermeier-Muth A, Muscholl E (1988) Short- and long-latency muscarinic inhibition of noradrenaline release from rabbit atria induced by vagal stimulation. J Physiol 401:277-293.
Hagbarth KE, Hallin RG, Hongell A, Torebjork HE, Wallin BG (1972) General characteristics of sympathetic activity in human skin nerves. Acta Physiol Scand 84:164-176.
Hagberg M, Kvarnstrom S (1984) Muscular endurance and electromyographic fatigue in myofascial shoulder pain. Arch Phys Med Rehabil 65:522-525.
Hales JR, Roberts RG, Westerman RA, Stephens FR, Fawcett AA (1993) Evidence for skin microvascular compartmentalization by laser-Doppler and photoplethysmographic techniques. Int J Microcirc Clin Exp 12:99-104.
149 Bibliography
Hall SC, Fazalbhoy A, Birznieks I, Macefield VG (2012) Biphasic effects of tonic stimulation of muscle nociceptors on skin sympathetic nerve activity in human subjects. Exp Brain Res 221:107-114.
Hallman DM, Lindberg LG, Arnetz BB, Lyskov E (2011) Effects of static contraction and cold stimulation on cardiovascular autonomic indices, trapezius blood flow and muscle activity in chronic neck-shoulder pain. Eur J Appl Physiol 111:1725-1735.
Hellström F, Roatta S, Thunberg J, Passatore M, Djupsjöbacka M (2005) Responses of muscle spindles in feline dorsal neck muscles to electrical stimulation of the cervical sympathetic nerve. Experimental Brain Research 165:328-342.
Henderson LA, Bandler R, Gandevia SC, Macefield VG (2006) Distinct forebrain activity patterns during deep versus superficial pain. Pain 120:286-296.
Henderson LA, Gandevia SC, Macefield VG (2007) Somatotopic organization of the processing of muscle and cutaneous pain in the left and right insula cortex: a single-trial fMRI study. Pain 128:20-30.
Henderson LA, Rubin TK, Macefield VG (2011) Within-limb somatotopic representation of acute muscle pain in the human contralateral dorsal posterior insula. Hum Brain Mapp 32:1592-1601.
Hervey G (1983) The functions of pain. In: The neurobiology of pain:Symposium of the Northern Neurobiology Group(W Winlow, A. V. H., ed), p 399 Leed, United Kingdom: Manchester University Press.
Hirche H, Schumacher E, Hagemann H (1980) Extracellular K+ concentration and K+ balance of the gastrocnemius muscle of the dog during exercise. Pflugers Arch 387:231-237.
Hjemdahl P, Fagius J, Freyschuss U, Wallin B, Daleskog M, Bohlin G, Perski A (1989) Muscle sympathetic activity and norepinephrine release during mental challenge in humans. Am J Physiol Endocrinol Metab 257:E654–E664.
150 Bibliography
Hnik P, Holas M, Krekule I, Kuriz N, Mejsnar J, Smiesko V, Ujec E, Vyskocil F (1976) Work-induced potassium changes in skeletal muscle and effluent venous blood assessed by liquid ion-exchanger microelectrodes. Pflugers Arch 362:85- 94.
Hodges PW, Ervilha UF, Graven-Nielsen T (2008) Changes in Motor Unit Firing Rate in Synergist Muscles Cannot Explain the Maintenance of Force During Constant Force Painful Contractions. The Journal of Pain 9:1169-1174.
Hofbauer RK, Rainville P, Duncan GH, Bushnell MC (2001) Cortical Representation of the Sensory Dimension of Pain. Journal of Neurophysiology 86:402-411.
Holstege G (1988) Direct and indirect pathways to lamina I in the medulla oblongata and spinal cord of the cat. Prog Brain Res 77:47-94.
Hong Y, Abbott FV (1994) Behavioural effects of intraplantar injection of inflammatory mediators in the rat. Neuroscience 63:827-836.
