A Study of Vagus Nerve Stimulation Issues and Techniques for Hemodynamic Control

A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA By

George Daniel O’Clock, Jr.

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

THESIS FACULTY ADVISOR: A.G. Talkachova (E.G. Tolkacheva)

THESIS CO-ADVISOR: B.H. KenKnight

May, 2018

© GEORGE DANIEL O'CLOCK, JR. 2018

Acknowledgements I would like to thank the members of the thesis committee, Dr. Alena Talkachova for her guidance, insight, and patience; Dr. Victor Barocas who gave me some very good advice as I began this odyssey at the U of MN; and Dr. Bruce KenKnight, who opened the door for me during a Cardiovascular Device course when he looked at my class paper and said, "George, it looks like you have a thesis." This committee is part of the group of more than 30 people who have mentored me for the past 55 years. They include Dr. Warren Warwick (dec.), Department of Pediatrics, U of MN Medical School, Minneapolis, MN; Dr. Björn E.W. Nordenström (dec.), Head of Diagnostic Radiology, Karolinska Inst., Stockholm, Sweden; Dr. Xin Yuling, Chief Specialist of Thoracic Surgery, Friendship Hospital, Beijing, PRC; Dr. Stuart R. Taylor, Dept. of Pharmacology, Mayo Graduate School, Rochester, MN; Dr. Lyle D. Feisel, Dean of Engineering, SUNY Binghamton, Binghamton, NY; Dr. William Hixon (dec.) and Cyrus Cox, Electrical Engineering, South Dakota School of Mines & Technology, Rapid City, S.D., Dr. Mark Lyte, Health Sciences Center, Texas Tech, Abilene, TX, Dr. Robert M. Williams (dec.), Economics Dept., UCLA, Los Angeles, CA; Dr. Donovan Nielsen, Biological Sciences, Minnesota State University-Mankato, Mankato, MN; Robert C. Dixon (dec.), RCD&A Consulting, Colorado Springs, CO, Carl Hildebrand (dec.), Perkin-Elmer, Norwalk, CT, Gene Turechek (dec.), Eastman Kodak, Rochester, NY; R.J. Dauphinee (dec.), TRW Systems, Redondo Beach, CA, family members, and two very supportive parents. Without the generous help and support they all gave, I could not have achieved my goals, and could not have arrived at this place in my lifetime. i Abstract

Approximately 75 million adults in the U.S. have hypertension. With appropriate adjustments, neuromodulation therapy, or more specifically vagus nerve stimulation (VNS), can provide cardiovascular condition and performance improvements for this segment of the U.S. population. In designing and developing VNS devices and protocols for cardiovascular disorders; the needs and preferences of the patient and the health care practitioner who treats the patient must be considered. They should be the in the group of primary design drivers for both the device and the protocol. With the patient and health care practitioner in mind, it appears that both implanted and non-invasive VNS approaches will have their own unique roles in treating cardiovascular disease. Under that scenario, new and more comfortable VNS implant designs and protocols can benefit from non- invasive VNS (NI-VNS) device and protocol advances. NI-VNS can provide a relatively safe, convenient, and effective way to obtain useful clinical study information to help define and establish proposed mechanisms-of- action associated with VNS regulation and control of blood pressure and heart rate. Also, VNS device development can be assisted and advanced even further with new and improved design tools. Included in that category are modeling/simulation tools and techniques that can help provide insights into the responses and sensitivities associated with VNS-cardiovascular- neurological interfaces and associated functional relationships.

ii TABLE OF CONTENTS

List of Tables ...... vi

List of Figures ...... vii

1. Introduction ...... 1 1.1 Vagus Nerve-Parasympathetic Nervous System Relationship . . . . . 1 1.2 Vagus Nerve Stimulation (Mechanical) ...... 2 1.3 Vagus Nerve Stimulation (Electrical) ...... 3 1.4 Recent Vagus Nerve Stimulation Implant Clinical Results for Heart Disease Applications ...... 4

2. Background ...... 7

3. Aim, Objectives, and Mission ...... 10

4. Methods ...... 12

4.1 NI-VNS Clinical Study ...... 12 4.2 Model and Simulation of VNS Device-Neurological- Cardiovascular System Interface: Block Diagram ...... 13 4.3 Model and Simulation of VNS Device-Neurological- Cardiovascular System Interface: Mathematical Basis ...... 16 4.4 Graphical Tools ...... 17 iii 5. Results ...... 18 5.1 Clinical Study Results: How They Relate to the Simulation . . . . . 18 5.1.1 VNI-VNS Hemodynamic Parameter Improvement Comparisons Between NI-VNS and VNS Implants: BP and HR ...... 18 5.1.2 Comparison of Clinical Results (This Study and Literature) and Simulation Results ...... 20 5.1.3 NI-VNS Hemodynamic Improvement Comparisons Between NI-VNS and VNS Implants: BP vs. HR, BP & HR vs. Time . . . 22 5.2 VNS Modeling/Simulation Results ...... 25 5.2.1 Comparison of Phase-Lock Loop and Nerve Conduction Model With Windkessel and Conventional Nerve Conduction Models ...... 25 5.2.2 Validation/Relevance of the Phase-Frequency Lock Loop Model ...... 28 5.2.3 Simulated Response to a Therapeutic Input ...... 28 5.2.4 The Effects of Feedback Strength on Simulated Responses . . . 31

6. Discussion ...... 34 6.1 Overview of Clinical Study and Simulation Implications ...... 34 6.2 Critique of Model and Simulation ...... 36 6.3 Interpretation ...... 39 6.3.1 VNS Design Verification Issues ...... 39 6.3.2 VNS Design Validation ...... 39 6.3.3 Mechanism-of-Action Issues ...... 40

iv 7.0 Conclusions ...... 41 7.1 NI-VNS vs.VNS Implants ...... 41 7.2 VNS Implant Design Improvement Considerations ...... 42 7.3 Improvements for the Hemodynamic Regulation-Control Model ...... 42 7.4 Mechanism-of-Action Clues ...... 43

References ...... 46

v List of Tables

Table I. Summary of VNS Implant Clinical Studies for Heart Disease Treatment Up to 2014 ...... 6

Table II. NI-VNS Hemodynamic Improvement Results for a Single Subject With Hypertension: All Results Achieved in Less Than Two Weeks of Treatment ...... 19

vi List of Figures

Figure 1. Pneumatic/mechanical system for the introduction of pneumatically driven trapezoidal pressure pulses to the thoracic system (including the left and right vagus nerves) for cardio- pulmonary benefits ...... 3

Figure 2. Cyberonics (now LivaNova) Vagus Nerve (VNS) device implanted on the left side vagus nerve using a programmable pulse generator ...... 8

Figure 3. One form of transcutaneous VNS (t-VNS) ...... 9

Figure 4. First order phase-lock loop cardiac model ...... 15

Figure 5. 2 D Scatter plots and linear regression line plots of systolic BP vs. HR ...... 23

Figure 6. Moving average of systolic BP shown along with the moving average of HR over a relatively long time frame ...... 24

Figure 7. Oscilloscope upper trace showing simulated baroreceptor activity (murine model), and lower trace showing multiplier output from the simulation ...... 26

vii Figure 8. Oscilloscope display of BP (upper trace) and VNS device excitation (lower trace) ...... 27

Figure 9. Simulated oscilloscope display showing an arrhythmia (upper trace) and the result of applying a therapeutic signal that corrects the arrhythmia problem (lower trace) ...... 29

Figure 10. Before-and-after NI-VNS treatment line graph of systolic and diastolic BP vs. time for 4 to 6 NI-VNS treatments ...... 30

Figure 11. Murine simulation plots of HR vs. VNS implant frequency for different levels of neurological feedback ...... 31

