Algorithms for Patient-specific Treatment Planning in Cerebral Palsy Based on the Muscle and Tendon Architecture - Short Term Follow-up of a Botulinum Toxin Intervention in Children With Spastic Cerebral Palsy
Phase: NA
Conditions: Spastic Cerebral Palsy
Sponsor: Universitaire Ziekenhuizen KU Leuven

The short-term impact of Botulinum Neurotoxin-A on 
muscle morphology and gait in children with spastic cerebral 
palsy  
NCT number: NCT05126693 
Protocol date: May 10, 2022 
Study protocol 
This study will include participants of two different projects. Both projects are approved by the local ethics 
committee of the University Hospitals Leuven, Belgium (s59945 TAMTA project, s62187 and s62645 3D-MMAP 
project) (Ethische Commissie Onderzoek KU Leuven/UZ Leuven). Written informed consent will be obtained from 
the parents or legal guardian. Participants will be included between May 2019 and June 2022. 
 
Participants 
Children aged between 3-11 years, GMFCS levels I-III with SCP, scheduled for BoNT-A in the MG and/or ST, as 
part of a multilevel treatment, will be included. Participants will be recruited via two parallel prospective studies. 
Both studies will investigate the effect of integrated BoNT-A treatment on muscle morphology and gait, while 
one study will also evaluate the effects on spasticity (s59945 TAMTA project). The selection criteria for the 
participants are similar in both studies. The exclusion criteria are: any signs of dystonia or ataxia in the lower 
limbs, severe muscle weakness or poor selectivity in the plantar flexors or knee flexors and cognitive problems 
that would impede the measurements, intrathecal baclofen pump, previous muscle surgery in the lower limbs 
or selective dorsal rhizotomy, previous bony corrections in the lower limb within two years before assessments, 
previous BoNT-A treatment within six months before the baseline assessment or within six months before the 
pre-BoNT-A 3DGA. Additionally, the physical therapy frequency has to be consistent for at least three months 
before inclusion. The participants will be measured twice, before and 8-10 weeks after BoNT-A treatment, to 
evaluate the short-term effects. All children (of both studies) will receive 3DfUS assessments and a 3DGA with 
clinical examination of the lower limbs. A subset of children (i.e. the participants of one of the two studies) will 
undergo an additional instrumented spasticity assessment in order to evaluate the BoNT-A-induced tone 
reduction. During the follow-up, all children will receive usual care including intensive physical therapy, orthotic 
management, and oral tone reduction if needed. 
 
An age- and functional severity-matched (group-matching based on age and GMFCS level) control group, meeting 
the same inclusion criteria as the intervention group, but not receiving any intervention apart from their usual 
care, will be included. The participants of the control group will be recruited via Cerebral Palsy Reference Center 
of the University Hospitals Leuven, Belgium and the Inkendaal Rehabilitation Hospital Vlezenbeek, Belgium. All 
children eligible for inclusion will be measured, however the final data selection will be based on accurate 
matching with the participants in the intervention group. The participants in the control group will undergo 
3DfUS measurements only, with an interval of 8-10 weeks between the two assessments.  
 
 
 

Procedure 
The most affected leg, based on the most recent clinical examination (highest modified Ashworth score for the 
ankle and knee joints), will be assessed. Measures of body mass, height, total leg length and lower leg length will 
be taken. A standardized anamnesis will be performed to request treatment history and usual care details. 
 
Muscle selection for BoNT-A injection will be primarily based on the most recent clinical examination and 
multidisciplinary evaluation of the 3DGA data. BoNT-A, Onabotulinum toxin A (Botox®, Allergan) injections will 
be applied by a pediatric orthopedic surgeon under general (mask) anesthesia, guided by ultrasonography or 
palpation. A dose of 100 units of BoNT-A will be diluted in 5ml of saline. The BoNT-A dilution will be injected at 
multiple sites, in areas with the largest concentration of motor endplates in the muscles selected for treatment. 
A maximum of 50 units will be injected per site. BoNT-A is suggested to be more effective when part of an 
integrated approach, i.e., combined with casting, orthotic wear and physical therapy. Therefore, lower leg casts 
and removable upper leg casts will be applied as indicated.  
 
All additional study assessments (3DfUS, clinical examination, instrumented spasticity assessment and 3DGA) will 
be performed by three trained assessors. The pre- and post-assessments will always be performed by the same 
assessor. 
 
Assessments 
1. Muscle morphology 
Muscle morphology of the MG and distal compartment of the ST muscle will be assessed by 3DfUS. This 
technique, combining B-mode ultrasonography with three-dimensional motion tracking, is proven valid and 
reliable in muscle morphology assessments in children with SCP in previous studies. 
 