Horeyseck G, Jänig W (1974a) Reflex activity in postganglionic fibres within skin and muscle nerves elicited by somatic stimuli in chronic spinal cats. Exp Brain Res 21:155-168.
Horeyseck G, Jänig W (1974b) Reflexes in postganglionic fibres within skin and muscle nerves after noxious stimulation of skin. Experimental Brain Research 20:125-134.
Hulliger M (1984) The mammalian muscle spindle and its central control. Rev Physiol Biochem Pharmacol 101:1-110.
Hunt SP, Pini A, Evan G (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328:632-634.
Iggo A, Steedman WM, Fleetwood-Walker S (1985) Spinal processing: anatomy and physiology of spinal nociceptive mechanisms. Philos Trans R Soc Lond B Biol Sci 308:235-252.
151 Bibliography
Ito H, Sugiyama Y, Mano T, Okada H, Matsukawa T, Iwase S (1996) Skin sympathetic nerve activity and event-related potentials during auditory oddball paradigms. J Auton Nerv Syst 60:129-135.
Iwase N, Takata S, Okuwa H, Ogawa J, Ikeda T, Hattori N (1984) Abnormal baroreflex control of heart rate in normotensive young subjects with a family history of essential hypertension. J Hypertens Suppl 2:S409-411.
Iwase S (2009) [Clinical application of skin sympathetic nerve activity]. Brain Nerve 61:243-253.
Iwase S, Sugiyama Y, Miwa C, Kamiya A, Mano T, Ohira Y, Shenkman B, Egorov AI, Kozlovskaya IB (2000) Effects of three days of dry immersion on muscle sympathetic nerve activity and arterial blood pressure in humans. Journal of the Autonomic Nervous System 79:156-164.
Jacquet YF, Lajtha A (1973) Morphine action at central nervous system sites in rat: analgesia or hyperalgesia depending on site and dose. Science 182:490-492.
Jacquet YF, Lajtha A (1974) Paradoxical effects after microinjection of morphine in the periaqueductal gray matter in the rat. Science 185:1055-1057.
Janig W (1985) Systemic and specific autonomic reactions in pain: efferent, afferent and endocrine components. Eur J Anaesthesiol 2:319-346.
Janig W (2006) The Integrative Action of the Autonomic Nervous System - Neurobiology of Homeostasis. Cambridge Cambridge University Press.
Janig W, McLachlan EM (1992) Characteristics of function-specific pathways in the sympathetic nervous system. Trends Neurosci 15:475-481.
Jiang C, Lin Y, Yu N (2011) Multi-Scale Surface Electromyography Modeling to Identify Changes in Neuromuscular Activation with Myofascial Pain. IEEE
Johansson H (1988) Rubrospinal and rubrobulbospinal influences on dynamic and static gamma-motoneurones. Behav Brain Res 28:97-107.
152 Bibliography
Johansson H, Djupsjobacka M, Sjolander P (1993) Influences on the gamma- muscle spindle system from muscle afferents stimulated by KCl and lactic acid. Neurosci Res 16:49-57.
Johansson H, Sjo ̈lander P, Sojka P, Wadell I (1989) Reflex actions on the gamma- muscle spindle systems of muscles acting at the knee joint elicited by stretch of the posterior cruciate ligament. Neuro Orthoped 8:9-21.
Johansson H, Sjolander P, Sojka P (1986) Actions on gamma-motoneurones elicited by electrical stimulation of joint afferent fibres in the hind limb of the cat. J Physiol 375:137-152.
Johansson H, Sojka P (1985) Actions on gamma-motoneurones elicited by electrical stimulation of cutaneous afferent fibres in the hind limb of the cat. J Physiol 366:343-363.
Johansson H, Sojka P (1991) Pathophysiological mechanisms involved in genesis and spread of muscular tension in occupational muscle pain and in chronic musculoskeletal pain syndromes: A hypothesis. Medical Hypotheses 35:196-203.
