3D-Microscopic Muscle Architecture in Cerebral Palsy

Overview

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.

Background: Altered muscle properties in CP have been reported by our own as well as other research groups, macroscopically and microscopically. Hence, key experts in the field acknowledge that further progress in the understanding of the pathogenesis of altered muscle properties will be gained by performing studies that combine macro- and microscopic evaluations of muscle structures, to define different disease presentations. Previous research also showed that a variety of treatment modalities, such as muscle stretching, strengthening, or tone reduction, have beneficial effects on neuromuscular symptoms and on certain muscle properties. However, treatment outcomes are not always satisfying and seem to be muscle and patient-specific. Recent developments in instrumented assessment of these clinical symptoms highlighted their heterogeneity. By applying these novel instrumented assessments, we found that the emergence of different neuromuscular phenotypes was found, such as distinct classes of spasticity based on muscle activation patterns measured during passive stretches at different velocities. While our results highlight that these phenotypes react differently to treatment, their etiology remains unknown. These findings suggest that patient-specific treatment can be improved when tuned to more entirely defined phenotypes. A comprehensive description of the intrinsic muscle properties, including microscopic as well as macroscopic features, is an important next step to further delineate the phenotypes, such that they can support the clinical decision making. The pathogenesis of altered muscle growth in CP children remains inconclusive. There is no doubt that mature muscles adapt in response to altered use patterns30, suggesting that hampered muscle growth is a secondary process. However, many studies were restricted to children older than 2 to 4 years, and are therefore unable to systematically address the etiology of muscle deformities31. There is an urgent need to study microscopic muscle properties in young children with CP, at different ages. Also, a complete longitudinal delineation of altered muscle growth, covering an extended malleable period of childhood, is still missing. Moreover, therapy for children with CP in Western countries generally starts at a very young age and includes physiotherapy, stretching casts, orthoses and BTX injections, which are all directed at the muscle. The diverse treatment histories of the participants from previous studies most likely influenced the development of certain muscles. Of particular interest is treatment with BTX injections, which became a first-line CP therapeutic intervention to treat focal spasticity, by means of chemodenervation. While BTX treatment results in reduced muscle tone, increased joint Range Of Motion \[ROM\] and improved gait, an increasing number of publications have raised concern that BTX may compromise muscle growth. Human studies on microscopic properties post BTX are rare. So far, there are no in vitro studies on CP muscles that have investigated the direct action of BTX on adult muscle stem cells. This is actually needed, since stroke and CP patients are different (in age, growth impact and number of BTX sessions) and animal studies suggest different mechanisms of intramuscular changes post BTX between juvenile and mature muscles. Hence, there is an urgent need to thoroughly investigate the potential impact of BTX on muscle atrophy and integrity in CP using a prospective study design. Indeed, research and clinics could benefit from a more detailed and longitudinal analysis of muscle samples, where it would be possible to characterize the short- and long-term effects of BTX injections on muscle tissue and on muscle adult stem cells. Furthermore, different mechanisms underlying the altered muscle growth may also interact. Whilst the diversity of phenotypes is likely to reflect the interplay of several factors, and different pathways have been suggested as presumed key players in the pathogenesis of altered muscle properties and neuromuscular symptoms, little understanding remains on the predominant effect in specific circumstances. Literature suggests a close interplay between the ECM and SCs, such that the composition and mechanical properties of the ECM regulate SC activity and renewal; and conversely, SCs dictate ECM composition. More CP-related research is necessary to determine the importance of alterations in SCs, ICs, and ECM components, and their interplay. The etiology of CP may also be relevant to understand the pathogenesis of altered muscle properties. Although prematurity and hypoxic-ischemic injury, placental insufficiency, and prenatal infection are well-recognized causes of CP, more than 30% of the children lack traditional risk factors. For many of these cases, a genetic base to their condition is suspected. Indeed, current estimates indicate that as many as 