Quantitative Response of Healthy Muscle Following the Induction of Capsaicin
Phase: EARLY_PHASE1
Conditions: Central Sensitisation
Sponsor: Toronto Rehabilitation Institute

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Quantitative Response of Healthy Muscle Following the Induction of Capsaicin: An 
Exploratory Randomized Controlled Trial 
Valerie Evans, MASc (C)1,2, B.Eng; Michael Behr, MASc, B.Eng1,3; Kei Masani, PhD, MEd, 
BEd2,3; Dinesh Kumbhare, MD, PhD, FRCPC, FAAPMR1,2,3 
 
1Department of Medicine, Division of Physical Medicine and Rehabilitation, University of 
Toronto, Toronto, Ontario, Canada  
2Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, 
Toronto, Ontario, Canada 
3Toronto Rehabiliation Institute, Universtiy Health Network, Toronto, Ontario, Canada 
 
 
 
Date: June 25,2019 
 
 
Corresponding Authour: 
Dinesh Kumbhare, MD, PhD, FRCPC, FRCPC, FAAPMR 
Associate Professor, Department of Medicine 
Division of Physical Medicine and Rehabiliation 
University of Toronto 
Toronto Rehabiliation Institute 
550 University Ave. 
Toronto, ON M5G 2A2 
P: 416-597-3422 x 4612 
Email: dinesh.kumbhare@uhn.ca 

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Abstract 
 
Background: Myofascial pain syndrome (MPS) is a prevalent chronic pain disorder primarily 
characterized by myofascial trigger points (MTrP). There is limited knowledge on the 
pathophysiology and mechanisms underlying MTrP and its development. Research has 
previously demonstrated the identification of MTrPs using ultrasound and vibration 
sonoelastography, although there is some contradictory evidence regarding if MTrPs present as 
hyper or hypoechoic regions. Electromyography (EMG) investigations of MTrP have 
demonstrated that MTrP are usually located proximal to innervation zones where the peak 
surface EMG signals are obtained from. Central sensitization has been proposed as the primary 
mechanism underlying MTrP development. Central sensitization is associated with 
hyperexcitability of neuronal responses to normal or noxious stimuli. There is a need for a study 
that measures ultrasound image textural changes and motor unit activity responses in the muscle 
following sensitization. The purpose of this study is to determine whether sensitizing healthy 
muscle using capsaicin induces a regional change in image texture variables within the specific 
and surrounding muscles, as well as the motor unit frequency and amplitude changes  that 
accompany them.This is an exploratory trial that aims to provide preliminary evidence on 
whether central sensitization is a direct cause of taut band and MTrP development.  
Methods: The study conforms to the Consolidated Standards of Reporting Trials 
recommendations. Ethical approval will be sought from the University Health Network (UHN) 
Research Ethics Board. This proposed study is a single centered, factorial, randomized placebo-
controlled trial with two independent variables, depth of capsaicin application and dose of 
capsaicin, for a total of four treatment arms and two control treatment groups.  

3 
 
Discussion: This will be the first study that assesses the B-mode ultrasound image texture of 
induced sensitized muscles, and will provide more evidence on muscle motor unit activity and 
regional changes of central sensitization. Findings from this study may support one of few 
hypotheses proposed delineating the involvement of central sensitization in the development of 
trigger points.  
Trial Registration:  
Name: National Institutes of Health ClinicalTrials.gov. 
Identifier: NCT03944889  
Date of Registration: May 07 2019  
URL: https://clinicaltrials.gov/ct2/show/NCT03944889 
 
