ALTERED HEMODYNAMIC, NEUROHUMORAL, AND BRAIN METABOLITE PROFILES IN CHILDREN WITH AND CHRONIC NAUSEA

BY

ASHLEY LYNNE WAGONER

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS & SCIENCES

In Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

NEUROSCIENCE

May, 2016

Winston-Salem, North Carolina

Approved by

Carolanne E. Milligan, Ph.D., Chair

Debra I. Diz, Ph.D., Co-Advisor

Stephen J. Walker, Ph.D., Co-Advisor

John E. Fortunato, M.D., MD Advisor

Hossam A. Shaltout, Ph.D.

David L. Mack, Ph.D.

ACKNOWLEDGEMENTS

I want to begin by thanking my thesis committee for their unwavering support, both personally and professionally, during my graduate career. Each member of my committee has had an impact on my research focus and helped me keep sight of my goals throughout my time at Wake Forest.

To my advisor Dr. Debra Diz, thank you for inviting me to join your lab group and encouraging me to stay focused when graduate school seemed overwhelming. I cannot thank you enough for being my advocate and friend during my graduate career. It was always clear you had my best interest as student at heart and for that I’m eternally grateful. To Dr. Steve Walker, thank you for providing a fantastic environment to flourish in as a scientist where I was surrounded by people who taught me something new everyday and for encouraging me to not be afraid to try new scientific approaches to long standing problems. Thank you for introducing me to Dr. John Fortunato and encouraging me to become a part of the American Heart Association Clinical Trial, which is the sole reason this thesis was possible. When I look back on the first tilt tests in 2012, I would have never thought those Wednesday mornings would turn into my dissertation. Thank you, Dr. Fortunato, for trusting me with your patients and their families and giving me a true clinical perspective of research. Also, thank you for staying in touch throughout the last 5 years between time zones and different states. I always looked forward to catching up with you during our conference calls. I attribute much of my success to your willingness to work with me as a graduate student even when you were just getting started. Without your mentorship and guidance this work would not have been possible.

ii Thank you, Dr. Milligan, for facilitating the truly collaborative environment within the neuroscience program. My graduate career is a true testament of collaboration within and outside of the neuroscience program and I am grateful that I had the opportunity to do so. To Dr. Hossam Shaltout, thank you for teaching me how to do noninvasive autonomic recordings in humans and helping me understand the baroreflex.

Your expertise in this subject is incomparable and adds exceptional value to my research and the HVRC as a whole. To Dr. David Mack, thank you for being a fantastic teacher and mentor for my stem cell projects and beyond. Your guidance from across the country made a big impact on my outlook of science and helped shape my experience at Wake

Forest.

Finally, I would like to thank my family and friends who have shown me incredible support and love throughout my time as a graduate student. I have been fortunate to be a member of multiple scientific communities at Wake Forest that have brought me wonderful friends and colleagues that offered advice, support, and laughter on a daily basis. I am also proud to call Winston-Salem my home, as it provided me with a sense of community and life-long friendships in and outside of science. To all my amazing friends, thank you for reminding me that graduate school is a (huge) part of my life, but not my entire life. To my partner, Michael, thank you for reminding me to not take myself too seriously and to remember to always have fun. The impact you’ve had on my life during graduate school is indescribable and I couldn’t imagine myself on this journey without you by my side. To my parents, Jan and David, my brother Alexander, my grandparents Ruth, Jay, and Lucille, thank you for always believing in me even when

I didn’t, and always encouraging me to strive for greatness. This is all for you.

iii TABLE OF CONTENTS

LIST OF FIGURES AND TABLES………………………………………...... vi

LIST OF ABBREVIATIONS…………………………………………………………..viii

ABSTRACT……………………………………………………………………………….x

CHAPTER ONE: INTRODUCTION

The Autonomic Nervous System………………………………………………….4

Integration of Autonomic and Neurohumoral Systems for Control of CV & GI Function……………………………………………………16

Inflammation: Cause or Consequence of Autonomic and Neurohumoral Imbalance………………………………………………………...31

Rationale and Aims………………………………………………………………41

References………………………………………………………………………..44

CHATER TWO:

DISTINCT NEUROHUMORAL BIOMARKER PROFILES IN CHILDREN WITH HEMODYNAMICALLY-DEFINED ORTHOSTATIC INTOLERANCE MAY PREDICT TREATMENT OPTIONS………………………………………………………………...69 Published in American Journal of Physiology: Heart & Circulatory Physiology 310(3): H416-425, 2016

CHAPTER THREE:

HEAD UPRIGHT TILT EVOKES EXAGGERATED ARGININE VASOPRESSIN IN CHILDREN WITH CHRONIC NAUSEA AND ORTHOSTATIC INTOLERANCE…………………………………….102 Submitted to The American Journal of Gastroenterology

CHAPTER FOUR:

IMPAIRED AUTONOMIC RESPONSES IN CHILDREN WITH ORTHOSTATIC INTOLERANCE ASSOCIATE WITH ELEVATED MARKERS OF INFLAMMATION IN THE DORSAL MEDULLA…136 Intended submission to Autonomic Neuroscience: Basic & Clinical

iv CHAPTER FIVE: SUMMARY AND CONCLUSIONS

Summary of Findings…………………………………………………………...168

HUT-induced hemodynamic and autonomic changes in OI and nausea……….172

Neurohumoral compensation for impaired pressure control…………….178

Neurohumoral actions involved in chronic nausea……………………………..185

The role of inflammation in autonomic dysfunction during orthostasis…….….188

Neurohumoral and inflammatory interactions in cardiovascular pathology……193

Limitations……………………………………………………………………...199

Concluding Remarks……………………………………………………………204

References……...……………………………………………………………….208

SCHOLASTIC VITA…………………………………………………………………..218

v LIST OF FIGURES AND TABLES

CHAPTER ONE

FIGURE 1: Autonomic responses to orthostasis……………………………….....6

FIGURE 2: Neurohumoral changes in response to orthostatic challenge……….16

FIGURE 3: Biochemical pathway and actions of the Renin-Angiotensin System…………………………………………………………..…..18

CHAPTER TWO

TABLE 1: Patient Demographics………………………………………………..78

TABLE 2: Correlations between neurohumoral biomarkers and hemodynamics at supine position and in response to HUT………………………….81

FIGURE 1: Orthostatic changes in SBP, DBP, HR upon HUT in children……………………………………………………………..79

FIGURE 2: Orthostatic changes in NE, Epi, AVP, Renin, Aldo, Ang II, and Ang-(1-7) in children……………………………………………….80

FIGURE 3: Orthostatic changes in SBP, DBP, and HR in children in 5 diagnostic HUT(+) subgroups………………………………………………….82

FIGURE 4: Orthostatic changes in NE, Epi, AVP, Renin, Aldo, Ang II, and Ang-(1-7) in children in 5 diagnostic HUT(+) subgroups………….85

CHAPTER THREE

TABLE 1: Patient Demographics……………………………………………....114

TABLE 2: Supine and HUT measurements of autonomic indices……………..115

FIGURE 1: Orthostatic changes in NE, Epi, and AVP in non-responders versus responders…………………………………………………………118

FIGURE 2: Orthostatic changes in Renin, Aldo, Ang II, Ang-(1-7) in non-responders versus responders………………………………...119

FIGURE 3: Orthostatic responses in MAP and HR in non-responders versus responders on HUT……………………………………………...... 120

vi FIGURE 4: Orthostatic changes in AVP and MAP in subjects separated by experience of nausea and hemodynamic response to HUT……….120

CHAPTER FOUR

TABLE 1: Supine Metabolite and Neurotransmitter concentrations…………...149

FIGURE 1: Hemodynamic responses to upright challenge in HUT(-) and HUT(+) subjects…………………………………………………………….146

FIGURE 2: Expected autonomic responses to upright challenge in HUT(-) and HUT(+) subjects…………………………………………………..148

FIGURE 3: Myoinositol and total Choline were significantly higher in the dorsal medulla of HUT(+) subjects ……………………………………...150

FIGURE 4: 1H-MRS spectra, acquired from the dorsal medulla in a HUT(-) (age 16, male) and HUT(+) (age 16, female) subject, showing mIns, Cho, Cr, NAA metabolites……………………………………………...150

FIGURE 5: Regression maps of the correlation summation for the descriptive data…………………………………………………………………153

CHAPTER FIVE

FIGURE 1: Schematic of brain inflammation contributing to OI……………...194

FIGURE 2: Autonomic dysfunction leads to systemic and brain inflammation 195

FIGURE 3: Sympathetic activation and exaggerated NE cause nausea and inflammation in POTS……………………………………………..196

FIGURE 4: The role of autonomic dysfunction in OH and in systemic and brain inflammation……………………………………………197

vii LIST OF ABBREVIATIONS

1H-MRS……….. Proton Magnetic Resonance Spectroscopy a7nAChR……… a7 nicotinic ACh receptor ACE…………… angiotensin converting enzyme ACh……………. acetylcholine ACTH………….. adrenocorticotropic hormone Aldo……………. aldosterone Ang I…………… angiotensin I Ang II………….. angiotensin II Ang-(1-7)………. angiotensin-(1-7) ANS……………. autonomic nervous system AP……………… area postrema AT1……………. angiotensin type 1 receptor AT2…………… angiotensin type 2 receptor AVP…………… arginine vasopressin BP……………… BPM…………… beats per minute BPV……………. blood pressure variability BRS……………. baroreflex sensitivity CRH……………. corticotropin-releasing hormone CRP……………. C-reactive protein CV……………… cardiovascular CVLM…………. Caudal ventrolateral medulla DBP……………. diastolic blood pressure DCS……………. discrete correlate summation DM…………….. dorsal medulla DMNV………… dorsal motor nucleus of the vagus ENS……………. enteric nervous system Epi……………… epinephrine FGID…………… functional gastrointestinal disorders GI……………… gastrointestinal GSH……………. glutathione HFα…………….. high frequency alpha HPA……………. hypothalmo-pituitary-adrenal HR……………… heart rate HRV……………. heart rate variability HUT……………. head upright tilt IL………………. interleukin LFα…………….. low frequency alpha

viii MAP……………. mean arterial pressure mIns……………. myoinositol NA……………. nucleus ambiguus NAA………….. N-acetyl aspartate NAAG………… N-acetyl aspartate glutamate NE……………. norepinephrine NET…………… norepinephrine transporter NTS…………… nucleus of the solitary tract OH…………….. orthostatic OI……………… orthostatic intolerance P/S…………….. POTS/Syncope PBN……………. parabrachial nucleus POTS………….. postural orthostatic syndrome PVN…………… paraventricular nucleus PVT……………. parasympathetic vagal tone RAS……………. renin-angiotensin system rMSSD…………. Root mean square of successive beat-to-beat interval duration RRI…………….. R-R interval RVLM…………. rostral ventrolateral medulla SAP…………….. systolic arterial pressure SBP…………….. systolic blood pressure SDMAP………… standard deviation of mean arterial pressure SDNN………….. standard deviation of beat-to-beat intervals SON……………. supraoptic nucleus SVB…………….. sympathovagal balance SVT…………….. sympathetic vagal tone tCho…………….. total choline tCr……………… total creatine TNF……………. tumor necrosis factor V1……………… vasopressin 1 receptor V2……………… vasopressin 2 receptor

ix ABSTRACT

Ashley Lynne Wagoner

ALTERED HEMODYNAMIC, NEUROHUMORAL, AND BRAIN METABOLITE PROFILES IN CHILDREN WITH ORTHOSTATIC INTOLERANCE AND CHRONIC NAUSEA

Dissertation under the direction of

Debra I. Diz, Ph.D., Professor Stephen J. Walker, Ph.D., Associate Professor

Orthostatic Intolerance (OI) is a type of autonomic dysfunction affecting

>500,000 adults and children in the United States. Upon standing, subjects with OI have impaired blood pressure (BP) regulation that leads to debilitating symptoms of dizziness, fatigue, nausea, and fainting. The development of OI is poorly understood and a variety of cardiovascular responses occur with standing including exaggerated tachycardia, hypotension, and syncope. OI is associated with functional gastrointestinal (GI) symptoms, specifically chronic nausea, in children and exaggerated neurohumoral responses in adults. The heterogeneity in symptoms of autonomic dysfunction highlights the importance of defining phenotypic profiles to ensure effective management and treatment of the patient-specific cardiovascular (CV) and gastrointestinal (GI) symptoms.

To do this, it is critical to identify key factors that regulate autonomic outflow in supine and upright positions in OI. To this end, our goals were to define phenotypes of OI and chronic nausea in children based upon measures of hemodynamic and neurohumoral responses to orthostatic challenge and to evaluate the relationship between supine cerebral metabolites to autonomic function.

x Using a head upright tilt table test (HUT) we evaluated hemodynamic responses and circulating components of the renin-angiotensin system, catecholamines, and arginine vasopressin (AVP) in children with chronic nausea and OI symptoms.

Hemodynamic profiles revealed 4 specific subtypes of OI, which demonstrated diverse tilt-induced neurohumoral changes. The distinct differences observed provide evidence for the usefulness of HUT and neurohumoral profiling in diagnosing and directing effective treatment options in children with OI.

In response to HUT 40% of subjects had HUT-induced nausea within 15 minutes of standing and an exaggerated increase in AVP that was independent of OI status. The greatest increase of AVP was observed in subjects with HUT-induced nausea and a positive HUT. Together, these observations suggests different mechanisms of AVP release are occurring in children with chronic nausea and OI such that baroreflex dysfunction may not be the sole cause of AVP release in these subjects.

Lastly, proton magnetic resonance spectroscopy was used to measure cerebral metabolite concentrations within the dorsal medulla. Subjects with OI demonstrated expected autonomic impairments upon HUT and had higher levels of metabolites indicative of glial inflammation. This is the first demonstration that neuroinflammation, previously implicated in autonomic pathology, accompanies the autonomic dysfunction observed children with OI. Collectively these findings provide practical information relevant to better diagnosis and treatment of OI in the clinical setting and elucidate novel mechanisms for potential causes of autonomic dysfunction to direct future hypothesis- driven studies.

xi CHAPTER ONE

INTRODUCTION

Orthostatic stresses are common daily events for adults and children that require prompt adjustments of blood pressure (BP) and volume to prevent venous pooling in the lower limbs. Orthostatic intolerance (OI), effecting >500,000 adults and children, occurs when compensatory mechanisms fail to maintain BP when in the upright position as a consequence of varying degrees of autonomic nervous system (ANS) dysfunction1.

Various cardiovascular responses occur with OI2-4 including exaggerated increases in heart rate (HR), hypotension, and syncope of which all are associated with secondary symptoms of dizziness, fatigue, and nausea1. Despite decades of research, the etiology underlying OI is not fully understood. OI in adults is also associated with neurodegenerative disorders5, 6, hypertension7, 8, coronary heart disease9, and heart failure10. However, it is unknown whether the development of OI during childhood progresses to these severe comorbid problems in adulthood. Thus, early detection, proper management, and directed treatment options are imperative for the health and quality of life of children, as well as prevention of adult-onset disorders.

Comorbid GI symptoms, including nausea and abdominal pain, present in all OI subtypes11-15. Even though symptoms are not often recorded during patient evaluation for

OI, studies have shown increased evidence for OI as an underlying mechanism for GI symptoms in both children and adults12, 16. In fact, ~80% of children with chronic nausea and orthostatic symptoms tested positive for OI, suggesting that imbalances in the ANS affect both CV and GI systems chronically12. Abnormal myoelectrical changes in the gut occur in the upright position as well directly associating autonomic dysfunction during

1 standing with altered GI motility that can elicit chronic GI symptoms12, 17, 18. In addition to autonomic imbalances, patients with OI and functional GI disorders (FGIDs), respectively, have heightened stress and anxiety. Inflammation is implicated in central regulation of autonomic function, GI disorders, and stress and anxiety and could play a role in the manifestation of symptoms in children.

Therapeutic agents including α-1 agonists19, 20, β-blockers21, fludrocortisone13, and desmospressin22 have been used to treat specific OI subgroups with similar efficacy.

Although suboptimal, these therapies have shown to treat secondary symptoms including nausea and lightheadedness, which improves the quality of life of patients with OI. For example, fludrocortisone was reported to improve GI symptoms and increase school attendance in children, but did not prevent the autonomic impairments observed during standing as evoked by head upright tilt (HUT)23. Thus, the pathophysiological changes associated with OI and GI symptoms need to be considered in order to provide optimal treatment strategies in these populations.

The studies contained in this dissertation provide evidence of distinct neurohumoral profiles in OI subtypes and nausea, respectively, as well as central inflammation in the brain stem area key to BP regulation. The following sections discuss autonomic pathology and central mechanisms involved in regulation of BP under normal and pathological conditions and during orthostatic challenge. Regulation of GI function is also affected by similar mechanisms24 and is discussed in conjunction with CV regulation. The integration of neurohumoral actions in autonomic regulation is discussed relative to BP homeostasis and autonomic performance upon orthostatic challenge. In addition to their involvement in BP regulation, neurohumoral mechanisms are involved in

2 inflammation in the brain, cardiovascular system, and GI tract in response to stress25. The role of inflammation in these areas may contribute to altered central regulation of autonomic function and provide evidence of novel contributors to autonomic imbalance observed in OI and direct future studies in children with OI.

3 1. The Autonomic Nervous System

The ANS is a part of the peripheral nervous system that controls involuntary physiological functions26. There are two opposing branches of the ANS, the sympathetic and parasympathetic systems, which work together to maintain homeostasis of visceral functions. The parasympathetic system stimulates functions of “rest and digest” which occur when the body needs to return to a resting state. The sympathetic system stimulates functions for “fight or flight,” by increasing energy in certain tissues to respond to environmental stressors. Both systems are regulated by reflex arcs between sensory and motor neurons in the viscera and autonomic brainstem nuclei26.

The sympathetic and parasympathetic systems work in concert for regulation of the cardiovascular system and maintenance of cardiac output. Activation of the sympathetic nervous system increases cardiac output and vasoconstriction to elevate HR and BP27. Alternatively, the parasympathetic system exerts tonic inhibition to maintain control over the heart, specifically to decrease cardiac output and lower HR and BP.

Activity of the vagus , which starts in the medulla and innervates the heart, is often used to assess parasympathetic tone. Indices of vagal function include heart rate variability (HRV), a measure of parasympathetic vagal tone to the heart, and baroreflex sensitivity (BRS) for control of heart rate in response to changes in arterial pressure27.

Both sympathetic and parasympathetic systems project efferents to the gastrointestinal tract providing extrinsic control over GI motility. Additional intrinsic neural control provided by the enteric nervous system (ENS) exists within the GI tract.

The extrinsic control is essential for almost all secretory and motor functions of the

4 digestive tract by modulating the ENS28. Thus, dysfunction of the ANS not only affects cardiovascular performance, but also regulation of GI motility.

The Baroreceptor Reflex

Arterial BP is regulated by feedback control systems that work together over time to determine BP and blood pressure variability (BPV) in response to environmental stimuli29. The baroreflex is a rapid negative feedback reflex involving widespread integration in the central nervous system and both autonomic systems to stabilize and maintain BP and blood volume30. Changes in BP are sensed within the aortic arch and carotid sinus by mechanosensitive high-pressure baroreceptors. Cardiopulmonary baroreceptors, often referred to as low-pressure baroreceptors, have nerve endings located in the heart, vena cava, and pulmonary vasculature, which convey information about changes in central blood volume to the brain. The studies presented in this thesis focus on the baroreflex for control of HR and BP in response to orthostatic challenge, where venous pooling in lower limbs occurs immediately and adjustments are provided by BP regulatory mechanisms to return BP above the diaphragm back to normal31, 32.

To effectively minimize BPV, the baroreflex modulates parasympathetic and sympathetic activity to the heart. Acute changes in BP stimulate arterial high-pressure baroreceptors and produce excitatory activity along the vagus and glossopharyngeal . The corresponding afferent activity is transmitted to the nucleus of the solitary tract (NTS) in the dorsal medulla. The NTS is essential to the integrity of the baroreflex arc as lesions to this autonomic center in the brainstem eliminate baroreflex responses33.

Additional input to the NTS comes from mechanosensitive fibers representing changes in blood volume. From the NTS, low- and high-pressure baroreceptor inputs are integrated

5 centrally and relayed to second order hypothalamic and autonomic brainstem nuclei involved in two diverging effector pathways controlling parasympathetic and sympathetic activity33, 34.

Autonomic Physiology of Upright Posture

Transition from supine recumbancy to upright

position or standing is followed by pooling of blood

almost immediately shifting over 500 milliliters of

blood below the diaphragm. The occurrence of

venous pooling results in decreased BP at the level

of the high-pressure baroreceptors in the heart35, 36.

To return BP to the head and neck to normal, arterial

baroreceptor responses to orthostasis occur rapidly

with increased HR occurring within one or two beats

in healthy individuals. Low-pressure receptors

detecting decreased filling of central venous

Figure 1. Autonomic responses to circulation act in concert with arterial baroreceptors orthostasis. Decreased BP upon standing leads to decreased BR input to compensate during standing, but are not essential to the NTS, resulting in various essential downstream effects. “+” stands for excitatory and “-“ stands for all orthostatic adjustments. Normally arterial for inhibitory. Hatched arrows indicate change in activity in baroreceptors tonically inhibit the caudal response to standing. HR, heart rate; SVR, systemic vascular resistance; ventrolateral medulla (CVLM) via NTS. The CVLM CO, cardiac output is home to vasopressor medullary neurons that inhibit sympathetic activity via GABAergic projections to the rostral ventrolateral medulla (RVLM). At the same time, the NTS activates glutamatergic projections to

6 parasympathetic pre-ganglionic neurons in the dorsal motor nucleus of the vagus

(DMNV) and nucleus ambiguus (NA) to maintain reduced cardiac output, heart rate and blood pressure under normal conditions27, 37-40.

As shown in the Figure 1, the decrease in BP that occurs with upright posture removes the tonic inhibition resulting in decreased vagal outflow and increased sympathetic activation. Decreased vagal activity to the heart and increased sympathetic preganglionic outflow leads to increased HR, systemic vascular resistance, and overall cardiac output. Since the heart cannot pump blood that it does not receive, the vascular resistance stimulated by sympathetic activation is a key modulator in maintaining BP during standing41, 42. Additionally, neurohumoral activity is altered by postural changes and specific mechanisms are discussed later on.

In the following studies, continuous BP and HR were acquired using a noninvasive arterial pressure dual-finger cuff to allow for calculation of autonomic indices of spontaneous baroreflex sensitivity (BRS), HRV, BPV, parasympathetic vagal tone (PVT), sympathetic vagal tone (SVT), and sympathovagal balance (SVB).

Spontaneous BRS is calculated by frequency-domain analysis via power spectra densities of systolic arterial pressure (SAP) and R-R intervals (RRI) at 1000 Hz. RRIs represent the time in milliseconds between R-waves in the QRS complex as measured by echocardiogram. BRS is reflected by low-frequency (LFα) and high-frequency alpha

(HFα) indices and are representative of the square roots of the ratios SAP and RRI43. LFα calculations represent mostly sympathetic tone, but previous reports show it also measures vagal function as well44. HFα is an indicator of parasympathetic vagal outflow regulated by the baroreflex. The power of RRI spectra in the LF and HF range (LF RRI

7 and HF RRI) is calculated using normalized units and the ratio is used as a measure of

45 SVB . BPV is calculated using the power of SAP spectra, LFSAP, or the standard deviation of MAP (SDMAP)46. Time domain parameters can also be used to measure

BRS and HRV. The SD of beat-to-beat intervals (SDNN) and the root mean square of successive beat-to-beat differences in beat-to-beat interval duration (rMSSD) are both representative of HRV. As shown in previous studies, BRS can be calculated over a time/sequence domain. This method uses the quantification of sequences of 3 beats (n) in which SAP consecutively increases (up sequences) or decreases (down sequences), which are accompanied by changes in the same direction of the RRIs of the subsequent beats

(n+1)47, 48. Chapters Three and Four present autonomic measurements using these methods in supine and during standing to evaluate abnormal postural changes in baroreflex function in children with nausea on HUT and with OI.

Autonomic Control of GI function

The GI tract possesses over one hundred million enteric neurons integral to intrinsic and autonomous control of GI functions of digestion, nutrient absorption and waste elimination. However, the central and autonomic systems provide extrinsic neuronal inputs that regulate and modulate intrinsic functions of the ENS28. Both sympathetic and parasympathetic branches have a role in autonomic modulation on GI activity. Furthermore, higher CNS centers that influence homeostatic control, including the hypothalamus, can also modulate GI activity24, 49, 50.

At the level of the gut, gastric smooth muscle motility is controlled in concert by release of peptides and transmitters by the ENS and modulation of transmitter release via autonomic nerve innervation28. Autonomic regulation of GI motility arises from

8 sympathetic outflow from the spinal cord and parasympathetic tone from the DMNV.

Sympathetic outflow from the thoracolumbar spinal cord is predominantly inhibitory to the gut smooth muscle and mucosal secretion, slowing GI motility and secretion.

Additionally, sympathetic efferent fibers to GI vasculature regulate neurally mediated vasoconstriction and therefore, overall GI blood flow24, 51-54.

The parasympathetic system has a more complex influence of GI activity, exerting both excitatory and inhibitory control in the gut regulating GI motility and inflammation. The vagus nerve supplies parasympathetic innervation to the stomach, small intestine, and proximal colon to regulate vasovagal reflexes of digestion49. The function of vagal neurons depends on the target organ as well as NTS neuromodulation, which provides the most prominent source of synaptic inputs to GI-projecting DMNV neurons. In addition to receiving CV and respiratory afferents, the NTS also receives vagal sensory inputs from the thoracic and abdominal viscera55. In fact, at the level of the diaphragm, 40% of fibers in the vagus nerve are afferent C-fibers from nodose ganglia in the gut11, 56. These afferent fibers project to the NTS, DMNV, and area postrema, which in turn modulate motor output to the GI tract. Types of sensory information relayed from the gut to the NTS specifically include distention or pressure, location of event, and stimulus modality55. The effects of gut-brain activity of inflammation in the GI tract are discussed later on in this chapter.

Central regulation of the ANS

In addition to medullary control of autonomic outputs, the central autonomic network involves several other interconnected areas throughout the brain57. This network is critical in moment-to-moment involuntary control of visceral function, homeostasis,

9 and adaptation to external stimuli as well as integration of body sensation with emotional and goal-related autonomic responses. Thus, central regulation of BP and GI motility modulated by brain structures throughout the neuraxis are essential to proper involuntary visceral functions58, 59.

While lower brainstem function is involved in baroreflex control of HR and BP, respiration, and GI function, the upper brainstem integrates pain afferents and behavior responses to stress. The area postrema is a circumventricular organ located in a dorsal medulla outside the blood brain barrier. This highly vascularized region receives neural inputs from the periphery and responds to circulating neurohormones60-64. Projections from the area postrema modulate actions of the NTS, DMNV, CVLM, RVLM, and parabrachial nucleus (PBN), all of which are involved in autonomic regulation of BP65, 66.

This region also serves as the emetic center of the brain stem that evokes nausea and vomiting when stimulated by altered GI motility or motion sickness. The PBN is a major relay center in the pons that receives inputs from spinal cord regions conveying visceral, nociceptive, and thermoreceptive information to the hypothalamus, amygdala, and thalamus. The PBN and other nuclei in the tegmentum also participate in control of GI motility in the lower GI tract and respiration58, 67. In the midbrain, the periacqueductal gray provides the interface between forebrain structures and the lower brain stem, participating in pain modulation associated with cardiovascular responses, responses to stress, GI pain, and other adaptive functions67, 68.

Most brainstem areas involved in involuntary visceral functions relay information to forebrain regions. The hypothalamus acts as a visceromotor pattern generator for circadian rhythms, food and water intake, neuroendocrine responses, and BP

10 regulation58,69. Changes in blood volume or osmolarity, and external stress trigger autonomic and endocrine responses modulated by the paraventricular nucleus (PVN) in the hypothalamus69, 70. Unlike any other brain site, the PVN has a closed reflex loop with both sympathetic and parasympathetic systems. The PVN receive autonomic afferents from the trigeminal pars caudalis and NTS representing both branches of the ANS71.

Neurons in the PVN project to the DMNV as well as directly to the spinal cord to regulate CV function72. The PVN also contains magnocelluar neurons that modulate stress, sodium intake, and GI functions via release of neurohormones including arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) involved in the hypothalamo-pituitary-adrenal (HPA) axis73, 74.

Insular and anterior cingulate cortical areas also contribute to both sympathetic and parasympathetic modulation via the hypothalamus and brainstem. The insular cortex regulates interoceptive and cognitive properties associated with autonomic performance58,75. Specifically, the right anterior insula integrates viscerotopic information from the dorsal medulla and emotional and cognitive processing to produce the conscious experience of bodily sensation75. Alternatively, the ventral anterior cingulate is part of the

“default mode network” of the brain and provides extensive connections to the insula, prefrontal cortex, amygdala, hypothalamus, and brainstem76, 77. Both brain regions have been targeted in neuroimaging studies investigating central regulation of autonomic dysfunction in adults with autonomic and neurodegenerative disorders78-82.

11 ANS dysfunction and disorders

Under normal conditions, the sympathetic and parasympathetic branches of the

ANS maintain a dynamic balance and make rapid adjustments based on changing environmental stimuli83-85. Impaired baroreflex responses and subsequent sympathovagal balance is implicated in various pathological conditions and may be an important mechanism underlying the development of OI, functional GI symptoms and comorbidities of both types of conditions. Additionally, differential activity of sympathetic and parasympathetic tone occurs in OI subtypes, postural orthostatic tachycardia syndrome (POTS), (OH), and neurally mediated syncope11, 41, 86. Understanding the underlying mechanisms resulting in heterogenous autonomic responses to orthostasis is necessary to direct maintenance and treatment strategies.

