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.
