Effects of Transcranial Direct Current Stimulation (tDCS) and Transcranial Ultrasound (TUS) on the Perception of Pain and Functional Limitations Due to Osteoarthritis of the Knee
Conditions: Chronic Pain, Osteoarthritis
Sponsor: Spaulding Rehabilitation Hospital



Detailed Protocol 4 
This study is to be funded by the National Institutes of Health (Highland Instruments SBIR). 
Highland Instruments is a company focused on developing neurostimulation technologies for 
treatment of neural pathologies. 
 
 
II. 
SPECIFIC AIMS 
 
In this study, we aim to assess the effects of tDCS in combination with TUS for the treatment of 
pain and functional limitations in subjects with OA of the knee.  We will explore Aim 1 Sub-aims 
1.1- 1.4 with a parallel design (active tDCS+ active TUS (n=32), and sham tDCS+sham TUS 
(n=32)). The specific aims of this experiment are as follows: 
 
Aim 1: Assess the preliminary therapeutic effects of tDCS in conjunction with TUS in 
chronic OA knee pain. 
Sub Aim 1.1: Safety of tDCS+TUS in subjects with OA of the knee.  
We hypothesize that tDCS in conjunction with TUS can be applied safely in OA subjects. To 
test this we will implement a comprehensive battery of safety and neurological tests in OA 
subjects following fixed doses of tDCS in conjunction with TUS provided over 
 
for 10 days, 20 minutes/day. These assessments include EEG recordings, 
Visual Analog Mood Scale (VAMS), Mini Mental Status Exam (MMSE), 4-Choice Reaction 
Time, adverse events (AE) questionnaire and Neurological and physical examinations.  We 
will compare active tDCS+ active TUS (n=32), and sham tDCS+sham TUS (n=32), inactive 
transducers.  Follow-up evaluations will continue, while no further treatment is given, for 8 
weeks (at 1, 2, 4, and 8 weeks) after the last stimulation. 
 
Sub Aim 1.2 Effects of tDCS+TUS in pain perception in subjects with OA of the knee.  
We hypothesize that noninvasive tDCS in conjunction with TUS can be effective in decreasing 
the perception of pain in subjects with OA of the knee. To test this, we will use sensitive, 
currently validated outcomes for chronic pain subjects. A rater blind to the treatment arm will 
administer the following tests: Visual Analog Score (VAS) for Pain, Pain/Medication Diary, 
Von Frey assessment, and pain pressure tests (PPT) with algometer following stimulation 
detailed above, in SubAim 1.1. We will compare the stimulation conditions as above. Follow-
up evaluations will continue, while no further treatment is given, for 8 weeks (at 1, 2, 4, and 8 
weeks) after the last stimulation. 
 
Sub Aim 1.3 Effects of tDCS+TUS in biomechanical function in subjects with OA of the knee.  
We hypothesize that tDCS in conjunction with TUS can be effective in improving the 
biomechanical function in subjects with OA of the knee. To test this, we will compare the 
impact of active tDCS in conjunction with active TUS to the other stimulation conditions, as 
above, on markers of biomechanical function. We will use a biomechanical data acquisition 
and analysis system to specifically assess subject’s knee biomechanical functionality and 
response to the intervention. For this purpose we will use a set of integrated sensors including 
accelerometers, gyroscopes, force sensors, and motion-capture cameras. Follow-up 
evaluations will continue, while no further treatment is given, for 8 weeks (at 1, 2, 4, and 8 
weeks) after the last stimulation. 

Detailed Protocol 5 
 
Sub Aim 1.4. Effects of tDCS+TUS in the quality of life in subjects with OA of the knee. 
We hypothesize that tDCS in conjunction with TUS can be effective in improving the quality 
of life (QOL) in subjects with OA of the knee. To examine this aim, we will implement the 
Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), as this is 
recommended as one of the most sensitive and specific instruments for quality of life 
assessment in OA [68] and physical functioning assessment including a wide range of daily 
activities, such as bathing and getting in or out of the car. We will also use a generic health 
status measure - the Medical Outcomes Study 36-Item Short Form (SF-36) [68]. We will 
compare the stimulation conditions as above. Follow-up evaluations will continue, while no 
further treatment is given, for 8 weeks (at 1, 2, 4, and 8 weeks) after the last stimulation. 
 

