Optimizing Ultrasound-induced Anti-inflammation in Human Subjects
Phase: NA
Conditions: Healthy Subjects
Sponsor: University of Virginia

Ultrasound anti-inflammation in human subjects   Version Date: 18 February 2025 
 
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Study Title: Optimizing Ultrasound-induced Anti-inflammation in 
Human Subjects 
 
NCT number: NCT05685108 
 
Version Date: 18Feb2025 
 

Ultrasound anti-inflammation in human subjects   Version Date: 18 February 2025 
 
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OPTIMIZING ULTRASOUND-INDUCED ANTI-
INFLAMMATION IN HUMAN SUBJECTS 
 
Short title: Ultrasound anti-inflammation in human subjects 
 
Protocol Number: 1.4 
 
National Clinical Trial (NCT) Identified Number: NCT05685108 
 
Principal Investigator: Mark D. Okusa, MD, FASN 
 
Funded by: National Institutes of Health (NIH) and Division of 
Nephrology, Department of Medicine, University of Virginia 
  
Version Date:  18 February 2025 
 
 
Confidentiality Statement 
The information contained in this document is the property of the University of Virginia, Division of 
Nephrology; Center for Immunity, Inflammation & Regenerative Medicine. Distributing, copying, or 
disclosing information contained in this document requires prior authorization from the University of 
Virginia, Division of Nephrology; Center for Immunity, Inflammation & Regenerative Medicine, except 
that this document may be disclosed to the University of Virginia Institutional Review Board for Health 
Sciences Research, and representatives of the U.S. Food and Drug Administration or other 
regulatory agencies as required by law>.   

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Key Roles and Study Governance 
Sponsor  
UVA Department of Medicine  
 
Principal Investigator 
 
Mark D. Okusa, MD, FASN; Division of Nephrology, Center 
for Immunity, Inflammation & Regenerative Medicine, 1300 
Jefferson Park Avenue – West Complex, 5th floor, 
Charlottesville, VA 22903-0133, Telephone: 434924.2187, 
Fax: 434.982.5575, Email: mdo7y@virginia.edu 
 
Sub-Investigators at the 
University of Virginia  
John A. Hossack, BS, PhD; 
Biomedical 
Engineering, 
Electrical 
and 
Computer 
Engineering, MR5 Rm 2121, 
415 
Lane 
Road, 
Charlottesville, VA 22908, 
Telephone: 
434.243.5866, 
Fax: 434.982.3870, Email: 
jh7fj@virginia.edu 
 
William 
T. 
Nash, 
PhD; 
Division 
of 
Nephrology, 
Center 
for 
Immunity, 
Inflammation, 
& 
Regenerative 
Medicine, 
1307 Lane Rd, Pinn Hall 
Charlottesville, VA 22903, 
Telephone: 
434.924.9628, 
Email: wtn3wq@virginia.edu 
 
Faeq Husain Syed, MD; 
Division 
of 
Nephrology; 
Hospital 
Drive/Outpatient 
Clinics 
Building 
McIntire 
Wing, 5th Floor, Room 5485 
Charlottesville VA 22908, 
Telephone: 
434.243.9322, 
Email: yev5bn@virginia.edu 
 
 
Biostatistician: 
Jennie Z. Ma, PhD; Department of Public Health Sciences, 
P O Box 800717, Charlottesville, VA 22908, Telephone: 
434.243.5778, 
Fax: 
434.243.5787, 
Email: 
jzm4h@virginia.edu 
 
Authors: 
 
N/A 
 
 
 

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STATEMENT OF COMPLIANCE ................................................................................... 6 
ABBREVIATION ............................................................................................................ 7 
1 
PROTOCOL SUMMARY ..................................................................................... 8 
1.1 
SYNOPSIS .......................................................................................................... 8 
1.2 
SCHEMA ...........................................................................................................11 
2 
INTRODUCTION ................................................................................................13 
2.1 
STUDY RATIONALE ............................................................................................13 
2.2 
BACKGROUND ...................................................................................................13 
2.3 
RISK/BENEFIT ASSESSMENT ..............................................................................14 
3 
OBJECTIVES AND ENDPOINTS .......................................................................15 
4 
STUDY DESIGN .................................................................................................18 
4.1 
OVERALL DESIGN ..............................................................................................18 
4.2 
JUSTIFICATION FOR DOSE ..................................................................................19 
4.3 
END OF STUDY DEFINITION ................................................................................19 
5 
STUDY POPULATION .......................................................................................19 
5.1 
INCLUSION CRITERIA .........................................................................................19 
5.2 
EXCLUSION CRITERIA ........................................................................................20 
5.3 
JUSTIFICATION FOR STUDY POPULATION ............................................................21 
5.4 
LIFESTYLE CONSIDERATIONS .............................................................................21 
5.5 
SCREEN FAILURES ............................................................................................22 
5.6 
STRATEGIES FOR RECRUITMENT AND RETENTION ...............................................22 
6 
STUDY INTERVENTION ....................................................................................22 
6.1 
DESCRIPTION OF STUDY INTERVENTIONS ...........................................................23 
6.2 
DOSING AND ADMINISTRATION ...........................................................................24 
6.3 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY ........................................27 
6.4 
STUDY INTERVENTION COMPLIANCE ...................................................................27 
6.5 
REGISTRATION, RANDOMIZATION AND BLINDING..................................................27 
6.6 
CONCOMITANT THERAPY ...................................................................................28 
7 
STUDY CLOSURE, STUDY INTERVENTION DISCONTINUATION AND 
PARTICIPANT DISCONTINUATION OR WITHDRAWAL ............................................28 
7.1 
STUDY DISCONTINUATION AND CLOSURE ...........................................................28 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL ...................................................28 
7.3 
DOSE-LIMITING TOXICITY ...................................................................................29 
7.4 
PROCEDURES FOR DISCONTINUATION OF STUDY INTERVENTION ..........................29 
7.5 
LOST TO FOLLOW-UP ........................................................................................29 
8 
STUDY ASSESSMENTS AND PROCEDURES .................................................29 
8.1 
CLINICAL ASSESSMENTS ...................................................................................29 
8.2 
RESEARCH SPECIMEN COLLECTION ...................................................................30 
8.3 
CORRELATIVE STUDIES .....................................................................................31 
8.4 
PARTICIPANT REPORTED OUTCOMES .................................................................31 
9 
DATA AND SAFETY MONITORING PLAN ........................................................31 
9.1 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS ............................................31 
9.2 
UNANTICIPATED ADVERSE DEVICE EFFECTS .......................................................35 
9.3 
REPORTING EVENTS TO PARTICIPANTS ..............................................................35 

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9.4 
EVENTS OF SPECIAL INTEREST ..........................................................................35 
9.5 
REPORTING OF PREGNANCY ..............................................................................35 
9.6 
UNANTICIPATED PROBLEMS ...............................................................................35 
9.7 
DATA BREACH ..................................................................................................36 
9.8 
PROTOCOL DEVIATION ......................................................................................36 
9.9 
PARTICIPANT WITHDRAWALS/DROPOUTS PRIOR TO STUDY COMPLETION .............37 
10 
STATISTICAL CONSIDERATIONS ...................................................................37 
10.1 
STATISTICAL HYPOTHESES ................................................................................37 
10.2 
SAMPLE SIZE DETERMINATION ...........................................................................38 
10.3 
POPULATIONS FOR ANALYSES ...........................................................................39 
10.4 
STATISTICAL ANALYSES .....................................................................................39 
11 
REGULATORY AND OPERATIONAL CONSIDERATIONS ..............................41 
11.1 
REGULATORY AND ETHICAL CONSIDERATIONS ....................................................41 
11.2 
DATA HANDLING AND RECORD KEEPING .............................................................44 
11.3 
PUBLICATION AND DATA SHARING POLICY ..........................................................44 
11.4 
CONFLICT OF INTEREST POLICY .........................................................................45 
12 
REFERENCES ...................................................................................................46 
13 
APPENDICES ....................................................................................................49 
13.1 
SCHEDULE OF ACTIVITIES (SOA) ........................................................................49 
13.2 
REPORTING TABLE ............................................................................................49 
13.4 
PROTOCOL AMENDMENT HISTORY .....................................................................50 
 
 
 

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STATEMENT OF COMPLIANCE 
The trial will be conducted in accordance with applicable United States (US) Code of 
Federal Regulations (CFR). International Conference on Harmonisation Good Clinical 
Practice (ICH GCP) guidelines will be incorporated consistent with institutional practice. 
The Principal Investigator will assure that no deviation from, or changes to the protocol 
will take place without documented approval from the Institutional Review Board (IRB), 
except where necessary to eliminate an immediate hazard(s) to the trial participants. All 
personnel involved in the conduct of this study have completed Human Subjects 
Protection and GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials 
will be submitted to the IRB for review and approval. Approval of both the protocol and the 
consent form must be obtained before any participant is enrolled. All changes to the 
consent form will be IRB approved; a determination will be made regarding whether a new 
consent needs to be obtained from participants who provided consent, using a previously 
approved consent form. 
 
 
For single site UVA investigator-initiated trials without an IND/IDE
 
 
 
Sponsor-Investigator 
 
______________________  
_______________________  
_________ 
Name (print)  
 
 
Signature 
 
 
 
Date 
Principal Investigator 
 
______________________  
_______________________  
_________ 
Name (print)  
 
 
Signature 
 
 
 
Date 

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ABBREVIATION 
AE 
Adverse Event 
AKI 
Acute kidney injury 
CAP 
Cholinergic anti-inflammatory pathway 
CFR 
Code of Federal Regulations 
CMP 
Clinical Monitoring Plan 
CONSORT Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DSMB 
Data Safety Monitoring Board 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
ELISA 
Enzyme-linked immunosorbent assay 
FCCF 
Flow Cytometry Core Facility 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
IDE 
Investigational Device Exemption 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISPTA 
Spatial-peak temporal-average intensity 
LPS 
Lipopolysaccharide 
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
Mechanical index 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NSAIDs 
Non-steroidal anti-inflammatory drugs 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
UVA 
University of Virginia 
 
 
 
 

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1 
PROTOCOL SUMMARY 
1.1 
Synopsis  
Title: 
Optimizing ultrasound-induced anti-inflammation in human 
subjects 
Study Description:  
 
 
 
 
 
Objectives & 
Endpoints: 
 
This study is designed to begin testing the hypothesis that 
pulsed ultrasound stimulation can be used effectively in human 
subjects to control pathogenic inflammatory responses through 
the stimulation of the cholinergic anti-inflammatory reflex 
pathway, a neuroimmune feedback response. The overall goal 
of this project is to determine which, if any, ultrasound 
stimulation protocols are able to restrict the inflammatory 
response of immune cells collected from healthy subjects post-
ultrasound stimulation. Subjects will receive ultrasound and 
then immune cells will be isolated from their blood and treated 
ex vivo with inflammatory stimuli to test their inflammatory 
capacity. 
 
Objectives 
Endpoints 
Primary 
 
• 
Objective 1: Determine 
ultrasound intensities within 
FDA approved guidelines 
that limit the inflammatory 
response  
• 
Objective 2: Determine 
whether targeting the 
spleen and/or cervical 
vagus nerve with 
ultrasound are effective 
methods for anti-
inflammatory ultrasound 
delivery 
(Same for both Obj. 1 & Obj. 2) 
Luminex assays and/or 
individual enzyme-linked 
immunosorbent assays 
(ELISAs) to measure cytokine 
concentration in the 
supernatants from ex vivo 
cultures. Peripheral blood 
immune cells from pre- and 
post-ultrasound samples will be 
cultured with inflammatory 
stimuli, such as 
lipopolysaccharide (LPS), for 24 
hours prior to collection of the 
supernatants. 
Secondary 
 
Determine the impact of 
ultrasound stimulation on the 
distribution of immune cells in 
the blood of human subjects. 
Flow cytometry analysis of 
white blood cells. 
Safety assessment 
 
Verify ultrasound stimulation 
does not produce any 
appreciable discomfort in 
healthy human subjects. 
Survey of participants’ state of 
mind and physical sensations 
(i.e., any new experience since 
receiving ultrasound stimulation 
such as pain, loss of sensation, 
and shifts in thought patterns or 
attitude). 
Study Population: 
 
A total of 55 participants will be recruited for this study. Subjects 
should be in overall good health without acute or chronic 
medical conditions, of any gender and demographic group, 
between the ages of 25 and 50 years, and preferably local to 
the Virginia/Washington D.C. area. 

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Description of 
Sites/Facilities 
Enrolling 
Participants: 
All enrollment and work associated with this study will be 
performed at the University of Virginia (UVA) in Charlottesville, 
Virginia, by UVA personnel associated with this project.   
 
Description of 
Study Intervention: 
The intervention used in this study is a pulsed ultrasound 
stimulation targeted to the spleen or cervical vagus nerve. 
Ultrasound technology has been used clinically for many years 
with a variety of applications, but our goal of targeted stimulation 
of a neuroimmune anti-inflammatory response is novel. 
 
