Altered corticospinal excitability of scapular muscles in individuals with shoulder impingement syndrome.

The purpose of this study is to assess and compare corticospinal excitability in the upper and lower trapezius and serratus anterior muscles in participants with and without shoulder impingement syndrome (SIS). Fourteen participants with SIS, and 14 without SIS were recruited through convenient sampling in this study. Transcranial magnetic stimulation assessment of the scapular muscles was performed while the participants were holding their arm at 90 degrees scaption. The motor-evoked potential (MEP), active motor threshold (AMT), latency of MEP, cortical silent period (CSP), activated area and center of gravity (COG) of cortical mapping were compared between groups using the Mann-Whitney U tests. The SIS group demonstrated following significances, higher AMTs of the lower trapezius (SIS: 0.60 ± 0.06; Comparison: 0.54 ± 0.07, p = 0.028) and the serratus anterior (SIS: 0.59 ± 0.04; Comparison: 0.54 ± 0.06, p = 0.022), longer CSP of the lower trapezius (SIS: 62.23 ± 22.87 ms; Comparison: 45.22 ± 14.64 ms, p = 0.019), and posteriorly shifted COG in the upper trapezius (SIS: 1.88 ± 1.06; Comparison: 2.76 ± 1.55, p = 0.048) and the serratus anterior (SIS: 2.13 ± 1.02; Comparison: 3.12 ± 1.88, p = 0.043), than the control group. In conclusion, participants with SIS demonstrated different organization of the corticospinal system, including decreased excitability, increased inhibition, and shift in motor representation of the scapular muscles.

1 Introduction

Shoulder impingement syndrome (SIS) is the most common shoulder disorder accounting for at least 40% of all shoulder problems [1]. Multiple factors contribute to SIS and one of them is scapular dyskinesis [2, 3]. Scapular dyskinesis indicates that the position and movements of the scapula on the thoracic cage are aberrant. Typically, the scapula rotates upward and externally and tilts posteriorly during shoulder elevation. With adequate scapular movements, acromion would move away from the humeral head during shoulder movement. However, in patients with SIS, the angles of scapular upward rotation and posterior tilt are insufficient, which may lead to impingement of subacromial tissues [3, 4].

Scapular movements and position are controlled by scapular muscles. Patients with SIS show different amplitude and timing of scapular muscle activation during movements. Through monitoring the electromyography (EMG) signals, previous studies found decreased muscle activation of the lower trapezius and the serratus anterior, and exaggerated muscle activation of the upper trapezius during shoulder elevation in athletes or individuals with SIS [5, 6]. Cools et al. [7] also found that athletes with SIS demonstrated delayed muscle activation of the upper and middle trapezius muscles compared to athletes without symptoms.

Different motor patterns may result from reorganization of the central nervous system. Although there is no structural damage in the central nervous system in patients with musculoskeletal injuries, chronic pain and prolonged symptoms are believed to alter the central nervous system [8]. The reorganization of the central nervous system may be also associated with the lack of treatment effectiveness and persistence of symptoms in some patients [9-11]. Using transcranial magnetic stimulation (TMS), recent studies have found that individuals with musculoskeletal pain, such as low back pain [12-14], chronic ankle instability [15], rotator cuff tendinopathy [16] and anterior cruciate ligament injury [17], demonstrated discrepancies in organization of the corticospinal systems, including changes in the location of the center of gravity of a cortical motor map, lower corticospinal excitability, and higher cortical inhibition [11-17]. Ngomo et al. [16] further demonstrated lower corticospinal excitability in the affected infraspinatus when compared with the non-painful side, and a relationship between the duration of pain and differences in corticospinal excitability in patients with unilateral rotator cuff tendinopathy. However, some of these variables were not reported in TMS studies of shoulder muscles such as differences in active motor threshold [12] and in intracortical inhibition [13], and it is unclear whether there are differences in the corticospinal system of other scapular muscles in patients with SIS. Therefore, the purpose of this study is to assess and compare the corticospinal excitability, intracortical inhibition and motor representation of the upper and lower trapezius, and the serratus anterior in participants with and without SIS.

2 Materials and methods

2.1 Setting and participants

Fourteen participants with unilateral SIS (7 males, 7 females; age: 24.64 ± 2.13 years old) and 14 sex, age, and hand-dominance matched participants without current shoulder or neck pain (7 males, 7 females; mean age: 23.64 ± 2.37 years old) were recruited from the local community through convenient sampling in this study (effect size: 0.724, alpha level: 0.05, power: 0.8, calculated based on the TMS data of the upper trapezius from our pilot study). The participants were only included if they were aged between 20–45 years. The inclusion criteria for the SIS were 1) chronic shoulder pain or tenderness on the greater tuberosity of the humerus for at least 3 months; 2) painful arc between 60∘and 120∘with active shoulder elevation; 3) positive in two of the following impingement tests: Neer impingement test, Hawkins-Kennedy impingement test, and empty can test, examined by a licensed physical therapist [18]. The exclusion criteria were: 1) a history of shoulder dislocation or traumatic injuries; 2) a history of shoulder surgery in the past six months; 3) a history of neurological diseases; 4) a history of seizure; 5) metal implants in head, neck, or chest and 6) other contraindications for receiving TMS assessment [19]. All participants were informed and signed a consent form before commencement of the study. Demographic data including weight, height, and the intensity of the most pain experienced during movement (using visual analogue scale) were collected before corticospinal excitability assessment. This study was approved by the Institutional Review Board of Taipei Veterans General Hospital (2012-07-009A), in the spirit of the Helsinki Declaration. This experiment was conducted at the Musculoskeletal and Sports Sciences Laboratory, National Yang Ming Chiao Tung University, and the Neurological Institute, Taipei Veterans General Hospital, Taiwan.

2.2 Corticospinal excitability assessment

The TMS (MagStim 200 stimulator, MagStim Company, Wales, UK) was used to assess the corticospinal excitability in three scapular muscles. To record the muscle activity of the upper and lower trapezius and serratus anterior through surface EMG (Neuropack M1 MEB-9200, Nihon Kohden, Tokyo, Japan), two EMG electrodes were placed 2.0 cm apart and parallel to the direction of muscle fibers on each muscle belly [20]. The location of electrodes were at the medial 1/3 of the line connecting the spinous process of the 7th cervical vertebra and the acromial angle for the upper trapezius; and at the midpoint from the point between the spinous processes of the 7th and 8th thoracic vertebra, to the root of the scapular spine for the lower trapezius. For the serratus anterior, the participant was asked to lift their arm forward to 90°, and the electrodes were placed horizontally along the axillary line at the level of inferior angle of scapula, and in front of the latissimus dorsi [20]. The EMG signals were sampled at 1,500 Hz and high-pass filtered of 20 Hz. To determine the locations for stimulation on the scalp, participants were asked to wear a size-appropriate swimming cap on which the tester drew a grid with a scale of 1 cm. To match an individualized grid, the investigator first identified the center between the eyebrows and the inion to draw the Y-axis (anteroposterior axis) and identified external canals of the ears to draw the X-axis (mediolateral axis). The intersection of these two axes was the grid origin called Cz. Then, the gird of swimming cap was aligned with the individual grid, and a 12 cm*12 cm grid was over the tested hemisphere. The grid intersections were used to determine the location and guide the coil moves for stimulation.

