Keywords:
Muscles - Ultrasonography - Muscular Dystrophies - Architecture - Physical Functional
Performance
Palavras-chave:
Músculos - Ultrassonografia - Distrofias Musculares - Arquitetura - Desempenho Físico
Funcional
INTRODUCTION
Duchenne muscular dystrophy (DMD) is one of the most common X-linked recessively inherited
neuromuscular diseases, with an incidence of 1/3500-5000 live male births[1],[2]. It results from mutations in the gene encoding dystrophin, leading to its absence[3]. The clinical scenario is primarily characterized by proximal muscle weakness, which
in turn causes difficulty in standing from the supine position, frequent falls, and
motor delays. Although functional performance is maintained until the age of 6 years,
affected boys often lose ambulation by 12 years of age[4]. Eventually, death occurs due to cardiac and pulmonary complications within the
20s[5].
Molecular tests and muscle imaging methods have been used for many years to diagnose
and manage different neuromuscular diseases[6]. Muscle imaging methods such as magnetic resonance imaging (MRI) and ultrasound
(US) are generally used in the evaluation of the dystrophic process in DMD. Although
the above techniques are validated, painless, and radiation-fee methods to assess
muscle pathologies[7], US has certain advantages over MRI, such as being more child-friendly, cheaper,
more convenient, and more cost-effective[8],[9]. Of note, ultrasonographic data on muscle architecture play an important role in
the evaluation of motor function in relevant cases[10].
Due to the progressive nature of the disease, functional levels of children deteriorate
with age. Many studies evaluating parameters such as balance, muscle strength, and
pulmonary function in children found that children with low functional status had
worse outcomes[11],[12],[13]. Additionally, it was shown that muscle echo-intensity was associated with disease
severity and longitudinal changes of the dystrophic process in DMD[14],[15]. Only one study has evaluated muscle architecture in adult DMD patients[16], and there is no data in children at early stages with different functional levels.
Accordingly, the primary aim of this study was to investigate the effect of functional
level on lower limb muscle architecture in children with early stage DMD. We also
compared the ultrasonographic parameters of muscle architecture between DMD vs. typically
developing (TD) peers.
METHODS
Participants
This was a cross-sectional observational study with three groups, conducted between
May 2019 and March 2020 in a tertiary care university hospital. The study protocol
was approved by the Clinical Research Ethics Board (Protocol Number: KA-19022). Informed
consent form was obtained from the children and their families and all the procedures
were performed in accordance with the Declaration of Helsinki.
Children aged 5 to 10 years were included in the study if the diagnosis of DMD was
confirmed by genetic analysis, the functional level was of Grade 1 and 2 according
to Vignos Scale[17], had no comorbidities, were on corticosteroid treatment for at least for 6 months,
and if they were able to cooperate with the study instructions. TD children aged 5
to 10 years were enrolled if they did not have any musculoskeletal, cognitive, neurological,
or cardiopulmonary diseases. Children were excluded if they had undergone any surgery
or suffered a lower limb injury.
Assessments
Demographic characteristics (age and body mass index) were recorded before the evaluations.
Functional level, muscle strength, and motor performance evaluations were consecutively
performed by a physiotherapist (NB) with six years of experience in rehabilitation
of pediatric neuromuscular disorders. There was a 10-minute interval between tests,
which were completed within one hour.
Functional level of children with DMD was assessed using the Vignos Scale[17], which has high intraclass correlation[18]. The scale has 10 grades, with Grade 1 denoting the best functional level and Grade
10 the worst. More specifically, children who are able to walk and climb stairs without
assistance are categorized as Grade 1, while children who can walk and climb stairs
with the aid of handrail are categorized as Grade 2.
Muscle strength evaluation was done for the knee extensors and ankle plantar flexors
using a hand-held dynamometer (Commander, Jtech Medical Industries, Main Midvale,
UT) at a standardized test position. For knee extensors, children were assessed in
the sitting position with the hip and knee flexed at 90° and the dynamometer placed
at ankle joint. Ankle plantar flexors were tested in the supine position with the
hip in neutral and the dynamometer placed around the metatarsals[19]. Three maximal voluntary isometric contractions of both legs were performed and
the mean values of the force used in the three repetitions were recorded bilaterally.