Orthostatic hypotension is a key manifestation of autonomic dysfunction resulting in a decrease greater than 25 mm Hg for systolic and greater than 10 mm Hg for diastolic

BP. The failure to compensate for venous pooling reflects structural or functional sympathetic denervation or impairment of baroreflex regulation of sympathetic outflow.

These impaired autonomic responses can occur as quickly as 3 minutes in upright position or have delayed onset after 3 minutes4, 87. In either case, secondary symptoms including nausea, visual blurring, abdominal pain, and fatigue, accompany OH. In fact,

OH occurs is 15% of syncope presentations88. Adults with OH also report supine hypertension and wide swings in BP, implicating chronic decreased HRV as an underlying mechanism86, 89, 90. Furthermore, OH is a primary manifestation of pure

12 autonomic failure, multiple system atrophy, and Parkinson’s and can occur secondary to diabetes and renal failure91.

Postural orthostatic tachycardia syndrome (POTS) is the most common presentation of OI, characterized by a significant HR increase of 40 beats per minute

(BPM) or HR greater than 120 BPM in children within 10 minutes of standing4, 41, 92.

Symptoms of cerebral hypoperfusion and sympathetic hyperactivity accompany POTS including impaired cognitive performance, dizziness, sweating, and nausea. The exaggerated sympathetic response also leads to palpitations, chest pain, and tremulousness41, 92-96. Pathophysiological mechanisms underlying POTS are heterogenous in nature3. Neuropathic POTS is characterized by impaired sympathetic vasoconstriction to the lower limbs. Volume dysregulation and hyperadrenergic subtypes of POTS implicate neurohumoral imbalances97, 98. Postural deconditioning by prolonged bed rest decreases the gain of vasoconstrictor response and the vestibulosympathetic response99-

102. A variety of comorbidities occur with POTS in adults including Ehlers-Danlos

Syndrome5, 103, chronic fatigue104-106, fibromyalgia107, and sleep disturbances104.

Neurally mediated syncope refers to a heterogenous group of conditions where a sudden decrease in autonomic activity occurs leading to a significant fall in BP, HR, and cerebral perfusion. Prolonged standing is a peripheral trigger for syncope where sympathetic vasoconstrictor nerve activity decreases and parasympathetic outflow increases resulting in decreased systemic vascular resistance and slowed HR4. Symptoms of hyperventilation, nausea, diaphoresis as well as cognitive slowing and visual disturbances often precede syncopal episodes108-110.

13 In both adults and children, OI and chronic GI symptoms are associated with altered autonomic activity to the heart, peripheral vasculature, and gut11. However, a high incidence of chronic unexplained nausea in the pediatric GI clinic could be accounted for by OI13, 111 and is investigated in Chapter Three. The experience of nausea is mediated by changes in gastric electrical activity, the ANS, and increased brain activity within multiple cortical and subcortical regions112-116. Robust changes in autonomic outflow occur with nausea causing gastric dysrhythmias that can be acute or chronic. In fact, blocking baro- and chemo- receptors by internal carotid ligation or carotid body denervation suppresses emesis117. Consistent with previous reports 114, 118, Farmer and colleagues reported increased sympathetic and decreased parasympathetic activity in nausea sensitive subjects induced by motion sickness112. Furthermore, sensory vagal inputs are thought to trigger gastrointestinal derived nausea sensation as well as sensitize feedback circuits contributing to the autonomic outflow116. It is also suggested that intensity of nausea experienced is associated with both central and autonomic outflow 115.

Thus autonomic imbalances impact GI motility, eliciting nausea and emesis regulated by both the NTS and area postrema119, 120.

Nonorthostatic symptoms of visceral pain, dysmotility, headache, and chronic fatigue accompany all subtypes of OI described previously3. Chronic nausea is also a public health concern with underlying autonomic complications121, 122. To date, the majority of research focuses on adults with OI and nausea as separate entities and not comorbid conditions. Both types of studies have described autonomic imbalances and neurohumoral responses to standing123-127 and motion-induced nausea112, 128, 129. However, there are few studies evaluating the role of neurohumoral compensation during

14 orthostasis in children with OI and/or nausea. Furthermore, no study to date has evaluated orthostatic neurohumoral responses as a cause of nausea experienced in upright position or just a consequence of impaired autonomic function.

15 2. Integration of autonomic and neurohumoral systems for control of CV & GI function

Neurohumoral responses to orthostasis

In addition to hemodynamic and autonomic responses, postural changes elicit alterations in neurohumoral mechanisms. The contribution of the humoral system to circulatory orthostatic adjustments Figure 2. Neurohumoral changes in response to orthostatic challenge. CO, cardiac output; SVR, systemic vascular resistance; + depends on the adequacy of SYMP, sympathetic activation; Na , sodium; H2O, water. blood volume circulation. Volume-regulatory mechanisms respond to decreased blood volume by initiating renal retention of water and sodium. Activation of humoral mechanisms is especially important during prolonged orthostasis38, 130.

The normal response to orthostatic challenge, as shown in Figure 2, consists of well-integrated neurohumoral changes that sustain BP. Normally, the decrease in BP at the level of the high-pressure baroreceptors is followed by an increase in sympathetic nerve activity to the heart and a vasculature response that also includes catecholamine release by the adrenal medulla and renin release by the kidney. The resulting vasoconstriction and increased HR serve to increase systemic resistance and cardiac output to return pressure in the upper body and head to normal. The increased

16 angiotensin II (Ang II) triggers release of two additional hormones, arginine vasopressin

(AVP) and aldosterone (Aldo) which over time promote increased thirst and sodium and water retention that aides in increasing blood volume. During orthostatic stress, activation of the renin-angiotensin system (RAS), catecholamine release, and AVP are necessary to combat imminent hypotension in the low volume state38.

Abnormal regulation or responses to both sympathetic activation and the RAS have been implicated in the pathogenesis of OI124, 130, 131. These neurohumoral components are released both centrally and peripherally and have actions in organ systems beyond the heart and vasculature including the gut. Furthermore, the effect of neurohormones released during orthostasis can also elicit changes in myoelectrical activity in the GI tract, which causes nausea and abdominal pain, among other GI symptoms128, 132-134. The following sections describe the major physiological roles of the

RAS, AVP, and catecholamines in relation to CV and GI mechanisms.

Role of RAS in cardiovascular homeostasis and gastric integrity

The RAS plays an important role in BP regulation under normal and pathophysiological conditions135. Angiotensin receptors are localized on brain regions involved in modulating both sympathetic and parasympathetic nervous system activity as well as in GI smooth muscle tissues136-138. Shown in Figure 3, the RAS pathway is a series of enzyme-substrate interactions that generates angiotensin peptides. The hepatic- derived precursor for the RAS, Angiotensinogen, is cleaved by the aspartyl protease enzyme renin into angiotensin I. Angiotensin I (Ang I) is processed by angiotensin converting enzyme (ACE) into Ang II, which is the primary effector of the RAS. Ang II facilitates various downstream effects by binding to angiotensin II type 1 (AT1) or type 2

17 135, 139, 140 (AT2) g-protein coupled receptors .

Alternatively, Angiotensin-(1-7) can also be

formed by cleavage of Ang I by various

endopeptidases or through cleavage of Ang II

by ACE2, a homolog of ACE141-144. The

actions of Ang-(1-7) primarily counteract Ang

II effects on CV function by acting on g-

protein coupled Mas receptors135, 141. The

balance between these two peptides in the Figure 3. Biochemical pathway and actions of the Renin-Angiotensin System. heart, vasculature, and brain is essential for CV Enzymes indicated by circles. Peptides indicated by rectangles. homeostasis and is implicated in the pathophysiology of various CV disorders, including hypertension and metabolic syndrome141, 145, 146.

The effects of the RAS in cardiovascular, fluid-electrolyte, and neuroendocrine homeostasis have been studied over decades in hopes to target therapeutic options for patients with cardiovascular disease. Ang II AT1 receptors and Ang-(1-7) mas receptors are abundant at each synaptic relay point of the sympathetic and parasympathetic systems139. As shown through systemic or central administration at various hypothalamic

(PVN, SON) and brainstem sites (NTS, DMNV, AP), Ang II elicits BP elevations via various mechanisms147-150. First, systemic administration of Ang II has shown to increase

BP by reducing vagally-mediated BRS for control of HR151, 152. Ang II-induced increases in BP are abolished by sympathectomy suggesting overactive sympathetic outflow to the heart as another mechanism of action153. Ang-(1-7) opposes effects of Ang II, eliciting

18 vasodilation and improved baroreflex function135, 154, 155. Decades of research has shown that the endogenous balance of Ang II and Ang-(1-7) mediates the pressor/depressor actions in brainstem nuclei involved in BP regulation. ACE inhibitors or AT1 receptor blockers are often prescribed for patients with Ang II-dependent hypertension because they improve BRS and promote increased BPV156. However, patients with resistant hypertension do not respond to these types of treatments, or a combination of treatments, suggesting there are other mechanisms underlying BP regulation and potential downstream effects of long-term elevations of Ang II, in addition to Ang-II mediated hypertension157, 158.

The RAS is also involved in the kidney’s role in BP regulation through alterations in sodium and water retention and excretion. Vasoconstrictive properties of Ang II are exerted in the kidney to prevent pressure-natriuresis135, 159. This reduces the kidney’s ability to compensate for increased BP by stimulating increases in sodium and water excretion. Additionally, stimulated release of Aldo from the adrenal gland occurs with increased Ang II. Since the primary role of Aldo is to maintain low extracellular potassium and guard against sodium loss, hyperaldosteronism is also a prevalent attribute of BP increases observed in hypertension160, 161. Again, Ang-(1-7) opposes Ang II effects, to increase sodium and water excretion serving to lower blood volume and subsequent

BP162-164.

Emerging evidence for RAS involvement in gastric tissues suggests a role of RAS in GI tissue integrity165 and GI blood flow. In fact, RAS receptor components have been localized in the esophagus166-168, stomach169, small intestine170 (Ewert 2006), and colon170, 171. Motility of gastric smooth muscle is critical to all functions of the GI tract

19 and dysrhythmias cause a variety of symptoms including abdominal pain and nausea.

Ang II is well known for producing smooth muscle contractions in the vasculature, but also demonstrates regulatory effects on GI musculature. Casselbrant and colleagues investigated Ang II actions in the human esophagus, reporting Ang II formed locally and centrally can target GI musculature and induce excitation, causing muscle contraction137.

Ang II-induced excitation has also been shown to mediate GI motility via AT1 receptors promoting both tonic contraction and increased frequency of spontaneous contractions172.

Furthermore, Ang II in the gut can also facilitate release of norepinephrine (NE) from sympathetic postganglionic axons, which may suppress excitatory transmission and muscosal secretion173. Although Ang-(1-7) offsets Ang II in many physiological settings, its role in GI motility has yet to be investigated.

Role of Catecholamines in sympathetic autonomic regulation

Endogenous catecholamines, NE and epinephrine (Epi), are active central neurotransmitters with actions in the peripheral autonomic nervous systems and ENS.

There are two types of receptors, alpha (α) and beta (β) that facilitate systemic effects of

NE and Epi. Furthermore, there are 2 subtypes of these receptors, α1, α2, β1, β2, which associate with g-proteins and second messengers that carry out downstream intracellular

174, 175 effects. NE binds to α1, α2, β1 receptors and Epi can bind to all four subtypes . While

NE is the main transmitter of the sympathetic system mediating regulation of the circulation, Epi levels correlate more closely with increases in pituitary-adrenocortical activity related to stress176, 177.

The CV system has extensive noradrenergic innervation, with the heart containing significantly higher NE concentrations compared to other organ systems. NE is an

20 exceptional clinical neurochemical biomarker of sympathetic innervation because it can be measured in human plasma and its metabolism detected by assays simultaneously measuring levels of NE and other compounds related to NE synthesis. Postganglionic sympathetic nerve endings and chromaffin cells in the adrenal medulla are responsible for releasing NE into the circulation. However, cotransmission of other compounds including

Epi, adenosine, acetylcholine (ACh) and dopamine occurs with stimulated release from sympathetic nerve endings176, 177. Stimulated release of NE from sympathetic ganglion causes CV smooth muscle cell contraction by binding to β1-adrenoceptors localized throughout the heart and vasculature178, 179. NE-stimulated vasoconstriction occurs systemically in arteries and veins through α1 and α2 receptors. Together these responses serve to increase cardiac output and systemic vascular resistance180, 181. Increases in HR are also stimulated by NE, but only acutely, as activated baroreceptors elicit vagally- mediated slowing of HR176.

Epi also plays a role in autonomic regulation to the heart, especially under conditions of stress. The adrenal medulla is responsible for 80% of Epi secretion in the circulation, as it contains phenylethanolamine N-methyltransferase that methylates NE to form Epi. Stimulated release of Epi occurs in response to global threats, spanning external physical, internal psychological, or metabolic stimuli177, 180, 182. Through adrenal venous release Epi rapidly reaches all cells of the body (with the exception of the brain) and low blood concentrations produce a wide variety of hormonal effects. Epi causes increased HR and smooth muscle contraction via β1-adrenoreceptors and arterial and venous vasoconstriction via β2-adrenoreceptors183. Interestingly, at low concentrations

Epi actually stimulates β2-receptor induced vasodilation in muscle and liver vasculature,

21 but vasoconstriction via α-receptors at high concentrations. Thus, although low concentrations are enough to elicit changes in vascular tone, high concentrations of Epi are needed to increase arterial pressure181, 182.

As discussed previously, sympathetic innervation of the GI tract affects blood flow, gastric tissue integrity, and GI motility. The roles of NE and Epi have not been directly investigated in the GI tract. However, α1 and β2 receptors are both found in GI tissues involved in motility and α1 receptors are found in sphincters of the stomach and

184-187 intestines . Since NE release is stimulated by increased sympathetic outflow and α1 receptors are present in the GI tract it is plausible that it has actions in this organ

52, 188-190 system . If so, NE acting on α1 receptors would increase vasoconstriction and decrease blood flow resulting in decreased gastric integrity and GI smooth muscle contraction. High concentrations of Epi may also induce vasoconstriction via β2 receptors.

The influence of Epi on GI function has been discussed in relation to gastric dysrhythmias and nausea, as increases were observed in response to visually-induced nausea17, 128.

Role of AVP in water homeostasis and blood pressure regulation

The neurohypophysial hormone AVP helps maintain CV homeostasis through various physiological regulatory processes including renal water absorption, systemic BP and vascular tone, baroreflex stimulation, and hormone secretion from the anterior pituitary, as well as modulation of social behavior and stress74, 191. The larger precursor protein prepro-AVP is cleaved to produce AVP and 2 other constructs, neurophysin 2 and copeptin192, 193. Neurophysin 2 is a carrier protein for both AVP and oxytocin. The functionality of copeptin is not well described, but it serves as a biomarker of AVP

22 release because its remains at more stable levels in the circulation21, 194. AVP is released from magnocellular neurons projecting from the PVN and SON of the hypothalamus to the posterior pituitary in response to changes in blood osmolality and BP. Parvocellular neurons of the PVN project to the anterior pituitary where AVP release induces secretion of stress-related hormones including adrenocorticotropic hormone (ACTH)195. The role of AVP in blood volume and BP regulation is discussed in the following section. Stress and inflammation in the context of neurohormones, especially AVP, and autonomic function is discussed later on in this chapter.

In addition to the pituitary projections, axons carrying AVP innervate other areas of the brain and spinal cord allowing AVP to exert actions systemically as well as locally in the brain196. Serum osmolality, representing the concentrations of osmolytes like sodium in the blood, is the most sensitive stimulus of AVP release. Under normal physiological conditions, glucose and urea readily cross neuronal cell membranes and do not stimulate AVP release. However, AVP release follows even as little as a 1% increase in plasma osmolality and acts on V2 receptors, which have the highest affinity for AVP compared to other vasopressin receptors. Specifically, AVP maintains body fluid homeostasis through the V2-receptor-cyclicAMP-aquaporin receptor 2 pathway as V2 receptors are mainly found in the collecting duct of the kidney where regulation of water retention occurs. When AVP binds to V2 receptors, a process termed antidiuresis occurs to trigger collecting duct cells to retain and shuttle water to kidney medullary circulation.

V2 receptors are also found in the vascular epithelium where AVP stimulates vascular dilation197-201.

23 Maintaining adequate amounts of water in the body is essential for blood volume and BP regulation. Although secretion of AVP is less sensitive to changes in BP and volume than its response to reduced osmolality, this peptide has repeatedly demonstrated vasoconstrictor properties in many vascular beds202, 203. AVP acts on V1a receptors located in the brain, smooth muscle cells, and adrenal cortex to increase vascular contraction, sympathetic nerve activity, and BRS to promote increases in BP204, 205. While low BP status can stimulate AVP release via both low-pressure baroreceptors and high- pressure baroreceptors, AVP/V1aR actions are necessary for basal BP maintenance. This is evident in V1aR-KO mice where baroreflex control of HR is significantly attenuated compared to WT mice206. Furthermore, neurons in the area postrema, activated by AVP, project to the NTS to modulate baroreflex output64. Specifically, AVP enhances the baroreflex via the area postrema64, 207 under pathophysiological conditions to adjust for impaired BP regulation204, 205, 208.

AVP involvement in BP regulation also occurs via activated V1a receptors which release nitric oxide (NO) and PGE-2. These two components subsequently induce release of renin. Renin synthesis from granule cells leads to production of Ang II and Aldo, which increase water and sodium absorption. Ang II itself can induce release of AVP dose-dependently209, 210 by acting on AT1-Rs in the PVN and SON. Furthermore, NO mediates a negative feedback system of Ang II-induced upregulation of AVP mRNA in those regions. V1b receptors, located in the anterior pituitary and adrenal cortex, facilitate

AVP-induced ACTH and catecholamine release, respectively, contributing to vascular contraction and BP increases observed in the pathophysiology of stress211.

24 Vasoconstrictive properties evoked by AVP also influence GI functions. In congruence with Ang II and NE mediated vasoconstriction in GI vasculature, AVP also has a role in splanchnic circulation and GI tissue integrity212. AVP synthesis has been shown to occur in rat GI tissue213 and vasopressin receptor mRNA expression localized in the GI tract214 implicates a local role of AVP in GI function. Specifically, V1a-R mediated vasoconstriction of vascular and GI tract reduces vascular bleeding due to injury. Additionally, increased GI motility in the stomach and duodenum occurs in response to systemic administration of AVP as well215. The majority of literature describing AVP’s role in the GI tract is a direct result of studies in context of GI disturbances and disorders compared to normal physiology and will be discussed in more detail in the following section.

Effects of Neurohumoral Dysregulation on CV & GI function

The localization of receptors and actions on autonomic nerves implicate alterations of RAS components, catecholamines, and AVP in many autonomic disorders, including OI and functional GI symptoms. Previous studies in adults with OI have shown direct relationships between hemodynamic and neurohumoral responses to standing109,

123-127, 216. Additionally, nausea has been associated with changes in endocrine, autonomic, and brain activity112, 114, 129, 217, 218. Nausea itself is a known stimulus for AVP release128. In addition to AVP, components of the RAS, including Angiotensin II (Ang

II), are known to provoke nausea via the area postrema and impact GI motility64, 137. This section will give an overview of these components in the pathogenesis of OI subtypes as well as involvement in GI functions that may lead to chronic functional GI disorders.

25 Varying mechanisms have been postulated to produce POTS pathology, including exaggerated catecholamine release and abnormal RAS responses to standing.

Specifically, the RAS has been identified as a key mediator in POTS subjects demonstrating blood volume depletion. This is unsurprising since the RAS regulates blood volume via Ang II and Aldo. However, low plasma renin and Aldo has consistently been observed with POTS despite low blood volumes127, 219, suggesting abnormalities in the kidney may be critical in the presentation of POTS associated with hypovolemia. The role of Ang II in POTS is associated with low blood volume as well with data suggesting increased Ang II is associated with low bioavailability of NO causing local dysregulation in blood flow220. Furthermore, Ang II is increased in POTS with little to no change in

Ang-(1-7) during orthostatic challenge125 suggesting Ang II is part of the compensatory mechanism intended to increase blood volume in hypovolemic POTS subjects, especially during orthostasis.

While the treatment of hypovolemia in POTS is still evolving, oral desmopressin, a synthetic version of AVP, has recently shown promise as it significantly attenuated tachycardia and improved symptoms of POTS22. Since this treatment does not stimulate

ACTH or cortisol release and has less effective vasodepressor actions221, 222, its mechanism of action in POTS enhances fluid retention. Fludrocortisone, an aldosterone agonist, is more commonly used as a treatment for POTS. This treatment acts to increase sodium retention and plasma volume expansion, but has adverse side effects as well as varied efficacy in adult and pediatric populations12, 13, 223.

In contrast, POTS patients with normal blood volume, termed “high flow” POTS, are characterized by reductions in total peripheral vascular resistance at supine position

26 and venous pooling in the lower limbs upon standing224. Both sympathetic denervation in lower limbs225 and/or cardiac sympathetic denervation226 have presented in these cases of

POTS. More recently small fiber neuropathy with C-fiber abnormalities in the skin were associated with myocardial postganglionic sympathetic innervation in POTS227. This suggests a loss of distal sympathetic tone may produce an exaggerated sympathetic response in highly innervated tissues like the heart. In these cases, β-blockers and α- agonists have been employed as treatments. However, as in the low-flow cases, blood volume regulation status is necessary to predict effectiveness of treatments.

Some POTS subjects have elevated NE concentrations during orthostasis suggesting that POTS is associated with a hyperadrenergic state92, 124. In these cases exaggerated NE release produces tachycardia and increased vascular resistance to compensate for venous pooling in the lower limbs. Several mechanisms of the hyperadrenergic state in POTS have been tested showing not only increased sympathetic outflow and NE release cause tachycardia, but augmentation of sympathetic signal due to impaired NE clearance or enhanced receptor sensitivity may be contributing factors224,

228. Decreased NE transporter (NET) protein expression has been reported in some POTS patients and epigenetic mechanisms in NET regulation are potentially important in these cases229. Since POTS is a heterogeneous disorder, studies evaluating these mechanisms have varied in their findings. For example, Goldstein and colleagues using cardiac extraction of 3H-labeled NE demonstrated that NE was not reduced in supine position.

Thus, exaggerated NE release during standing is more likely due to enhanced sympathetic activity to the heart rather than decreased uptake87. There are many comorbid symptoms that accompany POTS and could provide insight into the

27 manifestation of this disorder3, 121. Further studies are needed to produce better diagnostic approaches for tailored treatments for POTS.

Sympathoadrenal imbalances also present in vasovagal syncope in supine position during orthostatic challenge230, where Epi levels increased significantly before syncope with no observed difference in NE. Subjects with syncope have a significant drop in vascular resistance and mean arterial pressure and BP levels before syncope reach values that elicit significant hormonal effects229, 231. Thus increased Epi preceding syncope is not a simply a consequence of syncope, but a precursor and underlying pathophysiological mechanism of orthostatically-induced syncope230. The absence of NE response to standing has been consistently reported in both syncope and OH232-235. Syncopal subjects have low BP and low whole body NE spillover to plasma in supine and upright positions suggesting a failure of appropriate sympathetic responses to postural change. Key regulator proteins of tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis, and

NETs are lower in patients with low BP compared to subjects with normal BP. Thus, lower tyrosine hydroxylase reduces the amount of synthesized NE and decreases the need for NET recruitment to cell membranes232. Furthermore, NE infusion temporarily eliminated low BP and accompanying symptoms in a small cohort of OH subjects, providing evidence for cardiac and extra-cardiac sympathetic noradrenergic denervation236.

In addition to tachycardia upon standing, nearly 80% of POTS subjects have abdominal pain and 60% present with recurrent nausea and vomiting237. However, the reverse is also true. When children presenting in a pediatric GI clinic with recurrent GI pain underwent a battery of comprehensive autonomic testing, parasympathetic activity is

28 preserved, but a high percentage test positive for POTS, syncope, or OH12, 13, 16, 121.

Children with conditions of OI also produce abnormal gastric rhythms as measured by electrogastrography during upright tilt18 and in supine position after ingesting non- carbonate water. Both tachygastria and bradygastria can cause nausea and abdominal pain13, 134, which over time may cause gastroparesis238. Autonomic neuropathy is associated with both rapid239 and delayed240, 241 gastric emptying. More recently, altered gastric emptying has been implicated in POTS242, where one-third of POTS patients with chronic GI symptoms tested had either rapid or slowed gastric emptying. Since the sympathetic nervous system normally inhibits GI motility, it would be expected that

POTS subjects demonstrate more delayed than rapid gastric emptying. However, rapid gastric emptying could be resulting from sympathetic denervation to the GI tract52, bolstering the theory of reduced NE spillover in the lower extremities. While NE was not measured in these studies, it is possible that NE-induced vasoconstriction to the GI tract during orthostasis may exacerbate GI symptoms in POTS subjects.

In addition to increasing during low BP or volume, AVP increases are associated with nausea and gastric dysrhythmias. The relationship between AVP and nausea was first observed when drugs activating vasopressin pathways, such as chemotherapies243, apomorphine, and cholecystokinin subsequently evoked nausea244-246. AVP secretion also occurs in healthy volunteers reporting nausea after ingesting large volumes of water247. In fact, peripheral infusion of AVP produces both nausea and gastric dysrhythmias in healthy subjects128. Interestingly, injection of epinephrine has also shown to elicit gastric dysrhythmias, but not as severely as observed with AVP248. Central and peripheral release of AVP causes nausea via actions on the AP, the brain center for nausea and

29 vomiting. Projections from the AP to the NTS also elicit increased autonomic outflow during nausea and vomiting64, providing evidence for specific brain circuit activity during these experiences.

The majority of studies investigating the mechanisms underlying nausea use visual-vestibular mismatch, which has shown to increase sympathetic and decrease parasympathetic activity in the gut causing gastric dysrhythmias112, 116, 249. Gastric dysrhythmias occur minutes prior to and during the experience of nausea112, 116, 249.

Disturbed gastric rhythms potentially due to decreased vagal and splanchic afferent activity stimulate both nausea experience and AVP release 17. However, the directionality between nausea and AVP is unclear where either may be the cause or consequence of the other. Recently, Farmer and colleagues112 evoked nausea in healthy subjects using a motion video, which resulted in decreased parasympathetic and increased sympathetic activity in nausea-sensitive subjects. Nausea experienced by these subjects was preceded by activation of the amygdala, which regulates autonomic and endocrine function, and the anterior cingulate, a region of the brain associated with sympathetic activation.

Subjects with visually-induced nausea also had elevated AVP blood levels post-video, which positively correlated with mean nausea scores post-video. Although the relationships between AVP, BP, and nausea are apparent, AVP has not specifically been investigated in subtypes of OI where it may influence the compensatory mechanisms for blood pressure/volume that may exacerbate nausea and/or gastric dysrhythmias.

30 3. Inflammation: cause or consequence of autonomic and neurohumoral imbalance

Inflammation is the vascular response to foreign pathogens, designed to protect against injurious stimuli and to initiate healing. In response to injury, inflammation induces a variety of actions including vasodilation, increased blood flow to the affected tissue, increased vascular permeability, and production of cytokines250. The brain plays a pivotal role in detecting and modulating inflammation through two main pathways, the sympathetic nervous system and HPA axis. In response to stress or injury, the ANS also releases neurohumoral products, such as NE, Ang II and AVP251-253. In addition to their pressor effects these neuropeptides, as well as many others, influence regulation of the

HPA axis, which releases glucocorticoids that exert innate and adaptive immunity254.

Moreover bi-directional communication impacts all inflammatory processes as shown when inflammation in the gut impaired central processing and cognitive performance255.

Prolonged activation of inflammatory pathways is implicated in various disease states in the brain and periphery and these inflammatory states should be considered a risk factor for autonomic dysfunction256-258.

As discussed previously, the pathophysiology of autonomic dysfunction can be caused by a plethora of neuronal and endocrine alterations. These alterations can exacerbate or compensate for the dysfunction depending on severity and specificity of the diagnosis. Furthermore, autonomic dysfunction and GI disturbances are also associated with chronic inflammation in the CNS259-261. Thus, increased central inflammation could be a contributing factor to autonomic dysfunction observed in OI as well as chronic GI symptoms. However, the cause of the inflammation is unclear. Alternatively, autonomic dysfunction exhibiting exaggerated neurohormone release or impaired vagal function

31 could cause prolonged inflammatory states. The following section will discuss normal autonomic and endocrine responses involved in immune function as well as the relationship between inflammation and neurohumoral factors in CV and GI regulation.

Autonomic pathology of inflammation

The ANS plays a critical role in physiological regulation of inflammation under normal conditions and in response to acute and chronic environmental stressors. Both branches of the ANS have specific roles in regulation of inflammation. However, most research has been focused on the sympathetic system and noradrenergic regulation of the immune system. This is most likely due to the extensive sympathetic postganglionic innervation in primary and secondary immune organs and tissues262, 263, including the thymus, spleen, and lymphoid nodes and the expression of α and/or β receptors on almost all immune cells264, 265. Preganglionic sympathetic neurons also innervate the adrenal gland and induce the release of primarily Epi from adrenal chromaffin cells into the circulation. Both NE and Epi act on adrenoreceptors on immune cells, which subsequently activate signaling pathways that alter their cellular functions265, 266.