Detailed Protocol 6 
 
IV. 
SUBJECT SELECTION 
 
We will recruit up to 94 subjects for this study, with the intention of randomizing exactly 64 
subjects with OA of the knee for this study. Once we randomize 64 subjects, we will stop recruiting 
subjects.  Subjects will need to meet all of the following inclusion criteria and none of the following 
exclusion criteria: 
 
 
Inclusion Criteria: 
1. Able to provide informed consent to participate in the study. 
2. Subjects between 18-85 years old. 
3. Diagnosis of chronic osteoarthritis with pain of either knee as self-reported.  
Prior to beginning baseline procedures this must be confirmed by medical records or a 
letter from the subject’s physician, or diagnosis by a study staff licensed physician. This 
document may either be brought in by the subject or retrieved by study staff after the subject 
signs a medical release form. If the subject is unable to obtain the medical record, a 
diagnosis by a study staff licensed physician will be accepted. After consent, if the subject 
fails to provide the necessary documentation, or if it cannot be retrieved by study staff, the 
subject will be considered a screen fail.  
4. Existing knee pain of at least 3 on a 0-10 VAS scale on average over the past 6 months. 
5. Pain of at least 3 on a 0-10 VAS scale on average over the week prior to the first stimulation 
session.  
6. Pain resistant to common analgesics and medications for chronic pain used as initial pain 
management such as Tylenol, Aspirin, Ibuprofen, Soma, Parafon Forte DCS, Zanaflex, and 
Codeine. 
7. Having the ability to feel pain as self-reported. 
 
Exclusion Criteria: 
1. Pregnancy or trying to become pregnant in the next 6 months. 
2. History of alcohol or drug abuse within the past 6 months as self-reported. 
3. Contraindications to transcranial brain stimulation or TUS, e.g. implanted brain medical 
devices or implanted brain metallic devices. 
4. Unstable medical conditions (e.g. uncontrolled diabetes, uncompensated cardiac issues, 
heart failure or chronic obstructive pulmonary disease). 
5. Epilepsy. 
6. Use of carbamazepine within the past 6 months as self-reported. 
7. Suffering from severe depression (with a score of >30 in the Beck Depression Inventory). 
8. History of unexplained fainting spells as self-reported. 

Detailed Protocol 7 
9. Head injury resulting in more than a momentary loss of consciousness.  
10. History of neurosurgery as self-reported. 
 
 
V. 
SUBJECT ENROLLMENT 
 
A total of 64 subjects, from 18 to 85 years of age, with a diagnosis of Osteoarthritis and pain of 
the knee will be randomized in this study. The safety of tDCS in pregnant populations (and 
children) has not been assessed, and therefore pregnant women (and children) will be excluded. 
Women of childbearing potential will be required to take a urine pregnancy test during the 
screening process.  
 
Potential subjects will be identified by the following sources: 
 
1. Attending physicians or therapists may refer their patients to the study. We will provide 
the physicians, therapists and clinics with study information sheets and flyers. Prospective 
subjects will be encouraged to contact the study co-investigators. 
2. Flyers posted in public areas across the Boston-land region, in the outpatient specialist 
clinics, public posting boards (i.e., digital signage) and other private locations with given 
permission. 
3. Internet, email and newspaper advertisements. 
4. Advertising posted in public transportation (e.g., the subway-“The T”). 
5. Via the Partners Healthcare Research Patient Data Registry (RPDR). 
6. Possible subjects might also be identified though their medical records (Meditech, LMR, 
etc.) and their physicians might be asked to inform the subjects about the study.  
7. Presentations to outside sites (such as support groups, main rehabilitation centers from 
Spaulding and Partners Network, and others important institutions of the Massachusetts 
area, nursing/assisted living centers etc.) 
8. Attending public forums, conferences, or events (such as walk-a-thons and association 
events) at which the co-investigator will distribute IRB approved recruitment materials. 
 
