3D-Microscopic Muscle Architecture in Cerebral Palsy

Condition(s) studied

Study summary

The focus of this study is to understand and define the mechanisms of the altered muscle development and growth on a microscopic level within a long-term perspective in children with cerebral palsy and to relate these findings to muscle macroscopic properties defined by muscle imaging, to neuromuscular symptoms and to treatment.

This study aims to (1) evaluate intrinsic microscopic muscle properties of young growing children with CP, and (2) to evaluate these muscle properties in relation to macroscopic properties, neuromuscular symptoms and to treatment.

Improved understanding of changes in microscopic muscle properties, and how they relate to macroscopic properties and to the neuromuscular symptoms as well as how they are influenced by treatment, has the potential to delineate CP phenotypes prone to intervention and to optimize treatment protocols or develop new treatments, leading to new avenues for improving function in CP.

The method to study microscopic muscle properties involves analysis of muscle biopsies (histological / immunohistochemistry analysis, SC and IC culture, gene expression). Biopsies will be collected using the minimally invasive percutaneous needle microbiopsy technique, suitable for collecting repeated samples over time in the same individual while still leading to sufficient tissue of good quality for subsequent analysis1,2. For the children with CP, the local hospital's tradition of applying general anesthesia for delivering BTX injections will be exploited to collect the muscle samples prior to the BTX session and the one-year follow-up, or general anesthesia planned for orthopedic surgery or diagnostic imaging such as MRI, etc. For the collection of the samples 3 months before the BTX session, as well as 3 months and 6 months after BTX injections, the common approach for microbiospy collection will be applied, with local sedation on the skin (Rapydan©) and fascia (Xylocaine©) and local anesthesia by using Kalinox© (nitrous oxide in oxygen) under supervision of the University Hospital PROSA team. Biopsies of TD muscles will be collected in children with no history of neurological disorder, nor musculoskeletal problems at the level of the gastrocnemius or semitendinosus, at the time of upper limb orthopedic or trauma surgery and thus always under general anesthesia. Ultrasound guided percutaneous muscle biopsy has been performed in children (2 months-18 years)3,4 and has proven to be safe and well-tolerated. A pilot study (S61110) was conducted to confirm that the microbiopsy technique is suitable for the analysis of microscopic muscle properties and is well-tolerated in children with CP.

Two specific research goals are planned, with hypotheses emerging from literature.

Eligibility

Primary outcome measure(s)

• Muscle fiber size — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Cross sectional area in µm of the fiber. Muscle fiber size will be assessed on cryosection stained with an antibody cocktail specific to laminin, myosin heavy chain (MHC)-I, MHC-IIA, MHC-IIB, MHC-embryonal and immunofluorescent detection will be done using appropriate secondary antibodies.
• Muscle fibre type proportion — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  For each muscle, muscle fiber proportion will be assessed on cryosection stained with an antibody cocktail specific to laminin, myosin heavy chain (MHC)-I, MHC-IIA, MHC-IIB, MHC-embryonal and immunofluorescent detection will be done using appropriate secondary antibodies.
• Capillary density — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Capillaries /mm2 fiber and capillary to fiber ratio. Capillaries will be detected in-situ. Capillaries will be assessed using CD31 staining.
• Satellite cell density — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Number of satellite cells/mm2. Satellite cells will be detected in-situ. Co-localization of satellite cells will be assessed using Pax7 staining in conjunction with MHC-I/laminin, DAPI and CD31. Abundance of satellite cell content (Pax7+/DAPI+) will be counted and their association with the different fiber types will be identified.
• Myogenic differentiation potential by means of fusion index for SC (satellite cells) — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  SCs will be isolated by Fluorescence-Activated Cell Sorting (FACs) based on the presence of surface markers CD56.Behaviour of SCs will be analysed using a cell proliferation assay and a myogenic or adipogenic differentiation assay.
• Myogenic differentiation potential by means of fusion index for MABs (mesoangioblasts) — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  MABs will be isolated by Fluorescence-Activated Cell Sorting (FACs) based on the presence of surface markers ALP.
• Overall change in muscle belly length of the medial gastrocnemius muscle and the semitendinosus muscle — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Estimation of the muscle belly length by 3DfUS. Muscle volume will be normalized to anthropometric growth.
• Overall change in muscle volume of the medial gastrocnemius muscle and the semitendinosus muscle — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Estimation of the muscle belly volume by 3DfUS. Muscle volume will be normalized to anthropometric growth.
• Overall changes in muscle activation patterns — Through a study participation of 1,2 years depending on the trajectory: 1 evaluation moment at baseline, 1 follow-up 1,2 years after, or 5 evaluations: 3 months pre, baseline, 3 months after, 6 months after and 1,2 years after baseline
  Estimation of the muscle activation patterns using instrumented spasticity assessment.

Trial sites (1)

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