Sympathetic nerve activity modulates both initiation and effector phases of an immune response. Previous studies have shown both pro- and anti- inflammatory activity after stimulated NE release. In denervation experiments in mouse model of arthritis, the sympathetic system demonstrated proinflammatory effects during early phases while adrenergic anti-inflammatory effects were observed at later stages267. However, the effect of catecholamines on initial immune responses is determined by the available concentration of NE in the local milieu as well as availability of specific adrenoreceptor subtypes. β2 receptors are the predominant adrenoreceptors located on immune cells

32 which, when activated by high concentrations of NE, promote the Th2-driven antibody phenotype and suppress cytotoxic immune responses (T-cell, NK-cell, macrophage activity)268, 269. This shifts the balance of cytokine release toward anti-inflammatory factors including interleukins, IL-4 and IL-10. Additionally, catecholamines inhibit pro- inflammatory cytokines, tumor necrosis factor-α (TNF-α) and IL-1, from being produced.

In contrast, low concentrations of NE trigger release of TNF-α and IL-1, mediated by α- adrenergic receptors270, 271. During chronic inflammation the local innervation pattern of sensory and sympathetic nerve fibers changes with a shift toward proinflammatory sensory nerves. Thus, chronic release of NE may deplete stores over time, resulting in low NE concentrations that elicit chronic inflammation.

The parasympathetic nervous system also modulates inflammation, acting as an anti-inflammatory neural circuit272. The vagus nerve is known to play an important role in regulating inflammation to prevent tissue damage from excessive inflammatory responses. Specifically, the vagus nerve senses peripheral inflammation and projects this sensory information to the area postrema and NTS. Subsequent increases in vagal outflow to lymphoid organs, mediated by acetylcholine, inhibit proinflammatory cytokine production273, 274. The celiac ganglia receive cholinergic vagal inputs, which alter activity to the splenic nerve and to B-receptors on T-lymphocytes via NE signaling. T- lymphocytes are the immune cells that stimulate macrophages via the a7 nicotinic ACh receptor (a7nAChR) to produce ACh, which in turn reduces levels of inflammation275.

Both human and animal studies provide evidence to support the anti-inflammatory vagal pathway. Studies in mice have demonstrated decreased cytokine responses from stimulation of the vagus nerve and administration of a7nAChR agonists276. In contrast,

33 increased cytokine responses were shown following endotoxin administration and in a7nAChR subunit knockout mice277, 278. Heart rate variability (HRV) is the autonomic parameter most often used in human studies because it is a reliable index of cardiac vagal function and can be measured using noninvasive methods. In fact, HRV also has an inverse relationship with central and peripheral inflammatory markers.

Numerous retrospective and prospective large cohort studies have shown decreased HRV associates with higher levels of C-reactive protein (CRP)279, 280 and IL-6281-284. This relationship stands after controlling for age and sex, as well as sympathetic activity281, 284, among many other variables. These studies highlight the importance of vagal outflow against chronic inflammation and provide an easily accessible, non-invasive parameter for testing.

Vagal nerve stimulation has been employed in human studies as well.

Interestingly, vagus nerve stimulators are used to treat various disorders including inflammatory bowel disease285-287, chronic pain288, 289, epilepsy290, and depression291, 292.

This strategy has also been used specifically to modulate GI inflammation, motility and nociception293. Additionally, preliminary evidence suggests that the vagus nerve can rapidly respond to increased pro-inflammatory cytokines in the periphery, as shown when vagal activity increased 3 minutes after IP injection of TNF-α in rats294. While promising, vagal nerve stimulators can be invasive and expensive and are often secondary and tertiary to more safe treatment options 285.

Under normal conditions sympathetic and parasympathetic nervous systems balance inflammatory homeostasis in the periphery. However, clear associations between disorders with underlying autonomic dysfunction and inflammation have been

34 demonstrated suggesting when unregulated, inflammation is damaging to tissues in the brain and CV system. For example, significant inverse relationships between HRV and

IL-6 have been shown in patients with hypertension, type 1 diabetes, type 2 diabetes, and sepsis295-297. Patients with OI have impaired vagal reflex, as measured by HRV and

BRS298, but the role of inflammation has not been investigated specifically in adults or children with OI. Autonomic imbalance is also associated with both acute and chronic stress299, which ignite inflammatory responses centrally and peripherally. From a therapeutic standpoint, the knowledge that the vagus nerve assists in the regulation of homeostasis via modulation of inflammation provides rationale for the vagus nerve to be a therapeutic target in a variety of conditions, including GI inflammation.

The HPA axis and anti-inflammatory actions of Arginine Vasopressin

Central regulation of inflammation occurs via the HPA axis, which plays a vital role of homeostasis in response to stress. Normally, the HPA axis releases CRH and

ACTH in response to stress which act on the adrenal cortex to release cortisol. In turn, cortisol induces metabolic effects that inhibit the actions of stress on the brain and prevents inflammation. However, dysfunction of the HPA axis often results in a chronic inflammatory state due to the deficiency of corticosteroids and reduced negative feedback to the hypothalamus191. Inflammation caused by impaired HPA responses is known to exacerbate cardiovascular pathology associated with atherosclerosis, hypertension, and metabolic syndrome25, 300-302. Moreover, autonomic function and overall cardiovascular health are compromised in stress-related psychological disorders, including depression, post traumatic stress disorder (PTSD), and anxiety303-305, each significantly increasing the risk of cardiac morbidity and mortality306-310. Psychosomatic symptoms of stress and

35 anxiety often occur in patients with OI and functional GI disorders, respectively, due to chronic experience of symptoms and debilitated quality of life23, 311-314. Thus, inflammation due to chronic stress is also a major factor in the pathophysiology of cardiovascular disease and autonomic disorders and may contribute to autonomic dysfunction in OI.

Pro-inflammatory cytokines released in the periphery cross the blood brain barrier through circumventricular organs activating the HPA axis and subsequently stimulate release of both CRH and AVP315. These hormones act synergistically to induce release of

ACTH, which stimulates release of cortisol to reduce inflammation. AVP can act independently of CRH to release ACTH via V1b receptors located in the pituitary as well. In fact, studies in mice administered V1b antagonists ACTH did not increase in response to stress, suggesting CRH alone is not enough to elicit ACTH release74. Thus,

AVP plays an important role in acute stress responses and is necessary to stimulate

ACTH in response to inflammatory signals in the brain. AVP is also synthesized in the periphery where it exerts autocrine and paracrine effects in response to stress.

Specifically, chromaffin cells in the adrenal medulla can synthesize AVP independently in response to acute or chronic stress. In turn, AVP regulates release of cortisol from the adrenal gland via V1a and V1b receptors316, 317. V1a receptors are located on the adrenal cortex as well, which allows AVP to stimulate hypertrophy and hyperplasia resulting in increased production of Aldo and cortisol. Levels of cortisol determine anti-inflammatory actions of the HPA axis74, 318.

However, excessive production of either AVP or cortisol due to abnormal HPA axis may also contribute to a chronic inflammatory state. AVP can act as a pro-

36 inflammatory peptide by stimulating release of prolactin, another pro-inflammatory peptide that exacerbates inflammation. AVP also stimulates release of cytokines, increases T cell actions, and augments mixed lymphocyte response, all of which worsen inflammation319. Thus, the relationship between neurohormones in the HPA axis and inflammation is dependent the extent and duration of the release in response to stress.

Central inflammation causes autonomic dysfunction

Neuroinflammation is associated with various neurological and neurodegenerative diseases with comorbid autonomic dysfunction including multiple sclerosis, Alzheimer’s,

Parkinson’s, and Multiple System Atrophy320-323. Microglia, the immune cells of the brain, carry out immune responses and mediate inflammation in the brain. However, over activation of microglia often leads to production of neurotoxic cytokines that are damaging to neurons324, 325. In humans, evidence of metabolite markers of inflammation in the brain are emerging as magnetic resonance spectroscopy (MRS) is being used more frequently to assess cell-specific changes in the brain326. Although causation cannot be established using MRS, elevated markers of inflammation in autonomic cortical and noncortical areas associate with symptom severity327 and impaired cognitive function328 as well as dyslipidemia329, 330, which is a crucial factor in hypertension, obesity, and metabolic syndrome.

Microglia are activated in brainstem areas in hypertensive adults and studies reducing microglia cell numbers showed reductions in BP331. Pro-inflammatory cytokines, TNFα, IL-1β, produced in the periphery and centrally are present in the brain in patients with hypertension suggesting a direct role of inflammation in blood pressure regulation259, 260, 315, 332. Increased inflammation in the brain has also shown to induce

37 synthesis of renin, Aldo, AVP, and Ang II, which facilitate increases in BP.

Alternatively, increased concentrations of anti-inflammatory cytokines including IL-10 and IL-1 receptor antagonist, reduce hemodynamic and humoral indices in rats with heart failure333 and infusions decreased hypertensive responses to stress in healthy rats334.

Thus, microglial activation in the brain may exacerbate neurohumoral release, which in turn affects blood pressure regulation. In contrast, neurohormones can induce inflammation, facilitating a cycle of activation that over time produces serious downstream effects that impair autonomic control of the vasculature in the heart and GI tract.

Canonical actions of Ang II in the brain include increased sympathetic activity causing vasoconstriction and increased BP, as well as increased inflammation. Current studies exploring the role of Ang II and inflammation reported that Ang II increased permeability of the blood brain barrier, resulting in inflammation of cerebral microvasculature335. In addition to allowing passage of inflammatory cytokines from the periphery, Ang II induces inflammation centrally via AT-1 receptors by increasing reactive oxygen species, IL-6, and TNF-α336. Studies inhibiting Ang II via lesions in the circumventricular organ of the hypothalamus prevented increases in BP in response to low dose Ang II and the activation/infiltration of T-cells was averted258. The therapeutic implications of Ang II in brain inflammation have been validated in a variety of animal models using AT1 receptor blockade to show amelioration of stress, anxiety, ischemia, and brain inflammation337, 338. In contrast, Ang-(1-7) has anti-inflammatory effects in the brain showing the importance of the balance of Ang II and Ang-(1-7) peptides not only

38 for autonomic outflow but for integrity of neuronal tissues responsible for central mechanisms of BP regulation339.

GI pathology & inflammation

Autonomic imbalance and stress have similar effects on the GI tract as on the brain and CV system in regards to inflammation. Stress and anxiety produce circulating cytokines that affect GI function, ranging from changes in gut flora species to impaired

GI motility340. Patients with functional GI disorders have increased vigilance for bodily sensations, which produces heightened stress and anxiety when symptoms are experienced that facilitate unhealthy neuroendocrine responses341, 342. These neuroendocrine responses are actively involved in inflammation, as described previously, that exacerbate symptoms and lead to chronic inflammatory states.

The maintenance of proper gastric blood flow is imperative for prevention of mucosal damage by reactive oxygen species343. The role of Ang II in regulation of fluid and electrolyte homeostasis and regional blood flow are also implicated in inflammation and mucosal damage in GI tissues. Consistent with its actions in the cardiovascular system, Ang II elicits vasoconstriction in the gut reducing blood flow to GI tissues and inducing signaling cascades resulting in cellular damage and inflammation344, 345. In contrast to Ang II, Ang-(1-7) has opposing effects in the gut as it is well established that

Ang-(1-7) has anti-inflammatory properties which counteract pro-inflammation induced via Ang II- mediated vasoconstriction in the brain, heart, and vasculature. This is also evident in the GI tract as Ang-(1-7) administration provided gastroprotection against lesions induced by cold-restraint stress in rats and increased gastric blood flow346, 347.

39 Furthermore, the human gastric mucosa expresses renin and ACE, further implicating the

RAS in mechanisms of gastric integrity348.

Alternatively the microbiome of the GI tract can modulate nerve structure and function and communicate with the brain via similar neurohumoral and vagal pathways349-351. While it can be assumed that increased sympathetic activity in the periphery increases inflammation in the cardiovascular and GI systems, the ENS also modulates inflammation in local GI tissues independently352. Locally, neuropeptides synthesized in the ENS and in immune cells of GI tissues mediate proliferation of mucosoal lymphocytes, affect cytokine production, and influence intestinal permeability49. Enteric-specific glial cells have been more recently identified as active elements in GI and especially intestinal immune responses353, 354. Moreover, inflammatory signals in the ENS cotransmit with sensory and motor signals within the vagus nerve to modulate activity in the brain via the bidirectional gut-brain axis (GBA).

For example, colitis in rats caused an increase in production of cytokines in the GI tract, which reduced the number of DMNV neurons projecting to the stomach. Intestinal inflammation also diminished DMNV cell proliferation, increased apoptosis, and altered calcium resonses to glutamate355. In contrast, vagus nerve stimulation has shown to be effective in models of intestinal inflammation in animals, while data for this modality’s effectiveness in humans is lacking287, 356.

40 4. Rationale & Aims

Ongoing studies in our laboratory focus on understanding factors that alter baroreflex sensitivity for control of heart rate during upright posture and produce chronic clinical symptoms of OI and functional GI disorders. OI can manifest from a variety of autonomic imbalances, which produce heterogeneous populations of patients with unknown pathophysiology of their disorder2-4. There are several pharmacotherapies available to treat patients with OI, but most are suboptimal and produce varied effects subject-to-subject13, 19-22.

Previous studies in adults with OI have aimed to define neurohumoral profiles in

OI subtypes to identify potential avenues for treatments. In response to HUT, OI in adults has been associated with exaggerated neurohumoral responses including elevated catecholamines, Ang II, and AVP123, 125, 216, 261, 357. These responses differ among OI subtypes, highlighting the possibility of differential mechanisms related to specific hemodynamic profiles. Thus, the physiological and pathophysiological changes associated with OI need to be considered in order to provide optimal treatment strategies in these populations. Although children present with varying degrees of autonomic dysfunction in OI12, 13, 16, neurohumoral profiles have not been described in this population and may provide evidence for different maintenance strategies for chronic symptoms. Based on this, we hypothesize that children with OI undergoing HUT will demonstrate distinct neurohumoral responses that associate with specific BP and HR changes upon standing.

Comorbid GI symptoms associated with OI were first reported in the 1990’s358,359.

Since then others have empirically evaluated this relationship in adults and children

41 revealing that autonomic dysfunction is an underlying cause of OI and associated symptoms of dizziness, abdominal pain, and nausea13, 16, 18. Previous studies in our laboratory and others have reported up to 80% of children presenting in the pediatric GI clinic with chronic nausea and at least one OI symptom test positive on a HUT for OI12.

Interestingly, neurohormones released during orthostasis to compensate for initial decreases in BP control also act on central sites that induce nausea360. However, the impact of orthostatic challenge during HUT on the production of nausea has yet to be explored in regards to neurohumoral mediators. Therefore, we hypothesize that children with an abnormal HUT will have a higher incidence of nausea and an exaggerated neurohumoral response compared to those with a normal HUT.

The majority of evidence suggests an imbalance in ANS activity is fundamental in development of OI and comorbid GI symptoms, but the initial cause of dysfunction is unknown. Autonomic imbalance, as observed in OI, is also linked to systemic inflammation273, which can affect both cardiovascular and GI function. Furthermore, studies of adults with autonomic associated disorders, including MSA327 and heart failure81, have shown increases in central inflammation in brain areas associated with autonomic function and blood pressure regulation using MRS. Patients with chronic orthostatic and GI symptoms experience heightened anxiety and stress23, 313, which increases inflammation and the risk of cardiovascular disease as shown in stress-related psychological disorders310. Thus, inflammation may also contribute to altered autonomic activity in these subjects, but has yet to be associated specifically with OI or OI subtypes.

Nevertheless, MRS has not been employed in children with normal or abnormal autonomic function and may provide an innovative approach to understanding autonomic

42 dysfunction observed in OI. We hypothesize that children with OI will demonstrate characteristic brain metabolite profiles in the dorsal medulla that will correlate with autonomic function. To test this, we evaluated autonomic performance in upright position and collected supine brain metabolites localized in the dorsal medulla.

Accordingly, the specific aims are as follows:

Aim 1: Better define the relationships between neurohumoral phenotypes and

hemodynamic profiles in children with orthostatic intolerance during supine

position and upright posture as evoked by head upright tilt

Aim 2: Characterize the clinical phenotype of chronic nausea and OI by defining

hemodynamic and neurohumoral profiles in children with HUT-induced nausea

Aim 3: Define brain metabolite profiles in children with OI and investigate the

relationship between profiles and autonomic function in supine and upright

positions

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68 CHAPTER TWO

DISTINCT NEUROHUMORAL BIOMARKER PROFILES IN CHILDREN WITH HEMODYNAMICALLY-DEFINED ORTHOSTATIC INTOLERANCE MAY PREDICT TREATMENT OPTIONS

By

Ashley L. Wagoner1,2, Hossam A. Shaltout2,3, John E. Fortunato2,4,5, Debra I. Diz1,2,4

1Neuroscience Graduate Program,

Wake Forest Graduate School of Arts & Sciences, Winston-Salem, NC

2Hypertension and Vascular Research Center,

Wake Forest School of Medicine, Winston-Salem, NC, USA

Departments of 3Obstetrics and Gynecology and 4General Surgery

Wake Forest School of Medicine, Winston-Salem, NC

5Department of Pediatrics,

Virginia Commonwealth University School of Medicine, Richmond, VA

Short Title: Neurohumoral Profile in Orthostatic Intolerance

The following manuscript was published in The American Journal of Physiology Heart

and Circulatory Physiology Volume 310, pages H416-H425 in February 2016.

Differences in formatting and organization reflect the requirements of the journal.

69 Abstract

Rationale: Studies of adults with orthostatic intolerance (OI) reveal altered neurohumoral responses to orthostasis, which provide mechanistic insight into the dysregulation of blood pressure control. Similar studies in children with OI providing a thorough neurohumoral profile are lacking.

Objective: Determine the cardiovascular and neurohumoral profile in adolescent subjects presenting with OI.

Methods and Results: Subjects ages 10-18 years were prospectively recruited if they exhibited ≥2 traditional OI symptoms and were referred for tilt testing (HUT).

Circulating catecholamines, vasopressin (AVP), aldosterone, renin and angiotensins were measured supine and after 15 minutes of 70 degree tilt. Heart rate and blood pressure were continuously measured. Of the 48 patients, 30 had an abnormal tilt. Subjects with an abnormal tilt had lower systolic, diastolic, and mean arterial blood pressures during tilt, significantly higher levels of vasopressin during HUT, and relatively higher catecholamines and angiotensin II during HUT than subjects with a normal tilt. Distinct neurohumoral profiles were observed when OI subjects were placed into groups defined by the hemodynamic response: postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension (OH), Syncope, and POTS/Syn. Key characteristics included higher HUT-induced norepinephrine in POTS and vasopressin in OH and syncope, and higher supine and HUT aldosterone in OH subjects.

Conclusions: Children with OI and an abnormal response to tilt exhibit distinct neurohumoral profiles associated with the type of hemodynamic response during orthostatic challenge. Elevated AVP levels in syncope and OH groups are likely an

70 exaggerated response to decreased blood flow not compensated by higher NE levels as observed in POTS subjects. These different compensatory mechanisms support the role of measuring neurohumoral profiles towards the goal of selecting more focused and mechanistic based treatment options for pediatric patients with OI.

71 New & Noteworthy

Distinct neurohumoral profiles were detected in blood collected during head upright tilt in children with POTS, OH, and Syncope revealing different compensatory mechanisms.

This first comprehensive neurohumoral profile in children with orthostatic intolerance provides a basis for future strategic treatment options in children.

72 Introduction

Orthostatic intolerance (OI) affects 500,000 Americans rendering them unable to maintain adequate blood perfusion during upright position (42). When standing, patients often experience light-headedness, sweating, and nausea (22, 42). An abnormal cardiovascular response to head-upright tilt (HUT) orthostatic challenge can be broadly grouped into: postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension

(OH), and syncope. Management and treatment of OI is typically dependent on the specific cardiovascular (CV) diagnosis with most treatments having variable effectiveness in adult and adolescent populations (4, 8, 13, 30, 39). The pathophysiological mechanisms responsible for these cardiovascular changes are not well defined as presentation and symptoms are often heterogenous (14). This heterogeneity leads to empirical treatment of symptoms without a evidenced based approach related to their underlying cause (3, 6).

In response to HUT, reduction in blood volume or pressure at the level of the heart or carotid arteries is associated with reflex increases in catecholamines, vasopressin

(AVP), renin, angiotensin II (Ang II), and aldosterone (Aldo) (21). Although there are many additional non-reflex regulators that influence these systems, previous studies in adults with OI reveal exaggerated neurohumoral responses to upright posture including elevated catecholamines (16, 24), Ang II (34, 35), and AVP (11, 25, 37, 54).

Neurohumoral responses also differ among OI subtypes, underscoring the possibility of differential mechanisms related to specific hemodynamic profiles. Adults with POTS have been observed to have elevated levels of plasma norepinephrine (NE) and epinephrine (Epi) at the time of orthostatic challenge suggesting disproportionally higher

73 sympathetic activation likely compensating for low blood pressure (16, 34, 58). Elevated supine Ang II is also associated with POTS subjects with hypovolemia (49). In adults with OH, greater supine AVP and endothelin-1 concentrations have shown to be predictive of decreases in systolic BP during HUT (37). Increases in AVP and Epi during

HUT have also been observed in both adult OH and vasovagal syncope subjects (23, 32,

37). OI is also observed in pediatric patients (12), but is particularly difficult to diagnose and treat as it is under-recognized, and testing such as HUT can be invasive for children.

Characterization of the neurohumoral response to orthostatic challenge in children with

OI has not been well described, but dysregulation of these systems likely plays a role in its pathogenesis (12, 13).

We hypothesized that children with OI undergoing HUT would demonstrate distinct neurohumoral responses in association with specific patterns of changes in blood pressure and heart rate. We further suggest that specific cardiovascular responses in OI subtypes such as, POTS, OH, and syncope, are associated with unique neurohumoral profiles. To test this, we measured circulating levels of Epi, NE, AVP, Aldo, renin, and angiotensins [(Ang-(1-7) and Ang II)] both in the supine position and during HUT comparing these findings to their hemodynamic response in pediatric subjects with OI.

74 Methods

Subjects

Forty-eight male and female subjects ages 10-18 years were recruited from the pediatric clinic at Wake Forest Baptist Medical Center if they experienced 2 or more symptoms consistent with OI, in which HUT was deemed necessary, for at least 2 months prior to testing. These OI symptoms included: nausea, dizziness, lightheadedness, abdominal pain/bloating, syncope, excessive sweating, or chronic fatigue. Patients were excluded if a metabolic, mechanical, or mucosal inflammatory finding was identified as the cause for their symptoms. All subjects were required to undergo a 45-minute HUT test during which blood was drawn to measure: Epi, NE, Ang II, Ang-(1-7), Aldo, renin, and AVP after 15 minutes in the supine position and after 15 minutes of HUT. The Wake Forest

Baptist Medical Center’s Institutional Review Board approved this study. Parents and children provided written consent/assent prior to study participation.

Head upright tilt (HUT)

Each subject was positioned supine on the tilt table for 15 minutes before HUT, followed by an immediate upright tilt from zero to 70 degrees for up to 45 minutes. Continuous measures of blood pressure (BP) and heart rate (HR) were obtained using noninvasive finger arterial pressure measurements and analyzed by BIOPAC acquisition software

(Santa Barbara, CA). An average of the BPs and HR in the last 5 minutes of the supine period were used for baseline measures. The average HUT BPs and HR were calculated in the 2 minutes before blood samples were collected (after 15 minutes of HUT) to compare to baseline supine BP and HR. After 45 minutes the tilt-table was brought back to the supine position for an additional 15 minutes. The test was terminated early for

75 excessive GI symptoms (nausea, abdominal pain), syncope, cardiac , or at the request of the patient for severe discomfort.

Patients were diagnosed as having an abnormal HUT [HUT(+)] if they met criteria for POTS, OH, syncope or POTS/Syncope (P/S). Otherwise, patients were defined as having a normal HUT [HUT(-)]. POTS was defined as either a HR greater than 120 bpm or ≥40 bpm increase in HR from mean supine HR in the first 10 minutes of

HUT, sustained for at least 2 minutes (46). OH was defined as a 25 mm Hg decrease in systolic BP (SBP) from mean supine SBP in the first 10 minutes of HUT sustained for a minimum of 2 minutes without an associated increase in HR (14). Syncope was defined as temporary loss of consciousness during 45-minute HUT. Subjects were classified as

POTS/Syncope if they met criteria for POTS as defined above followed by syncope at any time during the 45-minute HUT.

Measurements of blood hormones

Measures of Aldo, renin, and angiotensins were carried out in the CLIA certified

Hypertension Core Assay Laboratory at Wake Forest. Measures of catecholamines (Epi,

NE, dopamine) and AVP were carried out in the Pathology lab at Wake Forest Baptist

Health. Radioimmunoassays (RIAs) previously described for human studies were used to measure three angiotensin peptides (I, II, 1-7) (27, 28). Ang I was measured using a modified version of the Peninsula assay, Ang II using the Alpco (Windham, NH) and

Ang-(1-7) using an in-house antibody. AVP was measured using an ALPCO Diagnostics

RIA (Windham, NH). Serum Aldo and plasma renin were measured as published (34).

Epi, NE and dopamine were measured after whole plasma extraction by high

76 performance liquid chromatography. Dopamine was not detectable in plasma of any of the subjects.

Statistical Analysis

For continuous variables, means and standard error of the mean were calculated and for categorical variables counts and percentages were calculated. Two-way ANOVA tests were conducted to examine the effect of HUT on HUT(+) vs. HUT(-) groups, as well as

HUT(-), OH, POTS, syncope, and P/S groups. When appropriate, post-hoc t-tests and one-way ANOVAs were conducted to identify specific effects of HUT on hemodynamic and neurohumoral variables between and among groups. Unpaired t-tests were performed comparing HUT(+) and HUT(-) groups, followed by a Welch’s Correction if there were unequal variances. Mann-Whitney tests were conducted when data sets were not normally distributed. Paired t-tests were performed to compare supine to HUT measures within groups and Wilcoxon’s Correction tests were used when data sets were not normally distributed. One-way ANOVA followed by Tukey’s post hoc multiple comparisons tests was performed to define differences between HUT(-), POTS, OH, syncope, and P/S groups. Kruskal-Wallis tests were performed when group data were not normally distributed. Pearson’s correlation test was performed to compare neurohumoral and hemodynamic variables. Spearman’s correlation test was performed when data sets were not normally distributed. All statistical analyses were performed using GraphPad

Prism 5.0. Results with p<0.05 were considered statistically significant.

77 Results

Forty-eight patients, 36 females and 12 males, presenting with OI symptoms for at least 2 months prior underwent HUT testing. The median age (± SEM) was 15.2 ± 0.3 years.

Eighteen patients were HUT(-) and 30 patients were HUT(+) over the 45 min HUT. The

30 HUT(+) subjects met criteria for one of the four groups (POTS, [n=7], OH, [n=5], syncope [n=8] or P/S, [n=10]). There were no differences in age or weight between groups. Body mass index was significantly higher in HUT(-) subjects than HUT(+) subjects. As expected, HUT(-) subjects were able to sustain HUT significantly longer than HUT(+) subjects. Syncope and P/S subjects sustained significantly less time on

HUT compared to HUT(-) and POTS subjects were able to stay on HUT longer than syncope subjects (Table 1).

HUT(+) versus HUT(-) subjects

Hemodynamic measurements

There were no significant differences between HUT(+) vs. HUT(-) subjects for BP and

HR in the supine position (Figure 1). During tilt, HUT(+) subjects had significantly lower systolic BP (SBP) and diastolic BP (DBP) compared to HUT(-) subjects

(interaction, p<0.05; HUT(+) vs. HUT(-), p<0.0005) (Figure 1A-B). Similar patterns

78 were observed for mean arterial

pressure (MAP). Additionally, HUT(-)

subjects had a significant increase in

DBP upon HUT compared to supine

(p<0.05). No other significant BP

changes were observed from supine to

HUT in either group. Both groups had a

significant increase of HR from supine

to HUT, but there were no differences

at supine or HUT between groups

(Figure 1C).

Neurohumoral measurements

There were no significant differences

between HUT(-) and HUT(+) subjects

for neurohumoral measures in the

supine position (Figure 2). Plasma

levels of NE, Epi (Figure 2A-B), renin,

(Figure 2D) and Ang II (Figure 2F)

significantly increased from supine to

HUT for both HUT(-) and HUT(+) groups, but there were no differences in these hormones between groups during HUT.

Plasma AVP concentrations increased in both groups from supine to HUT (p<0.05 for

HUT(-); p<0.001 for HUT(+)), but were higher in HUT(+) vs. HUT(-) subjects during

79 HUT (p<0.05; interaction, p=0.08) (Figure 2C). There were no differences in Aldo or

Ang-(1-7) levels between or among groups (Figure 2E, G).

Correlative Assessment of Neurohumoral Biomarkers and Hemodynamics

Based on our findings, we applied regression analysis to further elucidate the association between these neurohumoral biomarkers and hemodynamics at supine and in response to

HUT considering all 48 subjects and HUT(+) and HUT(-) groups alone (Table 2).

Supine NE had a negative correlation with Epi and Ang-(1-7) in both supine and HUT positions and positive correlation with supine AVP. As expected, NE during HUT had a positive relationship with both supine and HUT HR. These relationships were also

80 observed in HUT(+) subjects, but not in HUT(-) subjects. HUT levels of NE correlate in a positive manner with supine AVP and are negatively correlated with HUT Ang-(1-7).

The negative relationship between HUT NE and HUT Ang-(1-7) was also observed in

HUT(-) subjects. Supine Epi correlated directly with Supine HR, while HUT Epi had an inverse relationship with MAP. HUT Epi also directly correlated with Ang-(1-7) in both supine and HUT positions considering all 48 subjects and HUT(+) alone (Table 2).

In addition to relationships with NE and Epi, AVP associated with MAP in supine and during HUT. In the supine position, AVP showed a positive correlation with MAP.

In contrast, during HUT a significant negative correlation between MAP and AVP during

HUT was observed. Both of these correlations were also observed in HUT(+) subjects, but not in HUT(-). Ang II in the supine position correlated directly with supine HR.