We anticipate subjects will be primarily recruited through the Spaulding Rehabilitation 
Hospital/Network (one of the largest rehabilitation centers in the US). SRH provides outpatient 
services to the Boston area so we anticipate our recruitment procedures will reflect the distribution 
of races/ethnicities in the surrounding communities. In addition, we will approach colleagues at 
the other Harvard teaching hospitals, including Brigham and Women’s Hospital (BWH), 
Massachusetts General Hospital (MGH) and outpatient clinics in the greater Boston area. 
Furthermore, we plan to advertise this clinical trial in the greater Boston area in a variety of media 
including on the Internet, via patient registries that are designed to promote research, and in local 
newspapers. We will also approach senior centers, assisted living facilities and support groups. 
 
Eligible subjects will contact, or give permission to be contacted, by a co-investigator to obtain 
more information about the study. At the first point of contact (usually a phone call), study co-
investigator will administer a phone-screening questionnaire. Once the phone screening process is 
complete, the information gathered by the co-investigator will be taken to the PI of the study for 

Detailed Protocol 9 
Once this information is collected, the co-investigator will consult with the PI regarding the 
eligibility of the subject, who will then give approval for the subject to come to our laboratory for 
the screening procedure.   
 
Visit 1  
Screening Visit – (Approx Time: 45 mins) 
 
Screening Procedures: 
At Screening, the PI and/or a co-investigator will once more conduct a review of 
inclusion/exclusion criteria to determine the subject’s eligibility for enrollment. Study procedures 
will be reviewed with the subject, and documentation of informed consent will be obtained.  
 
At Screening the following procedures will be completed: 
 
• 
Discuss study-specific procedures with the subject.  
• 
Review inclusion and exclusion criteria. 
• 
Obtain a signed and dated consent form. 
• 
Conduct a Demographics Survey, Brief Medical History and the Beck Depression 
Inventory. 
• 
Urine pregnancy exam (if applicable). 
 
Confirmation of Osteoarthritis  
Prior to the screening visit the subject will be asked if they can bring a document from their 
physician confirming the diagnosis of osteoarthritis of the knee. If the subject does not have it 
upon consenting, the subject will be asked to sign a medical release form allowing us to retrieve 
this document. If the subject is unable to obtain the medical record, a diagnosis by a study staff 
licensed physician will be accepted. This requirement has been outlined in the consent form. The 
baseline visit will only occur once confirmation of this diagnosis has been received. 
 
 
Visit 2 
Baseline Visit – (Approx Time: 2 hours) 
This visit might be completed on the same day as the screening visit if time allows and the subject 
agrees.  
 
Baseline assessments: 
 
• Single Leg Standing Balance Test 
• Step Test 
• Functional Reach Test 
• Timed Up and Go Test  
• Knee Proprioceptive Test 
• Knee Extensor/Flexor Force Test 
• Knee Range of Motion (ROM) 
• Visual Analogue Scale (VAS) for Pain 

Detailed Protocol 10 
• Pain/Medication Diary 
• Von-Frey Assessment 
• Pain Pressure Test (PPT) with algometer 
• Descending Noxious Inhibitory Control (DNIC) 
• Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) 
• Medical Outcomes Study 36-Item Short Form (SF-36) 
• Visual Analog Mood Scale (VAMS) 
• EEG recording 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Neurological and Physical Exam 
 
Visit 3  
 
Re-screening procedure-(Approx Time: 5 minutes) 
 
Prior to performing any assessments for visit 3, the subject will be re-screened to determine if they 
comply with the following inclusion and exclusion criteria: “Pain of at least 3 on a 0-10 VAS scale 
on average over the week prior to the first stimulation session”.  If the subject does not report a 
VAS of at least 3 on a 0-10 VAS scale on average over the week prior to the stimulation session 
they will be screened-out of the trial and provided with the reason. If the subject does report a VAS 
of at least 3 on a 0-10 VAS scale on average over the week prior to the stimulation session, then 
they will continue with the remainder of the study visits.   
 
1st Stimulation Visit – (Approx Time: 3.0 hours) 
This study will be scheduled ~1 week after the baseline measurements were done.  
 
There will be 10 stimulation visits to be completed over 2 consecutive weeks (ideally Monday to 
Friday of each week). The subject will receive either 20 minutes of active tDCS in conjunction 
with active TUS, or sham tDCS in conjunction with sham TUS per day for the course of their 
stimulation visits.  
 