The ultrasound device contains several key components. The 
central component is the transducer that sends and detects 
ultrasonic sound waves. These waves are generated via a 
piezoelectric phenomenon in which an electric current is applied 
to piezoelectric crystals within the transducer. These crystals 
vibrate in response to the electric current and convert the 
energy into sound waves with a frequency beyond human 
hearing (i.e., ultrasonic). When these sound waves interact with 
solid objects, such as tissue, they are absorbed, scattered, 
refracted, and reflected. The reflection and scattering allows 
sound waves to be returned to the transducer and detected in 
order to generate an image based on the timing and intensity of 
the returned signal. For our purposes of stimulation, the 
detection of these waves is a minor element that will only be 
used in targeting ultrasound bursts to the proper physical 
location within the subject. According to our hypotheses, the 
interaction of emitted ultrasound waves of the proper intensity 
with tissue (spleen or cervical vagus nerve) is capable of 
stimulating a physiological response that produces an anti-
inflammatory state. 
 
The other critical element of the device for our study is the 
programming control unit. This allows for control of the intensity 
of the ultrasound wave and will enable us to ensure that the 
device is used in a safe, controlled, and consistent manner.  
 
The Sequoia system allows for general programming of the 
ultrasound parameters and enables us to scale the total energy 
deposited within the area of ultrasound stimulation to prevent 
detrimental effects, such as heat-induced tissue damage. The 
range of intensities chosen for this study have all been used 
safely in humans before and the Sequoia system will help us 
ensure that these parameters are not exceeded. The ultrasound 
machine used in this study is commercially available. 
Study Duration: 
The study is estimated to take 18 months to complete (from 
enrollment to completion of data analyses). 
Participant 
Duration: 
Each participant will complete a total of 4−6 visits. Each visit 
should require minimal time to complete and take no more than 
1−2 hours of the participant’s time. The time between each visit 

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is flexible based on the participant’s availability and schedule, 
with the only restriction being the time between ultrasound 
stimulations (at least 14 days between ultrasound stimulations). 
This 14-day window is intended to allow any impacts of the 
previous ultrasound stimulation to dissipate. Realistically, a 
participant could complete all visits within 1 month, but the study 
will accommodate the scheduling requirements of the 
participants. 
 
 

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1.2 
Schema 
 
OBJECTIVE 1 (GROUP 1; n = 30) 
 
  
 
   
 
MI, mechanical index. 
 
 
 

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OBJECTIVE 2 (GROUP 2; n = 10) 
  
 
MI, mechanical index. 
 
A detailed description of the schemata is provided in section 6.2. 
 

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2 INTRODUCTION  
2.1 
Study Rationale  
Inflammatory conditions are an imminent threat to human health, happiness, and survival. 
Major examples of these include sepsis, bodily trauma (burns or organ damage), and 
autoimmune disorders such as lupus erythematosus or rheumatoid arthritis. Such 
conditions are common within the human population and it is likely that the majority of 
people will experience one or more of them during their lifetime. While the etiology and 
manifestation of these conditions may be varied, there is now the potential for therapies 
that intentionally trigger inherent anti-inflammatory mechanisms that are hard-wired into 
our physiology to ameliorate or even nullify the impacts of pathologic inflammation. These 
pathways are applicable to multiple inflammatory conditions and provide a means to 
exploit an aspect of our physiologic “source code” to combat excessive or inappropriate 
inflammation. While the most common procedure for triggering these effects is to stimulate 
the vagus nerve electrically, this strategy requires invasive surgery and does not lend itself 
to clinical treatment or repeated therapy.  
 
We discovered that pulsed ultrasound stimulation of mice can recapitulate the outcomes 
of electrical stimulation and protect mice from pathologic inflammation to a large degree 
(1, 2). The use of ultrasound technology has multiple advantages given that it 
noninvasively provides accurate imaging test of the body, thus allows targeted stimulation, 
and is widely accessible in routine clinical practice. Ultrasound stimulation is already used 
clinically for the treatment of pain, physical injuries, and tumor ablation (3-5). Thus, it is a 
far more viable option for clinical translation to ameliorate inflammatory conditions and has 
the potential to provide much needed relief to a broad spectrum of patients. To this end, 
we are seeking to better understand the mechanisms of and requirements for ultrasound-
induced anti-inflammation, as well as how this treatment impacts the human system. We 
hypothesize that both spleen-targeted and cervical vagus-targeted ultrasound will produce 
an anti-inflammatory effect that will be reflected in reduced cytokine production, and that 
this will have a threshold of efficacy dependent on the intensity of ultrasound delivered. 
We also hypothesize that there will be measurable fluctuations in the distribution of 
immune cells in the blood and will seek to characterize this as a secondary endpoint. The 
enhanced understanding gained by this study will help ensure that this powerful technique 
is used safely and appropriately to maximize the benefit to patients as well as provide 
input for future study and treatment design. 
 
2.2 
Background  
The cholinergic anti-inflammatory pathway (CAP) represents one of the best studied 
neuroimmune-modulatory pathways (6-9). The CAP has been elucidated as a way for the 
nervous system to restrict inflammation and rescue many of the detrimental effects of 
inflammatory disease. By stimulating the vagus nerve, the neurotransmitter acetylcholine 
is produced in distal tissues and can be detected by immune cells expressing nicotinic 
acetylcholine receptors. For reasons unknown, this interaction leads to an anti-
inflammatory state, in which immune cells produce lesser amounts of inflammatory 
cytokines (10, 11). Interestingly, since the receptors for acetylcholine are nicotinic, 
treatment of rodents or cells with nicotine can also induce anti-inflammation and improve 
the survival of mice with experimental sepsis (11, 12). This neuro-immune crosstalk has 
also been shown to protect mice from acute kidney injury (AKI) following renal ischemia-
reperfusion (13, 14). Additional work with mice showed that pulsed ultrasound stimulation 
recapitulates the renal protection observed following electrical vagus nerve stimulation (1, 
2, 15, 16). This showed pulsed ultrasound stimulation has the potential to modulate 

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immune cell distribution and cytokine production in vivo, providing indications of anti-
inflammation that likely contribute to the observed renal protection (7). Given the 
invasiveness and/or questionable efficacy of currently available vagus nerve stimulation 
protocols and devices, the potential for non-invasive ultrasound to induce the same effects 
in humans is an attractive and viable clinical approach to ameliorate inflammation in a 
controlled fashion. 
 
 Pre-Clinical Experience 
While vagus nerve stimulation has received a great deal of attention for its anti-
inflammatory capability, work with pulsed ultrasound-mediated anti-inflammation is less 
prevalent. Studies previously performed by the Okusa lab are some of the only ones that 
have used a stimulation protocol in mice that is similar to the approach used in this trial 
(mechanical index [MI] between 0.5 and 1.9, single treatment, short duration of ultrasound 
exposure). As stated, these studies have shown that ultrasound targeted to the flanks of 
mice is capable of providing protection from AKI and potentially sepsis (1, 2). However, 
protection is lost when the spleen is removed prior to ultrasound stimulation (2, 15). This 
reliance on an intact spleen strengthens the similarities between vagus nerve stimulation 
and ultrasound stimulation and provides a rationale for targeting the spleen in this study. 
In addition to this work, another group has tested the impacts of a range of ultrasound 
intensities targeted to the spleen and liver of mice (7). This study exhibited a “dose 
response” with increasing ultrasound intensities and demonstrated that the inflammatory 
and hyperglycemic effects of lipopolysaccharide (LPS) injection were ameliorated 
comparably to vagus nerve stimulation (7). Another pre-clinical study used a similar 
ultrasound protocol to investigate the impacts of the treatment on hyperglycemic 
myocardial ischemia-reperfusion injury and found that spleen-targeted ultrasound was 
able to reduce infarct size (17). In addition, that study also investigated the efficacy of 
cervical vagus (neck)-targeted ultrasound, given that it may have an effect similar to 
spleen-targeted ultrasound through upstream vagus nerve modulation. The investigators 
found that cervical vagus ultrasound stimulation was similarly effective compared to 
spleen-targeted stimulation (17). This provides rationale for our second study group in 
which we will compare the efficacy of ultrasound delivered to the spleen and neck of 
subjects. 
 
 Relevant Clinical Experience 
While the parameters selected for this study are within the Food and Drug Administration 
(FDA) limits for ultrasound imaging, there are no published studies that have explored the 
impacts of pulsed ultrasound stimulation for protecting human subjects from inflammatory 
conditions. While ultrasound technology has been used clinically for many years for 
purposes ranging from diagnostic imaging to treating sports injuries, it’s use as a 
therapeutic anti-inflammatory agent is essentially unexplored, aside from a single pre-
publication study (18) (clinicaltrials.gov: NCT03548116 & NCT03690466). This study 
employed a MI of 1.4 and targeted the spleen of study participants for a 3-minute 
stimulation period. The authors showed that this stimulation protocol was sufficient to 
downregulate cytokines and associated pathways in healthy subjects and patients with 
rheumatoid arthritis without compromising the adaptive immune response or any negative 
outcomes or side effects of note (18). 
 
2.3 
Risk/Benefit Assessment 
 Known Potential Risks  
While ultrasound carries risks related to thermal and mechanical effects, these are 
dependent on the amount of energy deposited to the tissue site over time. Tissue heating 

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can cause damage to cells, including nerve cells. Mechanical forces can lead to shear 
stress from fluid streaming effects within tissue and cavitation of gas bubbles due to rapid 
changes in pressure generated by ultrasound pulses. Both of these can damage cells or 
tissue structures if not managed properly. These elements have led to the FDA 
establishing the upper limit for MI at 1.9 (19). MI is calculated by dividing the peak negative 
pressure of the ultrasound wave by the square root of the wave frequency (20). Significant 
tissue damage could lead to both immediate and long-range risks if it results in pain, 
reduced organ function, and/or chronic injury. However, by limiting the exposure to and 
power of our ultrasound parameters and remaining within the FDA prescribed limits for 
imaging ultrasound, we do not view these as substantial risks in this study. We anticipate 
to  gain additional insights on tolerability and safety of the ultrasound procedure by 
measuring urinary AKI biomarkers and spleen measures post-stimulation. Furthermore, 
the cumulative maximal volume of whole blood drawn during the entire study period will 
not exceed 48 ml, which is far below the 45 CFR 46.110-defined blood drawing limits.  
 
 Known Potential Benefits  
The animal and human work outlined above capture the potential benefits of this study. 
By enhancing our knowledge of how ultrasound stimulation impacts the human system, 
we come closer to its effective use in the clinic, which will potentially provide relief from 
inflammatory conditions for millions of patients. The studies above exhibit benefits in the 
form of reduced pro-inflammatory cytokine production, reduced inflammation-associated 
tissue injury, and potentially reduced sepsis-related morbidity and mortality, if ultrasound 
stimulation is functioning similarly to vagus nerve stimulation (7, 8).  
 
 Assessment of Potential Risks and Benefits  
The potential risks in this study have been considered and managed in the design. Due to 
the wide use of ultrasound in the clinic for other procedures, there is a long track record 
of safe use that has informed our design. We have designed our intervention to minimize 
the total energy deposited at tissue sites through a pulsed wave procedure, with defined 
MI intensities and short total duration, and have ensured that we stay beneath the FDA 
prescribed threshold for imaging ultrasound with our chosen MI range (19). Thus, we view 
the potential for detrimental outcomes as low in this study. The potential benefits, on the 
other hand, are far-reaching and would be incredibly meaningful to patients suffering from 
acute or chronic inflammation, a substantial portion of which have life-threatening 
conditions. 
 
 
3 OBJECTIVES AND ENDPOINTS 
  
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
•  Objective 1: Determine 
ultrasound intensities within FDA 
approved guidelines that are able 
to limit the inflammatory response  
 
•  Objective 2: Determine whether 
targeting the spleen and/or 
cervical vagus nerve are effective 
Luminex and/or individual ELISA 
assays to measure cytokine 
production from peripheral blood 
immune cells collected before and 
after ultrasound stimulation and 
cultured with inflammatory stimuli. 
Blood for analysis will be collected 
at the baseline visits 1–2 and 
Evaluating cytokine 
production post-ex vivo 
stimulation with known 
inflammatory agents is a 
relatively simple means of 
assessing the behavior of 
immune cells and does not 
require the induction of 
inflammation in the human 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
methods for anti-inflammatory 
ultrasound delivery 
 
24−48 hours after each ultrasound 
stimulation. 
 
