Exploration of Blood Flow Regulation to Bone in Humans
Phase: NA
Conditions: Bone Blood Flow Regulation
Sponsor: Spaulding Rehabilitation Hospital

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 Page 1 of 13 Bone blood flow in SCI v7, May 24, 2022 
 
 
 
Exploration of potential alterations in bone perfusion  
after spinal cord injury 
 
Version 7 
05/24/22 
 
Clinicaltrials.gov # NCT04083794 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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 Page 2 of 13 Bone Blood Flow in SCI v7, May 24, 2022 
 
 
I. 
BACKGROUND AND SIGNIFICANCE  
Spinal cord injury (SCI) results in rapid and severe osteoporosis with decreases in bone mass of 
up to 45% within the first 3 years after injury. Although this osteoporosis is primarily attributed to 
loss of weight bearing and associated muscle contractions in the paralyzed limbs, there are 
important reductions in bone blood flow that impact bone metabolism and bone turnover. Bone is 
a highly vascularized organ with an extensive network of arteries, arterioles, and capillaries that 
is critical for bone development, maintenance, and repair; [1], [2] without sufficient and well-
regulated blood flow, bone cannot maintain its integrity. [3] Thus, reductions in bone perfusion 
may be a key contributor to bone loss post SCI. 
There is extensive arterial remodeling below the lesion that occurs soon after injury, leading to 
significant structural maladaptations such as decreased femoral artery diameter and increased 
wall thickness. [4], [5] These changes relate to significant alterations in both vasoconstrictor and 
vasodilatory response. For example, decreases in flow with leg dependency are greater in those 
with SCI, indicating enhanced myogenic vasoconstriction. [6], [7] Moreover, increases in leg blood 
flow with with reactive hyperemia are markedly less in those with SCI, indicating impaired 
myogenic vasodilatory capacity. [7]–[9] Hence, the extensive structural changes post SCI likely 
result in diminished flow to the leg due to both greater vasoconstriction and lesser vasodilation. 
This potentially leads to restricted flow to bone in those with SCI. Furthermore, these 
hypothesized alterations in vasoconstrictor and vasodilatory capacity may also relate to changes 
in functional blood flow responses in bone. Compressive loading increases bone metabolism 
requiring greater blood flow. However, if bone perfusion in response to loading is impaired in SCI 
due to structural changes, strategies to improve bone health based on loading could be largely 
ineffective. In addition, the structural changes occurring post injury, relate to alterations in nitric-
oxide (NO) mediated vasodilation, another potential key mechanism of maintaining appropriate 
perfusion to bone. While there is almost no research investigating NO vasodilation in bone in 
humans, a handful of studies suggest that in skeletal muscle, those with SCI have increased NO-
mediated vasodilation compared to controls.[10], [11] This implies that there is either increased 
NO production or similar peripheral NO that are unopposed by sympathetic vasoconstriction. 
However, given the lack of information on NO mediated vasodilation in the tibial vasculature, it 
would be prudent to first establish it potential role in healthy individuals prior to exploration in 
those with SCI. Afterwards, we will investigate the impact of peripheral vascular remodeling on 
bone perfusion after SCI. Furthermore, we propose to investigate the effect of mechanical loading, 
on bone perfusion in those with SCI compared to able-bodied individuals.  
A critical limitation to the study of bone blood flow in humans has been the lack of noninvasive 
assessments. The dense nature of bone makes it difficult to investigate perfusion and the 
techniques used to quantify circulation in other tissues are either difficult or impossible to apply to 
bone in vivo. This study will use a recently developed custom-made near infrared spectroscopy 
(NIRS) device that can non-invasively assess blood content in bone. [12] While NIRS has been 
used to assess perfusion in soft tissue, [13], [14] it has not been extensively used to assess 
perfusion in bone. However, NIRS has been shown to provide reproducible results in controlled 

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conditions, [15] and can track dynamic changes in bone perfusion. [16], [17] In our preliminary 
work, we used NIRS to assess physiological changes in blood content in the tibial cortical bone 
in humans. Here, we aim to extend our preliminary work by examining the impact of structural 
and functional vascular changes on bone perfusion in SCI and determine bone flow response to 
different loading conditions.   
II. 
SPECIFIC AIMS 
 
1) Determine if popliteal artery structural changes are reflected in altered myogenic control 
of tibial bone perfusion post SCI. 
Hypothesis: Compared to able-bodied, those with SCI will demonstrate greater popliteal artery 
wall thickness and smaller diameter that will relate to both greater myogenic vasoconstriction 
(in response to leg dependency) and lesser myogenic vasodilation (in response to reactive 
hyperemia) in tibial bone.   
2) Determine if popliteal artery structural changes are reflected in lesser increases in tibial 
bone perfusion during compressive loading post SCI.  
Hypothesis: Compared to able-bodied, those with SCI will demonstrate greater popliteal artery 
wall thickness and smaller diameter that will relate to smaller increases in bone perfusion with 
increasing loading conditions.  
3) Determine if nitric-oxide mediated vasodilation is present in the tibial bone vasculature of 
able-bodied. 
Hypothesis: In able-bodied, the tibial bone vasculature vasodilates in response to a nitric oxide 
donor.  
All able-bodied volunteers will complete both aim1 and aim2. Only a subset of volunteers with 
SCI – those with time since injury between 3 and 24 months post injury – will be included for aim 
2. The proposed study will investigate the control of bone perfusion in the SCI population as it 
relates to vascular remodeling and loading conditions.  
III. 
SUBJECT SELECTION  
Inclusion criteria  
We anticipate we will recruit 29 individuals (males and females) aged 18 to 40 years with injuries, 
American Spinal Injury Association Impairment Scale (AIS) A and B and C. For aim 2, only 
individuals with SCI with complete injuries between 3 and 24 months post injury will be included. 
In addition, we will recruit 29 healthy individuals (males and females) age and gender matched to 
enroll for this study.  
Exclusion criteria (for both able-bodied and SCI groups)  

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Clinical signs or symptoms of heart disease, hypertension, coronary disease, diabetes, other 
neurological disease, cancer, abnormal resting ECG, pregnant women, underweight and obese 
individuals (body mass index between 18.5 and 29.9), use of amphetamines (Ritalin, Adderall, 
Concerta) in the past 48 hours, tibial fracture or tibial stress fracture in the past 2 years.  
Healthy individuals should have no recent weight changes > 15 pounds, should not be allergic to 
nitroglycerin patches, capsules, tablets, ointments, or spray, should not use erectile dysfunction 
drugs, and should not use guanylate cyclase stimulators, such as riociguat. Concomitant use can 
cause hypotension. 
Healthy individuals should not use PDE-5 inhibitors, such avanafil, sisildenafil, tadalafil, vardenafil 
hydrochloride. Concomitant use can cause severe hypotension, syncope, or myocardial ischemia. 
Healthy indivduals should not have severe anemia. Nitroglycerin is contraindicated in patients 
with severe anemia (large doses of nitroglycerin may cause oxidation of hemoglobin to 
methemoglobin and could exacerbate anemia). 
Those with SCI will have no extreme spasticity to avoid spontaneous contractions and cannot be 
on baclofen due to its potential autonomic effects.  
IV. 
SUBJECT ENROLLMENT 
We will recruit from eligibile ASRT participants study (Partners IRB Protocol # 2013P000604. 
Additionally, we will reply on word of mouth and referrals from the spinal cord injury program, the 
outpatient therapy department, and the Exercise for Persons with Disabilities (ExPD) program at 
Spaulding Rehabilitation Network to enroll participants in this study. Flyers will be posted at SRH 
outpatient sites and/or given to pontential subjects along with a phone number to call if they are 
interested in learning more about the study. Our laboratory has been regularly enrolling SCI 
patients for research from the SRN. For the able-bodied participants, in the past, we have 
successfully recruited young males and females by posting flyers on college campuses and/or by 
posting on internet websites. The website ad has a short description of the project and a number 
to contact if they would like more information. When the potential subject makes contact with the 
study staff, we use a phone screening of health questions and a short description of the study. 
This process usually takes 15 to 20 minutes. If individuals meet eligibility requirements from the 
phone screening and are interested in participating, they are then scheduled for a physical 
screening. At that time, we mail a consent form to give opportunity to read and understand the 
specifics of the study.  
Research study staff will obtain volunteer consent in accordance with guidelines established by 
the Institutional Review Board during the study visit to the Cardiovascular Laboratory. Participants 
are encouraged to ask questions and are reminded that participation is strictly voluntary and will 
not affect their current or future care at Spaulding Rehabilitation Hospital or any of its affiliates.  
V. 
STUDY PROCEDURES 
Study Visit 