30% of CP cases may be genetic in nature. Hence, for at least part of the CP children, some muscle properties might also be genetically driven. Moreover, specific groups of patients with Hereditary Spastic Paraplegia (HSP) present with a very similar clinical picture as the bilaterally involved patients with CP (i.e. similar symptoms of spasticity, muscle weakness, reduced muscle control and altered muscle structure) Aim: Objective 1: To study MICROSCOPIC MUSCLE PROPERTIES in CP and define their onset at an early disease stage and their progress with growth and pathology, and to delineate their potential genetic nature (≈ WP 1). Firstly, muscle biopsies will be collected to define microscopic properties of two age-groups of CP and TD children (2-5 versus 6-9 years). It is hypothesized that (1) a lower percentage of contractile material, increased collagen content, reduced number of SCs and altered fiber size distribution are observed in CP compared to TD children, and (2) these differences are age- and muscle-specific. Secondly, since knowledge on early events and a follow-up of these events are lacking at the microscopic level, repeated biopsies will be collected in two CP groups (i.e. with and without BTX treatment history) , to study changes in the microscopic properties at different time-points pre- and post BTX injections. The microbiopsies under general anaesthesia (at the time of the BTX session and 1.2 year later, i.e. at the time of the repeated BTX-session), will be performed on the medial gastrocnemius and semitendinosus, while the microbiopsies under locale aneasthesia (at 3 months pre and 3 and 6 months post BTX) will be performed only on the medial gastrocnemius. It is hypothesized that (1) BTX-injection induce immediate altered structural muscle properties and molecular changes, which partly recover within 1.2 year, combined with (2) the trajectory of changes (such as collagen content and persistence of inflammatory cells) differs between BTX-naïve patients and patients with BTX treatment history, during the 1.2-year follow-up. Thirdly, while literature suggests a close interplay between the ECM, SCs and ICs, there is a lack of CP-related research on the interaction between microscopic properties. A potential key regulatory factors that determine altered microscopic muscle properties will be defined, in particular, the relative importance of adult stem cell alterations by defining their interaction to ECM abnormalities. It is hypothesized that (1) the misbalanced interplay between adult stem cells and ECM is related to altered muscle properties, (2) the degree of misbalance evolves over time and (3) is muscle-specific. Fouthly, recent literature suggests a genetic contribution to the pathology in about 30% of the children with CP, and subgroups of patients with HSP (SPG3a and SPG4) have identified genetic problems and present with similar clinical symptoms as the children with bilateral CP. This triggers a novel research pathway on the multi-lineage differentiation of induced pluripotent stem cells in a select group of patients with proven genetic mutation. It is hypothesized that that patient-specific cell modelling reflects genetic mutations, influencing the patient phenotype. Objective 2: To delineate INTEGRATED CP PHENOTYPES prone to treatment, by identifying relations between different levels of muscle alterations (macro- and microscopic) and neuromuscular symptoms. The focus will be primarily on the symptom spasticity and on BTX treatment. (≈ WP 2). Biopsy collection in the enrolled children of WP1 will always be preceded by ultrasound measures. As part of their routine clinical follow-up, these children also receive gait analysis and clinical assessments (using standard clinical scales) of neuromuscular symptoms before the treatment. Additionally, instrumented assessments of spasticity and strength will be performed. This allows integrated analysis on multiple datasets, to delineate integrated CP phenotypes. Firstly, relations between macro- and microscopic properties and spasticity will be explored. It is hypothesized that (1) macroscopic growth is significantly related to the number and behavior of SCs \& ICs, to fiber type properties, and indirectly to ECM abnormalities, (2) the macroscopic parameter EI is associated to microscopic muscular tissue integrity, and (3) muscle properties are different between muscles classified by their spasticity patterns. Secondly, guidelines will be developed to fine-tune BTX treatment to the integrated phenotypes. It is hypothesized that (1) BTX induces altered muscle volume and integrity but does not change the number of SCs and (2) BTX response is significantly correlated to specific baseline CP phenotype markers (in particular to muscle integrity). Methods/design: To understand and define the mechanism and time course of muscle properties, different studies with different study designs will be applied. These studies are organized in two work packages, each covering one of the two specific research goals.