Figure 12. Murine simulation plots of HR vs. VNS implant current intensity for different levels of neurological feedback ...... 33

viii 1. Introduction

1.1 Vagus Nerve-Parasympathetic Nervous System Relationship The vagus nerve is often described as a “mind-body connection;” that influences relaxation responses, pain mitigation, digestion, intestinal inflammatory response, for example, with a neural pathway that “wanders” from the medulla oblongata to the abdomen. The vagus nerve is the longest of the twelve cranial nerves and operates as a distributed command-control- communication network within the parasympathetic arm of the autonomic nervous system. To get some idea of where and how the vagus nerve fits in with nervous system form and function, we need to start with a macroscopic view. The major parts of the human nervous system consist of the central nervous system (brain and spinal cord); and the peripheral nervous system (myelinated sensory and motor neurons that branch out from the spinal cord to various appendages) that extend to and serve all parts of the body. Included in the peripheral nervous system is the sympathetic nervous system (prepares the body for activity, mediates fight or flight response, maintains homeostasis) and the parasympathetic nervous system (restoration, repair, feed, breed, rest, and digest). The sympathetic nervous system and the parasympathetic nervous system are part of the autonomic nervous system (ANS) that addresses involuntary functions within the body trunk (heart, lungs, pancreas, gastrointestinal tract, etc.) that do not require thought to take action. The autonomic nervous system is the primary neural mediator of physiological responses to internal and external stimuli. The sympathetic 1 nervous system regulates catabolic responses and the parasympathetic nervous system, with the vagus nerve (cranial nerve X of the parasympathetic nervous system) regulating anabolic responses (Teff, K.L., 2008). The right and left vagus nerves mediate parasympathetic heart activity, with the right vagus nerve innervating the sinoatrial node (heart’s pacemaker) and the left vagus nerve innervating the atrioventricular node (mediates heart electrical current and conduction velocity).

1.2 Vagus Nerve Stimulation (Mechanical) Mechanical compression of the vagus nerve for various nervous system disorders predates the application of an electrical approach by more than 90 years (Yuan, H. and Silberstein, S.B., 2015; Corning, 1884). The mechanical vagus nerve stimulation (VNS) approach, using hand, thumb, finger, and belt pressure, was popular in the 1800s (Waller, A., 1870). A mechanical non-invasive vagus nerve stimulation (NI-VNS) technique, that can be quite effective in controlling heart rate (HR) and reducing blood pressure (BP), utilizes a pneumatically pulsed vest (employing 1 Hz to 2.5 Hz trapezoidal shaped pulses) that stimulates the entire thoracic region, along with the right and left vagus nerve network on both sides of the neck and thorax (O’Clock, G.D., et al.., 2012). The pneumatically pulsed system (shown in Fig. 1) can reduce systolic BP in the range of 15 to 18 mmHg with just a few half hour treatments. After a delay of about four days, diastolic (BP) reductions of 6 to 8 mmHg begin to appear. BP variability was also significantly reduced. The effects on BP can last up to 15 days after the pneumatically pulsed vest treatments have been discontinued. 2

Figure 1. Pneumatic/mechanical system for the introduction of pneumatically driven trapezoidal pressure pulses to the thoracic system (including the left and right vagus nerves) for cardio-pulmonary benefits (lung water secretion, mucus transport, heart rate and blood pressure control) showing pump, vest, hoses; and a pneumotachometer for measurement or airflow and pressure at the mouth. A nose clip minimizes air flow leakage (from O’Clock, G.D., Lee, Y.W., Lee, J., & Warwick, W.J., 2012).

1.3 Vagus Nerve Stimulation (Electrical) VNS has been used as a therapeutic tool to treat various diseases associated with sympathovagal imbalance for many years. Although, as previously stated, therapeutic mechanical compression of the vagus nerve predates the electrical approach by more than 90 years, Waller mentions the possible application of non-invasive “galvanism,” or electrical stimulation in his papers. (Waller, A., 1870). Waller's results convinced him that the non- invasive mechanical effects he was observing involved vagal stimulation and 3 not just a carotid artery stimulation-response; as was the belief at that time. Interestingly enough, Waller describes the vagal system in terms that indicate he recognized the vagus nerve as a substantive part of a distributed command-control-communication network within the nervous system. In the 1880's, the American neurologist James Leonard Corning developed a "carotid fork" transcutaneous VNS device to administer the combination of bilateral carotid artery compression and electrical stimulation (using insulated sponge electrodes and direct current from a galvanic battery) for seizure control in humans (Corning, J.L., 1884). In the late 1930's, seizure control animal studies were done with Faradic stimulation (short duration electrical pulses) of 1 to 50 Hz, with 0.2 to 0.5 msec pulse durations, and peak voltage levels of 2 V (Yuan, H. and Silberstein, S.D., 2015). Human electrical VNS implant study activity for treatment-refractory epilepsy and for chronic treatment-resistant epilepsy increased in the early to mid 1990's. Surgical and therapeutic issues involving safety and tolerability for implanted VNS devices have been summarized by Ben-Menachem, E., et al. (2015). Cyberonics received the first FDA clearance for a surgically implanted electrical VNS device to treat refractory epilepsy in 1997 (Yuan, H. and Silberstein, S.D., 2015).

1.4 Recent Vagus Nerve Stimulation Implant Clinical Results for Heart Disease Applications From 2011 to 2014, pre-clinical data showed substantial benefits with the application of invasive implanted VNS devices for cardiovascular applications. Electrical stimulation of the vagus nerve appears to be cardio- 4 protective (Yamakawa, K., et al., 2014). However, the European Neural Cardiac Therapy for Heart Failure (NECTAR-HF) trial, using a Boston

Scientific implanted VNS device on the patients' right side, did not demonstrate improved cardiac function for heart failure, possibly due to the application of a sub-therapeutic dose in the study cohort (see Discussion section of Zannad, F., et al, 2014). In the 2014 results reported for the Autonomic Neural Regulation Therapy to Enhance Myocardial Function in Heart Failure (ANTHEM-HF) study (Premchand, R.K., et al., 2014), the application of Cyberonic’s implanted VNS device did show improved cardiac function in patients with chronic heart failure (improved ejection fraction and 6 minute walk distance) regardless of whether the device was implanted in the left or right side. Another study, designated as Increase of Vagal Tone in Heart failure (INOVATE-HF), completed by Medtronic’s acquisition (Biocontrol Medical) was considered to be disappointing. Using an insulated VNS cuff with its VNS implanted device, significant reductions in heart failure did not occur, even though some heart function and quality of life enhancement results were achieved (Gold, M.R., et al., 2016). Table I provides an overview of the differences in treatment parameters involved with the three VNS device implant clinical studies. The results that were achieved indicate that VNS for cardiovascular applications may need to be more carefully thought out and evaluated by considering a larger range of VNS options, treatment protocol techniques, treatment parameter choices, and more realistic outcome expectations. Although positive outcomes were achieved with VNS as shown in Table I, the conclusions reached and disappointments expressed by the 5 Table I. Summary of VNS Implant Clinical Studies for Heart Disease Treatment Up to 2014

Study Stimulation Frequency Duty On-Off Outcomes* Current (Hz) Cycle Time (mA) (sec.) 2.0 ± 0.6 10 17.5% 14 on, ~ 3.9 % mean HR reduction ANTHEM- 66 off at 6 months, HF improved LV ejection fraction of 4.5% absolute INOVATE- 3.5 to 5.5 < 1 – 2 25% Heart function.

quality of life HF (synched to – improvements R wave) achieved

NECTAR- 1.24 to 1.42 20 12.5% 10 on & Quality of life improvements HF 50 off achieved *Earlier studies that lacked a sham control group showed promise. But the three 6 to 9 month studies failed to meet some, or all, of their primary end-point objectives associated with cardiac remodeling and functional capacity for symptomatic heart failure patients. However, the 2015 ANTHEM-HF results did provide the best results, especially with the longer 12 month treatment ENCORE follow-up study. ENCORE demonstrated a 6.3% increase in ejection fraction and a 10% decrease in left ventricular end-systolic volume. medical profession with respect to the NECTAR-HF, ANTHEM-HF, and INOVATE-HF VNS implant clinical studies (Hindricks, G., 2014; Gold, M.R., 2016; Olshansky, B. 2016) indicate that unrealistic outcome expectations may have masked the true value of the benefits achieved, or that can be achieved, with VNS implants.