Participants were asked to hold the affected arm at 90-degree scaption (arm elevation in the scapular plane) during whole TMS assessment (Fig 1). This testing position was used because we found it difficult to elicit TMS responses in the resting position, and the 90-degree scaption is a functional position for the shoulder complex at which our target muscles would generate about 10–15% muscle activation for TMS assessment [24]. The orientation of the 70-mm figure-of-eight TMS coil was 45 degrees to the previously defined Y-axis while TMS pulses are delivered. The stimulation started at 70–80% of the maximum stimulator output (MSO) at an approximate grid intersection where the underlying cortex controls the targeted muscle, and the inter-stimulation interval was set as 10 seconds. The coil would be moved around the surrounding grid intersections to determine the most excitable location, or the hot spot, where the stimulation would trigger the highest motor-evoked potential (MEP). Once the hot spot was located, the stimulation intensity was reduced gradually with a decrement of 5% MSO to find the lowest stimulator intensity which could elicit the peak-to-peak amplitudes of MEPs equal or over 100μV in three out of six trials. The above test was performed with individual muscle and the lowest intensity at hot spot, represent as %MSO, for the target muscle is called active motor threshold (AMT).

Fig 1

TMS testing position.

The participant held the arm at 90-degree scpation elevation during TMS assessment.

The testing parameters to investigate the corticospinal system for scapular muscles in this study including AMT, amplitude of MEP (amplitude), cortical silent period (CSP), latency of MEP (latency), activated area of cortical mapping (mapping area), and center of gravity of mapping area (COG). Higher AMT indicates that corticospinal excitability is decreased and a stronger stimulation is needed to trigger the action potential by recruiting more motor units of a target muscle. For better measurement reliability, the MEP amplitudes were calculated by averaging the five highest peak-to-peak EMG values out of 10 single-pulse TMS trials tested at 120%AMT at the hot spot in each scapular muscle (Fig 2) [21]. A greater amplitude means increased corticospinal excitability. The latency of MEP reflects the conductivity of the corticospinal system and was calculated by the time difference between the TMS-induced artifact and the beginning of the waveform of MEP. CSP indicates the EMG activities were suppressed temporarily after MEP when the TMS pulse is delivered during active muscle contract. The definition of intracortical CSP was defined as the time from the end of MEP to the return of EMG activity. CSP represents the inhibition mechanisms of the corticospinal system [22]. To determine the mapping area which controls each scapular muscle, TMS assessment was performed using 120% AMT. The tested spots of mapping started at the hot spot and move to the next spot in the grid along the X-axis/ Y axis. The mapping area was the range that the continuous tested spots which showed latency, MEP over 11μV and CSP in the waveform, in two out of four trials [21, 23]. Lastly, the COG of this mapping area was calculated using the following equation (Xi: location in X-axis of tested point; Yi: location in Y-axis of tested point; MEPi: corresponding amplitude of MEP; n: numbers of tested point in the mapping), and reflects a spatial average of cortical presentation of a muscle between [23]:

Fig 2

A typical motor-evoked potential of upper trapezius.

2.3 Statistical analysis

Statistical analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, USA). Because we had a small sample size and our data were not normally distributed as shown by histograms, the non-parametric Mann-Whitney U tests were used to compare the differences in demographic data and all the testing parameters between the SIS and the comparison groups. A p value of less than 0.05 was considered as significant.

3 Results

The demographics of both the SIS and the comparison groups are listed in Table 1 and there was no significant difference in the demographics between the two groups. The testing session took around 3–4 hours. None of the participants complaint of pain during testing, but most of the participants reported fatigue during and after the measurement.

Table 1

Demographic data of participants (mean ± SD).

	Comparison group	SIS group	p-valueb
Participant Number (Male/Female)	14 (7/7)	14 (7/7)	-
Tested Side (Right/Left)	13/1	13/1
Age (year)	24.64 (±2.13)	23.64 (±2.37)	0.062
Height (cm)	166.57 (±9.30)	170.29 (±9.82)	0.394
Weight (kg)	60.79 (±13.54)	63.93 (±13.62)	0.550
BMIa	21.66 (±2.89)	21.83 (±2.52)	0.818
VAS pain scale (0–10)	-	5.57 (1.34)	-

aWeight / (Height)2

bMann-Whitney U test

*p-value < 0.05 indicates significant difference

SIS: Shoulder impingement syndrome; SD: Standard deviation; BMI: Body mass index; VAS: Visual Analogue Scale.

All the results of the testing parameters are included in Table 2 with the original raw data in S1 Data. The AMTs of the lower trapezius (p = 0.028) and the serratus anterior (p = 0.022) were significantly higher in the SIS group. Neither the peak-to-peak amplitude of MEP nor the latency of MEP between the two groups was significantly different in all three scapular muscles. The CSP durations of the lower trapezius were significantly longer in the SIS group than in the comparison group (p = 0.019).

Table 2

Comparisons of corticospinal excitability between the SIS and comparison groups (mean ± SD).

		Comparison group (n = 14)	SIS group (n = 14)	p-valuea
AMT (%MSO)	Upper Trapezius	0.49 ± 0.06	0.53 ± 0.05	0.062
	Lower Trapezius	0.54 ± 0.07	0.60 ± 0.06	0.028*
	Serratus Anterior	0.54 ± 0.06	0.59 ± 0.04	0.022*
MEP amplitude (mV)	Upper Trapezius	1.20 ± 0.98	1.41 ± 0.64	0.168
	Lower Trapezius	0.82 ± 0.52	1.00 ± 0.44	0.334
	Serratus Anterior	0.50 ± 0.33	0.61 ± 0.38	0.232
CSP (ms)	Upper Trapezius	60.02 ± 23.40	62.25 ± 34.66	0.520
	Lower Trapezius	45.22 ± 14.64	62.23 ± 22.87	0.019*
	Serratus Anterior	64.50 ± 21.20	83.94 ± 32.36	0.141
Latency (ms)	Upper Trapezius	8.32 ± 1.25	9.01 ± 1.75	0.395
	Lower Trapezius	11.11 ± 1.49	10.42 ± 1.78	0.280
	Serratus Anterior	12.71 ± 2.26	11.93 ± 3.57	0.783
Mapping Area (cm2)	Upper Trapezius	24.95 ± 5.26	22.45 ± 7.91	0.118
	Lower Trapezius	22.83 ± 6.80	19.67 ± 7.19	0.215
	Serratus Anterior	26.93 ± 10.20	24.48 ± 7.10	0.462
COG (x, y)	Upper Trapezius	(3.41 ± 0.83, 2.76 ± 1.55)	(3.23 ± 0.48, 1.88 ± 1.06)	(0.448, 0.048*)
	Lower Trapezius	(3.54 ± 0.75, 3.00 ± 2.02)	(3.08 ± 0.39, 1.94 ± 1.14)	(0.060, 0.066)
	Serratus Anterior	(3.67 ± 0.88, 3.12 ± 1.88)	(3.37 ± 0.66, 2.13 ± 1.02)	(0.270, 0.043*)

aMann-Whitney U test

*p-value < 0.05

SIS: Shoulder impingement syndrome; SD: Standard deviation; AMT: active motor threshold; MEP: Motor evoked potential; CSP: Cortical silent period; COG: Center of gravity of mapping area.

