Evaluation of Motor Unit Abnormalities After Experimentally Induced Sensitization Using Capsaicin: A Randomized, Double-Blinded, Placebo-Controlled Study
Phase: PHASE4
Conditions: Central Sensitisation, Motor Disorders
Sponsor: Toronto Rehabilitation Institute

Official Title: Evaluation of Motor Unit Abnormalities after Experimentally 
Induced Sensitization Using Capsaicin: A Randomized, Double-Blinded, Placebo-
Controlled Study 
 
NCT Number: Not Assigned 
 
Date of Document: October 1st, 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Abstract 
Central sensitization is a condition that represents a cascade of neurological adaptations, resulting 
in an amplification of nociceptive responses from noxious and non-noxious stimuli. However, 
whether this abnormality translates into motor output and more specifically, ventral horn 
abnormalities, needs to be further explored. Twenty healthy participants aged 20-70 will be 
randomly allocated to topical capsaicin or a placebo topical cream which will be applied onto their 
left upper back to induce a transient state of sensitization. Visual analogue scale (VAS) ratings of 
pain intensity and brush allodynia score (BAS) will be used to determine the presence of pain and 
secondary 
allodynia. 
Surface 
electromyography 
(sEMG) 
and 
intramuscular 
electromyography(iEMG) will be used to record motor unit activity from the upper trapezius and 
infraspinatus muscles before and twenty minutes after application of capsaicin/placebo. Motor unit 
recruitment and variability will be analyzed in the sEMG and iEMG, respectively. An independent 
t-test will be performed on the data. This preclinical evidence may provide some explanation for 
the influence of central sensitization on changes in movement patterns that occur in patients who 
have pain encouraging of further clinical investigation.   
Keywords: 
Central 
Sensitization; 
Surface 
Electromyography; 
Intramuscular 
Electromyography; Motor Unit; Recruitment 
 
1. Introduction 
Chronic pain is characterized by pain lasting longer than three months [1-3]. It is a common 
condition affecting 1 in 5 people [4] and is rising due to the aging population and increased 
prevalence of comorbid conditions such as diabetes [5,6] and obesity [7]. Chronic pain produces 

a significant socioeconomical burden with the annual estimated cost in Canada exceeding 6 billion 
dollars [4,8-11]. As such, the assessment and diagnostic efficacy of chronic pain is of vital 
importance to improving its management throughout society.  
Central sensitization describes a state of neuronal hyper-excitability in the central nervous 
system that may occur due to malfunction of spinal and supraspinal pain facilitatory and inhibitory 
circuits resulting in amplification of somatosensorial responses [12]. Beyond somatosensorial 
changes, alteration in motor function can also be present with pain, and may be a reflex of 
neuromuscular function impairment [13,14]. A normal afferent input and normal central 
processing circuitry is essential to deliver normal efferent output. However, the influence of the 
changes that occur within the dorsal horn on the ventral horn remain largely ill defined [15,13].  
Motor unit assessment is crucial in evaluating diseases and abnormalities within the ventral 
horn [16]. Activity of the ventral horn, where anterior horn cells reside, is very important for motor 
unit activation [16]. Surface EMG (sEMG) [17] and intramuscular EMG (iEMG) [18] can be used 
to assess the neural drive to muscles, by recording motor units to understand the effects of central 
sensitization on motor control and the ventral horn. Based on Henneman’s size principle, motor 
units should be recruited in the same order, with smaller units being recruited first [19]. This 
principle presents an opportunity to investigate if central sensitization creates abnormalities on the 
motor unit level. 
Previously, central sensitization has been induced in healthy subjects to examine its 
neurophysiological effects via capsaicin [20-22]. Capsaicin, a chilli pepper extract, can be used to 
effectively induce experimental transient states of central sensitization. [23-25,21,22]. The 
presence of expanded sensorial responses and the involvement of the spinal nociceptive system 

post capsaicin have been largely tested by means of quantitative sensory testing methods and 
electromyography (EMG) [25,21,22,24].  
Despite the usefulness of experimental capsaicin to better understand the sensorial 
abnormalities, its impact on motor function and motor unit recruitment are lesser studied. Evidence 
suggests that nociceptive input by peripheral capsaicin exerts a centrally-mediated inhibitory effect 
on motor function [26]. A decrease in root mean squared (RMS) amplitude during exercise at the 
time of peak sensitization was measured by needle EMG [26]. However, the effect that capsaicin-
induced sensitization has on individual motor units or on their recruitment patterns has not been 
previously examined. 
The purpose of this study is to determine whether topical capsaicin-induced sensitization has 
any influence on ventral horn activity. We hypothesize that capsaicin induces a change in 
individual motor unit activity, as well as the recruitment pattern of many motor units, and may 
affect motor unit activity at different segmental levels from the level of capsaicin application.  
2.0 Methods 
2.1 Study Participants 
Ethical approval was obtained from the Ethics Board of the Toronto Rehabilitation Institute, 
University of Toronto prior to commencing the study. This was in accordance with the World 
Health Organization statement of ethical principles for medical research involving human subjects 
[27].  
Twenty-three healthy participants, age between 20-70 years old, with no direct trauma to 
cervicothoracic region within the past 30 days, no past medical history of inflammatory disorders 
as rheumatoid arthritis, no neurodegenerative disorders such as Parkinson's disease nor motor 

