Physiological Response to the 6-Minute Frame Running Test in Children and Adults With Cerebral Palsy.

PURPOSE: To determine the physiological response and association to peak oxygen uptake of the 6-minute Frame Running test (6-MFRT) in persons with cerebral palsy (CP). METHODS: Twenty-four participants with CP, Gross Motor Function Classification System II/III/IV, performed the 6-MFRT. Distance, peak heart rate (HR peak ), peak respiratory exchange ratio (RER peak ), and peak oxygen uptake ( O 2peak ) were measured. RESULTS: HR peak ranged from 146 to 201 beats per minute, RER peak from 0.94 to 1.49, 6-MFRT distance from 179 to 1220 m and O 2peak from 0.62 to 2.18 L/min. HR peak was achieved in 63%, RER peak in 71%. A strong correlation was observed between 6-MFRT and O 2peak . CONCLUSIONS: The 6-MFRT represented a (near) maximum effort for 75% of the participants and the 6-MFRT can be used to estimate oxygen consumption on an individual basis.

Gene expressions in cerebral palsy subjects reveal structural and functional changes in the gastrocnemius muscle that are closely associated with passive muscle stiffness.

Cerebral palsy (CP) is a non-progressive motor disorder that affects posture and gait due to contracture development. The purpose of this study is to analyze a possible relation between muscle stiffness and gene expression levels in muscle tissue of children with CP. Next-generation sequencing (NGS) of gene transcripts was carried out in muscle biopsies from gastrocnemius muscle (n = 13 children with CP and n = 13 typical developed (TD) children). Passive stiffness of the ankle plantarflexors was measured. Structural changes of the basement membranes and the sarcomere length were measured. Twelve pre-defined gene target sub-categories of muscle function, structure and metabolism showed significant differences between muscle tissue of CP and TD children. Passive stiffness was significantly correlated to gene expression levels of HSPG2 (p = 0.02; R2 = 0.67), PRELP (p = 0.002; R2 = 0.84), RYR3 (p = 0.04; R2 = 0.66), C COL5A3 (p = 0.0007; R2 = 0.88), ASPH (p = 0.002; R2 = 0.82) and COL4A6 (p = 0.03; R2 = 0.97). Morphological differences in the basement membrane were observed between children with CP and TD children. The sarcomere length was significantly increased in children with CP when compared with TD (p = 0.04). These findings show that gene targets in the categories: calcium handling, basement membrane and collagens, were significantly correlated to passive muscle stiffness. A Reactome pathway analysis showed that pathways involved in DNA repair, ECM proteoglycans and ion homeostasis were amongst the most upregulated pathways in CP, while pathways involved in collagen fibril crosslinking, collagen fibril assembly and collagen turnover were amongst the most downregulated pathways when compared with TD children. These results underline that contracture formation and motor impairment in CP is an interplay between multiple factors.

Reduced mitochondrial DNA and OXPHOS protein content in skeletal muscle of children with cerebral palsy.

AIM: To provide a detailed gene and protein expression analysis related to mitochondrial biogenesis and assess mitochondrial content in skeletal muscle of children with cerebral palsy (CP). METHOD: Biceps brachii muscle samples were collected from 19 children with CP (mean [SD] age 15y 4mo [2y 6mo], range 9-18y, 16 males, three females) and 10 typically developing comparison children (mean [SD] age 15y [4y], range 7-21y, eight males, two females). Gene expression (quantitative reverse transcription polymerase chain reaction [PCR]), mitochondrial DNA (mtDNA) to genomic DNA ratio (quantitative PCR), and protein abundance (western blotting) were analyzed. Microarray data sets (CP/aging/bed rest) were analyzed with a focused query investigating metabolism- and mitochondria-related gene networks. RESULTS: The mtDNA to genomic DNA ratio was lower in the children with CP compared to the typically developing group (-23%, p=0.002). Out of five investigated complexes in the mitochondrial respiratory chain, we observed lower protein levels of all complexes (I, III, IV, V, -20% to -37%; p<0.05) except complex II. Total peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) messenger RNA (p<0.004), isoforms PGC1α1 (p=0.05), and PGC1α4 (p<0.001) were reduced in CP. Transcriptional similarities were observed between CP, aging, and 90 days' bed rest. INTERPRETATION: Mitochondrial biogenesis, mtDNA, and oxidative phosphorylation protein content are reduced in CP muscle compared with typically developing muscle. Transcriptional pathways shared between aging and long-term unloading suggests metabolic dysregulation in CP, which may guide therapeutic strategies for combatting CP muscle pathology. What this paper adds Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. Gene expression in CP muscle is similar to aging and long-term bed rest.

