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

Abstract

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.

Short-Term Effects of Botulinum Toxin-A Injection on the Medial Gastrocnemius Histological Features in Ambulant Children with Cerebral Palsy: A Longitudinal Pilot Study

“…Microbiopsies from the muscle belly of the MG were obtained using a disposable core biopsy instrument (Bard ® Mission™ Disposable Core Biopsy Instrument 16G, Bard Benelux, Olen, Belgium) under ultrasound guidance, as previously described [ 60 ]. A baseline biopsy was collected when the child was under general anesthesia just before BoNT-A (Botox ® , Allergan) injection ( Figure 7 ).…”

“…Fiber types were identified through myosin heavy chain (MHC) staining; capillaries with CD31; and satellite cells (SC) with PAX7 [ 41 , 60 ]. For capillaries and SC staining, slides were first fixed for 10 min in cold acetone and washed.…”

“…Both capillary and SC staining were additionally incubated for 1 min with DAPI (D1306, Thermo Fisher Scientific, Dilbeek, Belgium) and washed. All slides were then mounted using ProLong ® Gold antifade reagent (Molecular Probes, Thermo Fisher Scientific, Dilbeek, Belgium) [ 60 ].…”

The relation between macro- and microscopic intrinsic muscular alterations of the medial gastrocnemius in children with spastic cerebral palsy

“…Muscle biopsy collection and analysis were performed, as described previously (Deschrevel et al, 2023b). Briefly, percutaneous microscopic biopsies of the MG were collected via guided ultrasonography during procedures that required general anaesthesia (as specified above).…”

“…This is probably due to the difficulty of collecting muscle biopsies, especially in young children and more particularly in age-matched TD children. To partially encounter these methodological problems, we adapted an existing micro biopsy technique (Deschrevel et al, 2023b), and recently collected biopsies of the medial gastrocnemius (MG) in an extended group of children with CP and age-matched TD children. This allowed to comprehensively describe the microscopic MG properties of 46 ambulant children with CP, aged 2 to 9 years, and from 45 age-matched TD children (Deschrevel et al, 2023a).…”