Johansson M, Elam M, Rundqvist B, Eisenhofer G, Herlitz H, Lambert G, Friberg P (1999) Increased sympathetic nerve activity in renovascular hypertension. Circulation 99:2537-2542.
Jones AKP, Brown WD, Friston KJ, Qi LY, Frackowiak RSJ (1991) Cortical and Subcortical Localization of Response to Pain in Man using Positron Emission Tomography. Proceedings of the Royal Society of London Series B: Biological Sciences 244:39-44.
Kabat H, Magoun HW, Ranson JW (1953) Electrical stimulation of points in the forebrain and midbrain. The resultant alteration in blood pressure. Archs Neurol 34:931-955.
Kandel ER, Schwartz JH, Jessell TM (2000) Principles of Neural Science. Sydney: McGraw-Hill
153 Bibliography
Kaufman MP, Hayes SG, Adreani CM, Pickar JG (2002) Discharge properties of group III and IV muscle afferents. Adv Exp Med Biol 508:25-32.
Kaufman MP, Longhurst JC, Rybicki KJ, Wallach JH, Mitchell JH (1983) Effects of static muscular contraction on impulse activity of groups III and IV afferents in cats. J Appl Physiol 55:105-112.
Kaufman MP, Rybicki KJ (1987) Discharge properties of group III and IV muscle afferents: their responses to mechanical and metabolic stimuli. Circ Res 61:I60- 65.
Kaufman MP, Rybicki KJ, Waldrop TG, Ordway GA (1984) Effect of ischemia on responses of group III and IV afferents to contraction. J Appl Physiol 57:644-650.
Keay K, Bandler R (2002) Distinct central representations of inescapable and escapable pain: observations and speculation. Experimental Physiology 87:275- 279.
Keay KA, Bandler R (1993) Deep and superficial noxious stimulation increases Fos-like immunoreactivity in different regions of the midbrain periaqueductal grey of the rat. Neuroscience Letters 154:23-26.
Keay KA, Clement CI, Owler B, Depaulis A, Bandler R (1994) Convergence of deep somatic and visceral nociceptive information onto a discrete ventrolateral midbrain periaqueductal gray region. Neuroscience 61:727-732.
Keller DM, Cui J, Davis SL, Low DA, Crandall CG (2006) Heat stress enhances arterial baroreflex control of muscle sympathetic nerve activity via increased sensitivity of burst gating, not burst area, in humans. The Journal of Physiology 573:445-451.
Kellgren JH (1938) Referred Pains from Muscle. Br Med J 1:325-327.
Kellogg DL, Jr. (2006) In vivo mechanisms of cutaneous vasodilation and vasoconstriction in humans during thermoregulatory challenges. J Appl Physiol 100:1709-1718.
154 Bibliography
Kellogg DL, Jr., Pergola PE, Piest KL, Kosiba WA, Crandall CG, Grossmann M, Johnson JM (1995) Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission.
Kienbaum P, Karlssonn T, Sverrisdottir YB, Elam M, BG W (2001) Two sites for modulation of human sympathetic activity by arterial baroreceptors. J Physiol 531:861-869.
Kienbaum P, Karlssonn T, Sverrisdottir YB, Elam M, BG W (2001) Two sites for modulation of human sympathetic activity by arterial baroreceptors. J Physiol 531:861-869.
Knutson GA (2000) The role of the [gamma]-motor system in increasing muscle tone and muscle pain syndromes: A review of the Johansson/Sojka hypothesis. Journal of Manipulative and Physiological Therapeutics 23:564-572.
Koelbaek Johansen M, Graven-Nielsen T, Schou Olesen A, Arendt-Nielsen L (1999) Generalised muscular hyperalgesia in chronic whiplash syndrome.
Kuczmierczyk AR, Adams HE, Calhoun KS, Naor S, Giombetti R, Cattalani M, McCann P (1986) Pain responsivity in women with premenstrual syndrome across the menstrual cycle. Percept Mot Skills 63:387-393.
