Optimizing Rehabilitation for Phantom Limb Pain Using Mirror Therapy and Transcranial Direct Current Stimulation (tDCS)
Phase: NA
Conditions: Phantom Limb Pain, Amputation, Traumatic
Sponsor: Spaulding Rehabilitation Hospital

Detailed Protocol 1 
ORGANIZATION OF DETAILED PROTOCOL 
 
Title: Optimizing Rehabilitation for Phantom Limb Pain Using Mirror Therapy and transcranial 
Direct Current Stimulation (tDCS). 
 
Protocol # 2015P001065 
 
Date: 08/14/2019 
 

Detailed Protocol 2 

Detailed Protocol 3 
Sponsors 
 
This study is funded by the NIH (1R01HD082302-01A1) 
 
II. 
SPECIFIC AIMS 
 
The specific aims of this experiment are as follows: 
 
- 
Aim 1: To evaluate the efficacy of tDCS and mirror therapy (MT) as rehabilitative tools 
for the management of chronic PLP patients.  
We will explore the aim 1 in a randomized factorial controlled trial in which patients will be 
assigned to one of four groups: active tDCS and active MT; sham tDCS and active MT; 
active tDCS and sham MT (which consists of using a covered mirror for the therapy); and 
both sham tDCS and sham MT (covered mirror).  
 

Detailed Protocol 4 
The primary end point will be severity of pain measured by changes in PLP as indexed by a 
Visual Analog Scale (VAS). The study will require a total of 13 weeks to be completed. The 
first week, each patient will give daily reports of their pain using a VAS, accounting for their 
PLP experience in the past 24hs. This initial baseline week will be followed by 4 weeks of 
treatment (tDCS and/or MT) and a final 8-week follow-up period without any treatment to 
examine long-lasting effects of each therapy. Our hypothesis is that the combined 
intervention (tDCS and MT) will result in a greater pain reduction as compared with any 
therapy alone and no therapy. 
 
 
- 
Aim 2: To examine the mechanisms underlying PLP using two neurophysiological 
techniques.  
For each of the four groups described in aim 1, we will determine the neural correlates of the 
effect. We will use single-pulse and paired-pulse transcranial magnetic stimulation (TMS) to 
assess cortical mapping and cortical excitability changes associated with cortical 
reorganization. We will also use functional magnetic resonance imaging (fMRI) to assess 
brain changes, including the quantification of maladaptive cortical reorganization. Using 
fMRI, we will determine the changes in Blood-Oxygen-Level dependent activation between 
the sensory-motor cortex of the affected and unaffected side after the treatment. We will 
compare these neurophysiological parameters before and after treatment in each of the four 
groups. Our hypothesis is that the combined group (tDCS and mirror therapy) will have a 
greater activation than any therapy alone and also than the no therapy group (sham tDCS 
and covered mirror). This effect will also be correlated with pain reduction. 
 
IV. 
SUBJECT SELECTION 
 
In this study, we will recruit 132 subjects with phantom limb pain (PLP) of traumatic etiology 
(congenital or diabetic amputees will be excluded since these patients may have a different 
neuroplastic profile). The subjects will be recruited by 2 main neurorehabilitation study centers, 
one at neuromodulation center - Spaulding rehabilitation Hospital and one at University of Sao 

Detailed Protocol 5 
Paulo Brazil (IMREA). 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. Subject is older than 18 years. 
3. Unilateral lower limb amputation. 
4. 3 months of phantom limb pain (experienced regularly for at least once a week) after 
the amputated limb has completely healed.* 
5 Average pain of at least 4 on a numeric rating scale in the previous week (NRS; ranging 
from 0 to 10).  
6 If the subject is taking any medications, dosages must be stable for at least 2 weeks prior 
to the enrollment of the study.  
 
*The healing status of the limb will need to be confirmed by the subject’s physician or by the 
physician of the protocol Dr. David Crandell (Co-Investigator).  
 
