Spinal Excitability Changes and Transcutaneous Spinal Cord Stimulation in Lower Limb Amputees
Phase: NA
Conditions: Amputation, Phantom Limb Pain, Hoffman's Reflex
Sponsor: University of Pittsburgh

 
 
 
 
 
Neuromodulation in Lower Limb Amputees 
NCT 04543786 
Last document update: Mar. 17, 2022 
 
 

 
 
Background:  
Limb amputation results in an extreme form of peripheral nerve injury. Damage to peripheral 
nerves, such as with neuropathy, crush injuries, nerve transection, or limb amputation often results 
in chronic pain, which may be associated with altered excitability of spinal sensorimotor 
pathways. These spinal pathways become hyperexcitable due to a lack of sensory input, which 
causes tonic disinhibition of descending circuits and spontaneous activity in the dorsal root 
ganglia. Spinal excitability can be measured using the H-reflex, in which electrical stimulation of 
muscle spindle Ia afferents activates spinal motoneurons via the myotatic reflex, as well as the 
posterior root-muscle (PRM) reflex, which is elicited by transcutaneous stimulation over the dorsal 
roots and is considered to be half of the H-reflex, excluding the peripheral primary afferents, but 
with multiple root activation. Spinal excitability has not been measured in amputees but may offer 
a potential biomarker for phantom limb pain (PLP). Neuromodulation may restore normal spinal 
excitability and reduce PLP, thus offering the potential to improve the quality of life in individuals 
with a lower limb amputation. The results of this study will provide the foundation for future 
development of a neuroprosthesis to restore spinal excitability and reduce PLP in individuals with 
a lower limb amputation.  
Objectives:  
The overall goal of this work is to investigate the changes in the spinal cord resulting from limb 
amputation. Specifically, we measured spinal cord excitability using posterior root-muscle (PRM) 
reflexes in people with a transtibial amputation. We hypothesized that spinal reflexes would be 
hyperexcitable, indicated by lower thresholds to evoke the reflexes, because of the neuropathic 
pain state. We applied tSCS each day for 5 days, targeting the dorsal roots corresponding to the 
distal limbs. We hypothesized that, after 5 days of tSCS, spinal reflex hyperexcitability and PLP 
would decrease. 
Design:  
Participants underwent 5 testing and stimulation sessions in 1 week. 
We recruited individuals with a unilateral transtibial amputation and who experienced phantom 
limb pain. To measure spinal cord excitability, we studied PRM reflexes, which were recorded 
using electromyography (EMG) electrodes placed on the residual limb. We placed bipolar 
electromyography (EMG) electrodes on the lateral gastrocnemius (LG), medial gastrocnemius 
(MG), tibialis anterior (TA), and vastus lateralis (VL) muscles and a high-density EMG electrode 
grid across the putative gastrocnemius muscles. We positioned a ground electrode onto the 
patella of the residual limb. We recorded EMG data at a sampling rate of 4000 Hz and streamed 
the data into MATLAB. We delivered stimulation using a DS8R stimulator with a firmware update 
to allow frequencies up to 10 kHz.  
We attempted to elicit H-reflexes by electrically stimulating the tibial nerve of the residual limb. 
Stimuli consisted of a 1-ms long monophasic, cathodic, square wave pulse. We varied the 
stimulation amplitude to determine the thresholds for the M-wave as well as the maximum 
amplitude of the M-wave. We elicited the PRM reflex in the residual limb by electrically stimulating 
the spinal dorsal roots. We placed round adhesive electrodes paravertebrally of the T12-L1 
spinous processes. We placed return electrodes on each anterior superior iliac spine. We wrapped 

 
 