Test-retest reliability of quantitative muscle testing in DMD and TD children was
0.83-0.99 and 0.74-0.99, respectively[20].
Motor performance was assessed using timed tasks items that included rising from the
floor, the 10-meter walk test, ascending/descending four stair steps and the six-minute
walk test (6MWT)[21]. The completion time of the items except the 6MWT was recorded in seconds. The 6MWT
consists of fast walking in an indoor a course divided by a straight line 25 m long
with cones placed at the beginning and end of the line[22]. The total distance covered in the six minutes is recorded.
Ultrasonographic evaluations were performed by a single physiatrist (LÖ) with 20 years
of experience in musculoskeletal US using a 5-12 MHz linear probe (Logiq P5, GE Medical
Systems, Wisconsin, USA). For the measurements of the vastus lateralis (VL), the children
were in the supine position, with the knees in extension and the ankles in resting
position. For measurement of the medial gastrocnemius (MG), they were lying in prone
position with the legs extended and ankles at resting position, hanging from the side
of the examination bed. Minimal pressure was applied with probe on the thickest sides
of the both muscles to avoid muscle compression. For longitudinal imaging ([Figurse 1A, 1B and 1F]), the pennation angle (PA) was measured as the angle between the muscle fascicles
and the deep aponeurosis, and the fascicle length (FL) was measured as the distance
of fascicle path between the two aponeuroses. For axial imaging ([Figure 1C-1E]), the distance between deep and superficial aponeuroses was measured as muscle thickness
(MT)[23],[24].
Figure 1 Ultrasonographic measurements of the vastus lateralis and medial gastrocnemius muscles.
Longitudinal imaging for vastus lateralis in a typically developing child (A) and
a Grade 2 Duchenne muscular dystrophy child (B). Note the loss of clarity (?) for
visualization of the pennate structure (B). Axial imaging for muscle thickness measurement
of vastus lateralis (C). Axial imaging for medial gastrocnemius in a typically developing
child (D) and a Grade 1 Duchenne muscular dystrophy child (E). Note the increased
echogenicity (*) of the muscle due to fibroadipose infiltration (E). Longitudinal
imaging for fascicle length and pennation angle measurements of medial gastrocnemius
(F).sc: subcutaneous fat; VL: vastus lateralis; VI: vastus intermedius muscle; MT:
muscle thickness; MG: medial gastrocnemius; Sol: soleus muscle; FL: fascicle length;
PA: pennation angle.
Statistical analysis
The IBM Statistical Package for the Social Sciences (SPSS), version 20.0, was used
for statistical analyses. Kolmogorov-Smirnov test was used to determine normal distribution.
Demographic and ultrasonographic parameters were compatible with parametric conditions
while muscle strength and motor performance assessments with non-parametric conditions.
Descriptive statistics for quantitative data are given as mean±standard deviation
(SD) or as median and interquartile range (25th and 75th percentiles). Mann-Whitney U or independent sample t-test was used to compare children
with DMD with TD peers, as appropriate. Statistical significance was accepted when
p<0.05. Spearman coefficients were used for correlation analyses and classified as
very high (0.90-1.0), high (0.70-0.90), moderate (0.50-0.70), low (0.30-0.49), and
negligible (0-0.30)[25].
RESULTS
Thirty-one children with DMD and five TD peers were enrolled. Only one child with
DMD was excluded because of poor cooperation. Overall, data of 35 children (70 lower
limbs) were included in the analyses.
Mean age of children with DMD and that of TD peers was 8.83±1.55 and 8.10±1.12 years,
respectively. Mean body mass index of children with DMD was 17.06±2.05 kg/m2 and that of TD peers was 16.55±0.78 kg/m2. Demographic characteristics were similar between the two groups (all p>0.05). In
the DMD group, 15 children had functional level of Grade 1 and 15 had Grade 2.