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Once recruited for the study, the volunteers will visit the laboratory for a health screening session 
to further determine eligibility. This 30-minute screening session includes obtaining informed 
consent, health history, and obtainingt weight and tibial length measurements.  
Once volunteers complete the screening process and meet the study inclusion criteria they will 
continue the visit to investigate tibial perfusion in response to leg dependency, reactive 
hyperemia, and increasing loading conditions . The study visit will take approximately 3 hours for 
participants that complete both aims and approximately 1.5 hours for those with SCI that complete 
only aim 1.   
Detailed procedures 
At the beginning of the study visit, we will obtain measurements of the popliteal artery thickness 
and lumen diameter using Doppler ultrasound (SonoSite Edge II, Fujifilm SonoSite). Throughout 
the study for the first aim, we will measure whole leg blood velocity in the popliteal artery using 
Doppler ultrasound (Multidop T2, DWL). Throughout the protocol, volunteers will be instrumented 
for measurement of tibial blood flow using a NIRS system (see Measurements below). The 
measurements will be taken in the same leg.  
Assessing tibial perfusion in response to leg dependency and reactive hyperemia (able-bodied 
and volunteers with SCI) 
We will use leg dependency and reactive hyperemia to assess myogenic vasoconstriction and 
myogenic vasodilation, respectively. This approach will provide insight to the relation between 
popliteal artery structure and myogenic vasoconstrictor response (leg dependency) and myogenic 
vasodilator response (reactive hyperemia), respectively.  
Leg dependency (i.e. the limb is lowered below heart level such that the knee is flexed at 90deg) 
results in an increased arterial transmural pressure, thereby engaging myogenic vasoconstriction. 
[6] Past work showed that those with SCI have more pronounced decreases in blood flow during 
leg dependency compared to able-bodied. [7] Thus, myogenic vasoconstrictor responses might 
be enhanced in the paralyzed legs. In addition, reactive hyperemia (i.e. increased calf blood flow 
in response to sustained ischemia via proximal thigh cuff inflation) is significantly lesser in SCI 
compared to able-bodied. [7]–[9] Thus, the myogenic vasodilatory capacity of the arterieres in the 
legs of those with SCI might be impaired.  
Throughout this aim, we will monitor continuously calf blood flow and tibial bone perfusion in the 
same leg. With the subject in supine position, we will obtain a 5 minutes resting baseline, followed 
by 5 minutes with the limb lowered below heart level (knee flexed at 90 deg). After 10 minutes of 
recovery, we will us reactive hyperemia to increase blood flow to the leg. With the subject supine, 
a pressure cuff placed above the knee will be inflated to supersystolic values (>200 mmHg) for 
10 minutes. Data will be obtained for a 5 minutes resting baseline, 10 minutes during cuff inflation, 
and 5 minutes after cuff release.  

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Assessing tibial perfusion in response to exogenous nitric-oxide (NO) (able-bodied)  
We will use an exogenous NO donor to assess NO-mediated vasodilation in the tibial bone 
vasculature of able-bodied. We will use one tablet of sublingual nitroglycerine (Nitroglycerine 
0.4mg Sublingual table) as the NO donor. With the subject in supine position, we will obtain a 5 
minutes resting baseline, followed by one tablet dissolved under the tongue, for 15 minutes. 
Throughout, we will continuously monitor heart rate and blood pressure, and calf blood flow and 
tibial bone perfusion in the same leg.  
Assessing tibial perfusion in response to increasing loading conditions (able-bodied and 
individuals with SCI with time since injury between 3 and 24 months) 
This approach will define the relationship between popliteal artery structure and magnitude of 
bone perfusion during loading of different magnitudes.  
We will use a custom tibial loading device (see Measurements below) to provide three periods of 
static bone loading, in random order: 1) 10% of body weight, 2) 30% of body weight, 3) 50% of 
body weight. 
After 10 minutes of recovery, the volunteers will undergo the three periods of  cycling bone loading 
described below. The subjects will be seated with the leg secured in the boot of the tibial bone 
loading device. The knee will be maintained at 90 deg flexion. Bone loading will be applied across 
the tibia at the 3 magnitudes of loading. Tibial bone perfusion of the same leg will be monitored 
continuously. Each period of static loading will consist of a 5 minutes resting baseline, 10 minutes 
of loading, and 5 minutes of recovery.  
Measurements  
Tibial Blood Flow. A custom made bone optical spectropsopy device that uses near-infrared (NIR) 
light will be used to continuously monitor tibial perfusion. Near infrared spectroscopy (NIRS) 
provides a safe, non-invasive, and inexpensive alternative to analyze hemodynamic changes in 
the biological tissues using light in the range of 600 to 1000 nm. The system has been previously 
used in Protocol #2014P002003 without any side effects. The system has two detectors allowing 
estimation of hemoglobin changes in a two layer (skin and bone) model and uses a broadband 
light source and high-sensitivity spectrometers to yield full NIR spectral information. The 
instrument uses a white-light tungsten halogen lamp (Fostec DCRII, Dallas, TX) to deliver light 
through a large optical fiber bundle to the skin. Two detector fibers are coupled with spectrometers 
(Mini-spectrometer TG C9405CB, Hamamatsu Photonics, Japan), placed at 1 and 2 cm distances 
from the source. Both spectrometers allow measurement of the diffusely reflected light between 
650 nm and 800 nm with 5 nm resolution. The light source and the two detectors are attached to 
a custom made probe head placed on the skin surface directly over the tibia. Light propagates 
between the source and the detectors in “banana-shaped” sensitivity functions and penetrates 
shallower for more closely separated source-detector pairs and more deeply for wider separated 
pairs. Thus, the detector 1 cm from the source probes the relatively thin (2 mm) skin layer, 
whereas the detector 2 cm from the source probes deeper into tissue and is therefore sensitive 
to the underlying bone. Placing the probe on the anterior side of the middle third tibia where the 
bone lies directly under the skin minimizes the effects of soft tissue and local changes in skin 

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blood flow on the NIRS measurements. The extended modified Beer-Lambert law for a two-layer 
model will be used to determine hemoglobin content changes in tibial bone from changes in light 
absorption.  
Tibial Loading Device. The custom-made device allows for 
controlled, compressive loading of the bone. The system consists 
of a boot with 2 inch strap over the knee. Wires pull vertically down 
on the brace via a cam-type device on a motor (Lesson, 90V DC). 
The load is monitored by air pressure cylinders on either side of 
the boot and the peak load cannot exceed the pressure of the 
cylinders. The servo-controlled pressure adjusts the load such that 
it remains at the predetermined level.  This allows for full control of 
the magnitude and pattern of loading on the lower leg. This system 
can apply static or cycling loading on the tibia. This approach has 
been modeled after a mouse tibial loading model that has been 
successfully employed to examine bone adaptations under 
imposed physical loading conditions. The system is able-to non-
invasively apply loading in a physiological direction and axially 
loads a single skeletal element – the tibia. The system also allows 
for isometric muscle contractions in the calf (plantar flexion) that 
can be performed in synchrony with the compressive loading and 
provides a stimulus that more closely mimics walking. The leg of the volunteer is placed in the 
boot of the device and secured with the strap over the knee. This ensures that the knee is 
maintained at 90 deg flexion throughout loading. Bone loading will be applied axially along the 
tibia and maintained constant at the different loading magnitudes. The loading force will be 
monitored and recorded via the two load cells place on either sides of the boot 
 