the participant’s torso using Coban wrap and placed a small piece of foam between the tSCS 
electrodes and the back of the chair. The stimulation pulses to evoke the PRM reflex were 1-ms 
long monophasic, cathodic, square wave pulse. For the purpose of evoking PRM reflexes in the 
residual limb, we stimulated through the tSCS electrode ipsilateral to the residual limb only. We 
determined the stimulation threshold for evoking a PRM reflex in the gastrocnemius muscles, 
followed by the maximum PRM reflex amplitude. The maximum PRM reflex amplitude was at 
either the stimulation amplitude past which the magnitude of the PRM reflex no longer increased 
or the maximum stimulation amplitude tolerated by the participant. In this study, we did not 
exceed a stimulation amplitude of 180 mA. We varied the stimulation amplitude to obtain 
recruitment curves for the M-waves PRM reflexes. Specifically, we stimulated 15 amplitudes 
between 5 mA below threshold and 10-15 mA above the M-wave maximum (or PRM reflex 
maximum) if tolerated or up to 180 mA in a random order. Each amplitude was repeated four 
times and stimuli were delivered 10 s apart. 
We performed the pain pressure threshold test using an algometer. The pain pressure threshold 
test measures the minimum amount of pressure that the participant can tolerate at a specific 
location. We pushed the rubber tip (1 cm diameter) of the algometer onto the skin over muscle 
(not pushing on bone) on several locations of both the residual and intact limbs. The participant 
reported when the pressure became painful, at which point we removed the algometer and 
recorded the pressure magnitude. 
We asked participants to rate their PLP in the last 24 hours using a visual analog scale (VAS) from 
0 and 10, where 0 indicated no pain at all, and 10 indicated the worst pain imaginable. Participants 
completed the short-form McGill Pain Questionnaire to describe their pain prior to participation in 
the study as well as throughout the week. The McGill Pain Questionnaire evaluates the sensation, 
temporal changes, and strength of pain. The total McGill Pain Questionnaire score indicates the 
intensity and affect the pain has on their life, and a 5-point change in McGill Pain Questionnaire is 
considered clinically meaningful. 
We delivered bilateral tSCS for neuromodulation continuously for 30 minutes, with a break at 15 
minutes to inspect the stimulation site. The tSCS consisted of 1 ms long pulses with a 10 kHz 
carrier frequency, delivered at 30 Hz. We started with a low amplitude of stimulation 
(approximately 10-20 mA) and slowly increased the stimulation beyond PRM reflex threshold, 
according to the comfort of the participant.  
All data were collected while the participant sat comfortably in a chair. At the beginning of each 
day, the participant rated their PLP over the last 24 hours using the VAS. We marked the location 
of the stimulation and EMG electrodes with a permanent marker to ensure consistent placement 
across the 5 days. Every day, we performed the M-wave and PRM reflex measures, followed by 
high-frequency tSCS for 30 minutes. At the beginning of the first and fifth days, the participant 
completed the McGill Pain Questionnaire, and we performed the pain pressure threshold test. 
 
 

 
 
Study Protocol for study using transcutaneous spinal cord stimulation in lower limb amputees 
 
 
 
 
 
Before participant comes in: 
1. AO0 = control port on stimulator 
2. PO0 = trigger port on stimulator 
3. AO1 = DIGI port on TMSi 
4. TMSi cables: HDEMG, 2x BPEMG, IMU 
5. PW on stimulator = 1000 us; 100%, 10 us; Monophasic, negative; 10 V = 180 mA 
6. Make sure Stimulator output is disabled 
 
Pain Assessments: 
1. Give participant MPQ, GQPLA  
Day 1: ☐   
 
 
 
 
Day 5: ☐   
2. Give participant VAS 
 
Day 1: ☐  Day 2: ☐  Day 3: ☐  Day 4: ☐  Day 5: ☐   
3. Use algometer to get pain threshold in the following regions: 
Residual limb 
Day 1 (lbf) 
Day 5 (lbf) 
Bottom of stump 
 
 
Anterior 5 cm above stump 
 
 
Posterior 5 cm above stump  
 
 
Medial 5 cm above stump 
 
 
Lateral 5 cm above stump 
 
 
Anterior 10 cm above stump 
 
 
Posterior 10 cm above stump 
 
 
Medial 10 cm above stump 
 
 
Lateral 10 cm above stump 
 
 
Mid-quad 
 
 
Mid-hamstring 
 
 
Intact limb 
 
 
On heel 
 
 
Ball of foot 
 
 
Top of foot 
 
 
Back of ankle 
 
 
Mid-shin (TA) 
 
 
Mid-calf 
 
 
Mid-quad 
 
 
Mid-hamstring 
 
 
Left arm 
 
 
Mid-biceps 
 
 
Right arm 
 
 
Mid-biceps 
 
 
 

 
 
Prep Participant: 
1. Prep skin on amputated limb 
2. Place and tape EMG electrodes 
a. HDEMG: SOL; BP1: LG; BP2: MG; BP3: TA; BP4: Q 
b. Orientation of grid: _____________________________ 
3. Place IMU on lateral side of stump 
4. Place tibial nerve electrodes 
5. Wrap the leg in Coban 
6. Place tSCS electrodes: 2 round adjacent to T12 (L1 = belly button, L4 = IC); 2 rect on ICs 
7. Turn on TMSi 
 