La Rovere MT, Pinna GD, Raczak G (2008) Baroreflex sensitivity: measurement and clinical implications. Ann Noninvasive Electrocardiol 13:191-207.
Laine H, Knuuti MJ, Ruotsalainen U, Utriainen T, Oikonen V, Raitakari M, Luotolahti M, Kirvela O, Vicini P, Cobelli C, Nuutila P, Yki-Jarvinen H (1998) Preserved relative dispersion but blunted stimulation of mean flow, absolute dispersion, and blood volume by insulin in skeletal muscle of patients with essential hypertension. Circulation 97:2146-2153.
Levenson RW (2003) Blood, sweat, and fears: the autonomic architecture of emotion. Ann N Y Acad Sci 1000:348-366.
Lewis T (1942) Pain. London: McMillan.
155 Bibliography
Lindberg LG, Oberg PA (1991) Photoplethysmography. Part 2. Influence of light source wavelength. Medical & Biological Engineering & Computing 29:48-54.
Lloyd DPC, Chang HT (1948) Afferent fibres in muscle nerves. J Neurophysiol 11:199-207.
Lund JP, Donga R, Widmer CG, Stohler CS (1991) The pain-adaptation model: a discussion of the relationship between chronic musculoskeletal pain and motor activity. Can J Physiol Pharmacol 69:683-694.
Lund JP, Sadeghi S, Athanassiadis T, Caram Salas N, Auclair F, Thivierge B, Arsenault I, Rompre P, Westberg KG, Kolta A (2010) Assessment of the potential role of muscle spindle mechanoreceptor afferents in chronic muscle pain in the rat masseter muscle. PLoS ONE 5:e11131.
Macefield V, Sverrisdottir Y, Elam M, Harris J (2008) Firing properties of sudomotor neurones in hyperhidrosis and thermal sweating. Clinical Autonomic Research 18:325-330.
Macefield VG (2005) Physiological characteristics of low-threshold mechanoreceptors in joints, muscle and skin in human subjects. Clinical and Experimental Pharmacology and Physiology 32:135-144.
Macefield VG (2010) A role for the sympathetic nervous system in sympathetically maintained pain? Clinical Neurophysiology 121:996-997.
Macefield VG (2013) Discharge rates and discharge variability of muscle spindle afferents in human chronic spinal cord injury. Clin Neurophysiol 124:114-119.
Macefield VG, Gandevia SC, Bigland-Ritchie B, Gorman RB, Burke D (1993) The firing rates of human motoneurones voluntarily activated in the absence of muscle afferent feedback. J Physiol 471:429-443.
Macefield VG, Rundqvist B, Sverrisdottir YB, Wallin BG, Elam M (1999) Firing Properties of Single Muscle Vasoconstrictor Neurons in the Sympathoexcitation Associated With Congestive Heart Failure. Circulation 100:1708-1713.
156 Bibliography
Macefield VG, Sverrisdottir YB, Wallin BG (2003) Resting discharge of human muscle spindles is not modulated by increases in sympathetic drive. The Journal of Physiology 551:1005-1011.
Macefield VG, Wallin BG (1995) Effects of static lung inflation on sympathetic activity in human muscle nerves at rest and during asphyxia. J Auton Nerv Syst 53:148-156.
Macefield VG, Wallin BG (1999) Respiratory and cardiac modulation of single sympathetic vasoconstrictor and sudomotor neurones to human skin. J Physiol 516 ( Pt 1):303-314.
Madan I, Reading I, Palmer KT, Coggon D (2008) Cultural differences in musculoskeletal symptoms and disability. Int J Epidemiol 37:1181-1189.
Magoun HW, Atlas D, Ingersoll EH, Ranson SW (1937) Associated Facial, Vocal and Respiratory Components of Emotional Expression: An Experimental Study. J Neurol Psychopathol 17:241-255.
Maihofner C, Seifert F, DeCol R (2010) Activation of central sympathetic networks during innocuous and noxious somatosensory stimulation. NeuroImage In Press, Uncorrected Proof.