6 2. Background

The vagus nerve is a critical neurological element, affecting many physiological responses associated with regulatory activities of the nervous system. Many health disorders can be addressed by stimulating the vagus nerve electrically or mechanically. For more than 27 years, implanted VNS devices, with electric current outputs in the 1 to 3.5 mA range, have been developed for many health care applications, including epilepsy and heart disease (Penry, J.K. & Dean, J.C., 1990; Olshansky, B., et al., 2008; Rozman, J., et al., 2009; DeFerrari, G.M., et al., 2010; Premchand, R.K., et al., 2014). VNS can provide regulation and control of hormones, inflammation, appetite, disease, and various organ functions by chemical, electrical, mechanical, thermal, ultrasonic, or optical means. Responses to VNS (including mechanical and electrical) do not always occur, and do not always occur immediately. For instance, delayed patient responses to mechanical stimulation of the sinus and vagus nerve have occurred up to four hours after stimulation (Hill, I.G.W., 1932). Fig. 2 shows the direct (invasive) vagus nerve electrical stimulation method for a VNS device that is implanted, and lists some operating parameters appropriate for the human model. Placement of the cuff electrodes for the invasive VNS approach in heart disease applications is confined to a small region of the vagus nerve; employing pulsed current levels of approximately 2 mA at approximately 10 Hz (see Cyberonics Physician’s Manual for VNS Therapy, May, 2003).

7

Figure 2. Cyberonics (now LivaNova) Vagus Nerve (VNS) device implanted on the left side vagus nerve using a programmable pulse generator (capable of being programmed externally non-invasively), with a bipolar electrical lead, and helical electrodes wrapped around the vagus nerve. With this arrangement, both efferent and afferent nerve fibers may be stimulated. The device shown above can operate between 1 Hz and 30 Hz, with output currents up to 3.5 mA, and pulse widths of 130 to to 1000 microseconds (see: Cyberonics/LivaNova website (© 2007-2013), Cyberonics Physician’s Manual for VNS Therapy, May, 2003, and Cyberonics US Patent 8,600,505 B2, Dec. 3, 2003).

The Cyberonics (LivaNova) VNS implanted device serves as the VNS example for this study. The device stimulates the left or right vagus nerve using a programmable pulse generator. It operates at frequencies up to 30 Hz, with a maximum output current of 3.5 mA, pulse widths of 130 to 1000 microseconds, a bipolar electrical lead, and helical electrodes wrapped around the cervical vagus nerve. With respect to various belief systems involving right side vs. left side VNS issues (electrical approach); right side VNS appears to be no more or less dangerous than left side VNS. Right side and left side stimulation each have their peculiarities and differences; and right side stimulation is often 8 regarded as more problematic. However, although right-side VNS device implantation can promote various uncomfortable respiratory events (labored breathing, aggravation of asthma, etc.); left-side thoracic vagal stimulation can decrease the force of heart muscle contractions, and under certain conditions can cause cessation of heart beat (McGregor, A., et al., 2005). Many clinicians now feel that right side invasive VNS implant stimulation is no more risky than left side invasive VNS implant stimulation. The same conclusions have been expressed for transcutaneous right and left side VNS (Chen, M., et al., 2015). So far, low level mechanical and transcutaneous electrical stimulation of the right and left side have not produced any side- effect disasters. In fact, with respect to the invasive VNS implant approach, there are indications that greater improvements in some areas can be achieved with right side VNS (Premchand, R.K., et al., 2014). For the electrical NI-VNS approach in this study, the electrodes are placed on the surfaces of the neck, or neck and ear point (see Fig. 3), using pulsed peak currents of 0.1 to 0.2 mA at multiple frequencies in the range of

Figure 3. In this study, left ear NI-VNS is one form of transcutaneous VNS (t-VNS); The probe electrode is placed in the concha entry point to the ear canal for part of the treatment, and in the forward part of the cymba conchae crease for the second half of the treatment. The counter-electrode can be placed below the earlobe, on the side of the neck, in or near the neck crease (preferred), or under the jaw bone. 9 0.1 Hz to 150 Hz. Electrode placement in NI-VNS involves both art and science. Some placement variations may occur for different conditions or different people. Since the early 2000's, various forms of NI-VNS have shown promising results for pain relief. Some of the side-effect treatment issues for NI-VNS have been described as slight pressure or pain at the stimulation site, paresthesia (prickling or tingling), itching sensations, and skin reactions to conductive gels. However, the capabilities of NI-VNS go far beyond the management of pain and depression. In cardiovascular system response studies, NI-VNS has demonstrated the ability to promote a shift from sympathetic nervous system activity to parasympathetic activity (Clancy, J.A., et al., 2014; Clancy, J.A. 2012; Murray, A.R., et al. 2016). This is an important feature for BP and HR regulation. The potential benefits of VNS with respect to providing BP and HR regulation serves as motivation to develop modeling and simulation tools that can assist in the design and design optimization of VNS devices and associated treatment protocols. In this case, design tools (models, simulations, graphics) need to be developed or improved for the purposes of design verification, design validation, and achieving a better understanding of VNS mechanisms-of-action.

3. Aim, Objectives, and Mission

In this study, the aim is to address invasive VNS and NI-VNS characteristics and responses associated with the burgeoning field of 10 neuromodulation therapy, to help improve hemodynamic control and regulation in cardiovascular disease applications (subsections 1.3, 1.4, 5.1 and 7.1). The objectives are: 1) to acquire and utilize information from clinical data and simulation results that will help guide device and protocol design for new classes of VNS devices (section 2 and subsections 4.1, 5.1.2, 5.1.3, 5.2.1, 5.2.2, 6.3.2, and 7.2), 2) to develop improved model/simulation tools that can provide realistic responses representing interfaces and functional relationships between the cardiovascular system, nervous system, and VNS device (sub-sections 4.2, 5.1, 5.2, 6.2, and 7.3), and 3) to integrate the information obtained from clinical and modeling/simulation efforts to provide mechanism-of-action insights (subsections 5.2.2, 5.2.4 and 7.4). From a biomedical device design validation standpoint, the medical community prefers implants. This preference is driven, in part, by patient compliance and physician control issues. Part of the mission for this study is to make a contribution that will eventually help determine what features, parameters, and characteristics of NI-VNS are useful for improving or evolving the more invasive VNS implant approaches. Results comparisons will be made between the VNS implant and NI-VNS approaches. Another part of the mission is focused on obtaining information that will be useful for the incorporation of device/protocol design parameters and characteristics that improve therapeutic efficacy, enhance outcome stability/reliability, and mitigate adverse event possibilities. The results and conclusions reached will hopefully help to make a difference in life or death consequences and/or quality of life outcomes for many people burdened with cardiovascular related disorders and disease. Three important mission aspects to be addressed are: 1) Determine 11 which NI-VNS results from the literature, laboratory, and clinical studies can help to improve VNS implant device and protocol design (includes sections 1 and 2 and sub-sections 5.2.3, 6.1 and 6.3.1), 2) Provide some indication as to how easy or difficult would it be to make the appropriate changes to incorporate those improvements (includes sub-sections 6.1 and 6.3.2), and 3) provide information that can help to eventually understand VNS mechanisms-of-action (includes sub-sections 5.1.2, 5.2.4, and 6.3.3).