Record Impedances: 
1. Run reflex GUI (StimHreflexTMSiv15.m) 
2. Change name of file to “SubjectID_ DayX_Impedacnes_R1”, reset counter to 1 
Day 1: ☐  Day 2: ☐  Day 3: ☐  Day 4: ☐  Day 5: ☐   
 
tSCS threshold:  
3. Place foam behind participant’s back 
4. Make sure stimulator PW is 1000 us  
5. Change name of file to “SubjectID_DayX_tSCSthresh_ R1”, reset counter to 1 
6. Make sure GUI in PRM-reflexes mode 
7. Calibrate stimulator 
8. Enable stimulator  
9. Set stimulation amplitude to 10 mA 
10. Stimulate single pulses 
11. Increase in 5 mA increments until have PRM-reflex in 1 of 3 stimulations  
12. Note down tSCS threshold:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
 
tSCS PRM max: 
1. Change name of file to “SubjectID_DayX_tSCSmax_R1”, reset counter to 1 
2. Stimulate at 5 mA above threshold and observe PRM response 
3. Increase by 5-10 mA until have maximum amplitude 
4. Note down tSCS max:  

 
 
Day 1: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 2: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 3: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 4: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 5: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
5. Disable stimulator 
 
tSCS recruitment curve: 
1. Calibrate stimulator  
2. Enable stimulator  
3. Change name of file to “SubjectID_ DayX_tSCSRC_ R1”, reset counter to 1 
4. Set limits to below threshold and above max 
a. Amplitude range:  
Day 1: ______mA to ______mA, notes: __________________________________ 
Day 2: ______mA to ______mA, notes: __________________________________ 
Day 3: ______mA to ______mA, notes: __________________________________ 
Day 4: ______mA to ______mA, notes: __________________________________ 
Day 5: ______mA to ______mA, notes: __________________________________ 
5. Stimulate RC (default settings: 10 s between points, 4 reps each, 15 sweeps) 
*Note: this takes 10 minutes to complete – warn the participant 
6. 1.3x threshold: 
Day 1 = ______mA, Day 2 = ______mA, Day 3 = ______mA, Day 4 = ______mA, Day 5 = ______mA 
 
tSCS rate-dependent depression: 
1. Change name of file to “SubjectID_ DayX_tSCSRDD_R1”, reset counter to 1 
2. Set min and max amplitudes to 1.3x threshold 
3. Stimulate 5 pulses with the time between pulses equal to 10 s (0.1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
4. Stimulate 5 pulses with the time between pulses equal to 1 s (1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
5. Disable stimulator 

 
 
H-reflex threshold:  
1. Strap leg to put pressure on electrodes 
2. Change name of file to “SubjectID_ DayX_Hthresh_R1”, set counter to 1 
3. Make sure GUI in H-reflex mode 
4. Calibrate stimulator 
5. Enable stimulator 
6. Set stimulation amplitude to 5 mA, stimulate single pulse 
7. Increase in 1-5 mA increments until have H-reflex in 1 of 3 stimulations  
8. Note down H-reflex threshold:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
 
M-max on peripheral nerve: 
1. Change name of file to “SubjectID_ DayX_Mmax_R1”, reset counter to 1 
2. Set stimulation amplitude to 5 mA above H-reflex threshold 
3. Stimulate single pulse, observe motor response 
4. Stimulate at higher amplitudes until motor response no longer increasing 
a. Note: Pk-pk amp in EMGpk variable 
5. Note down M-max:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
6. Disable stimulator 
 
Peripheral recruitment curve: 
1. Calibrate stimulator 
2. Enable stimulator  
3. Change name of file to “SubjectID_ DayX_PeriphRC_R1”, reset counter to 1 
4. Set limits to below H-reflex threshold and above M-max 
a. Amplitude range:  
Day 1: ______mA to ______mA, notes: __________________________________ 
Day 2: ______mA to ______mA, notes: __________________________________ 
Day 3: ______mA to ______mA, notes: __________________________________ 
Day 4: ______mA to ______mA, notes: __________________________________ 
Day 5: ______mA to ______mA, notes: __________________________________ 

 
 
5. Stimulate RC (default settings: 10 s between points, 4 reps each, 15 sweeps) 
*Note: this takes 10 minutes to complete – warn the participant  
6. 1.3x threshold:  
Day 1 = ______mA, Day 2 = ______mA, Day 3 = ______mA, Day 4 = ______mA, Day 5 = ______mA 
 