Malliani A, Pagani M, Lombardi F, Cerutti S (1991) Cardiovascular neural regulation explored in the frequency domain. Circulation 84:482-492.
Mano T, Iwase S, Toma S (2006) Microneurography as a tool in clinical neurophysiology to investigate peripheral neural traffic in humans. Clinical Neurophysiology 117:2357-2384.
Mantyh PW, Peschanski M (1982) Spinal projections from the periaqueductal grey and dorsal raphe in the rat, cat and monkey. Neuroscience 7:2769-2776.
Marsden CD, Merton PA, Morton HB (1976a) Servo action in the human thumb. J Physiol 257:1-44.
157 Bibliography
Marsden CD, Merton PA, Morton HB (1976b) Stretch reflex and servo action in a variety of human muscles. J Physiol 259:531-560.
Martínez-Lavín M (2004) Fibromyalgia as a sympathetically maintained pain syndrome. Curr Pain Headache Rep 8:385–389.
Matre D, Arendt-Neilsen L, Knardahl S (2002) Effects of localization and intensity of experimental muscle pain on ankle joint proprioception. European Journal of Pain 6:245-260.
Matre DA, SinkjÊr T, Knardahl S, Andersen JB, Arendt-Nielsen L (1999) The influence of experimental muscle pain on the human soleus stretch reflex during sitting and walking. Clinical Neurophysiology 110:2033-2043.
Matre DA, Sinkjer T, Svensson P, Arendt-Nielsen L (1998) Experimental muscle pain increases the human stretch reflex. Pain 75:331-339.
Matthews PB (1972) Mammalian Muscle Receptors and their Central Actions. London: Edward Arnold.
Matthews PB (1988) Proprioceptors and their contribution to somatosensory mapping: complex messages require complex processing. Can J Physiol Pharmacol 66:430-438.
Matthews WB (1970) The clinical implications of the H reflex and of other electrically induced reflexes. Mod Trends Neurol 5:241-253.
McLachlan EM (2007) Diversity of sympathetic vasoconstrictor pathways and their plasticity after spinal cord injury. Clin Auton Res 17:6-12.
Melzack R, Stotler WA, Livingston WK (1958) Effects of discrete brainstem lesions in cats on perception of noxious stimulation. J Neurophysiol 21:353-367.
Mense S (1997) Pathophysiologic basis of muscle pain syndromes. Phys Med Rehab Clin N AM 8:23-53.
Mense S (2008) Muscle pain: mechanisms and clinical significance. Dtsch Arztebl Int 105:214-219.
158 Bibliography
Mense S, Skeppar P (1991) Discharge behaviour of feline gamma-motoneurones following induction of an artificial myositis. Pain 46:201-210.
Merskey H, Bogduk N (1994) Classification of chronic pain: descriptions of chronic pain syndromes and definitions of pain terms. IASP Press.
Messlinger K (1997) What is a nociceptor? Anaesthesist 46:142-153.
Nashold BS, Jr., Wilson WP, Slaughter DG (1969) Sensations evoked by stimulation in the midbrain of man. J Neurosurg 30:14-24.
Noll G, Wenzel RR, Binggeli C, Corti C, Luscher TF (1998) Role of sympathetic nervous system in hypertension and effects of cardiovascular drugs. Eur Heart J 19 Suppl F:F32-38.
Noll G, Wenzel RR, Schneider M, Oesch V, Binggeli C, Shaw S, Weidmann P, Luscher TF (1996) Increased activation of sympathetic nervous system and endothelin by mental stress in normotensive offspring of hypertensive parents. Circulation 93:866-869.
Nordh E, Hulliger M, Vallbo AB (1983) The variability of inter-spike intervals of human spindle afferents in relaxed muscles. Brain Res 271:89-99.
Nordin M, Fagius J (1995) Effect of noxious stimulation on sympathetic vasoconstrictor outflow to human muscles. The Journal of Physiology 489:885- 894.
