Central Sodium Sensing: Implications for Blood Pressure Regulation
Phase: NA
Conditions: Salt Sensitivity of Blood Pressure
Sponsor: University of Delaware

 
 
 
 
 
 
 
 
 
 
 
Central Sodium Sensing: Implications for Blood Pressure Regulation 
 
NCT Number: NCT05480722 
 
August 19, 2025 
 
 

STUDY PROTOCOL 
 
Figure 1. Study Design 
All subjects will complete 8 visits (see above). Visits 1, 3, and 4 will be at the UD Center 
for Biomedical and Brain Imaging (CBBI). Visits 2, and 5-8 will take place at UD’s 
Science, Technology & Advanced Research (STAR) Campus. 
 
VISIT 1: INFORMED CONSENT, CBBI (60 MINUTES) 
Informed consent will be obtained in person by a trained member of the research team 
at CBBI. All procedures and aspects related to the study including any potential risks 
will be described to the subject. The subject will read the informed consent form and the 
investigator will answer any questions that the subject may have prior to obtaining the 
subject’s written consent. As documentation of the informed consent the subject and the 
investigator performing the consent will sign the consent document. A copy of the 
signed consent form will be given to the subject. After giving consent, subjects will lie in 
the mock scanner for at CBBI to screen for claustrophobia. Lastly, subjects will be given 
a personal food information form and 3-day diet recall to record food intake (2 weekdays 
and 1 weekend). These will help our dietician prepare food for the controlled feeding 
portion of this study. Subjects must complete these forms before the Nurse Managed 
Primary Care Center screening (visit 2). Subjects will not be excluded for food allergies 
V1/2: 
Consenting & 
Screening
Randomized Diet: 
- Low salt (2.6g NaCl/day, 1 g Na+)
- High salt (18g NaCl/day, 7 g Na+) 
Randomized Infusion: 
- 3% NaCl Hypertonic Saline
- Furosemide & 3% NaCl Hypertonic Saline
Salt-Sensitivity Testing (OPTIONAL)
Diet 1
Diet 2
Diet Week Timeline
Day 1 (V5 & 7): Urine collection, 
& BP; pick up food & equipment 
(BP cuff, urine collection, 
accelerometer)
Consume Randomized 7-day Diet
Day 8: Record BP and 
collect 24hr urine (at 
home)
Day 9 (V6 & 8): Blood 
draw, urine collection, & 
BP; return equipment
Day 2: Begin diet 
& accelerometer 
(at home)
>4week 
washout
>1week 
washout
V3: MRI 
Study Visit 1
V4: MRI 
Study Visit 2
>1 week 
washout
MRI Testing
Infusion: 
Diet: 

or preferences, but we will exclude individuals with very specific dietary restrictions such 
as celiac disease, gluten sensitivity, if an individual is vegan, or indicates an eating 
disorder. We can accommodate vegetarians who eat dairy and eggs. 
 
VISIT 2: SCREENING, STAR CAMPUS (60 MINUTES) 
All participants will be screened at the Nurse Managed Primary Care Center in the 
Health Sciences Complex of STAR. This initial screening visit lasts approximately 1 
hour and involves answering an MRI screening form and a medical history 
questionnaire. Additionally, nurses will assess resting blood pressure, 12-lead 
electrocardiogram, height and weight, and obtain a venous blood and urine sample. The 
blood and urine sample will assess liver and kidney function, a lipid profile (i.e., 
cholesterol), a complete blood count, glucose, sodium, potassium, and chloride. 
Approximately 2 teaspoons of blood will be sampled. Investigators will review all 
information. Individuals with any signs or symptoms of disease (example: history of 
angina, chest pain, dizziness, or shortness of breath, etc.) will be excluded. Individuals 
with blood work within normal limits, and a resting electrocardiogram within normal limits 
will be accepted into the study. The results from the screening are reviewed by the 
registered nurse to determine if participants qualify for the study. Participants will 
receive a copy of the data collected on their vital statistics. Women taking hormonal or 
non-hormonal contraceptives will not be excluded. Additional exclusion criteria include 
allergy to sulfa drugs (Lasix/furosemide), claustrophobia, low blood pressure (systolic 
blood pressure <100mmHg and/or diastolic blood pressure <60 mmHg), high blood 
pressure (defined here as systolic blood pressure >130mmHg and/or diastolic blood 
pressure >80 mmHg), low potassium levels in the blood (<3.5 mmol/L), a history of 
cancer, diabetes, or any other chronic disease; a history of any heart disease; use of 
tobacco or nicotine products; nursing mothers; or a body mass index less than 
18.5kg/m2 or greater than 30kg/m2. Certain medications may also result in exclusion 
from participating; this will be determined by the NMPCC in consultation with the 
primary investigator on an individual basis. If one of the test results is abnormal, 
individuals will be referred to their personal provider. 
 