Peripheral rate-dependent depression: 
1. Change name of file to “SubjectID_periphRDD_DayX_R1”, reset counter to 1 
2. Set min and max amplitudes to 1.3x threshold 
3. Stimulate 20 pulses with the time between pulses equal to 10 s (0.1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
4. Stimulate 20 pulses with the time between pulses equal to 1 s (1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
5. Disable stimulator 
6. Close reflex GUI 
 
tSCS neuromodulation: 
1. Ask participant if they need a break or to use the washroom 
2. Set stimulator pulse width to 40 us; output to biphasic 
3. Change control of output to the dial 
4. Connect bilateral stimulation lead wires 
5. Open neuromod GUI (tSCSguiControlStimv6.m) 
6. Enable stimulator 
7. Set duration to 15 minutes (900 s), start stimulation, with low amplitude set on the dial 
8. Slowly increase stimulation as they get used to the feeling and contractions 
9. Set amplitude to  
Day 1: ______________________________________________ mA (engagement of back muscles) 
Day 2: ______________________________________________ mA (engagement of back muscles) 
Day 3: ______________________________________________ mA (engagement of back muscles) 
Day 4: ______________________________________________ mA (engagement of back muscles) 
Day 5: ______________________________________________ mA (engagement of back muscles) 
10. Check skin under stimulation electrodes 

 
 
11. Begin second stimulation (15 minutes (900 s)) 
Day 1: ______________________________________________ mA (engagement of back muscles) 
Day 2: ______________________________________________ mA (engagement of back muscles) 
Day 3: ______________________________________________ mA (engagement of back muscles) 
Day 4: ______________________________________________ mA (engagement of back muscles) 
Day 5: ______________________________________________ mA (engagement of back muscles) 
12. Disable stimulator 
13. Disconnect bilateral tSCS lead 
 
 
REPEAT REFLEXES – Make new folder 
 
Record Impedances: 
1. Run reflex GUI (StimHreflexTMSiv15.m) 
2. Change name of file to “SubjectID_ DayX_Impedacnes_R2”, reset counter to 1 
Day 1: ☐  Day 2: ☐  Day 3: ☐  Day 4: ☐  Day 5: ☐   
 
tSCS threshold:  
1. Make sure stimulator PW is 1000 us, set to control mode 
2. Place foam behind participant’s back 
3. Change name of file to “SubjectID_ DayX_tSCSthresh_R2”, reset counter to 1 
4. Make sure in PRM-reflexes mode 
5. Calibrate stimulator 
6. Enable stimulator  
7. Set stimulation amplitude to 10 mA 
8. Stimulate single pulses 
9. Increase in 5 mA increments until have PRM-reflex in 1 of 3 stimulations  
10. Note down tSCS threshold:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
 
tSCS PRM max: 
1. Change name of file to “SubjectID_ DayX_tSCSmax_R2”, reset counter to 1 
2. Stimulate at 10 mA above threshold and observe PRM response 
3. Increase by 5-10 mA until have similar amplitude in response as M-max 
4. Note down tSCS max:  

 
 
Day 1: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 2: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 3: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 4: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
Day 5: occurred at ____________mA in ____________ muscle. Max defined by: ______________ 
5. Disable stimulator 
 
tSCS recruitment curve: 
1. Calibrate stimulator 
2. Enable stimulator  
3. Change name of file to “SubjectID_ DayX_tSCSRC_R2”, reset counter to 1 
4. Set limits to below threshold and above max 
a. Amplitude range:  
Day 1: ______mA to ______mA, notes: __________________________________ 
Day 2: ______mA to ______mA, notes: __________________________________ 
Day 3: ______mA to ______mA, notes: __________________________________ 
Day 4: ______mA to ______mA, notes: __________________________________ 
Day 5: ______mA to ______mA, notes: __________________________________ 
5. Stimulate RC (default settings: 10 s between points, 4 reps each, 15 sweeps) 
*Note: this takes 10 minutes to complete – warn the participant 
6. 1.3x threshold: 
Day 1 = ______mA, Day 2 = ______mA, Day 3 = ______mA, Day 4 = ______mA, Day 5 = ______mA 
 
tSCS rate-dependent depression: 
1. Change name of file to “SubjectID_DayX_tSCSRDD_R2”, reset counter to 1 
2. Set min and max amplitudes to 1.3x threshold 
3. Stimulate 5 pulses with the time between pulses equal to 10 s (0.1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
4. Stimulate 5 pulses with the time between pulses equal to 1 s (1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
5. Disable stimulator 

 
 