 
Exclusion Criteria: 
1. Pregnancy or trying to become pregnant in the next 2 months. 
2. History of alcohol or drug abuse within the past 6 months as self-reported. 
3. Presence of the following contraindication to transcranial direct current stimulation and 
transcranial magnetic stimulation  
- 
Ferromagnetic metal in the head (e.g., plates or pins, bullets, shrapnel) 
- 
Implanted neck or head electronic medical devices (e.g., cochlear implants, vagus 
nerve stimulator)  
4. Head injury resulting in permanent neurological deficits, such as cognitive or motor 
deficits, as self-reported.* 
5. Unstable medical conditions (e.g. uncontrolled diabetes, uncompensated cardiac issues, 
heart failure or chronic obstructive pulmonary disease) 
6. Uncontrolled epilepsy or prior seizures within the last 1 year 
7. History of unexplained fainting spells or loss of consciousness as self-reported during the 
last 2 years.  
8. History of neurosurgery, as self-reported. 
9. Mirror Therapy within 3 months prior to enrollment 
 
* permanent neurological deficit that may interfere with the assessments. 
 
The safety of tDCS in pregnant population (and children) has not been assessed and therefore 
pregnant women (and children) will be excluded. Women of child-bearing potential will be 
required to take a urine pregnancy test during the screening process.  

Detailed Protocol 6 

Detailed Protocol 7 
 
V. 
STUDY PROCEDURES  
 
We will recruit 132 participants with major lower unilateral limb amputation and phantom limb 
pain in the chronic phase. 
   

Detailed Protocol 8 
The patients will be assigned to one of four groups: the first will receive active tDCS and active 
MT; the second will receive sham tDCS and active MT; the third will receive active tDCS and 
sham MT; and the fourth will receive both sham tDCS and sham MT. Subjects and outcome 
evaluators will be blinded to the intervention assigned. The study will be done for a total of 13 
weeks.  The study procedures will be done in the same way in both study centers. Data collection 
will be performed according to the IRB approved protocol by trained and capable research staff. 
The Neuromodulation Center (Spaulding Rehabilitation Hospital) will be responsible for 
providing training and responsible for monitoring of the study, including maintaining regular 
contact with the University of Sao Paulo site through telephone contact. This phone contact will 
be logged and stored at the Regulatory binder. The data will be collected by staff members (PI, 
co-investigators and study staff) familiar with the protocol and the tDCS and TMS technology. 
The care, safety, and comfort of the subjects will be under the supervision of a physician and 
therapist at each trial site. The local IRB at each respective institution will be responsible for 
overviewing the conduct of the study trial locally. Both IRB institutions approved the research 
protocol. Unidentifiable data from the local institution in Brazil will be shared with the main site 
(Boston) via IRB approved data base RedCap. There will be no sharing of PHI between the sites. 
 
The subjects randomized to receive sham tDCS and/or sham MT will have the opportunity to 
enroll into an open label portion of the study at the conclusion of their participation in the 
randomized portion of the trial, including 10 sessions of tDCS combined with MT and additional 
questionnaire data. 
 
Study Outline 
 
Pre-screening Procedures: 
During the pre-screening process, the subject will contact a co-investigator usually via a phone 
call. During this call, the co-investigator will discuss in greater depth the details of the study, 
explain the study procedures and encourage the subject to ask questions. In the privacy of the 
laboratory, the co-investigator will ask the subject questions from the following: 
1) Phone screening questionnaire 
 
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 – (Approximate Time: 1 hour) 
 
Screening Procedures: 
At Screening the PI and/or a co-investigator will conduct, once more 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.  

Detailed Protocol 9 
• 
Review inclusion and exclusion criteria. 
• 
Obtain a signed and dated consent form. 
• 
Conduct a Demographics Survey, Brief Medical History and Beck depression inventory. 
• 
fMRI screening questionnaire*. 
• 
Urine pregnancy exam (if applicable). 
 
* If the subject is not eligible for an fMRI at the time of the study we will not perform this 
assessment as fMRI is not required for study eligibility and it is only used as a secondary 
outcome. 
 