Luminex technology allows the 
simultaneous measurement of 
multiple cytokines via a flow 
cytometry-based assay. This 
essentially leverages the principles 
of flow cytometry to perform 
multiple ELISA tests in a single 
assay. This analysis will enable us 
to assess the concentration of a 
broad range of cytokines in the 
supernatants from our ex vivo cell 
stimulations. This will provide 
robust data for evaluating the 
efficacy of ultrasound in limiting the 
inflammatory capacity (i.e. cytokine 
production) of immune cells. 
Additional ELISA assays may be 
necessary if we decide there are 
additional cytokines we would like 
to measure that do not have 
Luminex reagents available, but 
Luminex analysis will be the 
primary method for determining 
cytokine production. 
 
subjects enrolled in the study. 
Thus, this provides us with an 
effective strategy to assess 
the efficacy of ultrasound with 
minimal risk of discomfort or 
adverse events in the 
subjects. The use of 
immediate baseline analysis 
and subsequent post-
ultrasound sample collection 
will allow for the use of each 
subject as their own control in 
a standardized assay. This 
will provide us with the 
opportunity to identify the 
impacts of ultrasound on the 
inflammatory response for 
each individual subject and 
will yield the most consistent 
and reliable data for analysis. 
Secondary 
 
 
Determine the impacts of 
ultrasound stimulation on the 
distribution of immune cells in the 
blood of human subjects. 
Flow cytometry analysis of blood 
cells. This will be performed using 
a portion of the same blood 
samples collected for the primary 
objective (samples from immediate 
baseline and after each 
ultrasound). 
By using cell surface markers to 
identify individual immune cell 
populations in subjects’ blood, we 
will be able to quantify the 
abundance of populations of 
interest and track any ultrasound-
induced changes in their relative 
distribution. This is a relevant 
measure both separate from and 
related to the cytokine production 
measurements in the primary 
objective. Changes in the 
distribution of immune cells could 
alter the ability of subjects to 
respond to certain types of 
infection or other disruptions to 
homeostasis. While anti-
This endpoint was chosen 
since it supplements the 
primary endpoint and is 
achievable without additional 
sample collection. It provides 
an additional level of 
understanding to the results 
and context for interpretation. 
Further, results from this 
endpoint will provide 
information for forming 
hypotheses for follow-up 
studies. 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
inflammation is the goal, tracking 
this measurement will also inform 
potential side effects and situations 
that clinicians should be aware of 
when considering ultrasound 
therapy. Altered prevalence of 
immune cells will also provide 
context for analysis of the cytokine 
data. If there are reductions in the 
production of specific cytokines 
post-ultrasound, this will help us 
assess if the reduction is due to 
effects on the cells directly or if a 
reduction in cell numbers in the 
culture condition could explain the 
difference. 
 
Safety evaluation  
 
 
Verify ultrasound stimulation does 
not produce any appreciable 
discomfort in healthy human 
subjects or kidney stress or 
damage. 
Survey of participants’ state of 
mind and physical sensations (i.e., 
any new experience since 
receiving ultrasound stimulation 
such as pain, loss of sensation, 
and shifts in thought patterns or 
attitude). Information will be 
collected at each visit after the 
baseline visit and may include a 
final follow-up survey if responses 
during the study warrant additional 
attention. 
 
This endpoint will be achieved with 
a simple survey of patient 
feedback in which we will enquire 
about their overall experience 
post-ultrasound. We will enquire if 
they have experienced any 
noticeable changes in thought, 
mood, or sensation. This will be 
designed as an open-response 
inquiry to encourage honest, 
undirected feedback and track any 
concerns the subjects may have 
during the course of the study. 
 
Assessment of urinary AKI 
biomarkers to assess kidney stress 
potentially related to ultrasound 
stimulation. Optional spot urine 
samples for biomarkers will be 
collected at Visits 1–4 (i.e., 
immediately before ultrasound 
stimulation, and directly after and 
This endpoint has been 
included because, although 
we do not anticipate any 
adverse responses to the 
ultrasound stimulation, the 
use of ultrasound to stimulate 
what we believe to be a 
neuroimmune regulatory 
pathway is a novel approach. 
The potential neurological 
element is of interest and we 
want to confirm that we are 
targeting inflammatory 
mechanisms without any 
detrimental patient 
experience or kidney 
stress/damage. 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
24−48 hours after ultrasound 
stimulation). 
 
 
4 STUDY DESIGN  
4.1 
Overall Design 
This is a single-site pilot study that contains 2 major Groups to address questions about 
both dose response (Objective 1) and delivery route (Objective 2) for pulsed-ultrasound 
induction of anti-inflammation in humans. Thus, healthy subjects have been chosen as the 
study population for this analysis in order to determine therapeutic potential and possible 
best practices. In this small-scale study, subjects will be used as their own control for data 
analysis. 
 
Group 1 of the study (Objective 1) will be used to assess dosing thresholds for induction 
of anti-inflammation. We will test four different levels of ultrasound intensity (“doses”) as 
well as sham ultrasound treatment to determine which of them are capable of producing 
an anti-inflammatory effect. The doses will be defined in terms of MI and each subject will 
be randomly assigned to receive two different MI doses of ultrasound. The MIs to be tested 
are sham, 0.6, 1.0, 1.4, and 1.8. In animal studies (1, 2), a MI of 1.2 was sufficient to 
generate a protective anti-inflammatory response and we have used this as a guide while 
keeping in mind the MI limit previously set by the FDA of 1.9 (19). Each subject will receive 
two separate doses of ultrasound stimulation (one per treatment visit, two treatment visits) 
with a MI difference of 0.8. Subgroup 1 will be tested at MI = 0.6 & 1.4, Subgroup 2 will 
receive MI = 1.0 & 1.8, and Subgroup 3 will receive MI = sham US & 1.4. The two doses 
will be administered in separate visits with at least 14 days between each stimulation to 
allow any effects of the first stimulation to dissipate. We assume that 14 days represent 
an acceptable duration of time to dissipate any effects of ultrasound stimulation. This 
assumption is based on experimental data from the Okusa lab indicating that there is no 
significant efficacy of ultrasound-induced anti-inflammation in mice when ultrasound 
stimulation is applied ≥7 days before induction of ischemia-reperfusion injury (1). We have 
doubled this period to 14 days to ensure ample time is given for the effects of the first 
treatment to wane off. However, since there are no human data available for the duration 
of ultrasound-induced anti-inflammation, we have opted to perform two reciprocal 
stimulation set-ups within each group and subgroup. For example, in Group 1 Subgroup 
1, five participants will receive the lower MI dose in the first visit and the lower MI dose in 
the second visit while the other five participants will receive the doses in the reverse order. 
This will allow us to compare the relative effects of each MI as a first dose vs. as a second 
dose to verify that there is no residual impact of receiving a prior ultrasound treatment. 
 
Group 2 of the study (Objective 2) will be used to test the efficacy of ultrasound delivered 
to the spleen versus targeted to the cervical vagus nerve at the neck. In animal studies, 
splenectomy prior to delivery of ultrasound to the flank removes the bulk of protection 
offered by the treatment (2, 15), thus we believe this is a central tissue for generating the 
anti-inflammatory effect. However, human subjects vary widely in size and shape in the 
abdominal region and localization of the spleen may vary somewhat between individuals, 
so this may not be the ideal location for consistent ultrasound stimulation delivery in 
humans. Since we believe this treatment may be functioning through a vagus nerve-
mediated response and electrical stimulation of the vagus nerve produces anti-

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inflammation (10, 11), we will target both the spleen and cervical vagus nerve in separate 
treatment sessions for this subject group. The same reciprocal setup is used in this spleen- 
vs- neck-targeting group as in Group 1. Five participants will receive spleen ultrasound in 
the first treatment while the other five will receive neck ultrasound as the first treatment, 
with 14 days being allowed between treatment sessions. The ultrasound stimulations will 
be performed at a uniform MI of 1.4 for spleen vs. neck testing. 
 
Sample and data collection will be the same for both groups and will consist of research 
blood draws of ~3 ml prior to each treatment visit and 24−48 hours after each treatment 
visit. The interval of 24−48 hours was chosen based on experimental data from the Okusa 
lab showing that the efficacy of the ultrasound-induced anti-inflammation wanes in a time-
depended manner when ultrasound stimulation is applied ≥3 days before induction of 
ischemia-reperfusion injury (1). The blood will be divided into two portions with one being 
used for flow cytometry analysis of immune cell populations and the other used for setting 
up ex vivo stimulation assays to measure cytokine production. Analysis of each stimulation 
condition (stimulation 1 and 2, respectively) will be compared to the immediate baseline 
values (baseline 1 or 2, respectively) from the same subject. We will quantify the difference 
between the means of the baseline and treatment, for example as fold change or percent 
of baseline, for each subject’s samples. The magnitudes of difference will then be treated 
as an additional test statistic and will be grouped by treatment protocol (e.g., all MI = 0.6 
differences from baseline will be compiled into one group, all MI = 1.0 will be compiled into 
a group, etc.). The compiled magnitudes will be compared to those from the other 
treatment conditions to assess the relative impact of each ultrasound stimulation protocol. 
We believe this will yield the most relevant and reliable data for this study. 
 
4.2 
Justification for Dose 
As stated above, we have chosen our stimulation MI based on animal studies and FDA 
guidance for diagnostic ultrasound. The target sites for ultrasound delivery have also been 
chosen based on animal studies and current knowledge regarding the potential 
mechanism of ultrasound-induced anti-inflammation. 
 
4.3 
End of Study Definition 
Primary completion date is the date that the final participant was examined or received an 
intervention for the purposes of final collection of data for the primary outcome, whether 
the clinical study concluded according to the pre-specified protocol or was terminated. In 
the case of clinical studies with more than one primary outcome measure with different 
completion dates, this term refers to the date on which data collection is completed for all 
of the primary outcomes defined as the final date for the collection of data for the primary 
endpoint. 
 
Study completion date is the date the final participant was examined or received an 
intervention for purposes of final collection of data for the primary and secondary outcome 
measures and adverse events (AE; for example, last participant’s last visit), whether the 
clinical study concluded according to the pre-specified protocol or was terminated. 
 
5 STUDY POPULATION 
5.1 
Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria: 
1. Male or female, aged 25−50 years 

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2. Provision of signed and dated informed consent form 
3. Able to comprehend the study goals and procedures, stated willingness to comply 
with all study procedures, and availability for the duration of the study 
4. Considered English proficient so that the subject can follow verbal commands 
during the ultrasound procedure  
5. In good general health, as evidenced by medical history 
6. Laboratory results indicating normal blood count and adequate organ function per 
the following criteria: 
System 
Laboratory Value 
Hematological 
 
White blood cell count 
≥ 4.00 k/μL 
Platelets 
≥ 150 k/μL 
Hemoglobin 
≥ 11.0 g/dL 
Renal 
 
Estimated glomerular filtration rate 
≥ 60 mL/min/1.73 m2 
Blood urea nitrogen 
≤ 1.5 x ULN 
Hepatic 
 
AST and ALT 
≤ 2.5 x ULN 
Other 
 
Fasting blood sugar 
<126 mg/dL 
Hemoglobin A1c 
< 6.5% 
 
7. Agreement to adhere to Lifestyle Considerations (see Section 5.4) throughout 
study duration. 
 
5.2 
Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation in 
this study: 
 
1. Chronic medical conditions, including cancer (in remission or active cancer), 
cerebrovascular disease, chronic kidney disease, heart conditions (such as heart 
failure, coronary artery disease, cardiomyopathies), lung disease, liver disease, 
hypertension, diabetes mellitus type 1 and 2, human immunodeficiency virus 
infection, primary immunodeficiencies, solid organ or hematopoietic cell 
transplantation, tuberculosis, and cystic fibrosis, autoimmune disorders (e.g., 
rheumatoid arthritis, inflammatory bowel disease), sickle cell anemia or other 
anemia syndromes 
2. Mean systolic and diastolic blood pressure at screening ≥160 and ≥100 mm Hg, 
respectively, or on non-selective beta-blockers and/or alpha-methyl dopa, or 
hypertension requiring more than two anti-hypertension medications 
3. Obesity (body mass index ≥30 kg/m2) 
4. Use of anti-inflammatory or immunomodulatory medication, such as non-steroidal 
anti-inflammatory drugs (NSAIDs), corticosteroids, or other immunosuppressants, 
within one week of receiving ultrasound delivery 
5. Use of anticoagulant drugs (e.g., coumadin, direct oral anticoagulants) or 
antiplatelet drugs (e.g., aspirin, clopidogrel) within one week of receiving 
ultrasound delivery 
6. Pregnancy, breastfeeding, or planning to become pregnant during the study 
7. Active bacterial or viral infection; febrile illness within 2 weeks  
8. Known allergic reactions to ultrasound gel 

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9. Treatment with another investigational drug or other intervention within 1 month 
10. Any vaccination received within 1 month 
11. Current smoker or nicotine use within 2 weeks 
12. Use of recreational drugs within 2 weeks 
13. History of arrythmia (e.g., clinically significant bradycardia, atrial flutter, atrial 
fibrillation, ventricular arrythmias) 
14. History of deep vein thrombosis or pulmonary embolism 
15. History of bleeding disorder 
16. History of seizure 
17. History of unilateral or bilateral vagotomy 
18. Participants with an implantable medical device, such as pacemaker, hearing aid 
implant, or any implanted electronic device 
19. Surgery or traumatic injury (e.g., visceral injury, cerebral injury) in the past 3 
months 
20. Prior surgery on thyroid or parathyreoid glands, esophagus, stomach, or spleen 
21. Participant considered by the Investigator, after reviewing medical and psychiatric 
history, physical examination, and laboratory evaluations, to be unsuitable for any 
other reason that may either place the patient at increased risk during participation 
or interfere with the interpretation of the study outcomes. 
 