VISITS 3 & 4: fMRI SCAN (2 hours/visit; CBBI)  
Subjects will be asked to log food and water intake for the 3 days leading up to the MRI 
visits on the AHA Sodium Tracker. Subjects will be asked to consume a diet containing 
2,300 mg of sodium each of these days. Subjects will consume their own food. Food will 
NOT be provided by the dietician for the 3 days leading up to the MRI visits. Subjects 
will be instructed to refrain from taking over the counter medications for 48 hours, and 
from vigorous aerobic exercise or alcohol for 24 hours prior to MRI scans. Subjects will 
come into the study visit after at least a four-hour fast. The MRI studies must be 
completed within 3 months of the screening. If we are unable to schedule both MRI 
visits within 3 months of screening, subjects will be asked to come to the Cardiovascular 
Physiology Lab again before the MRI visit for a blood draw to check if electrolyte levels 
are normal. The second MRI data collection must be performed no sooner than 1 week 
from the first MRI data collection. 
 
Figure 2: Timeline of Experimental Protocol 
 
Urine Sample Collection and Absorbent Brief or Condom Catheter (men only) 
All subjects will be asked to provide a urine sample. We will measure electrolytes, 
osmolality, and specific gravity as well as perform a pregnancy test for women. Only 
women with a negative pregnancy test will be allowed to continue with the study. If the 
test is positive, the participant will be informed and referred to their primary care 
Time in the MRI (minutes)
10
20
60
30
50
40
Baseline MRI - No Infusion 
(~20min)
Condition 1: 
Furosemide 
Bolus
Condition 2: 
Isotonic 
Saline Bolus
0
Randomized 
Crossover
IV Insertion & 
Set-up 
(~40min)
-40
MRI - Hypertonic Saline Infusion (30min)
IV Removal & 
Blood Draw 
(~10min)
Crossover 

provider. After subjects provide a urine sample, they will be asked to change into scrubs 
and put on the absorbent brief, both provided by the research team in a private 
changing room and bathroom. Subjects will be asked to urinate into the brief when they 
have the urge to urinate during the MRI. Male subjects will have the additional option of 
wearing a condom catheter instead of the absorbent brief. Likewise, male subjects will 
be asked to urinate while wearing the condom catheter when they have the urge to 
urinate during the MRI. 
 
Catheter Insertion & Blood Sample 
At CBBI, a registered nurse will insert one catheter into the arm of the participant, leaving 
a small flexible tube in their vein prior to the MRI scan. The catheter will be used for 
infusion of the saline and furosemide, and to draw blood samples. Blood samples will be 
obtained pre- and post-infusion to measure electrolytes, osmolality, hematocrit, and 
hemoglobin. Some of the blood sample will be stored in our -80C freezer (indefinitely) for 
future assessment of renin angiotensin aldosterone system hormones, inflammatory 
markers, and reactive oxygen species. About 40 mL of blood will be sampled from the 
intravenous catheter, pre- and post-infusion (80 mL total). The catheter will be removed 
at the end of the study. 
 
Furosemide Administration 
Each subject will undergo two separate MRI study visits (visit 3 and visit 4), one with 
and one without NKCC2 antagonism. Furosemide (NKCC2 antagonist) will be 
purchased through the Nurse Managed Primary Care Center from Heritage 
Pharmaceuticals through McKesson (link here). NKCC2 antagonism will be achieved by 
administering a bolus of furosemide (40mg in 4mL of 0.9% NaCl isotonic saline) over a 
2-minute period immediately prior to the hypertonic saline infusion. This timing will 
achieve peak NKCC2 blockade. A bolus of 0.9% NaCl (4mL) will be administered over a 
2-minute period during the control infusion, and the participant will be blinded to the 
condition. A registered nurse, under the off-site supervision of a cardiologist will 
administer the bolus of furosemide and administer the hypertonic saline infusion. 
 