4. Methods

4.1 NI-VNS Clinical Study This study includes a clinical component involving one hypertensive subject, with no valve problems, receiving NI-VNS therapy; and a modeling-simulation component of the VNS device-neurological- cardiovascular system interface, where the simulation was designed to respond to therapy. Hemodynamic (BP and HR) regulation and control issues were the primary interests. In order to obtain BP and HR data non-invasively, two identical commercially available function generators were used to provide frequency variable waveforms in the extremely low frequency and super low frequency ranges. The function generators were adjusted to have identical pulsed outputs with opposite polarities. With this configuration, the clinical study utilized low frequencies (≤ 150 Hz) and low current (0.1 to 0.2 mA peak) to supply various waveforms for NI-VNS stimulation. Gel covered stainless steel probe-electrodes (low NI medical grade) and 3M snap ECG counter- 12 electrodes were used for the NI-VNS clinical study. The contact locations involved: 1) the region of the left and right vagus nerves in one approach; 2) left ear points (providing indirect access to the left side vagus nerve) as shown in Fig. 3, and 3) a point on the neck just under the jaw for the counter-electrodes. Systolic BP, diastolic BP, and HR data were taken morning and evening over a 12 month time period. BP and HR line diagrams and scatter plots were generated from the data in order to compare the effects of invasive VNS and several different NI-VNS techniques on state- of-the-heart conditions and BP-HR variations over time. State-of-the-heart can be defined as the use of selected groups of inter- related cardiovascular and cardio-pulmonary parameters that remain relatively constant or regulated, under specific conditions, and over certain time intervals. The responses of state-of-the-heart parameters (BP, HR, relaxation times, ejection fraction, electrocardiogram parameters, blood velocity and vorticity, etc.) to VNS therapy provide very good indicators concerning the quality of the VNS device/protocol design effort and some information on function that relates to mechanisms-of-action. Although state-of-the heart can be described by multiple parameters, we are only considering BP and HR responses associated with NI-VNS (as shown in Figs. 5, 6 and 10 of the Results section).

4.2 Model and Simulation of VNS Device-Neurological-Cardiovascular System Interface: Block Diagram

The modeling/simulation effort for this work focuses on an active model for the heart (shown in Fig. 4), with multiplicative feedback control, similar to the feedback approach used in phase-lock and frequency tracking 13 applications. The cardiovascular-neural feedback system combines the features of a phase or frequency lock loop percussion-pressure and/or impulse-momentum pump control system that does useful work, with an information transmission capability that manages the work effort. Some of the information transmission and work management capabilities may be aided by short term or long term memory features associated with T-wave morphology (Tseng, G.N., 2012) or hemodynamic parameters (Olufsen, M.S., et al., 2008) as described in the literature. The primary loop in Fig. 4 includes the heart model, waveform shape adjust, barorceptor-arterial region feedback (FB), and multiplier. The primary, or most sensitive loop, can be changed from animal; where the carotid baroreflex appears to have the lower threshold and higher sensitivity (Lau, E., et al., 2016); to human. Considering human applications, the aortic baroreflex "appears" to have the lower threshold and higher sensitivity for normotensive subjects (Sanders, J.S., et al., 1989; Mancia, G., et al., 1982). However, it appears to be just the opposite for hypertensives (Sanders, J.S., et al., 1989). The model/simulation has a phase-lock loop form or structure. This configuration appears to be the most realistic with respect to: 1) the way the heart locks up on single or multiple frequency inputs, and 2) the phase-lock loop or frequency lock kinds of responses measured at cardiovascular system cellular aggregate and organ levels (Guevara, M.R., 1982; Naidu,

V.P.S., 2002) with both mechanical and electrical stimulation. The simulation utilizes the 1999-2003 Electronics Workbench simulation tool that provides an ability to model systems using analog circuit components and interfaces that are programmed digitally. Although much 14 more tedious to implement, the analog approach associated with Electronics Workbench allows much more flexibility for the application in this study compared with other more modern simulation tools such as Simulink.

Figure 4. First order phase-lock loop cardiac model (showing time response variables in a block diagramed Laplace format) coupled with carotid and aortic arch baroreceptor FB. Parasympathetic regulatory capabilities between BP and HR are modeled with an inverse relationship between VCO input voltage and output frequency (heart analog). The BP waveform shape adjust block provides the capability to adjust for certain differences in waveform shape, offset, and amplitude.

The initial inspiration for the cardiovascular system phase-lock loop or frequency-lock model comes from a 1959-1960 prize winning University of Utah undergraduate student paper by R.M. Gardner, which was subsequently published in a 1961 issue of Electrical Engineering. With respect to support for Gardner's ideas; a significant amount of research has shown that the cardiovascular-autonomic nervous system exhibits the properties of a phase- 15 lock loop at the cellular aggregate and organ levels (Guevara, M.R., 1982); Naidu, V.P.S., 2002); Holland, N.L, 2008), especially due to the way the system tends to lock up on single or multiple frequency inputs.

4.3 Model and Simulation of VNS Device-Neurological-Cardiovascular System Interface: Mathematical Basis A mathematical relationship for the BP-HR response function utilizes multiplication in the frequency domain. For this approach, the Fourier series output response signal (or BP analog, VBP (ɷ)), is a function of the heart analog input signal involving a voltage controlled oscillator (VCO) output waveform, expressed as a Fourier series with a peak voltage Vpk, that is multiplied by the first order low pass system transfer function of the carotid baroreceptor, H1(ɷ)):

where τO ≠ TO/2 and 2π /TO ≈ 1/τ1. The ratio τO/TO is the VCO or heart signal duty cycle, K1 is carotid gain, CO is an offset, ɸ is phase, and τ1 is the carotid system time constant. When the Fourier series spectral components of the VCO pulsed waveform (or heart signal) are multiplied by the carotid transfer function frequency response, the spectral components for the output waveform are 16 reduced much more than the DC level. The resulting output spectra ends up representing a sawtooth waveform with a significant offset, similar in structure to the waveforms of Figs. 7 and 8. Expanding on the previous paragraph along with the first paragraph of sub-section 4.2; and referring to Fig. 4 and equations (1) and (2), the heart and nervous system interconnects appear to function as a biologically closed loop phase or frequency locked energy-information control system, consisting of a modulator-demodulator (modem), with the modulator involving the multiplier (X)-carotid baroreceptor low pass filter (H1 (ɷ))-

VNS (V1(ɷ)) interfaces. The heart appears to serve as a signal enhancing and frequency translating repeater simultaneously transmitting information and doing useful work. The heart-carotid barorecptor interface serves as a demodulator that produces a pulsed signal format and provides wave- shaping for the output signal (VBP(ɷ)). And that signal is fed back into the modulator (multiplier).

4.4 Graphical Tools State-of-the heart monitoring for this study does not employ any complicated measurement techniques, such as microneurography and low frequency/high frequency (LF/HF) ratio transition data (Murray, A.R., et al. 2016), or complicated qualitative formats that are associated with many cardiovascular state diagram techniques (see Larsson, M., et al., 2009). From the standpoint of graphical tools, this study uses line diagrams (such as Figs. 6 and 10) that show state-of-the-heart BP and HR variations over time, as well as scatter plots (such as Fig. 5) that indicate state-of-the-heart changes involving transitions between sympathetic and parasympathetic dominance 17 during and after NI-VNS treatment. These graphical tools served as primary therapeutic BP-HR performance enhancement indicators associated with NI- VNS treatment results. Statistical methods (t-tests) were then employed to determine if the changes in BP and HR with NI-VNS treatments were statistically significant. The results were compared with those published in the research literature involving invasive VNS devices and NI-VNS devices in heart disease applications.

5. Results

The results can be viewed from the standpoints of hemodynamic parameter (BP, HR, ejection fraction (EF), etc.) "state-of-the-heart" improvement graphical tools. The results of this study will concentrate on BP and HR improvements (Table II); along with BP vs. time, BP and HR vs. time, and BP vs. HR comparisons before and after treatment (Figs. 5 and 6).

5.1 Clinical Study Results: How They Relate to the Simulation

5.1.1 VNI-VNS Hemodynamic Parameter Improvement Comparisons Between NI-VNS and VNS Implants: BP and HR A summary of the NI-VNS BP and HR data from the thesis clinical study is shown in Table II. Putting aside medical community implant preferences for the moment, the NI-VNS results in Table II indicate that the lower current, multiple frequency, larger duty cycle, non-surgical NI-VNS device can provide better improvements in hemodynamic regulation over 18 much shorter time periods compared with VNS implants.