5.3 
Justification for Study Population 
For the purposes of this pilot study, we want a population that is immunologically mature 
and without extensive age-associated immunological decline. Pregnancy is considered as 
exclusion criteria given that current guidelines recommend to limit MI less than 1 during 
obstetric diagnostic ultrasound imaging (21). The inclusion criteria are broad to allow for 
timely completion of enrollment with good representation of the general population without 
known medical conditions. Blood pressure will be measured three times in a seated 
position and averaged. Relevant hypertension in exclusion criteria is defined as grade 2 
or higher hypertension per 2020 International Society of Hypertension Global 
Hypertension Practice Guidelines (22) or when treated with non-selective beta-blockers 
and/or alpha-methyl dopa, hypertension requiring more than two antihypertension 
medications. Diabetes mellitus is defined as fasting blood sugar ≥126 mg/dL or 
hemoglobin A1c ≥6.5%, or use of diabetic medication (23) or if self-reported. The 
estimated glomerular filtration rate is calculated using the 2021 Chronic Kidney Disease 
Epidemiology Collaboration creatinine equation (24). Participants who get vaccinated or 
develop an active bacterial or viral infection after study enrollment would have to wait a 
minimum of 4 weeks before an ultrasound stimulation can be delivered. This 1-month time 
period should allow for any restoration of the immune system after vaccination or infection 
and, therefore, dissipate their impact on outcome measures after ultrasound stimulation. 
 
5.4 
Lifestyle Considerations 
During this study, participants are asked to: 
• 
Abstain from alcohol for 24 hours before the start of each visit  
• 
Participants who have used nicotine products in the past will be instructed that use 
of nicotine-containing products (including nicotine patches) will not be permitted 
while they are in the trial 
• 
Abstain from strenuous exercise for 24 hours before each blood collection 
• 
Abstain from NSAIDs, if any taken, before the start of each visit up to each blood 
collection based on current NSAIDs discontinuation recommendations (22). 
 

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If subjects become ill or require new medications or treatments during the course of the 
trial, the nature of the condition will be examined on a case-by-case basis to determine 
continued eligibility. If the illness or condition is not deemed to be linked to an effect of the 
ultrasound treatment and does not produce long term changes in the subject’s health 
status, future visits will be postponed until the condition resolves. Otherwise, the subject 
will be withdrawn from the trial. 
 
5.5 
Screen Failures 
Screen failures are defined as participants who consent to participate in the clinical trial 
but are not subsequently entered in the study. A minimal set of screen failure information 
is required to ensure transparent reporting of screen failure participants, to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements (for NIH 
studies) and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse 
event (SAE). We do not anticipate many screen failures for this study since our inclusion 
criteria is broad and the major restrictions are age range, general good health without 
known medical conditions, no nicotine use, and not pregnant. Information regarding these 
criteria will be communicated to potential subjects during consent acquisition and further 
screening should generally not be necessary.  
 
5.6 
Strategies for Recruitment and Retention 
The general strategy for timely recruitment will focus on spreading word within the UVA 
community (graduate students, personnel, patient areas) as a first strategy. We will 
generate e-mail and social media announcements and flyers to post around UVA grounds. 
If we fail to reach target enrollment or believe the process is moving too slowly, we will 
extend the reach of our recruitment materials via social media and communication with 
other nearby medical centers/hospitals. As a retention strategy, participants will be 
compensated for each blood draw session after screening they complete at a rate of 
$100/session. We will also encourage retention via maintaining contact with the study 
participants. Thus, the total compensation would come to $400 for participants that 
complete all study procedures. We believe this will encourage healthy volunteers to 
consider enrolling in and completing the study without providing undue financial incentive. 
 
• 
The anticipated accrual rate is 5 patients per month 
• 
The single-center study will be conducted at the UVA Kidney Center Clinic in 
Charlottesville, VA 
• 
Sources of participants and recruitment venues will include outpatient clinics, UVA 
campus, and general public 
• 
Potential participants will be invited via written invitations put out at public places, 
local flyers, and via social media channels 
• 
Measures to increase participant retention will include maintaining contact with the 
participants via phone and/or email with the aim to ensure participants are kept 
well-informed of the study’s progress, and to identify barriers encountered 
throughout the study to organize strategies to enhance retention  
• 
We do not have specific strategies for recruitment of women or underrepresented 
minority populations since this is a proof-of-concept pilot study with broad 
enrollment criteria. We will accept all willing participants who meet inclusion criteria 
and determine the gender/societal distribution upon study conclusion. 
 
6 STUDY INTERVENTION 

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6.1 
Description of Study Interventions  
The interventions performed in this study are designed to test the impacts of different 
ultrasound stimulation procedures on the inflammatory capacity of circulating immune 
cells. This consists of two main approaches: 1) varying ultrasound intensity (MI = sham, 
0.6, 1.0, 1.4, and 1.8,) and 2) varying the physical location targeted by the ultrasound 
stimulation (spleen-targeted vs. cervical vagus-targeted). The ultrasound machine used in 
this study is commercially available and the ultrasound transducer used in this study is 
paired with a clinically approved diagnostic scanner within current FDA regulatory limits of 
MI <1.9 and spatial-peak temporal-average intensity (ISPTA) <720 mW/m2. Depending on 
the imaging depth used, the focal depth and power setting will be adjusted to achieve the 
target MI at the desired tissue location. The manufacturer of the transducer also assures 
that the instantaneous skin contact surface does not heat up and that skin contact 
materials are skin compatible and that these surfaces can be adequately cleaned and 
sterilized by one of any already existing protocols in use at UVA Health for transducer-
based scanning. 
 
Description of device and parameters: 
o Device model: Acuson Sequoia 512 
o Description of each component: Clinical Acuson Sequoia 512 ultrasound 
system (Siemens Healthcare Diagnostics, Inc, Tarrytown, NY) with a paired 
curvilinear Acuson 4C1 transducer intended for spleen ultrasound and a 
linear Acuson 15L8 transducer intendent for neck ultrasound 
o Device settings and programming:  
 
for spleen ultrasound: frequency 3 MHz; burst MIs 0.6−1.8 (further 
description provided in 4.1); burst duration: 4 sec; burst application 
repetition frequency 10 sec; duration of exposure: 12 min 
 
sham ultrasound will consist of turning the US system on, locating 
the stimulation area, and holding the US transducer in place for 12 
minutes without delivery of higher-intensity pulses. 
 
for neck ultrasound: frequency 10 MHz; burst MI 1.4; burst duration: 
4 sec; burst repetition frequency 10 sec; duration of exposure: 12 
min 
o Technically, the Sequoia is no longer supported commercially – but the 
settings being used were part of an FDA compliant and approved software 
release during the commercial life of the device. No modifications are being 
made to the device and the machine’s user interface will not allow, under 
any circumstance, for the settings to be modified outside of FDA compliant 
conditions. The use is technically “off label” since the settings of interest 
were intended for use in combination with ultrasound contrast agent, and 
no contrast agent is being used in our protocol. However, it can be 
scientifically argued that use of these ultrasound settings without contrast 
agent represents a lower risk than when used in conjunction with a contrast 
agent, since those agents can contribute to tissue damage and injury. This 
ultrasound device has been used in the clinic for many years and its safety 
has been well established. We are not testing the safety of the device in 
this trial, but rather the efficacy of the ultrasound protocol for inducing an 
anti-inflammatory state. While we do not anticipate this treatment protocol 
will cause any harm to the participants, we will verify its safety by monitoring 
the physical and mental state of participants throughout the trial. 

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6.2 
Dosing and Administration 
As stated above, different doses (i.e., ultrasound intensities) will be delivered in one of the 
main groups. All doses chosen for this study were selected to cover a range of MI but is 
below the FDA established maximum MI threshold allowed for ultrasound diagnostic 
imaging. 
 
Procedure for ultrasound delivery: 
Following screening and enrollment, participants will be scheduled in non-fasting state for 
the first visit to collect blood for laboratory testing to confirm good health, perform baseline 
flow cytometry analysis, and set up ex vivo stimulation to assess cytokine production. 
Participants will be assigned to the study groups while attempting to keep equal 
representation of age, sex, and ethnicity in each, but we will aim to complete enrollment 
of Group 1 first (Group 1; n = 20: dose response; Group 2, n = 10: site of stimulation spleen 
vs. neck). Subgroup 1 of Group 1 (n = 10) will receive MI doses of 0.6 and 1.4. Subgroup 
2 of Group 1 (n = 10) will receive MIs of 1.0 and 1.8. Both subgroups will be divided in 
respect to the sequence in which the MI doses are delivered (higher dose first or lower 
dose first). Subgroup 3 will receive the sham treatment first and the 1.4 MI dose second.  
Optional spot urine samples will be collected prior to ultrasound stimulation to measure 
AKI biomarkers. No study-specific medications will be administered in this study. There is 
no strict timing schedule or requirements aside from the 14-day minimal interval between 
ultrasound stimulations, so it is not possible for doses to be delayed or missed. For Group 
2, 5 participants will receive the first ultrasound stimulation targeting the spleen and the 
second targeting the cervical vagus in the neck, both at MI of 1.4, while in the other 5 
participants the sequence will be flipped (first targeting the neck, then the spleen).  
 
To expose the splenic region of the abdomen, the participants will be asked to lie in the 
right lateral recumbent position (Figure 1A) or to sit upright (Figure 1B; depending on the 
ultrasonographic visibility of the spleen), with both arms above their head aiming. Second, 
ultrasound gel will be applied to the left high flank region and ultrasound will be performed 
using a standardized intercostal imaging approach to detect the spleen and assess its 
dimensions in the B mode setting (23). Third, the ultrasound probe will be positioned 
targeting the splenic hilum as the focal landmark for ultrasound delivery (Figure 1C) and 
an image will be electronically stored to document the position and the device settings.  
 
 
 

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Figure 1: Ultrasound of the splenic hilum 
 
Adapted from Amboss.com/de (24). Panels A and B show two representative participant 
positions to examine the spleen with ultrasound, respectively. Panel C shows the 
corresponding B-mode ultrasound image of the spleen and the splenic hilum (yellow 
dashed circle and arrow) in a healthy individual.  
 
Throughout the ultrasound procedure, the ultrasonographer will check the location of the 
probe using the imaging mode to ensure that the spleen hilus remains the target 
throughout the ultrasound stimulation and participants will be asked to breath shallowly to 
limit movement of the spleen. Approximately 30 minutes after the ultrasound stimulation, 
the participants will be asked to provide a second spot urine sample for AKI biomarkers. 
Participants will be scheduled for another visit 1 to 2 days after the first ultrasound 
stimulation to collect the third spot urine sample for AKI biomarkers and draw blood for 
flow cytometry analysis and stimulation of blood cells to assess cytokine production. In 
addition, B mode ultrasonography will be repeated to measure the spleen diameters. No 
additional ultrasound stimulation will be delivered. A subject experience survey will be 
performed prior to blood collection to determine if they are experiencing any noticeable 
effects of the stimulation. Once the survey and blood collection are complete, the next 
stimulation visit will be scheduled and the participant reminded that they can contact us at 
any point if they experience any changes that may be related to the procedures. Surveying 
and scheduling will be performed the same for all groups. 
 
For cervical vagus-targeted ultrasound, first the participants will be asked to lie down in 
supine position with the neck hyperextended. Second, ultrasound gel and the ultrasound 
probe will be placed on the left side of the neck in the transverse plane (Figure 2A) using 
the B mode image setting (25). Third, the ultrasound probe will be positioned over the 
major neurovascular bundle (region of interest: lateral margins of the anterior cervical 
region beneath the sternocleidomastoidei muscles) as the focal landmark for ultrasound 
delivery (Figure 2B) and an image will be electronically stored to document the position 
and the device settings. Ultrasound stimulation will be applied with the same procedure 
(pulse repetition and duration) as stated previously. Before each ultrasound delivery, the 
ultrasonographer will check the location of the probe using the imaging mode to ensure 

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that the major neurovascular bundle remains the target throughout the ultrasound 
stimulation. In addition, the ultrasonographer will assess the dimensions of the spleen in 
the B mode setting. Approximately 30 minutes after the ultrasound stimulation, the 
participants will be asked to provide a second spot urine sample for AKI biomarkers. 
 
Figure 2: Ultrasound of the cervical vagus nerve 
 
Adapted from Ottaviani et al. (26). Panel A shows a schematic representation of the left 
cervical region highlighting the conventional placement of the ultrasound probe to visualize 
the major neurovascular bundle. Panel B shows the corresponding B-mode ultrasound 
image including the central carotid artery, inferior jugular vein, vagus nerve (yellow dashed 
circle and arrow), and nearby muscles. CCA, central carotid artery; IJV, inferior jugular 
vein. 
 
Participants will be scheduled for the last visit 1 to 2 days after the second ultrasound 
stimulation to provide another spot urine sample for AKI biomarkers and draw blood for 
flow cytometry analysis and stimulation of blood cells to assess cytokine production. 
Furthermore, B mode ultrasonography will be repeated to measure the spleen diameters. 
No additional ultrasound stimulation will be delivered. A subject experience survey will be 
performed as above, but no additional visit scheduling will be required at this point. 30 
days after the last ultrasound stimulation, all participants will be recontacted via phone to 
report any new discomforts or health issues that developed within the last month. 
 