 
Keywords: myofascial pain syndrome; ultrasound; image texture; electromyography; central 
sensitization; trigger points 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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List of Abbreviations: 
US: Ultrasound 
EMG: Electromyography 
MPS: Myofascial pain syndrome 
MTrP: Myofascial trigger points 
SEA: Spontaneous endplate activity 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Background 
Myofascial pain syndrome (MPS) is a prevalent chronic pain disorder primarily 
characterized by myofascial trigger points (MTrP). MTrP are stiff taut bands of muscle 
distinguished by hypersensitivity on palpation and local twitch responses on snapping 
palpation.[1] A current clinical challenge in chronic pain care, applicable to myofascial pain, is 
early detection and effective treatment targeting the underlying pathophysiology.  
There is limited knowledge on the pathophysiology and mechanisms underlying MTrP 
and its development. Ultrasound studies have been able to characterize trigger points[32,45,46], 
however there are some contradictory findings on whether these areas are hyperechoic or 
hypoechoic[45,46]. More recently, texture feature analysis have been shown to differentiate 
between healthy and MPS as well as active and latent trigger points[32].  Trigger points are 
typically located within the region of the muscle belly, where there is a high distribution of 
motor endplates.[2,3] Seventy-one percent of acupoint locations in the body, which are regions 
associated with motor points, motor endplates, or major motor nerve pathways overlap with 
common MTrP.[4]  
Electromyography (EMG) investigations of MTrP have demonstrated that MTrP are 
usually located proximal to innervation zones where the peak surface EMG signals are obtained 
from.[3] Motor endplates at MTrP present with spontaneous endplate activity (SEA) associated 
with excessive acetylcholine release.[5,6] In latent and active trigger points, SEA is 
characterized by continuous low amplitude action potentials. Active MTrP, which are more 
painful clinically, elicit intermittent spikes in addition to SEA. This continuous activity is 
thought to overexert and hyper contract the muscle resulting in the presentation of a taut band.[7] 
Studies assessing the contractility of MTrP have found increased muscle fatigability in active 

6 
 
and latent MTrP relative to unaffected muscles. Furthermore, it has also been demonstrated that 
there is increased fatigability and EMG activity in active MTrP relative to latent ones.[8] This 
data suggests that perturbations in the efferent system involved in muscle control is a vital 
component of the MTrP pathophysiology.  
Central sensitization has been proposed as the primary mechanism underlying MTrP 
development. Central sensitization is associated with hyperexcitability of neuronal responses to 
normal or noxious stimuli.[9] Furthermore, this concept offers an explanation of the afferent, 
efferent and abnormal signal modulation at the spinal cord level.[9,10] Patients with myofascial 
pain present with hyperalgesia and decreased pain threshold, i.e., signs of central sensitization, 
particularly at the region of MTrP.[5,11] Experimental evidence has demonstrated that afferent 
nociceptive fibers at the region of MTrP are sensitized. Neurons in the dorsal horn segment 
corresponding to the location of MTrP are also sensitized, manifesting clinically as hyperalgesia 
within the dermatome affected by MTrP.[12] This is evidenced by Kim et al’s[12] findings 
whereby they reported transcutaneous electric stimulation increased MTrP pressure pain 
thresholds when applied at a remote region within the same dermatome. Srbley et al.[1] further 
presented evidence of a sensitization arc that maintains the MTrP contracture. They 
experimentally induced sensitization at a remote region in the dermatome wherein a latent MTrP 
is located using topical capsaicin, and subsequently found a decrease in pressure pain thresholds 
at the MTrP.  
Investigations assessing neuronal responsivity to noxious stimuli have demonstrated 
increased sensitization at the region of primary nociception and the development of a secondary 
nociceptive field.[13-15] This has been confirmed using EMG and quantitative sensory testing 
methods to assess the activity of sensory nociceptive, mechanoheat, and chemoreceptive nerve 

7 
 
fibers following the injection or application of capsaicin.[13-15]. Many other factors are likely 
involved in the chronic pain patient including disordered ascending and descending signals 
within the central nervous system, termed nocioplastic pain by the IASP.[18,19,49]  Dideriksen 
et al.[16] and Falla et al.[17] demonstrated that motor unit potentials reduce in amplitude 
following the injection of nociceptive hypertonic saline into the upper trapezius muscle. This is 
in line with the EMG characteristics of MTrPs cited earlier.[7,8] However,  other studies, such as 
the study directed by Birch et al. (2000), present contradictory results, indicating that no 
significant differences in motor unit discharge rates were found. Studies assessing the EMG 
activity of trigger points failed to locate the aberrant activity that characterizes MTrP using 
surface EMG but were able to demonstrate the presence of SEA, intermittent spikes in active 
MTrP, as well as activity associated with local twitch responses using intramuscular EMG 
recordings due to the method’s specificity.[5]  
 
In regards to MTrP structure, Shankhar et al. (2012) was able to identify taut bands of 
MTrPs using 3D and 2D ultrasound[47]. They discovered that the muscle fascicles surrounding 
the taut band presented a different appearance than the adjacent healthy muscles. The study 
identified MTrPs as hyperechoic regions, as in another study confirmed by Lewis et 
al.(1999)[46]. In contrast to these studies, Sikdar et al. (2008) noted that MTrPs appear as 
hypoechoic elliptical reigions within the ultrasound image[48]. Although there is some evidence 
on the changing structure of myofascial trigger points, to the best of our knowledge, there has 
been no evidence of how central sensitization affects muscle structure through image analysis. 
There is a need for a study that measures specific B-mode ultrasound textural changes within 
sensitized areas, as well as motor unit activity responses in the muscle following sensitization.  