Randomization: 
Once eligibility and consent have been approved and completed, randomization will occur using 
the randomized list generated by an automatic web-base randomization program 
(www.randomization.com). Subjects will be randomly assigned to one of the four groups in a 
1:1:1:1 allocation ratio. We will use stratified randomization methods with random block sizes of 
blocks of 4 and 8. Patients will be stratified based on their baseline pain levels (two strata: less or 
equal of average 6 in VAS or more than 6 in VAS) and by study center (IMREA or SRH-
Neuromodulation Lab). Randomization order will be kept in sealed envelopes; therefore subjects 
will get their assignment according to the order of entrance in the study (for instance, subject 1 
will be assigned the first envelope that will contain his/her assignment according to this block 
randomization list). 
 
Visit 2 
Baseline Visit – (Approximate 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: 
 
• Visual Analogue Scale (VAS) for Pain  
• Visual Analogue Scale (VAS) for Stump pain 
• Visual Analog Scale (VAS) for Phantom Limb Sensation  
• Visual Analog Scale (VAS) for Phantom Limb Telescoping  
• Adapted Groningen  Questionnaire After Arm Amputation 
• Pain and Medication use diary* 
• Beck Anxiety Inventory 
• Mini Mental State examination (MMSE) 
• Stroop Test 
• Health Survey SF-36 
• Single and paired pulse TMS 
• Side Effects Questionnaire for TMS 
 
Visit 3 
fMRI Evaluation – (Approximate Time: 1 hour) 
 

Detailed Protocol 10 
**Visits 1-3 may be completed on the same day or broken up into two or three separate visit as 
the subject prefers   
 
 
Visit 4 – 12 (Approx Time: 45 minutes) 
Intervention visits: tDCS + Mirror Therapy 
This visit will be scheduled within 1 week after the baseline measurements are done.  
 
In the first 2 weeks subjects will receive tDCS (*sham or active) and mirror therapy (sham or 
active). There will be 10 stimulation visits to be completed in these 2 weeks (ideally Monday to 
Friday, 5 days each week). The subject will receive 20 minutes either of active or sham tDCS per 
day and 15 min of active or sham mirror therapy sessions. The subjects will receive the 
intervention according to the prior randomized group. 
 
Before stimulation, the subject will complete the following assessments: 
• VAS Phantom Limb Pain 
• VAS for Stump pain 
• VAS for Phantom Limb Sensation  
• VAS for Phantom Limb Telescoping  
 
After each session, subjects will complete the following assessments:  
• Side Effects Questionnaire for tDCS 
• VAS Phantom Limb Pain 
• VAS for Stump pain 
• VAS for Phantom Limb Sensation  
• VAS for Phantom Limb Telescoping 
 
 
Visit 13 
Last day of tDCS session + Mirror Therapy – (Approx Time: 2 hours) 
 
Before stimulation, the subject will complete the following assessments: 
• VAS Phantom Limb Pain 
• VAS for Stump pain 
• VAS for Phantom Limb Sensation  
• VAS for Phantom Limb Telescoping 
 
After each session, subjects will complete the following assessments:  
• Visual Analogue Scale (VAS) for phantom limb pain 
• Visual Analogue Scale (VAS) for Stump pain 
• Visual Analog Scale (VAS) for Phantom Limb Sensation  
• Visual Analog Scale (VAS) for Phantom Limb Telescoping  
• Pain and medication use diary 
• Stroop Test 
• Beck Anxiety Inventory 

Detailed Protocol 11 
• Beck Depression Inventory 
• Side Effects Questionnaire for tDCS 
• Blinding Questionnaire 
• Single and paired pulse TMS 
• Side Effects Questionnaire for TMS 
 
After the first 2 Weeks combined intervention (sham/active tDCS plus sham/real mirror 
therapy) at the Neuromodulation Center in front of the therapist, subjects will continue 
mirror therapy for 10 additional sessions over the course of  ~ 2 weeks at home.  
 
 
2 weeks of home Mirror Therapy – (Approx Time: 15 min) 
 
During this 2 week period the co-investigator will try to contact the subjects 3 times per week to 
assess adherence to the MT treatment and acquire pain diary and medication changes data. We 
are going to try maximum 2 attempts per day. 
 