Significant positive relationships were also observed between Ang II and Renin, Ang II and Aldo, and Renin and Aldo in supine and HUT positions (Table 2).

81 HUT(+) subgroups versus HUT(-) subjects

Hemodynamic measurements

To explore differences among patients

with specific OI subgroup diagnoses

during HUT, we analyzed hemodynamic

and neurohumoral changes in POTS, OH,

syncope, and P/S compared to HUT(-)

subjects. There was a significant

interaction for SBP during HUT indicating

differences among groups at baseline and

in response to tilt. Specifically, subjects

with OH (p<0.0001), POTS (p<0.01), and

syncope (p<0.01) had significantly lower

SBP during HUT compared to HUT(-)

subjects, as well as OH subjects having

significantly lower SBP than P/S subjects

(p<0.01). The only subgroup to have a

significant effect from supine to HUT for

SBP was the syncope group, which had a

decrease in SBP with HUT

(p<0.05)(Figure 3A). There was also a significant interaction for DBP (p=0.04) and MAP (p=0.009) from supine to HUT, in which all subgroups had significantly lower DBP and MAP than HUT(-) patients

82 (p<0.0001)(Figure 3B). HUT(-) subjects had significantly higher SBP, DBP, and MAP during HUT compared to all other groups and OH subjects had significantly lower SBP and MAP than POTS, syncope, and P/S subjects (p<0.0001) (Figure 3). HUT(-), POTS, syncope, and P/S subjects had a significant increase in HR from supine to HUT.

Consistent with the clinical definition of POTS, the POTS group had significantly higher

HR during HUT than all other subgroups (p<0.01) and a greater increase in HR from supine to HUT (p<0.0001). Additionally, OH subjects had significantly lower HR than

HUT(-) and P/S subjects during HUT (p<0.05) (Figure 3C).

Catecholamine measurements

In the supine position, there were no significant differences among groups. Subjects with

POTS had significantly higher levels of NE than HUT(-) subjects during HUT (p<0.001).

POTS subjects had exaggerated increases in NE (p<0.001) and Epi (p<0.05) from supine to HUT and higher plasma levels of NE compared to all other abnormal HUT subgroups

(p<0.01). P/S subjects were the only other group to have a significant change from supine to HUT in these variables, with a significant increase in NE with HUT(p<0.05). OH

(p=0.1), syncope (p=0.2), and P/S (p=0.07) groups showed a trending increase in Epi levels from supine to HUT. HUT(-) subjects had a significant increase from supine to

HUT in NE and Epi (Figure 4A-B).

Vasopressin measurements

At baseline, there were no differences among groups in plasma AVP concentrations. OH

(p<0.05) and syncope (p<0.01) subjects had significantly higher AVP during HUT than

POTS and HUT(-) subjects. Syncope subjects also had a significant increase in AVP from supine to HUT (p<0.001) and OH had a variably high, but not statistically

83 significant increase in AVP (Figure 4C). These two subgroups did not show a significant decrease in BP during HUT, but both had lower BP during HUT compared with HUT(-) and OH had lower pressure than most other groups during HUT.

Renin-angiotensin-aldosterone system (RAS) measurements

There were no differences among groups for renin supine or during HUT. An increase in renin from supine to HUT was observed in HUT(-) and syncope subjects (p<0.01)

(Figure 4D). There was no change in serum Aldo from supine to HUT in any group. OH subjects had higher Aldo measurements than HUT(-) and POTS subjects in supine position and HUT(-), POTS, and syncope subjects during HUT (Figure 4E).

For Ang II, there were no differences in supine values among subgroups. HUT(-),

POTS, syncope, and P/S groups had a significant increase from supine to HUT. There was a trend for group differences (p=0.054) likely driven by the higher Ang II during

HUT in the P/S group relative to HUT(-) and syncope subjects (Figure 4F). There were no changes in Ang-(1-7) elicited by HUT and no differences among subgroups (Figure

4G).

84

85 Discussion

The objective of our study was to better characterize the neurohumoral phenotype and its relationship with hemodynamic changes to HUT in children with OI (14, 46). The neurohumoral profile included measurements of circulating catecholamines (Epi and

NE), the RAS axis, and AVP to provide a comprehensive panel for subjects with OI to test the effects of orthostatic challenge. Our methodology was unique in that the neurohumoral profile was measured during HUT as opposed to previous studies in adults in which sample collection was taken in the supine position alone, after completion of a

30-minute test, or after evoked HUT response (11, 19, 20, 34, 37). Our approach aimed to more accurately reflect real-time neurohumoral changes as they related to cardiovascular symptoms including HR and BP changes, especially for neurohumoral factors that are less stable and metabolized quickly (1).

As we have previously reported (12, 13), pediatric subjects presenting with various OI symptoms frequently demonstrated an abnormal HUT raising the possibility of a cardiovascular basis for their OI including gastrointestinal symptoms. The majority

(62.5%) of patients with reported orthostatic dizziness were HUT(+), consistent with findings in adults (36) in which autonomic dysfunction was associated with a high rate of symptoms of lightheadedness, dizziness, fatigue, nausea, and/or abdominal pain. While the majority of HUT(+) subjects in our study had POTS (17 of 30 OI subjects), consistent with other studies (14, 43, 46, 49, 53), over half of the POTS patients experienced syncope later in the HUT (10). Subjects with P/S or syncope sustained significantly less time on HUT compared to HUT(-) subjects. POTS subjects were able to endure the HUT for a longer time compared to syncope subjects. While there was no statistical

86 significance, P/S subjects were only able to sustain half as much time on HUT compared to POTS subjects (Table 1). This is most likely due to P/S subjects experiencing syncope after 10 minutes of HUT, compared to POTS subjects who were able to sustain the 45- minute HUT.

When all HUT(+) subgroups were analyzed collectively, an increase in hormone secretion from supine to HUT included AVP, NE, Epi, renin, and Ang II. Although Ang-

(1-7) has been reported to influence AVP and NE release and baroreflex function in animal studies (7, 17, 44, 57), there were no differences in Ang-(1-7) between groups. A negative relationship was observed between Ang-(1-7) and NE, consistent with evidence of neuromodulatory action of Ang-(1-7) on release of NE (7, 17, 48, 57). Overall, only

AVP was markedly elevated between HUT(+) and HUT(-) patients. These findings, taken in the context of decreased blood pressure upon HUT in OI, suggests that AVP release is in response to a reduction in blood volume and perfusion of heart and atrial vessels as well as carotid arteries. Whether the hormone is effectively contributing to an increase in vasoconstriction and volume expansion (5, 25, 54), albeit insufficient to maintain normal arterial pressure, is not clear.

To determine whether the hemodynamic pattern presented by specific OI phenotypes was associated with specific neurohumoral profiles, HUT(+) patients were divided into four diagnostic subgroups. Adult POTS patients compensate for reduction in blood perfusion during orthostatic challenge by increasing HR to prevent a decrease in blood pressure (38, 42, 43), while different compensatory mechanisms and neurohumoral patterns occur relative to adult OH and syncope subjects (3, 14, 23, 32, 55). Our findings in children were similar to adults in that POTS subjects showed elevation in NE

87 compared to HUT(-) subjects, consistent with a hyperadrenergic state (16, 34, 38). There was little to no change during HUT in renin or Aldo. The lack of an Aldo response in

POTS subjects is consistent with other studies in which an increase in Aldo secretion is absent despite increases in Ang II during HUT (34, 38, 49). Plasma Ang II increased significantly from supine to HUT in POTS subjects; however, Ang II levels were not statistically different from those of HUT(-) subjects. There are several theories of why this occurs, including hypovolemia (49), impaired adrenal AT-1 receptors (56), reduced

ACE2 activity (50), or altered sodium loading (47, 49). Interestingly, AVP was not significantly increased in the POTS subgroup during HUT. Both of these observations may reflect the fact that blood pressure and perhaps blood flow, at least in POTS subjects, is maintained during HUT via the increased HR in these subjects.

Furthermore, in the P/S subjects where BP was not maintained over the duration of the 45 min HUT, Ang II levels tended to be higher when compared to HUT(-) subjects.

AVP responses were variable in this subgroup. In contrast to the subjects with POTS alone, the NE response was not elevated. This may suggest that those with POTS who progress to syncope may have a disproportionate reliance on the RAS compared to other vasoconstrictor systems versus those with POTS alone who may rely on sympathetic nervous system activation. Traditionally, this distinction between POTS and P/S has not been made when analyzing neurohumoral changes in adults, but the unique hemodynamic and autonomic response to HUT may reflect impairment in the ability to sustain a vasoconstrictive response in those with P/S compared to POTS alone. Thus, our findings support the concept that subjects in this category require additional compensatory

88 mechanisms to maintain sufficient perfusion over the longer HUT timeframe, and perhaps in daily activity.

In contrast to POTS subjects, OH and syncope subjects had no change in NE, increases Epi, and exaggerated increases in AVP. There was a failure to significantly increase renin and Ang II in the OH subjects with HUT, while the syncope group showed normal renin and Ang II responses. Both groups had a trending increase in Epi and a failure to increase NE, which is consistent with previous reports of sympathoadrenal imbalance in OH and syncope subjects (18). These studies showed drastic increases in

Epi prior to syncopal episodes suggesting that vasodilation in neurocardiogenic syncope is, at least in part, humorally mediated (10, 18). Furthermore, intraveneous infusion of

NE maintained BP in OH subjects during head up tilt, temporarily eliminating OH (19).

Thus, the imbalance of NE and Epi, with higher circulating Epi, most likely contributes to vasodilation or impairments in peripheral vascular resistance by stimulating vasodilating β2-adrenergic receptors in skeletal blood vessels, which results in low

BP/cardiac output in OH and syncope subjects (15). However, in the current study, the sympathoadrenal imbalance was accompanied by a trend for exaggerated increases in

AVP in syncope and OH subjects, and increased Aldo in OH subjects.

Surprisingly, OH subjects exhibited significantly higher serum Aldo compared to

HUT(-) subjects, with higher levels in both the supine and HUT positions. All other groups had lower Aldo levels while supine with little to no increase from supine to HUT.

A trend for higher AVP in the supine position was also observed in the OH group. The higher AVP may be a reflection of a chronically lower BP as has been suggested when copeptin was used as an indirect measure of chronically elevated AVP (59). The increase

89 in AVP may be due to lower pressures in the OH group compared to the syncope group at the time of the hormonal measurement, given that syncope occurred later in the HUT. A combination of high Aldo and AVP has also recently been associated with hospitalized neonatal foals with clinical hypoperfusion, providing further evidence for these clinical biomarkers to identify subjects with volume depletion and/or poor tissue perfusion (9).

Considering AVP release is most sensitive to reduced serum osmolality followed by sensitivity to decreases in blood pressure, our findings are consistent with the reduction in effective BP and perfusion as the stimulus for release of this hormone. However, the trigger for this response is still unclear. Thus, while accompanied by an imbalance in NE and Epi, higher Aldo and AVP at baseline and the exaggerated increase in AVP in response to HUT in OH subjects appear to be compensating for the lack of response in multiple other vasoconstrictive and fluid retention systems.

The relationship between AVP and reduced blood pressure has been described in both animal and human studies in which increased secretion of plasma AVP is triggered by unloading of arterial baroreceptors in the hypotensive state (26, 54). This is similar to our observation following HUT in both OH and syncope subjects with the reduction in blood pressure. However, stimulation of AVP also occurs independently of the baroreflex

(41). Baroreflex sensitivity and heart rate variability are reduced during HUT in normal subjects and there is even greater suppression of these indices of blood pressure and heart rate regulation in those with OI and HUT(+) (45). In fact, AVP release is reportedly greater in situations when the baroreflex is impaired (41), which would be consistent with observed AVP responses during HUT given the negative correlation between AVP and

90 BP during the upright position in the HUT(+) subjects. Thus, the trigger for AVP release, low BP and exaggerated impairment in baroreflex, remains unclear in the current study.

The positive correlation observed between AVP and BP in the supine position was an unexpected finding. In light of the association between dysautonomia and hypertension, this does raise some concern for future elevated blood pressure in HUT(+) subjects (most notably, the OH subgroup with higher supine Aldo) (2, 40). For example, autonomic indices including decreased heart rate variability and baroreflex sensitivity are accepted as risk factors and indicators for the development and progression of cardiovascular problems such as hypertension and stroke (29, 31, 33). Future studies may include measuring copeptin levels to further elucidate this question. Copeptin, a stable component of AVP, is a marker of treatment effectiveness for OI, with lower levels of copeptin observed in patients effectively treated with vasoconstrictors (59). Whether OI,

Aldo, or AVP/copeptin may also serve as biomarkers for development of hypertension in this patient population warrants further attention particularly in light of the absence of longitudinal cardiovascular studies in these patients.

The findings in this study underscore the distinct nature of the HUT(+) hemodynamic subgroups raising the question as to the need for more specific treatment strategies based on the individual subgroup characteristics. The exaggerated release of several hormones in the face of minimally maintained or lower pressure in POTS or P/S may suggest that vascular responsiveness to the constrictors is impaired. In contrast, deficits in release of neurohumoral factors other than AVP appear to have a role in OH.

POTS patients do not have high levels of Aldo or AVP either supine or during HUT suggesting that volume expansion with the use of aldosterone-like drugs such as

91 fludrocortisone may be sufficient, particularly when coupled with increased NE, to mitigate their pronounced orthostatic response. In contrast, fludrocortisone may be less effective in OH patients based on observed increases in Aldo. The exaggerated AVP response in this group supports this possibility. In fact, fludrocortisone is not effective in

~35% of adolescents treated for OI (12). Therefore, prospective, placebo-controlled studies are needed to determine the potential role for other treatment modalities such as vasoconstrictor therapy (e.g., midodrine) in OH subjects (3, 39, 51).

The limitations of the study include: first, while HUT(-) subjects served as controls, all those studied with HUT had reported symptoms of OI prior to the study.

Thus, future study designs require HUT and neurohumoral assessment of asymptomatic, healthy controls. Second, the sample size was relatively small particularly when considering HUT(+) subgroups. In order to better define potential differences among patients with POTS, OH, syncope, P/S, and the correlative relationship between hemodynamic and neurohumoral measures, a larger cohort of OI patients is required. To better determine the feasibility of conducting such a study, understanding the prevalence of OI in the pediatric population will be necessary. The timing of blood collection also requires further consideration. As discussed earlier, neurohumoral markers were measured 15 minutes after HUT in order to optimally capture orthostatic symptoms.

However, by performing a 45-minute HUT, several patients with syncope did not manifest orthostatic and cardiovascular changes until later in the study. Therefore, changes in the neurohumoral profile in this subgroup may be more pronounced at different times during the HUT. Finally, while the hormone panel we measured was comprehensive, it did not include cortisol. Cortisol levels may be relevant as anxiety can

92 be a co-morbid symptom in pediatric patients with OI (52). Assessing cortisol levels during HUT may help delineate the potential role of physiological stress in these children.

In conclusion, a distinct neurohumoral profile related to OI subgroup diagnoses was demonstrated during HUT in pediatric patients presenting with orthostatic symptoms and GI symptoms. These findings support the need for better understanding of the relationship between these hormones and cardiovascular changes with orthostatic challenge. By studying these potential cause-and-effects in OI patients, it is anticipated that more focused and possibly more effective treatments can be started in this group of patients, thereby, leading to an evidenced based approach.

93 Perspectives

Since dysautonomia, which can manifest as OI, is a potential risk factor for cardiovascular disease, understanding the mechanisms involved in producing symptoms of OI is warranted, particularly at an early age. The heterogeneity of clinical presentation in OI patients, with symptoms ranging from syncope to migraine headaches to gastrointestinal complaints, favors a multidisciplinary approach to their care. In this study, we measured a panel of neurohormones in OI subjects. To determine the impact of orthostatic challenge, we measured these hormones both while supine and during HUT and attempted to define differences among specific HUT(+) subgroups. We demonstrated that elevated AVP was associated with lower blood pressure and that OH subjects have elevated Aldo independent of upright posture. Further evaluation of AVP, the RAS, and catecholamines in subjects with orthostatic symptoms may elucidate the mechanisms involved in the cause of these OI subtypes. It may further provide insight into their long- term cardiovascular prognosis including risk of hypertension and cardiovascular disease.

94 Acknowledgements

The authors thank Anya Brown and the Hypertension Core Assay Laboratory for

technical assistance.

Sources of Funding

This work was supported by the American Heart Association Clinical National Center

Clinical Research Program Grant (AHA12CRP94200029). Additional support from the

Farley Hudson Foundation (Jacksonville, NC) and the Hypertension and Vascular

Research Center of Wake Forest University is gratefully acknowledged.

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101 CHAPTER THREE

HEAD UPRIGHT TILT EVOKES EXAGGERATED ARGININE VASOPRESSIN RESPONSE IN CHILDREN WITH CHRONIC NAUSEA AND ORTHOSTATIC INTOLERANCE

By

Ashley L. Wagoner1, Hossam A. Shaltout1,2, Debra I. Diz1,3, John E. Fortunato1,4

1Hypertension and Vascular Research Center,

Wake Forest School of Medicine, Winston-Salem, NC, USA

Departments of 2Obstetrics and Gynecology and 3General Surgery

Wake Forest School of Medicine, Winston-Salem, NC

4Department of Pediatrics,

Virginia Commonwealth University School of Medicine, Richmond, VA

Short Title: Exaggerated Arginine Vasopressin Responses in Children with Chronic

Nausea

The following manuscript is under review for the American Journal of Gastroenterology

and represents the efforts of the first author. Differences in formatting and organization

reflect the requirements of the journal.

102 Study Highlights

1. WHAT IS CURRENT KNOWLEDGE

a. Children with chronic unexplained nausea also present with symptoms of

orthostatic intolerance (OI).

b. The neurohumoral response to orthostasis has not been described in

children with chronic nausea.

2. WHAT IS NEW HERE

a. Children with orthostatic-induced nausea have exaggerated increases in

vasopressin upon standing, independent of OI status.

b. Nausea profile scores correlate with impaired autonomic function during

tilt.

103 Abstract

Background & Aims: Chronic nausea is very common and debilitating in adults and children. When an organic or metabolic cause is not identified, nausea is often defined as idiopathic or functional. A single objective mechanism for functional nausea is difficult to define due to its heterogeneous presentation. We aimed to define a phenotype of chronic nausea associated with orthostatic intolerance in pediatric patients by assessing the relationship between hemodynamic and neurohumoral characteristics and GI symptoms.

Methods: Subjects ages 10-18 years meeting Rome III criteria for functional dyspepsia with nausea and symptoms of orthostatic intolerance underwent head-upright tilt table testing. All subjects completed a Nausea Profile Questionnaire before tilt test.

Circulating catecholamines, vasopressin, aldosterone, renin, and angiotensins were measured supine and after 15 minutes of 70 degree tilt. Heart rate and blood pressure were continuously measured allowing calculation of baroreflex sensitivity, heart rate variability, blood pressure variability, and sympathovagal balance.

Results: Twenty of 48 subjects (42%) developed nausea within the first 15 minutes of tilt

(responders). Plasma vasopressin increased by ~40-fold in responders compared to ~2- fold in subjects without tilt-induced nausea (non-responders). There was an inverse relationship between total and somatic Nausea Profile scores with baroreflex sensitivity and heart rate variability, and a positive relationship with sympathovagal balance.

Conclusions: This is the first demonstration in children that nausea induced by an acute orthostatic challenge is associated with exaggerated vasopressin release and that the outcome of a Nausea Profile Questionnaire is associated with impairments in

104 cardiovascular autonomic function. Whether the nausea is secondary to the vasopressin or orthostatic intolerance or vice versa, the findings reveal the possibility of an underlying impairment of autonomic and/or neurohumoral regulation responsible for secondary GI symptoms. Future studies are needed to determine the potential role of orthostatic intolerance in other functional GI disorders. ClinicalTrials.gov number,

NCT01617616.

105 Introduction

Chronic nausea is a highly individual and debilitating symptom occurring in both adults and children, which is often difficult to describe objectively. In the absence of a distinct organic cause, chronic nausea is often defined as functional in nature (1). The pathophysiological mechanisms responsible for nausea symptoms are heterogeneous and most likely multifactorial. When routine gastrointestinal (GI) diagnostic testing fails to identify a cause, treatment is tailored to alleviate the symptoms regardless of the etiology.

These empirical treatments have variable efficacy particularly since the root cause of the nausea, whether primary or the consequence of an alternative systemic problem, is often difficult to define (2,3).

The majority of studies on nausea and functional gastrointestinal disorders

(FGIDs) have been conducted in adults. However, children also present in the GI clinic with FGIDs, and dyspepsia in children is frequently associated with nausea (4). Our previous work demonstrated that children with dyspepsia in which nausea was the predominant symptom also experienced a high incidence of orthostatic intolerance (OI) including symptoms such as lightheadedness with standing. When studied with head upright tilt (HUT) table testing, 80% had an induced orthostatic response consistent with postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension, or syncope

(5) as well as impaired baroreflex sensitivity (BRS) (6). This is consistent with findings in both adults (7) and children (8,9) in which autonomic dysfunction was associated with functional dyspepsia. In a subsequent study, pediatric subjects with unexplained chronic nausea and orthostatic intolerance, verified by an abnormal HUT, reported reduction in

106 nausea after a trial of fludrocortisone suggesting the possibility that nausea may be a secondary symptom of autonomic dysfunction (10).

While the relationship between the autonomic nervous system and hormonal changes has been explored in patients with chronic nausea (7,11–14), a comprehensive panel evaluating the impact of orthostatic challenge during HUT has not been employed in a pediatric population. To explore the relationship between nausea with orthostatic intolerance and neurohumoral changes, we aimed to study the cardiovascular, gastrointestinal, and neurohumoral responses during HUT in pediatric subjects with nausea predominant dyspepsia. Specifically, our panel included: circulating epinephrine

(Epi), norepinephrine (NE), arginine vasopressin (AVP), aldosterone (Aldo), renin, and angiotensins [Ang II and Ang-(1-7)]. We hypothesized that subjects with an abnormal

HUT would have a higher incidence of nausea and an exaggerated neurohumoral response compared to those with a normal HUT.

107 Methods

Patients

Pediatric subjects (n = 48), ages 10-18 years, were recruited from the pediatric GI clinic at the Wake Forest Baptist Medical Center if they met Rome III criteria for childhood functional dyspepsia with nausea as the predominant symptom (1) and had reported symptoms of OI. Patients were excluded if a metabolic, mechanical, or mucosal inflammatory cause had been defined to explain their GI symptoms. This included a diagnosis of inflammatory bowel disease, celiac disease, liver or pancreatic disease, hiatal hernia, or bowel obstruction. All subjects were required to complete a 45-minute HUT test. Blood was taken after 15 minutes in the supine position and after 15 minutes of

HUT to measure: Epi, NE, Ang II, Ang-(1-7), Aldo, renin, and AVP. The Wake Forest

Baptist Medical Center’s Institutional Review Board approved this study. Parents and children provided written consent/assent prior to study participation. All the authors had full access to study data and approved submission of the manuscript.

Nausea assessment

Each patient completed the validated Nausea Profile (15) within two weeks of HUT testing. Symptoms of nausea, abdominal pain, and dizziness were recorded throughout the entire HUT. Patients who experienced nausea within the first 15 minutes of the HUT were defined as having tilt-induced nausea.

HUT

All tilt-table tests were performed at Wake Forest Baptist Medical Center. Subjects were brought to the laboratory having fasted 2 hours after a light breakfast consisting of a 200 kcal combination of juice and toast. Heart rate (HR) and pulse oximetry were monitored

108 continuously, with blood pressure (BP) monitored using an automated BP cuff every one- minute through standard intermittent methods. Each subject was positioned supine on the tilt table for 15 minutes followed by immediate upright tilt to from zero to 70 degrees for up to 45 minutes. After 45 minutes the tilt-table was brought back to the supine position for an additional 15 minutes. The test was terminated early for excessive gastrointestinal symptoms, syncope, cardiac arrhythmia, or at the request of the patient for severe symptoms.

Patients were diagnosed as having a positive HUT if they met criteria for postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension (OH), syncope or

POTS/Syncope (P/S). POTS was defined as either a HR greater than 120 bpm or ≥40 bpm increase in HR from mean supine HR in the first 10 minutes of HUT, sustained for at least 2 minutes(16,17). OH was defined as a 25 mm Hg decrease in systolic BP (SBP) from mean supine SBP in the first 10 minutes of HUT sustained for a minimum of 2 minutes without an associated increase in HR(18,19). Syncope was defined as temporary loss of consciousness during 45-minute HUT. Subjects were classified as P/S if they met criteria for POTS as defined above followed by syncope at any time during the 45-minute

HUT. Out of 48 subjects, 30 had a positive HUT(+) and were categorized as POTS,

[n=7], OH, [n=5], syncope [n=8] or P/S, [n=10] (20).

Assessment of autonomic nervous system balance by BRS

Continuous BP and HR were acquired from noninvasive finger arterial pressure measurements for a minimum of 5 minutes in subjects in the supine position, prior to the tilt, and continuously throughout the 70 degrees tilt. An average of the BPs and HR in the last 5 minutes of the supine period were used for baseline measures. The average HUT

109 BPs and HR were calculated in the 2 minutes before blood samples were collected (after

15 minutes of HUT) to compare to baseline supine BP and HR. SBP and RR intervals

(RRI) files were acquired (BIOPAC acquisition software, Santa Barbara, CA) at 1000 HZ and analyzed using Nevrokard BRS software (Nevrokard BRS, Medistar, Ljubljana,

Slovenia) followed by post-hoc analysis for autonomic function variables (heart rate variability (HRV), blood pressure variability (BPV) and baroreflex sensitivity (BRS))

(21). BRS was measured as low frequency (LF), high frequency (HF) alpha indices, Seq

ALL, while HRV was measured as power of RRI spectra in LF, HF range and their ratio

(LFRRI/HFRRI) and standard deviation of normal beat-to-beat interval (SDNN) and BPV as standard deviation of the mean arterial pressure (SDMAP) as published previously

(22,23).

Measurements of blood hormones

Measures of Aldo, renin, and angiotensins were carried out in the CLIA certified

Hypertension Core Assay Laboratory at Wake Forest. Measures of catecholamines (Epi,

NE, dopamine) and AVP were carried out in the Pathology lab at Wake Forest Baptist

Health. Blood for plasma peptides was collected in an EDTA tube containing a cocktail of protease inhibitors, centrifuged and plasma removed and frozen at -80° C. Plasma was extracted using Sep-Pak columns. The sample was eluted, reconstituted and split for the three angiotensin peptide (I, II and 1-7) RIAs previously described for human studies

(24,25). Ang I was measured using a modified version of the Peninsula assay, Ang II using the Alpco (Windham, NH) and Ang-(1-7) using an in-house antibody. Serum

Aldo and plasma renin were measured as published (26). Epi, NE and dopamine were measured after whole plasma extraction by high performance liquid chromatography.

110 Dopamine was not detectable in plasma of any of the subjects. AVP was measured using an ALPCO Diagnostics RIA (Windham, NH).

Statistical Analysis

For continuous variables, means and standard error of the mean (SEM) were calculated and for categorical variables counts and percentages were calculated. The Fisher’s exact test was used to evaluate associations among categorical variables. Two-way ANOVA tests were conducted to examine the effect of HUT on non-responders versus responder groups. When appropriate, t-tests and one-way ANOVAs were conducted to examine specific effects of HUT on hemodynamic and neurohumoral variables between and among groups. Unpaired t-tests were performed comparing non-responders versus responders groups, followed by a Welch’s Correction for unequal variances. Paired t-tests were performed to compare supine to HUT measures within groups. Mann Whitney tests

(unpaired t-tests) and Wilcoxon’s Correction tests (paired t-tests) were used when data sets were not equally distributed. Pearson’s correlation test was performed to compare neurohumoral and hemodynamic variables. Spearman’s correlation test was performed when data sets were not normally distributed. All statistical analyses were performed using GraphPad Prism 6.0. Results with p<0.05 were considered statistically significant.

111 Results

Forty-eight patients, 36 females and 12 males, median age (± SEM) 15.2 ± 0.3 years, with nausea predominant functional dyspepsia and OI symptoms underwent 45- minute HUT testing (Table 1). Of the 48 subjects, 30 had an abnormal cardiovascular response to HUT [HUT(+)] and 18 did not [HUT(-)].

HUT-associated Nausea

Although thirty-one subjects (64.5%) reported nausea at some point during the entire 45-minute HUT test, to more accurately compare the relationship between nausea and neurohormonal levels (measured 15 minutes after HUT), the definition of HUT associated nausea was limited to those subjects experiencing nausea ≤15 minutes after

HUT. Based on this definition, 42% of subjects experienced HUT associated nausea

(responders, n = 20) and 58% did not (non-responders, n = 28). There were no differences in age, weight, or BMI between non-responders and responders, or with respect to HUT(-) or HUT(+). The HUT(+) subjects were less able to sustain the 45- minute HUT procedure. HUT(+) responders were on the HUT for the least amount of time, which was significantly less than HUT(-) groups (Table 1).

Nausea Profile and Autonomic function

Nausea Profile questionnaires used to evaluate symptoms within the previous two weeks were administered prior to HUT. Nausea scores (out of 100) for each category for all 48 subjects are as follows (mean ± SEM): Total Nausea: 43 ± 3; Somatic Distress: 45

± 3; GI Distress: 56 ± 3; Emotional Distress: 29 ± 3. There were no differences in Nausea

Profile scores when subjects were grouped into responders and non-responders as determined with HUT. However, there was a trend for higher total Nausea Profile scores

112 between responders and non-responders (p = 0.07). This is not unexpected as the Nausea

Profile was completed before the HUT and nausea was the chief complaint of all subjects entering the study. Based on Fisher’s exact test, a positive HUT did not associate with nausea experienced on HUT (p = 0.5) (Table 1).