8 
 
The purpose of this study is to determine whether sensitizing healthy muscle using 
capsaicin, a chilli pepper extract, induces a regional change in ultrasound texture features of the 
targeted muscle, or of the muscles in that are in close proximity. This regional change will be 
accompanied by electromyography (EMG) recordings to confirm the presence of 
abnormality. Central sensitization will be induced using topical capsaicin and injectable 
capsaicin at three different concentrations. This is an exploratory trial that aims to provide 
preliminary evidence on whether central sensitization is a direct cause of taut band and MTrP 
development.  
Our specific research questions are followings: 
a. Does sensitization cause a significant regional change in the texture features of the 
targeted muscle or surrounding muscles? Our hypothesis is that that there will be a 
structural change within the sensitized or adjacent muscles. 
b. Does capsaicin induced sensitization elicit a change in motor unit action 
potential(MUAP) amplitude by approximately 20 to 25% within the region of 
experimentally induced central sensitization or in the region of texture feature changes? 
Our hypothesis for this portion of the study is that sensitization modifies the anterior horn 
cell activity which will be measured by the amplitude of the potentials and the change in 
texture features.  
c. Does capsaicin induced sensitization elicit continuous electrical activity as observed in 
MTrPs? This tests the hypothesis that sensitization creates the presence of continuous low 
amplitude action potentials. 
d. Does capsaicin induced sensitization influence the rate of recruitment of motor units in 
the muscle within the region induced? Does this occur within the regions identified by the 

9 
 
changes in texture features? The recruitment of motor units is normally expected to 
follow the Henneman size principle.[20-23] Our hypothesis for this portion of the study is 
that sensitization will cause an aberration in recruitment. If this is demonstrated,  then 
there is modification of the normal processing at both dorsal and ventral horns of the 
spinal cord. 
e. Is there a location dependent EMG response to capsaicin induced sensitization? In other 
words, do the texture features or EMG responses from muscles that lie anatomically 
distant from the location of the site of intramuscular capsaicin injection have changes 
from their baseline calculations? This would test the hypothesis that type III and IV 
afferents have an influence on the anterior horn cells that lie distant to the ones that are 
supplying muscle fibers in the vicinity of the stimulated sensory afferents. 
f. Is there a dose dependent EMG response of motor units to capsaicin induced 
sensitization? Here, the hypothesis of causality will be assessed in a preliminary manner. 
If there is a relationship then further experiments that more thoroughly assess causality 
will be needed. 
Methods & Design  
Study Design: 
The study conforms to the Consolidated Standards of Reporting Trials recommendations. 
Ethical approval is approved by the University Health Network (UHN) Research Ethics Board. 
This study is registered under the National Institutes of Health ClinicalTrials.gov. 
This proposed study is a single centered, factorial, randomized placebo-controlled trial 
with two independent variables, depth of capsaicin application and dose of capsaicin, for a total 
of nine treatment arms. The first between groups variable will be topical capsaicin application 

10 
 
and injectable capsaicin injection. Within each partition there will be three treatments: control, 
50 micrograms, 100 micrograms. One-hundred micrograms reported to be an effective high dose 
of capsaicin.[13] The control group will receive a topical skin lotion which is inert and has no 
sensitization effect. An equal number of participants will be allocated to each of the six treatment 
groups using an electronic randomization generator. Block randomization will be used to ensure 
equal allotment into each group. An alternate member of the research team will conduct the 
randomization schedule a priori. Participant allocation will be concealed by placing their 
assignment in an envelope and delivered to participants by the same member of the research 
team uninvolved in the measurement or randomization protocols. Participants and investigators 
will be blinded to the delivered dose; however, the type of capsaicin delivery cannot be blinded 
from either participants or investigators. The member of the research team conducting the 
randomization schedule and concealing allocation will have knowledge of and keep track of the 
doses contained in the containers and vials of the topical and injectable capsaicin, respectively. 
They will deliver the appropriate dose to the team member implementing the experimental 
protocol to ensure there is blinding with respect to dose. This individual will not be involved in 
participant recruitment.  
 