Randomization: 
Once eligibility and consent have been approved and completed, after all baseline evaluations and 
immediately before the first stimulation session, stratified blocked randomization will occur using 
the randomized list generated by an automatic web-based randomization program 
(www.randomization.com). A staff member who is not involved in any of the study procedures 
will be responsible for creating the randomization list and monitoring it during the study. 
Randomization will be stratified by baseline pain, using the median of Phase I as a cutoff (VAS of 
7). Subjects will be randomly assigned to treatment or placebo groups in a 1:1 allocation ratio. We 
will use permuted blocks of variable sizes (4, 6, and 8) in a random manner to minimize the risk 
of study staff guessing at the next allocation. Randomization order will be kept in sealed envelopes.    
  
 

Detailed Protocol 11 
Blinding: 
 
All subjects and investigators will be blinded to the group assignment except for the co-
investigators providing stimulation and staff involved in the randomization process (the latter 
would otherwise not be involved in study procedures). Only the staff involved in the randomization 
will be authorized to break the blind if there is a clinical need as requested by the PI, clinical care 
providers, Medical Monitor, or IMC. The patients will remain blinded until they have completed 
all visits (whether they complete all 16 visits or drop out early), or in the case of an adverse event 
necessitating un-blinding.  
 
If the subject receives a sham stimulation, he/she may re-enroll into an open label portion of the 
study, where he/she will receive 10 days of active tDCS/TUS.  Un-blinded co-investigators (e.g., 
who provided stimulation) will coordinate with subjects electing to enter into the open label phase, 
which will be conducted by the un-blinded study staff.  The open-label study will be conducted 
without informing the blinded assessors of the patient enrollment.  In the open label phase, we will 
complete the VAS assessments on Day 5 and Day 10 of stimulation. This collected data will not 
be analyzed with the main data collected in the study. This data will be used for exploratory 
analyses only. 
 
Before stimulation, the subject will complete a series of assessments: 
• Single Leg Standing Balance Test 
• Step Test 
• Functional Reach Test 
• Timed Up and Go Test  
• Knee Proprioceptive Test 
• Knee Extensor/Flexor Force Test 
• Knee Range of Motion (ROM) 
• Visual Analog Scale (VAS) for Pain 
• Pain/Medication Diary will be given to the subject   
• Von-Frey Assessment 
• Pain Pressure Test (PPT) with algometer 
• Descending Noxious Inhibitory Control (DNIC) 
• Visual Analog Mood Scale (VAMS) 
• Neurological and Physical Exam 
 
After stimulation subjects will complete the following assessments:  
• Visual Analog Scale (VAS) for Pain 
• Von-Frey Assessment 
• Pain Pressure Test (PPT) with algometer 
• Visual Analog Mood Scale (VAMS) 
• Descending Noxious Inhibitory Control (DNIC) 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Adverse Events Questionnaire (AEs) 

Detailed Protocol 12 
• EEG recording 
 
Visits 4 – 6  
Stimulation visits – (Approx Time: ¾ hour) 
 
During the next 3 stimulation sessions – the subject will receive either 20 minutes of active tDCS 
in conjunction with active TUS, or sham tDCS in conjunction with sham TUS.  
Pre-stimulation: immediately before stimulation, subjects will be asked to complete the following: 
• Visual Analog Scale (VAS) for pain 
 
After stimulation, the subject will complete a series of assessments: 
• Visual Analog Scale (VAS) for Pain 
• Visual Analog Mood Scale (VAMS) 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Adverse Events Questionnaire (AEs) 
 
Visit 7 
5th Day of stimulation visits – (Approx Time: 2 ¼ hours) 
 
Pre-stimulation: immediately before stimulation, subjects will be asked to complete the following: 
• Visual Analog Scale (VAS) for pain 
 
After stimulation subjects will complete the following assessments: 
 
• Single Leg Standing Balance Test 
• Step Test 
• Functional Reach Test 
• Timed Up and Go Test  
• Knee Proprioceptive Test 
• Knee Extensor/Flexor Force Test 
• Knee Range of Motion (ROM) 
• Visual Analog Scale (VAS) for Pain 
• Pain/Medication Diary 
• Von-Frey Assessment 
• Pain Pressure Test (PPT) with algometer 
• Descending Noxious Inhibitory Control (DNIC) 
• Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) 
• Visual Analog Mood Scale (VAMS) 
• EEG recording 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Adverse Events Questionnaire (AEs) 