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Beat-by-Beat Calf Blood Flow. We will use Doppler derived flow velocities to estimate whole limb 
blood flow through the popliteal artery. A continuous wave 4 MHZ Doppler probe (Multidop T2, 
DWL) is placed against the skin under the knee to isolate the popliteal artery proximal to the 
bifurcation. 
Popliteal Artery Structure. A linear 6- to 15- MHz Doppler ultrasound (SonoSite Edge II, Fujifilm 
SonoSite, Bothell, WA) will be used to measure popliteal artery thickness and lumen diameter. 
The measurements will be taken at the distal popliteal artery above 2cm above arterial take-off, 
with the subject in supine position with the knee flexed at 45deg. The arterial diameter will be 
measured on transverse images, with the ultrasound beam perpendicular to the artery. [18]  
Tibial Length. Tibial length will be measured in sitting position with a tape measure.  
Body Weight. Weight will be measured on a scale.  
VI. 
STATISTICAL ANALYSIS 
Sample Size 
Our prior work found a borderline significant increase (p=0.06) in tibial perfusion after exercise in 
able-bodied. Power analysis at 80% of these data showed that 17 subjects would have provided 
a p<0.05 increase in perfusion. Therefore, for this study, we based our sample size on this 
calculation. We do not expect any sex-related difference; moreover, the SCI population is 
comprised primarily of males. To ensure a sufficient sample size for NO-mediated vasodilation, 
we will aim to recruit an additional of 17 individuals in each group. Therefore, we will recruit 29 
individuals with SCI (men and women) and 29 healthy individuals (men and women) to participate 
in the study.  
Statistics 
Data from NIRS monitoring will be used to determine hemoglobin content changes in tibial bone 
from changes in light absorption on a moment-by-moment basis. A detailed description of the 
analysis can be found in our previous work. [12] Briefly, NIRS monitoring provides a change in 
hemoglobin content from one state to another. This is due to the fact that absolute intensity of the 
scattered light measured across the tibia cannot be used for an absolute measure of perfusion. 
However, as perfusion increases, there is a proportional increase in absorption (and vice versa). 
Thus, percent change in total hemoglobin detected reflects the relative change in perfusion.  
The main outcomes assessed will be popliteal artery thickness and lumen diameter, tibial 
perfusion, and whole leg blood flow. This project will employ a simple and straightforward 
statistical approach to compare responses within and between groups. Statistical significance will 
be set at 0.05, and power at 80%. Descriptive statistics will be presented including mean, median, 
standard deviation, and range. Differences/relations will be considered as statistically significant 
at alpha=0.05 level. For all parametric statistical tests, conformity of data to required statistical 
assumptions will be verified using standard methods. If assumptions for parametric tests do not 
hold, non-parametric alternatives will be employed. The analysis plan is written as though there 
are no distributional problems; assumptions for all analyses will be checked and analogous 
methods calling for fewer and/or weaker assumptions will be used, if called for. For the data from 
aim 1, unpaired t-tests will be used to assess changes in perfusion between groups and reactive 

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hyperemia/leg dependency. Since the two conditions result in distinct and opposite responses, 
they should be considered separately. For the data from aim 2, for the three passive loading 
conditions (10%, 30%, 50% body weight without associated muscle contractions), a 2x3 ANOVA 
will be used to assess changes in perfusion between groups across loading conditions.  
The results of this study will provide invaluable insights into bone perfusion in the paralyzed legs 
of those with SCI. For both aims, we anticipate that individuals with SCI will have less bone 
perfusion compared to able-bodied due to the extensive vascular maladaptations. Even if the 
results are not different between those with SCI and able-bodied, this study will have explored a 
potentially key contributor to SCI-related bone loss, with major implications in treating 
osteoporosis. 
VII. 
RISKS AND DISCOMFORTS 
The tibial bone loading device should pose minimal risks – the device applies loading at loads 
less than full body weight – from 10% to 50% body weight. This should pose minimal risk to 
volunteers with SCI since they will be less than 40 years of age and within 2 years of injury and 
so should have minimal risk of osteoporosis. Cuff inflation might result in muscle discomfort that 
will quickly go away after cuff release. In those with SCI, cuff inflation can also cause autonomic 
dyreflexia. The NIRS system is non-invasive and poses little risk to the patients. Initial trials 
showed minor reddening of the skin; however, this was temporary and it quickly went away. 
Nitroglycerin (0.4 mg Nitroglycerin sublingual) at the small doses given is considered low risk, and 
any higher risk patients are excluded during the eligibility process. Sublingual nitroglycerin may 
result in headaches, weakness, dizziness, lightheadedness, nausea, mild burning or tingling with 
the tablet in the mouth, and flushing (warmth, redness, or tingly feeling under the skin) as the 
body adjusts to the medication. Some serious but unlikely side effects include fainting and fast 
and irregular heart rate. Blood pressure and heart rate will be monitored continuously, and the 
nitroglycerin will be removed immediately if large declines in blood pressure (>20 mmHg) occur. 
VIII. 
POTENTIAL BENEFITS 
This research may help physicians and scientists explore the potential contribution of vascular 
maladaptations to SCI related bone loss, with major implications in treating osteoporosis. Subjects 
will not directly benefit from participation in this protocol.  
MONITORING AND QUALITY ASSURANCE 
The research coordinator will be responsible for monitoring the completeness of all data and 
source documents. The Principal Investigator will monitor the informed consent procedures in 
accordance with the Informed Consent Compliance Checklist of Partners HealthCare Systems 
HRQIP. The subjects data/protocol adherence will be monitored by the research coordinator at 
each step in the study including. Checklists and note pages are used to note any deviations or 
omissions from the protocols. The Cardiovascular Research Laboratory Medical Emergency 

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Safety Plan will be followed in the case of an adverse medical event. Any clinical/health related 
issues would be immediately presented to the subject (i.e. abnormal EKG, blood chemistries, etc) 
to determine appropriate notification (ie. current physician or appropriate specialist) .Based on 
the seriousness of the situation a physician may be contacted to provide clinical guidance on the 
appropriate course of action. Event assessment and follow-up will be done by the PI as needed 
on a case by case basis. 
This study will be conducted in compliance with the protocol, International Council for 
Harmonization/Good Clinical Practice requirements(ICH/GCP) and applicable state, local and 
federal regulatory requirements. 
 
Reporting to Partners IRB: 
Reports of unanticipated problems involving risks to subjects or others are to be submitted 
through Insight/eIRB within 5 working days/7 calendar days of the date the investigator first 
becomes aware of the problem. 
 