Participants 
Recruitment 
Participants will be recruited from UHN and the University of Toronto clinics, 
employees, volunteers, visitors, students, and external participants. UHN is a tertiary healthcare 
centre with eight hospitals located in Toronto, Canada and the University of Toronto is an 
academic institution with a main campus located in close proximity to the main UHN hospitals 

11 
 
in Toronto. Advertisements, flyers, and in person recruitment will be employed to collect the 
necessary sample size for the study. The recruitment procedures will run in line with the 
approved guidelines from the UHN research ethics boards. Participants will not be coerced into 
participating and will be informed that they have the right to withdraw at any time during the 
experimental procedures. They will also be informed they have the right to withdraw their data 
prior to publication. Participants will be briefed and consented on recruitment prior to 
commencing the study procedures. Participant treatment allocation will be recorded on a separate 
document until all of the data collection is complete and the data is analyzed. 
Eligibility Criteria 
Female or male participants who meet the following inclusion criteria will be included in 
the study: (1) participants are healthy with no past medical history (2) a visual analogue score 
below 3 indicating low pain severity, however, ideally who complain of no pain (3) right or left 
handed, (4) normal body mass index, (5) have sufficient command of the English language to 
provide informed consent and to understand the study protocols, (6) participants agree to sign a 
consent to volunteer for the research.  
Exclusion Criteria 
Participants who meet one or more of the following criteria will be excluded: (1) history 
of pain, (2) physical examination detection of myofascial trigger points, (3) participants present 
with a history of pain related disturbances such as poor sleep, cognitive disturbances, psychiatric 
disorders, (4) history of general medical disorder that may affect the outcome of the study such 
as diabetes mellitus, (5) and a history of cervical radiculopathies or (6)  history of inflammatory 
arthropathy.  
 

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Experimental Protocol 
This experimental protocol will be carried out at the Kumbhare Lab, Toronto 
Rehabilitation Institute to ensure any medical emergencies can be promptly cared for. Following 
the completion of the preliminary intake forms and participant debriefing, informed consent will 
be obtained. Each participant will be seated upright with their hands comfortably on their lap in a 
chair that has a high supportive back. They will be asked to relax their neck and shoulder 
muscles. A member of the research team will then apply the inclusion and exclusion criteria. The 
presence of a MTrP will be assessed by palpation of the upper trapezius muscle since this is the 
current method utilized in clinical practice. If there is a MTrP, then they will be asked to 
withdraw from the study since they do not satisfy the inclusion and exclusion criteria.  
Participants will sit on a chair with one arm extended downward, and with the other arm 
on an armrest of the chair. The extended arm will be fixed by a wristcuff and a chain down to the 
floor, with a loadcell between the floor and the end of chain. The loadcell will measure the 
exerted force. The anatomical location for ultrasound probe and electrode placement will be 
identified for each participant. The ultrasound probe will be placed on 3 muscles: the trapezius, 
the supraspinatus, and the infraspinatus. Two ultrasonic pictures will be taken of each. Care will 
be taken to image the region of the muscle that was not mechanically impacted by the insertion 
of the needle or injection of the substance. The area will then be cleaned with alcohol and water. 
The Delsys Galelio surface sensor, and the intramuscular needle electrode will be placed directly 
on the trapezius' identified area. Participants will be asked to gently contract their trapezius 
muscle. They will be instructed to perform a gradually increasing contraction in isometric 
condition, in a controlled manner with a monitor showing the exerted force as well as the target 
force. They will hold this contraction at 30% of their maximal voluntary contraction, and then 