Table II. NI-VNS Hemodynamic Improvement Results for a Single Subject With Hypertension: All Results Achieved in Less Than Two Weeks of Treatment**

*Confidence Level **Ylikoski, J., et al. (2017) report a reduction in sympathetic preponderance and HR reductions of 6.9% after several weeks of treatment for 75% of their sympathovagal imbalanced patients using NI-VNS at 0.8 mA and 25 Hz, with 60 to 90 minutes of treatment five days each week. (also see Clancy, et al. (20140 and Murray, et al. (2016) using 20-50 mA and 30 Hz for NI-VNS). 19 The differences in results achieved with NI-VNS compared with VNS implants would have to be associated with device design and output differences. Some of the basic differences between the NI-VNS device outputs employed in this study vs. those associated with VNS implants are: 1) the output stimulating currents associated with the NI-VNS approaches in this study are approximately 10 to 20 times lower than VNS implants, 2) there is much more frequency content in the NI-VNS device, 3) for most of the frequency content, the NI-VNS duty cycle is 50%, and 4) various bipolar formats are preferred as waveform structures for NI-VNS rather than the biphasic structures utilized with VNS implants. All of the NI-VNS results of Table II are clinically and statistically significant. However, with respect to confidence levels; it is well known that statistical significance is not necessarily clinical significance (Turk, D.C., 2000; Stommel, M. and Wills, C., 2004). The L-R NI-VNS reductions in mean BP shown in Table II can reduce the relative risk of coronary disease by at least 20% according to Goldman and Ausiello (2007) with a 14 % reduction in recurrent stroke incidence (Marmot, M. and Elliott, P., 2005). According to Palatini (2011) the 4.9% statistically significant L-R NI-VNS 1 reduction in mean HR and the statistically non-significant 5.2% left ear NI- VNS 3 reduction in mean HR can reduce the risk of cardiovascular death and all-cause mortality by at least 6%.

5.1.2 Comparison of Clinical Results (This Study and Literature) and Simulation Results The ANTHEM–HF and ENCORE studies (Table I) tend to support VNS frequencies below 20 Hz for heart disease. Also, below 5 Hz, some of 20 the literature indicates that enhanced murine nitric oxide (NO) production (or release) occurs for 5 V monophasic pulsed VNS waveforms at frequencies in the 2 to 5 Hz range for intestinal applications (Takahashi and Owyang, 1995). However, VNS frequencies in the 10 Hz to 30 Hz range appear to be the most trouble free for the murine simulation. The murine simulation indicated more intense HR and BP aberrations at frequencies of 5 Hz, and below. Under certain conditions for humans, significant increases or decreases in HR can occur with small increases in VNS current levels (Zaaimi, B., et al., 2007). Similar results occurred with the murine model simulation at high and low currents (see Fig, 12). Also, for relatively strong FB levels, significant lock-up differences (Window effects) can occur in the simulation with small changes (≤ 5%) in current. Window effects could account for some human trial inconsistencies. Window effects occur when, as frequency or current increases, the system responds to certain ranges of current or frequency, but does not respond in others, producing alternating ranges of response and non-response. Fig. 1 in Levy, M.N., et al. (1969) shows window effects for P-R and P-P intervals with small changes in VNS frequency administered to a canine model. The window effect often occurs in systems that have positive FB and/or certain kinds of nonlinearities; such as approximate dead zone, hysteresis, etc. But one of the most interesting aspects concerning how the simulation relates to clinical study data involves paradoxical effects in animal studies where under parasympathetic conditions, VNS (electrical) produces responses in HR that resemble sympathetic responses, as reported by Levy, M.N., et al., (1969) and Zaaimi, B., et al., (2007). Using the parasympathetic 21 nervous system dominated model of Fig. 4 in this study, the simulation exhibits the kinds of VNS paradoxical effects that Levy and Zaaini report (see Figs. 11 and 12). These kinds of responses will occur over certain current and frequency ranges with a multiplicative FB system.

5.1.3 NI-VNS Hemodynamic Improvement Comparisons Between NI-VNS and VNS Implants: BP vs. HR, BP & HR vs. Time Considering state-of-the-heart conditions, this study is interested in data that shows transitions between sympathetic and parasympathetic nervous system regulation, as shown in the scatter plot of Fig. 5. This kind of before-and-after NI-VNS treatment change in state-of-the heart can be easily shown using scatter plots for BP and HR; where the transition from sympathetic nervous system dominance (positive correlation between HR and BP) to parasympathetic nervous system dominance (negative correlation between HR and BP) is clearly evident. Fig. 5 shows BP as a function of HR during morning, afternoon, and evening measurements using scatter plots and regression lines. The linear regression line s lopes f or the scatter plots of Fig. 5 were calculated. Figs. 5(a) and (c)) yield a positive systolic BP vs. HR slope of + 0.4 ± 0.2 mmHg/bpm before NI-VNS treatment, indicating sympathetic nervous system dominance. On the other hand, after NI-VNS treatment (Figs. 5(b) and (d)), the slope of the regression line changed to - 0.6 ± 0.13 mmHg/bpm; indicating that NI-VNS produced a change or imbalance in the sympathetic and parasympathetic activities. In order to show sympathetic-parasympathetic transitions clearly, taking too much data can compromise the results. Many scatter plot efforts 22

(a) (b)

(c) (d) Figure 5. For left-right NI-VNS; the 2 D Scatter plots and linear regression line plots of systolic BP vs. HR show sympathetic dominance (positive relationship between BP and HR: for (a) and (c)) before NI-VNS treatment. A parasympathetic dominance. or inverse relationship between BP and HR is shown for (b) and (d)) after NI-VNS treatment. This switch in dominance can be seen on 2 D line graphs (see Fig. 6). But with respect to a visual aid, the strength or degree of this kind of neurological control switching is easier to see with scatter plots and regression lines. have failed to show a correlation between BP and HR because far too much data was taken over long periods of time, where too many state-of-the-heart changes have occurred. To avoid mixing data involving different heart 23 states, individual scatter plots should be limited to data taken over time frames of less than a month (preferably not much more than two weeks) for cardiovascular or cardio-pulmonary applications. A 2 D line graph can also show a state-of-the-heart change before and after an event; such as exercise, drug therapy, or as shown in Fig. 6, before, during and after NI-VNS treatment. The strength of the 2 D line graph is that it can show state-of-the-heart changes for specific times during and after treatment that are not as clear with scatter plots. Fig. 6 shows when the state- of-the-heart change occurred (which was on the first few days of treatment),

Figure 6 Moving average of systolic BP is shown along with the moving average of HR over a relatively long time frame (approximately 6 months). A state-of-the-heart change in amplitude and time duration variability occurs for both BP and HR at time 24, due to the introduction of electrical NI-VNS. and the abrupt transition from a long-wave chaotic BP vs. time characteristic before NI-VNS treatment, to a short-wave chaotic BP vs. time characteristic with much lower maximum BP and HR levels during and after NI-VNS 24 treatment. The scatter plot provides much more clarity with respect to sympathetic-parasympathetic transitions; but the 2D line plot provides more information with respect to the timing and degree of BP and HR changes both during and after treatment. The statistically and clinically significant results achieved with NI- VNS that are shown in Table II and Figs. 5 and 6 indicate that one series of NI-VNS treatments can provide near-term hemodynamic parameter regulation improvements in the range of 4.9% to 8.5% for systolic BP reduction, 3% to 7.6% for diastolic BP reduction, and 4.4 to 7.7% for HR reduction. The NI-VNS treatment protocols that accomplished these results require only 4 to 6 treatments of 12 to 16 minutes each, spaced a few days apart, over a time period of 10 to15 days. In comparison, the VNS implant requires a cyclical 24 hour protocol where the VNS device is actively stimulating ("on") a little over 4 hours during the day for several months; and appears to require many months to achieve a heart rate improvement of 4% or less.