Detailed Protocol 13 
• Neurological and Physical Exam 
 
Visits 8 – 11  
6-9th Day of Stimulation visits – (Approx Time: ¾ hour) 
 
Pre-stimulation: immediately before stimulation, subjects will be asked to complete the following: 
• Visual Analog Scale (VAS) for pain 
 
After stimulation, the subject will complete a series of assessments: 
• Visual Analog Scale (VAS) for Pain 
• Visual Analog Mood Scale (VAMS) 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Adverse Events Questionnaire (AEs) 
 
 
Visit 12 
10th Day of stimulation visits – (Approx Time: 2 ¼ hours) 
 
Pre-stimulation: immediately before stimulation, subjects will be asked to complete the following: 
• Visual Analog Scale (VAS) for pain 
 
After stimulation subjects will complete the following assessments: 
 
• Single Leg Standing Balance Test 
• Step Test 
• Functional Reach Test 
• Timed Up and Go Test  
• Knee Proprioceptive Test 
• Knee Extensor/Flexor Force Test 
• Knee Range of Motion (ROM) 
• Visual Analog Scale (VAS) for Pain 
• Pain/Medication Diary 
• Von-Frey Assessment 
• Pain Pressure Test (PPT) with algometer 
• Descending Noxious Inhibitory Control (DNIC) 
• Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) 
• Medical Outcomes Study 36-Item Short Form (SF-36) 
• Visual Analog Mood Scale (VAMS) 
• EEG recording 
• Mini Mental Status Exam (MMSE) 
• 4-Choice Reaction Time  
• Adverse Events Questionnaire (AEs) 

Detailed Protocol 16 
 
 
DESCRIPTION OF ASSESSMENTS:   
A rater blind to the treatment arm will administer the following tests (also used in our previous 
study [70]:  
 
Visual Analogue Scale (VAS) for Pain: The VAS is a common assessment used which asks subjects 
to self-reportedly measure their pain on a visual scale (i.e., unbearable to none).  This will help us 
to monitor changes in subjects’ pain levels when they come in for sessions. 
 
Pain/Medication Diary: To help monitor pain levels, as well as safety, subjects will keep a diary 
listing their daily medications and pain levels when not at the laboratory. 
 
Mechanical Detection Threshold (von-Frey MDT): This technique is adapted to evaluate light 
touch in small cutaneous regions [71]. The most used procedure is with the Von Frey monofilament 
(0.008g to 300g) via the method of limits. The examiner makes use of an ascending standardized 
sequence of varying stimulus intensities. In the case of ramping the stimulus up, the individual is 
asked to report when the sensation is first detected  
 
Single applications of standardized monofilaments will be applied first to a reference region 
(thenar region, ipsilateral to the most painful knee), using the smallest weighted monofilament, 
followed by sequentially larger monofilaments until sensation is reported [71]. Following the 
reference region, the most painful knee will be tested. The monofilaments must be applied 
perpendicular to the skin and bow for a period of approximately 1.5 seconds [71, 72]. The threshold 
will be taken as the lowest force that causes a light touch sensation 

Detailed Protocol 17 
 
Mechanical Pain Thresholds (von-Frey MPT): The evaluation of mechanical pain thresholds is 
performed using standardized, fine, blunt-tipped probes. The threshold to produce pain is recorded 
in this test. The force applied by the Von Frey monofilament is standardized by applying just the 
right amount of force to bow the monofilament for approximately 1.5 seconds [71, 72]. The 
subjects will be asked to say when they sense pain. The thenar region, ipsilateral to the most painful 
knee will be tested. Following the reference region, the most painful knee will be tested. The 
smallest monofilament that produces pain will be recorded.  
 
Fixed Intensity hyperalgesia evaluation (von-Frey hyperalgesia): To study hyperalgesia the 
previously registered smallest monofilament to produce pain in the thenar region will be used [71]. 
A standard stimulus of fixed intensity will be applied over the thenar region, ipsilateral to the most 
painful knee, and the subject will be asked to rate the sensation of pain using a visual analogue 
scale for pain. The process will be done 3 times and the geometric mean of the visual analogous 
scale will be calculated. Following the procedure, the most painful knee will be studied and the 
subject will be asked to rate the sensation of pain using a visual analogous scale for pain. The 
process will be done 3 times and the geometric mean of the visual analogous scale will be 
calculated. 
 