13 
 
perform a gradually decreasing contraction to rest. This will be performed four times for each 
type of study intervention and before as well as after each intervention, namely topical control 
cream, topical capsaicin at 50ug and 100 ug, injection of the superior aspect of the superior 
fascia followed by intramuscular injection with these two concentrations of capsaicin. The 
placement of these latter will be verified by ultrasound guidance. The intramuscular needle will 
then be removed and participants will be bandaged and cared for appropriately by the expert 
physician performing the experiment.Following this, ultrasound pictures will be taken again 
from the same muscles mentioned previously. Participants will be re-examined to determine if 
there were any adverse effects from the experimental procedures. If any occurred, then these will 
be carefully managed by the medical members of the research team and recorded. Participants 
will be asked to remain at the lab for an additional 30 minutes to ensure they are well prior to 
leaving (Figure 2).  
Central sensitization and measurement techniques 
Inducing central sensitization 
Central sensitization will be induced using capsaicin according to the established 
technique.[13,24,25] It induces increased pain severity at high doses relative to heat and elicits a 
dose dependent pain responses, allowing for more easily quantifiable measurements.[26] 
Capsaicin delivery induces both primary and secondary nociception suggesting that central 
mechanisms are involved in the nociceptive response, also termed as central sensitization.[13,14] 
Previous evidence has confirmed that capsaicin can sensitize nociceptive and mechanoheat 
receptors outside of the region of primary hyperalgesia, with speculation that it can also sensitize 
chemonociceptive receptors.[13] Therefore, capsaicin can effectively be used to induce central 
sensitization.  

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Capsaicin will be applied directly to the region of the innervation zone at the muscle 
belly to sensitize the neurons within the region of taut band development. Topical and 
intramuscular capsaicin will be used. The capsaicin formula will be compounded by a registered 
pharmacist. Topical capsaicin will be delivered in a cream and injectable capsaicin will be 
intermixed with saline prior to injection. The control group in the topical capsaicin arm will be 
treated with the cream base used during the experiment without added capsaicin and the injection 
arm will be injected with saline. A trained medical professional on the research team will apply 
the topical capsaicin or topical placebo treatments. The region of topical application will measure 
5cm squared in the dermatome zone location to cover an area of approximately 25cm2. A trained 
physician in physical medicine and rehabilitation will deliver injectable capsaicin using a 27-
gauge needle at the location of the superior fascia of the upper trapezius muscle with ultrasound 
guidance. The fascial layer was chosen as it is the region with the lowest pressure pain threshold 
relative to skin and muscle, suggesting a high density of nociceptors within the layer, and it 
possesses connections to the spinal cord.[27] Changes in muscle fascia properties and 
nociception at muscle fascia, compared to nociception at muscle or subcutaneous tissue, has also 
been implicated in the development of pain.[28,29] Furthermore, intramuscular capsaicin will 
also be injected using ultrasound guidance to avoid the superior or inferior fascia. 
To confirm the presence of central sensitization, brush allodynia will be used to detect 
mechanical hyperalgesia outside the region of primary nociception—region of topical placement 
or injection—which is the region of secondary hyperalgesia.[13-15] The size of the region of 
secondary hyperalgesia will be measured to characterize the extent of central sensitization. This 
will be accomplished using a tape measure and the perpendicular dimensions of the region of 
secondary hyperalgesia will be recorded in square centimeters. 

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Ultrasound analysis 
The Sonosite Ultrasound machine located at the Toronto Rehabilitation Institute clinic will be 
used. The ultrasound probe will be placed onto the trapezius before and after the sensitization is 
induced and an ultrasonic image will be captured. Texture feature analysis will be performed 
using MATLAB and the Signal Processing Toolbox. Analyses will include first order parameters 
such as mean and standard deviation of the pixel level values, as well as second order 
parameters, which provide details on the spatial distribution of pixel values. These include co-
occurance and run-length matrices, local binary pattern and blob analysis[Quant text ref].  
Measurement of motor unit activity using EMG 
The Delsys Trigno surface EMG system will be used in this experiment. The system 
offers the benefit of having a 4-pin mini-grid that can extract individual motor unit data, without 
causing participants discomfort. The analysis will also be complemented by the use of 
intramuscular EMG, since it offers high location specificity when measuring EMG activity, 
while only a local region is analzed. The intramuscular EMG data will be analyzed by a Cadwell 
Sierra Wave device. 
The anatomical location for electrode placement, along the C7-acromion line, will be 
identified for each participant. Baseline EMG measurements, as well as the ultrasonic images 
will be taken from the upper trapezius muscle to obtain the baseline motor unit recruitment 
curve. The trapezius muscle was chosen for study since it is a common site for MTrPs, given the 
high load cervical muscles carry as well as the general increase sedentariness that perpetuates 
poor posture and cervical muscle strain, leading to trigger point presentation. Furthermore, the 