The mapping area did not show significant differences between the two groups in all three scapular muscles (Table 2). The COGs of the mapping area of the upper trapezius (p = 0.048) and serratus anterior (p = 0.043) were significantly more posterior in the SIS group than those in the comparison group (p = 0.048, p = 0.043, Table 2 and Fig 3). There was no significant difference in the COG of the lower trapezius mapping area.

Fig 3

The COG in the SIS and comparison groups.

Red triangle: SIS group; blue circle: Comparison group. (A) upper trapezius; (B) serratus anterior.

4 Discussion

This study aimed to investigate whether there were differences in the corticospinal systems for scapular muscles in participants with SIS. Our results showed that the participants with SIS had lower contralateral corticospinal excitability (higher AMT) in the lower trapezius and serratus anterior, and higher contralateral intracortical inhibition (longer CSP) in the lower trapezius, and differences in contralateral cortical representation of the upper trapezius and serratus anterior. These findings reflected that SIS was associated with different organization of the corticospinal system of the scapular muscles, which should be concerned when managing people with SIS.

The AMTs of the lower trapezius and serratus anterior were significantly higher in the SIS group than those in the comparison group, which means that a stronger stimulation is needed to trigger the action potential by recruiting more motor units of lower trapezius and serratus anterior in participants with SIS. The present finding was similar to previous studies researching the shoulder disorders. Ngomo et al. [16] found that in patients with rotator cuff tendinopathy, the AMT of the infraspinatus at the affected side was significantly higher than that at the non-affected side. Similarly, Alexander [24] demonstrated that AMT of the lower trapezius was significantly higher in patients with non-traumatic shoulder instability compared to the healthy group. One of the speculated causes of decreased corticospinal excitability is prolonged pain that changes intrinsic neuronal membrane excitability [25, 26]. The chronic pain may inhibit the motor cortex excitability that allows the spinal motor system to respond to the pain as a protective mechanism [16, 27]. It has to be noted nevertheless that chronic pain does not necessarily have the same effect as acute pain. While previous studies of systemic review and meta-analysis showed reduced corticospinal excitability in the patients with experimental acute pain [28, 29], no consistence pattern on TMS variables was observed for the chronic pain conditions [30]. In addition, our participants with SIS may change their movement patterns to avoid moving their affected shoulder due to pain. This immobilization of shoulder may also decrease the cortical excitability even within a short period of time. Previous studies identified increased resting motor threshold (RMT) following 24-hour immobilization of the elbow and fingers [31], and decreased MEP amplitudes in participants who experienced 4-day finger immobilization [32]. Both increased RMT and decreased MEP amplitudes indicated decreased corticospinal excitability.

In this study, we did not find significant differences in latency between the SIS and comparison groups. MEP latency was considered a potential indicator to represent the function of corticospinal neural transmission and to detect stressed or damaged neuronal pathway [33, 34]. In patients with musculoskeletal dysfunction, such as those with SIS, we did not expect to see impaired corticospinal tract integrity or dysfunction, which may explain why there was no difference on MEP latency between the two groups.

Measuring CSP is widely used to investigate changes of neurophysiology in patients with different diagnoses, such as Epilepsies and Parkinson’s disease [35]. It can also be used to predict the motor and functional prognosis in patients with a stroke [35]. CSP is attributed by both the spinal and supraspinal inhibition mechanisms [36]. Specifically, the supraspinal mechanism contributes to the later and longer part of CSP and is medicated by gamma-aminobutyric acid (GABA) mediated inhibitory circuits [25]. In this study, the lower trapezius had longer CSP in the SIS group. This finding was similar to Bradnam et al. [26] who also found significantly longer CSP of the infraspinatus in patients with chronic shoulder pain when compared to healthy adults. This results indicated that the supraspinal inhibition may be increased due to chronic pain in participants with SIS.

We did not find differences in the mapping areas of the three scapular muscles between the SIS and comparison groups. However, the COG of the mapping area of the upper trapezius and the serratus anterior shifted posteriorly, closer to the Cz. Previous studies demonstrated different motor cortex organization represented by shifted COGs in patients with phantom limb pain after upper extremity amputation and in patients with recurrent low back pain [14, 37]. In addition, Tsao et al. found that the patients with recurrent low back pain demonstrated a more posteriorly and laterally located COG of the motor cortex as compared to the pain-free subjects, which were similar to our results [14, 38]. However, Ngomo et al. [16] did not find differences in COG locations of the infraspinatus in patients with rotator cuff tendinopathy. Since Ngomo et al. [16] compared COGs between hemispheres within participants instead of a comparison group, it is unclear if the COG position of the hemisphere controlling the unaffected shoulder would be different while COG at the other hemisphere changed with chronic shoulder pain. This might be a potential reason that our study showed different result regarding the COG assessment.

Boroojerdi et al. [39] showed that the COG of the hand muscles matched the activated cortex area during hand clench, recorded using task functional magnetic resonance imaging [fMRI]. Therefore, locating the COG through cortical mapping is considered as a reliable way to find the corresponding motor cortex area that controls specific muscles. Alexander [24] presented the COG coordinates of the upper trapezius (3.7 ± 0.7 cm, -0.5 ± 0.9 cm), the lower trapezius (3.7 ± 0.6 cm, -0.7 ± 0.7 cm), and the serratus anterior (3.8 ± 0.5 cm, -0.6 ± 0.8 cm) based on the data of three healthy participants. Compared to the control group of this study, our result demonstrated a similar medio-lateral orientation at the X-axis in the three muscles, but a more anterior anterio-posterior position at the Y-axis. We speculated that the differences in methodologies, such as the head shapes and muscle contraction levels, and the small sample size of the previous study might contribute to the inconsistent findings of COG locations in asymptomatic participants between studies.

In the present study, we found that the three tested muscles, the upper and lower trapezius and the serratus anterior demonstrated different neurophysiological changes. These inconsistencies might be attributed to the roles and characteristics of each muscle during arm elevation activities. The lower trapezius and serratus anterior are the prime movers and stabilizers during arm elevation and majority of the studies demonstrated decreased EMG activities in these two muscles in people with shoulder impingement or pathology [5, 6, 40]. In addition to being the scapular upward rotator, the upper trapezius also contributes to clavicle movements in the beginning of shoulder elevation and was found to be over-activated by showing increased EMG activities during shoulder elevation [5, 6, 40, 41]. Therefore, most corticospinal differences were observed in the serratus anterior and/or lower trapezius, including decreases in excitability and increases in inhibition. The differences in COG, however, did not follow this pattern. The COGs located more posteriorly in the upper trapezius and serratus anterior. These two muscles demonstrated similar characteristics in firing timing. The upper trapezius fired shortly before arm movements and the serratus anterior fired in the beginning of the arm movement at 53 ms, while the lower trapezius started to activate at 349 ms after the shoulder started to move [42]. Possibly the muscles that were recruited at the beginning, but not at later stage, of movement were more likely be associated with COG changes in participants with SIS.