Hypertonic Saline Infusion 
A 3% NaCl hypertonic saline will be infused at a rate of 0.15 ml·kg-1·min-1 into the 
participant’s arm for 30 minutes total while participants are in the MRI scanner. The 
hypertonic saline will be purchased from a manufacturer and will be prepared by the 
registered nurse. The volume of hypertonic saline in the tubing will be closely monitored 
by the registered nurse to ensure that participants are receiving the correct amount of 
saline. The volume infused and infusion rate is similar to previous studies infusing 
saline, which we have previously published (Farquhar et al AJP-Heart 2006). The 3% 
hypertonic saline is expected to raise blood sodium concentration and plasma 
osmolality approximately 1-2%. Blood samples will be taken pre- and post-infusion to 
measure changes in blood sodium concentration and osmolality.  
 
MRI Scan. The MRI scan will take approximately 1 hour (Figure 2). The subject will be 
free to stop at any time, either by indicating their desire to do so directly to the 
investigator or by squeezing a ball while in the MRI scanner that alerts the technologist 
who is running the machine. 
● MRI Screening Form. Participants will again fill out the MRI screening form prior to 
the scan to ensure they can safely undergo MRI. 
● Structural and BOLD MRI.  During all scans the subjects will wear disposable 
earplugs and headphones to reduce scanner noise. Participants may also be asked 
to wear a pulse oximeter on their finger to measure heart rate or a respiratory belt 
around their chest to measure breathing. Both devices are provided by Siemens with 
the MRI scanner, are MR-safe and MR-compatible, and interface with the MRI 
scanner software. Participants will also wear an FDA-approved MRI compatible 
blood pressure cuff on their middle finger throughout the scan that measures blood 
pressure through a Pulse Decomposition Analysis. This device is made through 
BioPac Systems and is MR-safe and MR-compatible.  
 
 
 
 

OPTIONAL COMPONENT OF STUDY (VISITS 5-8) 
VISITS 5 & 7: BLOOD DRAW, URINE COLLECTION, BP; & FOOD & EQUIPMENT 
PICKUP, STAR (30min/visit)  
Salt Sensitivity Assessment: Subjects will consume a low (2.6g NaCl/day, 1g Na+) and 
high (18g NaCl/day, 7g Na+) sodium diet for 7 days each (randomized, washout between 
diets). Twenty-four-hour BP (SunTech Oscar2) and urine, to confirm diet compliance, will 
be collected on day 8 of each diet week (Figure 2). A change of 24 hr mean BP of > 5 
mmHg, from the low to the high sodium condition will be considered a salt sensitive 
response. 
 
Participants will report to the CV lab of STAR on day 1 (Figure 1) of each randomized 
diet to 1) have a venous blood draw performed on them by a research team member 
trained in phlebotomy; 2) provide a urine sample; 3) have brachial blood pressure 
measured; and 4) pick up food, a 24hr BP cuff, and urine collection supplies. Blood 
samples will be obtained to measure electrolytes, osmolality, hematocrit, and 
hemoglobin. Some of the blood sample will be stored in our -80C freezer (indefinitely) 
for future assessment of renin angiotensin aldosterone system hormones, inflammatory 
markers, and reactive oxygen species. About 40 mL of blood will be collected from the 
butterfly needle insertion. Urine samples will be obtained to measure electrolytes, 
osmolality, and specific gravity as well as a pregnancy test for women. Diets will be 
prepared by a registered dietician on staff. These diets will be separated by a minimum 
4-week washout, but no greater than 6 months. Participants will drink water ad libitum 
but quantities will be recorded. Participants during different dietary control phases may 
be provided with Gatorade, juices and milk; in addition, the dietician will provide a 
detailed diet menu to allow for additional beverages such as coffee, decaffeinated 
coffee and/or tea that will not be provided to the participants. Physical activity will also 
be assessed using a physical activity monitor to evaluate activity patterns during each 
period of the controlled feeding study. Prior to starting the controlled salt diet, we will 
confirm that all female subjects are not pregnant by performing an over-the-counter 
pregnancy test. To accomplish this, participants will have to provide a small urine 
sample. 

VISITS 6 & 8: BLOOD DRAW, URINE COLLECTION, BP; & EQUIPMENT DROP-
OFF, STAR (30min/visit) 
Participants will report to the CV lab of STAR on day 9 (Figure 1) of each diet to 1) have 
a venous blood draw performed on them; 2) provide a urine sample; 3) have brachial 
blood pressure measured; and 4) drop off any left over food, the 24hr BP cuff, 
accelerometer and urine collection supplies. Blood samples will be obtained to measure 
electrolytes, osmolality, hematocrit, and hemoglobin. Some of the blood sample will be 
stored in our -80C freezer (indefinitely) for future assessment of renin angiotensin 
aldosterone system hormones, inflammatory markers, and reactive oxygen species. 
About 40 mL of blood will be collected from the butterfly needle insertion. Urine samples 
will be obtained to measure electrolytes, osmolality, and specific gravity as well as a 
pregnancy test for women. 
 