 Dose Escalation and Regimen 
While we will deliver different doses of ultrasound stimulation in this study, it is not 
technically a dose escalation study since there is no drug regimen. Each visit and/or 
treatment represents an independent point for end point data collection, so there is not a 
longitudinal element for determining the effect of a treatment administered over time.  
 
 Dose Modifications and Delays 
For Group 2, we will monitor data from Group 1 and determine if we still consider an MI of 
1.4 as the optimal stimulation for spleen vs. neck targeted US. If the data indicate that a 

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lower dose would be efficacious, we will evaluate the option of altering this element of 
Group 2. 
 
6.3 
Preparation/Handling/Storage/Accountability   
 Acquisition and Accountability 
The devices have already been obtained for prior research by the Hossack lab at UVA. In 
the event that new devices are required, these will be obtained from the same 
manufacturer, Sequoia Ultrasound devices will be programmed by Hossack lab personnel 
and checked prior to use on subjects for proper functioning. Devices will be stored in 
secured locations either within the Okusa lab or Hossack lab while not being used for 
treatments in the study. 
 
 Formulation, Appearance, Packaging, and Labeling 
The Clinical Acuson Sequoia 512 ultrasound system is manufactured by Siemens 
Healthcare Diagnostics, Inc, Tarrytown, NY, and is intended for diagnostic ultrasound 
imaging. It will be programmed by the Hossack lab. The paired Acuson 4C1 and 15L8 
probes are intended for diagnostic ultrasound imaging. 
 
 Product Storage and Stability 
As stated, the devices will be stored in secured lab locations while not in use and tested 
for proper function prior to use. There are no storage requirements for stability of this 
device. 
 
 Preparation 
Device parameters will be programmed and preset by Hossack lab personnel. Parameters 
will include frequency (MHz), burst MI, burst duration (seconds), burst application interval 
(seconds) and total ensonification duration (minutes – by watch or timer). The parameters 
will be double-checked by the ultrasonographer and documented by electronically storing 
the images prior to the initiation of ultrasound stimulation. 
 
6.4 
Study Intervention Compliance 
The only protocol requirements for compliance/adherence is the time allowed between 
ultrasound stimulations. This will be accomplished when scheduling subjects for treatment 
by verifying the date of their previous ultrasound stimulation and ensuring there is at least 
a 14-day period between ultrasound stimulation sessions. 
 
6.5 
Registration, Randomization and Blinding 
Registration: All participants must sign the consent form prior to determination of eligibility 
for this study. Registration will occur following verification of eligibility by the treating 
physician. Participants who are consented and accrued to the study should be registered 
in OnCore in accordance with the Clinical Trial Management System Policy via the UVA 
OnCore Resources link in Oncore. General guidelines are available in the OnCore User 
Manual and Data Entry Guide. Participants should receive their first study treatment within 
2 months of registration. 
 
Randomization: Participants 1-30 will be assigned to Group 1 (Schema 1.2 on p. 10-11). 
Once the Group 1 enrollment target is met, participants 31-40 will be assigned to Group 
2.  Randomization will be used to assign participants to subgroups within Group 1 (1:1) 
and to an order of treatments within Group 1 subgroups and Group 2 (1:1). Randomization 
will be stratified by gender and race. 
 

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Blinding: This study does not involve any blinding or masking procedures. Subjects will be 
told which treatment they are receiving. 
 
 Emergency Unblinding Procedures 
Not applicable. 
 
6.6 
Concomitant Therapy 
Not applicable. 
 
 Rescue Medicine/Supportive Care 
Not applicable. 
 
7 STUDY 
CLOSURE, 
STUDY 
INTERVENTION 
DISCONTINUATION 
AND 
PARTICIPANT DISCONTINUATION OR WITHDRAWAL 
 
7.1 
Study Discontinuation and Closure 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to regulatory 
authorities. If the study is prematurely terminated or suspended, the Principal Investigator 
(PI) will promptly inform the Institutional Review Board (IRB) and sponsor and will provide 
the reason(s) for the termination or suspension. Study participants will be contacted, as 
applicable, and be informed of changes to study visit schedule. 
  
Circumstances that would warrant termination or suspension include, but are not limited 
to 
• 
Determination of unexpected, significant, or unacceptable risk to participants  
• 
Change in funding status 
 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor and IRB. 
 
7.2 
Participant Discontinuation/Withdrawal  
Participants are free to withdraw from participation in the study at any time upon request. 
A participant’s study treatment would be discontinued for the following reasons: 
 
• 
Pregnancy 
• 
If any clinical AE, laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best 
interest of the participant 
• 
If the participant meets an exclusion criterion (either newly developed or not 
previously recognized) that precludes further study participation 
• 
Participant decision to withdraw from study treatment and/or the study for any 
reason 
• 
Loss to follow-up 
• 
A patient’s substantial inability to follow commends during ultrasound (e.g., inability 
to remain relatively still for several minutes) or non-compliance (e.g., multiple 
missed visits, inability and/or unwillingness to adhere to scheduled appointments) 
• 
Initiation of prohibited intervention or medication. 

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The reason for participant discontinuation or withdrawal from study treatment will be 
recorded on the case report form. Participants who sign the informed consent form 
and subsequently withdraw, or are withdrawn or discontinued from the study, will be 
replaced. Participants that withdraw from the study (not only from study treatment, but 
all study follow-up) will not be contacted for any further study visits. 
 
7.3 
Dose-Limiting Toxicity 
Not applicable. 
 
7.4 
Procedures for Discontinuation of Study Intervention 
Discontinuation from ultrasound stimulation does not mean discontinuation from the study, 
and remaining study procedures should be completed as indicated by the study protocol. 
If a clinically significant finding is identified (including, but not limited to changes from 
baseline) after enrollment, the investigator or qualified designee will determine if any 
change in participant management is needed. Any new clinically relevant finding will be 
reported as an AE. 
 
The data to be collected and procedures to be completed at the time of study intervention 
discontinuation are included in the schedule of assessments in section 13.1. 
 
7.5 
Lost to Follow-Up   
A participant will be considered lost to follow-up if he or she fails to return for any 
scheduled visits and is unable to be contacted by the study site staff.  
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit and 
ascertain if the participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will 
make every effort to regain contact with the participant including regular e-mails 
and/or phone calls until the participant either responds or the study period is 
closed.   
• 
These contact attempts should be documented in the study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up. 
 
8 STUDY ASSESSMENTS AND PROCEDURES 
8.1 
Clinical Assessments  
For screening purposes, patients will be asked to affirm that they are in good health with 
no acute or chronic medical conditions or ongoing use of anti-inflammatory medications. 
This affirmation will be confirmed by a review of the subject’s prior medical history and 
medical record review by a licensed clinician associated with the study and by additional 
lab tests. 
 
 Physical Exam  
 
• 
Physical examination A physical examination will be performed during 
subject screening to further verify good health and study eligibility. A physical 
exam will also be performed prior to the final blood draw to verify no new 
conditions have arisen in the subjects during the course of the study and 

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30 
ultrasound treatments. These will be completed by a trained physician. Height 
and weight will also be recorded at the time of physical examination. 
• 
Vital signs before and after each ultrasound stimulation (temperature [°C], 
pulse [beats per min], respiratory rate [breaths per min], systolic and diastolic 
blood pressure [both in mm Hg]).  
 
 Clinical Laboratory Assessments 
Laboratory assessments required for screening purposes will include i) a urine pregnancy 
testing (for women of childbearing potential will require urine pregnancy testing prior to 
enrollment and, ii) assessment of complete blood count (one EDTA 3.0 ml tube) and serum 
creatinine, blood urea nitrogen, blood glucose, AST, and ALT (one SST 3.5 ml tube). 
 
 Imaging 
Ultrasound imaging will be performed during target area acquisition prior to pulsed 
ultrasound delivery during treatment visits. This is accomplished by placing ultrasound gel 
on the target area and moving the ultrasound probe over the gel until the tissue target 
location has been identified. This will be performed by UVA personnel trained in the use 
of ultrasound. 
 
 Assessment of Adverse Events 
Each participant will be evaluated by a licensed clinician at each study visit. The NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 5 (if applicable) will 
be used for the characterization and grading of adverse events.  
 
 Other Clinical Assessments  
Demographics (race/ethnicity) and medical history, smoking status (current, former, 
never), hypertension, diabetes mellitus, medication (maintenance therapy, vitamins, 
supplements). 
 
8.2 
Research Specimen Collection 
 Tissue 
Not applicable. 
 
 Research Blood 
~12 ml of blood will be drawn each during the first stimulation visit and both post-
stimulation visits using EDTA blood collection tubes. Whole blood will be processed in the 
Okusa laboratory by centrifugation to pellet cells followed by chemical red blood cell lysis. 
After RBC lysis, the white blood cells will be pelleted again via centrifugation, counted, 
and separated into portions for flow cytometry analysis and plating for in vitro stimulation 
assays. Analyses will also include a complete blood count with differential measured in 
the UVA central laboratory. 
 
 Research Urine 
Participants will be asked to provide optional spot urine samples immediately prior to 
ultrasound stimulation, and ~30 minutes and 24−48 hours after ultrasound stimulation. 
Urine will be processed by centrifugation. Supernatants will be flash frozen, stored at -
80°C, and thawed immediately prior to analysis.  
 
Biomarkers of interest will include neutrophil gelatinase–associated lipocalin (NGAL) and 
the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor binding 
protein ([TIMP-2]•[IGFBP7]). NGAL is the most extensively investigated biomarker for 

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31 
diagnosing AKI estimating severity of AKI, and it has been proposed as a distal tubule 
damage biomarker (27, 28). TIMP-2 and IGFBP7 are 21- and 25-kDa proteins that are 
secreted in the early phase of tubular damage (e.g., in the context of ischemia (29) or 
sepsis (30)) by the tubular epithelial cells and are implicated in G1 cell cycle arrest, which 
is thought to be a part of the protective mechanisms cells use when exposed to stress 
(31). In contrast to tubular damage biomarkers (e.g., NGAL), TIMP-2 and IGFBP7 can be 
released in response to non-injurious, noxious stimuli (29). For this reason, both 
biomarkers are often referred to as kidney stress biomarkers (32). [TIMP-2]•[IGFBP7] 
have been incorporated in the first diagnostic test for AKI approved by the FDA 
(Nephrocheck, Astute Medical, San Diego, CA, USA).  
 
 Stool 
Not applicable. 
 
8.3 
Correlative Studies 
White blood cells will be used for flow cytometry and cytokine production assays to be 
performed in the Okusa lab. A panel of fluorescently tagged antibodies will be developed 
to identify individual immune cell populations by surface marker expression patterns. 
Cytokine production analysis will be accomplished by plating 2x105 – 1x106 cells per well 
and adding inflammatory compounds, such as lipopolysaccharide (LPS), to stimulate 
cytokine production. After a 24-hour stimulation period, supernatants will be collected from 
the culture wells and stored at -20 degrees Celsius for future use in Luminex assays to 
determine the concentration of specific cytokines. Any leftover specimen will be discarded. 
 
8.4 
Participant Reported Outcomes 
At post-treatment collection visits, subjects will be surveyed to ensure they are not 
experiencing any unanticipated discomfort or changes in mood or sensation. This will be 
a simple binary response ("yes” there are noticeable changes post-treatment or “no” there 
are no discernable changes). If subjects respond that they are experiencing any 
divergences from normal day-to-day life then they will be asked for additional information 
in the form of a description of their experience. At enrollment and the baseline visit, 
subjects will also be informed/reminded that they can send feedback and concerns to 
study personnel at any time via e-mail and be provided with contact information. 30 +/- 2 
days after the last ultrasound stimulation, all participants will be recontacted via phone to 
report any new discomforts or health issues that may have developed within the last 
month. 
 
 
9 DATA AND SAFETY MONITORING PLAN  
9.1 
Adverse Events and Serious Adverse Events  
 Definition of Adverse Events (AE) 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 
(a)). 
 Definition of Serious Adverse Events (SAE) 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the 
view of either the investigator or sponsor, it results in any of the following outcomes:  
• 
death,  
• 
a life-threatening adverse event,  
• 
inpatient hospitalization or prolongation of existing hospitalization,  

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• 
a persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions, or  
• 
a congenital anomaly/birth defect.  
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse. A 
planned medical or surgical procedure is not, in itself, an SAE 
 Definition of a Suspected Adverse Reaction 
Any adverse event for which there is a reasonable possibility that the treatment caused 
the adverse event.  Suspected adverse reaction implies a lesser degree of certainty about 
causality than adverse reaction, which means any adverse event caused by the treatment. 
 Classification of an Adverse Event 
9.1.4.1 Severity of Event 
All AEs will be assessed by the study clinician using a protocol defined grading system. 
AEs will be graded and classified as follows: 
• 
Grade 1, Neutral – an event will be considered neutral if there is a noticeable 
change that may reasonably stem from the treatment protocol, but this change has 
no impact on the subject’s daily life or enjoyment of activities. 
• 
Grade 2, Mild – an event will be classified as mild if the detected change has some 
impact on the subject’s daily life, but does not impact overall health or prevent 
performance of tasks or activities. 
• 
Grade 3, Significant – an event will be classified as significant if it results in 
disruption of a subject’s daily life or good-health status. 
• 
Grade 4, Severe – an event will be classified as severe if it is considered life-
threatening or debilitating to the subject and prevents functioning in daily-life or is 
deemed a concern to long-term health or physical capability.   
9.1.4.2 Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to study intervention assessed by 
the clinician who examines and evaluates the participant based on temporal relationship 
and his/her clinical judgment. The degree of certainty about causality will be graded using 
the categories below. In a clinical trial, the study product must always be suspect.  
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and 
other possible contributing factors can be ruled out. The clinical event, including 
an abnormal laboratory test result, occurs in a plausible time relationship to study 
intervention administration and cannot be explained by concurrent disease or other 
drugs or chemicals. The response to withdrawal of the study intervention 
(dechallenge) should be clinically plausible. The event must be pharmacologically 
or phenomenologically definitive, with use of a satisfactory rechallenge procedure 
if necessary. 
 