The present study suggested that neurophysiological changes existed in people with SIS, but there were some limitations of our methodologies. Our TMS data was recorded and analyzed without EMG activity normalization. No pre-stimulus EMG activation was recorded as the background control. This might impact interpretation of our data. We did measure EMG activation of the three target muscles (upper and lower trapezius and serratus anterior) in 12 participants (six in each group) in the preliminary study, and found no group difference in muscle activation during scaption. This however did not fully eliminate the possible effect of the group differences in EMG patterns of recruitment on our findings. Future research should consider using EMG activation or M wave to normalize the TMS data. Since the levator scapulae has similar anatomical position with the upper trapezius, there might be EMG signal crosstalk from the levator scapulae that influenced assessment results. A fine wired EMG may be used for eliminating this crosstalk in the future. During the TMS assessment, participants were asked to maintain shoulder elevation in a fixed angle and the overall assessment took 3 to 4 hours. Thus, the testing results may be influenced due to fatigue toward the end of the assessment. Furthermore, participants might experience tiredness or fail to concentrate throughout the prolonged experimental procedure, which was also a potential factor influencing corticospinal excitability. A more efficient testing procedure or sufficient resting intervals should be considered in the future design. In addition to these limitations, all our participants experienced pain while or after performing overhead shoulder activities, but showed none or very mild symptoms during other daily tasks. It remains unclear how the patterns and frequency of pain may change corticospinal excitability and result in motor cortex reorganization. Furthermore, we did not record the history of sports for our participant. The intensity or duration of sports training may also lead to motor cortex reorganization. Since we only recruited participants aged between 20 and 45 years old, the brain plasticity at this age may impact the motor cortex organization patterns in a way that is different from other age groups after chronic pain. Finally, our results could not indicate whether the changes happen at spinal or cortical levels based on our methodologies. A study design using twin pulses of TMS might help to clarify the issue.

In conclusion, this is the first study investigating changes in the corticospinal system in scapular muscles in participants with SIS. Our results suggested the corticospinal alteration in this specific population. Future studies may need to investigate whether rehabilitation treatment could reverse these changes in the corticospinal system and whether these changes are related to the effectiveness of treatment.

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As you will see, both reviewers have raised substantial concerns about the manuscript. If you believe that you can address these concerns, I would recommend revising.

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In particular you will need to provide additional data on the following:

1) the fact that EMG was not controlled during the scapular movements and all the differences between groups could come from the absence of EMG control. Higher EMG activity in one group could have led to in differences in AMT or CoG. You need to provide EMG activity of the three muscles during TMS testing.

2)Testing hotspot, AMT and mapping of three different muscles would certainly take 3-4 hours. Did participants report pain and fatigue during testing?

3) The location of EMG electrodes are not specified. Considering that the lower trapezius and serratus anterior are difficult to locate and test using EMG, more details are need in the methods on how the electrodes were placed and how authors did confirm positioning over these muscles. Also, a lot of cross-talk could come from adjacent muscles.

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Reviewer #1: No

Reviewer #2: Partly

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Reviewer #1: PONE-D-21-33464: Altered Corticospinal Excitability of Scapular Muscles in Individuals with Shoulder Impingement Syndrome

General comments:

Yi-Fen Shih et al. compared TMS variables of three scapular muscles (serratus anterio, superior and inferior trapezius) in participants with and without impingement syndrome. They observed significant differences between multiple variables (e.g. AMT, CoGy). I have several methodological concerns that could make the results un-interpretable.

• TMS was tested during shoulder scaption and EMG was not controlled during this movement. All the differences between groups could come from the absence of EMG control. Indeed, higher EMG activity in one group could result in differences in AMT or CoG. Authors need at least to provide EMG activity of the three muscles during TMS testing.

• The TMS session was really ambitious. Testing hostpot, AMT and mapping of three different muscles would certainly take 3-4 hours. Did participants report pain and fatigue during testing?

• The location of MEG electrodes are not specified. Considering that the lower trapezius and serratus anterior are difficult to locate and test using EMG, more details are need in the methods on how the electrodes were placed and how authors did confirm positioning over these muscles. Also, a lot of cross-talks could come from adjacent muscles.

• Data are not all available in a public repository. Only means and a measure of dispersion are available in the Figures.

Specific comments:

Introduction

- Ln 40-41: I don’t think that we can assure that scapular dyskinesis is the leading factor of SIS. I would be more appropriate to indicate that this is one of the factor.

- Ln 53: Typo with reference (7)

- Ln 55: I don’t think we can use the term muscle kinetics that refer more to the biochemistry of muscle contraction rather than muscle activation. I think motor control may be a more appropriate term. Also the sentence is a bit trivial, it is clear that scapular dyskinesis suggest alteration of scapular motor patterns. Dyskinesis means modification of movement.

- Ln 63: Typo: recently should be recent

- Ln 64-66: Authors should use the term lower rather than decrease since it is cross-sectional studies. Decrease/increase infers that pain induced this effect, but we cannot conclude this from cross-sectional studies. Other studies also showed lower in intracortical inhibition level of trunk muscles in chronic low back pain compared to pain-free controls 1,2.

- Ln 77: What do authors mean by corticospinal inhibition? Do they mean intracortical inhibition? Typo: motor representation rather than presentation.

Material and methods

- Ln 78: How the effect size was calculated?

- Ln 95-98: It is important to provide more details about the exact locations of the EMG electrodes for each muscle tested. How do authors ensure that the EMG electrodes recorded muscle activity from the muscles targeted? Specifically, considering that the trapezius inferior and serratus anterior may be difficult to locate and palpate, what techniques did authors use to ensure proprer electrode placement?

- Ln 109: Why using a 90-degree scaption during TMS testing?

- Ln 117: type: grid rather than grip

- Ln 113-121:

o Did authors tested motor threshold before finding the hotspot? The order in which this is presented let is supposed this. If so, this is problematic.

o It seems like hostpsot and AMT of each muscle was measured independanlty. This takes usually a lot of time. Can authors provide how long it took to measure hotspot and AMT of each individual muscle?

- Ln 127-129: Why did only the 5 fist MEP amplitude were averaged? This is unconventional.Ln 135

- Please use references when potential mechanisms for TMS variables are stated (e.g. 3).

- Ln 139-141: Why a 11 µV MEP amplitude is used as cut-off? Why not using the same cut-off as for the AMT?

- The total duration of the session should be mentioned. This is a really long and ambitous session with three muscles tested for hotspot, AMT and mapping. How did the participants cope with such a long session? Did they report pain or fatigue? If so, how fatigue and pain may affect the results?

Results:

- It is critical to control the level of EMG that was present during TMS. Differences between groups may be driven by an increase EMG activity during the scaption movement. For exmaple, for mapping, a previous study showed that CoG was different at rest vs. in activity4. Was there differences in EMG activity for the three muscles during TMS?

- 1 Massé-Alarie, H., Beaulieu, L. D., Preuss, R. & Schneider, C. Corticomotor control of lumbar multifidus muscles is impaired in chronic low back pain: concurrent evidence from ultrasound imaging and double-pulse transcranial magnetic stimulation. Exp Brain Res 234, 1033-1045, doi:10.1007/s00221-015-4528-x (2016).