Immobilization leads to reduced stretch reflexes but increased central reflex gain in the rat.

Plastic adaptations are known to take place in muscles, tendons, joints, and the nervous system in response to changes in muscle activity. However, few studies have addressed how these plastic adaptations are related. Thus this study focuses on changes in the mechanical properties of the ankle plantarflexor muscle-tendon unit, stretch reflex activity, and spinal neuronal pathways in relation to cast immobilization. The left rat hindlimb from toes to hip was immobilized with a plaster cast for 1, 2, 4, or 8 wk followed by acute electrophysiological recordings to investigate muscle stiffness and stretch reflex torque. Moreover, additional acute experiments were performed after 4 wk of immobilization to investigate changes in the central gain of the stretch reflex. Monosynaptic reflexes (MSR) were recorded from the L4 and L5 ventral roots following stimulation of the corresponding dorsal roots. Rats developed reduced range of movement in the ankle joint 2 wk after immobilization. This was accompanied by significant increases in the stiffness of the muscle-tendon complex as well as an arthrosis at the ankle joint at 4 and 8 wk following immobilization. Stretch reflexes were significantly reduced at 4-8 wk following immobilization. This was associated with increased central gain of the stretch reflex. These data show that numerous interrelated plastic changes occur in muscles, connective tissue, and the central nervous system in response to changes in muscle use. The findings provide an understanding of coordinated adaptations in multiple tissues and have important implications for prevention and treatment of the negative consequences of immobilization following injuries of the nervous and musculoskeletal systems.NEW & NOTEWORTHY Immobilization leads to multiple simultaneous adaptive changes in muscle, connective tissue, and central nervous system.

Multi-frequency bioimpedance: a non-invasive tool for muscle-health assessment of adults with cerebral palsy.

Muscle contracture development is a major complication for individuals with cerebral palsy (CP) and has lifelong implications. In order to recognize contracture development early and to follow up on preventive interventions aimed at muscle health development, non-invasive, and easy to use methods are needed. The aim of the present study was to assess whether multi-frequency Bioimpedance (mfBIA) can be used to detect differences between skeletal muscle of individuals with CP and healthy controls. The mfBIA technique was applied to the medial gastrocnemius muscle of n = 24 adults with CP and n = 20 healthy controls of both genders. The phase angle (PA) and the centre frequency (fc) were significantly lower in individuals with CP when compared to controls; PA: - 25% for women and - 31.8% for men (P < 0.0001); fc: - 5.6% for women and - 5.2% for men (P < 0.009). The reactance (Xc) and the extracellular resistance (Re) of skeletal muscle from individuals with CP were significantly higher when compared to controls; Xc: + 9.9% for women and + 28.9% for men (P < 0.0001); Re: + 39.7% for women and + 91.2% for men (P < 0.0001). The present study shows that several mfBIA parameters differ significantly between individuals with CP and healthy controls. Furthermore, these changes correlated significantly with the severity of CP, as assessed using the GMFCS scale. The present data indicate that mfBIA shows promise in terms of being a useful diagnostic tool, capable of characterizing muscle health and its development in individuals with cerebral palsy.

An acoustic myography functional assessment of cerebral palsy subjects compared to healthy controls during physical exercise.

Individuals with cerebral palsy (CP) participate in reduced levels of physical activity and spend an increased amount of time in a sedentary state compared with healthy control subjects. Whether this in part can be explained by impaired muscle function is still unclear. The aim of the present study was to elucidate differences in muscle fibre recruitment during treadmill exercise between CP subjects and healthy age-, sex- and BMI-matched controls. This is a case-control study. Acoustic myography (AMG), a method recording fibre use and efficiency from contracting muscles, was applied during a period of treadmill exercise. The recorded AMG parameters revealed that the CP subjects had a significantly lower initial S-score (spatial summation) than the controls (P < 0.01). However, the T-score (temporal summation) and the E-score (efficiency) showed no significant differences between individuals with CP and the healthy control subjects. The present findings indicate that CP subjects use a higher degree of spatial summation (more fibres recruited) to keep up the same speed during treadmill exercise when compared to healthy matched control subjects. Our results suggest that individuals with CP have a tendency to recruit far more muscle fibres during bouts of exercise than healthy individuals. This may partly explain why CP subjects experience premature fatigue.