Visit 14: 
Last day of mirror therapy - (Approx Time: 3 hours) 
This visit will be scheduled in the last MT home session and the following assessments will be 
performed. This visit can be divided in two days if time allows and the subject agrees.  
 
Subject can perform the last mirror therapy session either at home or at the Neuromodulation 
Center. 
 
After the MT session, subject will complete the following assessments: 
• Visual Analogue Scale (VAS) for phantom limb pain 
• Visual Analogue Scale (VAS) for Stump pain 
• Visual Analog Scale (VAS) for Phantom Limb Sensation  
• Visual Analog Scale (VAS) for Phantom Limb Telescoping  
• Pain and Medication uses diary 
• Stroop Test 
• Health Survey SF-36  
• Patients’ Global Impression of Change (PGIC) scale 
• Beck Anxiety Inventory 
• Beck Depression Inventory 
• Single and paired pulse TMS 
• Side Effects Questionnaire for TMS 
• fMRI evaluation 
 
Visit 15: fMRI Evaluation – (Approximate Time: 1 hour) 
 
**Visits 14 & 15 may be completed on the same day or broken up into two separate visit as the 
subject prefers  
 

Detailed Protocol 12 
Visit 16:  
First Follow Up Visit – (Approx Time: 1 hour) 
This visit will be scheduled ~ four weeks (+/- 5 days) after the last MT home session and the 
following assessments will be performed: 
• Visual Analogue Scale (VAS) for phantom limb pain  
• Visual Analogue Scale (VAS) for Stump pain. 
• Visual Analog Scale (VAS) for Phantom Limb Sensation  
• Visual Analog Scale (VAS) for Phantom Limb Telescoping  
• Beck Anxiety Inventory 
• Beck Depression Inventory 
• Pain Medication uses diary 
 
**Visit 16 may be completed by terms of phone interview as the subject prefers. 
 
Visit 17: 
Last Follow Up Visit - (Approx Time: 2 hours) 
This visit will be schedule ~eight weeks (+/- 5 days) after the last MT home session and the 
following assessments will be performed: 
• Visual Analogue Scale (VAS) for phantom limb pain  
• Visual Analogue Scale (VAS) for Stump pain 
• Visual Analog Scale (VAS) for Phantom Limb Sensation  
• Visual Analog Scale (VAS) for Phantom Limb Telescoping  
• Pain Medication uses diary. 
• Stroop Test. 
• Health Survey SF-36.  
• Beck Anxiety Inventory 
• Beck Depression Inventory 
• Single and paired pulse TMS 
• Side Effects Questionnaire for TMS 
• Blinding questionnaire 
 
**After visit 14 until the end of the trial participation, the co-investigator will try to contact the 
subjects 3 times per week to acquire pain diary and medication changes data. We are going to try 
maximum 2 attempts per day.  
 
The schedule for the subjects can be seen in table 1, which illustrates the procedures that will be 
performed at each visit: 
 
 
Table 1: Visits Schedule 
 
 
Consent 
and 
Screening 
Basel
ine 
fMRI 
Visit 
Interventions Session 
fMRI 
Visit 
Follow-Up 

Detailed Protocol 13 

Detailed Protocol 14 

Detailed Protocol 15 
observed in the mirror to their phantom limb and to keep their attention focused on the task. 
Instructions will be explained verbally, demonstrated by a therapist, and performed by the 
subject in front of the therapist during the first two weeks (the MT sessions will be scheduled 
back-to-back with the tDCS sessions). After this training, subjects will continue MT 
everyday for 2 more weeks at home. Subjects will be instructed to stop MT if it intensifies 
their pain, and to document if this happens 
• Sham Mirror Therapy (Covered MT): Subjects will be asked to perform movements in the 
same way that the active group but with a covered mirror. 
 
 
Figure 1: Schematic view of the experiment in time points: baseline, procedures, subjects 
enrolled intervention, first follow-up and second follow up. 
 
DESCRIPTION OF ASSESSMENTS:   
A rater blind to the treatment arm will administer the following tests: 
 
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). We will 
use a VAS to determine subjects’ pain scores. Subjects will rate their pain from 0 – indicating no 
pain at all, to 10 – indicating the worst pain felt. This scale is also colored, from green (at 0) to 
red (at 10), as a visual indicator of pain. This assessment tool is frequently used in many research 
studies evaluating pain levels [15, 27, 34, 35]. 
 