- 2 Massé-Alarie, H., Flamand, V. H., Moffet, H. & Schneider, C. Corticomotor control of deep abdominal muscles in chronic low back pain and anticipatory postural adjustments. Exp Brain Res 218, 99-109, doi:10.1007/s00221-012-3008-9 (2012).

- 3 Ziemann, U. et al. TMS and drugs revisited 2014. Clin Neurophysiol, doi:10.1016/j.clinph.2014.08.028 (2014).

- 4 Masse-Alarie, H., Bergin, M. J. G., Schneider, C., Schabrun, S. & Hodges, P. W. "Discrete peaks" of excitability and map overlap reveal task-specific organization of primary motor cortex for control of human forearm muscles. Hum Brain Mapp 38, 6118-6132, doi:10.1002/hbm.23816 (2017).

-

Reviewer #2: In current manuscript authors have assess and compare the corticospinal excitability, corticospinal inhibition and motor presentation of the upper and lower trapezius, and the serratus anterior in participants with and without SIS.In general manuscript is written well but for better presentation of the manuscript i would like authors to comment so following points

Abstract:

Page 3, Ln 23:Mention sampling technique used for recruitment of participants

Page 3, Ln 28: A sentence on how the data was analyzed will be useful

Page 3, Ln 29-33: Provide p-values for the results

Page 3, Ln 35: Shift “in” motor presentation

Introduction:

Page 5, Ln 64: The term “individuals with musculoskeletal pain” is very broad. Please be more specific about the population in which the changes were reported. Were these studies done on people with shoulder pain or any musculoskeletal pain at any site?

Page 5, Ln 64: Unclear what “changes in the center of gravity of a cortical map” means.

Methods:

Page 6, Ln 77: Please provide details on participant recruitment i.e., how and from where were the participants recruited for the study?

Page 6, Ln 78: Please provide more details about the inclusion criteria of participants. What was the age group that was targeted? The criteria about age group appears for the first time in the second last paragraph of the discussion section. This is needs to be mentioned in the methods section. Was pain intensity, pain duration, cause of impingement: traumatic or non-traumatic and bilateral or unilateral involvement used to screen eligible participants?

Page 6,Ln 80-83:Please provide references for the cluster of tests used for the inclusion of patients.

Page 6, Ln 82: Add “examined” before “by a licensed…” and replace “physical therapy” with “physical therapist”.

Page 8, Ln 108, 109: Was the arm held in scaption decided based on side affected or contralateral to testing hemisphere?

Page 10, Ln 150: Please provide the justification for using a non-parametric Mann-Whitney U test. Please provide information on whether the data was normally distributed.

Results:

Page 11 Ln 155: Please change the tense to past tense in Ln 155

Table 1 shows that participants pain intensity was collected using VAS scale, but this has not been mentioned in the methods section. Please provide more details about participant characteristics such as side of affected shoulder, pain duration (acute or chronic condition), does the VAS pain rating represent pain at rest or pain during movement, pain on the day of testing or average pain in past week/month.

Discussion:

Page 12 Line 170-172: Recheck sentence structure and rephrase. Include which side corticospinal excitability is increased or decreased i.e., ipsi- or contralateral. Please include the clinical implication of the findings in the first paragraph of the discussion section.

Page 15, 234: Replace “studies” with “study”

Page 17, Line 254-269: Please include some suggestions on how the limitation listed for the current study can be improved in future research.

In table two on line 387 please mention the test for the P valuse as done in line 380 for table 1.

Please improve the figure 2 ,3A and 3B quality as currently they arr blurry.

**********

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13 Jan 2022

Reviewer #1

General comments:

1. Yi-Fen Shih et al. compared TMS variables of three scapular muscles (serratus anterio, superior and inferior trapezius) in participants with and without impingement syndrome. They observed significant differences between multiple variables (e.g. AMT, CoGy). I have several methodological concerns that could make the results un-interpretable. TMS was tested during shoulder scaption and EMG was not controlled during this movement. All the differences between groups could come from the absence of EMG control. Indeed, higher EMG activity in one group could result in differences in AMT or CoG. Authors need at least to provide EMG activity of the three muscles during TMS testing.

Ans: Thank you for pointing out this issue. Because this investigation was conducted between 2014 and 2015 when using EMG activity to normalize the TMS data was less popular, we did not collect the EMG data when measuring TMS. We added discussion regarding the lack of EMG control in the limitations: “Our TMS data was recorded and analyzed without EMG activity control. This might impact some of the TMS variables such as AMT and CoG.” (P16 Ln271-274)

2. The TMS session was really ambitious. Testing hostpot, AMT and mapping of three different muscles would certainly take 3-4 hours. Did participants report pain and fatigue during testing?

Ans: Thank you for raising this question. The whole testing session took around 3-4 hours and most of the participants reported not much pain, but fatigue. This information is added in the Results (P10, Line 169-170) and discussed in the limitations. (P17 Ln277-283)

3. The location of MEG electrodes are not specified. Considering that the lower trapezius and serratus anterior are difficult to locate and test using EMG, more details are need in the methods on how the electrodes were placed and how authors did confirm positioning over these muscles. Also, a lot of cross-talks could come from adjacent muscles.

Ans: Thank you for reviewer’s suggestion. The details of the EMG electrode locations are added in the 2.2 Corticospinal excitability assessment, Procedure (P6, Ln101-107).

4. Data are not all available in a public repository. Only means and a measure of dispersion are available in the Figures.

Ans: Raw data are now available in supplement 1.

Specific comments:

Introduction

5. Ln 40-41: I don’t think that we can assure that scapular dyskinesis is the leading factor of SIS. I would be more appropriate to indicate that this is one of the factor.

Ans: Thank you for reviewer’s suggestion. The sentence is revised accordingly:” Multiple factors contribute to SIS and one of them is scapular dyskinesis (P3 Ln40).”

6. Ln 53: Typo with reference (7)

Ans: Thank you for reviewer’s reminder. This typo has been corrected. (P5 Ln 51)

7. Ln 55: I don’t think we can use the term muscle kinetics that refer more to the biochemistry of muscle contraction rather than muscle activation. I think motor control may be a more appropriate term. Also the sentence is a bit trivial, it is clear that scapular dyskinesis suggest alteration of scapular motor patterns. Dyskinesis means modification of movement.

Ans: Thank you for reviewer’s comment. We have deleted the redundant sentence.

8. Ln 63: Typo: recently should be recent

Ans: Thank you for reviewer’s reminder. This typo is corrected. (P4 Ln59)

9. Ln 64-66: Authors should use the term lower rather than decrease since it is cross-sectional studies. Decrease/increase infers that pain induced this effect, but we cannot conclude this from cross-sectional studies. Other studies also showed lower in intracortical inhibition level of trunk muscles in chronic low back pain compared to pain-free controls 12.

Ans: Thank you for reviewer’s valuable suggestion. The description is revised accordingly, using “lower” and “higher” instead of decreases or increases. (P4 Ln63-64)

10. Ln 70: What do authors mean by corticospinal inhibition? Do they mean intracortical inhibition? Typo: motor representation rather than presentation.