Pain pressure tests (PPT): PPT will be determined using blunt pressure delivered by a 1-cm2 hard-
rubber probe using a FDA approved device (commander algometer – JTECH medical). During 
testing a series of discrete pressures are applied to a reference area, the thenar area ipsilateral to 
the most painful knee. Following the reference region testing, the most painful knee will be tested. 
The subject will let the investigator know when he/she feels any pain, and at that time the procedure 
will be stopped, and the value will be recorded. This procedure will be repeated 3 times. The test 
will take approximately 7-10 min to complete. 
 
Descending Noxious Inhibitory Control (DNIC): Endogenous pain modulation is commonly 
evaluated in the laboratory using DNIC testing paradigms. These procedures incorporate a 
conditioning stimulus (a noxious stimulus that evokes DNIC activation) and a test stimulus (a 
noxious stimulus used to evaluate the analgesic response to the conditioning stimulus - this will be 
the PPT). This study will evaluate DNIC in pain patients using pressure as the test stimulus, and 
cold water as the conditioning stimulus. Pressure will be delivered using the same device as for 
the evaluation of PPT. DNIC will be induced approximately 1-min later by having subjects 
immerse their hand into a water bath maintained at 10-12˚C for approximately 1 min. Parallel to 
the last 30s of DNIC conditioning (cold water immersion), the same test stimulus will be reapplied 
(PPT procedure). DNIC will be evaluated as the mean difference in pain rating of the test stimulus 
applied before and during the conditioning stimulus. This value would typically be treated as a 
continuous variable if measured repeatedly in a clinical trial because it is unlikely that descending 
analgesic systems are either “on” or “off”, but rather function within a range of magnitudes across 
subjects. 
 
EEG recording: One week prior to stimulation, on the first day of stimulation, at the end of each 
stimulation week, and during each follow up visit, EEG recordings will be taken to monitor brain 
activity and saved for post recording evaluation.  

Detailed Protocol 18 
 
Neurological and Physical Exam: Trained study staff will conduct baseline and follow-up 
neurological examinations focused to ensure subject safety one week prior to stimulation, on the 
first day of stimulation, at the end of each stimulation week, and during each follow up visit [73]. 
 
Visual Analog Mood Scale (VAMS): This is a self-assessment scale in which subjects rate their 
own emotions, including anxiety, depression, stress, and sleepiness along a 100 mm line.  
 
Mini Mental Status Exam (MMSE): This is a sensitive, valid and reliable 30-point questionnaire 
that is used extensively in clinical and research settings to measure cognitive impairment. This 
instrument will be used as a brief screening of cognitive abilities. The MMSE will be administered 
at the baseline evaluation and every subsequent visit. 
 
4-Choice Reaction Time: Patients will be seated in front of a computer screen placed at eye level. 
One of four possible circles, each 4 cm in diameter, will be presented horizontally spaced on the 
screen and aligned above the appropriate response keys. The stimuli will be generated and response 
times recorded using Superlab pro v2.0 software (Cedrus Corporation, San Pedro, Ca). The 
patients have to push the key aligned with the circle that appeared using only the index finger of 
the dominant hand. The circle will not disappear until the correct button is pushed. The time 
between the appearance of the circle and the push of the correct response key will be defined as 
the choice reaction time. The 4-choice reaction time will be administered at the baseline evaluation 
and every subsequent visit. 
 
Adverse Effects Questionnaire: At each session after stimulation begins, subjects will complete a 
questionnaire to evaluate potential adverse effects of stimulation (headache, neck pain, mood 
alterations, and seizures) on a 5-point scale. The scale will also be administered at the follow-up.  
The subjects will be asked whether they have experienced any side effects in an open-ended 
manner and they will then be specifically asked about headache, neck pain, scalp pain, scalp burns, 
tingling, skin redness, sleepiness, trouble concentrating, and acute mood change. If any side effects 
are reported, the degree of relatedness to the intervention will be assessed.  
 
We will be investigating adverse effects using open-ended questions. An AE can be any 
unfavorable and unintended sign, symptom, or disease temporally associated with the use of a 
treatment, whether or not considered related to the product. 
 