5.2 VNS Modeling/Simulation Results

5.2.1 Comparison of Phase-Lock Loop and Nerve Conduction Model With Windkessel and Conventional Nerve Conduction Models The model and simulation used in this study provide similar cardiovascular waveform responses (see Fig. 7) as those observed with the more commonly used Windkessel models. Compared with Windkessel models, the phase-lock loop model, with an active heart analog, appears to provide the widest variety of realistic responses to various inputs with 25 system parameter and signal level changes. For example, in order for the simulated baroreceptor activity response of Fig. 7 to show a normal baroreceptor response or BP response with well

Figure 7 The oscilloscope upper trace shows simulated baroreceptor activity (murine model), and the lower trace shows the multiplier output from the simulation. The upper trace is very similar to the Windkessel simulation of BP and baroreceptor activity from Holland (2008).

defined ramp waves riding on top of the offset, the simulated ANS coupling to the cardiovascular system needs to be relatively weak, the baroreceptor transfer function time constant needs to be relatively low. Some interesting results occur when the interface between the VNS device and the nervous system incorporates simulated nerve fiber conductivity modulation feature, as described by Swadlow, Kocsis, and Waxman, (1980), and Grinberg, et al, (2008). These papers provide strong support for nerve fiber conductivity modulation, and they provide an excellent overview on how higher currents can be carried along the near surface of the nerve fascicle/perineurium bundle, influencing conductivity in other regions of the nerve (such as the action potential). More of this kind of information can be extracted with the phase-lock loop approach than the standard Windkessel model/simulation provides. 26 From the standpoint of simulated VNS response, depending upon the heart parameters and level of ANS-heart feedback, HR can respond with a sympathetic or parasympathetic dominance. Fig. 8 shows a sympathetic-like response where the HR is in its normal "state" with no VNS input; and the HR then responds to the VNS input with an increase that is locked on to and follows the VNS output frequency. Getting to the "core" of the model and simulation validation issue, with respect to most Windkessel models, the Windkessel heart is modeled as a passive device (integrator) or as an independent source with no active FB control. Although, for a limited time frame, the heart can sustain life

Figure 8 Oscilloscope display of BP (upper trace) and VNS device excitation (lower trace) where the upper trace represents a range of approximately 80 mmHg to 130 mmHg, at a typical murine HR of 300 to 600 bpm; and the lower trace peak pulse represents a potential of approximately 0.25 V that is associated with a 2.5 mA simulated VNS stimulation current. independently (such as a non-innervated transplant), the heart in a normal cardiovascular system is far from being a passive independent system. Also, the cardiovascular-autonomic nervous system interface appears to involve multiplication (van de Vooren, H., et al., 2007), neural modulation 27 (Swadlow, Kocsis, and Waxman, 1980); Grinberg, et al,, 2008), and phase or frequency locking of an active heart (Guevara, M.R., Glass, L., Shrier, 1982; Naidu, V.P.S., 2002). This combination does not fit well with standard Windkessel models.

5.2.2 Validation/Relevance of the Phase-Frequency Lock Loop Model Looking at model and simulation validation issue from another point of view; electrocardiogram spectral variations have been attributed to the enhancement of related parasympathetic activity and synchronization or entrainment of heart rate (Holland, N.L, 2008; Yasuma, F. and Hayano, J., 2004; O’Clock, G.D., et al., 2012). To address these spectral characteristics realistically, the model requires some kind of phase or frequency lock loop with respect to the cardiovascular system and its neurological interface. Considering synchronization, entrainment, and locking phenomena; it has been suggested that the heart is a frequency variable oscillator in a phase- lock loop with a multiplicative physical coupling between the nervous system and the cardiovascular system (Holland, N.L., 2008; Naidu, V.P.S., 2002; Guevara, M.R., Glass, L., and Shrier, 1982).

5.2.3 Simulated Response to a Therapeutic Input

When incorporated into a simulation, many models will show responses due to change of parameters, representing a transition from a normal state to a diseased or wounded state. But they often do not respond to a therapeutic input with the introduction of an exogenous source. The simulation in this study demonstrates a therapeutic effect (Fig. 9) with the

28 introduction of an external VNS source to an arrhythmia condition. The arrhythmia was produced by a simulated nervous system or heart condition, and is similar to the murine arrhythmia or premature ventricular contraction shown in Annoni, et al. (2015).

The simulated therapeutic effect occurred at frequencies of 20 Hz to 26 Hz, duty cycles of 4% to 6%, and VNS current levels of 5 mA to 8 mA which matches fairly closely with therapeutic results appearing in the literature. What is interesting about Fig. 9 is that, in this simulation, a

(a)

(b) Figure 9 The simulated oscilloscope display of (a) shows an arrhythmia (upper trace) similar to those shown in Annoni, et al. (2015). The simulated heart beat aberration of (a) was produced by the use of cardiovascular parameters and nervous system inputs that tend to produce unstable responses. The display of (b) shows the result of applying a therapeutic signal that has a high enough energy to correct the arrhythmia problem. narrow pulse signal generated by the nervous system, that involves only

29 0.5% of the heart output energy, produces a heart beat aberration. But the heart beat aberration requires a therapeutic VNS output energy that is approximately 3.3% of the heart output energy in order to stabilize the heart beat. In this case, in the simulation, it requires almost 7 times more energy to re-stabilize the cardiovascular-neural interface and heart beat than it does to produce the original heart beat aberration. One of the simplest ways to show the dramatic effects NI-VNS can have on BP is to compare systolic and diastolic BP (see Fig. 10) on a line diagram before, during, (the arrow in Fig. 10 marks the starting point) and after administering a number of treatments. After NI-VNS treatment, there is a significant reduction (approximately 50%) in peak-to-peak systolic BP during a specific time frame, along with a reduction by a factor of 4 in the time frame involving maximum-to-minimum BP variations.

↑ Figure 10 Before-and-after NI-VNS treatment line graph of systolic and diastolic BP vs. time for 4 to six NI-VNS treatments showing changes in peak and peak-to-peak systolic BP over a 16 day time frame (the arrow points to the time NI-VNS treatment started). 30 5.2.4 The Effects of Feedback Strength on Simulated Responses Fig. 11 represents two opposite conditions that were simulated. In an extreme case, parasympathetic nervous system control can be made so weak that, at mid-range, it produces an almost linear positively correlated HR vs. VNS frequency characteristic (blue line). A strong parasympathetic nervous

Figure 11 Murine simulation plots of HR vs. VNS implant frequency where, in an extreme case (blue line), weak parasympathetic feedback can initially promote a positive correlation (seemingly sympathetic) between VNS input frequency and HR, and progress toward an unrealistic HR with further increases in VNS current intensity. The red line shows a more realistic case for parasympathetic feedback that is much stronger, with the correlation between VNS input frequency and HR becoming negative (more on the parasympathetic side) after initially exhibiting a positive characteristic. system feedback produces a more regulated HR vs. VNS frequency characteristic with upper and lower limits (red line). As Gierthmuehlen, M., et al (2016) and Brack et al. (2004) indicate, HR either remains relatively constant, or decreases, with both murine and 31 rabbit left vagal VNS frequency increases. However, different levels of simulated parasympathetic FB can promote responses to VNS input frequency variations that are more like those associated with sympathetic responses (see human patient data from Mirkovic, T., et al., 2012). This kind of response, involving positive correlations between VNS frequency and HR occurring over small ranges of VNS input frequency, are shown in Fig. 11. With respect to simulated VNS electric current intensity vs. HR, very weak parasympathetic FB can promote extreme increases in HR as VNS electric current increases (red line, Fig. 12). Some of this kind of behavior can be observed at low VNS current levels in the literature (Kember, G., et al. 2014). On the other hand, enhanced levels of parasympathetic FB can promote a more extended response (blue line, Fig. 12). As a result, it appears that well regulated VNS response characteristics for mid-range input frequencies and mid-range input currents, shown in Figs. 11 and 12, can occur over a wide range of ANS FB levels. There has been a significant amount of work showing that there are both nervous system and chemical interactions between parasympathetic and sympathetic nervous system elements (Levy, M.N., 1971). However, the simulation exhibits these kinds of seemingly interactive responses on its own; indicating that the sympathetic-like responses in the parasympathetic system environment may be due to phase-lock loop or frequency lock characteristics of the VNS-nervous system-cardiovascular system structure. Saku, K., et al. (2014) show murine HR only decreasing with a VNS applied voltage (0-8 V) and frequency (5 to 50 Hz); whereas Mirkovic, T., et al. (2012) show both increases and decreases in human HR at specific levels of current (0-2.5 mA) over specific frequency ranges (between 10 and 30 Hz). 32 Over time, Ippolito, et al. (2013) show increases in murine HR with chronic VNS at currents between 0.5 and 1.0 mA. Some of these parasympathetic-

Figure 12 Murine simulation plots of HR vs. VNS implant current intensity for very weak FB (red) and a more enhanced level of feedback (blue). At low and high electric current levels, this figure shows unrealistically high heart rates for low and high stimulating currents with these different FB levels. However, at mid-range, the VNS stimulated HR and change in HR are reasonable and controllable for a wide range of FB levels and VNS output current intensities. sympathetic response conflicts can be attributed to the differences that often occur between animal model and human studies (Shanks, N., et al., 2009). From information provided in the literature, and the results of this study; it may be important for VNS device and protocol designers and developers to recognize that with respect to parasympathetic FB variations and VNS operating conditions; VNS electrical stimulation can produce sympathetic-like responses within certain frequency ranges at both low and 33 high frequencies (from human patient data provided by Mirkovic, T., et al., 2012), at low VNS current levels (see Kember, G., et al. 2014), and at high current levels (see Fig. 12).