Ans: Thank you for reviewer’s reminder. We changed the “corticospinal inhibition” to “intracortical inhibition”, and the typo is corrected. (P4 Ln69-70)

Material and methods

11. Ln 78: How the effect size was calculated?

Ans: This effect size was calculated based on the AMT of the upper trapezius from our pilot study. This information is added. (P5 Ln 78)

12. Ln 95-98: It is important to provide more details about the exact locations of the EMG electrodes for each muscle tested. How do authors ensure that the EMG electrodes recorded muscle activity from the muscles targeted? Specifically, considering that the trapezius inferior and serratus anterior may be difficult to locate and palpate, what techniques did authors use to ensure proprer electrode placement?

Ans: Thank you for reviewer’s suggestion. The details of the EMG electrode locations are added in the 2.2 Corticospinal excitability assessment, Procedure (P6, Ln101-107).

13. Ln 109: Why using a 90-degree scaption during TMS testing?

Ans: Thank you for the question. We found it difficult to elicit TMS responses in the resting position, and the 90-degree scaption is a functional position for the shoulder complex at which our target muscles would generate about 10-15% muscle activation for TMS assessment. (P8, Ln119-122)

14. Ln 117: type: grid rather than grip

Ans: Thank you. This typo is corrected. (P9, Ln126)

15. Ln 113-121: Did authors tested motor threshold before finding the hotspot? The order in which this is presented let is supposed this. If so, this is problematic.

Ans: Thank you for raising this issue. We started with 70-80% of the maximum stimulator output (MSO) to find the hot spot first, and then gradually decreased the intensity to find the lowest stimulator intensity as the active motor threshold (AMT). The sentence is corrected to clarify the confusion. (P9, Ln126-131)

16. Ln 113-121: It seems like hostpsot and AMT of each muscle was measured independanlty. This takes usually a lot of time. Can authors provide how long it took to measure hotspot and AMT of each individual muscle?

Ans: Thank you for raising this concern. We did measure the hotspot and AMT independently for each muscle. Because the hotspots and AMTs of these three muscle are not far from each other, it was not too troublesome to do it one by one. It generally took us about an hour to do all three.

17. Ln 127-129: Why did only the 5 fist MEP amplitude were averaged? This is unconventional.

Ans: Thank you for the question. We average the five highest EMP out of 10 trials, not the first five. The sentence is corrected. (P9, Ln139-140)

18. Ln 135: Please use references when potential mechanisms for TMS variables are stated (e.g. 3).

Ans: Thank you for the suggestion. The reference is added. (P9 Ln148)

19. Ln 139-141: Why a 11 µV MEP amplitude is used as cut-off? Why not using the same cut-off as for the AMT?

Ans: Thank you for raising the question. This criterion came from Wassermann et al. (1992)’s protocol, and the reference is added. (P9 Ln 152)

20. The total duration of the session should be mentioned. This is a really long and ambitous session with three muscles tested for hotspot, AMT and mapping. How did the participants cope with such a long session? Did they report pain or fatigue? If so, how fatigue and pain may affect the results?

Ans: Thank you for the suggestion. The whole testing session took around 3-4 hours and most of the participants reported not much pain, but fatigue. This information is added in the Results (P10, Line 168--170) and discussed in the limitations. (P17 Ln277-283)

Results:

21. It is critical to control the level of EMG that was present during TMS. Differences between groups may be driven by an increase EMG activity during the scaption movement. For exmaple, for mapping, a previous study showed that CoG was different at rest vs. in activity. Was there differences in EMG activity for the three muscles during TMS?

Ans: Thank you for raising the concern. We realized the importance of EMG activity control for the TMS data. Because this investigation was conducted between 2014 and 2015 when using EMG activity to normalize the TMS data was less popular, we did not collect the EMG data when measuring TMS. We added discussion regarding the lack of EMG control in the limitations: “Our TMS data was recorded and analyzed without EMG activity control. This might impact some of the TMS variables such as AMT and CoG.” (P18 Ln322-324)

Reviewer #2:

In current manuscript authors have assess and compare the corticospinal excitability, corticospinal inhibition and motor presentation of the upper and lower trapezius, and the serratus anterior in participants with and without SIS.In general manuscript is written well but for better presentation of the manuscript i would like authors to comment so following points

Abstract:

1. Page 3, Ln 23:Mention sampling technique used for recruitment of participants

Ans: Thank you for reviewer’s comment. We used convenient sampling (leaflets) to recruit participants. The information is added. (Abstract, Ln24)

2. Page 3, Ln 28: A sentence on how the data was analyzed will be useful

Ans: Thank you for reviewer’s suggestion. We used Mann-Whitney U tests to analyze the data. The information is added. (Abstract, Ln28)

3. Page 3, Ln 29-33: Provide p-values for the results

Ans: Thank you for reviewer’s suggestion. All p-values are added. (Abstract, Ln30-34)

4. Page 3, Ln 35: Shift “in” motor presentation

Ans: Thank you for reviewer’s reminder. The “in” is added. (Abstract, Ln35)

Introduction:

5. Page 5, Ln 64: The term “individuals with musculoskeletal pain” is very broad. Please be more specific about the population in which the changes were reported. Were these studies done on people with shoulder pain or any musculoskeletal pain at any site?

Ans: Thank you for reviewer’s comment. These included low back pain, chronic ankle instability, rotator cuff tendinopathy, and anterior cruciate ligament injury. The information is added. (P4, Ln60-61)

6. Page 5, Ln 64: Unclear what “changes in the center of gravity of a cortical map” means..

Ans: Thank you for reviewer’s reminder. We rephrased the sentence to clarify the confusion as” changes in the location of center of gravity…” (P4, Ln62)

Methods:

7. Page 6, Ln 77: Please provide details on participant recruitment i.e., how and from where were the participants recruited for the study?

Ans: Thank you for reviewer’s reminder. We used convenient sampling (leaflets) to recruit participants from the local community. The information is added. (P5, Ln77)

8. Page 6, Ln 78: Please provide more details about the inclusion criteria of participants. What was the age group that was targeted? The criteria about age group appears for the first time in the second last paragraph of the discussion section. This is needs to be mentioned in the methods section. Was pain intensity, pain duration, cause of impingement: traumatic or non-traumatic and bilateral or unilateral involvement used to screen eligible participants?

Ans: Thank you for reviewer’s suggestion. We add detailed inclusion criteria in the “participant” section. (P5 Ln79-84)

Page 6,Ln 80-83:Please provide references for the cluster of tests used for the inclusion of patients.

Ans: Thank you for the suggestion. The reference 16 is added.

Magee DJ, Manske RC. Orthopedic Physical Assessment: Elsevier - Health Sciences Division; 2021.

9. Page 6, Ln 82: Add “examined” before “by a licensed…” and replace “physical therapy” with “physical therapist”.

Ans: Thank you for reviewer’s suggestion. The sentence is revised accordingly. (P5 Ln83-84)

10. Page 8, Ln 108, 109: Was the arm held in scaption decided based on side affected or contralateral to testing hemisphere?

Ans: Thank you for raising the concern. The testing arm was the affected side. The sentence is revised to clarify the confusion. (P7 Ln117)

11. Page 10, Ln 150: Please provide the justification for using a non-parametric Mann-Whitney U test. Please provide information on whether the data was normally distributed.