Sequence variants in muscle tissue-related genes may determine the severity of muscle contractures in cerebral palsy.

Muscle contractures are a common complication to cerebral palsy (CP). The purpose of this study was to evaluate whether individuals with CP carry specific gene variants of important structural genes that might explain the severity of muscle contractures. Next-generation-sequencing (NGS) of 96 candidate genes associated with muscle structure and metabolism were analyzed in 43 individuals with CP (Gross Motor Function classification system [GMFCS] I, n=10; GMFCS II, n=14; GMFCS III, n=19) and four control participants. In silico analysis of the identified variants was performed. The variants were classified into four categories ranging from likely benign (VUS0) to highly likely functional effect (VUS3). All individuals with CP were classified and grouped according to their GMFCS level: Statistical comparisons were made between GMFCS groups. Kruskal-Wallis tests showed significantly more VUS2 variants in the genes COL4 (GMFCS I-III; 1, 1, 5, respectively [p < .04]), COL5 (GMFCS I-III; 1, 1, 5 [p < .04]), COL6 (GMFCS I-III; 0, 4, 7 [p < .003]), and COL9 (GMFCS I-III; 1, 1, 5 [p < .04]), in individuals with CP within GMFCS Level III when compared to the other GMFCS levels. Furthermore, significantly more VUS3 variants in COL6 (GMFCS I-III; 0, 5, 2 [p < .01]) and COL7 (GMFCS I-III; 0, 3, 0 [p < .04]) were identified in the GMFCS II level when compared to the other GMFCS levels. The present results highlight several candidate gene variants in different collagen types with likely functional effects in individuals with CP.

Tendinosis-like changes in denervated rat Achilles tendon.

BACKGROUND: Tendon disorders are common and lead to significant disability and pain. Our knowledge of the 'tennis elbow', the 'jumpers knee', and Achilles tendinosis has increased over the years, but changes in denervated tendons is yet to be described in detail. The aim of the present study was to investigate the morphological and biochemical changes in tendon tissue following two weeks of denervation using a unilateral sciatic nerve transection model in rat Achilles tendons. METHODS: Tendons were compared with respect to cell number, nuclear roundness, and fiber structure. The non-denervated contralateral tendon served as a control. Also, the expression of neuromodulators such as substance P and its preferred receptor neurokinin-1 receptor, NK-1R, was evaluated using real-time qRT-PCR. RESULTS: Our results showed that denervated tendons expressed morphological changes such as hypercellularity; disfigured cells; disorganization of the collagen network; increased production of type III collagen; and increased expression of NK-1R. CONCLUSION: Taken together these data provide new insights into the histopathology of denervated tendons showing that denervation causes somewhat similar changes in the Achilles tendon as does tendinosis in rats.

Muscle disuse caused by botulinum toxin injection leads to increased central gain of the stretch reflex in the rat.

Botulinum toxin (Btx) is used in children with cerebral palsy and in other neurological patients to diminish spasticity and reduce the risk of development of contractures. We investigated changes in the central gain of the stretch reflex circuitry in response to Btx injection in the triceps surae muscle in rats. Experiments were performed in 21 rats. Eight rats were a control group, and 13 rats were injected with 6 IU of Btx in the left triceps surae muscle. Two weeks after Btx injection, larger monosynaptic reflexes (MSR) were recorded from the left (injected) than the right (noninjected) L4 + L5 ventral roots following stimulation of the corresponding dorsal roots. A similar increase on the left side was observed in response to stimulation of descending motor tracts, suggesting that increased excitability of spinal motor neurons may at least partly explain the increased reflexes. However, significant changes were also observed in postactivation depression of the MSR, suggesting that plastic changes in transmission from Ia afferent to the motor neurons also may be involved. The data demonstrate that muscle paralysis induced by Btx injection is accompanied by plastic adaptations in the central stretch reflex circuitry, which counteract the antispastic effect of Btx.NEW & NOTEWORTHY Injection of botulinum toxin into ankle muscles causes increased gain of stretch reflex. This is caused by adaptive changes in regulation of transmitter release from Ia afferents and increased excitability of spinal motor neurons.