Alternate order of MRI (Visits 3 and 4) and Salt Sensitivity Testing (Visits 5-8). 
Subjects will be given the option to instead do the salt sensitivity testing (visits 5-8) first, 
followed by the MRI study visits (visits 3 and 4) if they have previously volunteered in an 
MRI study at the University of Delaware (and were not claustrophobic) or must begin 
with the salt sensitivity testing due to the logistics of scheduling.  
 
STUDY POPULATION AND RECRUITMENT 
We will recruit 50 normotensive men and women (18-45 years old), from the community 
surrounding the University of Delaware. Our study sample will be representative of the 
general population, including all races/ethnicities. Our strategies will include: Nextdoor 
app, classifieds, flyers posted locally, and targeted social media ads using ‘bump’ 
advertisement. The study will include all healthy participants with blood pressure 
between 100/60 to 130/80 mmHg. Individuals will not have any chronic diseases. 
 
Participants excluded from the study will include individuals with known cardiovascular 
disease, high blood pressure (defined here as systolic blood pressure >130mmHg 
and/or diastolic blood pressure >80 mmHg), low blood pressure (defined here as 
systolic blood pressure <100mmHg and/or diastolic blood pressure <60 mmHg), an 

allergy to sulfa drugs (Lasix/furosemide), low potassium in the blood (hypokalemia, <3.5 
mmol/L), metabolic diseases, cancer, kidney disease, a body mass index (BMI) outside 
of the range of 18.5 and 30 kg/m2, nicotine product consumer, pregnant or planning to 
become pregnant, and other chronic diseases. Participants with any conditions which 
would contra-indicate MRI: implant of pacemakers or pacemaker wires; artificial heart 
valve; brain aneurysm surgery; middle ear implant; non-removable hearing aid or 
jewelry; braces; cataract surgery or lens implant; implanted mechanical or electrical 
device; foreign metallic objects in the body such as bullets, BBs, shrapnel, or metalwork 
fragments; claustrophobia; uncontrollable shaking or the inability to lie still for 1.5 hours 
will be excluded.  
 
RISKS AND BENEFITS 
There are no known risks associated with obtaining height, weight, resting blood 
pressure, or urine analysis to test for pregnancy. 
 
Catheter Insertion & Butterfly Needle Insertion for Blood Draw 
Participants may have pain or bruising at the site where the catheter or butterfly needle 
is placed in the arm, and there is a small risk of infection. Fainting sometimes occurs 
during or shortly after blood is drawn.  
 
Condom Catheter 
The condom catheter is secured on the male penis by a self-adhesive latex. The self-
adhesive latex may increase male participant discomfort when removing the condom 
catheter. Additionally, there is a risk of an allergic reaction to the latex if male participants 
are allergic to latex. 
 
Hypertonic Saline Infusion 
The risks associated with infusing saline in the vein are low but can include damage to 
the vein, blood dilution, spasm or contraction of the vein, edema, high blood pressure, 
excess fluid in the lungs or brain, volume overload (too much fluid in the body), and heart 
failure. Adverse symptoms of these conditions include local arm discomfort where the 

catheter was inserted, shortness of breath, a headache, and/or fluid accumulation in the 
arms, legs, or lungs. 
 
None of these adverse symptoms are expected in healthy adults. Blood pressure may 
increase during the hypertonic saline infusion. Therefore, hypertonic saline will not be 
administered if resting blood pressure on the day of testing is above 140/90 mmHg. If 
blood pressure goes above 170/90 mmHg, we will stop the infusion. A controlled infusion 
of 3% NaCl has been safely performed in our lab and other labs.  
 
Hypertonic saline infusion studies have been previously used in our lab 
1. Greaney JL, Ray CA, Prettyman AV, Edwards DG, Farquhar WB. Influence of 
increase plasma osmolality on sympathetic outflow during apnea. Am J Physiol 
Regul Integr Comp Physiol, 299: R1096. 2010.  
2. Wenner MM, Rose WC, Delaney EP, Stillabower ME, Farquhar WB. Influence 
of plasma osmolality on baroreflex control of sympathetic activity. Am J Physiol 
Heart Circ Physiol, 293: H2313-H2319. 2007. 
3. Farquhar WB, Wenner MM, Delaney EP, Prettyman AV, Stillabower ME. 
Sympathetic neural responses to increased osmolality in humans. Am J Physiol 
Heart Circ Physiol, 291: H2181-H2186. 2006. 
4. Babcock MC, Brian MS, Watso JC, Edwards DG, Stocker SD, Wenner 
MM,  Farquhar WB. Alterations in dietary sodium intake affects cardiovagal 
baroreflex sensitivity. American Journal of Physiology-Regulatory, Integrative 
and Comparative Physiology, ajpregu.00002. 2018.  
 