Visual Analogue Scale (VAS) for Stump Pain: any painful sensation in the stump. Subjects will 
rate their stump pain from 0 – indicating no pain at all, to 10 – indicating the worst pain felt. The 
scale will include colors to help in identifying the correct response [15]. 
 
Visual Analog Scale (VAS) for Phantom Limb Sensation: all non-painful sensations in the 
amputated part of the limb. Subjects will be presented with a scale starting at 0- No phantom 
limb sensation, to 10 – Full sensation of the amputated limb. The scale will include colors to help 
in identifying the correct response [15]. 
 
Visual Analog Scale (VAS) for Phantom Limb telescoping: refers to the shrinking and retraction 
of the phantom towards the residual limb. Subjects will be presented with a scale starting at 0 - 
indicated that the phantom was enlarged, and 10 meant that the phantom was completely 
retracted into the stump the scale will include colors to help in identifying the correct response 
[15]. 

Detailed Protocol 16 
 
Adapted Groningen Questionnaire after Arm Amputation: This questionnaire is originally meant 
to obtain information’s concerning complaints that may be developed after arm amputation.  We 
adapted the current arm version for lower limb amputation. This questionnaire [36] has been 
used in several clinical trials assessing PLP.  
 
Pain and medication diary: To help monitor pain levels and medication use information, as well 
as safety. Subjects will be asked to record the number of phantom limb episodes on a daily basis, 
using a pain diary. They will record the intensity of the strongest episode as well as phantom 
limb sensation and stump pain on a colored visual analog scale included in the diary, where 0 
represents no pain at all and 10 represents the highest pain the patient has ever felt. Moreover 
subjects will record their current medications and dosages daily in a pain medication diary, until 
completion of the study. 
 
Beck Depression Inventory: This self-report inventory consists of 21 multiple-choice questions 
and is widely used method to classify depression severity. It assesses for the presence of several 
symptoms related to depression, such as irritability, hopelessness and decreased cognitive 
performance. Physical symptoms such as weight loss and fatigue are also included. This 
instrument has been used previously to evaluate depression severity in patients with phantom 
limb pain [37], as well as in other chronic pain conditions [31, 38, 39]. If the subject appears at-
suicidal risk (detected by the BDI questionnaire- as defined by a score of ≥ 1 on question 9), the 
medical coverage will be contacted immediately by research staff to provide further evaluation 
and action to protect the subject (i.e. if the subject needs to be escorted or referred to the MGH 
ED Acute Psychiatry Service). In addition, if a subject has moderate to severe depression 
(defined by a score of ≥ 20 in the Beck Depression Inventory), the investigator will strongly 
recommend the subject search for depression management centers and encourage them to speak 
to their primary care physician). If the BDI questionnaire is performed over the phone (visit 16) 
the same procedure described above will be performed over the phone by the research staff (the 
research staff will discuss with the medical coverage and follow his/her plan of action according 
to above while subject is on the phone). 
 
Beck Anxiety Inventory:  This self-report inventory consists of 21 multiple-choice questions 
about how the subject has been feeling in the last week, expressed as common symptoms of 
anxiety (such as numbness and tingling, sweating not due to heat, and fear of the worst 
happening). It is designed for an age range of 17–80 years old. Each question has the same set of 
four possible answer choices, which are arranged in columns and are answered by marking the 
appropriate one with a cross [40]. 
 
Mini Mental State examination (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. It will be 
used as a baseline evaluation [41]. 
 
Quality of Life Assessment (Short version of SF-36): The short version of the SF-36 health 
survey is used as a measurement of quality of life. It provides a profile of functional health and 
well-being scores. It is also used as a psychometrical index of physical and mental health. This 

Detailed Protocol 17 
instrument is widely used as a quality of life assessment in patients after an amputation and those 
suffering from phantom limb pain [42-45]. 
 