Are mechanically sensitive regulators involved in the function and (patho)physiology of cerebral palsy-related contractures?

Skeletal muscle tissue is mechanosensitive, as it is able to sense mechanical impacts and to translate these into biochemical signals making the tissue adapt. Among its mechanosensitive nature, skeletal muscle tissue is the largest metabolic organ of the human body. Disturbances in skeletal muscle mechanosensing and metabolism cause and contribute to many diseases, i.e. muscular dystrophies/myopathies, cardiovascular diseases, COPD or diabetes mellitus type 2. A less commonly focused muscle-related disorder is clinically known as muscle contractures that derive from cerebral palsy (CP) conditions in young and adults. Muscle contractures are characterized by gradually increasing passive muscle stiffness resulting in complete fixation of joints. Different mechanisms have been identified in CP-related contractures, i.e. altered calcium handling, altered metabolism or altered titin regulation. The muscle-related extracellular matrix (ECM), specifically collagens, plays a role in CP-related contractures. Herein, we focus on mechanically sensitive complexes, known as costameres (Cstms), and discuss their potential role in CP-related contractures. We extend our discussion to the ECM due to the limited knowledge of its role in CP-related contractures. The aims of this review are (1) to summarize CP-related contracture mechanisms, (2) to raise novel hypotheses on the genesis of contractures with a focus on Cstms, and (3) to stimulate novel approaches to study CP-related contractures.

Botulinum toxin injection causes hyper-reflexia and increased muscle stiffness of the triceps surae muscle in the rat.

Botulinum toxin is used with the intention of diminishing spasticity and reducing the risk of development of contractures. Here, we investigated changes in muscle stiffness caused by reflex activity or elastic muscle properties following botulinum toxin injection in the triceps surae muscle in rats. Forty-four rats received injection of botulinum toxin in the left triceps surae muscle. Control measurements were performed on the noninjected contralateral side in all rats. Acute experiments were performed, 1, 2, 4, and 8 wk following injection. The triceps surae muscle was dissected free, and the Achilles tendon was cut and attached to a muscle puller. The resistance of the muscle to stretches of different amplitudes and velocities was systematically investigated. Reflex-mediated torque was normalized to the maximal muscle force evoked by supramaximal stimulation of the tibial nerve. Botulinum toxin injection caused severe atrophy of the triceps surae muscle at all time points. The force generated by stretch reflex activity was also strongly diminished but not to the same extent as the maximal muscle force at 2 and 4 wk, signifying a relative reflex hyperexcitability. Passive muscle stiffness was unaltered at 1 wk but increased at 2, 4, and 8 wk (P < 0.01). These data demonstrate that botulinum toxin causes a relative increase in reflex stiffness, which is likely caused by compensatory neuroplastic changes. The stiffness of elastic elements in the muscles also increased. The data are not consistent with the ideas that botulinum toxin is an efficient antispastic medication or that it may prevent development of contractures.

3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling.

Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.

Acute exercise improves motor memory: exploring potential biomarkers.

We have recently shown that a single bout of acute cardiovascular exercise improves motor skill learning through an optimization of long-term motor memory. Here we expand this previous finding, to explore potential exercise-related biomarkers and their association with measures of motor memory and skill acquisition. Thirty-two healthy young male subjects were randomly allocated into either an exercise or control group. Following either an intense bout of cycling or rest subjects practiced a visuomotor tracking task. Motor skill acquisition was assessed during practice and retention 1 h, 24 h and 7 days after practice. Plasma levels of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), epinephrine, norepinephrine, dopamine and lactate were analyzed at baseline, immediately after exercise or rest and during motor practice. The exercise group showed significantly better skill retention 24h and 7 days after acquisition. The concentration of all blood compounds increased significantly immediately after exercise and remained significantly elevated for 15 min following exercise except for BDNF and VEGF. Higher concentrations of norepinephrine and lactate immediately after exercise were associated with better acquisition. Higher concentrations of BDNF correlated with better retention 1 h and 7 days after practice. Similarly, higher concentrations of norepinephrine were associated with better retention 7 days after practice whereas lactate correlated with better retention 1h as well as 24 h and 7 days after practice. Thus, improvements in motor skill acquisition and retention induced by acute cardiovascular exercise are associated with increased concentrations of biomarkers involved in memory and learning processes. More mechanistic studies are required to elucidate the specific role of each biomarker in the formation of motor memory.

