Histological analysis of the medial gastrocnemius muscle in young healthy children.

Introduction: Histological data on muscle fiber size and proportion in (very) young typically developing (TD) children is not well documented and data on capillarization and satellite cell content are also lacking. Aims: This study investigated the microscopic properties of the medial gastrocnemius muscle in growing TD children, grouped according to age and gender to provide normal reference values in healthy children. Methods: Microbiopsies of the medial gastrocnemius (MG) muscle were collected in 46 TD boys and girls aged 2-10 years subdivided into 4 age groups (2-4, 4-6, 6-8 and 8-10 years). Sections were immunostained to assess fiber type cross-sectional area (fCSA) and proportion, the number of satellite cells (SC), capillary to fiber ratio (C/F), capillary density for type I and II fiber (CFD), capillary domain, capillary-to-fiber perimeter exchange index (CFPE) and heterogeneity index. fCSA was normalized to fibula length2 and the coefficient of variation (CV) was calculated to reflect fCSA intrasubject variability. Results: Absolute fCSA of all fibers increased with age (r = 0.72, p < 0.001) but more in boys (+112%, p < 0.05) than in girls (+48%, p > 0.05) Normalized fCSA, CV and fiber proportion did not differ between age groups and gender. C/F was strongly correlated with age in boys (r = 0.83, p < 0.001), and to a lesser extent in girls (r = 0.37, p = 0.115), while other capillary parameters as well as the number of SC remained stable with increasing age in boys and girls. Discussion: This study provides reference values of histological measures in MG according to age in normally growing boys and girls. These data may be used as a reference to determine disease impact and efficacy of therapeutic approach on the muscle.

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

Botulinum toxin-A (BoNT-A) injection is known to exert beneficial effects on muscle tone, joint mobility and gait in children with cerebral palsy (CP). However, recent animal and human studies have raised the concern that BoNT-A might be harmful to muscle integrity. In CP-children, the impact of BoNT-A on muscle structure has been poorly studied, and inconsistent results have been reported. This study was aimed at determining the time course effect of a single BoNT-A administration on medial gastrocnemius (MG) morphology in CP-children. MG microbiopsies from 12 ambulant and BoNT-A-naïve CP-children (age, 3.4 (2.3) years, ranging from 2.5 to 7.8 years; seven boys and five girls; GMFCS I = 5, II = 4 and III = 3) were collected before and 3 and 6 months after BoNT-A treatment to analyze the fiber cross-sectional area (fCSA) and proportion; capillarization; and satellite cell (SC) content. Compared with the baseline, the fCSA decreased at 3 months (-14%, NS) and increased at 6 months (+13%, NS). Fiber size variability was significantly higher at 3 months (type I: +56%, p = 0.032; type IIa: +37%, p = 0.032) and 6 months (type I: +69%, p = 0.04; type IIa: +121%, p = 0.032) compared with the baseline. The higher type I proportion seen at 3 months was still present and more pronounced at 6 months (type I: +17%, p = 0.04; type IIx: -65%, p = 0.032). The capillary fiber density was reduced at 3 months (type I: -43%, NS; type II: -44%, p = 0.0320) but normalized at 6 months. There was a non-significant increase in SC/100 fibers at 3 months (+75%, NS) and 6 months (+40%, NS) compared with the baseline. These preliminary data suggest that BoNT-A induced alterations in the MG of children with CP, which were still present 6 months after BoNT-A injection but with signs of muscle recovery.

Histological analysis of the gastrocnemius muscle in preschool and school age children with cerebral palsy compared with age-matched typically developing children.

Inconsistent alterations in skeletal muscle histology have been reported in adolescents with cerebral palsy (CP) and whether alterations are present in young children and differ from older children is not yet known. This study aimed to define histological alterations in the medial gastrocnemius (MG) of ambulant CP (gross-motor classification system, GMFCS I-III) stratified in two age groups (preschool children, PS: 2-5 and school age children, SA: 6-9-yr old) compared with age-matched typically developing (TD) children. We hypothesized that alterations in muscle microscopic properties are already present in PS-CP and are GMFCS level specific. Ultrasound guided percutaneous microbiopsies were collected in 46 CP (24-PS) and 45 TD (13-PS) children. Sections were stained to determine fiber cross-sectional area (fCSA) and proportion, capillary, and satellite cell amount. Average absolute and normalized fCSA were similar in CP and TD, but a greater percentage of smaller fibers was found in CP. Coefficient of variation (CV) was significantly larger in PS-CP-GMFCS I-II and for type I fiber. In SA-CP, all fiber types contributed to the higher CV. Type IIx proportion was higher and type I was lower in PS-CP-GMFCS-III and for all SA-CP. Reduced capillary-to-fiber ratio was present in PS-CP-GMFCS II-III and in all SA-CP. Capillary fiber density was lower in SA-CP. Capillary domain was enhanced in all CP, but capillary spatial distribution was maintained as was satellite cell content. We concluded that MG histological alterations are already present in very young CP but are only partly specific for GMFCS level and age.NEW & NOTEWORTHY Inconsistent histological alterations have been reported in children with cerebral palsy (CP) but whether they are present in very young and ambulant CP children and differ from those reported in old CP children is not known. This study highlighted for the first time that enhanced muscle fiber size variability and loss of capillaries are already present in very young CP children, even in the most ambulant ones, and these alterations seem to extend with age.

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.