Hypertonic saline infusion studies performed in other labs 
1.  Stachenfeld NS, DiPietro L, Palter SF, and Nadel SR. Estrogen influences osmotic 
secretion of AVP and body water balance in postmenopausal women. Am J 
Physiol Jan; 274(1):R187-95, 1998. 
2.  Stachenfeld NS, Mack GW, Takamata A, DiPietro L, and Nadel ER. Thirst and fluid 
regulatory responses to hypertonicity in older adults. Am J Physiol Sep;271(3 pt 
2): R757-65), 1996.  

Furosemide 
There are several risks of infusing furosemide although the adverse reactions are 
uncommon. Furosemide is a widely used medication. References 1 (n=199) & 3 (n=8) 
below used an identical dosage that we propose (40mg). The infusion of furosemide could 
lead to gastrointestinal, allergic, central nervous system, blood and heart reactions. Blood 
pressure may decrease after the furosemide bolus. If blood pressure goes below 90/50 
mmHg after furosemide is given, we will stop the study visit. Symptomatic hypotension 
leading to dizziness or fainting may also result. Furosemide is a diuretic and increases 
the urge to urinate and can make participants feel thirsty. Because of this, it is likely that 
participants will need to use the restroom during the MRI scan. When subjects need to 
use the restroom during the MRI scan, they will be asked urinate into the absorbent brief 
they will be wearing. This is a normal response, but it may cause a feeling of 
embarrassment. If participants choose to come out of the MRI scan to use the restroom, 
the study will be concluded because the effect of the hypertonic saline and furosemide 
will have worn off by the time the participant is put back into the MRI. Participants will still 
be compensated and allowed to proceed with the other study visits. 
 
Pregnancy: Furosemide falls under Pregnancy Category C. At higher than maximal 
recommended human doses, rodent studies have shown unexplained fetal harm. There 
are no adequate and well-controlled studies in pregnant women. Although there are no 
established guidelines at this time regarding furosemide and pregnancy, participants will 
be informed that there is risk.  If the participant may be pregnant or if the participant does 
not want to expose themselves to this risk, they will be excluded from participating in this 
study. Female subjects will only be permitted to participate upon a negative pregnancy 
test.  
 
Furosemide infusion studies performed in other labs 
1. Chun TY, Bankir L, Eckert GJ, Bichet DG, Saha C; Zaidi S, Wagner MA, Pratt JH. 
Ethnic differences in renal responses to furosemide. Hypertension, 52(2):241-8. 
2008.  

2.  Musso CG, Reynaldi J, Vilas M, De Miguel R, Imperiali N, Algranati L. Fractional 
excretion of K, Na, and Cl following furosemide infusion in healthy, young and very 
old people. Int Urol Nephrol. 42:273-277. 2010 
3. van Meyel JJ, Smits P, Russel FG, Gerlag PG. Diuretic efficiency of furosemide 
during continuous administration versus bolus injection in healthy volunteers. 
Clinical Pharmacology and Therapeutics. Apr;51(4):440-444. 1992. 
 
Magnetic Resonance Imaging (brain structure and activation): 
MRI does not use any ionization radiation and it does not use radioactivity, however, there 
are risks associated with MRI scans. Potential risks are listed below. 
 
● Metal: The MRI scanner produces a constant strong magnetic field, which may cause 
any metal implants, clips, or implanted medical devices within the body to shift position 
or malfunction. Participants will not be allowed to participate in this study if they have 
any implanted metal, clips or devices. Participants will be screened to make sure that 
it is safe for them to enter a strong magnetic field. Participants will be asked to provide 
as much information as they can, for example if they had surgery in the past, so that 
we may decide whether it is safe for them to be a participant. Metallic objects brought 
into the MRI environment can become hazardous projectiles and can also interfere 
with the data quality. To minimize this risk, metal earrings, other piercings, necklaces 
and any other metal in contact with their body must be removed prior to the study. 
Participants must also remove all items from their pockets, including coins, electronics 
(including cell phones and hearing aids) and wallets. Participants must remove belts 
with metal buckles, and they may be asked to change into a gown that we will provide 
if their clothing contains significant metal, including metal underwire bras. 
● Pregnancy: Exposure to MRI scanning might be harmful to an unborn child. Although 
there are no established guidelines at this time regarding MRI and pregnancy, 
participants will be informed that there is a possibility of a yet undiscovered pregnancy 
related risk.  If the participant may be pregnant or if the participant does not want to 
expose themselves to this risk, they will be excluded from participating in this study. 