Autonomic function and spontaneous BRS were determined using spectral analysis and sequence methods. There were no differences in autonomic indices between responders and non-responders in supine or HUT position (Table 2). There were also no associations between baseline autonomic recordings and Nausea Profile scores. However, regression analysis revealed significant associations between Nausea Profile scores and autonomic indices during HUT. Total Nausea Profile scores negatively correlated with

HUT measures of BRS (all sequences, r = -0.33, p ≤ 0.01) and HRV (SDNN, r = -0.3, p <

0.05). There was a positive relationship between total Nausea Profile scores and measures of sympathovagal balance (LF RRI/ HF RRI, r = 0.32, p < 0.05) during HUT.

Somatic distress-specific Nausea Profile scores showed similar patterns of associations with autonomic indices as total nausea scores. GI distress-specific Nausea Profile scores negatively correlated with HUT HRV as measured by SDNN (r = -0.31, p < 0.05).

Emotional distress negatively associated with HUT BRS (r = -0.29, p < 0.05).

113 Non- A B C D Responders responders HUT(-) HUT(+) HUT(-) HUT(+) p-value n 28 12 16 20 6 14 NS Sex (M/F) 7/21 1/11 6/10 5/15 2/4 3/11 Age (yrs) 15 ± 0.4 15 ± 0.5 15 ± 0.6 15.4 ± 0.4 15.1 ± 0.7 15.5 ± 0.5 NS Weight (kg) 61.4 ± 3.1 63.5 ± 5.5 59.8 ± 3.6 57.7 ± 3.1 62.2 ± 4.6 55.7 ± 3.8 NS BMI 23.3 ± 0.9 25.1 ± 1.9 22 ± 0.8 21.5 ± 1.1 24.2 ± 2.2 20.2 ± 1.1 NS Nausea 38 ± 3$ 37 ± 6 39 ± 5 49 ± 4$ 41 ± 8 52 ± 5 $p=0.07 Profile score a vs b p<0.05 Time on 45.4 ± 30.3 ± a vs d 36.8 ± 2.2 30.6 ± 3.4 45.8 ± 0.4c 24 ± 3.7d HUT (mins) 0.2a 2.9b p<0.001 c vs d p<0.01 Experienced nausea >15 11 5 6 n/a n/a n/a mins on HUT (n)

Table 1. Patient demographics. Non-responders did not experience nausea during the first 15 minutes of HUT. Responders experienced HUT during the first 15 minutes of HUT. All values are expressed in exact number (n) or mean ± SEM. Fisher’s exact test determined HUT response does not associate nausea experienced during HUT. Unpaired t-tests were used to assess differences between non- responders and responders. One-way ANOVAs were used to assess differences between groups A-D. A trend was observed considering Nausea Profile score between non-responders and responders. Subjects in group A were on the HUT for a significantly longer time compared to group B and D. Subjects in group D were in upright position for significantly fewer minutes than groups A and C.

114 Non- Responders responders n=20 n=28 Supine HUT Supine HUT Parasympathetic Vagal Tone 27.3 ± 3.3 7.8 ± 0.9 25.9 ± 3.7 7.9 ± 2.1 (HFα, ms/mm Hg) Baroreflex Sensitivity 24.2 ± 2.7 7.7 ± 0.8 22.7 ± 2.7 6.2 ± 1.1 (Seq All, ms/mm Hg) Heart Rate Variability 76.3 ± 7.3 46.5 ± 4.2 78.7 ± 6.7 41.1 ± 3.4 (SDNN, ms) Blood Pressure Variability 3.2 ± 0.1 3.8 ± 0.2 3.4 ± 0.3 4 ± 2.2 (SDMAP, mm Hg) Sympathovagal 1.3 ± 0.23 6.4 ± 0.9 1.0 ± 0.16 5.5 ± 7.1 Balance (SVB)

Table 2: Supine and HUT measurements of autonomic indices. Blood Pressure Variability was the only parameter that did not significantly change from supine to HUT. (mean ± SEM) (ms, millisecond; mm Hg, millimeters of mercury)

115 Neurohumoral Response to HUT

For neurohumoral measurements, there were no differences between groups at supine position (p > 0.05). Both groups exhibited increases in NE, Epi (Figure 1A, B),

Renin and Ang II (Figure 2A, C) from supine to HUT, but there were no differences between responders and non-responders. A trend was observed for greater Epi concentrations in responders during HUT (p=0.06) (Figure 1B). Both responders and non-responders exhibited an increase in AVP from supine to HUT (p=0.0001).

Responders had a ~40-fold increase in AVP compared to a ~2-fold increase observed in non-responders (Figure 1C). There were no changes in Aldo or Ang-(1-7) levels observed from supine to HUT and no between group differences at either position (p > 0.05, Figure

2B, D).

Arterial pressure (systolic, diastolic, mean) was not statistically different in responders versus non-responders during HUT. There were no differences in any hemodynamic measurement in the supine position between responders and non- responders. However, responders showed a trend towards lower mean arterial pressure

(MAP) during HUT compared to non-responders (p = 0.057) (Figure 3A). The failure to significantly increase MAP during HUT in both groups, as typically observed in a normal population likely reflects the presence of HUT(+) subjects in both responders and non- responder groups. HR increased significantly for both responders and non-responders and there was no difference during HUT (Figure 3B).

To further explore the relationship between HUT status and nausea status on

HUT, we separated subjects into 4 groups, as follows: [A] non-responders + HUT(-), [B] non-responders + HUT(+), [C] responders + HUT(-), [D] responders + HUT(+) (Table

116 1). There were no differences in baseline measurements for neurohormones among groups (p > 0.05). Responders + HUT(+) had an exaggerated increase in AVP upon

HUT which was significantly higher than non-responders + HUT(-) subjects (Figure 4A).

There was an observed trend for group differences for Epi with responders + HUT(+) subjects having the highest Epi levels during HUT (p = 0.1, data not shown). For Ang II, all groups except non-responders + HUT(-) had a significant increase in Ang II (HUT effect, p = 0.0002). The two-way ANOVA revealed a significant overall group difference

(p = 0.02), but there were no specific group differences following post-hoc analysis (p >

0.05, data not shown). All 4 groups had a significant increase in NE from supine to HUT positions, while non-responders + HUT(-) and responders + HUT(+) had a significant increase in renin upon HUT. There were no group differences or HUT effects considering

Ang-(1-7) and Aldo (p > 0.05).

Supine MAP was not different among groups. Both HUT(+) groups had significantly lower MAP than both HUT(-) during HUT, with responders + HUT(+) having the lowest MAP during HUT (Figure 4A). As expected, there were no differences between groups for supine HR and all groups had a significant increase upon HUT (data not shown). These expected group differences confirm that low blood pressure is not necessarily the cause for nausea in all subjects.

117 A B Norepinephrine Epinephrine 600 300 ** NR ** R 400 200 *** pg/mL 200 pg/mL 100 ** 0 0 Supine HUT Supine HUT C Arginine Vasopressin ## 100 *** 80

60

40 pg / ml 20 *

0 Supine HUT

Figure 1. Orthostatic changes in NE (A), Epi (B), and AVP (C)in non-responders versus responders. For neurohumoral measurements, there were no differences between groups at supine position. Both groups exhibited similar increases in NE, Epi, AVP, Renin and Ang II (A-D, F) from supine to HUT. There were no changes in Aldo or Ang-(1-7) levels observed from supine to HUT (E, G). Both groups exhibited significantly higher AVP during HUT. However, responders had a ~40-fold increase in AVP compared a ~2-fold increase observed in non-responders (C). (NR, non-responders; R, responders; pg, pictogram; mL, milliliter;)(supine versus HUT, *p<0.05, **p<0.01, ***p<0.001), (non- ## responders versus responders, p<0.01)

118 A B Renin Aldosterone 5 9 *** NR 4 R 8 3 7

2 6

** ng/dL pg/mL 1 5

0 4 Supine HUT Supine HUT C D Angiotensin II Angiotensin-(1-7) 60 20

*** 18 40 *** 16 14 pg/mL pg/mL 20 12

0 10 Supine HUT Supine HUT

Figure 2. Orthostatic changes in Renin (A), Aldo (B), Ang II (C), Ang-(1-7) (D) in non-responders versus responders. For neurohumoral measurements, there were no differences between groups at supine position. Both groups exhibited similar increases in Renin and Ang II (A, C) from supine to HUT. There were no changes in Aldo or Ang-(1-7) levels observed from supine to HUT (B, D). (NR, non-responders; R, responders; pg, pictogram; ng, nanogram; mL, milliliter; dL, deciliter) (supine versus HUT, **p<0.01, ***p<0.001)

119 A B Mean Arterial Pressure Heart Rate 95 120 90 NR 110 R *** 85 100 80 90 ***

mm Hg 75 80 70 70 beats / minute 65 60 Supine HUT Supine HUT

Figure 3. Orthostatic responses in MAP (A) and HR (B) in non-responders versus responders on HUT. There were no differences between groups for MAP (A) or HR (B) during supine or HUT positions. Both groups had similar increases in HR upon HUT (B). (NR, non-responders; R, responders; mm Hg, millimeters of Mercury; BPM, beats per minute) (supine versus HUT, ***p<0.001)

A B Mean Arterial Pressure Arginine Vasopressin # ## 150 ## 120 # Supine ### ** HUT 100 100 * 80 mm Hg

pg / ml 50

60 0 NR R NR R NR NR R R HUT(-) HUT(+) HUT(-) HUT(+)

Figure 4. Orthostatic changes in AVP (A) and MAP (B) in subjects separated by experience of nausea and hemodynamic response to HUT. There were no differences among groups for baseline measures of AVP or MAP. Responders + HUT(+) had an exaggerated increase in AVP upon standing which was significantly higher than non-responders and HUT(-)(B). Both HUT(+) groups had significantly lower MAP during HUT compared to HUT(-) groups(D). (NR, non-responders; R, responders; millimeters of Mercury; pg, pictogram; mL, milliliter) (supine versus HUT, *p<0.05, # ## ### **p<0.01) (between group differences, p<0.05, p<0.01, p<0.001)

120 Discussion

OI is not commonly assessed when considering FGIDs such as chronic nausea in adults or children. HUT testing has recently been shown to evoke nausea in a high incidence of pediatric subjects presenting with nausea predominant functional dyspepsia

(5,10). Our objective in this study was to characterize the clinical phenotype of chronic nausea and OI by defining the relationship among the hemodynamic, neurohumoral and nausea responses to HUT using standard protocols (16,18). Our results showed the following associations in children presenting with chronic nausea and OI: 1) HUT evoked symptoms of nausea; 2) most neurohormones tested increased with orthostatic challenge, but only elevated AVP differentiated responders from non-responders; 3) HUT(+) responders demonstrated the most pronounced AVP increase, which was associated with lowest MAP; 4) Nausea Profile scores collected prior to HUT associate with impaired

BRS, HRV, and sympathovagal balance on HUT. Although our findings do not prove causality, the hemodynamic and neurohumoral changes associated with OI may be contributing factors to nausea symptoms experienced in daily life.

Our methodology was unique in that the neurohumoral profile was measured in the supine position and during HUT in pediatric patients to more accurately relate real- time changes between the onset of nausea and cardiovascular symptoms and hormone levels. This differs from previous work in adults using HUT in which sample collection was taken in the supine position alone or after completion of a 30-minute test (26–29).

Our provocative challenge to evoke nausea was also novel using HUT compared to other studies in which visual stimuli were used to induce motion sickness associated nausea and AVP (11,30–32). By using HUT, we also observed that the duration of orthostatic

121 challenge may be relevant to onset of GI symptoms. For instance, HUT evoked nausea in 42% of subjects within 15 minutes, which increased to 64.5% by the end of the 45- minute HUT. GI symptoms have been previously reported to occur later in the HUT

(5,33,34), perhaps resulting from a more dramatic cardiovascular response to prolonged standing, i.e., insufficient blood perfusion culminating in hypotension and syncope. As suggested previously (10,35), the longer duration of HUT may unmask the GI- predominant symptoms accompanying syncope as opposed to the ability of the shorter duration test to detect OI. For future studies it will be necessary to collect blood samples at later time points during HUT in addition to the 15-minute time point to better delineate the impact on the neurohumoral response for those who experience delayed GI symptoms.

To objectively assess a patient’s nausea and determine its role as a screening tool, we administered a nausea questionnaire – Nausea Profile – to all subjects before HUT testing. The Nausea Profile, validated for use in adults (15), is also a reliable measure in adolescents (36). There were no differences in Nausea Profile scores between non- responders and responders suggesting that while the questionnaire can quantitate nausea, it is unable to predict or differentiate the HUT responder vs. the non-responder phenotypes. Interestingly, the total and somatic Nausea Profile scores negatively correlated overall with markers of autonomic function including BRS and HRV. The GI- specific distress scores negatively correlated with HRV as measured by SDNN. This is consistent with previous studies highlighting impaired/decreased HRV in patients with functional dyspepsia, nausea, and abdominal distress (11–13,32). This may indicate an underlying disorder of the autonomic nervous system not detected simply by using HUT.

122 These findings also support the notion of studying the use of other questionnaires for this patient population such as the Composite Autonomic Symptom Score (COMPASS-31), which includes more extensive scoring of both autonomic and GI symptoms (37).

To determine the impact of HUT on key neurohumoral changes in these pediatric subjects, we examined a comprehensive panel of hormones including: circulating Epi,

NE, AVP, Aldo, renin, and angiotensins [Ang II and Ang-(1-7)]. AVP, Epi, and Ang II have all been shown to induce nausea via central nervous system sites of action as well as influencing gastric motility. They also increase in response to cardiovascular changes associated with HUT in adults (38–40). Both responder and non-responder groups had expected increases in NE, Epi, Renin, Ang II, and AVP during HUT versus supine posture. However, when comparing responders versus non-responders, the most distinct and significant difference occurred with AVP.

To better understand the impact of a positive HUT on responders and non- responders, we compared differences in neurohormones among the four HUT phenotypes: HUT(+) responders, HUT(-) responders, HUT(+) non-responders, and

HUT(-) non-responders. For AVP, responders with a positive HUT had the greatest response. Responders with a negative HUT also demonstrated an increase in AVP, but it was less pronounced. Despite not being statistically significant, the level of AVP in this group was above the expected AVP concentrations under normal physiological conditions (<2pg/mL) (41,42). Consistent with our previous study (20), non-responders with a positive HUT had a significant increase in AVP during HUT compared to supine, but again, not as pronounced as responders with a positive HUT. Thus, a clear association was shown between nausea and AVP for subjects tested, which has not

123 previously been reported in children. While OI seems to be a dominant trigger for increased AVP, and thereby, nausea, it is not clear if it is the only factor for the AVP response as evidenced by the responders with a negative HUT.

Release of AVP from the posterior pituitary can be stimulated through many complex physiological mechanisms (41,43,44). Serum osmolality, a measurement not included in this study, is the most sensitive stimuli of AVP secretion (43). Additional stimuli of AVP release include inflammation (45–47) and stress (44,48). We did not measure cortisol or inflammatory markers in the current study, but have previously reported that nausea symptoms associate with both anxiety symptoms and OI in children

(36). High anxiety and stress associated with nausea in these subjects can be one possible contributing factor for higher plasma AVP levels during HUT. The role of serum osmolality, stress hormones such as cortisol, and inflammatory markers as they relate to

AVP and nausea warrants further study.

Changes in blood volume and blood pressure also stimulate AVP release (49).

While AVP plays a minor role in blood pressure regulation in healthy subjects (50), it has an important vasopressor role in disease states including autonomic failure and hypotension (44,51–53). AVP has been shown to enhance baroreflex control of HR, as a small dose of AVP improves BRS (54), but there were no associations between AVP levels and indices of BRS observed in this study. Although both responders and non- responders demonstrated a significant increase in heart rate with HUT, there were no differences in either blood pressure or heart rate between responders and non-responders in either supine or HUT positions indicating that blood flow alone was not the sole cause of the nausea. As expected, when separated into the four phenotypes, responders and

124 non-responders with a positive HUT, independent of nausea, demonstrated lower blood pressure than those with a negative HUT. Both non-responders and responders exhibited expected decreases in autonomic function and BRS in response to HUT, but once again, there were no differences between groups suggesting that autonomic dysfunction alone wasn’t the primary etiology of their nausea. Previous studies have shown significant differences in these autonomic indices in subjects experiencing nausea. However, the provocative challenge in our study (HUT) was different than those using motion sickness or visual-vestibular mismatch models, which could explain the observed difference in autonomic response (7,11,13,32). For our study, lower blood pressure and impaired baroreflex stimulating AVP release are limited to subjects with a positive HUT only, suggesting at least two distinct nausea phenotypes distinguished based on their HUT response. This finding is consistent with a current study reporting comorbid conditions, including nausea, are equally present in children and adults with and without OI (55).

The positive relationship between AVP and nausea has previously been described in adults (12,56). Nausea itself is a known stimulus for AVP release, as shown by AVP infusion studies (39), where exogenous AVP produced nausea and gastric dysrhythmias

(39,40,57,58). Furthermore, AVP release has been shown to mediate changes in myoelectric activity and overall gastric motility across species (12,59–61). In a recent study using a motion video to evoke nausea in healthy subjects, those with visually- induced nausea had decreased ghrelin and elevated AVP blood levels post-video, which positively correlated with mean nausea scores post-video (13). Thus, the mechanisms leading to AVP release in our subjects may be multifactorial and related to autonomic pathology with associated OI as well as nausea itself. Our current study does provide

125 evidence that the combined effect of OI and nausea has the most pronounced impact on

AVP release as evidenced by the highest plasma AVP concentrations were observed in responders with a positive HUT. These data support the need for future studies measuring the timing of the relationship between GI symptoms, gastric myoelectrical activity, and

AVP more closely in subjects with functional GI symptoms and OI. While there was an acute increase in AVP in responders during HUT, baseline supine values were not different among groups. Copeptin, a stable C-terminal co-peptide of AVP, was recently shown to be an predictor of the effectiveness of β-blocker treatment for OI and to indirectly reflect the status of plasma osmotic pressure during daily life (62). Further study to determine whether copeptin would be a marker of chronic AVP release is therefore warranted, particularly in subjects with orthostatic nausea. Better understanding of these associations may serve to identify potential biomarkers for chronic nausea similar to that of OI (62–64). Whether AVP or copeptin reflect a causative or responsive marker, or provides insight into mechanisms, requires continued study.

Although not observed directly in our study, nausea has also been associated with significant autonomic changes. Subjects experiencing nausea induced by motion sickness demonstrate increased sympathetic and decreased parasympathetic activity. In a separate study, subjects experiencing nausea had increased sympathetic activity after systemic injection of Epi (12,32,56,65). Our findings are consistent considering the observed trend for higher Epi in responders with a positive HUT compared to the other three groups.

This suggests a disproportionate increase in sympathetic activation in these subjects.

Additionally, Ang II, a vasoconstrictor peptide, significantly increased from supine to

126 HUT for all responders as well as non-responders with a positive HUT. Ang II is involved in cardiovascular function and increases are associated with impaired BRS and

HRV, which occur in HUT(+) subjects (6). Increases in Ang II observed in responders +

HUT(-) may contribute to nausea experienced on HUT via actions on the area postrema

(66), increased neuronal excitation in smooth muscle tissue (67–69) or reduction of gastric blood flow via microvasculature vasoconstriction (70). Future studies are necessary to delineate the impact of increased Ang II and Epi in children with chronic nausea with or without a positive HUT.

This study has several limitations. First, all subjects included in this study presented in the clinic with nausea and had reported symptoms of OI prior to the study.

Thus, better understanding of HUT response in asymptomatic, healthy controls is necessary. Second, the sample size was relatively small particularly when separatied into

4 groups based upon nausea and hemodynamic responses to HUT. The optimal timing of blood collection also requires further consideration, as 22% of our subjects did not manifest nausea symptoms until later (>15 minutes) in the 45-minute test. As discussed earlier, neurohumoral markers were measured 15 minutes after HUT in order to optimally capture the profile during the timeframe when cardiovascular instability is defined (~10 minutes). Therefore, differences in the neurohumoral profile in nausea subgroups may be more pronounced at different times during the HUT. Finally, while the hormone panel we measured was comprehensive, it did not include copeptin as discussed above, cortisol or inflammatory markers. Cortisol levels may be relevant as anxiety can be a co-morbid symptom in pediatric patients with FGIDs and orthostatic symptoms (36,71).

Inflammatory cytokines, including IL-1 and IL-6, have been associated with AVP release

127 in the CNS, which could exacerbate the stress response to nausea (45). Assessing these types of markers during HUT may help delineate the potential role of physiological stress in these children.

In conclusion, these studies demonstrate for the first time a relationship between nausea during HUT and the neurohumoral profile, specifically AVP, in a pediatric patient population presenting with an otherwise functionally defined condition, nausea predominant dyspepsia, and orthostatic symptoms. The patient population was particularly unique as subjects presented to the gastroenterologist for symptoms whose root cause may be due to an underlying cardiovascular/autonomic disorder. These results suggest that the mechanism underlying AVP release and nausea symptoms for some subjects may be due to the abnormal hemodynamic changes during HUT. However,

AVP release in those with HUT associated nausea (responders) and a normal HUT may be due to factors unrelated to a cardiovascular etiology. Thus, more than one mechanism may be at play in this group of patients underscoring the importance of meticulously defining differential phenotypes of nausea and functional GI disorders. Finally, the complex nature and multisystem involvement underlying the etiology of the GI symptoms in this patient population strongly supports the need to obtain a more comprehensive history as well as for a multidisciplinary approach to their diagnosis and management.

128 Acknowledgements

The authors thank Anya Brown and the Hypertension Core Assay Laboratory for technical assistance.

Sources of Funding

This work was supported by the American Heart Association Clinical National Center

Clinical Research Program Grant (AHA12CRP94200029). Additional support from the

Farley Hudson Foundation (Jacksonville, NC) and the Hypertension and Vascular

Research Center of Wake Forest University is gratefully acknowledged.

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135 CHAPTER FOUR

IMPAIRED AUTONOMIC RESPONSES IN CHILDREN WITH ORTHOSTATIC INTOLERANCE ASSOCIATE WITH ELEVATED MARKERS OF INFLAMMATION IN THE DORSAL MEDULLA

By

Ashley L. Wagoner1,2, John Olson2, Brian Westwood2,

John E. Fortunato2,5, Debra I. Diz1,2,4, Hossam A. Shaltout2,3

1Neuroscience Graduate Program,

Wake Forest Graduate School of Arts & Sciences, Winston-

Salem, NC

2Hypertension and Vascular Research Center,

Wake Forest School of Medicine, Winston-Salem,

NC, USA

Departments of 3Obstetrics and Gynecology and 4General Surgery,

Wake Forest School of Medicine, Winston-Salem, NC

5Department of Pediatrics,

Virginia Commonwealth University School of Medicine, Richmond, VA

Short Title: Brain Metabolite Profile in Children with Orthostatic Intolerance

The following manuscript is intended for submission to

Autonomic Neuroscience: Basic & Clinical.

136 Abstract

Background: Children with orthostatic intolerance (OI) have exaggerated decreases in heart ratevariability (HRV) and suppression of baroreflex sensitivity (BRS) with standing. The aim of this study was to identify potential central biomarkers for OI in the dorsal medulla, a brainstem area that plays a critical role in baroreflex and blood pressure regulation.

Methods: We used proton (1H) magnetic resonance spectroscopy (1H-MRS) to quantify markers of neuronal and glial integrity in children with OI compared with asymptomatic controls. Eighteen subjects ages 10-18 years were evaluated for blood pressure, heart rate and autonomic function in supine and upright positions. All subjects underwent 1H-MRS scans of dorsal medulla on a clinical 3T magnet while supine an average of 2 weeks following orthostatic challenge (standing or head upright tilt, HUT)

Results: Of the 18 subjects, 11 tested positive for OI [HUT(+)] and 7 did not (controls).

HUT(+) subjects exhibited expected hemodynamic and autonomic responses to HUT, including higher HR and lower HRV and parasympathetic tone during standing compared to control subjects, as well as a significant decrease in BRS. HUT(+) subjects had higher myoinositol (mIns) and total choline (tCho), markers of glial inflammation, than control subjects. Total Cho and mIns concentrations did not correlate with indices of autonomic function measured in the supine position. Univariate correlative analysis showed significant inverse relationships between upright parasympathetic vagal tone (PVT) and

BRS to tCho and mIns respectively.

Conclusions: Children with OI have higher mIns and tCho in the dorsal medulla while supine that may be predictive of impairment in parasympathetic tone and BRS upon

137 upright posture. This first report that OI in children [HUT(+)] is associated with elevated mIns and Cho, markers of inflammation in a variety of neuropathies, raises the intriguing possibility that the autonomic dysfunction observed while standing in these subjects reflects inflammation as an underlying mechanism.

138 Introduction

Autonomic dysfunction manifesting as orthostatic intolerance (OI) affects more than 500,000 Americans1. In both adults and children OI is characterized by the inability to sustain blood pressure (BP) upon standing or during head upright tilt (HUT) testing and is often accompanied by debilitating symptoms including dizziness, nausea, and fatigue1, 2. Significant decreases in heart rate variability (HRV) and baroreflex sensitivity

(BRS)3 as well as exaggerated release of neurohormones in response to standing4 are known to occur in OI subjects compared to subjects with a negative HUT. These responses most likely represent underlying factors of cardiovascular (CV) instability and the secondary symptoms experienced in daily life4, 5. Blood pressure regulation via the baroreflex requires widespread central and autonomic integration for proper modulation of parasympathetic and sympathetic activity to the heart and vasculature6, 7. While hemodynamic and neurohumoral profiles have been described in OI in children, central mechanisms involved in the pathogenesis of impaired autonomic regulation in OI have yet to be studied.

Proton magnetic resonance spectroscopy (1H-MRS) is a powerful non-invasive tool used to investigate neurochemical alterations in the brain8-12 and has been applied to disorders of autonomic function including multiple system atrophy (MSA)13,

Alzheimer’s14, Parkinson’s15, Huntington’s16, amyotrophis lateral sclerosis17, and multiple sclerosis18. This technique utilizes high-resolution anatomic imaging to quantify neurotransmitters and metabolites in discrete regions of interest in the brain.

Neurochemical compounds detected by 1H-MRS provide cell type specific information including neuronal integrity (N-acetyl aspartate, NAA), glutamate metabolism (N-acetyl

139 aspartate glutamate, NAAG), cell cycle turnover (creatine and phosphocreatine, tCr), cell membrane integrity (total choline including glycerophosphorylcholine + phosphorylcholine, tCho) and glial inflammation (myoinositol, mIns; tCho)9-12, 19-23.

This technique is established in adults as well as in children and differences in metabolites in a variety of brain regions throughout development have been described previously24. However, 1H-MRS has yet to be employed in children targeting brainstem regions related to autonomic function and may provide an innovative approach to understanding cardiovascular instability observed in OI. Furthermore, if metabolite profiles are predictive of autonomic performance in upright position, 1H-MRS could be used to detect biomarkers of autonomic dysfunction and provide a noninvasive approach to diagnosis and development of treatment strategies in children.

In the current study, we hypothesized that children with OI would exhibit characteristic brain metabolite profiles in the dorsal medulla (DM) that correlate with autonomic function. To test this, we measured continuous HR and BP during supine and standing positions to allow calculation of autonomic indices. Using 1H-MRS, 11 supine metabolites/neurotransmitters were measured in the dorsal medulla (DM) of children with

OI, as defined by HUT, and asymptomatic controls.

140 Methods

Patients

We studied eighteen male and female subjects ages 10-18 years recruited from either the pediatric clinic at Wake Forest Baptist Medical Center or from the community of

Winston-Salem, NC. All subjects completed an upright position challenge (either standing or head upright tilt testing) to establish cardiovascular groupings. Patients with cardiovascular disease, malignancy, psychiatric illness, neurodevelopmental delay, or other co-morbid conditions as well as individuals with pacemakers, implanted devices, or any other MRI contraindications were excluded from this study. The Wake Forest Baptist

Medical Center’s Institutional Review Board approved this study. Parents and children provided written consent/assent prior to study participation.

Orthostatic Challenge & Head Upright Tilt

Asymptomatic controls were recruited from the community and HUT(+) subjects were recruited through pediatric gastroenterology and cardiology clinics at Wake Forest

Baptist Health. All control subjects underwent a 10-minute standing challenge for collection of hemodynamic and autonomic measurements in supine (5 minutes) and upright positions (10 minutes). All subjects recruited from pediatric clinics at Wake

Forest underwent HUT, which consisted of 15 minutes in supine position followed by immediate upright tilt to from zero to 70 degrees for up to 45 minutes. After 45 minutes the tilt-table was brought back to the supine position for an additional 15 minutes. The test was terminated early for excessive GI symptoms (nausea, abdominal pain), syncope, cardiac arrhythmia, or at the request of the patient. Continuous measures of blood pressure (BP) and heart rate (HR) were obtained using noninvasive finger arterial

141 pressure measurements and analyzed by BIOPAC acquisition software (Santa Barbara,

CA). An average of the BPs and HR in the last 5 minutes of the supine period were used for baseline measures. The average upright BPs and HR were calculated in the 2 minutes prior to test termination.

Patients were diagnosed as having an abnormal HUT [HUT(+)] if they met criteria for postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension

(OH), or syncope. Otherwise, patients were defined as having a normal HUT [HUT(-)].

POTS was defined as either a HR greater than 120 bpm or ≥40 bpm increase in HR from mean supine HR in the first 10 minutes of HUT, sustained for at least 2 minutes25, 26. OH was defined as a 25 mm Hg decrease in systolic BP (SBP from mean supine SBP in the first 10 minutes of HUT sustained for a minimum of 2 minutes without an associated increase in HR26. Syncope was defined as temporary loss of consciousness during upright position.