Stroop test: In this task the subject is presented with names of colors written in the same color or 
in a different color, thus on the one hand the word names a color (red) and is written in another 
color (blue). In this task, the automatized behavior (reading) is in conflict with the desired 
response (naming the color). The Subject has to inhibit/suppress the automatic response of 
reading and naming the color the word is written in. The Stroop is one of the most commonly 
used tools for determining attentional problems, also used to assess executive function and 
working memory [46, 47]. Stroop test will be applied as a preliminary safety assessment of 
cognitive changes from baseline to post and follow-up visits.  
 
Side Effects Questionnaire for tDCS: At each stimulation session, subjects will complete a 
questionnaire to evaluate potential adverse effects of tDCS (tingling, burning sensation, 
headache, neck pain, mood alterations) and mirror therapy (anxiety, grief, dizziness) on a 4-point 
scale (None, mild, moderate and severe). 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 on a 5-point scale. This type of adverse events questionnaire 
has been used frequently in our previous tDCS studies [31], including in patients with phantom 
limb pain [48]. 
 
Side Effects Questionnaire for TMS: At each TMS assessment session, subjects will complete a 
questionnaire to evaluate potential adverse effects of rTMS (headache, neck pain, itching and 
redness at the site of stimulation) on a 5-point scale [49]. 
 
tDCS blinding questionnaire: after the stimulation session, subjects will complete a 
questionnaire to determine if our blinding methods were effective. We are using a 30s sham 
montage, just as we use in our other trials, keeping the device on the subject for the duration of 
the session [50]. 
 
Patient’s Global Impression of Change scale: subjects will complete a questionnaire to assess 
their perception of change (if any) in the activity limitations, symptoms, emotions and overall 
quality of life after their participation in intervention’s visits of the trial [51]. 
 
Single and Paired Pulse TMS: Subjects will undergo several sessions of TMS to assess cortical 
excitability and cortical reorganization. For TMS assessment we will use Magstim BiStim² TMS 
device (Magstim ®). We will study the most distal muscle (i.e. biceps or quadriceps) of the 
amputated limb. In addition, if MEP is not obtained in the leg, the MEP will be assessed in the 
first dorsal interosseous. We will initially investigate changes in cortical excitability evaluating 
the motor evoked potential (MEP) and the resting motor threshold (MT); we will use the same 
methods as in our previous study [52], as well as short-interval intracortical inhibition (SICI) and 
intracortical facilitation (ICF) using the paired-pulse technique. We will investigate both 
contralateral and ipsilateral primary motor cortex (in relation to the amputated limb). We will 
investigate the resting motor threshold (MT), measured following the technique as described by 

Detailed Protocol 18 
Rossini et al., where resting MT will be definssed as the lowest stimulus intensity to evoke a 
MEP of 50 μV in five of 10 trials in the relaxed muscle [53]. We will record this in both 
primary motor cortices when possible. For the MEP study, we will initially adjust TMS intensity 
to achieve a baseline MEP in the selected muscle of about 1 mV peak-to-peak amplitude before 
intervention. Stimulation intensity will be kept constant for each subject throughout the 
evaluation sessions. The MEPs will be recorded and stored in a computer for off-line analysis. 
We will record 10 MEPs for each time point (immediately before and after the tDCS treatment 
for both motor cortices) and average their peak-to-peak amplitude and area-under the-curve. The 
short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) will be measured 
using the paired-pulse technique [54], in both primary motor cortices. A suprathreshold test 
stimulus adjusted to an MEP amplitude of 1 mV will be preceded by a subthreshold conditioning 
stimulus (CS; 80% MT) at interstimulus intervals of 2, 3, 6, 9, 10, and 12 ms, which samples 
inhibitory (2, 3ms, SICI) and excitatory (9, 10, 12 ms, ICF) windows, respectively. Ten stimuli 
will be applied at each interval in a randomized order. The percentage of inhibition or facilitation 
for each ISI before and after treatment will be calculated. Cortical mapping: eight stimulations at 
120 % stimulus of rMT intensity (posterior to anterior current) will be delivered to each of 
fifteen sites forming a 3 x 5 grid, with a constant 1.5 cm distance between sites, over the primary 
motor cortex (M1). At each stimulation site, the peak-to-peak amplitudes of the recorded motor 
evoked potentials (MEPs) will be measured and averaged offline. 
The map CoG will be computed for the medio-lateral (x) coordinates using the formula: CoGx = 
(Σxi *MEPi) / ΣMEPi where MEPi represents the mean amplitude of the MEPs produced at one 
site. The MV will be calculated as the sum of the average MEP amplitude at each active site, 
where an active site was defined as a site at which the mean MEP amplitude was at least 
0.05mV. 
 