Data regarding muscle strength and motor performance are shown in [Table 1]. Muscles strength (except for the dominant side ankle plantar flexors) and motor
performance were worse in children with DMD compared with TD children (all p<0.05).
DMD children with Grade 1 functional level had higher knee extensor strength and better
motor performance compared with those with Grade 2 (all p<0.05).
Table 1
Strength and functional assessment results (median, 25th-75th range).
|
|
DMD
|
p-valuec
|
TD peers (n=5)
|
p-valued
|
|
Grade 1 (n=15)
|
Grade 2 (n=15)
|
|
Strength (D)a
|
15.73 (13.80-21.00)
|
9.30 (6.30-10.80)
|
<0.001*
|
32.50 (31.86-45.75)
|
0.001*
|
|
Strength (ND)a
|
16.50 (13.20-19.50)
|
8.80 (6.86-12.00)
|
<0.001*
|
32.00 (28.79-34.65)
|
<0.001*
|
|
Strength (D)b
|
26.33 (24.23-29.97)
|
26.10 (22.80-28.80)
|
0.9
|
32.00 (24.30-37.40)
|
0.1
|
|
Strength (ND)b
|
23.00 (21.27-27.37)
|
26.50 (21.00-30.50)
|
0.5
|
32.00 (28.79-34.65)
|
0.007*
|
|
Rising from floor (sec)
|
4.75 (3.01-5.25)
|
13.75 (9.00-33.92)
|
<0.001*
|
2.09 (1.10-2.30)
|
<0.001*
|
|
10-m walk (sec)
|
7.56 (5.88-8.19)
|
8.45 (7.43-11.42)
|
0.01*
|
5.95 (4.97-6.22)
|
0.005*
|
|
Ascending 4 steps (sec)
|
2.39 (2.08-2.92)
|
8.83 (3.66-11.44)
|
<0.001*
|
1.76 (1.05-2.17)
|
0.001*
|
|
Descending 4 steps (sec)
|
2.15 (1.83-2.75)
|
4.40 (2.78-7.76)
|
0.007*
|
1.63 (1.25-1.84)
|
0.003*
|
|
6-min walk test (m)
|
443.00 (410.00-473.00)
|
350.00 (300.00-390.00)
|
<0.001*
|
580.00 (538.00-595.50)
|
<0.001*
|
n: number of participants; DMD: Duchenne muscular dystrophy; D: dominant; ND: non-dominant;
TD: typically developing; aMuscle strength of knee extensors; bMuscle strength of ankle plantar flexors; *p<0.05; cGrade 1 vs. Grade 2; dDMD vs. TD peers.
Ultrasonographic measurements are shown in [Table 2]. Muscle architecture parameters regarding VL could not be obtained from 3 children
(20%) with Grade 1 and 6 children (40%) with Grade 2 DMD because of excessive muscle
echogenicity. FL of the VL muscle and MT and FL of the MG muscle were lower in children
with DMD of Grade 1 compared to Grade 2 (all p<0.05). MT and VL values of the VL muscle
and MT, FL, and PA values of the MG muscle were higher in children with DMD compared
to TD peers (all p<0.05).
Table 2
Ultrasonographic measurements of the subjects’ lower limbs (mean±SD).