16 
 
trapezius muscle has been used frequently in previously published work, which analyze motor 
unit parameters, power spectrum and amplitude measurements.[31,32,33] 
sEMG Analysis 
After the identification for electrode placement and the captured baseline ultrasonic 
images, the area will be cleaned with alcohol and water. The Delsys Galelio sensor will be 
placed directly on the identified area. The EMG sensor (bandwidth of 20-450Hz ) consists of 4 
metal contacts for detecting the signal at the skin surface. Both the right and left sides of the 
trapezius will be measured.  
The Delsys Trigno system, which includes sensors, the Trigno base station, and the 
EMGworks software will be used to decompose the acquired EMG signals into individual motor 
units. The following parameters will be analyzed: the firing times and frequency of individual 
motor units, motor unit action potential amplitude and shapes[36-38], the root mean square value 
(RMS) of each channel and of the entire signal[16], the coefficient of variation for force 
steadiness[39], and the centroid of the EMG signal in the cranial-caudal and the medial-lateral 
directions[16]. This will be repeated for each contraction. 
 
Intramuscular EMG analysis 
A 27g monopolar electromyography needle will be placed into the midbelly of the upper 
fibers of the trapezius muscle using ultrasound guidance. The intramuscular EMG signals will be 
measured using Cadwell Sierra Wave. Since this system does not offer motor unit 
decomposition, we will use the intramuscular EMG analysis to provide us with more details of 
the gross EMG measurements such as the overall RMS value, the coefficient of variation for 
force steadiness, and the centroid measurements. 

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Determination of Central Sensitization:  
Before measuring outcomes, approximately 20 minutes after capsaicin application we 
will analyze the area of secondary hyperalgesia for each study subject, then determine whether 
central sensitization has in fact been induced. The presence of central sensitization is confirmed 
by expansion of the receptive field and area of secondary hyperalgesia beyond the 25 cm2 initial 
application area. If it has, the study participant will undergo the rest of the experimental 
methodology. If it has not, the capsaicin will be applied once more and the region of secondary 
hyperalgesia will be remeasured in 20 minutes later. The above described process for confirming 
the presence of central sensitization will take place again. If the study participant has not had 
sensitization at this stage they will be withdrawn from the study. 
Primary Outcomes 
The primary outcome measures:  
1. Textural regional changes 
2. Recruitment and Amplitude regional changes  
These will be recorded for each arm of the study, please see figure 1. 
Secondary outcomes: 
1. A record of adverse events  
2. Safety evaluation The investigating physician monitors the patient clinically for signs of 
distress. The methods and timing for assessing, recording, and analyzing safety 
parameters. Any signs of distress noted by investigating physician will be assessed 
clinically and appropriately managed. The information will be recorded on the patient’s 
data form. The study team will discuss all incidents and any potential causal link to the 
study interventions. Patients will be provided with a contact number for the principal 

18 
 
investigator in order to report any changes that occur during or after the study. Our 
publication will include a list of any adverse events or intercurrent illness encountered. 
Any patient who experiences an adverse event will be triaged to their family physician or 
local emergency department as necessary. They will be contacted after to obtain 
information on the course of their treatment and the outcome of the event. 
3. Sample size calculation.  
Using GPower V3.0.10 (Dusseldorf, Germany), considering a medium effect size 
(Hedge’s G) of 0.5 and a moderate correlation coefficients (0.5) among repeated 
measures, sample size calculation determined that a minimum of 70 participants would 
be required to detect differences via repeated measures ANOVA ( two measurement as 
before and after among 7 groups) with 80% power and an alpha of 0.05. We aim to 
recruit 84 participants to account for potential attrition (~20%) of participants given the 
study duration and frequency of follow-up visits.  
 
We plan to recruit equal number of men and women. To date, there are no reported sex 
differences in induction of Central Sensitization using Capsaicin among males and 
females with neurological impairments. 
 