Reporting Unanticipated Problems that are Adverse Events 
Any unanticipated untoward or unfavorable medical occurrence, including abnormal sign (for 
example, abnormal physical exam or laboratory finding), symptom, or disease, that indicates 
that the research places subjects at increased risk of physical or psychological harm than 
previously known or recognized are to be submitted through Insight/eIRB as an Other Event, 
Adverse Event. The investigator must provide the following information in the report: 
(1) a detailed description of the adverse event; 
(2) the basis for determining that the event is unexpected in nature, severity, or frequency; 
(3) the basis for determining that the event is related or possibly related to the research 
procedures; 
(4) the basis for determining that the research places subjects at an increased risk of harm (i.e., 
a serious adverse event); and 
(5) whether any changes to the research or other corrective actions are warranted. 
NOTE: The PHRC does not accept sponsor IND/IDE safety reports describing adverse events 
that have occurred at sites other than those subject to this policy unless the report is of an 
incident that is: 
(1) serious; (2) unexpected or unanticipated; (3) related to the investigational drug/device; and 
(4) suggests that subjects are at an increased risk of harm and as such warrants changes in the 
research, consent process, or informing subjects. IND/IDE safety reports that warrant changes 
are to be submitted through Insight/eIRB as an Amendment. 
 
Reporting Unanticipated Problems that are not Adverse Events 
All other unanticipated incidents, experiences, information, outcomes, or other problems that 
indicate that the research places subjects at an increased risk of physical, psychological, 
economic, legal, or social harm than was previously known or recognized are to be submitted 
through Insight/eIRB as an 
Other Event. The investigator must provide the following information in the report: 
(1) a detailed description of the unanticipated problem; 
(2) the basis for determining that the problem is unexpected; 
(3) the basis for determining that the problem indicates that the research places subjects at an 
increased risk of harm; and 
(4) whether any changes to the research or other corrective action are warranted. 
 

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Reporting to the National Institute of Arthritis and Muscloskeletal and Skin Disease 
(NIAMS) and Safety Officer (Dr.Ralph Marino). 
 
This protocol has funding from NIAMS. NIAMS has appointed a Saftey Officer, Dr. Ralph Marino,  
to act in an advisory capacity to the NIAMS to monitor participant safety, evaluate the progress of 
the study, to review procedures for maintaining the confidentiality of data, the quality of data 
collection, management and analyses. 
 
The Principal Investigator (PI)  will report unanticipated problems (UPs), serious adverse events 
(SAEs) and protocol violations that impact participant safety to the NIAMS and the Safety 
Officer, (through the Exective Secretary, KAI-Research) within 48 hours of the PI becoming 
aware of the event. KAI facilitates the review of the expedited reporting of these events by the 
Safety Officer. The Safety Officer will review the Safety Reports submitted by the PI to KAI twice 
a year. KAI facilitates the review of these reports by the Safety Officer and the NIAMS, and 
provides the final review comments to the PI when the review is complete. During the course of 
the study, ad hoc meetings may be called by the NIAMS or Safety Officer at any time to discuss 
potential safety issues. 
 
Definitions: 
Adverse event means any untoward or unfavorable medical occurrence in a human subject, 
includingany abnormal sign (for example, abnormal physical exam or laboratory finding), 
symptom, or disease,temporally associated with the subject’s participation in the research, 
whether or not considered relatedto the subject’s participation in the research (modified from the 
definition of adverse events in the 1996. International Conference on Harmonization E-6 
Guidelines for Good Clinical Practice). 
 
Serious adverse event means any event temporally associated with the subject’s participation in 
research that meets any of the following criteria: 
• 
results in death; 
• 
is life threatening (places the subject at immediate risk of death from the event as it 
occurred); 
• 
requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
results in a persistent or significant disability/incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
any other adverse event that, based upon appropriate medical judgment, may jeopardize 
the subject’s health and may require medical or surgical intervention to prevent one of 
the outcomes listed above (examples of such events include allergic bronchospasm 
requiring intensive treatment in the 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). 

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Possibly related to the research means there is a reasonable possibility that the adverse event, 
incident,experience or outcome may have been caused by the procedures involved in the 
research (modified from the definition of associated with use of the drug in FDA regulations at 
21 CFR 312.32(a)). Reasonable possibility means that the event is more likely than not related to 
participation in the research or,in other words, there is a >50% likelihood that the event is 
related to the research procedures. 
 
X. 
REFERENCES 
[1] 
M. L. Brandi and P. Collin-Osdoby, “Vascular biology and the skeleton,” J. Bone Miner. 
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10.1359/JBMR.050917. 
[2] 
M. Brookes and R. G. Harrison, “The vascularization of the rabbit femur and tibio-fibula,” 
J. Anat., vol. 91, no. 1, pp. 61–72, Jan. 1957. 
[3] 
J. T. Lacey, “NON-UNION OF FRACTURES: AN EXPERIMENTAL STUDY,” Ann. Surg., 
vol. 89, no. 6, pp. 813–848, Jun. 1929. 
[4] 
D. H. J. Thijssen et al., “Time course of arterial remodelling in diameter and wall 
thickness above and below the lesion after a spinal cord injury,” Eur. J. Appl. Physiol., vol. 112, 
no. 12, pp. 4103–4109, Dec. 2012, doi: 10.1007/s00421-012-2400-2. 
[5] 
C. R. West, A. Alyahya, I. Laher, and A. Krassioukov, “Peripheral vascular function in 
spinal cord injury: a systematic review,” Spinal Cord, vol. 51, no. 1, pp. 10–19, Jan. 2013, doi: 
10.1038/sc.2012.136. 
[6] 
K. Okazaki et al., “Vasoconstriction during venous congestion: effects of venoarteriolar 
response, myogenic reflexes, and hemodynamics of changing perfusion pressure,” Am. J. 
Physiol. Regul. Integr. Comp. Physiol., vol. 289, no. 5, pp. R1354-1359, Nov. 2005, doi: 
10.1152/ajpregu.00804.2004. 
[7] 
M. Kooijman, M. de Hoog, G. A. Rongen, H. J. M. van Kuppevelt, P. Smits, and M. T. E. 
Hopman, “Local vasoconstriction in spinal cord-injured and able-bodied individuals,” J. Appl. 
Physiol. Bethesda Md 1985, vol. 103, no. 3, pp. 1070–1077, Sep. 2007, doi: 
10.1152/japplphysiol.00053.2007. 
[8] 
M. Kooijman, G. A. Rongen, P. Smits, and M. T. E. Hopman, “Preserved alpha-
adrenergic tone in the leg vascular bed of spinal cord-injured individuals,” Circulation, vol. 108, 
no. 19, pp. 2361–2367, Nov. 2003, doi: 10.1161/01.CIR.0000096480.55857.3C. 
[9] 
R. s. Khakha, Z. Bloomer, D. Bain, G. Nicholson, A. Gall, and M. Ferguson-Pell, 
“Differences in the reactive hyperemia response of bone in able bodied and spinal injured 
persons using near infrared spectroscopy,” Orthop. Proc., vol. 88-B, no. SUPP_II, pp. 305–305, 
May 2006, doi: 10.1302/0301-620X.88BSUPP_II.0880305c. 