|
VL muscle
|
DMD
|
|
TD peers (n=10)
|
p-valueb
|
|
Grade 1 (n=24)
|
Grade 2 (n=18)
|
p-valuea
|
|
MT (cm)
|
2.03±0.33
|
2.26±0.47
|
0.07
|
1.90±0.72
|
0.002*
|
|
FL (cm)
|
6.45±1.32
|
7.93±0.98
|
<0.001*
|
6.04±0.84
|
0.006*
|
|
PA (degree)
|
18.78±3.80
|
19.41±3.52
|
0.5
|
19.09±2.88
|
0.9
|
|
GM muscle
|
DMD
|
|
TD peers (n=10)
|
p-valueb
|
|
Grade 1 (n=30)
|
Grade 2 (n=30)
|
p-valuea
|
|
MT (cm)
|
1.83±0.32
|
2.18±0.48
|
0.002*
|
1.36±0.14
|
<0.001*
|
|
FL (cm)
|
3.47±0.67
|
4.27±0.99
|
0.001*
|
2.97±0.36
|
<0.001*
|
|
PA (degree)
|
30.87±4.20
|
30.97±5.33
|
0.9
|
25.25±2.67
|
<0.001*
|
SD: standard deviation; n: number of limbs; DMD: Duchenne muscular dystrophy; MG:
medial gastrocnemius; VL: vastus lateralis; MT: muscle thickness, FL: fascicle length;
PA: pennation angle; TD: typically developing. *p<0.05. aGrade 1 vs. Grade 2, bDMD vs. TD peers.
Correlations among ultrasonographic measurements, muscle strength, and motor performance
are shown in [Table 3]. Concerning the VL muscle, FL was negatively correlated with muscle strength and
6MWT and positively correlated with time for rising from the floor and ascending four
stair steps (all p<0.05). Concerning the MG muscle, FL was negatively correlated with
the 6MWT and positively correlated with time for rising from the floor and ascending
four stair steps. Low correlations were found between VL MT and the 10-meter walk
test and between MG MT and time for rising from the floor and ascending four stair
steps (all p<0.05).
Table 3
Correlations between ultrasonographic and functional assessment results in children
with Duchenne muscular dystrophy.
|
VL (n=21)
|
MT (D)
|
MT (ND)
|
FL (D)
|
FL (ND)
|
PA (D)
|
PA (ND)
|
|
Strength (D)a
|
-0.13
|
-0.22
|
-0.38
|
-0.46*
|
-0.08
|
0.13
|
|
Strength (ND)a
|
-0.11
|
-0.23
|
-0.40
|
-0.44*
|
-0.07
|
0.07
|
|
Rising from the floor
|
0.30
|
0.24
|
0.62*
|
0.47*
|
0.24
|
0.17
|
|
Ten-meter walk test
|
0.44*
|
0.26
|
0.29
|
0.13
|
0.11
|
0.29
|
|
Ascending four steps
|
0.29
|
0.27
|
0.43*
|
0.37
|
0.33
|
0.10
|
|
Descending four steps
|
0.13
|
0.08
|
0.07
|
-0.19
|
0.06
|
0.13
|
|
Six-minute walk test
|
-0.23
|
-0.24
|
-0.45*
|
-0.27
|
-0.10
|
-0.26
|
|
MG (n=30)
|
MT (D)
|
MT (ND)
|
FL (D)
|
FL (ND)
|
PA (D)
|
PA (ND)
|
|
Strength (D)b
|
0.01
|
0.03
|
0.23
|
0.23
|
-0.09
|
-0.05
|
|
Strength (ND)b
|
-0.05
|
-0.03
|
0.34
|
0.31
|
-0.24
|
-0.19
|
|
Rising from the floor
|
0.42*
|
0.28
|
0.46*
|
0.52*
|
-0.02
|
-0.05
|
|
Ten-meter walk test
|
0.07
|
0.01
|
0.24
|
0.25
|
0.00
|
0.07
|
|
Ascending four steps
|
0.37*
|
0.28
|
0.41*
|
0.47*
|
0.00
|
0.05
|
|
Descending four steps
|
0.27
|
0.23
|
0.16
|
0.08
|
0.11
|
0.20
|
|
Six-minute walk test
|
-0.30
|
-0.17
|
-0.39*
|
-0.36
|
0.07
|
-0.06
|
n: number of participants; D: dominant; ND: non-dominant; DMD: Duchenne muscular dystrophy;
MG: medial gastrocnemius; VL: vastus lateralis; MT: muscle thickness; FL: fascicle
length; PA: pennation angle. Muscle strength of knee extensorsa and ankle plantar flexorsb were presented. *p<0.05.