Ultrasound assessments will be performed while the participant will be placed in prone position. The lower leg 
will be supported by a triangular cushion to allow slight knee flexion (approximately 25 degrees) and avoid bi-
articular stretch on the MG and ST. The ankle will be in resting position without fixation. The joint angles will be 
measured by goniometry. 
Large amounts of acoustic transmission gel will be used to assure smooth movement of the transducer and 
optimal contact between the transducer and skin. An additional curved gel pad, the Portico, will be used to 
reduce muscle deformation during the acquisition. Muscle images will be recorded by Stradwin software (version 
6.0; Mechanical Engineering, Cambridge University, Cambridge, UK) while moving the transducer from muscle 
origin to tendinous insertion. The acquisitions will be repeated in case of bad quality images, movement or 
muscle contraction or when the bony landmarks or muscle borders are missed.  
After data collection, the muscle images will be processed by a trained assessor using the Stradwin software 
(version 6.0; Mechanical Engineering, Cambridge University, Cambridge, UK). Muscle volume (in millimeters) will 
be estimated by the cubic planimetry technique based on segmentations that will be manually drawn along the 
inside of the muscle borders in 5-10% of the transverse 2D muscle images. Additionally, echogenicity intensity, 
as a proxy of muscle quality, will be based on the average of the interpolated reconstruction of the muscle and 
will be expressed as a gray value on an 8-bit scale (values between 0-255), whereby higher values (whither 
images) will indicate reduced muscle quality. Muscle belly lengths will be calculated as the Euclidean distance 
between the muscle origin (MG: posterior surface of medial femoral condyle, ST: ischial tuberosity) and the 
muscle-tendon junction. The tendon length of the MG will be calculated between the muscle-tendon junction 
and tendinous insertion. The MTU length of the MG will be calculated by adding the muscle belly to the tendon 
length. 
 
In order to compare children with different body dimensions, muscle volume will be normalized to the product 
of body mass*height, and muscle and tendon lengths will be expressed as a percentage of total leg length. 
 
The absolute muscle-specific growth rates for muscle volume during the period between the two assessments 
will be calculated by the following formulas:  
 
growth rate, muscle volume = 𝑚𝑢𝑠𝑐𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) 𝑃𝑂𝑆𝑇− 𝑚𝑢𝑠𝑐𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) 𝑃𝑅𝐸 
𝑎𝑔𝑒 (𝑚𝑜𝑛𝑡ℎ𝑠) 𝑃𝑂𝑆𝑇− 𝑎𝑔𝑒 (𝑚𝑜𝑛𝑡ℎ𝑠) 𝑃𝑅𝐸
 

The primary outcomes will include the normalized muscle volume and echogenicity intensity of the MG and distal 
compartment of the ST and the absolute muscle volume growth rates.  
 
2. Gait analysis 
Gait, over ground barefoot walking at a self-selected speed, will be assessed by 3DGA in a 10 to 12 camera 
(Oxford Metrics, Oxford, UK) gait lab (Vicon Motion Systems, Oxford, UK). Twelve reflective markers will be 
placed on anatomical landmarks according the Vicon Plug-In-Gait lower limb model. Ground reaction forces will 
be measured with force plates (Advanced Mechanical Technology Inc., USA) that are embedded in the walkway. 
During the 3DGA, kinematic data (joint angles for ankle, knee and hip, and segmental orientation of pelvis and 
foot), kinetic data (joint moments and power) and surface EMG data of the medial gastrocnemius, soleus, tibialis 
anterior, rectus femoris, vastus lateralis, the medial and lateral hamstrings and gluteus medius will be collected 
for each leg. The kinematic data of the most affected leg (same as for the 3DfUS and instrumented spasticity 
assessments) and a selection of spatial-temporal parameters will be included in this current investigation, but 
the kinetic and EMG data will not be included in any of the analyses. 
 
Gait cycles will be identified and manually marked using the Vicon Nexus software (Oxford Metrics, Oxford, UK). 
All trials will be exported and imported to a custom-made Matlab script (The MathWorks, 
Natick, MA, USA, 2020b). All gait cycles will be judged on quality. Trials will be excluded in case of artefacts, gaps 
in data or indications of inaccurate marker placement. Furthermore, the knee varus-valgus angle will be 
evaluated and trials with absolute knee varus-valgus ROM exceeding 15 degrees, a knee valgus angle exceeding 
-10 degrees, or trials that are not representative of the participants’ gait pattern will be excluded. As many good-
quality steps as possible, will be used to create average trials for the ankle and knee joint kinematics. 
The GPS, and specific kinematic gait parameters that are found to be sensitive to detect BoNT-A-induced changes, 
will be extracted per joint. Additionally, the spatial-temporal parameters cadence, walking velocity and stride 
length will be calculated. Finally the full kinematic waveforms will be exported for pre-post comparison. 
 
The primary outcomes will include the kinematic waveforms, BoNT-A-sensitive kinematic gait parameters, GPS 
and spatial-temporal parameters.  
 