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[10] 
L. M. Brewster et al., “Effects of circulating extracellular microvesicles from spinal cord-
injured adults on endothelial cell function,” Clin. Sci., vol. 134, no. 7, pp. 777–789, Apr. 2020, 
doi: 10.1042/CS20200047. 
[11] 
R. Brown, D. Celermajer, V. Macefield, and M. Sander, “The Effect of Nitric Oxide 
Inhibition in Spinal Cord Injured Humans with and without Preserved Sympathetic Control of the 
Vasculature,” Front. Neurosci., vol. 10, p. 95, Mar. 2016, doi: 10.3389/fnins.2016.00095. 
[12] 
A. E. Draghici et al., “Near infrared spectroscopy for measuring changes in bone 
hemoglobin content after exercise in individuals with spinal cord injury,” J. Orthop. Res. Off. 
Publ. Orthop. Res. Soc., May 2017, doi: 10.1002/jor.23622. 
[13] 
D. A. Boas, C. E. Elwell, M. Ferrari, and G. Taga, “Twenty years of functional near-
infrared spectroscopy: introduction for the special issue,” NeuroImage, vol. 85 Pt 1, pp. 1–5, 
Jan. 2014, doi: 10.1016/j.neuroimage.2013.11.033. 
[14] 
M. Ferrari and V. Quaresima, “A brief review on the history of human functional near-
infrared spectroscopy (fNIRS) development and fields of application,” NeuroImage, vol. 63, no. 
2, pp. 921–935, Nov. 2012, doi: 10.1016/j.neuroimage.2012.03.049. 
[15] 
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“A near infrared instrument to monitor relative hemoglobin concentrations of human bone tissue 
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10.1063/1.3398450. 
[16] 
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hemodynamics of the human patella bone by diffuse optical spectroscopy,” Physiol. Meas., vol. 
34, no. 8, pp. 839–857, Aug. 2013, doi: 10.1088/0967-3334/34/8/839. 
[17] 
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Bone by Hybrid Broadband (600-1200 nm) Diffuse Optical and Correlation Spectroscopies,” 
PloS One, vol. 11, no. 12, p. e0168426, 2016, doi: 10.1371/journal.pone.0168426. 
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2006, doi: 10.1016/j.jvs.2005.11.026. 
 
 

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 Page 1 of 13 Bone Blood Flow V11, March 24, 2021 
 
 
 
 
Exploration of blood flow regulation to bone in humans 
 
Version 11 
 
03/24/21 
 
 
 
 
Clinicaltrials.gov # NCT04083794 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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 Page 2 of 13 Bone Blood Flow V11, March 24, 2021 
 
 
I. 
BACKGROUND AND SIGNIFICANCE  
 
All tissues of the human body require adequate perfusion to provide oxygen and nutrients to meet 
metabolic demands. It has been long recognized that the arterial system in bone is of 
overwhelming importance, adequate circulation being necessary for nearly all skeletal functions, 
including development, growth, homeostasis, and repair. [1], [2] There is an extensive network of 
arteries, arterioles, and capillaries that supplies blood flow to bone and without adequate 
circulation bone cannot maintain its integrity. [3] For example, inadequate flow has been 
associated with bone loss,[4] impaired growth,[5] and delayed fracture healing.[6]  
 
Despite the appreciation of the importance of blood circulation to bone health, the regulation of 
blood flow to bone has been relatively unstudied in humans and the mechanisms that regulate 
bone blood flow are not well understood. The majority of work in bone perfusion has been in 
various animal models (mice, rats, rabbits, dogs, cats, etc.) with the preponderance of data 
suggesting that there are key systemic effectors of acute changes in bone perfusion. One of the 
potential factors controlling blood flow regulation is the sympathetic nervous system, the bone 
vasculature being innervated by a rich network of sympathetic nerves. Although no studies in 
humans have been conducted, most of animal data demonstrates that sympathetic activation 
reduces blood flow to bone. Application of norepinephrine decreases blood flow to both intact 
bone [7], [8] and isolated bone. [9] Likewise, stimulation of sympathetic nerves decreases flow to 
bone [7]  via alpha-adrenergic receptor activation.[9] Moreover, the smooth muscle of the 
arterioles in bone respond as expected to infused vasodilators and vasoconstrictors.[10], [11] 
Hence, the sympathetic innervation of bone vasculature serves a functional purpose in the control 
of blood flow. If this were not the case, independent of the link between bone metabolism and 
bone flow, the arterial network in bone would act as a simple pressure passive system. Here, we 
propose to investigate the role of vascular sympathetic activity in blood flow regulation in humans.  
 
A critical limitation to the study of bone blood flow in humans has been the lack of noninvasive 
assessments. The dense nature of bone makes it difficult to investigate perfusion and the 
techniques used to quantify circulation in other tissues are either difficult or impossible to apply to 
bone in vivo. We recently developed a custom-made near infrared spectroscopy (NIRS) device 
that can non-invasively assess blood content in bone. [12] While NIRS has been used to assess 
perfusion in soft tissue,[13], [14] it has not been extensively used to assess perfusion in bone. 
However, NIRS has been shown to provide reproducible results in controlled conditions,[15] and 
can track dynamic changes in bone perfusion.[16], [17] In our preliminary work, we used NIRS to 
assess physiological changes in blood content in the tibial cortical bone in humans. Although our 
preliminary work is intriguing, it provided insufficient insight to regulation because this study was 
unable to disentangle the various possible contributors to bone perfusion. Thus, we aim to extend 
our preliminary work by examining the response of tibial perfusion to vascular sympathetic activity 
and systemic perfusion pressure in healty individuals via NIRS monitoring.  In addition, we will 
contrast these responses with those seen in individuals with spinal cord injury (SCI). SCI 
represents a human ‘model’ of chronic reduced loading with loss of sympathetic regulation below 
the level of injury that alters control of bone perfusion. Contrasting the response of tibial perfusion 
to vascular sympathetic activity and systemic perfusion pressure in able-bodied individuals and 
individuals with SCI will set the basis for future work that will define whether alterations in 
perfusion may relate to pathophysiologies of bone.  
 
Hypothesis  
 

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 Page 3 of 13 Bone Blood Flow V11, March 24, 2021 
 
 
We hypothesize that bone blood perfusion decreases proportional to increases in leg vascular 
sympathetic outflow. 
 
 
II. 
SPECIFIC AIMS 
 
1) Determine the effect of increased vascular sympathetic activity with maintained blood 
pressure on tibial perfusion. 
 
2) Determine the effect of increased vascular sympathetic activity with increased systemic 
pressure on tibial perfusion.  
 
3) Compare the changes in tibial perfusion in response to systemic factors between able-
bodied and those with SCI. 
 
Knowledge of tibial perfusion in response to increased sympathetic outflow, with or without 
changes in systemic pressure, will allow us to determine the sympathetic contribution to bone 
blood flow.  
 
III. 
SUBJECT SELECTION  
 
Inclusion criteria  
 
It is anticipated that 34 healthy males and 34 healthy females aged 18 to 40 years will be recruited 
and enrolled for this study.  
 
 
Also, we anticipate we will recruit 17 males with SCI, aged 18 to 40 years, with injuries American 
Spinal Injury Association Impairment Scale (AIS) A and B and C, with full hand function. We will 
only enroll males with SCI since the SCI population is predominantly male (about 75%). 
 
Exclusion criteria (for both able-bodied and SCI groups) 
Clinical signs or symptoms of heart disease, hypertension, coronary disease, diabetes, other 
neurological disease, cancer, recent weight change >15 pounds, abnormal resting ECG, 
pregnant and/or breastfeeding women, underweight and obese individuals (body mass index 
between 18.5 and 29.9), use of amphetamines (Ritalin, Adderall, Concerta) in the past 48 hours, 
tibial fracture or tibial stress fracture in the past year.  
 
In addition, those with SCI will have no extreme spasticity to avoid spontaneous contractions 
and cannot be on baclofen due to its potential autonomic effects. 
 
 
IV. 
SUBJECT ENROLLMENT 
 
In the past, we have successfully recruited young males and females by posting flyers on college 
campuses and/or by posting on internet websites. The website ad has a short description of the 
project and a number to contact if they would like more information. When the potential subject 

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 Page 4 of 13 Bone Blood Flow V11, March 24, 2021 
makes contact with the study staff, we use a phone screening of health questions and a short 
description of the study. This process usually takes 15 to 20 minutes. If individuals meet eligibility 
requirements from the phone screening and are interested in participating, they are then 
scheduled for a physical screening. At that time, we mail a consent form to give opportunity to 
read and understand the specifics of the study.  
 