H-reflex threshold:  
1. Change electrodes to tibial nerve electrodes, put strap on the leg 
2. Change name of file to “SubjectID_ DayX_Hthresh_R2”, set counter to 1 
3. Make sure GUI in H-reflex mode 
4. Calibrate stimulator 
5. Enable stimulator 
6. Set stimulation amplitude to 5 mA, stimulate single pulse 
7. Increase in 1-5 mA increments until have H-reflex in 1 of 3 stimulations  
8. Note down H-reflex threshold:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
 
M-max on peripheral nerve: 
1. Change name of file to “SubjectID_DayX_Mmax_R2”, reset counter to 1 
2. Set stimulation amplitude to 5 mA above H-reflex threshold 
3. Stimulate single pulse, observe motor response 
4. Stimulate at higher amplitudes until motor response no longer increasing 
a. Note: Pk-pk amp in EMGpk variable 
5. Note down M-max:  
Day 1: occurred at ____________mA in ____________ muscle 
Day 2: occurred at ____________mA in ____________ muscle 
Day 3: occurred at ____________mA in ____________ muscle 
Day 4: occurred at ____________mA in ____________ muscle 
Day 5: occurred at ____________mA in ____________ muscle 
6. Disable stimulator 
 
Peripheral recruitment curve: 
1. Calibrate stimulator 
2. Enable stimulator  
3. Change name of file to “SubjectID_ DayX_PeriphRC_R2”, reset counter to 1 
4. Set limits to below H-reflex threshold and above M-max 
a. Amplitude range:  
Day 1: ______mA to ______mA, notes: __________________________________ 
Day 2: ______mA to ______mA, notes: __________________________________ 
Day 3: ______mA to ______mA, notes: __________________________________ 
Day 4: ______mA to ______mA, notes: __________________________________ 
Day 5: ______mA to ______mA, notes: __________________________________ 

 
 
5. Stimulate RC (default settings: 10 s between points, 4 reps each, 15 sweeps) 
*Note: this takes 10 minutes to complete – warn the participant  
6. 1.3x threshold:  
Day 1 = ______mA, Day 2 = ______mA, Day 3 = ______mA, Day 4 = ______mA, Day 5 = ______mA 
 
Peripheral rate-dependent depression: 
1. Change name of file to “SubjectID_periphRDD_DayX_R2”, reset counter to 1 
2. Set min and max amplitudes to 1.3x threshold 
3. Stimulate 20 pulses with the time between pulses equal to 10 s (0.1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
4. Stimulate 20 pulses with the time between pulses equal to 1 s (1 Hz) 
Day 1 notes: __________________________________ 
Day 2 notes: __________________________________ 
Day 3 notes: __________________________________ 
Day 4 notes: __________________________________ 
Day 5 notes: __________________________________ 
5. Disable stimulator 
6. Close reflex GUI 
 
Notes: 
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Statistical Analysis Plan 
We tested for normality using the Shapiro–Wilk test and assessed the homogeneity of variance 
using Levene’s test. A p-value < 0.05 was considered significant.  
Grouped threshold data contain the mean threshold across all channels of the HD-EMG grid. We 
performed a linear correlation between the PRM reflex threshold for each electrode on the HD-
EMG grid with its impedance value. We expressed reflex thresholds in units of charge (µC), 
obtained by multiplying the stimulation amplitude (in mA) by the pulse width (1 ms). To compare 
the PRM reflex thresholds over 5 days, we performed a one-way analysis of variance (ANOVA) 
with Bonferroni post-hoc tests. 
We created recruitment curves for the PRM reflexes, M-waves, and F-waves by plotting the mean 
peak-to-peak amplitude of the response from each electrode on the HD-EMG grid as a function of 
stimulation amplitude. The slope of the recruitment curve corresponded to the slope of the line 
between the inflection points across the steepest part of the curve.  
We determined the change in PPT over time by subtracting the PPT at each location on Day 1 
from Day 5. We normalized the PPT at each location to the maximum PPT value recorded for each 
participant. We expressed the change in PPT on a scale between -1 and 1, where -1 indicated a 
maximal decrease in PPT, 1 indicated a maximal increase in PPT, and 0 indicated no change in 
PPT. We also compared the average PPT across all tested locations on each limb between day 1 
and day 5 using a paired t-test.  
We used repeated measures ANOVA to compare the mean VAS score from all participants across 
the 5 days. A decrease in the VAS score by 50% and at least 1 point is considered clinically 
meaningful. We summated the responses from each subsection of the MPQ to obtain a total 
score. A clinically meaningful decrease in MPQ score is a 5-point decrease.