Assessment of autonomic nervous system balance by BRS for control of HR, HRV, and

BPV

We collected continuous measures of BP and HR throughout the upright challenges, which allowed for post-hoc analysis of autonomic function variables including heart rate variability (HRV), blood pressure variability (BPV), baroreflex sensitivity (BRS), parasympathetic tone (PVT; high frequency alpha, HFα), sympathetic tone (ST; low frequency, LFα), and sympathovagal balance (SVB, LF/HF RR interval). The analysis techniques have been validated in human subjects previously3, 27. Continuous BP and HR were acquired from noninvasive finger arterial pressure measurements for a minimum of

5 minutes in subjects in the supine position, prior to upright position, and continuously

142 throughout the upright position. SBP and RR intervals (RRI) files acquired (BIOPAC acquisition software, Santa Barbara, CA) at 1000 HZ were analyzed using Nevrokard

SA-BRS software (Nevrokard SA-BRS, Medistar, Ljubljana, Slovenia) for measures of

BRS (as LF, HF alpha indices, Seq BRS), HRV (as power of RRI spectra in LF, HF range and their ratio (LFRRI/HFRRI), standard deviation of beat-to-beat interval (SDRR) and the root mean square of successive beat-to-beat differences in R-R interval duration

(rMSSD)) and BPV (as power of SBP spectra calculated as LFSAP and standard deviation of the mean arterial pressure (SDMAP)) as published previously28, 29.

Magnetic Resonance Imaging and Proton (1H)-MR spectroscopy

All subjects underwent 1H-MRS scans of the dorsal medulla (DM) on a clinical 3T magnet while supine after HUT testing (average two weeks following). A Siemens

SKYRA ultrashort superconducting 3T Magnet (Siemens, Erlangen, Germany) was used to acquire images with a gradient system 45mT/m and 200T/m/sec for each axis. High- resolution T1-weighted structural scans were obtained using an inversion recovery 3D spoiled gradient echo sequence (matrix size=256 x 256; field of view=24cm; 1.5 mm sections, no gap; 128 slices; in-plane resolution=0.94mm). High-resolution T1-weighted images were used for voxel localization of 1H-MRS and proton spectra were acquired from the DM of all subjects. The single-voxel 1H-MRS was performed using point resolved spectroscopy pulse sequence in LC Model software (TE = 35ms, voxel size =

10x7x20mm). Water suppression correction was applied when necessary.

We collected measurements of 11 metabolites including alanine (Ala), total creatine (tCr, creatine and phosphocreatine), glutamate (Glu), glutamine (Gln), total choline (tCho, phosphocholine and glycerophosphorylcholine), glutathione (GSH),

143 myoInositol (mIns), N-acetyl aspartate (NAA) and NAA-glutamate (NAAG). LC Model quantification expresses errors in metabolite quantification in % standard deviation (SD), representing the 95% confidence intervals of the estimated concentration30, 31. Absolute values of metabolites were used for analysis when less than <50% the standard deviation.

Analysis was not conducted when there were less than three useable metabolite measurements per group. Total Cr was not used for normalization ratios because a trending difference was observed between HUT(+) and control subjects.

Statistical Analysis

For continuous variables, means and standard error of the mean were calculated and for categorical variables counts and percentages were calculated. Two-way ANOVA tests were conducted to examine the effect of upright position or HUT on HUT(+) vs. control groups. When appropriate, post-hoc t-tests were conducted to identify specific effects of standing on hemodynamic and autonomic variables between and among groups.

Unpaired t-tests were performed comparing HUT(+) and control groups, followed by a

Welch’s Correction if there were unequal variances. Mann-Whitney tests were conducted when data sets were not normally distributed. Paired t-tests were performed to compare supine to HUT measures within groups and Wilcoxon’s Correction tests were used when data sets were not normally distributed. Pearson’s correlation test was performed to compare metabolite and hemodynamic variables. Spearman’s correlation test was performed when data sets were not normally distributed. These statistical analyses were performed using GraphPad Prism 6.0 (San Diego, CA). Results with p<0.05 were considered statistically significant.

144 For assessment of complex relationships among the variables studied, the entire data set was analyzed by correlate summation analysis (CSA). CSA is a data mining method designed to find the variables that are most covariant within all variables being studied, relative to clustering, as described previously32-34. Discrete correlate summation

(DCS) specifically compares differences in correlations between two groups for each variable versus all of the others relative to the mean shift. Alternatively to principle component analysis, this approach provides a simple means of ranking overall covariance qualified to data clustering using an online Microsoft Excel template

(https://en.wikipedia.org/wiki/Correlate_summation_analysis). For control and HUT(+) groups, a correlation matrix was constructed for each variable. Variables with less than 5 data points per group were excluded from DCS analysis. Correlation matrices were transformed into linear probability matrices and the log mean ratio (lgmr) was calculated for each variable. The correlate summation was then multiplied by the log mean ratio, to yield the discrete mean-correlate summation (DCSx). Thus, DCSx is the product of the totaled absolute value of the logarithm of the p-value ratios between two groups' correlations to a given variable and its absolute value of the logarithm of the group mean ratios. The higher the DCSx the more covariant the variable compared to all other variables. To control for age and gender, multiple regression analysis was conducted in

Instat3 software (GraphPad Software, Inc., San Diego, CA) for the top 2 upright autonomic measurements (PVT and BRS) and top 2 supine metabolite variables (mIns and tCho) identified by DCS.

145 Results

Eighteen subjects, 6 male and 12 female, ages 10-18 underwent upright challenge, continuous BP and HR recording, and 1H-MRS. Of the 18 subjects, 11 were HUT(+) and

7 were controls. The median age (± SEM) was 15.5 ± 0.59. The 11 HUT(+) subjects met the criteria for one of three OI subtypes, POTS (n=6), OH (n=2), syncope (n=3). There was no difference in BMI between controls and HUT(+) groups (20.7 ± 1.4 vs. 20.2 ±

1.7). HUT(+) subjects were significantly older than controls subjects (15.7 ± 0.6 vs. 13.1

± 1.0 years, p = 0.02).

Hemodynamic and Autonomic measurements

There were no differences between groups for supine measurements of mean arterial pressure (MAP) or HR (Figure 1). HUT(+) and control subjects had significant increases in MAP upon standing (p < 0.05, Figure 1A). Both groups also had increases in

HR upon standing with HUT(+) subjects having significantly higher HR during upright position than controls (p = 0.004, Figure 1B).

A B Mean Arterial Pressure Heart Rate 150 150 ### **** control 100 * * 100 ** HUT(+)

mm Hg 50 50 beats / minute 0 0 Supine HUT Supine HUT

Figure 1. Hemodynamic responses to upright challenge in control and HUT(+) subjects. (A) Both control and HUT(+) groups had significant increases in MAP upon standing. (B) HUT(+) had an exaggerated increase in HR upon standing. HUT(+) subjects had significantly higher HR than controls during HUT. (mm Hg, millimeters of Mercury) [gray bars = HUT(+); white bars = controls] [Two-way ANOVA; Supine vs standing values, p<0.05*, p<0.01**; p<0.0001****; controls vs HUT(+), p<0.001###].

146 At baseline there were no differences between groups for autonomic parameters of BRS, HRV, BPV, PVT, SVT, or SVB (Figure 2). HUT(+) subjects had a significant decrease in BRS (all sequences, p < 0.01) and a trend was observed for group differences

(p = 0.07). Control subjects had an expected decrease in BRS that did not reach statistical significance (Figure 2A). For HRV as measured by SDNN, HUT(+) subjects had a significant decrease from supine to standing which was also significantly lower than

HRV for controls (Figure 2B). There were no group differences in supine and upright measures of BPV, but HUT(+) subjects had a significant increase upon standing (Figure

2C). HUT(+) subjects also had a significant reduction in PVT, as measured by HFα, upon standing that was significantly lower than controls during upright position (Figure 2D).

There were no differences between groups at either position for SVT, as measured by

LFα, and HUT(+) subjects had a significant reduction upon standing (Figure 2E).

HUT(+) subjects had a significant increase in SVB upon standing that was significantly higher than control subjects (Figure 2F).

147 A B Baroreflex Sensitivity Heart Rate Variability 30 100 ## control 80 20 HUT(+) 60

40 * Seq all 10

SDNN (ms) 20

(ms / mm Hg) ** 0 0 Supine HUT Supine HUT C D Blood Pressure Variability Parasympathetic Vagal Tone 8 40

6 30 ##

* α 4 20 HF

2 10

(ms / mm Hg) ** SDMAP (mm Hg) 0 0 Supine HUT Supine HUT E F Sympathetic and Vagal Tone Sympathovagal Balance 40 # 10 ** 30 8

α 6 20 LF 4 10 ** LF %/HF % 2 (ms / mmHg) (ms / mm Hg) 0 0 Supine Standing Supine Standing

Figure 2. Expected autonomic responses to upright challenge in control and HUT(+) subjects. (A) HUT(+) subjects had a significant reduction in BRS from supine to standing. (B) HUT(+) had a significantly greater reduction in HRV from supine to standing. (C) There were no differences between groups during supine or standing positions for BPV, although there was a trend for group differences during standing (p=0.07). HUT(+) subjects had a significant increase in BPV upon standing. (D) HUT(+) had a significant reduction in parasympathetic tone (HFα) upon standing that was significantly lower than controls during upright position. (E) For LFα, there were no difference between groups at either position, but HUT(+) subjects had a significant reduction upon standing. (F) HUT(+) subjects had a significant increase in sympathovagal balance upon standing that was significantly higher than control subjects. [gray bars = HUT(+); white bars = controls] [Two-way ANOVA; Supine vs HUT values, p<0.05*, p<0.01**; control vs HUT(+), p<0.05#, p<0.01##].

148 Supine metabolite measurements

Metabolite concentrations (millimolar) for control and HUT(+) subjects collected

in supine position and were not significantly different between groups are reported in

Table 1. Trends were observed for lower NAA and GSH in HUT(+) subjects (p = 0.1).

HUT(+) subjects had significantly higher mIns and tCho in the DM than control subjects

(Figure 3A-B). Representative DM spectral scans from one control and one HUT(+)

subject are displayed in Figure 4.

Metabolite/Transmitter Control HUT(+) p-value

Alanine 1.9 ± 0.08 (3) 2.6 ± 0.4 (6) 0.13

Creatine (tCr, Cr + PCr) 4.4 ± 0.7 (7) 5.4 ± 0.2 (11) 0.14

Glutamate (Glu) 4.9 ± 0.6 (5) 5.4 ± 0.6 (9) 0.64

Glutamine (Gln) 3.3 ± 0.7 (3) 3.5 ± 0.2 (7) 0.73

Glu+Gln 6.2 ± 1.2 (6) 7.6 ± 0.7 (11) 0.27

Glutathione (GSH) 4.2 ± 2.1 (3) 2.0 ± 0.2 (8) 0.10

N-acetyl aspartate (NAA) 3.9 ± 0.5 (3) 3.0 ± 0.2 (7) 0.10

NAA-Glutamate (NAAG) 4.9 ± 0.8 (7) 5.4 ± 0.3 (11) 0.60

NAA+NAAG 6.7 ± 1.0 (7) 7.4 ± 0.2 (11) 0.39

Table 1. Supine Metabolite and Neurotransmitter concentrations. All concentrations reported in millimolar (mM) followed by n’s in parentheses for each measurement per group. (p<0.05*)

149 A B Myoinositol Total Choline 10 2.5 * 8 * 2.0

6 1.5 mM mM 4 1.0

2 0.5

0 0.0 control HUT(+) control HUT(+)

Figure 3. Myoinositol (A) and total Choline (B) were significantly higher in the dorsal medulla of HUT(+) subjects. All concentrations reported in millimolar (mM). [Controls, n = 7; HUT(+), n = 11; p<0.05*]

Figure 4. 1H-MRS spectra, acquired from the dorsal medulla in a control (age 16, male) and HUT(+) (age 16, female) subject, showing mIns, Cho, Cr, NAA metabolites. Data collected by Siemens 3T scanner and analyzed via Siemens 3T Skyra to display these spectra. (ppm, parts per million)

150 Regression analysis and CSA of age, metabolites, and autonomic parameters

An important goal for this study was to determine whether there was a relationship between metabolite concentrations and autonomic parameters, as previously shown in a rat model of hypertension33 and in humans13. We used DCS, a type of correlation summation analysis, to understand the relative degree of correlation among variables between control and HUT(+) groups. Regression maps compare age, BMI, and supine measurements of mIns, tCho, Glu, NAA+NAAG, tCr metabolites to supine

(Figure 5A) and standing (Figure 5B) autonomic measures separately to prevent overlapping associations between the autonomic parameters at the different time points.

These regression maps illustrate the concept behind DCS by grouping correlations to the upper left quadrant sorted by largest to smallest DCSx score.

No specific clustering was observed when comparing age, BMI, supine autonomic parameters and supine metabolites (Figure 5A). Expected correlations between supine autonomic parameters (PVT and HRV, BRS, ST, and SVB) were identified by DCS.

Positive correlations between metabolites were observed between mIns and tCho, as well as tCr compared to mIns, tCho, and NAA+NAAG. Age had an inverse relationship with

SDMAP and HR and positive correlation with BMI, but no relationships were observed between age and metabolites.

The regression map for standing autonomic measurements, age, BMI, and supine metabolite concentrations illustrated more distinct clustering (Figure 5B). PVT and BRS in upright position had the largest DCSx scores demonstrating the greatest covariance compared to other variables between groups. Expected correlations were also observed between PVT, BRS, SVB, and SVT. DCS also revealed significant negative correlations

151 with PVT and BRS during upright position compared to both mIns and tCho. Thus, the differences in mIns and tCho concentrations in the DM are associated with upright PVT and BRS. Age had an inverse relationship between upright BPV.

Multiple regression analysis was used to evaluate the strength of autonomic and metabolite variables in a linear model controlling for age and gender. Analysis was limited to the top 2 autonomic variables and metabolites, ranked by DCS. When controlling for age and gender, mIns was negatively associated with upright BRS

(F(4,10) = 4.22, p = 0.03) and tCho was negatively associated with upright PVT (F(4,10)

= 3.1, p = 0.04). Thus, even though age was significantly different between control and

HUT(+) groups, it does not account for the differences observed in metabolites, BRS, or

PVT.

152

A

B

Figure 5. Regression maps of the correlation summation for the descriptive data. (A) Regression map for age, BMI, supine metabolites and supine autonomic measurements. (B) Regression map for age, BMI, supine metabolites, and autonomic measurements collected in standing position. [All shaded areas are p < 0.05 with the variables sorted from left to right and top to bottom according to ranking for the discrete correlate summation. Each box has a “-“ or “+” indicating the direction of the significant correlation between variables. There are three shades indicated in the regression map, the darkest (black) indicates the correlation has a p < 0.001, the medium shade of gray indicates p <0.01, and the lightest gray indicates p < 0.05.]

153 Discussion

The objective of this study was to define brain metabolite profiles in children with dysautomia manifesting as OI and investigate the relationship between these profiles and autonomic function in supine and upright positions. Using 1H-MRS, significantly higher mIns and tCho, markers of glial inflammation and myelin degradation, were observed in

HUT(+) subjects. As expected, HUT(+) subjects had impaired autonomic responses to standing including significant decreases in BRS, HRV, and PVT from supine to upright position. Although no associations were observed in supine autonomic measurements, upright BRS and PVT had inverse relationships with medullary concentrations of mIns and tCho, suggesting glial inflammation and/or myelin degradation in the DM may contribute to an underlying mechanism of autonomic dysfunction in children with OI.

Our study is the first report of altered brain metabolites in children with OI and first report to associate orthostatically-induced autonomic dysfunction to biomarkers of neuroinflammation.

Autonomic regulation upon orthostatic challenge is known to be impaired in adults and children with OI35-38. In this study, control subjects demonstrated normal autonomic responses to standing while autonomic responses were impaired in HUT(+) subjects. Both HUT(-) and HUT(+) groups had similar increases in MAP, but HUT(+) subjects had significantly higher HR in upright position. This is most likely due to

HUT(+) subjects primarily presenting with postural orthostatic tachycardia syndrome

(POTS), which is characterized by increases in HR ≥40 BPM or HR ≥120BPM within 10 minutes of standing25, 26. HUT(+) subjects also had significant decreases in BRS, HRV,

SVT, and PVT and significant increases in BPV and SVB from supine to standing. While

154 decreases in both SVT and PVT were observed, SVB was significantly higher in HUT(+) subjects highlighting a clear imbalance in sympathovagal activity. This may have occurred because the measure of SVT (LFα) employed is also in part representative of vagal tone39, 40 and the decrease observed could be attributed to the impaired vagal response. In future studies, employing direct measurements of sympathetic activity, such as microneurography41, 42, could elucidate the impact of sympathetic impairments during upright challenge more specifically. Autonomic impairments observed were characteristic of a positive HUT test and were expected in HUT(+) subjects, as this is how they are diagnosed with OI.

1H-MRS has not previously been used to evaluate metabolite concentrations in the medulla of children and few adult 1H-MRS studies have measured metabolites in brainstem regions13, 15, 43, 44. The DM is a major hub of autonomic nuclei responsible for blood pressure regulation6, 45 and was the target brain region in the current study. In this brain region, tCho and mIns were the only two metabolites significantly higher in

HUT(+) versus control subjects. A recent study in adults with MSA, a neurodegenerative disorder effecting autonomic function and movement, investigated metabolite differences within the entire pons and medulla respectively. NAA and tCho were significantly lower and mIns was higher in the medulla and pons. While higher mIns/Cr in the pons associated with symptom severity measured by the unified MSA rating scale, no direct physiological measurements were collected13.

Choline-containing metabolites are present in all brain cell membranes and serve as markers of structural integrity as they are involved directly in phospholipid synthesis and degradation reflecting cell proliferation. These compounds can be detected by 1H-

155 MRS with concentrations presenting 2-3 fold higher in glia than in neurons46-48. Elevated tCho is associated with ischemic damage to myelin as well as glial inflammation21, 22.

HUT(+) subjects had significantly higher tCho than control subjects, of which concentrations were consistent with previous reports of tCho in healthy children ages 5-

18 years old24. Our finding differs from the MSA cohort where tCho was significantly lower in the pons and medulla. However, MSA subjects also had lower NAA, a marker of neuronal integrity and density, implying there may be fewer healthy neurons in this brain region13. In the current study, a trend was observed for lower NAA. The sample size for

NAA was lower than mIns and tCho and most likely reflects lower statistical power.

While our hypothesis was not confirmed in this study, the trend for lower NAA is consistent with previous reports of lower NAA concentrations associating with impaired

BRS in adults. Lower NAA in children with OI may indicate impaired neuronal function, which could result in increased glial activation and myelin degradation. Conducting these studies in a larger cohort may help determine if NAA concentrations in children with OI fit the metabolite profiles of MSA and other disorders previously described in the literature. Future 1H-MRS studies should also investigate the relationship between NAA and tCho, especially in children, as there could also be an age dependent ratio of these two metabolites.

Myoinositol is an osmolyte found only in glia and astrocytes and its primary role is to maintain glial cell volumes46. When elevations are detected by 1H-MRS, mIns serves as a marker of glial activation indicative of inflammation49. Significantly higher mIns was observed in the DM of HUT(+) subjects. Elevated cerebral mIns has been related to systemic inflammation, as measured by C-reactive protein50, as well as in dyslipidemia

156 and metabolic syndrome in adults51, 52. Since glial inflammation in the brain is known to be neurotoxic, mIns has been proposed as a biomarker of neurological disorders associated with neuroinflammation including Alzheimer’s14, 53 and a marker for cognitive vulnerability in cognitively normal adults51, 52. Elevated mIns has not been reported in children and adolescents so it is unclear whether it could be an early marker for cognitive decline in that population. However, as demonstrated in our findings, inflammation in the

DM could play a role in the autonomic impairments observed in OI.

After defining metabolite profiles in OI, we aimed to investigate relationships between autonomic function during orthostasis and supine metabolites. DCS was first employed to identify autonomic and metabolite variables with the greatest correlation shift relative to the mean differences observed between groups. Measures of BRS and

PVT during standing had the largest amount of covariance detected between control and

HUT(+) groups and negatively correlated with supine tCho and mIns. The relationships between those variables were explored using MRA. Once corrected for age and gender, tCho had an inverse relationship with upright PVT and mIns had an inverse relationship with BRS in upright position. This suggests the effect of inflammation, as indicated by elevated tCho and mIns, may be specifically contributing to the impaired BRS and PVT occurring in upright position in HUT(+) subjects.

In addition to modulating parasympathetic and sympathetic activity to the heart, the vagus nerve plays an important role in regulating inflammation and prevention of tissue damage54, 55. Inflammation is a well-established component of autonomic disorder pathology in metabolic syndrome56, 57 and hypertension58-61. Recently adults with orthostatic hypotension, a subtype of OI, were shown to have increased concentrations of

157 fibrinogen, a marker of systemic inflammation, and carotid artery IMT62. Although this study only considered subjects with OH, it suggests irregular blood pressure regulation during orthostasis is associated with increased systemic inflammation.

Anti-inflammatory properties mediated by vagal activity have been previously reported with many studies showing inverse relationships between vagal function and inflammatory markers of CRP and IL-663-69. Alternatively, activated glia produce pro- inflammatory cytokines that can directly and indirectly modulate neuronal activities70.

Since appropriate baroreflex and parasympathetic responses correlate with lower levels of inflammatory markers, the impaired autonomic pathology seen in our study could suggest decreased inhibition of inflammation resulting in increased markers of glial activation

(tCho and mIns). Alternatively, increased glial inflammation in the DM may induce greater actions of pro-inflammatory cytokines modulating vagal activity to the heart and potentially exacerbating abnormal autonomic actions71. While HUT(+) subjects in the current study presented with high concentrations of metabolites indicating inflammation and impaired autonomic activity during standing, the direction of causality remains unclear and should be tested in future studies. Since trends were observed for lower NAA and GSH, these metabolites should be evaluated in larger cohorts to determine the role of neuronal integrity (NAA) and antioxidants (GSH) in children with OI as well as mIns and tCho. If NAA is lower in the medulla, as shown in MSA, decreased neuronal integrity could also induce glial activation resulting in higher concentrations of mIns.

This study has limitations that should be considered before future studies are conducted. First, we recruited subjects with OI symptoms for HUT testing which resulted in HUT(+) patients with different subtypes of OI being grouped together. Also, the

158 overall sample size was small which limited our ability to do statistical analysis considering subgroups. Future studies should aim to recruit a larger number of subjects for HUT(+) and control groups so metabolite profiles for specific OI subcategories can be explored. Although our age range was small (10-18 years), there was a significant difference in ages of control and HUT(+) groups. Age did not correlate with any metabolite or autonomic parameters that were significantly different between groups, but age-matched controls are necessary for validation of our results in this age range of children. There are also limitations considering 1H-MRS approaches. The effects of physiological motion due to respiration may have impacted our recordings from the dorsal medulla and measures of metabolites and transmitters know to have roles in the

DM were unable to be measured. Although water suppression improved spectral quality, the SD limitation was 50% and lower % SD should be targeted in studies with larger sample sizes. In addition to the DM, future studies could target other central areas of autonomic function including the insular cortex and paraventricular nucleus of the hypothalamus.

In conclusion, this study demonstrated the utility of 1H-MRS evaluation of the

DM and provides evidence for neuroinflammation associated with autonomic function in children with OI. While causality could not be established in our study, there is a relationship between glial inflammation in the DM and CV impairments observed in

HUT(+) subjects. Given that supine mIns and tCho were predictive of impaired PVT and

BRS responses to tilt, 1H-MRS methods could be used as an alternative to tilt when testing treatment options for children with OI. Furthermore, autonomic dysfunction observed while standing may implicate inflammation as an underlying mechanism of the

159 CV pathology of OI. Although it is unclear whether high mIns and tCho represent chronic or acute inflammation, relationships between autonomic function and metabolites observed provide preliminary data that should be evaluated in larger cohorts of children with multiple subtypes of OI and asymptomatic controls.

16 0 Acknowledgements

The authors thank Anya Brown and the MRI Center at Wake Forest Baptist Health.

Sources of Funding

Support for this project was provided by Centers for Integrative Medicine and

Hypertension & Vascular Research at Wake Forest Baptist Health, the American Heart

Association (AHA12CRP9420029) and the Farley Hudson Foundation.

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167 CHAPTER FIVE

SUMMARY AND CONCLUSIONS

1. Summary of Results

Autonomic dysfunction manifesting as OI affects individuals of all ages and is associated with development of severe neurodegenerative diseases1, 2 as well as cardiovascular disease including hypertension in adults3. In recent years greater acknowledgement of this disorder by physicians has led to increased diagnosis with reports indicating as many as 3 million adults and children have dysautonomia with OI.

OI is characterized by various autonomic response patterns to standing, which lead to complex and heterogeneous phenotypes4-6. While the underlying cause of OI is difficult to determine, characterization of adults with specific OI subtypes has provided a basis for better diagnosis, management, and treatment. However, it is unknown whether the persistence of OI symptoms and autonomic dysfunction from childhood will lead to hypertension, multiple system atrophy, or other complex disorders in adulthood.

In addition to irregular cardiovascular responses, OI is accompanied by chronic

GI symptoms of abdominal pain and nausea, cognitive impairments associated with executive function and visual disturbances, and psychosomatic symptoms of anxiety and increased stress6-10. Moreover, all of these symptoms are associated with autonomic imbalances independent of BP regulation, but it is unknown whether orthostasis is the driving factor of these symptoms. Thus, identification of factors that may influence autonomic control of BP and HR during standing is imperative to advance our

168 understanding of OI and its risk factors so that more effective pharmacotherapies can be developed.

The neurohumoral system plays a critical role in the modulation of BP and blood volume under normal conditions and during orthostatic challenge11. Although they have many non-reflex functions, exaggerated responses in RAS, catecholamines, and AVP are observed in adults with OI during upright position12-16. Different neurohumoral responses to standing have been documented in all subtypes of OI in adults and used to provide evidence of underlying mechanisms of the disorder and for better treatment options.

However, neurohumoral profiles have not been described in pediatric populations of OI.

In Chapter Two, we used a unique methodology to obtain a comprehensive panel of neurohormones and define neurohumoral biomarker profiles in children with OI. Data from this study demonstrated distinct neurohumoral responses during HUT associated with the specific hemodynamic changes upon standing, coinciding with OI subtypes. Our findings in POTS subjects were consistent with the hyperadrenergic state observed in adults13,17. However, we report new evidence of impaired RAS-associated vasoconstrictor release and exaggerated AVP release upon standing in OH and syncope subjects. These findings underscore the distinct nature of HUT(+) subgroups and provide evidence for more specific treatment strategies based on individual group characteristics.

Chronic nausea is a common symptom in adults and children and presents as OI in as much as 80% of affected individuals18. During orthostasis, abnormal gastric rhythms occur in OI subjects, which can elicit nausea and changes in GI motility19-21.

Interestingly, neurohormones involved in regulation of BP also act on central nervous system sites associated with GI function and presentation of nausea and abdominal pain22,

169 23. In Chapter Three we report the evaluation of the impact of HUT on the experience of nausea and associated neurohumoral responses in children with chronic nausea and at least one symptom of OI. We also evaluated the usefulness of the Nausea Profile questionnaire validated in adults24 in predicting autonomic dysfunction on HUT in these children. This study was the first to demonstrate abnormal neurohumoral changes to orthostatic challenge contemporaneously with HUT-induced nausea in children.

Specifically, AVP was significantly higher in subjects with HUT-induced nausea and the highest in subjects with a positive HUT and nausea during HUT. Thus, more than one mechanism may be at play in this group of patients with heterogeneous presentations of

OI and nausea. The data support the necessity of collecting more comprehensive patient history and suggests a multidisciplinary approach will aid in diagnosis and management of nausea and OI, respectively.

Blood pressure regulation is impaired upon standing in children with OI and is associated with exaggerated neurohormones in the circulation in response to standing, as shown in Chapters 2 and 3. However, baroreflex regulation for HR and BP control, as well as release and subsequent actions of neurohormones, involve central mechanisms occurring in cardioregulatory centers, including the dorsal medulla. Previous studies have utilized non-invasive MRS to evaluate neurochemical alterations in specific brain areas associated with autonomic function, including the medulla, pons25, and insular cortex26, to provide insights into whether abnormal central regulation accompanies diseases such as diabetes27, Parkinsonism28 or heart failure26. In Chapter Four, we used MRS to determine brain metabolite profiles in the dorsal medulla of children with OI and the data reveal evidence of inflammation and myelin degradation. Elevated mIns and tCho were

170 predictive of impaired HRV and BRS responses during upright position, providing novel evidence of neuroinflammation in the brain associated with impaired vagal function observed in OI.

Collectively these studies demonstrate a novel approach to diagnosis and management of OI and reveal potential biomarkers for autonomic dysfunction upon standing. The importance of understanding autonomic dysfunction in children is emphasized by the impaired quality of life experienced by these individuals, and the various serious disorders that are associated with OI in adulthood. Our data were collected in a non-invasive manner, which is especially important for future studies in children with OI. Therefore, we conclude that the non-invasive collection of neurohumoral and metabolite profiles may represent an attractive approach for early diagnosis and evaluation of effective treatment options for children with OI and comorbid

GI symptoms.