We will recruit SCI participants from the outpatient therapy department and the Exercise for 
Persons with Disabilities (ExPD) program at Spaulding Rehabilitation Network. Flyers will be 
posted at SRH outpatient sites and/or given to potential subjects along with a phone number to 
call if they are interested in learning more about the study. Our laboratory has been regularly 
enrolling SCI patients for research from the SRN. For the able-bodied participants, in the past, we 
have successfully recruited young males and females by posting flyers on college campuses 
and/or by posting on internet websites. The website ad has a short description of the project and 
a number to contact if they would like more information. When the potential subject makes contact 
with the study staff, we use a phone screening of health questions and a short description of the 
study. This process usually takes 15 to 20 minutes. If individuals meet eligibility requirements 
from the phone screening and are interested in participating, they are then scheduled for a 
physical screening. At that time, we mail a consent form to give opportunity to read and 
understand the specifics of the study. 
 
Research study staff will obtain volunteer consent in accordance with guidelines established by 
the Institutional Review Board during the study visit to the Cardiovascular Laboratory. Participants 
are encouraged to ask questions and are reminded that participation is strictly voluntary and will 
not affect their current or future care at Spaulding Rehabilitation Hospital or any of its affiliates.  
 
 
V. 
STUDY PROCEDURES 
 
Study Visit 
 
Once recruited for the study, the volunteers will visit the laboratory for a health screening session 
to further determine eligibility. This 30-minute screening session includes obtaining informed 
consent, health history, and obtaining height and weight measurements.  
 
Once volunteers complete the screening process and meet the study inclusion criteria they will 
continue the visit to investigate the effect of sympathetic outflow to tibial perfusion. The study visit 
will take approximately 3.5 hours to complete. The screening visit and the study visit may occur 
on the same day or on different days.  
 
 
Detailed procedures 
 
Throughout the protocol, able-bodied volunteers will be fully instrumented for measurements of 
tibial blood flow, muscle/bone sympathetic activity, popliteal blood flow velocity, ECG, beat-by-
beat arterial pressure, brachial arterial pressure, and respiration (see Measurements below). All 
those with SCI enrolled in this study will have complete injuries and thus vascular sympathetic 
control is disrupted below the level of injury. Hence, in individuals with SCI the arterial network in 
bone might act as a simple pressure passive system. Given the disrupted vascular sympathetic 
control, in those with SCI we will not record muscle/bone sympathetic activity.  
 
In able-bodied, we will use direct microneurographic measures of nervous activity to the lower leg 
vasculature in the common peroneal nerve as a measure of sympathetic activity to the bone 

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vasculature of the tibia throughout the protocol. Our assumption is that peroneal nerve 
sympathetic activity reflects vasomotor output to both muscle and bone of the lower leg. Though 
this assumption is not testable prior to having data in hand, we assume that Hilton’s law prevails. 
According to Hilton’s law, bone innervation derives from the peripheral nerves of the muscle that 
overlies the bone. The common peroneal nerve primarily innervates the fibula, but also the deep 
peroneal branch innervates the tibialis anterior which overlies the tibia. Though we acknowledge 
this is potentially inexact, our sympathetic nervous activity measure should broadly reflect 
sympathetic outflow to the lower leg.  
 
We will use three approaches to increase sympathetic outflow to the leg vasculature: head tilt, 
cold pressor test, and isometric handgrip exercise (IHE) to fatigue. In those with SCI, we expect 
that bone vasculature will respond as a pressure passive system. 
 
Subjects will lie supine throughout the study protocol. After instrumentation, the subject will 
perform three maximal voluntary contractions (MVC) on a handgrip  dynamometer. This is to 
determine 30% MVC for sustained isometric exercise. Afterwards, an adequate nerve recording 
will be obtained. To ensure that a good nerve recording is obtained, the subjects will be asked to 
perform a Valsalva maneuver, which results in an expected increase in sympathetic activity. 
Invented by Antonio Maria Valsalva in the 17th century, the Valsalva maneuver, a prolonged 
expiration against a closed glottis, is widely used because of its safety and ease of quantification. 
This voluntary act of straining creates transient changes in intra-thoracic and intra-abdominal 
pressure with concomitant increases in sympathetic activity. To achieve this, subjects will be 
asked to breathe through a tube with a small vent in the glottis to prevent them from increasing 
mouth instead of chest pressure. A pressure gauge attached to this tube will allow the subject 
maintain a target pressure of 40 mmHg for 15 seconds. Data will be obtained for 1 minute prior 
to the maneuver, 1 minute after, with 2 minutes of recovery.  
 
Assessing tibial perfusion in response to increased sympathetic outflow with maintained blood 
pressure 
 
This approach will provide insight to the relation between moment-by-moment bone perfusion and 
beat-by-beat increases in vascular sympathetic activity without alterations in systemic pressure. 
During head-up tilt, decreases in central blood volume and circulatory adjustments are 
proportional to the angle of the head-up tilt [20]. Sympathetic activity increases and muscle blood 
flow decreases significantly by a tilt angle of 30 degrees [21]. In addition, head-down tilt to 6-8 
deg results in a decrease in sympathetic activity [22]. Hence, increased levels of head-up tilt 
provide a strong stimulus for active vasoconstriction in the leg, while maintaining system pressure 
constant. This provides a stimulus that progressively increases sympathetic activity with a 
maintained systemic perfusion pressure.  Data will be obtained for a 5 minutes resting baseline, 
followed by 25 minutes of continuous tilt of successive 5 minutes periods at 10 deg head-down 
tilt, 0 deg tilt, 10 deg head-up tilt, and 20 deg head-up tilt.  
 
 
Assessing tibial perfusion in response to increased sympathetic outflow and increased systemic 
pressure. 
 
The purpose of this approach is to provide insight to the relation between bone perfusion and 
progressive increases in sympathetic outflow accompanied by increases in systemic pressure. 

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 Page 6 of 13 Bone Blood Flow V11, March 24, 2021 
Cold pressor test and sustained isometric handgrip will be used to increase sympathetic nervous 
activity, with increased systemic pressure.  
 
The cold pressor test provokes a nociceptive response that causes increases in sympathethic 
activity, blood pressure, and heart rate [23]. Past work showed that these responses are water 
temperature dependent. Graded increases in sympathetic activity, accompanied by parallel 
increase in arterial pressure, were seen during immersion at 0 and 7oC, but not at 14oC [24]. 
 
A 5 minutes resting baseline will be obtained prior to the subject performing the cold pressor test. 
Afterwards, participants will undergo a cold pressor task, consisting of immerion of a hand for 3 
min in 0oC water. This will be followed by 3 minutes of recovery data collection.  
 
Isometric handgrip to fatigue (IHE) results in time-dependent, progressive increases in peroneal 
nerve sympathetic outflow and blood pressure [25]. With sustained handgrip forces greater than 
25% of maximal voluntary contraction, sympathetic activity increases progressively to end 
exercise. Hence, IHE provides a strong stimulus for active vasoconstriction in the leg that 
contributes to the exercise pressor response.  
 
A 5 minutes resting baseline will be obtained prior to the subject performing IHE at 30% MVC 
sustained to fatigue. An oscilloscope will be calibrated to provide the subject visual feedback on 
force generation; subjects will be verbally encouraged to maintain force throughout; fatigue will 
be determined by the investigators as maximal perceived rating of exertion accompanied by 
inability to maintain target force for more than 3 sec, despite continued effort. 
 
In those with SCI, given the lack of vascular sympathetic control and thus no increase in 
sympathetic outflow during tilt, cold pressure, and isometric handgrip exercise, we expect that 
bone blood flow will respond as a pressure passive system and hence will track blood pressure 
response.  
 