Determinants of Frame Running Capacity in Athletes With Cerebral Palsy to Improve Training Routines and Classification Strategies: A Cross-sectional Observational Study.

OBJECTIVES: The aim of the study were to (1) investigate what physical and physiological parameters are most important for Frame Running capacity, a parasport for individuals with ambulatory difficulties, and (2) determine whether Frame Running capacity can be predicted in athletes with cerebral palsy. DESIGN: Athletes with cerebral palsy ( N = 62, Gross Motor Classification System I-V; 2/26/11/21/2) completed a 6-min Frame Running test. Before the 6-min Frame Running test, muscle thickness, passive range of motion (hip, knee, ankle), selective motor control, and spasticity (hip, knee, ankle) were measured in both legs. In total, 54 variables per individual were included. Data were analyzed using correlations, principal component analysis, orthogonal partial least square regression, and variable importance in projection analysis. RESULTS: The mean 6-min Frame Running test distance was 789 ± 335 m and decreased with motor function severity. The orthogonal partial least square analysis revealed a modest degree of covariance in the variables analyzed and that the variance in the 6-min Frame Running test distance could be predicted with 75% accuracy based on all the variables measured. Variable importance in projection analysis indicated hip and knee extensor spasticity (negative effect), and muscle thickness (positive effect) arose as the most important factors contributing to Frame Running capacity. CONCLUSIONS: These results are an important resource to enable optimization of training regimes to improve Frame Running capacity and contribute to evidence-based and fair classification for this parasport.

No inflammatory gene-expression response to acute exercise in human Achilles tendinopathy.

Although histology data favour the view of a degenerative nature of tendinopathy, indirect support for inflammatory reactions to loading in affected tendons exists. The purpose of the present study was to elucidate whether inflammatory signalling responses after acute mechanical loading were more pronounced in tendinopathic versus healthy regions of human tendon and if treatment with non-steroidal anti-inflammatory medications (NSAID's) reduces this response. Twenty-seven tendinopathy patients (>6 months) were randomly assigned to a placebo (n = 14) or NSAID (Ibumetin NYCOMED GmbH Plant Oranienburg Germany (600 mg) × 3/day/1 week) group (n = 13) in a double-blinded-fashion. Tendon biopsies were taken from the painful and a healthy region of the same tendon 2 h after 1 h running. Gene-expression of several targets was analysed in the sampled Achilles tendon biopsies. The mRNA for TGF-ß, collagen-I and collagen-III were significantly higher expressed, and decorin, CTGF, IL-6 and IL-10 were significantly lower expressed in the tendinopathic versus healthy tendon area. Only IL-10 was lower in expression in experiments with NSAID administration, while all other determined parameters were unaffected by NSAID. All ultrasonographic outcomes were unchanged in response to acute exercise and not influenced by NSAID. The signalling for collagen and TGF-beta was upregulated after acute loading in tendinopathic tendon. In contrast to the hypothesis, inflammatory signalling was not exaggerated in tendinopathic tendon 2 h after acute mechanical loading.

Altered gene expression levels of genes related to muscle function in adults with cerebral palsy.

Cerebral palsy (CP) is the most common cause of movement disorders in children. Next generation sequencing (NGS) studies have previously shown that expression levels are fundamentally different in children with CP compared to typically developing (TD). However, given that children are in full development, we might expect gene expression levels to change once maturity is reached. Therefore, the main purpose of this study was to investigate gene expression levels of 93 target genes in adults with CP using NGS on muscle biopsies of the gastrocnemius, taken from 22 participants (n = 12 adults with CP; n = 10 TD adults). Subsequently, we carried out NGS of the mitochondrial genome to identify mtDNA variants, and additionally we studied the mitochondrial content using transmission electron microscopy images of the gastrocnemius muscle. Finally, we compared systemic ion levels between TD adults and adults with CP. Differential gene expression levels were found in genes involved in muscle contraction (MYH1 and MYBPC2), mitochondrial function kATP5J, CYCS and NDUFB6), calcium handling (CAMK2B and ATP2A), metabolism (LPL), muscle signaling (MYC, CREB1, ACVR2B, LMNA and TRIM54), and ECM (TNC). There was no statistical significant difference between CP and TD for mtDNA variant frequencies and mitochondrial content. The ion levels of Ca2+, Na+ and K+ were statistically significantly reduced while the Cl- levels were significant increased in adults with CP compared to TD adults. These results highlight that most transcriptional differences are related to muscle function in adults with CP and that mitochondrial function might be altered but not mitochondrial content.