neurone diseases as amyotrophic lateral sclerosis, or other neuromuscular disorder will be recruited 
for this study. Also, we will include subjects that have a normal body mass index (18.5 – 24.9) 
[28] and have a pain visual analogue scale (VAS) below 3 indicating low pain severity [29]. Since 
prevalence of neck pain in the general population is high, mild pain or aches are not necessarily 
related to an abnormality of the underlying muscle [30]. Participants have to be able to 
communicate in English. Participants will be excluded if they had persistent pain for more than 3 
months. This will be determined by the physician on our research team (DK). 
 
2.2 Dataset Collection 
An initial screening will be performed to assess eligibility in the study. Each participant will 
be seated upright with their hands comfortably on their lap and asked to relax their neck and 
shoulder muscles. The physician member of the research team will then assess the patients’ pain 
intensity by visual analogue scale (VAS). VAS ranges from 0 to 100 mm which 0 mm reflecting 
no pain at all and 100 mm representing the worst imaginable pain [29]. Following this, brush 
allodynia, a clinical technique used to identify pain due to a stimulus that does not normally 
provoke pain, will be performed to confirm presence of central sensitization [12]. To map out 
borders of secondary allodynia, subjects will be instructed to recognize a distinct alteration in the 
sensation perception such as increased burning, intense pricking, or an unpleasant sensation, and 
that location will be marked [31]. Brush allodynia score (BAS) will be calculated as the distance 
between the farthest points marked on the superior and inferior axis multiplied by the distance 
between the farthest points marked on the medial and lateral axis as previously described by 
Cavallone et. al. [31]. The VAS and the presence of central sensitization by means of BAS will be 
assessed at baseline (pre) before the induction of sensitization and twenty minutes after (post).  

Upon successful screening of inclusion and exclusion criteria, participants will have their left 
side area of skin (overlying the upper trapezius and infraspinatus muscles) cleansed with alcohol 
preparation pads and water. The skin will be abraded with ‘3M Red Dot’ abrasive strips before 
application of the surface electromyogram (Trigno Galileo sensors, Delsys Inc.). Electrodes will 
be placed in 4 areas: the muscle belly of the upper trapezius, and the infraspinatus, as well as 
reference electrodes on C7 and the acromion. These electrodes are 4 channel EMG sensors and 
have their signals filtered from 20 – 450 Hz. The sEMG recordings are wirelessly transmitted to 
the Trigno base station, which relays and compiles the data to Neuromap (Delsys Inc.) for signal 
analysis. A monopolar needle electrode will be inserted into the upper fibers of trapezius muscle 
and its reference will be placed at the mid-clavicle point. Using this setup intramuscular recordings 
of single motor units will be performed using an Excaliber, Natus Medical clinical 
electrodiagnostic machine. 
2.2.1 Electromyograms recordings before application of intervention 
Participants will then be instructed to perform horizontal shoulder abductions from 0° to 90° 
and then from 90° to 0° for 1 minute. The study subject will be verbally cued to move their arm 
every 2 seconds. sEMGs were recorded during this time. Upon completion of this task, a 
monopolar intramuscular needle electrode will be placed directly into the upper trapezius muscle. 
Participants will then be instructed to gently contract their trapezius muscle by shrugging their 
shoulder, enough to recruit only the first motor unit in that region. Visual feedback of the signal 
will be given to the subject to ensure that only the first motor unit was activated for the movement. 
iEMG recordings will be recorded for 30 seconds at a sampling frequency of 6 kHz. 
2.2.2 Application of intervention 

Participants will receive either a dose of 2.5 ml (75µg/ml) capsaicin cream (treatment, 
Zostrix brand) or skin lotion (placebo) which is inert and causes no sensitization effects. 
Participants will also blinded to the delivered treatment, using concealed containers for the creams. 
The location of application will be a 10 cm by 10 cm square on trapezius muscle which extends 
from T3 to T8 on the left side that all recordings were conducted. 
After collection of the baseline sEMG and iEMG recordings, a trained medical professional 
will apply the capsaicin / placebo cream directly to the region of skin in a standardized 10 cm x 10 
cm square at the spinal levels T3-T8, to sensitize the nociceptive afferents within that region. A 
twenty-minute waiting period will be used to enable the sensitizing effects of capsaicin to take 
effect.  
 