In able-bodied, tibial blood perfusion, an adequate nerve recording, and popliteal flow will be 
obtained. These time series will allow derivation of relations between nerve activity and tibial 
perfusion, and between nerve activity and popliteal flow. Nerve activity will serve as an index of 
vasoconstriction activity to the entire leg, while tibial perfusion and popliteal flow will represent a 
measure of downstream leg vascular responses. If nerve activity is not obtained, the subjects will 
still perform the procedures described above. In that case, the relationship between tibial blood 
perfusion and popliteal flow will be assessed.  
In those with SCI, tibial blood perfusion and popliteal flow will be obtained. The relationship 
between tibial blood perfusion and popliteal flow will be assessed. These approaches will provide 
insight to the proportionality between sympathetic outflow and bone blood flow, as well as the 
hypothesized pressure passive bone vasculature in those with SCI. 
 
Subjects may be asked to repeat any of the study assessments if there is difficulty 
obtaining results that meet our quality standards or there are equipment issues that 
arise. 
 
Measurements  
 
Tibial Blood Flow. A custom made bone optical spectropsopy device that uses near-infrared (NIR) 
light will be used to continuously monitor tibial perfusion. Near infrared spectroscopy (NIRS) 
provides a safe, non-invasive, and inexpensive alternative to analyze hemodynamic changes in 
the biological tissues using light in the range of 600 to 1000 nm. The system has been previously 

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used in Protocol #2014P002003 without any side effects. The system has two detectors allowing 
estimation of hemoglobin changes in a two layer (skin and bone) model and uses a broadband 
light source and high-sensitivity spectrometers to yield full NIR spectral information. The 
instrument uses a whitelight tungsten halogen lamp (Fostec DCRII, Dallas, TX) to deliver light 
through a large optical fiber bundle to the skin. Two detector fibers are coupled with spectrometers 
(Mini-spectrometer TG C9405CB, Hamamatsu Photonics, Japan), placed at 1 and 2 cm distances 
from the source. Both spectrometers allow measurement of the diffusely reflected light between 
650 nm and 800 nm with 5 nm resolution. The light source and the two detectors are attached to 
a custom made probe head placed on the skin surface directly over the tibia. Light propagates 
between the source and the detectors in “banana-shaped” sensitivity functions and penetrates 
shallower for more closely separated source-detector pairs and more deeply for wider separated 
pairs. Thus, the detector 1 cm from the source probes the relatively thin (2 mm) skin layer, 
whereas the detector 2 cm from the source probes deeper into tissue and is therefore sensitive 
to the underlying bone. Placing the probe on the anterior side of the middle third tibia where the 
bone lies directly under the skin minimizes the effects of soft tissue and local changes in skin 
blood flow on the NIRS measurements. The extended modified Beer-Lambert law for a two-layer 
model will be used to determine hemoglobin content changes in tibial bone from changes in light 
absorption.  
 
Sympathetic Microneurography. Multiunit, post-ganglionic muscle sympathetic nerve activity will 
be recorded with a tungsten microelectrode in the peroneal nerve only in the able-bodied 
population. The peroneal nerve will be mapped at the fibular head via low voltage transcutaneous 
electrical stimuli. Subsequently, the microelectrode is inserted manually through the 
unanesthetized skin and initially provides low voltage electrical stimuli (1-4 volts) to help locate 
the nerve. When the microelectrode enters a nerve fascicle innervating muscle, involuntary 
twitches are induced. The microelectrode is then used to record sympathetic efferent nerve 
outflow to calf muscle. This procedure is safe and the principal investigator has performed over 
1000 experiments since 1991 without complication. The raw nerve signal will be amplified, 
rectified and integrated for visual inspection during experiments (Nerve Traffic Analyzer, Model 
662c-3, University of Iowa Bioengineering, Iowa City, IA) and the amplified raw signal will be 
directly sampled at 20 kHz for further signal processing. 
 
Beat-by-Beat Calf Blood Flow. We will use Doppler derived flow velocities to estimate whole limb 
blood flow through the popliteal artery. A continuous wave 4 MHZ Doppler probe (Multidop T2, 
DWL) is placed against the skin under the knee to isolate the popliteal artery proximal to the 
bifurcation. 
  
Electrocardiogram. A standard 4-lead electrocardiogram will be used to obtain continuous heart 
rate.  
 
Arterial Pressure. The arterial waveform in a finger of the hand will be derived on a beat-to-beat 
basis by the Finapres Blood Pressure System (Finapres, Ohmeda) with brachial oscillometric 
blood pressure (Dash 2000, GE) as a standard measure of arterial pressure.  
 
Respiration. A respiratory belt will be placed around the chest for breathing depth and frequency. 
This signal does not provide exact tidal volumes and will only be used to monitor breathing.  
 
Following the removal of insturments, the able-bodied volunteers will be asked to complete the 
online MSNA questionnaire administered via REDCap, if the patient had the electrode placed 

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beneath the surface of their skin. If the subject does not have access to the internet, a paper 
copy of the MSNA questionnaire will be mailed to his/her address. 
 
VI. 
STATISTICAL ANALYSIS 
 
Sample Size 
 
Our prior work found a borderline significant increase (p=0.06) in tibial perfusion after exercise in 
able-bodied. Power analysis at 80% of these data showed that 17 subjects would have provided 
a p<0.05 increase in perfusion. Therefore, for this study, we based our sample size on this 
calculation. However, we will recruit equal numbers of men and women to explore potential sex-
related differences. Also, due to individual anatomic differences and day-to-day variability in 
success, microneurographic recordings can be obtained in approximately two out of three 
volunteers. To ensure successful sympathetic nerve recordings in 17 individuals, we will need to 
recruit ~30% more subjects. Thus, we will recruit 34 healthy males, 34 healthy females, and 17 
males with SCI. 
 
Statistics 
 
Data from NIRS monitoring will be used to determine hemoglobin content changes in tibial bone 
from changes in light absorption on a moment-by-moment basis. A detailed description of the 
analysis can be found in our previous work. [12] Briefly, NIRS monitoring provides a change in 
hemoglobin content from one state to another. This is due to the fact that absolute intensity of the 
scattered light measured across the tibia cannot be used for an absolute measure of perfusion. 
However, as perfusion increases, there is a proportional increase in absorption (and vice versa). 
Thus, percent change in total hemoglobin detected refects the relative change in perfusion.  
 
The main outcomes assessed will be tibial perfusion, whole leg blood flow, and sympathetic 
activity. Descriptive statistics of the time series of these outcomes will include mean, median, 
standard deviation, and range. Differences/relations will be considered statistically significant at 
alpha = 0.05 level.  
 
For cold pressor test, isometric handgrip exercise, and head tilt, in able-bodied we will assess the 
relationship between dependent increases in sympathetic outflow and changes in tibial perfusion. 
Since it is unclear whether absolute or relative changes in sympathethic outflow may best relate 
to relative changes in tibial flow, both will be explored as within subject predictors. Sex-related 
differences in response will be assessed simply as group differences in the individual slopes of 
the relation between tibial flow and sympathetic activity.  
 
For cold pressor test, isometric handgrip exercise, and head tilt, in those with SCI we will assess 
the relationship between changes in mean arterial pressure and changes in tibial perfusion.  
 
 
VII. 
RISKS AND DISCOMFORTS 

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The proposed study will utilize cardiovascular monitoring techniques with minimal risks. The 
attachment and removal of ECG electrodes and inflation of the blood pressure cuff may cause 
mild discomfort. Recordings from the peroneal nerve with a microelectrode may cause the minor 
discomfort of muscle weakness and/or pins and needles sensation, which resolve spontaneously 
within a few days. The immersion of the hand in ice-cold water may cause mild to moderate 
discomfort. The risks associated isometric handgrip exercise are minimal, but may cause mild to 
moderate discomfort. The NIRS system is non-invasive and poses little risk to the patients. Initial 
trials showed minor reddening of the skin; however, this was temporary and it quickly went away. 
The tilt test may cause subjects to feel dizzy, nauseous, sweaty or pale and may increase heart 
rate or decrease blood pressure; however, this is temporary and it goes away in a few minutes 
after the subject are brought back to neutral position. 
 