● Inner ear damage: MRI scanning produces loud noises that can cause damage to 
the inner ear if appropriate hearing protection is not used. Earplugs and/or 
headphones will be provided to protect participant’s ears.  
● Claustrophobia: When a participant is inside the MRI scanner, the “bore” of the 
scanner will surround the part of their body that is being scanned. For example, if a 
participant was getting an MRI of their knee, the participant would be positioned so 
that their knee was centered in the bore of the scanner. In the case of fMRI, we are 
interested in brain activity, and the participant’s head will be centered inside a close-
fitting scanning coil positioned in the bore of the scanner. If the participant feels 
anxious in confined spaces, they will have the option to discontinue the study. We will 
have subjects try our “mock” scanner to evaluate their comfort level with the enclosed 
space of the magnet bore. If the participant decides to participate and begins to feel 
claustrophobic, they will be able to tell us via the intercom or the squeeze ball and we 
will discontinue the study immediately. 
● Burns: In rare cases, contact with the MRI transmitting and receiving coil, conductive 
materials such as wires or other metallic objects, or skin-to-skin contact that forms 
conductive loops may result in excessive heating and burns during the experiment. 
The operators of the MRI scanner will take steps, such as using foam pads, when 
necessary, to minimize this risk. Tattoos with metallic inks can also potentially cause 
burns. In addition, please let the MRI operator know immediately if you experience 
any heating or burning sensations during a scan. The scanning session will be 
stopped as soon as the participant tells the operator.  
● Nerve or muscle stimulation. While the scanner is operating, there is a small chance 
that the rapidly changing magnetic fields could cause a slight tingling sensation or a 
muscle twitch, usually felt in the upper arms or torso. While these sensations may be 
startling, they are not dangerous or a health risk, and they have no lasting 
consequences. The sensations should stop when the scan ends. Because these 
sensations may nevertheless be distracting or even possibly uncomfortable, 
participants will be asked to squeeze the signal bulb to alert the scanner operator if 
they feel tingling or muscle twitching, and we will immediately stop the scan. 

Participants will then have the opportunity to choose to withdraw from the study or to 
continue.  
● Other Risks. Besides the risks listed above, there are no other known risks from the 
magnetic field or radio waves at this time. Although functional MRI scanning has been 
used for more than 20 years, long-term effects are unknown. 
 
Seven-day diets:  
There are minimal risks associated with consuming a 7-day low salt diet and 7-day high 
salt diet. During the high salt diet, blood pressure may rise to higher-than-normal levels, 
and subjects might experience slight hand swelling, frequent urination from increased 
water intake, bloating, nausea and/or vomiting during the dietary salt manipulation. There 
is no increased risk for women regardless of phase of menstrual cycle and diets should 
not affect menstruation. 
 
7-day accelerometer, 24-hour blood pressure monitoring & urine collection:  
There are no known risks to wearing an accelerometer or continuous blood pressure 
monitor on the upper arm.  During waking hours, blood pressures are taken every 20 
minutes, while at night they are taken every 30 minutes.  Minor discomfort may be 
experienced when the cuff inflates, and it may wake subjects during sleep. The 24-hour 
blood pressure cuff and 24-hour urine collection may disrupt normal daily living. There 
are no known risks to providing a urine sample. 
 
STEPS TAKEN TO MINIMIZE RISK 
Catheter Insertion & Butterfly Needle Insertion for Blood Draw 
Standard safety precautions will be taken when drawing blood samples and placing the 
IV catheter and butterfly needle. To minimize the risk of infection associated with blood 
collection, only sterile, single use catheters and butterfly needles will be used. To 
minimize the risk of bruising, proper blood collection technique will be utilized.  
 
 
 

Condom Catheter 
There is nothing we can do to minimize the risk of discomfort when removing the condom 
catheter. We will ask male participants if they have a known latex allergy. If they are 
allergic to latex, they will not be permitted to use the condom catheter and will have to 
use the absorbent brief. 
 