2.2.3 Electromyograms recordings after application of intervention 
To confirm the presence of central sensitization, brush allodynia will be used to detect 
mechanical allodynia outside the region of the primary nociception – region of topical placement 
– which is the region of secondary allodynia [25]. Upon confirmation of central sensitization, in 
participants with application of topical capsaicin, participants will be entered into the experimental 
arm of the study. 
2.3 Study Procedures for EMG Analyses 
This section outlines the procedures for the EMG analysis. For the sEMG data, the pre- 
recording motor units will be matched with an algorithm implemented in MATLAB (Mathworks, 
version 2018a), described in section 2.3.1, in order to determine the aberration of recruitment 
pattern after treatment.  

For the iEMG data, the pre- and post-recording motor units will also be matched using an 
algorithm implemented in MATLAB (Mathworks, version 2018a), described in 2.3.2. The 
variability of that motor unit shape will be compared before and after capsaicin and between 
groups. 
 
2.3.1 
 Surface EMG  
Recorded sEMGs will be analyzed in Neuromap [32], which provides an average template, 
average amplitude, and order of recruitment of each motor unit detected and isolated from the raw 
signal using Delsys’ motor unit decomposition algorithm [33]. Each motor unit template will then 
be analyzed based on their “overall match” in shape and amplitude. 
Shape Analysis 
The motor unit signal that will be obtained from M electrode contacts at T consecutive time 
samples will be assembled into an M x T matrix. This matrix will then be compressed into a single 
vector to create a motor unit “signature” [34]. This will be done by taking the recordings from all 
electrode contacts at a single time step and concatenating it to the recordings from all contacts at 
the next time point. This concatenation will be repeated to the time T to create the signature (Figure 
1). This process will be performed for the pre- and post-recording motor units for each participant.  
The shape of each detected pre-recording motor unit will be compared to the shape of each 
post-recording motor unit via the cross-correlation function, xcorr, in Matlab (Mathworks,2018a). 
This function returns a normalized value between 0 – 1 between each pre- and post-recording 
motor unit. Cross-correlation values between pre- and post-recording motor unit pairs that are 

below a preset threshold of 0.8 [35] will be set to 0 as the pair is unlikely to be from the same 
motor unit. 
Amplitude Analysis 
The motor unit average amplitudes obtained from the Neuromap software will be used for 
calculating an amplitude ratio between pre- and post-recording motor units. This amplitude ratio 
will be calculated for each pre/post motor unit pair to determine similarities in the amplitude. 
Similar to the shape analysis, amplitude ratios that are below a preset threshold of 0.8 or above 1.2 
are set to 0. This ratio will be normalized to be within a scale from 0 – 1 to allow for comparison 
between shape and amplitude using the following equation (Eq.1) when the amplitude ratio is 
above one. 
 
 
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝐴𝑚𝑝. 𝑅𝑎𝑡𝑖𝑜 =  1 −𝐴𝑚𝑝. 𝑅𝑎𝑡𝑖𝑜 
(1) 
 
 The numbers from the shape analysis and amplitude analysis, which are not set to 0, will be 
averaged together to express an “overall match” between pre- and post- motor units from each 
participant. As mentioned, based on Henneman’s size principle, motor units should be recruited in 
the same order with smaller units being recruited first and subsequent motor units are recruited as  
more force is needed [19]. To observe whether this principle is upheld or violated, the 
reorganization of the recruitment will be identified by how much earlier or later the similar motor 
units were recruited. In order to quantify the recruitment, the difference in the order of recruitment 
between the pre and post recording will be calculated. Recruitment order of motor units will be 

determined through a custom automated search algorithm (Algorithm 1), which will determine the 
best match between pre- and post-recording motor units.  
Algorithm 1: Automated Motor Unit Matching method 
Input: Matrix of averaged values from shape and amplitude analysis 
Output: Matched pre-post motor units 
1: 
   loop through each pre-motor unit (row) and each post-motor unit (col)   
2: 
   if value at current location is the highest in that row and column, and the number   
is not equal to zero 
3: 
      Record match between the pre- and post- motor unit, and set the row and col  
 
      to 0 (i.e. remove the pre- and post- motor unit from further pairing) 
4: 
Return matched pre-post motor unit pairs 
 
 
 
 
The average recruitment order difference for each motor unit will be obtained for each 
participant. Following this, for each participant, the recruitment difference for all of their motor 
units will be averaged, to obtain one vector of average recruitment changes per person. This final 
matrix containing numbers on a continuous scale, will be used for statistical testing. See Appendix 
A for a detailed explanation of the matrix set up.  
2.3.2 Intramuscular EMG analysis using template matching 
Recorded iEMGs will be thresholded using the median absolute deviation estimate approach 
(Eq.2) to find the peak location of motor unit action potentials (MUAPs) [36]. 