Oliveira LR, de Melo VU, Macedo FN, Barreto AS, Badaue-Passos D, Jr., Viana dos Santos MR, Dias DP, Sluka KA, DeSantana JM, Santana-Filho VJ (2012) Induction of chronic non-inflammatory widespread pain increases cardiac sympathetic modulation in rats. Auton Neurosci 167:45-49.
Parmer RJ, Cervenka JH, Stone RA (1992) Baroreflex sensitivity and heredity in essential hypertension. Circulation 85:497-503.
Partanen JV, Ojala TA, Arokoski JPA (2010) Myofascial syndrome and pain: A neurophysiological approach. Pathophysiology 17:19-28.
159 Bibliography
Paulson PE, Minoshima S, Morrow TJ, Casey KL (1998) Gender differences in pain perception and patterns of cerebral activation during noxious heat stimulation in humans. Pain 76:223-229.
Pedersen J, Sjolander P, Wenngren BI, Johansson H (1997) Increased intramuscular concentration of bradykinin increases the static fusimotor drive to muscle spindles in neck muscles of the cat. Pain 70:83-91.
Powley TL (1999) Central Control of Autonomic Functions - The Organization of the Autonomic Nervous System. In: Fundamental Neurosciene (Zigmond, M. J. et al., eds) London: Academic Press.
Rendell MS, Johnson ML, Smith D, Finney D, Capp C, Lammers R, Lancaster S (2002) Skin blood flow response in the rat model of wound healing: expression of vasoactive factors. J Surg Res 107:18-26.
Rendell MS, Milliken BK, Finnegan MF, Finney DE, Healy JC, Bonner RF (2000) A comparison of the microvascular response in the healing wound in the spontaneously hypertensive and non-hypertensive rat. Int J Surg Investig 2:17- 25.
Reyes del Paso GA, Garrido S, Pulgar n, Duschek S (2011) Autonomic cardiovascular control and responses to experimental pain stimulation in fibromyalgia syndrome. Journal of Psychosomatic Research 70:125-134.
Ro JY, Capra NF (2001) Modulation of jaw muscle spindle afferent activity following intramuscular injections with hypertonic saline. Pain 92:117-127.
Rotto DM, Kaufman MP (1988) Effect of metabolic products of muscular contraction on discharge of group III and IV afferents. J Appl Physiol 64:2306- 2313.
Rubin TK, Henderson LA, Macefield VG (2010) Changes in the spatiotemporal expression of local and referred pain following repeated intramuscular injections of hypertonic saline: a longitudinal study. J Pain 11:737-745.
160 Bibliography
Rybicki KJ, Kaufman MP, Kenyon JL, Mitchell JH (1984) Arterial pressure responses to increasing interstitial potassium in hindlimb muscle of dogs. Am J Physiol 247:R717-721.
Rybicki KJ, Waldrop TG, Kaufman MP (1985) Increasing gracilis muscle interstitial potassium concentrations stimulate group III and IV afferents. J Appl Physiol 58:936-941.
Sabido F, Milazzo VJ, Hobson RW, 2nd, Duran WN (1994) Skeletal muscle ischemia-reperfusion injury: a review of endothelial cell-leukocyte interactions. J Invest Surg 7:39-47.
Sainburg RL, Poizner H, Ghez C (1993) Loss of proprioception produces deficits in interjoint coordination. J Neurophysiol 70:2136-2147.
Sato A, Sato Y, Schmidt RF (1984) Changes in blood pressure and heart rate induced by movements of normal and inflamed knee joints. Neuroscience Letters 52:55-60.
Sato T, Nishinaga M, Kawamoto A, Ozawa T, Takatsuji H (1993) Accuracy of a continuous blood pressure monitor based on arterial tonometry. Hypertension 21:866-874.
Schobel HP, Ringkamp M, Behrmann A, Forster C, Schmieder RE, Handwerker HO (1996) Hemodynamic and sympathetic nerve responses to painful stimuli in normotensive and borderline hypertensive subjects. Pain 66:117-124.