4. Statistical analysis plan  
Baseline participants’ characteristics including demographics will be analyzed using 
appropriate descriptive statistics. Mean and standard deviation will be calculated for 
continuous variables. Categorical variables will be presented as numbers and percentages. 
To test the change in ultrasound images from baseline to experimental conditions, the 

19 
 
texture features will be computed in MATLAB and principal component analysis will be 
computed to reduce the variables. A repeated measures MANOVA will be applied for 
each intervention group. To test the change in EMG within and between groups a 
repeated measurement ANOVA will be applied considering each intervention group as 
factor. The dependent variable would be considered as pain intensity, duration, and area 
of pain in separate analysis.  Data distributions would be first checked with the Shapiro-
Wilk to conform the normal distribution of the data.  Additionally, we will test for the 
homogeneity of variance using Mauchly’s test as well as correcting the degrees of 
freedom if necessary [40]. If the assumption of normality not met, for all skewed data an 
appropriate transformation will be used. We will construct univariate and multivariable 
linear regression models of differences in EMG amplitude (differences between after and 
before) as a function of intervention groups when adjusted for important covariates. Due 
to small sample size in each group, there is not possible to adjust for several covariates. 
Therefore, in a univariate model we will find the most important ones. For initial 
selection of relevant variables, all variables with a p-value≤0.2 in the univariate analysis 
will be selected and retained in the multivariable analysis.   
All Statistical analyses will be conducted with SAS for Windows (version 9.3; SAS 
Institute, Inc, Cary, NC). Using a two-sided test a P-value ≤ 0.05 will be considered as 
statistically significant.   
 
Discussion 
This will be the first study that assesses the effect of capsaicin induced central 
sensitization on ultrasound texture features and muscle motor unit activity. Findings from this 

20 
 
study may support one of few hypotheses proposed delineating the involvement of central 
sensitization in the development of trigger points. A prominent hypothesis in the literature is the 
integrated hypothesis. The integrated hypothesis suggests that unaccustomed eccentric activity or 
submaximal to maximal concentric muscle exertion leads to muscle fiber damage, segmental 
hypercontraction within the muscle fibers, and ischemia.[6] The resultant damage and ischemia 
at the muscle instigates an inflammatory biomarker cascade that potentiates the activity of motor 
neurons, subsequently increasing the release of acetylcholine and inducing muscle contraction, 
and sensitizes sensory neurons.[6,41] The sensitization of peripheral sensory and motor neurons 
is thought to contribute to the sensitization of dorsal horn neurons in the associated spinal 
segment, which likely influences efferent neuronal activity at the muscle. From this hypothesis, it 
would be expected to observe uncoordinated motor unit activity and activation given that muscle 
fiber damage has occurred and motor units may be differentially potentiated or sensitized. The 
findings from studies inducing acute nociception in the muscle support this hypothesis.[16,42] 
Hypertonic saline injections have been shown to reorganize muscle activity within the 
muscle.[42] Previous findings also suggest that motor unit discharge frequency changes variably 
among motor units, although predominantly decreasing, as a consequence of external 
nociception.[16] This study should elucidate whether a nociceptive stimulus that causes central 
sensitization induces uncoordinated motor unit activation and differential discharge patterns 
among the units. The integrated hypothesis presupposes that the cause of myofascial trigger 
points is exogenous and induces endogenous changes in muscle. The Cinderella and the 
neurogenic hypothesis suggest otherwise.  
The Cinderella hypothesis suggests sustained low threshold motor unit activation 
metabolically overloads oxidative muscle fibers, leading to muscle fiber damage or ‘ragged 