171 2. HUT-induced hemodynamic and autonomic changes in OI and nausea

Orthostasis is associated with decreases in BP at the level of the high-pressure baroreceptors and decreases in blood volume at the level of the low-pressure baroreceptors as a result of increased blood flow below the diaphragm. Initial venous pooling and decreased arterial pressure is immediately followed by increased sympathetic activation to the heart that increases HR and to blood vessels that increases vascular resistance. In healthy subjects BRS and HRV, indices of vagal function, decrease initially and then rapidly compensate for the venous pooling bringing BP to the upper body back to normotensive conditions29, 30. However, subjects with OI have significantly greater reductions in BRS and HRV upon standing, which lead to increased BPV and overall impaired regulation of BP. These impairments persist until individuals return to supine position31. To date, the mechanisms underlying baroreflex dysfunction and impaired autonomic outflow associated with OI are poorly understood.

Autonomic dysfunction manifesting as OI can present in a variety of hemodynamic patterns. Subjects with OI can generally be sorted into one of three categories: postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension

(OH), and syncope4-6. Preliminary data collected in our laboratory revealed a high incidence of children presenting in the pediatric GI clinic with chronic nausea who tested positive on a HUT for OI. Additionally, children with POTS had greater MAP during supine position when compared to HUT(-) subjects suggesting that children with POTS may be at risk for developing hypertension in adulthood32. One of our goals was to confirm the prevalence of OI in chronically nauseous children and another was to

172 evaluate the usefulness of chronic nausea and OI symptoms as predictors of autonomic instability.

Consistent with our previous studies18, 19, 63% of subjects presenting with chronic nausea and symptoms of OI tested positive on a tilt table test [HUT(+)] (Chapter Two).

As a group these subjects had significantly higher HR and lower BP upon standing.

Specifically, SBP and DBP were significantly lower during HUT, with no observable change in DBP occurring for HUT(+) subjects, as opposed to the expected increase in

DBP reflecting the increased vascular tone that occurs in normal subjects. HUT(+) subjects exhibited heterogeneous hemodynamic responses resulting in four OI phenotypes: POTS, OH, syncope and POTS/Syncope (P/S). Expected hemodynamic patterns were observed for POTS, OH, and syncope, as this is how children are typically diagnosed. However, children with chronic nausea present with multiple types of OI and must undergo HUT to be diagnosed.

The phenotype of P/S was unexpected as it is not a traditional diagnosis of OI.

Subjects who met the criteria for POTS presented with tachycardia within the first 10 minutes of HUT and the majority were able to sustain upright posture for the full 45 minute HUT. In contrast, P/S subjects presented with tachycardia in the first 10 minutes followed by a syncopal episode at an average time point of 25 minutes into tilt. Our data show that SBP and DBP did not change from supine to HUT in P/S subjects.

Furthermore, HR was significantly lower in subjects with P/S compared to POTS alone and BP was higher in P/S than in OH subjects. A combination of POTS and syncope on

HUT has been described in the literature33, but has not been investigated empirically to delineate the combinatorial response from POTS, syncope, or OH alone. Since HUT was

173 terminated earlier than 45 minutes if subjects experienced severe symptoms and requested for it to end or if presyncope/syncope occurred, some POTS subjects with severe symptoms may have experienced syncope later during HUT if they remained on

HUT. Alternatively, POTS subjects without syncope may have a more intact vasoconstrictor and cardiac response, which allows them to remain in upright position without losing consciousness. When syncope occurs following POTS it suggests that the tachycardia in response to an impaired baroreflex is not enough to compensate for decreased BP for long periods of time. Thus, it is possible that these subjects present with a novel form of OI and future studies should consider this phenotype when evaluating patients.

In Chapter Three we measured hemodynamic and autonomic responses associated with HUT-induced nausea in children to evaluate the impact of orthostasis on the experience of nausea. Of the 48 subjects, 64.5% experienced nausea at some point during the 45 minute HUT. To more closely correlate nausea experienced to timing of blood sampling and cardiovascular changes during HUT, we limited our responder group to patients who experienced nausea before 15 minutes of HUT. This resulted in 42% of subjects experiencing HUT-induced nausea (responders). In contrast to results in Chapter

Two, hemodynamic responses in children with HUT-induced nausea did not differ from those without nausea on HUT. The absence of hemodynamic differences was likely because responder and non-responder groups were composed of both HUT(-) and

HUT(+) subjects. Thus, OI-associated cardiovascular symptoms that meet the definition of HUT(+) accompany nausea in only 29% of the subjects. Another dissociation between

HUT-induced nausea and cardiovascular symptoms is notable given that OI

174 cardiovascular responses are defined within 15 minutes of tilt and a greater percentage of subjects experienced nausea at time points later in the HUT test. This is interpreted to indicate that GI-associated symptoms of OI may take longer to manifest than cardiovascular responses and should be taken into account for future studies evaluating orthostatically-induced nausea. Whether the GI symptoms are a secondary to the prior altered cardiovascular responses in the OI subjects also displaying nausea on tilt, or are a distinct response is not possible to discern in our study.

We also separated subjects based on the combination of OI status and nausea response to evaluate the contribution of OI to nausea. As expected, hemodynamic differences were dependent on the OI status independent of nausea on HUT where

HUT(+) groups, regardless of nausea, had significantly lower BP during HUT than

HUT(-) subjects. Furthermore, when we considered OI subgroups, we observed that each subtype was composed ~50/50 with responders and non-responders. Our findings are consistent with a recent study by Chelimsky and colleagues34, which reported symptoms associated with OI, including chronic nausea, are prevalent in children with and without a

HUT(+) and evaluation on tilt is necessary for proper diagnosis in addition to extensive medical history of symptoms.

Previous studies have shown increased sympathetic and decreased parasympathetic activity are associated with nausea35, 36. Measuring continuous HR and

BP during our studies enabled us to calculate indices of BRS, HRV, parasympathetic tone

(PVT), and sympathovagal balance (SVB) during supine and upright positions as well as changes from supine to HUT. In contrast to previous studies, no significant autonomic differences were observed between responder and non-responder groups. However, our

175 study is the first to use HUT to induce nausea. Previous studies used visual-vestibular mismatch stimuli and motion sickness to induce nausea in healthy patients35-38, which may induce nausea by a different mechanism. Nevertheless, increased sympathetic activity is known to impact GI motility and cause gastric dysrhythmias leading to the experience of nausea, but this was not specifically observed in our study. Interestingly

Nausea Profile scores representing all three domains of nausea (GI, emotional, somatic) had a negative relationship with upright measurements of BRS and HRV. Although there were no differences between groups for autonomic parameters and Nausea Profile scores, this relationship suggests that a questionnaire to evaluate symptoms may aid in predicting autonomic impairments in larger cohorts. Since subjects in our study presented with nausea and no observable changes in autonomic function within 15 minutes of standing, observing autonomic changes at the exact moment of nausea may be more effective for evaluating the specific contribution of autonomic function in nausea during orthostasis.

In Chapter Four, HUT was used to diagnose OI and a standing challenge for 10 minutes was used to verify healthy autonomic function in HUT(-) subjects. In this study,

HUT(-) subjects were true controls with no accompanying secondary symptoms such as chronic nausea or OI symptoms. HUT(+) subjects had significantly higher HR and SVB than HUT(-) subjects as well as significantly lower HRV, PVT, and SVT. HUT(+) subjects also had a significant decrease in BRS from supine to HUT, which was not observed in HUT(-) subjects. All of these differences were expected as OI is characterized by autonomic impairments during upright challenge. However, in this study

HUT(+) subjects were slightly (~2 years), but significantly, older than HUT(-) subjects.

While it is well established that BRS decreases with the aging process over larger age

176 ranges, the issue in the current study is whether the slightly older age related to differences in Tanner stage (an indicator of puberty) and whether the age difference had an impact on the indices of autonomic function measured in our study39-42. Since OI is associated with autonomic imbalances and HUT(+) were older in this study, correlative and multiple regression analyses were used to show that age did not contribute to these differences. Furthermore, when controlling for age or gender the associations between upright BRS and HRV and brain metabolites were not affected.

It is imperative that future studies use continuous measurements of HR and BP to calculate autonomic function in supine and HUT positions along with notations about movement or external factors that may effect recordings. Expected hemodynamic and autonomic changes were observed considering HUT(+) subjects, but the 42% of subjects with HUT-induced nausea did not demonstrate altered autonomic function compared to those without nausea. While we used the average HR and BP in the 2 minutes prior to syncopal episode or blood draw for neurohormones, these measurements did not always coincide with the timing of nausea experienced on HUT and future studies should aim to match the time of nausea with measures of autonomic performance. In addition, a 45- minute tilt was useful in eliciting novel hemodynamic changes and an increase in the proportion of children experiencing nausea, which would not have been observed if the

HUT ended at the recommended time necessary for development of traditional cardiovascular symptoms. Thus, when investigating children with GI symptoms on HUT, a longer duration should be used for more accurate evaluation.

177 3. Neurohumoral compensation for impaired blood pressure control

The heterogeneous nature of OI subgroups suggests that the impairments of BP control cannot be localized to one specific point of origin and other factors are most likely involved in the presentation of symptoms. While as a group HUT(+) subjects had lower BRS and HRV upon standing, there were also alterations in humoral factors involved in cardiovascular regulation, including angiotensin peptides, arginine vasopressin, Aldo, and catecholamines that may contribute to the autonomic impairments observed in OI patients during HUT. In fact, studies consistently show altered circulating neurohumoral concentrations at supine and after upright challenge in adults with POTS, syncope, and OH, respectively12, 13, 15, 43. Although OI is under-recognized in children, they have also been shown to present with similar patterns of autonomic instability18, 44-46.

In Chapter Two we aimed to define neurohumoral phenotypes in children, hypothesizing that children with OI undergoing HUT would demonstrate neurohumoral responses in association with specific patterns of changes in BP and HR.

To date, neurohumoral profiling has been conducted using blood samples collected at supine position or after 30 minutes of standing12, 15, 47-49. Our study utilized novel time sampling to more accurately reflect neurohumoral changes in response to cardiovascular instability on HUT by collecting blood samples no later than 15 minutes into HUT. Chapter Two described distinct neurohumoral changes that track with hemodynamic profiles of each OI subtype. These neurohumoral mechanisms are exaggerated in various instances, but are not successfully compensating for the hemodynamic impairments during HUT.

178 Across all of the subtypes of OI, neurohumoral characterisitics of POTS have been the most studied. Adults with POTS have presented with elevated NE in supine position and exaggerated increases in NE after upright HUT, consistent with a hyperadrenergic state17. Additionally, POTS subjects with low blood volume have elevated Ang II and low plasma renin activity and Aldo16, 50, 51. However, these studies did not collect samples during HUT when the hemodynamic impairments were occurring.

Since tachycardia in POTS occurs within the first 10 minutes6, collecting blood samples after a 30-minute HUT would not fully capture the neurohumoral response when the cardiovascular instability is actually occurring.

In the current studies, children presenting with POTS had significant elevations in NE, Epi, and Ang II from supine to HUT. NE was the only component significantly higher in POTS subjects during HUT relative to all other OI groups, which showed comparable increases in Epi and Ang II. AVP, renin, Aldo, and Ang-(1-7) did not increase significantly in POTS subjects. Since NE is a marker of sympathetic activation, exaggerated increases in POTS indicate overactivation of the sympathetic system working to compensate for the decreases in BP upon standing. However, once BP reaches a steady state, HR remains at high rates, which implicate sympathetic activation in the genesis of trademark symptoms of OI/POTS including extreme sweating, dizziness, and nausea.

As expected, NE during HUT position positively correlated with HR in HUT position considering all 48 subjects and HUT(+) subjects alone. Interestingly, NE also inversely correlated with Ang-(1-7) in supine and HUT positions. Since Ang-(1-7) is known to modulate NE release43, 52-54, this raises the question of the role in Ang-(1-

179 7)/Ang II balance in POTS. Even though Ang II was not significantly higher in POTS than other groups, there was a significant increase in Ang II with standing while Ang-(1-

7) did not change. This could be a result of decreased ACE2 activity to convert Ang II to

Ang-(1-7) in POTS, which has been shown previously and thought to contribute to imbalance of angiotensin peptides in POTS55. Furthermore, Ang-(1-7) may increase NO bioavailability necessary for proper vasodilation, which is decreased in POTS as well51.

Since Ang II did not significantly increase in POTS subjects, it is unclear whether NE release alone is the responsible mechanism for the vasoconstriction in POTS. Thus, prevention of symptoms during orthostasis via Ang-(1-7) supplementation could ultimately cause syncope or hypotension as opposed to tachycardia.

We did not collect samples to measure blood volume in our study so our POTS group may be heterogeneous in that regard. Previous studies have tried to address the conundrum of low Aldo and renin in POTS subjects with higher Ang II, but have not measured AVP as an index of volume retention16, 50, 51. We showed that AVP does not increase upon standing in POTS. Since increased Ang II normally triggers release of

AVP and Aldo56-58, the absence of increases in both suggest the majority of our subjects do not have low blood volume upon standing in the face of exaggerated sympathetic activation. Moreover, Ang II is the only additional neurohormone that could be contributing to vasoconstriction and to elevate blood volume in POTS because AVP and

Aldo did not increase upon standing.

Treatments for POTS have targeted mechanisms of blood volume expansion as well as adrenergic receptor actions to improve symptoms. Interestingly, desmopressin which is a synthetic version of AVP, was used to treat POTS and showed to attenuate

180 tachycardia and improved symptoms of POTS59. Of note, this particular study did not describe the blood volume status of POTS subjects testing. Aldo agonist treatment with fludrocortisone is another traditional treatment for children and adults with OI18, 60. While it has shown suboptimal efficacy in studies evaluating multiple subtypes of OI, it may prove to be more effective in POTS as it promotes volume expansion through Aldo mechanisms of action. In addition, copeptin, a peptide co-released with AVP, was recently shown to be lower in POTS subjects in supine position who responded to treatment with metoprolol (β-blocker) and was higher in subjects that responded to treatment with midodrine (α-agonist)61, 62. This study suggests that copeptin could serve as a predictor of the effectiveness of therapies such as metoprolol and midodrine62 treatments for POTS subjects with normal or high blood volumes. While we did not collect data to evaluate blood volume, POTS subjects in our study did not demonstrate elevated AVP at baseline or during HUT, consistent with reports of hyperadrenergic states. Our data confirm that treatments promoting volume expansion should be more effective in improving quality of life and attenuating negative symptoms of POTS in children, as seen in adults. Moreover, more studies are required to evaluate the safety of these treatments over the long term and to identify specific mechanisms involved in ameliorating primary and secondary symptoms of OI.

In contrast to POTS, subjects with OH have significantly lower BP and no significant increase in HR upon standing with no statistically significant increase in catecholamines. The lack of an increase in NE and Epi is consistent with previous studies suggesting impairments of baroreflex control of HR and sympathetic activation contribute to significant decreases in BP upon standing that do not correct until returned

181 to supine6, 63, 64. Additionally, two neurohormones, AVP and Aldo, showed characteristic profiles in the OH subjects in our study. AVP was significantly higher in OH subjects upon standing compared to HUT(-) and POTS subjects. In this case, AVP may be acting as a vasoconstrictor since other vasoconstrictive factors including catecholamines and

Ang II are not elevated. Given the failure to mount an increase in DBP upon standing in these subjects, any vasoconstriction provided by AVP is not increasing systemic vascular resistance sufficiently to maintain overall MAP. Interestingly, Aldo was significantly higher at supine position compared to HUT(-) and POTS subjects and higher in HUT position compared to HUT(-), POTS, and syncope subjects. This implies that OH subjects already have elevated Aldo to compensate for decreased BP, and may explain why fludrocortisone is reported to be ineffective in treating OH. Furthermore, elevated

Aldo at supine position is a risk factor for hypertension57, 65 and increases the likelihood that children with OH might develop this disorder earlier in life than the general population. Longitudinal studies are required to determine if this is the case.

Syncope results from loss consciousness due to decreased cerebral blood flow and overall decrease in HR and BP that, under normal circumstances, would elicit compensatory neurohumoral responses6, 66. In our study AVP, renin, and Ang II significantly increased upon standing in syncope subjects. These components were most likely acting to increase vasoconstriction systemically to maintain pressure and, therefore, blood flow to the brain. Consistent with previous reports67, 68, NE levels did not increase.

However, those studies demonstrated elevated Epi at baseline and immediately prior to syncopal episodes, which was not observed in our study. Because syncope did not always coincide with the timing of the blood draw, our measurements may not fully reflect the

182 neurohumoral profile in all syncope subjects. Thus, the sympathoadrenal imbalance reported previously was not observed in this study specifically. Future approaches should aim to collect additional blood samples immediately prior to syncopal episodes to obtain a greater understanding of the role of NE and Epi in syncope.

In congruence with OH subjects, AVP was significantly higher in syncope subjects compared to HUT(-) and POTS subjects. In both cases, AVP may prove to be the last ditch effort of the humoral system to maintain BP. However, over time AVP alone is not enough to prevent syncope. Hemodynamic presentations of OH and syncope initially resemble one another, but neurohumoral profiles in our study were able to discriminate between them as indicated by higher Aldo at all times in OH subjects.

Previous studies have shown increased endothelin-1 (ET-1) in OH subjects during supine position compared to those with syncope, providing evidence for use of this biomarker in discriminating between the two phenotypes prior to HUT12. We did not measure ET-1, which is a potent vasoconstrictor. ET-1 elevations in OH could contribute to their ability to maintain more effective cerebral perfusion to prevent syncope during standing. Again, overcompensation of vasoconstrictor peptide during supine position most likely contributes to the large swings in BPV observed in OH subjects and should be addressed by a physician for prevention of hypertension and other metabolite disorders associated with impaired BP regulation64.

The neurohumoral profile of P/S subjects resembled responses in POTS and syncope, but was less pronounced. For example, NE significantly increased in P/S subjects, but was still significantly lower than POTS subjects alone and comparable to

NE concentrations of syncope. Ang II also significantly increased in P/S subjects with

183 concentrations comparable to POTS subjects. These results suggest that P/S subjects are more reliant on Ang II-mediated vasoconstriction to maintain BP as opposed to sympathetic activation via NE. However, these alterations in neurohumoral responses do not prevent subjects from experiencing syncope later on during HUT. Thus, our findings suggest a novel hemodynamic and neurohumoral phenotype of a combinatorial presentation of POTS and syncope that requires additional compensatory mechanisms to maintain sufficient perfusion over a longer timeframe. Additional studies should aim to further define neurohumoral responses at multiple time points during HUT before pharmacotherapy recommendations are suggested.

The information obtained in these studies on neurohumoral and hemodynamic responses to HUT in children with or without chronic nausea can be used to identify more effective treatment strategies and improve quality of life by ameliorating secondary symptoms of OI. Based on these findings, we suggest that the efficacy of specific treatments applied to OI patients can be improved if they are based on their hemodynamic performance on HUT. However, more frequent blood sample collection is needed to accurately reflect neurohumoral profiles of patients with syncope and P/S as well as assessment of blood volume status, which may aid in interpretation of neurohumoral results.

184 4. Neurohumoral actions involved in chronic nausea

Autonomic function is not commonly assessed when considering functional GI disorders, as most patients would see a gastroenterologist for symptoms of chronic nausea who may not be familiar with comorbid symptoms of OI. However, HUT has been shown to evoke nausea in a high percentage of pediatric patients presenting with chronic nausea19. Emerging evidence also suggests comorbid symptoms occur with or without OI, but a HUT is still necessary to delineate symptoms34. Interestingly, neurohumoral components released during orthostatic challenge have actions beyond those of BP regulation to the heart. Vasopressin-69, AT-170, and adreno- receptors71, 72 are abundant in GI vasculature, mucosal, and muscular tissues as well as in brain sites responsible for emetic responses22, 23, 73. Thus, we hypothesized that neurohumoral responses to orthostasis would associate with the experience of nausea during HUT.

In Chapter Three, neurohumoral responses associated with HUT-induced nausea were described revealing comparable elevations in all neurohormones between groups except for AVP. Specifically, 40% of subjects experienced HUT-induced nausea within

15 minutes of HUT and exhibited a 40-fold increase in AVP from supine to HUT. For our groupings we limited the nausea experience to 15 minutes to more closely associate neurohumoral profile to blood sampling and nausea. MAP did not differ at supine or

HUT positions between responders and non-responders, suggesting that AVP release was more closely associated with nausea than BP regulation. Interestingly, 11 subjects experienced nausea after 15 minutes of HUT. When including those subjects in the responder group, the differences in AVP did not change. This is most likely due to the inability of the blood sampling to reflect nausea at a later time point.

185 A trend was observed for lower MAP in responders, which led us to evaluate the role of OI and nausea together to differentiate between groups. For these comparisons, the 48 subjects in the study were separated into 4 groups: HUT(+) responders, HUT(+) non-responders, HUT(-) responders, and HUT(-) non-responders. When considering the four groups, BP was significantly lower in both groups of HUT(+) subjects as compared with HUT(-) subjects. This approach allowed us to assess the independent contributions of HUT-induced nausea and a positive HUT on neurohormones. Interestingly AVP was significantly higher in HUT(+) responders than HUT(-) non-responders and comparable increases in AVP were observed in HUT(-) responders and HUT(+) non-responders.

Clearly, the combination of HUT-induced nausea and low BP had the greatest effect on

AVP release. However, AVP increased to concentrations higher than normal physiological levels (<2pg/mL)74 in patients with nausea and a negative HUT and non- responders with a positive HUT. Consistent with data presented in Chapter Two, subjects with a positive HUT and no nausea experience had elevated AVP upon standing. This evidence shows that AVP release is related to nausea, independent of OI status. Although the directionality of this relationship is unclear, AVP may be elevated during daily life in these subjects and serve as one mechanism underlying the nausea during upright posture.

As reviewed in Chapter One, AVP increases have long been associated with nausea experiences and low BP75, 76. However, our studies were the first to show the relationship between exaggerated AVP during orthostatically-induced nausea in children.

The role of AVP in inducing nausea in these subjects needs further study, as the specific mechanism is remains unclear. AVP release could be causing increased vasoconstriction in GI tissues that decreases GI tissue integrity and leads to increased ROS, as observed

186 with Ang II77, 78. Additionally, gastric dysrhythmias are elicited with AVP administration to produce nausea and vomiting regulated by the area postrema22, 23, 79. Ang II also acts on the area postrema, which sends projections to the NTS modulating baroreflex and respiratory function during emesis23. Although we did not see differences in Ang II between responders and non-responders, two-way ANOVA results showed significant group differences when those groups were separated by OI status. However, no significant group differences were found after post-hoc analysis. In addition to AVP and

Ang II, infusions of Epi have also been shown to elicit nausea and gastric dysrhythmias in healthy adults80. The patterns of Epi release observed were similar to AVP, with

HUT(+) responders having the highest concentrations of Epi, but were not significant.

Future studies comparing children with chronic nausea to asymptomatic controls may reveal more direct neurohumoral responses to HUT-induced nausea than our mixed groups.

It is clear that neurohumoral responses to orthostasis are associated with the induction of nausea during standing. However, it is unclear which factor occurs first, as low BP and nausea are both associated with AVP release and AVP can elicit nausea independent of changes in BP. Alternatively, AVP infusions demonstrate detrimental effects on GI motility that can cause nausea as well75, 80-82. Our study did not show causality between nausea and AVP, but provides a basis for using HUT to induce nausea emulating nausea experienced in daily life in the context of specific neurohumoral changes during standing. Ongoing studies are needed to investigate whether copeptin could be used as a predictive marker of nausea on HUT, given its longer half-life in the circulation that may reflect chronic increases in AVP.

187 5. The role of inflammation in autonomic dysfunction during orthostasis

In Chapter Four, we used 1H-MRS to measure brain metabolites that serve as markers of neuronal and glial integrity, inflammation, cell cycle turnover, and glutamate metabolism in the dorsal medulla. MRS is a non-invasive procedure conducted in an MRI scanner that is able to detect tissue concentrations of these brain markers of inflammation and cellular integrity that would otherwise be difficult to sample in human subjects83-87.

This method has previously been used to evaluate specific neurochemical differences in neurological and cardiovascular disorders with associated autonomic impairments, including heart failure26, hypertension27, Alzheimer’s88, multiple system atrophy25, multiple sclerosis89, amyotrophic lateral sclerosis90. However, prior studies did not directly measure autonomic function in addition to brain metabolites.

In our pilot study described in Chapter Four a cohort of 18 pediatric subjects ages

10-18 was evaluated for autonomic instability using HUT or a 10-minute standing challenge and then underwent MRS scanning to measure brain metabolites in supine position. We observed increased mIns and tCho, markers of glial inflammation and myelin degradation91-93, in HUT(+) compared to HUT(-), indicative of potentially damaging neuroinflammation in the dorsal medulla of children with OI. Interestingly, these two metabolites did not associate with any index of autonomic function in supine position, but demonstrated strong inverse relationships with upright measures of BRS and parasympathetic vagal tone (PVT). Since BRS and PVT were significantly lower in

HUT(+) subjects, our data may implicate inflammation and its detrimental effects on myelin and neural tissues as contributors to impairments in BP regulation by effecting baroreflex circuitry. Although we cannot draw conclusions on causality between

188 impaired baroreflex responses and neuroinflammation, the discussion that follows provides speculation on the interaction of these components and their downstream effects based on previous studies of neuroinflammation.

The CNS and ANS have critical roles in detection and modulation of inflammation through two major pathways, the HPA axis and balance of sympathovagal activity94. When these pathways are altered due to disease, homeostasis of inflammation is altered often causing increased pro-inflammatory cytokine production and subsequent tissue damage in various organ systems including the brain, heart, GI tract, and associated vasculature95, 96. Cardiovascular disease is associated with increased pro-inflammatory cytokines that act systemically impacting vasculature function and centrally in the hypothalamus and medullary areas95, 97-99. As shown in heart failure patients and post- myocardial infarction, pro-inflammatory cytokines released by activated microglia in the

PVN significantly increased sympathoexcitation causing increased HR and vasoconstriction100. Although microglial-activated inflammation has been shown to negatively affect the baroreflex, this relationship has not been evaluated in regards to impaired BP regulation in adults or children with OI.

Both branches of the ANS are involved in immune system regulation101.

Increases in sympathetic activity have been shown to occur following administration of pro-inflammatory cytokines into the PVN, suggesting that central inflammation causes increased HR and vasoconstriction through this pathway102. HUT(+) subjects in our study had significantly lower sympathetic tone as measured by LFα, but this measurement does, in part, reflect vagal tone103. Sympathovagal balance was significantly higher and HFα and HRV were lower in HUT(+) subjects suggesting the

189 differences observed in LFα most likely represent differences in vagal tone between groups. Taken together, autonomic function in HUT(+) subjects indicate the sympathovagal imbalance is due to lower PVT and potentially higher sympathetic activation. Although we do not obtain a direct measure of sympathetic activation and do not know for certain of its role in this case, the majority of HUT(+) subjects in Chapter

Four met the criteria for POTS, which is characterized by exaggerated sympathetic activation during standing. Furthermore, if neuroinflammation in the dorsal medulla was causing initial autonomic dysfunction, previous evidence suggests we would have observed significantly greater sympathetic activation. Thus, it is possible that increased sympathetic activation has a role in elevated markers of inflammation, but this was not directly demonstrated in this study.

In contrast to the sympathetic system, parasympathetic vagal outflow has anti- cholinergic inflammatory actions via activation of nACha7 receptors found on macrophages104. The existence of this pathway has been demonstrated in human and animal models, where electrical vagal stimulation has increased neuroprotection and decreased TNFα levels in circulation105, 106. Moreover, in humans elevated systemic inflammatory markers IL-6 and CRP are associated with decreased HRV and vice versa107. Vagal activity also inhibits release of pro-inflammatory cytokines and protects against inflammation-induced tissue damage. In our study, HFα and HRV were significantly lower in HUT(+) subjects compared to HUT(-) subjects upon standing.

HUT(+) subjects also demonstrated a significant decrease in BRS upon standing, which is another indicator of vagal function. Upright PVT and BRS both negatively correlated with markers of inflammation, mIns and tCho. Thus, in HUT(+) subjects the anti-

190 inflammatory modulation by the vagus nerve may no longer be inhibiting inflammatory processes, which would lead to increased systemic inflammation. When pro- inflammatory factors are released in the periphery due to injury they can cross the blood brain barrier and modulate central mechanisms by acting on the PVN to alter BP regulation95. However, the specific action of crossing the blood brain barrier has not been directly associated with impaired PVT, so it remains unclear whether decreased PVT is the primary cause of neuroinflammation in the dorsal medulla of HUT(+) subjects.

The HPA axis also plays a vital role in homeostasis of stress and immune function108. Although we did not collect physiological markers of stress or anxiety, our group has previously reported increased anxiety in children with OI and chronic nausea as determined using standard State/Trait Anxiety Questionnaires10. Increased stress and activation of the HPA axis is associated with decreased HRV and overactivation of the sympathetic system in acute and chronic inflammatory states. These autonomic patterns also set the stage for pro-inflammatory cytokines to damage various organ systems. This is evident in patients with high stress and anxiety, which are at greater risk for developing high blood pressure and atherosclerosis. These traits should be carefully monitored and considered when treating subjects with OI, as they may be exacerbating symptoms or masking the underlying cause of dysfunction. Furthermore, neurohumoral components involved in OI are also involved in the HPA axis and inflammation and should be considered as well.

1H-MRS also detected trends for lower NAA and GSH between HUT(-) and

HUT(+) subjects. NAA is a marker of glutamate metabolism and neuronal/axonal integrity and decreases are associated with decreased neuronal density and impaired

191 neuron function109. GSH is an antioxidant that acts in the brain to deactivate ROS in cells and prevent downstream ROS effects of tissue damage110. Trends observed for these markers are consistent with our findings of elevated mIns and tCho since microglial activation is neurotoxic and ROS are key signaling molecules involved in inflammation111, 112. Our findings are also consistent with previous studies in which higher mIns was accompanied by lower NAA in hippocampi of Alzheimer’s patients113,

114. Future studies are needed to further investigate the potential relationships between

NAA and GSH in blood pressure regulation.