• 
Probably Related – There is evidence to suggest a causal relationship, and the 
influence of other factors is unlikely. The clinical event, including an abnormal 
laboratory test result, occurs within a reasonable time after administration of the 
study intervention, is unlikely to be attributed to concurrent disease or other drugs 
or chemicals, and follows a clinically reasonable response on withdrawal 
(dechallenge). Rechallenge information is not required to fulfill this definition. 

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• 
Possibly Related – There is some evidence to suggest a causal relationship (e.g., 
the event occurred within a reasonable time after administration of the trial 
medication). However, other factors may have contributed to the event (e.g., the 
participant’s clinical condition, other concomitant events). Although an AE may rate 
only as “possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related”, as 
appropriate. 
 
• 
Unlikely to be related – A clinical event, including an abnormal laboratory test 
result, whose temporal relationship to study intervention administration makes a 
causal relationship improbable (e.g., the event did not occur within a reasonable 
time after administration of the study intervention) and in which other drugs or 
chemicals or underlying disease provides plausible explanations (e.g., the 
participant’s clinical condition, other concomitant treatments). 
 
• 
Not Related – The AE is completely independent of study intervention 
administration, and/or evidence exists that the event is definitely related to another 
etiology. There must be an alternative, definitive etiology documented by the 
clinician. 
 
9.1.4.3 Expectedness 
The research team will be responsible for determining whether an adverse event (AE) is 
expected or unexpected. An AE will be considered unexpected if the nature, severity, or 
frequency of the event is not consistent with the risk information previously described for 
the study intervention. 
 
 Abnormal Laboratory Values 
Women with a positive pregnancy testing will be excluded from enrollment or from further 
participation. Subjects with abnormal laboratory values as the criteria below will be 
excluded from enrollment or from further participation: 
 
System 
Abnormal Laboratory Value 
Hematological 
 
White blood cell count 
< 4.00 k/μL 
Platelets 
< 150 k/μL 
Hemoglobin 
< 11.0 g/dL 
Renal 
 
Estimated glomerular filtration rate 
< 60 mL/min/1.73 m2 
Blood urea nitrogen 
> 1.5 x ULN 
Hepatic 
 
AST and ALT 
> 2.5 x ULN 
Other 
 
Fasting blood sugar 
≥ 126 mg/dL 
Hemoglobin A1c 
≥ 6.5% 
 
 Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to 
the attention of study personnel during study visits and interviews of a study participant 
presenting for medical care, or upon review by a study monitor. 

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All AEs including local and systemic reactions not meeting the criteria for SAEs will be 
captured on the appropriate case report form (CRF). Information to be collected includes 
event description, time of onset, clinician’s assessment of severity, relationship to study 
product (assessed only by those with the training and authority to make a diagnosis), and 
time of resolution/stabilization of the event. All AEs occurring while on study must be 
documented appropriately regardless of relationship. All AEs will be followed to adequate 
resolution. 
 
Any medical condition (including a laboratory abnormality) that is present at the time that 
the participant is screened will be considered as baseline and not reported as an AE. 
However, if the study participant’s baseline medical condition worsens at any time during 
the study, it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the 
duration of the event at each level of severity to be performed. AEs characterized as 
intermittent require documentation of onset and duration of each episode. 
Study clinicians will record all reportable events with start dates occurring any time after 
informed consent is obtained until 30 days after the last day of study treatment. At each 
study visit, the investigator will inquire about the occurrence of AE/SAEs since the last 
visit. Events will be followed for outcome information until resolution or stabilization. 
 
 Adverse Event Reporting 
AEs must be recorded into the University of Virginia OnCore and case report forms per 
the following guidelines (Table 2). 
 
Table 1 
 
 Serious Adverse Event Reporting 
 
The study clinician will report to the sponsor any serious adverse event, whether or not 
considered study intervention related, including those listed in the protocol or investigator 
brochure and must include an assessment of whether there is a reasonable possibility that 
the study intervention caused the event.  
 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the 
site investigator deems the event to be chronic or the participant is stable. 
 
Table C: Low Risk Studies  
Reporting requirements for AEs that  that occur within 30 days of the last protocol specified treatment/intervention 
 
Grade 1-2 
Grade 1-2 
Grade 3 
Grade 4 
 Expected  
Unexpected  
 
 Expected or 
Unexpected  
Expected or  
Unexpected 
 
Without 
hospitalization 
With 
hospitalization 
Unrelated 
Unlikely 
Not required 
 
Not required 
Not required  
Not required 
 
15 days 
Possible 
Probable 
Definite 
Not required 
Not required 
 
30 days 
 
15 days 
 
(24-hrs)* 
15 days 
*Enter into  UVA OnCore database  within 24 hours if unexpected and definitely related to protocol specified treatment 
Hospitalization defined as an inpatient hospital stay or prolongation of a hospital stay equal to or greater than 24 hours 

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• 
Internal Event Resulting in death that is deemed DEFINITELY related to (caused 
by) study participation 
• 
Report to the UVA IRB-HSR within 24 hours. Report within 24 hours using 
IRB Online and a phone call. 
 
• 
Internal, Serious, Unexpected, Probably or Possibly Related 
o Report to the UVA IRB-HSR within 7 days from the time the study team 
receives knowledge of the event. Timeline includes submission of signed 
hardcopy of AE form.  Report using IRB Online.   
 
9.2 
Unanticipated Adverse Device Effects 
 Definition of Unanticipated Device Effect (UADE) 
Unanticipated adverse device effect is any serious adverse effect on health or safety or 
any life-threatening problem or death caused by, or associated with a device, if that effect, 
problem, or death was not previously identified in nature, severity, or degree of incidence 
in the investigational plan or application (including a supplementary plan or application), 
or any other unanticipated serious problem associated with a device that relates to the 
rights, safety, or welfare of subjects (21 CFR 812.3(s)). 
 
 Reporting of UADE 
Single Site Studies (UVA-IRB-HSR is the IRB of record) 
• 
Report to the UVA IRB-HSR within 10 calendar days of the study team receiving 
knowledge of the event.  Report using IRB Online. 
 
9.3 
Reporting Events to Participants 
If there is any new information relevant to the participant’s willingness to continue to 
participate in the study, such as if there is substantial reason to believe new risks of the 
study treatment have been identified that were not included on the consent form that the 
participant originally signed, the study team will contact the participant to discuss this 
information. If the participant is still receiving study treatment, the study team will present 
the participant with an updated consent form and confirm that he or she wants to continue 
receiving study treatment. The Sponsor will determine whether new risks are applicable 
to participants who are in follow-up, whether participants need to be notified, and whether 
re-consenting is required. 
 
9.4 
Events of Special Interest 
Not applicable. 
 
9.5 
Reporting of Pregnancy 
Not applicable. 
 
9.6 
Unanticipated Problems 
 Definition of Unanticipated Problems (UP) 
The Office for Human Research Protections (OHRP) considers unanticipated problems 
(UPs)(may include a data breach) involving risks to participants or others to include, in 
general, any incident, experience, or outcome that meets all of the following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research 
procedures that are described in the protocol-related documents, such as the 
Institutional Review Board (IRB)-approved research protocol and informed 

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consent document; and (b) the characteristics of the participant population being 
studied; 
• 
Related or possibly related to participation in the research (“possibly related” 
means there is a reasonable possibility that the incident, experience, or outcome 
may have been caused by the procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
• 
This definition could include an unanticipated adverse device effect. Please refer 
to section 9.2.1 for the definition of an unanticipated adverse device effect. 
 
 Unanticipated Problem Reporting 
• 
Report UPs that are not adverse events, protocol deviations, or data breaches (see 
section 9.7 for reporting for data breaches) to the UVA IRB-HSR within 7 calendar 
days from the time the study team receives knowledge of the event.  Report using 
the Unanticipated Problem Report form. 
• 
Report UPs that are SAEs in accordance with the guidelines for SAE reporting. 
 
 Reporting Unanticipated Problems to Participants 
If during the course of the study there is an unanticipated problem that affects current or 
past participants, affected participants will be contacted if needed. 
 
9.7 
Data Breach 
 Definition of Data Breach 
An unauthorized acquisition, access, or use of protected health information (PHI) that 
compromises the security or privacy of such information. 
 
 Reporting a Data Breach 
• 
Report to the UVA Corporate Compliance and Privacy Office as soon as possible 
and no later than 24 hours from the time the incident is identified. Report by 
telephone. 
 
• 
Report to InfoSec if the breach involves electronic data. Report as soon as possible 
and no later than 24 hours from the time the incident is identified. Refer to the 
following for details:  http://security.virginia.edu/report-information-security-
incident. 
 
• 
Report to UVA police if the breach includes such things as stolen computers.  
Report by telephone. 
 
 
9.8 
Protocol Deviation 
 Definition of Protocol Deviation 
A protocol deviation is defined as any change, deviation, or departure from the study 
design or procedures of a research project that is NOT approved by the institution’s IRB 
prior to its initiation or implementation, OR deviation from standard operating procedures, 
Good Clinical Practices (GCPs), federal, state or local regulations. Protocol violations may 
or may not be under the control of the study team or UVA staff. These protocol violations 
may be major or minor violations. 
 

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 Reporting of a Protocol Deviation 
It is the responsibility of the site investigator to use continuous vigilance to identify and 
report deviations. All deviations must be addressed in study source documents, reported 
to the Data Coordinating Center.   
• 
Report to the UVA IRB-HSR major deviations within 7 calendar days from the time 
the study team received knowledge of the event. Report using the Protocol 
Deviation and Protocol Exception Reporting Form. 
 
• 
For minor deviations, please reference the IRB-HSR for tips for recording minor 
deviations 
 
 
9.9 
Participant Withdrawals/Dropouts Prior to Study Completion 
Participants who withdraw consent and those dropping out of the study secondary to an 
AE will be reported to the IRB of record according to IRB guidelines.  
 
10 STATISTICAL CONSIDERATIONS  
The goal of this pilot study is to generate proof-of-concept data as well as identify 
stimulation parameters that are most effective. Thus, we are not using blinding, arm 
switching, or other complex study designs that would require additional statistical 
consideration. The proposed statistical comparisons and data analyses are described in 
details in the sections below. The proposed tests we are using are appropriate for the type 
of comparisons we are performing and use of participant immediate baseline values as a 
normalization method will simplify the group analyses, hopefully limiting the subject-to-
subject variability. 
 
10.1 Statistical Hypotheses 
 
• 
Primary Efficacy Endpoint(s):  
Null Hypothesis 1: ultrasound treatment does not alter the ability of immune cells to 
produce cytokines. 
Hypothesis 1: ultrasound treatment will reduce the capacity of immune cells to produce 
inflammatory cytokines 
This will be tested by comparing subject immediate baseline data to ultrasound 
treatment data using 1-way ANOVA tests with all final data from each subject.  
 
Null hypothesis 2: ultrasound treatment does not alter the ability of immune cells to 
produce cytokines. 
Hypothesis 2: There is a ultrasound “dose threshold” that determines the efficacy of 
ultrasound to limit cytokine production by immune cells. 
This will be tested as a dose response effect using all data gathered by the end of the 
trial. 
 
Null Hypothesis 3: Splenic ultrasound stimulation will have greater efficacy for 
reducing cytokine production from immune cells than ultrasound targeted to the 
cervical vagus nerve. 
Hypothesis 3: Splenic and cervical vagus-targeted ultrasound will be equally effective 
at inducing reductions in cytokine production from immune cells. 

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This will be tested using 1-way ANOVA tests to compare cytokine production between 
immediate baseline, spleen-targeted, and cervical vagus-targeted data for each 
subject and across all subjects once all data has been collected. 
  
• 
Secondary Efficacy Endpoint(s): 
Null Hypothesis: ultrasound stimulation has no impact on immune cell distribution in 
the blood. 
Hypothesis: ultrasound stimulation will produce significant changes in the abundance 
of circulating immune cell populations in the blood. 
This will be tested by comparing immediate baseline data ( baseline 1 or baseline 2) 
from each subject to their respective stimulation samples using 1-way ANOVA tests 
once all data from a subject has been gathered. 
 