Ans: Thank you for raising the concern. A non-parametric test was used because of the small sample size and some our data were not normally distributed as shown by histograms. This information is added. (P10, Ln162-163)

Results:

12. Page 11 Ln 155: Please change the tense to past tense in Ln 155

Ans: Thank you. The typo is corrected accordingly. (P10, Ln168)

13. Table 1 shows that participants pain intensity was collected using VAS scale, but this has not been mentioned in the methods section. Please provide more details about participant characteristics such as side of affected shoulder, pain duration (acute or chronic condition), does the VAS pain rating represent pain at rest or pain during movement, pain on the day of testing or average pain in past week/month.

Ans: Thank you for the suggestion. The information is added in the Methods (chronic pain for at least 3 months, the most pain experienced during movement etc.) (P5, Ln80-90) and Table 1.

Discussion:

14. Page 12 Line 170-172: Recheck sentence structure and rephrase. Include which side corticospinal excitability is increased or decreased i.e., ipsi- or contralateral. Please include the clinical implication of the findings in the first paragraph of the discussion section.

Ans: Thank you for reviewer’s suggestion. We revised the paragraph as suggested “Our results showed that the participants with SIS had lower contralateral corticospinal excitability (higher AMT) in the lower trapezius and serratus anterior, and higher contralateral intracortical inhibition (longer CSP) in the lower trapezius, and changes in contralateral cortical representation of the upper trapezius and serratus anterior. These findings reflected that SIS was associated with reorganization of corticospinal system of the scapular muscles, which should be concerned when managing people with SIS.” (P12 Ln185-190)

15. Page 15, 234: Replace “studies” with “study”

Ans: Thank you. The “studies” is replaced by the “study”. (P16 Ln270)

16. Page 17, Line 254-269: Please include some suggestions on how the limitation listed for the current study can be improved in future research.

Ans: Thank you for reviewer’s suggestion. We add some information regarding how to improve the study methods in the “limitation”. (highlighted, P16 Ln 270-293)

17. In table two on line 387 please mention the test for the P valuse as done in line 380 for table 1.

Ans: Thank you for the suggestion. The information is added in Table 2.

18. Please improve the figure 2 ,3A and 3B quality as currently they arr blurry.

Ans: Thank you for reviewer’s suggestion. We re-do the figures with better resolutions. (Figure 2 and 3)

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4 Feb 2022

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: Yes

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Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-21-33464: Altered Corticospinal Excitability of Scapular Muscles in Individuals with Shoulder Impingement Syndrome

General comments:

I thank authors for considering my comments. However, there are some authors’ responses for which I am not convinced and other elements I would like them to address.

- About EMG control, in 2014-2015, it was well known that EMG background amplitude (representing the net motoneuronal excitability) was critical to control in TMS research considering the MEP amplitude represent the excitability at both cortical and spinal levels. Authors may control for EMG by measuring pre-stimulus EMG background to determine if it was similar between groups for each muscle tested. For example, if participants of the SIS group recruit more the lower trapezius during the task, it may explain the longer duration of the CSP or the difference in AMT. Further analyses are required to ensure that the results are not due to different patterns of recruitment in the different groups.

- I encourage authors to review the literature and report TMS studies comparing patients with and without musculoskeletal pain for cortical and corticospinal control. For example, differences in AMT 1 and in intracortical inhibition 2. Considering that these variables were not reported in TMS studies of shoulder muscles, it may help for the discussion of the results.

- About the use of the 5 highest MEP out of 10, why did the authors used these methods? Could they use references and discuss how it could impact on the results?

- Ln. 64-67: In this sentence, it should be clear that the difference was with the non-painful side, and not with a control group.

- About sample size calculation: what was the alpha and beta levels used?

- About the position of the electrodes, authors should provide a reference for these electrode sites.

- About the justification of the scaption position, please refer to a study for the EMG level stated (10-15% MVC).

- Ln 202-203: Please refer to systematic reviews and meta-analysis (e.g. 3,4) and it is important to be cautious to assume that chronic pain has the same effect as acute pain. A systematic review of chronic pain did not find any consistent pattern on TMS variables 5 conversely to experimental acute pain which reduces corticospinal excitability 3,4.

- Ln 231-232: Authors can discuss/described a bit more the findings of Tsao et al. that showed a difference in CoG position in the M1 map in low back pain compared to a painfree group 6,7.

-

- 1 Strutton, P. H., Theodorou, S., Catley, M., McGregor, A. H. & Davey, N. J. Corticospinal excitability in patients with chronic low back pain. J Spinal Disord Tech 18, 420-424, doi:00024720-200510000-00008 [pii] (2005).

- 2 Massé-Alarie, H., Beaulieu, L. D., Preuss, R. & Schneider, C. Corticomotor control of lumbar multifidus muscles is impaired in chronic low back pain: concurrent evidence from ultrasound imaging and double-pulse transcranial magnetic stimulation. Exp Brain Res 234, 1033-1045, doi:10.1007/s00221-015-4528-x (2016).

- 3 Burns, E., Chipchase, L. S. & Schabrun, S. M. Primary sensory and motor cortex function in response to acute muscle pain: A systematic review and meta-analysis. Eur J Pain, doi:10.1002/ejp.859 (2016).

- 4 Rohel, A. et al. The effect of experimental pain on the excitability of the corticospinal tract in humans: A systematic review and meta-analysis. Eur J Pain 25, 1209-1226, doi:10.1002/ejp.1746 (2021).

- 5 Parker, R. S., Lewis, G. N., Rice, D. A. & McNair, P. J. Is Motor Cortical Excitability Altered in People with Chronic Pain? A Systematic Review and Meta-Analysis. Brain Stimul 9, 488-500, doi:10.1016/j.brs.2016.03.020 (2016).

- 6 Tsao, H., Danneels, L. A. & Hodges, P. W. ISSLS prize winner: Smudging the motor brain in young adults with recurrent low back pain. Spine (Phila Pa 1976) 36, 1721-1727, doi:10.1097/BRS.0b013e31821c4267 (2011).

- 7 Tsao, H., Galea, M. P. & Hodges, P. W. Reorganization of the motor cortex is associated with postural control deficits in recurrent low back pain. Brain 131, 2161-2171, doi:10.1093/brain/awn154 (2008).

-

Reviewer #2: Thanks for addressing my comments.Authors have satisfactorily replied to my concern and I am happy to endorse the paper.I only have a minor aesthetic comment about the quality of figure 3 A & B .It would be nice to improve the quality for overall readership of the article.

**********

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Reviewer #1: Yes: Hugo Massé-Alarie

Reviewer #2: No

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21 Feb 2022

Reviewer #1

General comments:

1. I thank authors for considering my comments. However, there are some authors’ responses for which I am not convinced and other elements I would like them to address. About EMG control, in 2014-2015, it was well known that EMG background amplitude (representing the net motoneuronal excitability) was critical to control in TMS research considering the MEP amplitude represent the excitability at both cortical and spinal levels. Authors may control for EMG by measuring pre-stimulus EMG background to determine if it was similar between groups for each muscle tested. For example, if participants of the SIS group recruit more the lower trapezius during the task, it may explain the longer duration of the CSP or the difference in AMT. Further analyses are required to ensure that the results are not due to different patterns of recruitment in the different groups.