Functional magnetic resonance imaging (fMRI): to quantify maladaptive cortical reorganization 
before and after treatment. Each fMRI exam will be performed at the Boston University Medical 
Center, Center of Biomedical Imaging; this is a fee-for-service, and they are not engaged in the 
research. The BU Medical Center has an extensive and expanding inventory of state-of-the-art 
imaging facilities. One of the core technologies being developed and used at the center are 
Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS). Additionally for this visit an 
fMRI survey  (BU Safety Screening Form )form will be filled out by the radiation technician or 
study investigator (NINDS CDE) [26]. 
We decided to conduct a longitudinal design sessions (baseline and post-treatment). To minimize 
the limitations of this longitudinal analysis we will use an algorithm of initialization in the 
FreeSurfer processing stream [55].  
 
The fMRI sessions will be conducted using the following stimuli and task detailed below and in 
Figure 2.  
 

Detailed Protocol 19 
 
 
Figure 2. The scheme shows tasks performed during fMRI sessions. 
 
We based this design in an experiment investigating activation of motor networks associated 
with observation and imagination of the lower limbs movements [56]. The stimuli will consist of 
the movement of the lower limb (non amputated limb), followed by the same movement but now 
the participant will be able to observe his/her leg moving and the leg image in the mirror through 
a online video.  
The following three conditions will be therefore investigated: 
1. Movement of the leg (MOV-LEG): the subjects will perform movements of the non 
amputated leg. Each participant will be instructed about the type and pace of the movements. 
2. Movement of the leg observing the mirror (MIR-LEG): the subjects will perform the same 
movements from the previous condition, looking the mirror image of the intact leg in an 
online video. Each participant will be instructed about the type, pace and video. 
3. Rest condition: the subjects will rest during this period and will be instructed to not perform 
any kind of movements during this period. 
 
Each of these conditions will have the same length – 20 seconds. The fMRI session will have  
four runs that will contain  6 blocks (6 of each condition). The order of the conditions will be 
randomized. For the analysis we will determine the ROI of interest (primary sensory motor 
cortex of the lower limbs) and first determine the ratio of activation between the MOV-LEG and 
MIR-LEG (in the respective ROI of interest – contralateral to the respective leg). Using this 
coefficient we will then compare across the different conditions. Our hypothesis is that the 
combined group (tDCS and mirror therapy) will have a greater activation than any therapy alone 

Detailed Protocol 20 
and also the no therapy group (sham tDCS and covered mirror). This effect will also be 
correlated with pain reduction. 
 
VI. 
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.  
 
Sample size calculation: 
To calculate the sample size for our primary outcome PLP as measured by a visual analogue 
scale (VAS), we used the study from Soler et al. as a reference ‘‘Effectiveness of transcranial 
direct current stimulation and visual illusion on neuropathic pain in spinal cord injury”. Using 
VAS as the basis for the calculation and considering results from this study: a mean and standard 
deviation of 5.2 (±1.5) in experimental group (tDCS + visual illusion) and 6.4 (±1.6) in control 
group (visual illusion), and using a bidirectional alpha of 0.05 and power of 80%, we would need 
a total sample of 108 subjects (27 subjects in each group). Additionally, estimating a 
conservative attrition rate of 20%, our sample size would be 132 subjects. 
 Although we calculated the sample for our primary aim, it is important to underscore that power 
calculation will be adequate also for our secondary aim measuring neurophysiological outcomes. 
In fact, neurophysiological outcomes usually have less variability thus need smaller sample sizes 
to show significant differences. In fact as shown in our preliminary data and also from data from 
other studies [58, 59] effect sizes from TMS data will be larger and for the fMRI, we expect 
BOLD changes in sensory motor cortex around 3 to 5% (as shown by previous studies [56]) – 
therefore a sample of 7 to 8 subjects will be enough to detect significant changes. 
 