VIII. 
POTENTIAL BENEFITS 
 
This research may help physicians and scientists better understand how the nervous system 
controls bone perfusion in humans. Subjects will not directly benefit from participation in this 
protocol.  
 
MONITORING AND QUALITY ASSURANCE 
 
The research coordinator will be responsible for monitoring the completeness of all data and 
source documents. The Principal Investigator will monitor the informed consent procedures in 
accordance with the Informed Consent Compliance Checklist of Partners HealthCare Systems 
HRQIP. The subjects data/protocol adherence will be monitored by the research coordinator at 
each step in the study including. Checklists and note pages are used to note any deviations or 
omissions from the protocols. The Cardiovascular Research Laboratory Medical Emergency 
Safety Plan will be followed in the case of an adverse medical event. Any clinical/health related 
issues would be immediately presented to the subject (i.e. abnormal EKG, blood chemistries, etc) 
to determine appropriate notification (ie. current physician or appropriate specialist) .Based on 
the seriousness of the situation a physician may be contacted to provide clinical guidance on the 
appropriate course of action. Event assessment and follow-up will be done by the PI as needed 
on a case by case basis. 
 
This trial will be conducted in compliance with the protocol, International Council for 
Harmonization/Good Clinical Practice requirements(ICH/GCP) and applicable state, local and 
federal regulatory requirements. 
 
Reporting to Partners IRB: 
Reports of unanticipated problems involving risks to subjects or others are to be submitted 
through Insight/eIRB within 5 working days/7 calendar days of the date the investigator first 
becomes aware of the problem. 
 
 
Reporting Unanticipated Problems that are Adverse Events 
Any unanticipated untoward or unfavorable medical occurrence, including abnormal sign (for 
example, abnormal physical exam or laboratory finding), symptom, or disease, that indicates 
that the research places subjects at increased risk of physical or psychological harm than 
previously known or recognized are to be submitted through Insight/eIRB as an Other Event, 
Adverse Event. The investigator must provide the following information in the report: 

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 Page 10 of 13 Bone Blood Flow V11, March 24, 2021 
(1) a detailed description of the adverse event; 
(2) the basis for determining that the event is unexpected in nature, severity, or frequency; 
(3) the basis for determining that the event is related or possibly related to the research 
procedures; 
(4) the basis for determining that the research places subjects at an increased risk of harm (i.e., 
a serious adverse event); and 
(5) whether any changes to the research or other corrective actions are warranted. 
NOTE: The PHRC does not accept sponsor IND/IDE safety reports describing adverse events 
that have occurred at sites other than those subject to this policy unless the report is of an 
incident that is: 
(1) serious; (2) unexpected or unanticipated; (3) related to the investigational drug/device; and 
(4) suggests that subjects are at an increased risk of harm and as such warrants changes in the 
research, consent process, or informing subjects. IND/IDE safety reports that warrant changes 
are to be submitted through Insight/eIRB as an Amendment. 
 
Reporting Unanticipated Problems that are not Adverse Events 
All other unanticipated incidents, experiences, information, outcomes, or other problems that 
indicate that the research places subjects at an increased risk of physical, psychological, 
economic, legal, or social harm than was previously known or recognized are to be submitted 
through Insight/eIRB as an 
Other Event. The investigator must provide the following information in the report: 
(1) a detailed description of the unanticipated problem; 
(2) the basis for determining that the problem is unexpected; 
(3) the basis for determining that the problem indicates that the research places subjects at an 
increased risk of harm; and 
(4) whether any changes to the research or other corrective action are warranted. 
 
 
Reporting to the National Institute of Arthritis and Muscloskeletal and Skin Disease(NIAMS) and 
Safety Officer (Dr.Ralph Marino) 
 
This protocol has funding from NIAMS. NIAMS has appointed a Saftey Officer, Dr. Ralph Marino,  
to act in an advisory capacity to the NIAMS to monitor participant safety, evaluate the progress of 
the study, to review procedures for maintaining the confidentiality of data, the quality of data 
collection, management and analyses. 
 
The Principal Investigator (PI)  will report unanticipated problems (UPs), serious adverse events 
(SAEs) and protocol violations that impact participant safety to the NIAMS and the Safety 
Officer, (through the Exective Secretary, KAI-Research) within 48 hours of the PI becoming 
aware of the event. KAI facilitates the review of the expedited reporting of these events by the 
Safety Officer. The Safety Officer will review the Safety Reports submitted by the PI to KAI twice 
a year. KAI facilitates the review of these reports by the Safety Officer and the NIAMS, and 
provides the final review comments to the PI when the review is complete. During the course of 
the study, ad hoc meetings may be called by the NIAMS or Safety Officer at any time to discuss 
potential safety issues. 
 
Definitions: 
Adverse event means any untoward or unfavorable medical occurrence in a human subject, 
includingany abnormal sign (for example, abnormal physical exam or laboratory finding), 

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symptom, or disease,temporally associated with the subject’s participation in the research, 
whether or not considered relatedto the subject’s participation in the research (modified from the 
definition of adverse events in the 1996. International Conference on Harmonization E-6 
Guidelines for Good Clinical Practice). 
 
Serious adverse event means any event temporally associated with the subject’s participation in 
research that meets any of the following criteria: 
• 
results in death; 
• 
is life threatening (places the subject at immediate risk of death from the event as it 
occurred); 
• 
requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
results in a persistent or significant disability/incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
any other adverse event that, based upon appropriate medical judgment, may jeopardize 
the subject’s health and may require medical or surgical intervention to prevent one of 
the outcomes listed above (examples of such events include allergic bronchospasm 
requiring intensive treatment in the 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). 
 
Possibly related to the research means there is a reasonable possibility that the adverse event, 
incident,experience or outcome may have been caused by the procedures involved in the 
research (modified from the definition of associated with use of the drug in FDA regulations at 
21 CFR 312.32(a)). Reasonable possibility means that the event is more likely than not related to 
participation in the research or,in other words, there is a >50% likelihood that the event is 
related to the research procedures. 
 
Deviation means any alteration/modification to the PHRC-approved protocol without prospective 
PHRC approval. The term protocol encompasses all PHRC-approved materials and documents 
including the detailed protocol, protocol summary, consent form, recruitment materials, 
questionnaires, and any other information relating to the research study. 
 
Unanticipated problem involving risks to subjects or others means any incident, experience, 
information, outcome, or other problem that is unexpected and related to the research, and that 
indicates that the research places subjects or others at an increased risk of physical, 
psychological, economic, legal, or social harm than was previously known or recognized. 
Unexpected means that the incident, experience, or outcome is unexpected (in terms of nature, 
severity or frequency) given the (a) research procedures that are described in the protocol-
related documents, such as the IRB-approved research protocol and informed consent 
document and (b) the characteristics of the study population being studied. 
 
Unexpected adverse event means any adverse event occurring in one or more subjects 
participating in a research protocol, the nature, severity, or frequency of which is not consistent 
with either: (1) the known or foreseeable risk of adverse events associated with the procedures 
involved in the research that are described in (a) the protocol-related documents, such as the 
IRB-approved research protocol, any applicable investigator brochure, and the current IRB-
approved informed consent document, and (b) other relevant sources of information, such as 

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product labeling and package inserts; or (2) the expected natural progression of any underlying 
disease, disorder, or condition of the subject(s) experiencing the adverse event and the 
subject’s predisposing risk factor profile for the adverse event. 
 
 
 
 
 
 
 
 
X. 
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