6. Discussion

6.1 Overview of Clinical Study and Simulation Implications Addressing the aim, objectives, and mission of this study as they relate to invasive VNS and NI-VNS characteristics, responses, and implications; at this point in time, hemodynamic regulation/control results achieved in the NI-VNS clinical study appear to be as good as or better than results achieved with VNS implants. The VNS simulation indicates that in order to minimize the possibility of encountering instabilities, heart rate aberrations, and window effects; VNS implant operating parameters involving frequencies of 10 to 20 Hz, current levels in the range of 1.5 to 2.5 mA, duty cycles of approximately 10%, a biphasic waveform, and an electrode cuff ; all appear to provide an appropriate combination to achieve reliable and reproducible results. On the other hand, the NI-VNS clinical study effort produced very good results with respect to BP and HR regulation (both from the standpoints of mean BP-HR reduction and variability), using peak currents of 100 µA, frequencies in the range of 0.1 Hz to 150 Hz, duty cycles of approximately 50%, and a variety of bipolar waveforms. The NI-VNS electrodes were placed either on an ear point and lower jaw, or on each side of the throat. In comparing the VNS implant and NI-VNS results, it would appear that VNS implant design might profit by considering bipolar 34 waveforms, more frequency content, lower currents, and other electrode configurations. The lower currents and wide range of frequencies utilized in the NI- VNS approach appear to promote a high level of consistency in response, no significant risks, no adverse events, and very low costs. For comparison, the costs associated with a VNS implant are approximately $25,000 to $30,000 (Donovan, C.E. 2006), and there are surgical risks and discomfort issues. The difference in near-term costs between a VNS implant and NI-VNS appear to be somewhere between 5-to-one and 8-to-one. Regarding clinical study implications, comparisons of the NI-VNS results of Table II with the VNS implant results of Table I indicate that a multiple frequency approach should be seriously considered for VNS implants in hemodynamic regulation applications. Since the multiple frequency approach, in the range of 0.1 Hz to 150 Hz, provides much better hemodynamic regulatory control over much shorter periods of time compared with the single frequency technique, this advantage by itself supports the idea that incorporation of multiple frequencies deserves some serious consideration for VNS implants. Multiple frequencies would be relatively easy to implement considering the significant advances in integrated circuit technology and interconnects that have been made. Also, considering long term efficacy for many health issues, VNS is often considered as not being curative. But, under some conditions, chronic cyclic nerve stimulation has proven to have beneficial electrophysiological effects with respect to action potential duration, repolarization, myocardial conduction velocity, etc. (Lee, S.W., et al., 2016). From the standpoint of certain heart conditions, could VNS implantation approach a curative result 35 if applied early enough or if applied in some kind of sequenced combination with NI-VNS? The answer to that question goes beyond the boundaries of this study; and that combination conflicts with current design validation requirements dominated by medical profession preferences. But, along with the advantages shown in the preceding paragraph, the question should be kept in mind as the VNS approach and medical attitudes evolve.

6.2 Critique of Model and Simulation Simulation information can assist the device/protocol design effort by: 1) identifying the range of parameter types and parameter values that can produce the results achieved in the VNS clinical environment, 2) determine where instabilities and inconsistencies might occur, and 3) provide some idea of how much better (or worse) those results might be with device/protocol parameter variations. However, all models have weaknesses and short-comings when applied to a simulation effort. The model developed for this study is no exception. One of the more significant weaknesses of the model is that it represents a distributed cardiovascular-neural system with its various interconnects using a lumped system format. This approach produces significant controllability and observability problems. A significant amount of activity is hidden within the lumped parameter structure. A hybrid approach would be more realistic. The hybrid simulation could involve a lumped element approach for the electrical excitation system of the heart, diode switching circuits for valve action, and distributed networks for parts of the vascular system and nervous system. The feedback system model shown in Fig. 4 is often referred to as a phase-lock loop as it relates to the cardiovascular-nervous system interface. 36 From the standpoint of rigor, referring to that diagram as a phase-lock loop is not accurate. The model is often designated as a phase-lock sub-system in the research literature due to certain spectral response characteristics observed with electrocardiograms, and the cardiovascular system's ability to synchronize with respiratory signals. However, Fig. 4 is not a classical phase-lock loop by any stretch of the imagination. There are a number of significant differences between Fig. 4 and a classical phase-lock system including: 1) cardiovascular physiology requires that the relative positions of the low pass filter (carotid baroreceptor analog) and the voltage controlled oscillator (heart analog) must be reversed compared to their positions in a conventional phase-lock loop, 2) the error signal (multiplier output that drives the voltage controlled oscillator (VCO)) is not filtered, and the error signal itself is not small. In fact, at times the error signal is huge compared with error signals associated with conventional phase-lock loop or feedback control servo systems, 3) the primary loop signals do not have the same form at various points around the primary loop, 4) depending upon the FB level, the system sometimes behaves as a frequency-lock loop, 5) the analog representation for the aortic arch baroreceptor FB intensity levels can induce substantial changes in the VCO output frequency that vary significantly from the input signal frequency, 6) the output waveform has a substantial offset (which is the analog representation of the diastolic BP level), 7) when incorporated into a simulation, this model's loop gain is only around 2,300. Typical loop gains for a conventional phase-lock loop control and tracking system in communication or information transmission systems are two to three orders of magnitude higher. 8) if neural modulation is to be considered as a substantive conduction mechanism, the vagus nerve-aortic arch 37 baroreceptor feedback could involve multiplication, or a combination of multiplication and addition rather than just simple addition, 9) there is no active frequency translation or conversion in the feedback loops of Fig. 4 when making the transition from the baroreceptor sensors to the nervous system, and from the nervous system back to the heart, and 10) in the Fig. 4 FB model, the baroreceptors are modeled as passive elements similar to many low-pass baroreceptor models published in the literature (Moin, L., et al., 2017; Mahdi, A., et al., 2013; Le Rolle, V., et al., 2008). These passive representations have similar modeling and simulation shortcomings for baroreceptor representation as the passive representations for the heart impose in many Windkessel models. With some adjustments in the heart analog, a more active baroreflex model similar to van de Vooren, H., et al. (2007) could provide a more realistic baroreceptor reflex response for follow-on work. These short-comings do not seem to produce serious simulated cardiovascular system waveform and response discrepancies; but they do need to be thought out for more rigorously structured VNS-neurological- cardiovascular system simulation efforts. However, this study indicates that any attempt to analyze the VNS- neurological-cardiovascular system and its interfaces will require some differences in technique compared with standard Windkessel approaches. And even with those non-standard approaches, a more rigorously structured model and simulation will not be complete enough under conventional phase-lock loop or frequency-lock loop analysis and formulations. Although the model/simulation of Fig. 4 is credible and can be useful, it is a "work in progress," with significant room for improvement. 38 6.3 Interpretation

6.3.1 VNS Design Verification Issues Referring to sub-section 6.1, the bipolar frequency variable (~50% duty cycle ) NI-VNS waveform utilized in the clinical part of this study is significantly different compared with the low duty cycle biphasic waveforms used in VNS implants and other NI-VNS devices. The results achieved with the waveform used in this study strongly indicate that VNS designers need to re-evaluate the biphasic waveform choices that dominate present VNS design. Also, a number of simulated VNS-cardiovascular system-neural system response instabilities agree with a number of statements made by those in the design and development of VNS implants. In general, for the single frequency approach, VNS frequencies should be confined to less than 20 Hz for cardiovascular BP-HR regulation applications.

