Fine-needle percutaneous muscle microbiopsy technique as a feasible tool to address histological analysis in young children with cerebral palsy and age-matched typically developing children.

Cerebral palsy (CP) is a heterogeneous group of motor disorders attributed to a non-progressive lesion in the developing brain. Knowledge on skeletal muscle properties is important to understand the impact of CP and treatment but data at the microscopic levels are limited and inconsistent. Currently, muscle biopsies are collected during surgery and are restricted to CP eligible for such treatment or they may refer to another muscle or older children in typically developing (TD) biopsies. A minimally invasive technique to collect (repeated) muscle biopsies in young CP and TD children is needed to provide insights into the early muscle microscopic alterations and their evolution in CP. This paper describes the protocol used to 1) collect microbiopsies of the medial gastrocnemius (MG) and semitendinosus (ST) in CP children and age-matched TD children, 2) handle the biopsies for histology, 3) stain the biopsies to address muscle structure (Hematoxylin & Eosin), fiber size and proportion (myosin heavy chain), counting of the satellite cells (Pax7) and capillaries (CD31). Technique feasibility and safety as well as staining feasibility and measure accuracy were evaluated. Two microbiopsies per muscle were collected in 56 CP (5.8±1.1 yr) and 32 TD (6±1.1 yr) children using ultrasound-guided percutaneous microbiopsy technique. The biopsy procedure was safe (absence of complications) and well tolerated (Score pain using Wong-Baker faces). Cross-sectionally orientated fibers were found in 86% (CP) and 92% (TD) of the biopsies with 60% (CP) and 85% (TD) containing more than 150 fibers. Fiber staining was successful in all MG biopsies but failed in 30% (CP) and 16% (TD) of the ST biopsies. Satellite cell staining was successful in 89% (CP) and 85% (TD) for MG and in 70% (CP) and 90% (TD) for ST biopsies, while capillary staining was successful in 88% (CP) and 100% (TD) of the MG and in 86% (CP) and 90% (TD) for the ST biopsies. Intraclass coefficient correlation showed reliable and reproducible measures of all outcomes. This study shows that the percutaneous microbiopsy technique is a safe and feasible tool to collect (repeated) muscle biopsies in young CP and TD children for histological analysis and it provides sufficient muscle tissue of good quality for reliable quantification.

Botulinum Toxin Treatment of Adult Muscle Stem Cells from Children with Cerebral Palsy and hiPSC-Derived Neuromuscular Junctions.

Botulinum neurotoxin type-A (BoNT) injections are commonly used as spasticity treatment in cerebral palsy (CP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised, and the BoNT effect on muscle stem cells remains not well defined. This study aims at clarifying the impact of BoNT on growing muscles (1) by analyzing the in vitro effect of BoNT on satellite cell (SC)-derived myoblasts and fibroblasts obtained from medial gastrocnemius microbiopsies collected in young BoNT-naïve children (t0) compared to age ranged typically developing children; (2) by following the effect of in vivo BoNT administration on these cells obtained from the same children with CP at 3 (t1) and 6 (t2) months post BoNT; (3) by determining the direct effect of a single and repeated in vitro BoNT treatment on neuromuscular junctions (NMJs) differentiated from hiPSCs. In vitro BoNT did not affect myogenic differentiation or collagen production. The fusion index significantly decreased in CP at t2 compared to t0. In NMJ cocultures, BoNT treatment caused axonal swelling and fragmentation. Repeated treatments impaired the autophagic-lysosomal system. Further studies are warranted to understand the long-term and collateral effects of BoNT in the muscles of children with CP.