10.2 Sample Size Determination 
This is a pilot study designed to gather descriptive data that helps characterize the impacts 
of ultrasound and its potential to reduce inflammatory responses. Given that our 
comparisons are well-defined across groups and we will be mostly using baseline data as 
a comparator to treatment data from the same individuals to account for within-subject 
variation, the data will be considered as independent and analyzed using the traditional 
methods. Nevertheless, the within-subject correlation will be assessed (see Section 10.4). 
 
We will be collecting 2 types of outcome measures: cytokine concentrations in 
supernatants and count/percentage of immune cells in blood samples. Each subject’s cells 
will be tested in replicates and the means of the replicate values will be used for evaluating 
individual subject’s responses. The means of each subject’s replicate samples will be 
combined with that from the other subjects of the same group for assessing differences 
between treatment conditions. If there is too much variation in the absolute values from 
each subject, we will normalize each measure to its respective subject’s baseline as a 
surrogate test statistic for assessing differences between treatment conditions. 
 
For testing the impact of ultrasound dose on cytokine production and immune cell 
abundance, the null hypothesis is that there is no difference between immediate baseline 
and stimulation. We hypothesize that increasing ultrasound MIs will yield increasing anti-
inflammation. For testing the efficacy of spleen vs cervical vagus targeting, the null 
hypothesis is that they will yield differential efficacy for inducing anti-inflammation. We 
hypothesize that both stimulation sites should produce similar anti-inflammation effects. 
 
For each test, we are using a type I error rate of α = 0.05 and a power of 80%. Since we 
plan to test a broad panel of cytokines and have not finalized our selection of the individual 
cytokines to include, it is difficult to predict the means for each. However, using our 
experience with mice and the similarity of much of this analysis to our mouse studies, we 
are assuming a variance of 20% for our calculations. Thus, using an online sample size 
calculator 
for 
determining 
ANOVA 
sample 
sizes, 
(https://homepage.univie.ac.at/robin.ristl/samplesize.php?test=anova), 
we 
have 
determined that a sample size of 10 subjects per group should be sufficient for this pilot 
analysis and the identification of variations of interest within the collected data. 
 
Due to the relative simplicity of our treatment protocol, data collection, and smaller sample 
size for recruitment, we do not anticipate significant impacts from dropout, withdrawal, or 
missing data on our study power. 
 

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 Randomization and Measures to Minimize Bias 
This study does not include randomization or blinding elements and measurements will all 
be made by analyzing samples with appropriate equipment. Immune cell population data 
will be gathered using flow cytometers and cytokine data will be gathered using similar 
technology (Luminex MagPix). Thus, there is no bias in raw data recording since values 
will be generated based on the machines’ detection and will simply be recorded and 
compiled by the researchers, bypassing subjectivity in the measurements. All participants 
and samples will be handled the same throughout the study aside from the differences in 
the stimulation parameters. Sample collection, processing, and analysis will all be 
standardized to discourage procedural variations that may impact data values. The fact 
that there is no separate control group and each participant will be used as their own 
baseline will also help prevent bias in measurement and analysis. 
 
10.3 Populations for Analyses 
Cytokine production analysis, dataset 1: each subject will have their own individual dataset 
of cytokine measurements that will be analyzed independently. 
 
Cytokine production analysis, dataset 2: all subjects in from Objective 1/Group 1 will be 
included in the dataset to determine the effects of ultrasound stimulation intensity on 
inflammation. 
 
Cytokine production analysis dataset 3: all subjects from Objective 2/Group 2 will be 
included to determine the efficacy of splenic- vs. cervical vagus-targeted ultrasound. 
 
Immune cell population analysis dataset 1: each subject will have their own individual 
dataset of cytokine measurements that will be analyzed independently. 
 
Immune cell population analysis dataset 2: all subjects in from Objective 1/Group 1 will be 
included to determine the effects of ultrasound stimulation intensity on inflammation. 
 
Immune cell population analysis dataset 3: all subjects from Objective 2/Group 2 will be 
included to determine the impact of splenic- vs. cervical vagus-targeted ultrasound. 
 
10.4 Statistical Analyses 
10.4.1 General Approach 
Data will be presented as individual points for each measurement and calculation 
performed. Each value obtained from an individual’s samples will be plotted as a discrete, 
independent data point when displayed in graphical formats. Means, medians, and 
standard deviations will be calculated and presented in table format. The response will be 
defined as the difference between post-ultrasound intervention (stimulation 1 or 
stimulation 2, respectively) and immediate baseline (baseline 1 or baseline 2, 
respectively). The potential within-subject correlation in the response from two sets of 
ultrasound stimulations will be evaluated in the linear mixed effects model and quantified 
using the intra-class correlation (ICC). If ICC is significantly different from zero, then the 
clustering effect at the subject level will be accounts for using the linear mixed effects 
model. 
 
Additional details regarding the statistical approach are found in the sections above and 
below. Briefly, all statistical tests will use p ≤ 0.05 as the significance cut-off level unless 
multiple comparison testing requires this be adjusted. All tests will be two-tailed. Given our 
smaller sample sizes, the normality of each group will be interrogated using the Lilliefors 

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test. The outcome of this test will then determine the details of the subsequent analysis 
and tests performed. If there are points that we suspect to be outliers, we will consider 
using the Grubbs test to verify or assuage our suspicions and ensure the highest quality 
data is included in our analyses. 
 
10.4.2 Analysis of the Primary Efficacy Endpoint(s) 
For both primary endpoint objectives 1 & 2: Cytokine production levels from ex vivo 
supernatants will be quantified as pg/ml concentrations. Each participant will have their 
own individual concentration data sets. Each of the 4 blood samples from a participant 
(baseline 1, stimulation 1, baseline 2, stimulation 2) will be assessed for cytokine 
production. The cytokine concentrations will be determined by Luminex analysis. Thus, an 
individual’s data set will consist of presenting the individual data points and the means 
and/or medians of the values from each sample. To compare baseline and ultrasound 
stimulation conditions for each participant, ANOVA or non-parametric equivalent statistical 
tests will be performed. In addition, the means of the ultrasound stimulation conditions will 
be normalized to the respective immediate baseline data from the same participant (e.g., 
fold change from baseline or percent of baseline). Normalized data will be compiled by 
experimental group to assess the magnitude of divergence from baseline for each 
stimulation. The experimental groups are: Objective 1 - 1) spleen, MI = 0.6; 2) spleen, MI 
= 1.0; 3) spleen, MI = 1.4; 4) spleen, MI = 1.8; Objective 2 - 5) spleen, MI = 1.4; 6) neck, 
MI = 1.4. All groups will be compared to each other in an ANOVA/non-parametric analysis 
and multiple comparison tests will be performed to identify where any differences lie. The 
appropriate tests will be chosen once we have the data and can properly assess the 
statistical assumptions that can be made.  
 
 
10.4.3 Analysis of the Secondary Endpoint(s) 
The secondary endpoint analysis is fully independent from analysis of the primary 
endpoint. The analyses will be performed in parallel and are meant to assess completely 
separate immunological characteristics of circulating immune cells. Immune cell flow 
cytometric data will be obtained by isolating cells from the blood samples and staining 
them with fluorescent antibodies to identify individual immune cell populations prior to 
running through a flow cytometer. The population characteristics will be presented as cell 
numbers per ml of blood (calculated from # of cells detected via flow cytometry and volume 
of blood run through the cytometer), percent of total events detected, and percent of total 
CD45+ cells detected. For each participant, they will have their own individual data sets, 
as above for the primary endpoint. Each blood sample from a participant (baseline 1, 
stimulation 1, baseline 2, stimulation 2) will be assessed for immune cell populations. 
Thus, an individual’s data set will consist of presenting the individual data points and the 
means and/or medians of the values from each sample. To compare baseline and 
ultrasound stimulation conditions for each participant, ANOVA or non-parametric 
equivalent statistical tests will be performed. In addition, the means of the ultrasound 
stimulation conditions will be normalized to the respective baseline data from the same 
participant (e.g., fold change from baseline or percent of baseline). Normalized data will 
be compiled by experimental group to assess the magnitude of divergence from baseline 
for each treatment. The experimental groups are: Objective 1 - 1) spleen, MI = 0.6; 2) 
spleen, MI = 1.0; 3) spleen, MI = 1.4; 4) spleen, MI = 1.8; Objective 2 - 5) spleen, MI = 1.4; 
6) neck, MI = 1.4. All groups will be compared to each other in an ANOVA/non-parametric 
analysis and multiple comparison tests will be performed to identify where any differences 
lie. The appropriate tests will be chosen once we have the data and can properly assess 
the statistical assumptions that can be made. 

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10.4.4 Safety Analyses and Safety Evaluation 
The only intervention in this study is a brief, non-invasive ultrasound stimulation that we 
do not anticipate to have appreciable detrimental impacts on subject health or day-to-day 
life. Tolerability and safety will be assessed based on patient experience responses during 
visits and voluntary communication from subjects in the event they experience an event 
they are concerned about and measurement of urinary AKI biomarkers. The safety 
evaluation involved in this study is collection of subject experience responses and spot 
urine collection. Urinary biomarker levels before and after ultrasound stimulation will be 
compared using the Wilcoxon Signed-Rank test. 
 
10.4.5 Baseline Descriptive Statistics 
The entire study is descriptive in nature and baseline data constitutes a significant portion 
of the analysis datasets. 
 
10.4.6 Planned Interim Analyses 
Not applicable. 
 
10.4.7 Sub-Group Analyses 
This is a pilot trial to gather descriptive data. Sub-group implications that are present in 
the datasets will be used to inform future analysis and study designs, but is not a primary 
concern in this study. 
 
10.4.8 Tabulation of Individual Participant Data 
Individual participant data will be assessed independently for part of the analysis, but will 
not necessarily be tabulated. 
 
10.4.1 Safety evaluation 
The safety evaluation involved in this study is collection of subject experience responses 
for identification of AEs and verification of ultrasound stimulation safety and feasibility.  
 
11 REGULATORY AND OPERATIONAL CONSIDERATIONS 
11.1 Regulatory and Ethical Considerations 
 Informed Consent Document  
Consent forms will be written in accord with federal regulations and will be reviewed and 
approved by the UVA IRB-HSR prior to use. Signed consent forms and other research 
records will be retained in a confidential manner. 
 
 Consent Procedures and Documentation 
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continues throughout the individual’s study participation. 
Consent forms will be Institutional Review Board (IRB)-approved and the participant will 
be asked to read and review the document. A member of the study team will explain the 
research study to the participant and answer any questions that may arise. A verbal 
explanation will be provided in terms suited to the participant’s comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research 
participants.  Participants will have the opportunity to carefully review the written consent 
form and ask questions prior to signing. The participants should have the opportunity to 
discuss the study with their family or surrogates or think about it prior to agreeing to 

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42 
participate. The participant will sign the informed consent document prior to any 
procedures being done specifically for the study. Results from procedures completed prior 
to consent for standard of care purposes may be used for research purposes. Participants 
must be informed that participation is voluntary and that they may withdraw from the study 
at any time, without prejudice. A copy of the informed consent document will be given to 
the participants for their records. The informed consent process will be conducted and 
documented in the source document (including the date), and the form signed, before the 
participant undergoes any study-specific procedures. The rights and welfare of the 
participants will be protected by emphasizing to them that the quality of their medical care 
will not be adversely affected if they decline to participate in this study. 
 
 Confidentiality and Privacy   
Participant confidentiality and privacy is strictly held in trust by the participating 
investigators, their staff, and the sponsor(s) and their interventions. This confidentiality is 
extended to cover testing of biological samples and genetic tests in addition to the clinical 
information relating to participants. Therefore, the study protocol, documentation, data, 
and all other information generated will be held in strict confidence. Consents will be 
maintained in a confidential manner in accordance with the code of federal regulations 
and HIPAA. When possible, specimens will be coded with study-specific IDs (not MRN or 
name). No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor. All research 
activities will be conducted in as private a setting as possible. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. 
The clinical study site will permit access to such records. 
 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept 
in a secure location for as long a period as dictated by the reviewing IRB, Institutional 
policies, or sponsor requirements. 
 
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the UVA School of Medicine (SOM). 
Individual participants and their research data will be identified by a unique study 
identification number. The study data entry and study management systems used by 
clinical sites and by UVA SOM research staff will be secured and password protected.  
To further protect the privacy of study participants, the research team may apply for a 
Certificate of Confidentiality. This certificate protects identifiable research information from 
forced disclosure. It allows the investigator and others who have access to research 
records to refuse to disclose identifying information on research participation in any civil, 
criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help 
achieve the research objectives and promote participation in studies by helping assure 
confidentiality and privacy to participants. 
 
 Future Use of Stored Specimens and Data  
This study does not include plans to store any subject specimens.  

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 Safety Oversight 
The principal investigator, Prof. Mark D. Okusa, MD, FASN, will serve as the safety 
monitor for this study. Any study under the purview of the University of Virginia HSR-IRB 
is subject to review of UVA documents. Studies are chosen for Post-approval Monitoring 
(PAM) either a) at random or b) requested by a study team member or any member of the 
IRB-HSR.   
 