Ans: Thank you for reviewer’s suggestion. Unfortunately the raw EMG set has been deleted, we were unable to retrieve it for further analysis. Some of the participants (6 in each group) were tested in the preliminary study for EMG activation of the three target muscles during scaption, and we found no group difference in EMG activities of the upper and lower trapezius and serratus anterior (p values ranging from 0.26 to 0.99). This information was added in the final paragraph of the Discussion. (Line 278-284)

2. I encourage authors to review the literature and report TMS studies comparing patients with and without musculoskeletal pain for cortical and corticospinal control. For example, differences in AMT 1 and in intracortical inhibition 2. Considering that these variables were not reported in TMS studies of shoulder muscles, it may help for the discussion of the results.

Ans: Thank you for reviewer’s valuable suggestion. References are added as suggested. (P4 Ln60-69)

3. About the use of the 5 highest MEP out of 10, why did the authors used these methods? Could they use references and discuss how it could impact on the results?

Ans: Thank you for pointing out the question. This method was chosen for better measurement reliability based on the Cavaleri et al.’s review, and this information is added. (P8 Ln142-145)

4. Ln. 64-67: In this sentence, it should be clear that the difference was with the non-painful side, and not with a control group.

Ans: Thank you for reviewer’s suggested. The sentence is revised accordingly to avoid the confusion. (P4 Ln65)

5. About sample size calculation: what was the alpha and beta levels used?

Ans: Thank you for reviewer’s reminder. The information is added (effect size: 0.724, alpha level: 0.05, power: 0.8). (P5 Ln79-80)

6. About the position of the electrodes, authors should provide a reference for these electrode sites.

Ans: Thank you for reviewer’s reminder. The reference of placing these EMG electrodes was added. (P6 Ln104 & P7 Ln111)

7. About the justification of the scaption position, please refer to a study for the EMG level stated (10-15% MVC).

Ans: Thanks for raising the question. This information is added. (P7 Ln124-125, reference 24)

8. Ln 202-203: Please refer to systematic reviews and meta-analysis (e.g. 3,4) and it is important to be cautious to assume that chronic pain has the same effect as acute pain. A systematic review of chronic pain did not find any consistent pattern on TMS variables 5 conversely to experimental acute pain which reduces corticospinal excitability 3,4.

Ans: Thank you for the valuable suggestion. This information is added. (P13 Ln205-209)

9. Ln 231-232: Authors can discuss/described a bit more the findings of Tsao et al. that showed a difference in CoG position in the M1 map in low back pain compared to a painfree group 6,7.

Ans: Thank you for reviewer’s suggestion. We add this information in the discussion. (P15 Ln239-241)

- 1 Strutton, P. H., Theodorou, S., Catley, M., McGregor, A. H. & Davey, N. J. Corticospinal excitability in patients with chronic low back pain. J Spinal Disord Tech 18, 420-424, doi:00024720-200510000-00008 [pii] (2005).

- 2 Massé-Alarie, H., Beaulieu, L. D., Preuss, R. & Schneider, C. Corticomotor control of lumbar multifidus muscles is impaired in chronic low back pain: concurrent evidence from ultrasound imaging and double-pulse transcranial magnetic stimulation. Exp Brain Res 234, 1033-1045, doi:10.1007/s00221-015-4528-x (2016).

- 3 Burns, E., Chipchase, L. S. & Schabrun, S. M. Primary sensory and motor cortex function in response to acute muscle pain: A systematic review and meta-analysis. Eur J Pain, doi:10.1002/ejp.859 (2016).

- 4 Rohel, A. et al. The effect of experimental pain on the excitability of the corticospinal tract in humans: A systematic review and meta-analysis. Eur J Pain 25, 1209-1226, doi:10.1002/ejp.1746 (2021).

- 5 Parker, R. S., Lewis, G. N., Rice, D. A. & McNair, P. J. Is Motor Cortical Excitability Altered in People with Chronic Pain? A Systematic Review and Meta-Analysis. Brain Stimul 9, 488-500, doi:10.1016/j.brs.2016.03.020 (2016).

- 6 Tsao, H., Danneels, L. A. & Hodges, P. W. ISSLS prize winner: Smudging the motor brain in young adults with recurrent low back pain. Spine (Phila Pa 1976) 36, 1721-1727, doi:10.1097/BRS.0b013e31821c4267 (2011).

- 7 Tsao, H., Galea, M. P. & Hodges, P. W. Reorganization of the motor cortex is associated with postural control deficits in recurrent low back pain. Brain 131, 2161-2171, doi:10.1093/brain/awn154 (2008).

Reviewer #2

1. Thanks for addressing my comments. Authors have satisfactorily replied to my concern and I am happy to endorse the paper. I only have a minor aesthetic comment about the quality of figure 3 A & B. It would be nice to improve the quality for overall readership of the article.

Ans: Thank you for reviewer’s valuable comment. We further improved the quality of fig 3A & B as suggested.

Submitted filename: Response to Reviewers_R2_0221.docx

Click here for additional data file.

23 Mar 2022

28 Mar 2022

Reviewer #1: General comments:

I thank the authors for considering my comments. I would urge authors to be cautious in the language used for data interpretation. Differences between groups coming from cross-sectional studies cannot be interpreted as “changes”, “reorganisation” or “modification”. Only a longitudinal study allows to conclude this. Authors should change these terms used throughout the manuscript. Also, I think the English should be revised before publication of the manuscript.

Answer: Thanks for the suggestions. The manuscript has been proof read by an English native speaker, and terms such as reorganization, modification, changes have been revised according to your suggestion throughout the manuscript (highlighted).

Specific comments:

Ln 32 : Considering this is a cross-sectional study, it is not possible to conclude in a “reorganisation” of the corticospinal system since it is not known if this is a predisposing factors or a modification which occurs because of pain. Authors should use a the term “different organisation” or “suggest a reorganisation”

Answer: Thanks for the suggestion. The sentence is revised as “participants with SIS demonstrated different reorganization of the corticospinal system…”. (Ln34-35)

Ln 279-280: I would be careful in the language used to interpret the findings. The sentence “The present study proved that neurophysiological changes existed in people with SIS, but there were some limitations of our methodologies.” should be modified to “ The present study suggest […]”.

Answer: Thanks for the suggestion. The sentence is revised as “ The present study suggested […]”. (Ln 280)

Ln 320: “suggest” instead of “supported”

Answer: Thanks for the suggestion. The sentence is revised accordingly. (Ln 310)

Submitted filename: Responses to reviewers R3_0325.docx

Click here for additional data file.

3 May 2022

Altered Corticospinal Excitability of Scapular Muscles in Individuals with Shoulder Impingement Syndrome

PONE-D-21-33464R3

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Reviewer #1: The authors addressed all my comments in the revised version of the manuscript.

I would like to thank the authors for adressing my comments.

Reviewer #3: The authors have addressed all queries raised by the reviewers. The authors have performed multiple revisions and I think the work is important. These are difficult muscles to target via TMS so the work will be of interest to groups trying to attempt this type of stimulation for shoulder muscles.

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Reviewer #1: Yes: Hugo Massé-Alarie

Reviewer #3: No

6 May 2022

PONE-D-21-33464R3

Altered Corticospinal Excitability of Scapular Muscles in Individuals with Shoulder Impingement Syndrome

Dear Dr. Shih:

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