Sherrington C (1900) The muscular sense. New York: Macmillan.
Siddall PJ, Cousins MJ (2004) Persistent pain as a disease entity: implications for clinical management. Anesth Analg 99:510-520.
Sikandar S, Dickenson AH (2012) Visceral pain: the ins and outs, the ups and downs. Curr Opin Support Palliat Care 6:17-26.
Simons DG, Mense S (1998) Understanding and measurement of muscle tone as related to clinical muscle pain. Pain 75:1-17.
161 Bibliography
Sinkjaer T, Andersen JB, Larsen B (1996) Soleus stretch reflex modulation during gait in humans. J Neurophysiol 76:1112-1120.
Sorensen J, Graven-Nielsen T, Henriksson KG, Bengtsson M, Arendt-Nielsen L (1998) Hyperexcitability in fibromyalgia.
Staud R (2008) Heart rate variability as a biomarker of fibromyalgia syndrome. Future Rheum 3:475–483.
Stohler CS, Zhang X, Lund JP (1996) The effect of experimental jaw muscle pain on postural muscle activity. Pain 66:215-221.
Sugenoya J, Iwase S, Mano T, Sugiyama Y, Ogawa T, Nishiyama T, Nishimura N, Kimura T (1998) Vasodilator component in sympathetic nerve activity destined for the skin of the dorsal foot of mildly heated humans. J Physiol 507 ( Pt 2):603- 610.
Sugiura Y, Terui N, Hosoya Y (1989) Difference in distribution of central terminals between visceral and somatic unmyelinated (C) primary afferent fibers. J Neurophysiol 62:834-840.
Sundlof G, Wallin BG (1977) The variability of muscle nerve sympathetic activity in resting recumbent man. The Journal of Physiology 272:383-397.
Sundlof G, Wallin BG (1978) Human muscle nerve sympathetic activity at rest. Relationship to blood pressure and age. The Journal of Physiology 274:621-637.
Svensson P, Graven-Nielsen T, Matre D, Arendt-Nielsen L (1998) Experimental muscle pain does not cause long-lasting increases in resting electromyographic activity. Muscle & Nerve 21:1382-1389.
Svensson P, Houe L, Arendt-Nielsen L (1997) Bilateral experimental muscle pain changes electromyographic activity of human jaw-closing muscles during mastication. Exp Brain Res 116:182-185.
162 Bibliography
Svensson P, Miles TS, Graven-Nielsen T, Arendt-Nielsen L (2000) Modulation of stretch-evoked reflexes in single motor units in human masseter muscle by experimental pain. Experimental Brain Research 132:65-71.
Takeuchi S, Iwase S, Mano T, Okada H, Sugiyama Y, Watanabe T (1994) Sleep- related changes in human muscle and skin sympathetic nerve activities. Journal of the Autonomic Nervous System 47:121-129.
Tank J, Jordan J, Diedrich A, Stoffels M, Franke G, Faulhaber H-D, Luft FC, Busjahn A (2001) Genetic Influences on Baroreflex Function in Normal Twins. Hypertension 37:907-910.
Thrasher TN (2002) Unloading arterial baroreceptors causes neurogenic hypertension. Am J Physiol Regul Integr Comp Physiol 282:R1044-1053.
Thrasher TN (2004) Baroreceptors and the long-term control of blood pressure. Exp Physiol 89:331-335.
Thrasher TN (2005) Baroreceptors, baroreceptor unloading, and the long-term control of blood pressure. Am J Physiol Regul Integr Comp Physiol 288:R819-827.
Thunberg J, Ljubisavljevic M, Djupsjobacka M, Johansson H (2002a) Effects on the fusimotor-muscle spindle system induced by intramuscular injections of hypertonic saline. Experimental Brain Research 142:319 - 326.