The purpose of Post-approval Monitoring audits is to ensure that documentation of clinical 
research studies is of the highest quality, verify protocol adherence, and ensure that all 
Federal and local rules concerning clinical research are being fulfilled.  Post-approval 
monitoring is done by staff within the office of the Vice President for Research (VPR) in 
accordance with their Standard Operating Procedures. The conduct of an on-site review 
may include but is not limited to: 
• 
requests for progress reports from investigators, 
• 
examinations of research records, including signed informed consent documents, 
protocol modifications, and unexpected, serious, and/or related adverse 
experience reports, 
• 
contacts with research subjects, or 
• 
observation of the consent process and/or research procedures. Examples of 
when observation of the consent process could occur are: 
• 
Full board IRB determines during review of a project that a conflict of 
interest exists such that the informed consent process should be observed 
by a neutral party; 
• 
IRB is made aware of a complaint or concern with regard to the informed 
consent process; or 
• 
IRB determines as a result of the monitoring process that the consent 
process is insufficient and education/training is required for conduct of 
consent. 
 
 Site Monitoring 
Any study under the purview of the University of Virginia HSR-IRB or HSR-SBS is subject 
to review. Studies are chosen either a) at random or b) requested by a study team member 
or any member of the IRB-HSR and the DSMC.  
The purpose of audits is to ensure that documentation of clinical research studies is of the 
highest quality, verify protocol adherence, and ensure that all Federal and local rules 
concerning clinical research are being fulfilled. A study will be triggered for an audit once 
3 patients have been registered in OnCore. 
Semi-annual auditing is required for all High-risk studies. For high-risk studies, if findings 
are satisfactory after two reviews, protocols will be audited once a year. Any time findings 
are unsatisfactory, auditing will return to the original schedule. 
 
 Quality Assurance and Quality Control 
Each clinical site will perform internal quality management of study conduct, data and 
biological specimen collection, documentation and completion according to institutional 
policies. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system 
and data QC checks that will be run on the database will be generated. Any missing data 
or data anomalies will be communicated to the site(s) for clarification/resolution. 
 

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Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable 
regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing 
Practices (GMP)).  
 
 
11.2 Data Handling and Record Keeping  
 Data Collection and Management Responsibilities  
Data collection is the responsibility of the clinical trial staff at the site under the supervision 
of the site investigator. The investigator is responsible for ensuring the accuracy, 
completeness, legibility, and timeliness of the data reported.  
All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data.   
Studies using Oncore: Data will be collected using a password-protected, centralized 
electronic case report form called ON-line Clinical Oncology Research Environment = 
Oncore.] Data recorded in the electronic case report form (eCRF) derived from source 
documents should be consistent with the data recorded on the source documents.  
Cellular data will be collected using high dimensional spectral cytometers, such as the 
Cytek Norhtern Lights or Aurora, and cytokine data will be collected with the Magpix 
analyzer housed in the Flow Cytometry Core Facility (FCCF). These machines output data 
in the .fcs format which can be analyzed by flow cytometry analysis software, for example 
FlowJo. Cytokine data will be processed by the FCCF and returned to investigators in a 
spreadsheet format. Data will be analyzed using a combination of computational 
spreadsheet software such as Microsoft Excel, statistical software such as Prism, and/or 
statistical programming languages such as R. All samples will be immediately deidentified 
following collection and referred to by their experimental identifiers during processing, data 
collection, and analysis. A classified and secured record key that associates subjects with 
their experimental identifiers will be maintained in a single location that is firewalled, 
encrypted, and password protected. There should be no need to reference this document 
aside from quality control to ensure that the proper baseline control samples are 
associated with the proper treatment samples for statistical analysis purposes. 
 
 Study Records Retention  
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonisation (ICH) region and 
until there are no pending or contemplated marketing applications in an ICH region or until 
at least 2 years have elapsed since the formal discontinuation of clinical development of 
the study intervention. These documents should be retained for a longer period, however, 
if required by local regulations. No records will be destroyed without the written consent 
of the sponsor, if applicable. It is the responsibility of the sponsor to inform the investigator 
when these documents no longer need to be retained. Record retention will be in accord 
with device and HIPAA regulations. 
 
11.3 Publication and Data Sharing Policy 
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations: 
 

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The research team for this study is small and unified in vision. All investigators and 
personnel with a significant role in the design, completion, and/or analysis of the study will 
be included as authors in the event that results are published. 
 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination 
of NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results 
Information Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and 
results information from this trial will be submitted in accordance with regulations and any 
active contractual obligations. In addition, every attempt will be made to publish results in 
peer-reviewed journals.   
  
11.4 Conflict of Interest Policy 
Although this is a pilot study designed to collect proof of principle data for future use and 
study design (if the results support the hypotheses), the independence of this study from 
any actual or perceived influence is still critical. Therefore, any actual conflict of interest of 
persons who have a role in the design, conduct, analysis, publication, or any aspect of 
this trial will be disclosed and managed. Furthermore, persons who have a perceived 
conflict of interest will be required to have such conflicts managed in a way that is 
appropriate to their participation in the design and conduct of this trial. The study 
leadership in conjunction with the institution has established policies and procedures for 
all study group members to disclose all conflicts of interest and will establish a mechanism 
for the management of all reported dualities of interest.  
 
 

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prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-
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Gigliotti JC, Huang L, Bajwa A, Ye H, Mace EH, Hossack JA, et al. Ultrasound 
Modulates the Splenic Neuroimmune Axis in Attenuating AKI. J Am Soc Nephrol. 
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Zachs DP, Offutt SJ, Graham RS, Kim Y, Mueller J, Auger JL, et al. Noninvasive 
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Experimental Hypertensive and Diabetic Nephropathy. Hypertension. 2020;76(6):1906-
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C, et al. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. 
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Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, et al. Nicotinic 
acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 
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Inoue T, Abe C, Sung SS, Moscalu S, Jankowski J, Huang L, et al. Vagus nerve 
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14. 
Tanaka S, Abe C, Abbott SBG, Zheng S, Yamaoka Y, Lipsey JE, et al. Vagus 
nerve stimulation activates two distinct neuroimmune circuits converging in the spleen to 
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Inoue T, Abe C, Kohro T, Tanaka S, Huang L, Yao J, et al. Non-canonical 
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Burks SR, Nguyen BA, Tebebi PA, Kim SJ, Bresler MN, Ziadloo A, et al. Pulsed 
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2015;33(4):1241-53. 
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Charles EJ, Tian Y, Zhang A, Wu D, Mehaffey JH, Gigliotti JC, et al. Pulsed 
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injury. J Thorac Cardiovasc Surg. 2021;161(4):e297-e306. 
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Graham RS, Zachs DP, Cotero V, D’Agostino C, Ntiloudi D, Kaiser CRW, et al. 
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2022). 2020. 
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Sen T, Tufekcioglu O, Koza Y. Mechanical index. Anatol J Cardiol. 2015;15(4):334-
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21. 
Tirada N, Dreizin D, Khati NJ, Akin EA, Zeman RK. Imaging Pregnant and 
Lactating Patients. Radiographics. 2015;35(6):1751-65. 
22. 
Younan M, Atkinson TJ, Fudin J. A Practical Approach to Discontinuing NSAID 
Therapy Prior to a Procedure. Practical Pain Management. https://paindr.com/wp-
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Discontinuing-NSAID-Therapy-Prior-to-a-Procedure.pdf (accessed March, 17, 2022). 
2013. 
23. 
Chow KU, Luxembourg B, Seifried E, Bonig H. Spleen Size Is Significantly 
Influenced by Body Height and Sex: Establishment of Normal Values for Spleen Size at 
US with a Cohort of 1200 Healthy Individuals. Radiology. 2016;279(1):306-13. 
24. 
Sonographische Untersuchung der Milz [Sonographic evaluation of the spleen]. 
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https://www.amboss.com/de/wissen/Sonografische_Untersuchung_der_Milz/ (accessed 
March 26, 2022). 
25. 
Giovagnorio F, Martinoli C. Sonography of the cervical vagus nerve: normal 
appearance and abnormal findings. AJR Am J Roentgenol. 2001;176(3):745-9. 

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26. 
Ottaviani MM, Wright L, Dawood T, Macefield VG. In vivo recordings from the 
human 
vagus 
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ultrasound-guided 
microneurography. 
J 
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2020;598(17):3569-76. 
27. 
Ostermann M, Zarbock A, Goldstein S, Kashani K, Macedo E, Murugan R, et al. 
Recommendations on Acute Kidney Injury Biomarkers From the Acute Disease Quality 
Initiative Consensus Conference: A Consensus Statement. JAMA Netw Open. 
2020;3(10):e2019209. 
28. 
Paragas N, Qiu A, Zhang Q, Samstein B, Deng SX, Schmidt-Ott KM, et al. The 
Ngal reporter mouse detects the response of the kidney to injury in real time. Nat Med. 
2011;17(2):216-22. 
29. 
Emlet DR, Pastor-Soler N, Marciszyn A, Wen X, Gomez H, Humphries WHt, et al. 
Insulin-like growth factor binding protein 7 and tissue inhibitor of metalloproteinases-2: 
differential expression and secretion in human kidney tubule cells. Am J Physiol Renal 
Physiol. 2017;312(2):F284-F96. 
30. 
Yang QH, Liu DW, Long Y, Liu HZ, Chai WZ, Wang XT. Acute renal failure during 
sepsis: potential role of cell cycle regulation. J Infect. 2009;58(6):459-64. 
31. 
Zarbock A, Kellum JA. Remote Ischemic Preconditioning and Protection of the 
Kidney--A Novel Therapeutic Option. Crit Care Med. 2016;44(3):607-16. 
32. 
Katz NM, Kellum JA, Ronco C. Acute Kidney Stress and Prevention of Acute 
Kidney Injury. Crit Care Med. 2019;47(7):993-6. 
 
 
 

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13 APPENDICES 
13.1 Schedule of Activities (SoA) 
 
Procedures 
Screening/Baseline 1 
, Day -2 to -1 
Study Visit 1, Day 1 
Study Visit 2  
Day 2 (+1) day 
Study Visit 3/ 
Baseline 2 
Day 14 
Study Visit 4 
Day 15 (+1) day 
Final Study Visit 5 
(via phone) 
Day 30 +/-2 days  
Informed consent 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
Group assignment 
X 
 
 
 
 
 
Administer study 
intervention (ultrasound 
stimulation) or sham 
treatment 
 
X 
 
 
 
 
Administer study 
intervention (ultrasound 
stimulation) 
 
 
 
X 
 
 
Diagnostic ultrasonography 
 
X 
X 
X 
X 
 
Concomitant medication 
review 
X 
X--------------------------X 
 
Physical exam  
X 
 
 
X 
 
 
Vital signs 
X 
X 
 
X 
 
 
Height 
X 
 
 
 
 
 
Weight 
X 
X 
X 
X 
X 
 
Hematology a  
X 
Xb 
Xb 
Xb 
Xb 
 
Serum chemistry c 
X 
 
 
 
 
 
Pregnancy test d 
X 
 
 
 
 
 
Survey form 
 
X--------------------------X 
X 
Adverse event review and 
evaluation 
 
X--------------------------X 
X 
Urinary AKI biomarkers e,f 
 
X 
X 
X 
X 
 
Other assessments (flow 
cytometry analysis of 
immune cell populations, 
ex vivo stimulation assays 
to measure cytokine 
production) 
 
X 
X 
X 
X  
 
a:   Complete blood count. 
b:   Complete blood count with differential. 
c:    Serum creatinine, BUN, AST, ALT, fasting blood sugar, hemoglobin, A1c. 
d:   Urine pregnancy test (women of childbearing potential). 
e:   Spot urine sample (optional). 
f:   Optional urine samples will be collected at Visits 1 to 4 (i.e., prior to 
ultrasound stimulation, and ~30 minutes and 24 hours after ultrasound 
stimulation). 
 
 
13.2 Reporting Table 
Single-site study; section not applicable. 
 
 
 
 
 

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13.4 Protocol Amendment History 
 
Version 
Date 
Description of Change  
Brief Rationale 
1.1 
September 
2, 2022 
Refinement of exclusion criteria 
To 
improve 
quality 
and 
participant safety  
1.2 
January 29, 
2023 
Incorporation of AKI biomarkers in 
urine, complete blood count with 
differential sent to 
the UVA 
laboratory prior to ultrasound 
stimulation 
and 
post-treatment 
visit, respectively; incorporation of 
B 
mode 
ultrasonography 
performed at post-treatment visits 
To  gain additional insights 
on 
safety 
of 
ultrasound 
stimulation 
1.3 
May 
15, 
2024 
a) 
Added 
additional 
control 
subgroup to Objective 1; b) 
Refinement of criteria required 
before ultrasound stimulation can 
be delivered; c) weight data 
collection at Visits 1 to 4; d) 
timepoints of urine collection as 
noted in 8.2.3 (Research Urine) 
were not consistent with the ones 
given 
in 
13.1 
(Schedule 
of 
Activities)  
a) 
Review 
of 
the 
data 
collected so far indicates a 
sham treatment would be 
useful for increasing the 
confidence in our analysis 
and 
conclusions 
b) 
To 
improve participant safety 
and interpretability of results; 
c) to see whether ultrasound 
stimulation has any effects 
on weight; d) correction of 
urine collection timepoints in 
“Schedule of Activities”