21 
 
fiber’ presentation and metabolic changes such as ischemia, hypoxia, and insufficient ATP.[7,41] 
This may result in disturbed calcium homeostasis and the development of the muscle contracture 
known as the trigger point. Low threshold motor unit activity was demonstrated in a number of 
articles assessing the effect of continued muscle contraction and psychologically demanding 
tasks—muscle overexertion and persistent psychological stress are theorized triggers for chronic 
pain and trigger point development.[43,44] The sustained contracture in the muscle maintains the 
hypoxic and acidic environment, leading to peripheral and central sensitization of neurons in the 
associated spinal segment.[7] This suggests an endogenous and exogenous cause for the trigger 
points. Under the premise of the Cinderella hypothesis, it would be expected to observe sustained 
low threshold motor unit activity with central sensitization.  
The neurogenic hypothesis is distinct from the integrated and Cinderella hypotheses as it 
poses trigger point development is a result of endogenous central sensitization.[1] Central 
sensitization would result from persistent nociceptive input from other peripheral mechanic or 
systemic pathologies leading to neurogenic inflammation, such as trauma or endocrine disease. 
Subsequently, inflammatory and algogenic substances would be released from peripheral 
nociceptors onto tissue and sensitizing the region. Sensitization of motor and sensory neurons 
innervating somatic muscle may lead to the phenomenon of trigger points. Ultimately, the 
neurogenic hypothesis stipulates the trigger point is a secondary outcome central sensitization, 
which is the primary pathology. Textural changes throughout the induced muscle, as well as the 
regional muscles would support this hypothesis. Continuous low threshold motor unit activity 
would also be an anticipated observation as neurons will be increasingly sensitized. It would also 
be expected to observe increased low threshold motor unit activity in remote muscles that present 

22 
 
with signs of central sensitization, e.g. hyperalgesia, however this is outside the scope of this 
article.   
Limitations and Conclusions 
Overall, the findings from this study should present preliminary evidence to inform 
central sensitization’s effects on motor unit activity. Results may provide plausibility for the 
aforementioned hypotheses. A limitation of this study is the degree of invasiveness needed to 
increase the specificity of the EMG measurements, however, the trade-off between invasiveness 
and specificity is necessary to ensure we capture a sufficient effect size with a feasible sample 
size. Additionally, the experimental protocol will be carried out on healthy participants with 
induced central sensitization to determine the effects of this phenomenon on muscle activity. 
Although these results are not directly generalizable to participants with myofascial pain, this 
study’s results should provide insight as to whether central sensitization does perturb muscle 
activity and structure, and inform further investigations into the pathophysiology of myofascial 
pain. Another limitation of this study in light of the integrated and Cinderella hypotheses is that 
sensitization will be induced prior to the assessment of EMG activity at motor neurons. 
However, it is important to understand whether central sensitization is a direct cause of aberrant 
motor unit activity, as a fraction of the population develops myofascial trigger points following 
precipitating events such as injury, stress, or poor posture. The presence of central sensitization 
at a specific severity or ‘tipping point’ may predispose some individuals to developing trigger 
points—or induce trigger points with increased severity—and the mechanisms described by the 
integrated or Cinderella hypotheses may lead to the manifestation of the trigger point. These 
hypotheses need not be mutually exclusive, but rather may be complementary or present as 
alternate etiologies to the development of the trigger point. It is possible the anatomical changes 

23 
 
resulting from each hypothesis leads to a different manifestation of trigger points that appear 
clinically equal. Further studies assessing central sensitization markers and EMG activity of 
muscle innervated by spinal segments remote from the region of primary sensitization are needed 
to elucidate the overarching pathophysiology of trigger point presentation.  
 
Acknowledgements: N/A 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

24 
 
Declarations 
Ethics approval and consent to participate: 
The study conforms to the Consolidated Standards of Reporting Trials recommendations. 
Informed consent will be obtained from all subjects participating in this study. Ethical approval 
was approved by the University Health Network (UHN) Research Ethics Board(Approval 
Number: 18-6163). The approval form is included in the supplementary files of this submission. 
This study is registered under the National Institutes of Health ClinicalTrials.gov. 
Consent for publication: 
Not applicable. 
Availability of data and material: 
The datasets used and/or analyzed during the study will be available from the corresponding 
author upon reasonable request. 
Trial Status:  
Protocol version and date: Version 2, May 1,2019  
Recruitment: Starting June 2019- November 2019 
The Delsys Trigno EMG machine has been ordered. The pharmacy has approved the intended 
capsaicin formula that will be used in the experiment. Participants will be recruited from June 
2019- November 2019 
Competing interests: 
The authors declare that they have no competing interests. 
Funding: 
This study is being conducted with no external funding and was instead funded from the Toronto 
Rehabilitation Institute. 

25 
 
Authours’ Contribution: 
All authors (VE, KM, DK) contributed to the study design; drafted, reviewed and finalized the 
study protocol; critically revised the manuscript and approved the final manuscript.  
 
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Figures Legend: 
Figure 1. Flow chart of methods  
Figure 2. Proposed experimental protocol timeline 
 
 
 
 
 
 
 
 
 
 

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