Examples of AEs are as follows: 
• 
Subjective symptoms offered by or elicited from the subject  
• 
Objective signs observed by the Investigator or other study personnel  
 
All concurrent diseases that occur after the start of the study, including any change in severity or 
frequency of preexisting disease will be investigated in several domains including: seriousness, 
severity, length of duration, and if any causal relationship exists with the intervention [30]. 
 
Single Leg Standing Balance Test: We will record the time for which a subject is able to stand 
unsupported on one foot while looking straight ahead with hands on hips [74]. During this test, the 

Detailed Protocol 19 
subject will be standing on a force platform. We will also use an accelerometer/gyroscope sensors 
worn by the subject (e.g., on the subjects’ ankle, above the most painful knee (thigh and shank), 
and/or on the back (~level L5)) and a Motion Capture system to record subject kinematics during 
the movements described above [75, 76] [77, 78]. No video images will be saved during the 
experiment, only positional coordinates of the subjects’ body parts that are extracted in real-time 
from the video and/or sensor will be saved. Subjects will be informed regarding video equipment 
and informed consent will be obtained.   
 
Step Test: Subject will be asked to stand unsupported with the feet parallel to each other and a 
block 5 cm directly in front of them. Subject will then be advised which leg is the stepping leg and 
asked to place the whole foot onto the block, then return it fully down to the floor. This procedure 
will be repeated as fast as possible. One completed step will comprise placing the foot fully onto 
the block and then on the floor [79]. During this test, the subject will be standing on a force 
platform. We will also use motion analysis system (e.g., motion capture camera, 
accelerometers/gyroscopes, force platform) to record subject kinematics during the movements. 
 
Functional Reach Test: Subject will be instructed to stand next to, but not touch the wall, and 
position the arm that is closer to the wall at 90 degrees of shoulder flexion with a closed fist. The 
subject will be asked to reach forward as far as they can without taking a step, keeping their hands 
in a fist shape. Hand position will be recorded. The difference between the start and end position 
will be the reach distance [74]. During this test, the subject will be standing on a force platform. 
We will also use motion analysis system (e.g., motion capture camera, accelerometers/gyroscopes, 
force platform) to record subject kinematics during the movements. 
 
Timed Up and Go Test: We will measure the time that the subject takes to rise from a standard 
arm chair, walk to a tape mark 3-meter away, turn around, walk back to the chair, and sit down 
[74]. Quantitative, objective metrics of gait function will include stride length and gait speed [75] 
[80, 81]. We will also use motion analysis system (e.g., motion capture camera, 
accelerometers/gyroscopes, force platform) to record subject kinematics during the movements. 
 
Knee Proprioceptive Test: Proprioceptive acuity will be assessed by the ability to reproduce self 
positioning of the leg with eyes closed [74]. The subjects will be seated on a chair, with hips and 
knees at 90°; the full extension of the leg will represent 180°. The subject’s leg will be passively 
moved and the angle of flexion will be measured at a randomly selected position with a goniometer 
that will be attached to the subject’s leg, this should take approximately 5 seconds. Then the 
subject’s knee will be returned to its original position by the examiner. The subject will then be 
asked to reproduce the criterion angle and to maintain it for approximately 5 seconds; this angle 
will also be measured with a goniometer. The difference between the criterion and reproduced 
angle will be taken as the measure of proprioception acuity. The procedure will be done 3 times. 
The average of the three procedures will be taken as the proprioceptive acuity for the knee [79]. 
 
Knee Extensor/Flexor Force Test: The subject will be seated on a chair with hips and knees flexed 
to 90 degrees and the maximal voluntary hip flexion and lower leg flexo-extension will be 
individually measured with a JTech Commander Manual Muscle Testing MMT Device (JTECH 
medical) on both legs. We will integrate the recorded data with that recorded via the motion 

Detailed Protocol 20 
analysis system (e.g., motion capture camera, accelerometers/gyroscopes, force platform) to assess 
subject kinematics during the movements from the other tests (see above). 
 
Knee Range of Motion (ROM): The knee range of motion (flexion/extension angle) will be 
measured with a goniometer. [82] The examiner will flex the subject’s knee, measuring the 
maximum knee flexion obtained. The end of the range of knee flexion occurs when resistance is 
felt and attempt to overcome the resistance cause additional hip flexion. This procedure is validated 
and standardized as described by Norkin and White [83]. 
 
Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC): The WOMAC is a 
self-reported questionnaire that measures three main components of the impact of OA on quality 
of life: pain (0-20 score), stiffness (0-8 score) and functional limitation (0-68 score). Physical 
functioning assessments include a wide range of daily activities, such as bathing and getting in or 
out of the car. 
The Medical Outcomes Study 36-Item Short Form (SF-36): It measures 8-health related concepts 
using 36 questions and provides a profile of functional health and well-being scores. It also 
provides a psychometrical index of physical and mental health (note, as stated earlier in this and 
the subsequent phase of the research the administration of all exams will be completed by a rater 
blinded to the treatment arm). 
 
VII. 
BIOSTATISTICAL ANALYSIS 
 
Data forms and questionnaires will be coded in a standardized manner, and double-entered into 
our database. Digital measures/recordings will be similarly tracked in our database and regularly 
backed up. Analyses will be conducted using standard statistical software such as SAS and Matlab. 
We will use REDCAP as a database (http://projectredcap.org/), and personnel entering the data 
will be blinded.  
 
Sample size calculations and data analysis: 
 
Aim 1:  Data forms and questionnaires will be coded in a standardized manner, and double-entered 
into our database. Digital measures/recordings will be similarly tracked in our database and 
regularly backed up. Analyses will be conducted using standard statistical software such as SAS 
and Matlab.  
 
For planning sample size calculation for this study, we utilized the OARSI recommendations for 
the management of knee OA. In this recommendation [84], a panel of experts reviewed evidence 
for the most commonly used pharmacological and non-pharmacological interventions for OA pain, 
giving effect sizes for all these interventions. Examples of non-pharmacological interventions are: 
education, physical therapy, aerobic exercise, knee brace, footwear, thermal modalities, TENS and 
acupuncture. Examples of pharmacological treatments are: acetaminophen, NSAIDs, injections 
with corticosteroids and treatment with glucosamine. In this review, effect sizes vary from 0.06 to 
0.72. In fact the largest effect size was shown with injection of corticosteroids. Based on these 
effect sizes and the effect size we found in our pilot data (1.4), we set the effect size for our sample 
size calculation considering a effect size smaller or equal than our pilot data and larger than the 

Detailed Protocol 21 
larger effect size from this meta-analysis (0.72) [84]. For this particular protocol, we plan a sample 
size of 64 participants. We considered the same sample per group as four times the subjects in 
Phase 1: 32 subjects per group. This will be enough to detect a difference in effect size of 0.9 and 
thus an effect size that is superior than other treatments used in OA according to a large guideline 
on OA management [84], though we expect a larger effect size for pain. However it is important 
to be able to detect significance with smaller effect sizes in the Phase 2 work, as some of the 
neurophysiological outcomes require additional power. Note that the main aim here would still be 
the comparison of active ESStim vs. sham ESStim. 
 
All analyses will be conducted according to the principle of intention-to-treat (using regression-
based single imputation method). Intention-to-treat (ITT) population will include all randomized 
subjects. We will also perform an additional sensitivity analysis in which we will use the method 
of multiple imputation. The primary outcomes will be pain changes as indexed by VAS scores, 
biomechanical function changes as indexed with the motion analysis suite (i.e., our biomechanical 
data acquisition and analysis system), and QOL changes. Differences between the groups will be 
tested using Student's t-test, and additionally we will adjust for important baseline variables and 
test the time effect in general mixed longitudinal models. This model will be used for the primary 
and secondary outcomes (dependent variable (one model for each variable): VAS scores, 
biomechanical function changes as indexed with the motion analysis suite, and QOL changes, and 
independent variables will be treatment group and time of assessment (we will also adjust for 
baseline pain levels in this model)). Secondary analyses will be conducted in an exploratory 
manner (no correction for multiple comparisons). We will also conduct an EEG analysis to 
correlate EEG data with pain measures.  Similar analysis will be conducted for the adverse effects 
measuring continuous outcome and for the categorical outcomes we will use Fisher’s exact test. 
In both cases, for safety analysis we will use uncorrected p-value to increase the likelihood of 
detecting detrimental adverse effects. 
 
 

Detailed Protocol 23 
 

Detailed Protocol 24 

Detailed Protocol 25 

Detailed Protocol 26 

Detailed Protocol 27