Statistical Analysis 
 
For aim 1: 
The primary outcome is PLP indexed by VAS. PLP will be analyzed using intensity of pain over 
time. To analyze these data, we will initially build a mixed ANOVA model in which the 
dependent variable will be the outcome of PLP (such as VAS) and the independent variables will 
be group (active tDCS-MT; sham tDCS-MT; active tDCS and covered MT; sham tDCS- covered 
MT), time (baseline and after treatment and follow-up) and the interaction group*time. In 
addition we will add the random variable ID to account for within subject’s variability and the 
repeated measures on time. We will initially perform this full model and, subsequently, post-hoc 
comparisons with corrections for multiple comparisons will be carried out initially to test our 
main hypotheses. Furthermore we will test the correlation in improvement between outcomes 
using Pearson’s correlation tests. P-values for secondary and exploratory outcomes will be 
determined without corrections for multiple comparisons. Finally we will apply a path analysis 
[60, 61] to the primary outcome data to determine if pain reduction associated with the combined 
intervention (tDCS and MT) is due to direct effects versus indirect effects through improvement 
in secondary outcomes. We propose that a direct effect of tDCS and MT on pain can be assumed 
if the treatment effect cannot be explained by changes in psychological or functional outcomes. 
Statistical models for pain will be developed using covariates that include site center, baseline 

Detailed Protocol 21 
pain, psychological changes, functional changes, and the covariate treatment (main effect of 
treatment). To complete the path analysis separate regression models will be run to model the 
effects’ of treatment on each outcome alone including all the secondary outcomes. Analyses of 
the secondary outcomes will be conducted in an exploratory manner (no correction for multiple 
comparisons). For the intention-to-treat analysis, we will use a conservative method and assume 
that participants will not improve from the last measured point. But we will also perform a 
sensitive analysis for the missing data using other methods such as completers only.  
 
For aim 2: 
The secondary outcomes are other pain measurements, psychological, neuropsychological and 
quality of life measurements and neurophysiological markers (as indexed by TMS and fMRI). 
Functional neuroimaging data will be analyzed using both Freesurfer image analysis suite 
combined with the FS-FAST functional data analysis package (Massachusetts General Hospital, 
Charlestown, MA) (http://surfer.nmr.mgh.harvard.edu/) and with Brain Voyager QX 1.9 
software package (Brain Innovation, Maastricht, Netherlands). Briefly, a high resolution 
anatomical T1-weighted anatomical volume is used to reconstruct a cortical surface mesh and 
then inflated to allow viewing of sulcal activation. The functional preprocessing includes 3-D 
motion correction using the first volume alignment, high pass filtering to remove temporal linear 
trends, correction for slice time acquisition and spatial smoothing (Gaussian kernel, 5.0 mm 
FWHM). Subjects are screened for excessive motion (defined as a maximum tolerance of 3mm 
in any direction). Following co-registration of the preprocessed functional image with the high-
resolution anatomical, data is transformed into standard Talairach space. Voxel time courses for 
each subject are fit by a general linear model (GLM) for statistical analysis. Each experimental 
condition is modeled by a boxcar regressor matching the condition time course. These boxcar 
regressors are then smoothed by a canonical hemodynamic response function [62]. Individual 
subject maps for each contrast of interest is generated by projecting the volume of significance 
values resulting from the GLM onto the reconstructed cortical surface mesh for each hemisphere 
in each subject. Statistical significance is set at a standard threshold criterion of p<0.05 corrected 
for multiple comparisons using a cluster-size threshold adjustment [63]. 

Detailed Protocol 22 

Detailed Protocol 23 

Detailed Protocol 24 

Detailed Protocol 25 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Detailed Protocol 26 
 
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Detailed Protocol 27 
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Detailed Protocol 28 
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Detailed Protocol 29 
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