! ! ! Physiological Studies of the Vestibulosympathetic Reflex in Humans Elie Hammam, BMedSci (Hons I) School of Medicine University of Western Sydney Supervisor Prof. Vaughan Macefield Co-Supervisor Prof. Kenny Kwok A thesis submitted to the University of Western Sydney in candidature for the award of Doctor of Philosophy, 2014 ! ! "! ! ! ! STATEMENT OF AUTHENTICATION I, Elie Hammam, declare that this thesis is based entirely on my own independent work, except for sections which were performed in collaboration with colleagues as acknowledged in the study and resulted in the publication of the journal articles shown below. To the best of my knowledge this project does not contain material previously submitted in fulfillment of the guidelines and requirements for the award of Doctor of Philosophy in the School of Medicine, University of Western Sydney, and has not been submitted for qualifications at any other academic institution. Elie Hammam ! ! ! ! #! ! ! ! ACKNOWLEDGEMENTS Undertaking the highest scholarly exercise a University offers has certainly been a long, arduous, but nevertheless a fulfilling journey. Now completed, reflection has allowed me to appreciate that what I have achieved is merely a credit to my efforts. I am deeply indebted to the guidance of my mentors, encouragements from friends and support from family. First and foremost, I wish to acknowledge my stellar supervisor Vaughan Macefield who has never shied from supporting me throughout my candidature. Vaughan, from the very beginning you believed in me and never ceased to impart your knowledge, skills and wisdom. You have ensured all throughout my candidature that I get a holistic development in preparation to a life with academic excellence. You are a highly accomplished professor but yet grounded on humility and selflessness; and that is what makes you a special individual. Words cannot sum up what you have done for me nor express my gratitude, so I will simply say - thank you. I have had the privilege to be surrounded by a number of professors that have mentored me and saw my candidature and academic skills come to fruition. Philip Bolton you are a gentleman and a scholar. Thank you for committing to regularly making the trip down from Newcastle and more importantly for all of your scientific contribution and personal influence that has helped shape my progress as a scientist and a person. PhD supervision is not a one-man effort, but a two-man task. To my co-supervisor, Kenny Kwok, thank you for your efforts in ensuring funding and infrastructure whenever and wherever needed. Your dedication, professionalism and thoroughness set a standard of work ethos to aim for. ! ! $! ! ! ! “He who walks with the wise grows wise” Proverbs 13:20. Matthew Barton, it’s a blessing to have walked this journey with you. We met as colleagues and parted as life- long friends. Thank you for all your support. Finally, this journey would have been meaningless without my family. Especially, my mum thank you for your love, unreserved support and sacrifices that shaped the man I am today. To my brothers, thank you for always believing in me, keeping me on track and seeing me grow one step at a time. Last but not least, the best outcome from these past 4 years is meeting my fiancé, Jen. Jen you were not a person to lean on because you made leaning unnecessary. You appreciate my lame jokes and sense of hunour; you are an amazing individual, thank you for your commitment and dedication in supporting me in everything that I do – I love you. I dedicate this thesis to my family ! ! %! ! ! ! ABSTRACT I have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, GVS influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. To further identify the source of vestibular input in the generation of vestibulosympathetic reflexes, I conducted a series of studies using sinusoidal linear acceleration of seated subjects (head vertical) to physiologically stimulate the vestibular system. In Study I & II, I tested the hypotheses that selective activation of one set of otolithic organs - those located in the utricle, which are sensitive to displacement in the horizontal axis - could entrain muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA). Cross-correlation analysis revealed for the 10 subjects in Study I a marked entrainment of SSNA for all types of movements: vestibular modulation was 97±3 % for movements in the X-axis and 91±5 % for displacements in the Y-axis. Furthermore, Study II revealed partial entrainment of MSNA to the sinusoidal stimulus: vestibular modulation was 32±3 % for displacements in the X-axis and 29±3 % in the Y-axis; these