Thunberg J, Ljubisavljevic M, Djupsjöbacka M, Johansson H (2002b) Effects on the fusimotor-muscle spindle system induced by intramuscular injections of hypertonic saline. Experimental Brain Research 142:319-326.
Thunberg J, Lyskov E, Korotkov A, Ljubisavljevic M, Pakhomov S, Katayeva G, Radovanovic S, Medvedev S, Johansson H (2005) Brain processing of tonic muscle pain induced by infusion of hypertonic saline. European Journal of Pain 9:185-194.
Traub RJ, Sengupta JN, Gebhart GF (1996) Differential c-fos expression in the nucleus of the solitary tract and spinal cord following noxious gastric distention in the rat. Neuroscience 74:873-884.
163 Bibliography
Trombetta IC, Somers VK, Maki-Nunes C, Drager LF, Toschi-Dias E, Alves MJ, Fraga RF, Rondon MU, Bechara MG, Lorenzi-Filho G, Negrao CE (2010) Consequences of comorbid sleep apnea in the metabolic syndrome--implications for cardiovascular risk. Sleep 33:1193-1199.
Trott JR (1976) The effect of low amplitude muscle vibration on the discharge of fusimotor neurones in the decerebrate cat. J Physiol 255:635-649.
Tsuboi H, Ohno O, Ogawa K, Ito T, Hashimoto H, Okumura K, Satake T (1987) Acetylcholine and norepinephrine concentrations in the heart of spontaneously hypertensive rats: a parasympathetic role in hypertension. J Hypertens 5:323- 330.
Tucker K, Butler J, Graven-Nielsen T, Riek S, Hodges P (2009) Motor unit recruitment strategies are altered during deep-tissue pain. J Neurosci 29:10820- 10826.
Vallbo AB (1970) Response patterns of human muscle spindle endings to isometric voluntary contractions. Acta Physiol Scand 80:43A.
Vallbo AB (1971) Muscle spindle response at the onset of isometric voluntary contractions in man. Time difference between fusimotor and skeletomotor effects. J Physiol 218:405-431.
Vallbo AB, Hagbarth KE (1967) Impulses recorded with micro-electrodes in human muscle nerves during stimulation of mechanoreceptors and voluntary contractions. Electroencephalogr Clin Neurophysiol 23:392.
Vallbo ÖB, Hagbarth KE (1968) Activity from skin mechanoreceptors recorded percutaneously in awake human subjects. Experimental Neurology 21:270-289.
Victor RG, Leimbach WN, Jr., Seals DR, Wallin BG, Mark AL (1987) Effects of the cold pressor test on muscle sympathetic nerve activity in humans. Hypertension 9:429-436.
Vierck CJ, Whitsel BL, Favorov OV, Brown AW, Tommerdahl M (2013) Role of primary somatosensory cortex in the coding of pain. Pain 154:334-344.
164 Bibliography
Vyskocil F, Hnik P, Rehfeldt H, Vejsada R, Ujec E (1983) The measurement of K+e concentration changes in human muscles during volitional contractions. Pflugers Arch 399:235-237.
Wall P (1979) On the relation of injury to pain. Pain 6:253-264.
Wallin BG, Charkoudian N (2007) Sympathetic neural control of integrated cardiovascular function: Insights from measurement of human sympathetic nerve activity. Muscle & Nerve 36:595-614.
Wallin BG, Elam M, Morris J, Gibbins I (1997) Cutaneous Sympathetic Nerve Activity in Humans. In: Autonomic Innervation of the Skin, The Autonomic Nervous System Amsterdam: Harwood Academic Publishers.
Wallin BG, Esler M, Dorward P, Eisenhofer G, Ferrier C, Westerman R, Jennings G (1992) Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans. The Journal of Physiology 453:45-58.
Yap E (2007) Myfascial pain - An overview. Ann Acad Med Singapore 36:43-48.
Yezierski RP (1988) Spinomesencephalic tract: projections from the lumbosacral spinal cord of the rat, cat, and monkey. J Comp Neurol 267:131-146.
165