 
 
𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑=  10 × 𝑀𝑒𝑑𝑖𝑎𝑛(|𝑋|)
0.6745
 
(2) 
 
Each detected peak, along with 12.5 ms before and after will be stored and used for analyses. 
These detected MUAPs will then be clustered to determine the number of motor units present in 
the recording. In particular, the first detected peak and its surrounding environment (i.e. the 
MUAP) forms the first motor unit template. Subsequent detected MUAPs will be compared to the 
first template using cross correlation. If the cross correlation is more than 0.85, it will be 
determined as the same MUAP and added to that motor unit cluster and the template will be 
updated. However, if the cross-correlation is less than 0.85, it will be classified as a different motor 
unit and a new template cluster will be created for comparison.  
This process will be repeated until all of the detected MUAPs in the pre- and post-recording 
are classified. The MUAP cluster with the highest number of MUAPs, for the pre-recording, will 
be compared with all MUAP clusters found in the post-recording for each participant. The MUAP 
cluster in the post that has the highest cross-correlation value will be considered “matched.” 
Participants that do not have matched MUAPs with cross-correlation values above or equal to 0.95 
will not be used for statistical analysis. 
2.4 Statistical Methods 
2.4.1 Surface EMG 
An independent samples t-test will be performed on the difference in recruitment position 
using treatment as the factor for the trapezius muscle. In particular, the statistical test will compare 

post-pre capsaicin recruitment differences against post-pre placebo recruitment differences. The 
average difference in recruitment order will be used to avoid bias of having multiple data points 
per participant that correspond to different motor units.  
 
2.4.2 Intramuscular EMG 
From the intramuscular EMG algorithm mentioned in section 2.3.2, the resultant matrices will 
be the variances for the pre-recording motor unit trains. The statistical model used will assess the 
difference between the pre- and post- recording motor units for each capsaicin and placebo as well 
as a between group comparison (Post-Pre capsaicin motor unit variance vs. Post-Pre placebo motor 
unit variance). These analyses would provide insight into our hypothesis that sensitization induced 
by capsaicin effects upon the motor unit characteristics. Either an independent samples t-test or a 
Kruskal-Wallis H test will be performed, depending on how many participants’ pre and post motor 
units match up. 
3 Sample Size Calculation 
The minimum sample size required per group to detect whether a significant difference exists 
between two means for one dependent variable was calculated with a confidence level of 95%, a 
80% statistical power level and 5% error. A mean detectable difference of 5 for the dependent 
variable, mean motor unit recruitment change, and an estimated standard deviation of 3 was used. 
The estimated sample size was 7 people per group; however, considering a 20% drop out rate, the 
suggested sample size was 9 people per group. This analysis was completed using PASS Software, 
version 15.  

 
4 Conclusions 
This may provide some insights for the neurophysiological influence of central sensitization 
on changes in efferent responses and movement patterns that occur in patients with persistent pain. 
However, the clinical implication of this experimental findings on the movement properties needs 
to be further investigated. 
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Figure 1: Process of creating the motor unit signature from a set of recordings. Each column of 
the matrix corresponds to the recordings from all electrode contacts at a single time step up to the 
time T. The columns are then concatenated to create a single vector corresponding to motor unit 
signature. 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
APPENDIX A 
 
MUPRE MUPOST 
Difference 
Array 
1 
9 
8 
2 
8 
6 
4 
13 
9 
5 
16 
11 
6 
15 
9 
8 
19 
11 
14 
18 
4 
Supplementary Figure 1: Pre/Post Difference 
In order to compare the reorganization in recruitment, the difference in order of recruitment was 
calculated and put into an array. This difference array was calculated for each person, and an 
average difference in recruitment per person was analysed using a t-test. The reason for having 
an average per person was to avoid any bias by having multiple data points per person that 
correspond to different motor units. Assumptions of normality and homogeneity of variances 
were verified from the Shapiro- Wilk test (p=0.187) and Levenes Statistic(p=0.081), therefore a 
t-test can be used. 
 
Average Difference per person (for T test USE): 
= 𝐴𝑣𝑒𝑟𝑎𝑔𝑒(𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒𝑠)𝑝𝑒𝑟 𝑝𝑒𝑟𝑠𝑜𝑛 
= (8) + (6) + (9) + (11) + (9) + (11) + (4)
7
 
= 8.286