were significantly smaller than those evoked in SSNA. In addition, in Study III I examined the capacity for the vestibular utricle to modulate muscle sympathetic nerve activity (MSNA) during sinusoidal linear acceleration at amplitudes below perceptual threshold. Subjects (n=16) were exposed to a range of amplitudes presented in a quasi-random order (1.25, 2.5, 5, 10, 20 and 30 mG), at a constant frequency of 0.2 Hz. Cross-correlation analysis revealed potent sinusoidal modulation of MSNA even at accelerations subjects could not perceive (1.25-5 mG). The modulation index showed a positive linear increase with acceleration amplitude, such that the modulation was significantly higher (25.3 ± 3.7 %) at 30 mG than at 1.25 mG (15.5 ± 1.2 %). Finally, in Study IV I sought to better ! ! &! ! ! ! understand how the brain differentiates between head-only movements that do not require changes in vasomotor tone in the lower limbs from body movements that do require vasomotor changes. As a result, I tested the hypothesis that neck movements modulate MSNA in the lower limbs of humans. Subjects (n=10) lay supine, at rest, during sinusoidal stretching of neck muscles (100 cycles, 35o peak to peak at 0.37 ± 0.02 Hz) and during a ramp-and-hold (17.5o for 54 ± 9 s) static neck muscle stretch, while their heads were held fixed in space. Cross-correlation analysis revealed cyclical modulation of MSNA during sinusoidal neck muscle stretch (modulation index 45.4 ± 5.3%), which was significantly less than the cardiac modulation of MSNA at rest (78.7 ± 4.2%). Overall, by using slow sinusoidal physiological stimuli, evidence accumulated throughout my doctoral candidature emphasizes the role of the utricle, through the vestibulosympathetic reflex, in control of the peripheral vasculature. Moreover, these vestibulosympathetic reflexes can be evoked below perceptual threshold. In addition, through dynamic stimuli of neck proprioceptors my findings also indicate that sensory endings in the neck, as well as vestibular inputs, contribute to cardiovascular control in awake humans via their projections to the vestibular nuclei. ! ! '! ! ! ! !TABLE OF CONTENTS STATEMENT OF AUTHENTICATION 2 ACKNOWLEDGEMENTS 3 ABSTRACT 5 TABLE OF CONTENTS 7 TABLE OF FIGURES 9 TABLE OF TABLES 11 Chapter 1 INTRODUCTION 12 1.1 Vestibular System 13 1.1.1. Structure and physiology 1.2. The Autonomic Nervous System 18 1.2.1. General 1.2.2. Sympathetic control of blood pressure 1.2.3. Recording sympathetic nerve activity in humans 1.2.4. Afferent and efferent pathways of the baroreflex 1.2.5. Physiological implication 1.2.5.1. Compensation 1.2.5.2. Limitations of the predominant mechanism 1.3. The vestibulosympathetic reflex (VSR) and cardiovascular control 32 1.3.1. Vestibulosympathetic reflex in animals 1.3.2. Vestibulosympathetic reflexes in humans 1.3.2.1. Caloric stimulation 1.3.2.2. Head-down neck flexion method 1.3.2.3. Off vertical-axis rotations (OVAR) 1.3.2.4. Galvanic vestibular stimulation 1.3.2.5. Linear acceleration 1.4. Aims 55 ! ! (! ! ! ! Chapter 2 GENERAL METHODS 59 2.1. Subjects 60 2.2. Experimental Protocol 61 2.2.1. Motion Simulator 2.2.2. Neck Table 2.2.3. Microneurography 2.2.4. Recording procedures 2.2.5. Other measured parameters 2.3. Analysis 71 Chapter 3 RESULTS 74 Modulation of skin sympathetic nerve activity (SSNA) by the vestibular utricle Chapter 4 RESULTS 84 Modulation of muscle sympathetic nerve activity by the vestibular utricle Chapter 5 RESULTS 95 Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub- perceptual sinusoidal linear acceleration in humans Chapter 6 RESULTS 110 Modulation of muscle sympathetic nerve activity by neck proprioceptors Chapter 7 GENERAL DISCUSSION 123 REFERENCES 141 APPENDIX 161 ! ! )! ! ! ! TABLE OF FIGURES Chapter 1 INTRODUCTION Figure 1.1 Drawing of the anatomical structure of the labyrinth 14 Figure 1.2 Morphological polarization maps for saccular and utricular maculae in different species 15 Figure 1.3 Anatomical outline of the macula 16 Figure 1.4 Direct MSNA recording from an awake human subject 23 Figure 1.5 The afferent/efferent pathways of the baroreflex 25! Figure 1.6 The physiological effects of postural changes from the supine to standing position 28 Figure 1.7 Orthostasis related activation of the baroreflex (feedback mechanism) increases MSNA and leads to vasoconstriction 31! Figure 1.8 Blood pressure responses to nose-up tilt in chloralose-anesthetized and paralysed cats before and after transection of the CN VIII 33! Figure 1.9 A schematic diagram outlining the direction of GVS 42! Figure 1.10 Baroreceptor influenced MSNA is influenced by vestibular