6.3.2 VNS Design Validation As indicated in sub-section 6.1, VNS design validation requirements are currently dominated by the medical profession. Patient compliance issues, monitoring/control, and minimization of "hands on" requirements for treatment will favor the VNS implant approach over NI-VNS for heart disease applications. Also, even considering invasive/non-invasive VNS complementary possibilities, design validation will most likely continue to be dominated by the current medical profession's preference for implants. The "signals" from the medical community and the clinical results from this study indicate that it may be prudent for NI-VNS interests to consider a miniaturized version of the present NI-VNS hardware to be superficially 39 implanted. The implanted device and electrodes could provide much lower current levels in the region of the vagus nerve compared with the higher currents associated with VNS implant electrodes that are clamped around the vagus nerve. In this manner, the superficially implanted NI-VNS hardware could serve in a complementary or stand-alone mode, and would be in an acceptable "form" to meet the preferences of medical doctors.

6.3.3 Mechanism-of-Action Issues From the standpoint of "function," both the literature and simulation indicate that under parasympathetic dominance, sympathetic-like VNS responses can occur over certain ranges of VNS frequency and electric current with different levels of parasympathetic FB for both human and different animal models. For VNS mechanism-of-action studies, the human and animal responses may relate quite well to one another, and better than we realize. The problem of conflicting results or different results between human and animal studies may be more associated with "reproducibility" and "repeatability" rather than function, along with a lack of attention to state-of-the-heart variations that can place significant restrictions on clinical data acquisition (protocol, quantity of data, timing, etc.). To avoid instabilities and HR aberrations, the simulation indicates that ANS coupling to the cardiovascular system prefers a weak level of feedback. The simulation results also indicate that the VNS implant spiral cuff may be reducing the effect of certain elements of ANS feedback, along with possibly promoting some form or nerve fiber or nerve bundle conductivity modulation. The spiral cuff placement may contribute to a stabilizing and regulating influence with respect to the VNS-cardiovascular-nervous system 40 interface. However, these assertions need to be explored in more detail before conclusive statements can be made.

7.0 Conclusions

7.1 NI-VNS vs.VNS Implants Reviewing some of the comments from sections 5 and 6, and considering the Aim, Objectives, Mission, and Implications; compliance issues, monitoring/control, and the minimization of “hands on” requirements in administering treatment will most likely favor the invasive VNS implant approach over non-invasive VNS techniques for heart disease patients who need heart regulation assistance. And even considering non- invasive/invasive complementary possibilities, the complementary treatment feature may still not be acceptable from the standpoint of compliance and “hands on” issues. This is a political reality that the medical community imposes on medical device design validation (i.e. Did we design the right device?). From the standpoint of near-term impact; the usefulness and value of the non-invasive effort may be associated with information it can provide on waveform structure, frequencies, frequency range coverage, neural modulation, stimulation, current intensities, treatment/stimulation duration, and reliability/stability issues that may be relevant and applicable for VNS heart disease and heart regulation applications in general. However, ignoring the dominant effects of the medical community on medical device design validation and focusing on control and regulation of BP, HR, and hemodynamic variability; for a number of near-term and long- 41 term hemodynamic control purposes, NI-VNS provides significantly better results when compared with VNS implants. In fact, the simulation indicates that NI-VNS can have a beneficial impact on heart beat aberrations. However, with respect to immediate response to heart beat aberrations, the VNS implant approach can react much faster to individual aberration events.

7.2 VNS Implant Design Improvement Considerations Addressing the combination of Aim, Objective, and Mission issues; the NI-VNS clinical study results indicate that incorporating multiple frequencies and certain kinds of bipolar waveforms with VNS implants would be relatively easy to do (IC chip technology offers significant adaptability advantages), and could promote enhanced hemodynamic performance over much shorter time frames (days rather than months). Also, the NI-VNS results indicate that short-term "reactive" capabilities of the VNS implant might be combined with a periodic therapeutic protocol (multi-mode approach) to provide advantages offered by both NI-VNS and VNS implants when medical community attitudes are more receptive to this possibility.

7.3 Improvements for the Hemodynamic Regulation-Control Model This model for the heart-nervous system-VNS interface (that responds to therapeutic inputs) appears to be (so far) the first to go beyond the conventional Windkessel approach, and actually utilize a phase-lock loop model with an "active" heart. Many authors have mentioned the phase-lock loop model in their research papers and theses, but they have not provided a functioning phase-lock loop simulation that responds to therapy. In order to 42 meet the requirements of the analytical effort associated with VNS in hemodynamic regulation, the more realistic model that also addresses therapy issues is critically important in order to provide analytical tools that can assist in VNS device/protocol design improvement. A more realistic model would have portions the vascular and nervous system modeled using a distributed system structure, frequency conversion in going from heart pressure pulses to nervous system action potentials, an active baroreceptor reflex configuration, and a hybrid lumped-distributed element heart model that offers more flexibility compared with the 555 timer (VCO) used in the simulation for this study.

7.4 Mechanism-of-Action Clues Although there is much to be done with respect to characterizing VNS implant and NI-VNS mechanisms-of-action, a core view has been evolving. Animal and human studies have shown promise for the application of VNS implants to improve parasympathetic activity and enhance hemodynamic performance (Akdemir, B. and Benditt, D.G., 2016; Premchand, R.K., et al., (2014); Mirkovic, T, et al. (2012)). However, there appear to be some critical factors to address with respect to VNS implant electrode design and placement. In some animal studies (porcine), VNS implants appear to activate certain afferent vagus nerve fibers, which reflexively can inhibit cardiac parasympathetic efferent electrophysiological and hemodynamic effects (Yamakawa, K., et al., (2014)). On the other hand, in human studies, certain NI-VNS techniques (30 Hz, 10-50 mA tragus ear point stimulation, and 25 Hz, 0.8 mA lower ear point stimulation), appear to increase 43 parasympathetic activity and reduce sympathetic activity. In a number of publications, the combined results of these authors show NI-VNS induced shifts in the preponderance of sympathetic activity, decreases in the low- frequency to high-frequency ratio (LF/HF) of HR variability, reductions in resting heart rate, and improvements in ANS balance; by means of activating the afferent branch of the vagus nerve along with other sensory nerves (Deuchars, S.A, et al. (2017); Antonino, D., et al. (2017) Ylikoski, J., et al., (2017); Murray, J., et al., 2016; and Clancy, J. et al. (2014)). The one subject NI-VNS clinical results in this study appear to support these sympathetic- parasympathetic shifts and ANS balance capabilities reported for NI-VNS in the literature. Although this study is not going to provide substantial clarity to the many VNS mechanism-of-action issues and conflicts; the results indicate; 1) the interface between certain elements of the autonomic nervous system and the heart appears to be multiplicative (which helps to support a phase-lock loop model for the heart), 2) the sympathetic responses (that are like those in the literature) for a simulation that is based on parasympathetic elements provides further validation for the phase-lock loop model, 3) the phase-lock loop simulation shows a preference for weak to very weak ANS FB in order to provide stability and reliable operating responses associated with different ranges of VNS frequency and current, 4) The simulation indicates that there may be complementary or synergistic aspects with combined NI-VNS and VNS implant approaches, and 5) Changes in the state-of-the-heart can cause significant misinterpretation and confusion with mechanism-of-action assessment if parasympathetic dominated and sympathetic dominated data become "mixed." 44 The understanding of VNS mechanisms-of-action will require a more complete ANS model (involving both sympathetic and parasympathetic elements), more detailed VNS-nervous system interaction models, a model that incorporates some of the interaction features between sympathetic and parasympathetic elements at the cardiovascular interface, and improved simulation tools.

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