Hypertonic Saline & Furosemide Infusion 
The risks of the hypertonic saline and furosemide infusions will be minimized by having 
proper medical oversight of the procedure. A registered nurse, under the supervision of 
a cardiologist, will administer the infusions and monitor the participant throughout the 
entire protocol. We are using previously established hypertonic saline and furosemide 
doses that have shown to be safe for research (see references above). We will not 
administer furosemide if blood pressure is too low (hypotensive, <100/60 mmHg), if 
subjects are allergic to sulfa drugs (Lasix/furosemide), or if subjects have low potassium 
in the blood at their screening visit (hypokalemia, <3.5 mmol/L). Furosemide is a diuretic 
and increases the urge to urinate and can make participants feel thirsty. When subjects 
need to use the restroom during the MRI scan, they will be asked to urinate into the 
absorbent brief they will be wearing. This is a normal response, but it may cause a feeling 
of embarrassment.  
 
Following the conclusion of the infusion protocols, participants will be given sanitation 
wipes and asked to change back into their clothing and remove and dispose of the 
absorbent brief in the private changing room and bathroom. They will also be given water 
and a snack and will remain in the lab for at least 45 minutes for observation by the 
research nurse. We will leave the IV catheter in the arm and assess blood pressure and 
electrolytes during this time, to ensure the values are within clinically acceptable limits 
prior to the participant leaving the facility. In the unlikely case the person has symptoms, 
an abnormal blood pressure (e.g., < 90/50 mmHg or > 170/90 mmHg), and/or abnormal 
electrolyte levels (e.g., potassium level (< 3.5 mmol/L), the registered nurse will determine 
the appropriate course of action. The course of action will be determined by our post 
furosemide blood pressure and potassium decision charts (Figure 3). Both decision flow 

charts will be followed to determine appropriate course of action after the infusion. 
Subjects may be asked to remain at the MRI facility for further blood pressure and 
electrolyte observation (up to 2 hours after infusion). The additional blood draw (4mL) will 
be through the IV catheter already in place. Subjects may also be encouraged to eat a 
diet high in potassium for a few days if potassium is low (diet suggestions attached). In 
the highly unlikely case of abnormal electrolytes and symptoms, subjects may be referred 
to their primary care physician or sent to a local emergency department via ambulance. 
We do not expect any adverse effects in the well-screened subjects that will be tested as 
part of this protocol. 
 
 
Figure 3 Decision Charts. Post furosemide blood pressure decision chart (left). Post 
furosemide potassium decision chart (right). Both decision flow charts will be followed to 
determine appropriate course of action after the infusion. 
 
Magnetic Resonance Imaging (brain structure and activation) 
The fMRI protocol will be performed using an MRI scanner employing pulse sequences 
and hardware that have been approved by the FDA for human clinical use. The field 
strength is 3 Tesla and all relevant operating characteristics (RF power deposition, rate 
of change of the field gradients, coil design) fall within the limits of FDA guidelines for 
NMR exposure. Participants will be carefully screened to exclude those who may have 
metal in or on their bodies that cannot be removed (e g., bullets, metal filings, body 
piercings, etc.). MR facility rules strictly forbid staff from entering the magnet room 
carrying metal objects. The risk of claustrophobia is minimized by screening subjects for 

self-reported claustrophobia and making sure the subject is lying comfortably with head 
and neck supported and providing ear protection with headphones, a mirror to see out, a 
button to signal distress, and an intercom.  Scan time will be kept to a minimum.  Female 
participants will be given a urine pregnancy test immediately before the scanning period, 
and those with a positive result will not be scanned. Participants are given a squeeze ball 
to use in case of an emergency. Participants are informed that if they experience any 
unpleasant sensations or are otherwise uncomfortable, they can alert the MRI 
technologist via the squeeze ball and the technologist will stop the scan immediately.  
 
With regard to subject confidentiality, all records will be coded, stored in a locked cabinet, 
and kept strictly confidential. Samples stored in the freezer will all be coded; names will 
not appear on samples. Our database will be password protected, as is the computer that 
the database resides on. Subject names do not appear in the database, just a subject ID 
number. The list linking the ID number with the subject name is kept in a separate, locked 
filing cabinet. Thus, breaches of confidentiality are very unlikely.  
 
University policy requires that all study personnel complete human subjects training. 
Thus, the PIs, study coordinator, and graduate students involved in this data collection 
have all had this training. While the risk of medical emergency is very low, all subject 
personnel are certified in CPR. The Nurse Managed Health Center is located across the 
hallway from the research laboratory. An automated external defibrillator is located on 
site, and emergency numbers are posted on the phone and in the laboratory. Christiana 
Care Hospital, a 913-bed teaching hospital, is located 4 miles from the University.  
 