Our study was the first to demonstrate elevated neuroinflammatory markers detected by MRS associate with autonomic function during upright position. Most importantly, our findings demonstrate that metabolites of glial inflammation are predictive of decreased parasympathetic vagal tone and spontaneous baroreflex sensitivity upon standing. These data provide a foundation for the use of MRS in characterizing central mechanisms of OI and autonomic dysfunction in larger cohorts that could also show differential metabolite profiles in OI subtypes as observed with neurohumoral profiles.

192 6. Neurohumoral and inflammatory interactions in cardiovascular pathology

Neuroinflammation is induced by activated microglia, which serve as the immune cells of the brain111. In the study discussed in Chapter Four, increased markers of glial inflammation were observed in HUT(+) subjects characterized by impairments in autonomic function upon upright posture. While systemic inflammation as measured by

IL-6 and CRP levels is inversely related to vagal function107, 115, 116, studies have not shown direct evidence of increased neuroinflammation in response to decreased vagal tone. However, central pro-inflammatory cytokines in the PVN and dorsal medulla increase BP and sympathetic activity, as well as induce synthesis of vasoconstrictor hormones, renin, Aldo, and AVP96. This suggests the neurohumoral system associated with BP regulation and GI function is also intricately involved in inflammatory pathways.

Since we did not measure inflammatory markers and neurhomoral hormones in the same subjects, we can only postulate potential mechanisms involved between these factors, nausea, and cardiovascular function in OI. In the following section two general schematics are proposed, providing an outline of potential pathophysiological mechanisms of OI and chronic nausea that include hemodynamic, neurohumoral and inflammatory components. Mechanisms specific to POTS, OH, and syncope are also covered as they most likely differ based on their distinct hemodynamic and neurohumoral profiles.

First, increased glial inflammation in the dorsal medulla may initially impact the baroreflex causing decreased vagal function and/or increased sympathetic activation.

Increased sympathetic activation, as seen in POTS, leads to elevated circulating NE, which is known to produce pro-inflammatory responses peripherally. The subsequent

193 Figure 1. Schematic of brain inflammation contributing to OI. Inflammation in the brain induces synthesis of neurohumoral hormones involved in CV disease and decreased BRS, which lead to OI. Nausea may be secondary to these processes and could contribute to the chronic inflammatory state in the brain and peripherally. BBB, blood brain barrier. production of pro-inflammatory cytokines is associated with increased inflammation in the gut, which can cause disruptions in GI motility117. Afferents carrying information regarding the inflammatory state and GI motility in the gut project to the brain via the vagus nerve and modulate autonomic outflow118. While gastric dysrhythmias are associated with nausea independent of inflammation, inflammation in the gut can exacerbate GI symptoms to produce nausea and GI pain while facilitating a chronic inflammatory state in the brain119, 120.

Pro-inflammatory actions in the brain also increase synthesis of vasoconstrictor peptides involved in cardiovascular pathology including AVP, Ang II, Aldo, and renin96.

As described in previous sections, these components are all involved in regulation of the baroreflex and AVP and Ang II can induce nausea via altered GI blood flow and actions on the area postrema. Furthermore, decreased GI blood flow causes increased ROS and

194 inflammation in the gut that can influence neuronal firing from the DMNV to the gut, exacerbating the inflammatory state118. Since central inflammation can induce altered neurohumoral release and baroreflex impairments characteristic of OI, it is possible that elevated neuroinflammation is an initial contributor to the pathology of OI and chronic nausea through these mechanisms (Figure 1). However, this theory does not explain why or how OI manifests with various hemodynamic and neurohumoral subtypes.

Furthermore, the majority of studies in humans investigate this mechanism as if the autonomic dysfunction is the initiator of increased inflammation.

As depicted in Figure 2, Subjects

presenting with HUT(+) in our studies

demonstrated low BP, BRS, HRV, and

PVT indicative of impaired

sympathovagal balance, increased AVP,

and increased markers of glial

inflammation. In Chapter Two, decreased

BP in OI subjects associated with

exaggerated increases in AVP upon Figure 2. Autonomic dysfunction leads to systemic and brain inflammation. Autonomic Dysfunction in OI causes Increased AVP and standing. While nausea is associated with subsequent nausea and Inflammation in the periphery and in the brain. Dotted lines indicate imbalances in sympathovagal outflow35, the origin of the pathway. BBB, blood brain barrier. AVP was the only characteristic factor of

HUT-induced nausea in Chapter Three. AVP carries out pro-inflammatory actions by activating prolactin, which together worsens inflammation systemically121. AVP also acts on the gut and area postrema inducing gastric dysrhythmias and nausea22, 23.

195 Furthermore, inflammation induced by AVP can also cause increased GI inflammation, which in turn causes a plethora of GI symptoms including nausea and GI pain. Taken together, increased AVP release due to an impaired baroreflex could cause downstream effects of inflammation in the brain and gut. In this case, increased brain inflammation may exacerbate baroreflex impairments already present in OI. Whether inflammation in the dorsal medulla is the initiating event to impair the baroreflex to upright posture as the first insult is not known from our studies, but may be a consequence of prior infectious agents such as a virus as had been suggested122, 123. As shown in Chapter Three, exaggerated AVP is associated with nausea independent of OI status and its actions on the AP and on GI smooth muscle cells are known to alter GI motility. The primary finding in Chapter Two was that OI subtypes demonstrate different neurohumoral response to standing and these should be addressed in relation to inflammation as well.

In contrast to the OI group as a whole, POTS subjects had exaggerated release of NE during orthostasis, but did not demonstrate exaggerated AVP release. It is well established that Figure 3. Sympathetic activation and exaggerated POTS have increased sympathetic NE cause nausea and inflammation in POTS. Dotted lines indicate the origin of the pathway. BBB, activation and NE release that blood brain barrier. causes tachycardia upon standing17. However, both influence pro-inflammatory cytokine

196 production, providing a mechanism of which could contribute to increased inflammation observed in the dorsal medulla. As shown in Figure 3, increased sympathetic activation in

POTS causes release of NE, which serves to increase HR to aid in maintaining cardiac output during standing. Over time, increased NE can induce release of pro-inflammatory cytokines, which can cross the blood brain barrier through the circumventricular organs to modulate BP regulation in the PVN and dorsal medulla, facilitating the persistent sympathetic activation observed in POTS95.

OH and syncope

are characterized by low

BP and impaired

baroreflex regulation of

sympathetic outflow. In

our study, both groups

had an exaggerated

release of AVP from

supine to HUT, but

Figure 4. The role of autonomic dysfunction in OH and syncope in differed considering systemic and brain inflammation. OH and syncope subjects have decreased sympathetic outflow and Aldo, which was decreased BP and exaggerated increases in AVP. OH also has elevated Aldo at supine and HUT positions. Both hormones have various elevated during supine downstream effects that contribute to inflammation. Red dotted lines indicate the origin of the pathway. Black dotted lines indicate Aldo pathway. BBB, blood brain barrier. and HUT in OH subjects. Exaggerated elevations of AVP during standing in OH and syncope most likely serve to increase systemic vascular resistance for maintenance of cardiac output.

However as discussed previously, AVP has various downstream effects that could impact

197 the pathology of OI including increased systemic inflammation and nausea. Again, circulating pro-inflammatory cytokines can cross the blood brain barrier and negatively impact BP regulation. Recent studies show that adults with OH have increased markers of systemic inflammation as well124. Moreover, elevated Aldo causes oxidative stress and endothelial dysfunction in the peripheral and central vasculature associated with stroke and cardiovascular disease125. Thus, increased Aldo in children with OH may also demonstrate similar pathology, which may further link inflammation in the dorsal medulla to facilitation of baroreflex impairments (Figure 4).

To further explore the relationships between inflammation and neurohumoral markers involved in OI, it is imperative for future studies to measure both types of factors. Our data provide foundational support for the role of inflammation in the pathogenesis of autonomic dysfunction manifesting as OI, but causality between the two components has yet to be determined. To date, the cause of OI is still unknown and treatments remain suboptimal.

198 7. Limitations

Patient Selection

Many studies in children and adults with OI are retrospective, identifying risk factors using patient records in large cohorts of hundreds to thousands. In contrast, empirical studies aiming to evaluate hemodynamic or neurohumoral responses in OI include much fewer subjects, comparable to the 48 subjects included in Chapters Two and Three. Even so, conducting initial patient evaluation and tilt testing in children can be a time consuming and complicated process since parents are also involved in decision making, scheduling, and consent. In our study, we chose to separate the HUT(+) group into subtypes of OI, resulting in individual group numbers that were much smaller, impacting power for statistical analysis. Also, separating the 48 subjects by nausea experience and HUT response further reduced group sample size disproportionately, with the HUT(-) responders having only 6 subjects. Although this grouping did limit our statistical power, it was necessary for understanding our results and our interpretations and relative emphasis of the results took this into consideration. Lastly, patient recruitment for the study described in Chapter Four aimed to include 22 subjects, but 4 subjects did not have useable MRS spectral scans due to interference of imaging the targeted brain area as a result of orthodontic braces. Since braces are common in children within our target age range we need to be more vigilant with inclusion criteria for future imaging studies.

Another considerable limitation was the lack of asymptomatic controls in studies described in Chapters Two and Three, where all subjects presented in the pediatric GI clinic with chronic nausea and one symptom of OI. While HUT(-) subjects served as our

199 control for these studies, they were not true asymptomatic controls and the underlying cause of chronic nausea is still unknown. In contrast, Chapter Four included asymptomatic controls. Interestingly, after evaluation of control/HUT(-) autonomic profiles, one subject was redistributed to the HUT(+) group based on their autonomic response to 10-minute upright posture. This highlights the necessity of HUT for diagnosing OI and evaluation of control subjects, as some children may present without severe symptoms and still test positive for autonomic imbalances.

Sample collection

To our knowledge sample collection during HUT has not been utilized to characterize neurohumoral profiles in children. We chose to collect blood samples 5 minutes prior to HUT in supine position and 15 minutes after the initiation of HUT since the definition of OI based on cardiovascular responses is made within this time frame.

However, some subjects experienced severe cardiovascular symptoms prior to 15 minutes. In the case of a subject being presyncopal or having severe symptoms prior to

15 minutes we would collect a blood sample prior to termination of the HUT.

Unfortunately if subjects experienced syncope, tachycardia, or hypotension after the initial 15-minute time point, the blood sample would not reflect these hemodynamic alterations. Additionally, blood samples did not coincide with the 11 subjects who experienced HUT-induced nausea after the first 15 minutes of HUT. Thus, collecting more blood samples at different time points may help us more fully understand the neurohumoral profiles in subjects with syncope, P/S, or HUT-induced nausea during

HUT tests with longer durations.

200 Our neurohumoral panel was more comprehensive than previous studies in adults and the first conducted of this type in children. Although we gained great insight into neurohumoral responses to HUT, additional markers could have been collected that would have been helpful in further understanding our results. For example, cortisol measurements could have aided in understanding the role of physiological stress in children during HUT. By collecting cortisol we could have validated the use of State-

Trait anxiety questionnaires in this population as well. Furthermore, knowing the relationship between cortisol and AVP release would have given us a greater understanding the role of AVP in stress and inflammation in addition to BP regulation.

Collecting additional sample volume for the measurement of pro- and anti- inflammatory markers would have also proved interesting as they would be indicative of systemic inflammation. Having this information may have provided a stronger link between impaired vagal function and brain inflammation detected using the 1H-MRS in our patient population.

Head upright tilt in Children

Conducting clinical trials with children requires higher levels of validation of methodologies used to ensure proper patient care. Moreover, parents and children both have to understand the procedure and risks before consenting. Since children with OI may not experience severe secondary symptoms because they have adapted to remaining in upright position as little as possible, a 45-minute HUT could seem long and invasive.

Careful explanation of HUT procedure and the knowledge the patient would gain about their condition from the study was thoughtfully conveyed to each family. Additionally, collecting blood samples required an intravenous (IV) line in the forearm, which is very

201 unpopular in children (and their parents). A dual finger cuff for beat-to-beat measures of

HR and BP was on the same arm as the IV and an arm cuff for intermittent BP measurements was on the opposite arm. While the Wake Forest School of Medicine IRB approved our procedure, HUT alone could be considered invasive as it elicited severe and sometimes life-threatening cardiovascular impairments, but an IV most likely increased discomfort and overall stress in our subjects prior to the beginning of the HUT.

Magnetic Resonance Spectroscopy methodology in children

Our 1H-MRS protocol was much less invasive than the HUT as no IVs or BP cuffs were used during the scan. The only complication arising with children is the risk of movement in the scanner, which while a major problem for anatomical imaging is less of a problem for tissue metabolite profiles. Claustrophobia was also a concern, but this issue did not arise for anyone in our study. Our target region of interest in Chapter Four was the dorsal medulla, which sits immediately below the fourth ventricle. Since respiratory movement most likely caused the voxel location to move during changes in breathing, this area is difficult to image clearly for anatomic studies, but metabolite concentrations are less affected by movement. Water suppression techniques were used to increase detection/sensitivity for the metabolites126, 127, but variability in %SDs for metabolite detection indicated low reliability of measures of GABA and several other metabolites present in the dorsal medulla. A more powerful magnet (> than the 3T used in our studies) would improve the resolution of these hard to detect but important metabolites.

Information from other brain regions associated with autonomic function with differential metabolite profiles would be desirable to determine whether observations of inflammation are generalizable to other brain areas and should be included in future

202 studies. The insular cortex is an example of an area of great interest as preliminary data revealed increased network connectivity in the insular cortex of HUT(+) compared to

HUT(-) children, suggesting HUT(+) children have heightened awareness of bodily sensations associated with their disorder.

203 8. Concluding Remarks

The current studies were initiated as a result of the recognition that children presenting in the pediatric GI clinic with chronic nausea also had at least one symptom related to OI. Upon further investigation it was determined that an OI diagnosis could be made in ~80% of children with chronic nausea for which no functional GI mechanism could be identified. Moreover, standard treatments of OI improved GI symptoms and quality of life for the subjects identified as OI. However, the apparent heterogeneity of

OI coupled with chronic nausea proved that further understanding of neurohumoral and central mechanisms involved in BP regulation in children was warranted. Furthermore, uncovering factors that modulate baroreflex function during orthostasis is imperative to developing more effective treatments to prevent persistence of cardiovascular instability into adulthood.

In summary, we demonstrate the following:

1. Children with chronic nausea and at least one symptom of OI test positive for OI

in high incidence and present with various cardiovascular responses

representative of different phenotypes of autonomic dysfunction: POTS, OH,

syncope and POTS/Syncope.

2. Children meeting the diagnosis of POTS have exaggerated NE release during

HUT which is consistent with studies in adults with POTS. Pharmacotherapies

targeting volume expansion mechanisms are recommended for these subjects to

reduce the need for exaggerated heart rate to maintain cardiac output and arterial

pressure while standing.

204 3. Children diagnosed with OH have elevated Aldo at supine and HUT positions and

exaggerated AVP increases from supine to HUT. Those presenting with syncope

also had exaggerated increases in AVP and no catecholamine or Ang II

vasoconstrictor response. As opposed to volume expanders, vasoconstrictor

therapies may be more effective in ameliorating symptoms associated with OH

and syncope.

4. HUT-induced nausea was not associated with differential hemodynamic or

autonomic responses, but demonstrated exaggerated AVP upon standing

independent of OI status. Subjects with HUT-induced nausea and HUT(+) had the

greatest increase in AVP, suggesting multiple phenotypes of nausea exist, one

characterized by HUT-induced nausea with HUT(+) and another considering

nausea alone and elevated AVP alone.

5. Increased markers of glial inflammation are present in the dorsal medulla of

children with OI (with or without nausea) relative to subjects without OI or

nausea and are predictive of impaired baroreflex sensitivity and parasympathetic

vagal tone during upright position.

These findings have clinical implications for diagnosis and treatment of children with OI and chronic nausea that will impact the daily lives of patients and their families.

Chapter Two highlights the effectiveness of HUT in diagnosing children with chronic nausea with potentially severe autonomic instability. While the ultimate goal would be to identify a marker for OI in supine position, none of the neurohumoral markers studied were different between groups at baseline. Current studies are underway measuring

205 copeptin as an indicator of chronic elevation of AVP and perhaps volume depletion in OI and chronic nausea during supine position. In Chapter Three we described two phenotypes of nausea associated with elevated AVP, one of which may be related to impaired autonomic function. Finally, Chapter Four provided the first report of elevated brain inflammation in subjects with OI. Although this was conducted in a small cohort, brain metabolite markers of inflammation in supine position were predictive of autonomic impairments observed in OI subjects in the upright position. The relationship between central inflammation and autonomic function in this population requires further study for causality, but provides a foundation for the use of a relatively new imaging technique, 1H-MRS in children with severe neurological or cardiovascular disorders.

To conclude, we propose the following future assessments be implemented during HUT and 1H-MRS in larger cohorts to expand on our studies:

1. Measure cortisol as a marker of physiological stress to determine its relationship

to neurohormones released during orthostasis;

2. Collect samples for evaluation of blood volume to further delineate POTS

subjects based on their volume status;

3. Collect blood samples at multiple time points and especially during the

experience of nausea during a 45-minute HUT to capture the neurohumoral

profile at the time of symptom experience and cardiovascular instability;

4. Measure brain metabolites in additional brain areas associated with autonomic

function, including the insular cortex to determine if inflammation occurs in other

areas implicated in OI, and;

206 5. Conduct electrogastrogram recordings during supine and HUT positions in

children with chronic nausea to evaluate electrical changes in response to

orthostasis and neurohumoral profiles.

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217 SCHOLASTIC VITA

ASHLEY LYNNE WAGONER

EDUCATION

Ph.D., Neuroscience (2016) Wake Forest University, Winston-Salem, North Carolina, USA

Advisor: Debra Diz, Ph.D. Dissertation title: Altered Hemodynamic, Neurohumoral, and Brain Metabolite Profiles in Children with Orthostatic Intolerance and Chronic Nausea

B.S., Neuroscience (2011) Furman University, Greenville, South Carolina, USA

Advisor: David Hollis, Ph.D. Thesis title: Isolation and Molecular Characterization of REM2 in Rainbow Trout (Onchorynchus mykiss)

HONORS AND AWARDS

2010 Furman Advantage Award for Undergraduate Summer Research

2013, 2014 Student Gold Award in Clinical Science, Wake Forest School of Medicine Division of Surgical Sciences Research Day

2014 Poster Travel Award for Clinical Sciences, International Society for Hypertension, New Investigator Symposium, San Francisco, CA

2014 2nd Place Overall Poster Award, Wake Forest Institute for Regenerative Medicine Annual Retreat, Pinehurst, NC

2014, 2015 Wake Forest University Graduate School of Arts & Sciences Alumni Student Travel Award to travel to American Heart Association Council for Hypertension, San Francisco, CA (2014); Washington, DC (2015)

2015 Student Travel Award Recipient to attend the International Meeting of Autism Research, Salt Lake City, Utah

2015 Predoctoral Fellowship, Studies in Translational Regenerative Medicine, NIH-NIBIB T32 Program

2015 Student Silver Award in Clinical Science, Wake Forest School of Medicine Division of Surgical Sciences Research Day

218 POSITIONS AND COMMUNITY OUTREACH

2011 Volunteer, WFU Brain Awareness Council

2012 - 2016 Editor, The Neurotransmitter, newsletter for Western NC Chapter of SfN

2012 - 2015 Student Representative, Admissions Committee for Program in Neuroscience at Wake Forest University Graduate School

2013 - 2015 Steering Committee Coordinator, WFU Brain Awareness Council

2014 Summer Research Mentor, WFIRM Summer Scholars Program

2014 - 2015 Teaching Assistant, Neuroscience Graduate Course in Neuroanatomy

PROFESSIONAL MEMBERSHIPS

Society for Neuroscience, 2010-2015 Western North Carolina Society for Neuroscience, 2011-present International Society for Autism Research, 2012-2016 American Heart Association Council for High Blood Pressure, 2013-present International Society of Hypertension, 2014-present

MANUSCRIPTS

Hollis DM, Sawa Y, Wagoner A, Rawlings JS, Goetz FW. Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression. Comp Biochem Physiol B Biochem Mol Biol. (2012) Feb 161 (2):93-101.

Fortunato JE, Wagoner AL, Harbinson RL, D’Agostino, Jr. RB, Shaltout HA, Diz DI. Effect of Fludrocortisone Acetate on Chronic Unexplained Nausea and Abdominal Pain in Children with Orthostatic Intolerance. J Pediatr Gastroenterol Nutr. 2014 July; 59(1): 39-43.

Mack DL, Guan X, Wagoner A, Walker SJ, Childers MK. Disease-in-a-Dish: The Contribution of Patient-specific Induced Pluripotent Stem Cell Technology to Regenerative Rehabilitation. American Journal of Physical Medicine and Rehabilitation. 2014 Nov; 93 (11 Suppl 3);S155-68.

Tarbell SE, Shaltout HA, Wagoner AL, Diz DI, Fortunato JE. Relationship among nausea, anxiety, and orthostatic symptoms in pediatric patients with chronic unexplained nausea. Experimental Brain Research. 2014 Aug; 232(8):2645-50.

219

Wagoner AL, Shaltout HA, Fortunato JE, Diz DI. Distinct Neurohumoral Biomarker Profiles in Children with Hemodynamically-Define Orthostatic Intolerance. Am J Physiol Heart Circ Physiol 310: H416 –H425, 2016.

Wagoner AL, Shaltout HA, Diz DI, Fortunato JE. Head Upright Tilt Evokes Exaggerated Arginine Vasopressin Response in Children with Chronic Nausea and Orthostatic Intolerance. Am J Gastro. Under review.

Wagoner AL, Fortunato JE, Diz DI, Shaltout HA. 1H-Magnetic Resonance Spectroscopy Reveals Elevation of MyoInositol and other Markers of Inflammation in the Dorsal Medulla of Children with Orthostatic Intolerance. To be submitted to Autonomic Neuroscience: Basic & Clinical

PUBLISHED ABSTRACTS

Wagoner AL, Shaltout HA, D’Agostino RB, Diz DI, Fortunato JE. Relationship of Arginine Vasopressin and Blood Pressure in Patients with Orthostatic Intolerance. Hypertension, 2013;62:A261. American Heart Association Council for High Blood Pressure, New Orleans, LA, September 2013.

Fortunato JE, Wagoner AL, Shaltout HA, Diz DI. Increase in Arginine Vasopressin is Associated with the Reduction in Blood Pressure in Patients with Chronic Nausea and Orthostatic Intolerance. J Pediatr Gastroenterol Nutr, 2013; 57:S, e97. North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN), Chicago, IL, October 2013.

Shaltout HA, Wagoner AL, Fortunato JE, Diz DI. Impaired Hemodynamic Response to Head up Tilt in Adolescents Presenting Chronic Nausea and Orthostatic Intolerance. Hypertension. 2014;64:A513. American Heart Association Council for High Blood Pressure, San Francisco, CA, September 2014.

Wagoner AL, Shaltout HA, Diz DI, Fortunato JE. Novel Neurohumoral Responses to Head Upright Tilt Testing in Children with Chronic Nausea and Orthostatic Intolerance. Hypertension, 2014;64:A033. American Heart Association Council for High Blood Pressure, San Francisco, CA, September 2014.

Wagoner AL, Fortunato JE, Diz DI, Shaltout HA. 1H-Magnetic Resonance Spectroscopy Reveals Elevation of MyoInositol and other Markers of Inflammation in the Dorsal Medulla of Children with Orthostatic Intolerance. Hypertension, 2015;66:AP069. American Heart Association Council for Hypertension, Washington, DC, September 2015.

220 ABSTRACTS

Clinical Autonomic Abstracts Wagoner AL, Harbinson RL, Diz DI, Shaltout HA, Fortunato JE. Effect of Fludrocortisone Acetate on Chronic Unexplained Nausea and Abdominal Pain in Children with Dysautonomia. Western North Carolina Society for Neuroscience Research Day, Winston-Salem, NC, November 2012.

Wagoner AL, Harbinson RL, Diz DI , Shaltout HA, Fortunato JE. Effect of Fludrocortisone Acetate on Chronic Unexplained Nausea and Abdominal Pain in Children with Dysautonomia. Wake Forest University 13th Annual Graduate Student and Postdoctoral Research Day, Winston-Salem, NC, March 2013.

Wagoner AL, Shaltout HA, D’Agostino RB, Diz DI, Fortunato JE. Relationship of Arginine Vasopressin and Blood Pressure in Patients with Orthostatic Intolerance and Chronic Nausea. 21st Annual Division of Surgical Sciences Research Day, Winston Salem, NC, November 2013.

Wagoner AL, Shaltout HA, D’Agostino RB, Diz DI, Fortunato JE. Relationship of Arginine Vasopressin and Blood Pressure in Patients with Orthostatic Intolerance. International Society of Hypertension New Investigators’ Symposium, New Orleans, LA, September 2013.

Wagoner AL, Shaltout HA, Diz DI, Fortunato JE. Novel Neurohumoral Responses to Head Upright Tilt Testing in Children with Chronic Nausea and Orthostatic Intolerance. International Society of Hypertension New Investigators’ Symposium, San Francisco, CA, September 2014.

Wagoner AL, Shaltout HA, Diz DI, Fortunato JE. Novel Neurohumoral Responses to Head Upright Tilt Testing in Children with Chronic Nausea and Orthostatic Intolerance. 22nd Annual Division of Surgical Sciences Research Day, Winston Salem, NC, November 2014.

Wagoner AL, Fortunato JE, Diz DI, Shaltout HA. 1H-Magnetic Resonance Spectroscopy Reveals Elevation of MyoInositol and other Markers of Inflammation in the Dorsal Medulla of Children with Orthostatic Intolerance. 23rd Annual Division of Surgical Sciences Research Day, Winston Salem, NC, November 2015

Community Outreach Abstracts Severino AL, Wagoner AL, Haynes KA, Skelly MJ, Rose JH, Constantinidis C, Godwin DW. Wake Forest University's Brain Awareness Council: Evolution of Outreach to the Piedmont Triad Community. Society for Neuroscience Education Outreach Poster Session. Washington, DC, November 2014.

221 Beckelman B, Alberto G, Severino AL, Wagoner AL, Carr A, Ewin S, Shing E, Godwin DW. Wake Forest University's Brain Awareness Council: Evolution of Outreach to the Piedmont Triad Community. Society for Neuroscience Education Outreach Poster Session, Chicago, IL, October 2015.

Disease-in-a-dish Stem Cell Abstract Presentations: Wagoner AL, Mack DL, McKee EE, Walker SJ. Development of a Cell Culture Model for Enteric Nervous System Function in Children with Autism Spectrum Disorder. Wake Forest Institute for Regenerative Medicine 2014 Retreat, Pinehurst, NC, February 2014.

Wagoner AL, Mack DL, McKee EE, Walker SJ. Development of a Cell Culture Model for Enteric Nervous System Function in Children with Autism Spectrum Disorder. Wake Forest University 14th Annual Graduate Student and Postdoctoral Research Day, Winston-Salem, NC, March 2014.

Wagoner AL, Mack DL, McKee EE, Walker SJ. Modeling Enteric Nervous System Function in children with Autism Spectrum Disorder. Wake Forest University Neuroscience Graduate Student Research Day, April 2014.

Wagoner AL, Mack DL, Walker SJ. Enteric Nervous System Dysfunction in Autism Spectrum Disorder: Development of an in vitro Human Model System. International Meeting for Autism Research, Atlanta, GA, May 2014.

Valcarce-Aspegren M, Wagoner AL, McKee EE, Walker SJ. Induced pluripotent stem cells derived from children with Phelan-McDermid Syndrome for use as functional model of enteric nervous system. Wake Forest Institute for Regenerative Medicine Summer Scholars Research Day. (M Valcarce-Aspegren, Summer Scholar)

Chang YR, Mead I, Pendergraft S, Wagoner A, Bishop C. Differentiating induced pluripotent stem cells into human three-dimensional cerebral organoids to model the effect of cannabinoids on the developing brain. Wake Forest Institute for Regenerative Medicine Summer Scholars Research Day. (YR Chang, Summer Scholar)

Wagoner AL, Mack DL, McKee EE, Walker SJ. Modeling enteric nervous system function in children with Autism Spectrum Disorder. Society for Neuroscience, Washington, DC, November 2014.

Chang YR, Mead I, Pendergraft S, Wagoner A, Bishop C. Differentiating Induced Pluripotent Stem Cells into Cerebral Organoids. Tissue Engineering Regenerative Medicine International Society, Undergraduate Researcher Poster Session, Washington, DC, December 2014.

222 Wagoner AL, Mack DL, McKee EE, Walker SJ. A Novel Cost-Effective Approach to Derivation of Induced Pluripotent Stem Cells from Epstein-Barr Virus Immortalized β- Lymphoblastoid Cell Lines. International Meeting for Autism Research, Salt Lake City, UT, May 2015.

Wagoner AL, Mack DL, McKee EE, Walker SJ. Modeling Enteric Nervous System Function in children with Phelan McDermid Syndrome. International Meeting for Autism Research, Salt Lake City, UT, May 2015.

Wagoner AL, Mack DL, McKee EE, Walker SJ. A Novel Cost-Effective Approach to Derivation of Induced Pluripotent Stem Cells from Epstein-Barr Virus Immortalized β- Lymphoblastoid Cell Lines. North Carolina Engineering and Regenerative Medicine Conference and Innovation Summit, Winston-Salem, NC, October 2015.

Wagoner AL, Mack DL, McKee EE, Walker SJ. Modeling Enteric Nervous System Function in children with Phelan McDermid Syndrome. North Carolina Engineering and Regenerative Medicine Conference and Innovation Summit, Winston-Salem, NC, October 2015.

223