 
3. Spasticity assessment 
A subset of participants (n=14) will undergo an instrumented spasticity assessment in order to objectively 
evaluate the change in the hyperactive stretch reflex during passive stretch. This method has been previously 
described by Bar-On and colleagues  and is proven sensitive enough to detect treatment-induced changes. 
 
Four low-velocity (during 5 seconds) passive stretches of the plantar flexors and medial hamstrings will be 
performed over the full ROM. Next, four full ROM high-velocity passive stretches will be performed whereby the 
stretch will be performed as fast as possible. The interval between the stretch repetitions will be at least 7 
seconds to avoid post-activation depression. During the stretches, muscle activation will be recorded by surface 
EMG (Zerowire Cometa, Milan, IT) 
The raw EMG signals will be filtered by a 6th order zero-phase Butterworth bandpass filter (20-500 Hz) The root 
mean square envelope of the EMG signal (RMS-EMG) will be computed after application of a low-pass 30 Hz 6th 
order zero-phase Butter-worth filter. 
 
All data analyses will be carried out with MATLAB Software (The MathWorks,Natick, MA, USA, 2020b). The quality 
of the individual stretches will be checked using custom-made software. Stretch repetitions will be excluded in 
case of inconsistent stretch velocities, poor surface EMG data, out-of-plane movement or indications of 
insufficient participant relaxation (constant EMG activity of the antagonist). 
 
In order to quantify the alteration in muscle tone after BoNT-A treatment, the average RMS-EMG expressed in 
millivolts, will be calculated as the square root of the area underneath the RMS-EMG time curve, divided by the 
duration of the time interval, during the high-velocity stretch. The interval will start 200 milliseconds prior to the 
time point where maximum stretch velocity will be reached and ended at the time corresponding to 90% of the 
full ROM. This value will be referred to as the muscle activation during a high-velocity stretch. All BoNT-A-induced 
changes will be compared to previous reported SEM or BoNT-A-induced values. 

The average RMS-EMG expressed in millivolts during a high-velocity stretch will be used as the primary outcome 
to assess alterations in muscle tone. Additionally, the increase during the high-velocity stretch will be calculated 
by subtracting the muscle activation during a low-velocity stretch, which will be calculated following the same 
method. This value will be called the change in muscle activation and will be included as a secondary outcome.  
 
4. Clinical examination 
A standardized clinical examination including assessments of ROM, spasticity, selectivity and strength will be 
performed during the pre- and post-BoNT-A assessments of the intervention group and during the pre-
assessment of the control group. These data will be used to describe and compare both groups at baseline. The 
maximal dorsiflexion angle and popliteal angle (unilateral) will be assessed by goniometry. The level of spasticity 
will be assessed by the modified Ashworth scale and modified Tardieu scale. Muscle strength will be assessed by 
manual muscle testing, and selectivity will be assessed by the Selective Control Assessment of the Lower 
Extremity. The evaluations of strength and selectivity will not be included in the current investigation. 
 
5. Statistical Analysis 
Normality of data distribution will be checked by a Kolmogorov-Smirnov test (p<0.05). When most parameters 
are not normally distributed, non-parametric statistical tests will be performed. 
 
Participant characteristics, maximal dorsiflexion angle (measured with the knee extended), popliteal angle 
(unilateral), and normalized muscle morphology parameters of the intervention and control group will be 
compared at baseline by a Mann-Whitney U test. 
 
A Wilcoxon signed rank test will be used to evaluate (within) treatment effects in muscle morphology, 
instrumented spasticity assessment outcomes, spatial-temporal parameters of the 3DGAs (alpha = 0.05). A 
Bonferroni correction will be applied for the gait kinematic parameters per joint (ankle: alpha 0.05/4 = 0.0125, 
knee: alpha 0.05/3 = 0.0167). Gait parameters will be considered meaningful if statistically significant changes 
will be noted that are exceeding the SEM or MCID. 
 
Additionally, median difference scores (xpost – xpre) for muscle morphology parameters will be calculated in both 
the intervention and control group and between-group differences were tested by a Mann Whitney-U test. 
Additionally, the changes will be expressed as a percentage change of the baseline values for both groups. 
 
Paired t-tests of the ankle and knee kinematics over the entire gait cycle using SnPM analysis will be performed 
to identify treatment-induced changes in these sagittal plane motions. The averaged ankle and knee kinematic 
waveforms, consisting of 101 points will be imported to the SPM1d software (version 0.4, available for download 
at http://www.spm1d.org/) in Matlab (The MathWorks, Natick, MA, USA, version 2020b). Clusters will be formed 
if the critical threshold, based on random field theory, will be crossed (alpha = 0.05). In case of significant clusters, 
the p-value, details on the location and the duration (percentages of the entire gait cycle) will be reported. 
Clusters will be considered meaningful when their duration was exceeding 3% of the gait cycle and whether the 
differences between the means of the pre- and post-BoNT-A joint kinematics will be exceeding the respective 
SEMs.  
 
 
 

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