MRI ACQUISITION AND ANALYSIS AND STATISTICS 
MRI Acquisition 
We will use a 3T MRI Siemens Prisma Scanner and 64-channel head coil to obtain all 
brain images. Padding will be placed around the participant’s head to minimize head 
motion. Participants will be instructed to keep both their head and the rest of their body 
as still as possible.  
  

All participants will undergo a T1-weighted anatomical scan [repetition time (TR) = 2080 
ms; echo time (TE) = 4.6 ms; field of view = 210 x 210 mm; voxel size = 0.7 mm3; slice 
thickness = 0.7 mm] followed by two BOLD fMRI scans (baseline & infusion). Functional 
scans will be acquired using a multi-band gradient-echo echo-planar imaging (EPI) 
sequence [TR = 829 ms; TE = 40 ms; flip angle = 52°; field of view = 208 x 208 mm; 
voxel size = 2.0 mm3; slice thickness = 2.0 mm]. During functional scans, participants 
will be instructed to keep their eyes open and fixated on a white cross (on a black 
screen) to minimize the effects of eye motion and ensure the reliability and consistency 
of our results. These sequences are identical to those used in our group’s pilot study. 
 
MRI Analysis 
The preprocessing steps described here were also utilized in our group’s pilot study. 
Functional MRI images will be converted and analyzed using several software packages 
including MRIcroGL, AFNI (Analysis of Functional NeuroImages) and FSL (FMRIB 
Software Library). Initially, the 30-minute infusion fMRI scans will be split into an early 
phase (0-15 minutes) and late phase (15-30 minutes). Processing steps include: 1) de-
spiking of the resting-state fMRI scans to eliminate outliers, 2) slice-timing correction of 
the fMRI scans, 3) 3D rigid motion correction to a reference frame to correct for head 
motion in the resting-state data (we removed time points with head motion > 0.5 mm), 4) 
cleaning the fMRI data using ANATICOR (accounting for the 6 motion parameters, 
white matter, and CSF signal), 5) band-pass filtering (0.008 to 0.10 Hz) (42), 6) skull-
stripping of the T1-anatomical scan using FSL, 7) co-registration (i.e., alignment) of the 
T1-anatomical and functional scans, 8) normalization of all scans to the 1mm MNI152 
template, and 9) spatial smoothing of the resting-state data using a Gaussian kernel of 
full-width half maximum (FWHM) = 4mm. These smoothing dimensions align with 
recommendations to choose a Gaussian kernel of FWHM double the voxel size (which 
was 2 mm3 for this study) and are appropriate for smaller brain structures like the SFO 
and OVLT. 
 
The ROIs for this study will be defined as follows. The SFO and OVLT will be defined as 
a 2-mm radius spheres based on previous studies. The Montreal Neurological Institute 

(MNI) coordinates corresponding to the center of each ROI are: SFO: x = 0 mm, y = 2 
mm, z = -6 mm and OVLT: x = 0 mm, y = 2 mm, z = -12 mm. The location of these 
seeds has been verified using Duvernoy’s Atlas of the Human Brainstem and 
Cerebellum and these ROIs are consistent with those used in a pilot study conducted in 
our lab.  
 
Following pre-processing, we will conduct seed-to-seed functional connectivity analyses 
to assess the synchronization of the spontaneous low frequency fluctuations in the 
BOLD signal between our ROIs. We will calculate functional connectivity between the 
SFO and OVLT. Pearson correlations will be computed between the ROIs for the seed-
to-seed functional connectivity analysis. Pearson correlations will be converted to Z-
scores using a Fisher’s transform which will be statistically analyzed, as in our pilot 
study.  
 
Statistical Analysis 
Normality will be verified using Shapiro-Wilk tests. Normally distributed baseline 
characteristics will be reported as frequences and mean standard deviation. Two-way 
repeated measures analysis of variance (ANOVA) models will be used to assess 
changes in functional connectivity between baseline, the early phase (0-15 min) of the 
infusion, and the late phase (15-30min) of the infusion with and without furosemide 
prior. If data are non-normally distributed, aligned rank transformations (ARTs) will be 
conducted prior to performing two-way repeated measures ANOVAs. ARTs will be 
completed using ARTool2 (version 2.2.2) or ARTool integrated into R. Significant main 
effects of time and time*sex interactions will be followed with pairwise post-hoc 
comparisons corrected for multiple comparisons.