DISCUSSION
This study explored the effect of functional level on lower limb muscle architecture
in children with early stage DMD and compared it with that of TD peers. To our best
knowledge, this is the first US study involving children with different functional
levels demonstrating that the architecture of the VL and MG muscles is affected even
at early stages of the disease, and the changes might also be related with functional
deterioration.
Absence of dystrophin in DMD makes the sarcolemma fragile and easily damaged by stresses
that develop during muscle contractions[3]. Furthermore, sarcolemmal damage is accompanied by muscle fiber necrosis and inflammation,
and muscle tissue is replaced by fat and connective tissue[3],[16]. Indeed, muscle architectural parameters of VL could not be obtained in 20% of children
with Grade 1 and in 40% of children with Grade 2 DMD in our study. Gradually, this
replacement results in ‘pseudohypertrophy’ i.e., excessive increase in muscle size.
Of note, the muscle groups in which pseudohypertrophy occurs in children with DMD
are the quadriceps and plantar flexors[26],[27]. In this sense, and in agreement with the literature, the fact that MT was higher
in our DMD children could be due to pseudohypertrophy. Likewise, the reason why Grade
2 DMD children - who had worse functional level - also had a higher MT for MG than
Grade 1 children could be the inflammatory process mentioned earlier. Although the
MT of VL was higher in Grade 2 DMD than in Grade 1 DMD, the lack of statistical significance
might be due to missing/small data.
Concerning muscle architecture, Lovering et al.[28] reported that the increased muscle volume due to pseudohypertrophy may correlate
with higher PA and maintained FL in Mdx mice. On the other hand, it was found that
PA and FL were not different in adult DMD patients compared with healthy controls[16]. Our results have shown that the FL of VL and the PA and FL of MG were higher in
children with DMD and that the FL of both muscles increased with worsening functional
level. These inconsistencies may be due to heterogeneous patient populations with
different demographic and disease characteristics. Nonetheless, muscle architecture
seems to be impaired in children with DMD starting from early stages.
Outcome measures from muscle strength and motor performance are often taken into consideration
in the prognosis of DMD. Studies have found that these two measures are lower in children
with DMD even at the early stage[13],[29]. As expected, our findings revealed that the knee extensor and ankle plantar flexor
(only of the non-dominant leg) strength and motor performance were better in TD children
(vs. DMD). Between DMD groups, knee extensor strength and motor performance were worse
in Grade 2 than in Grade1. We attribute the indifference in ankle plantar flexor strength
between groups to the fact that distal muscle groups were less affected at the early
stages of disease.
MT negatively correlated with motor performance. In other words, increased MT was
associated with longer duration of timed performance tests. Similarly, one MRI study
in children with DMD reported worse timed performance test results despite increased
cross sectional area of triceps surae muscle[26]. These findings reaffirm the fact that the “pathological” hypertrophy (i.e. pseudohypertrophy)
caused by inflammatory fat infiltration is different than the normal physiological
development of TD peers. Additionally, similar to MT, increased FL was correlated
with weaker muscle strength and worse motor performance. Thus, low or no correlations
between muscle architecture and physical assessment parameters may be explained by
additional compensatory movements of children with DMD during functional tasks[30].
The major limitation of this study was its small sample size (especially for the TD
group). However, the uncertainty related with the current COVID-19 pandemic complicated
subject enrollment in several ways and the study had to be terminated earlier than
originally planned.
To summarize, in the light of our study results, two main conclusions can be drawn.
First, MT is increased and functional performance is decreased in children with DMD
and second, muscle architecture is affected even at the early stages of the disease.
Further studies that include children with a diagnosis of other neuromuscular diseases
and peers matched for physical activity level in addition to children in the advanced
stages of DMD are needed. Also, the effect of therapeutic approaches on muscle architectural
properties in DMD should be investigated in future clinical trials.