The Health Effects of 14 Weeks of Physical Activity in a Real-Life Setting for Adults with Intellectual Disabilities.

Background: The life expectancy of individuals with intellectual disabilities (ID) is reduced compared to the general population, and one of the main contributors to earlier death is inactivity. Aim: To investigate how 14 weeks of physical activity (PA) in a real-life setting affects cardiovascular fitness, body composition and bone health of adults with ID. Methods: Adults with ID were recruited into a PA-group (N = 52) or a control group (CON, N = 14). The PA-group participated in 14 weeks of PA, and body composition, cardiovascular fitness and bone health were assessed before and after the intervention. Outcomes and Results. Cardiovascular fitness and body composition improved from pre to post within the PA-group: Heart rates (HR) during the last 30 seconds of two increments of a treadmill test, were reduced (3.2 km/h: -4.4 bpm, p < 0.05; 4.8 km/h: -7.5 bpm, p < 0.001) and fat mass was reduced (-1.02 kg, p < 0.05). A between-group difference in favour of the PA-group, were observed in whole body bone mineral density (BMD) (0.024 g/cm2, p < 0.05). Conclusions and Implications. Fourteen weeks of PA performed in a real-life setting increased cardiovascular fitness, reduced fat mass and improved BMD in the weight-bearing skeleton in the PA-group. Increased and regular PA seems to be a promising tool to promote physical health in adults with ID.

3D synchrotron imaging of muscle tissues at different atrophic stages in stroke and spinal cord injury: a proof-of-concept study.

Synchrotron X-ray computed tomography (SXCT) allows 3D imaging of tissue with a very large field of view and an excellent micron resolution and enables the investigation of muscle fiber atrophy in 3D. The study aimed to explore the 3D micro-architecture of healthy skeletal muscle fibers and muscle fibers at different stages of atrophy (stroke sample = muscle atrophy; spinal cord injury (SCI) sample = severe muscle atrophy). Three muscle samples: a healthy control sample; a stroke sample (atrophic sample), and an SCI sample (severe atrophic sample) were imaged using SXCT, and muscle fiber populations were segmented and quantified for microarchitecture and morphology differences. The volume fraction of muscle fibers was 74.7%, 70.2%, and 35.3% in the healthy, stroke (atrophic), and SCI (severe atrophic) muscle fiber population samples respectively. In the SCI (severe atrophic sample), 3D image analysis revealed fiber splitting and fiber swelling. In the stroke sample (atrophic sample) muscle fiber buckling was observed but was only visible in the 3D analysis. 3D muscle fiber population analysis revealed new insights into the different stages of muscle fiber atrophy not to be observed nor quantified with a 2D histological analysis including fiber buckling, loss of fibers and fiber splitting.

Extracellular vesicle characteristics and microRNA content in cerebral palsy and typically developed individuals at rest and in response to aerobic exercise.

In this study, the properties of circulating extracellular vesicles (EVs) were examined in cerebral palsy (CP) and typically developed (TD) individuals at rest and after aerobic exercise, focusing on the size, concentration, and microRNA cargo of EVs. Nine adult individuals with CP performed a single exercise bout consisting of 45 min of Frame Running, and TD participants completed either 45 min of cycling (n = 10; TD EX) or were enrolled as controls with no exercise (n = 10; TD CON). Blood was drawn before and 30 min after exercise and analyzed for EV concentration, size, and microRNA content. The size of EVs was similar in CP vs. TD, and exercise had no effect. Individuals with CP had an overall lower concentration (∼25%, p < 0.05) of EVs. At baseline, let-7a, let-7b and let-7e were downregulated in individuals with CP compared to TD (p < 0.05), while miR-100 expression was higher, and miR-877 and miR-4433 lower in CP compared to TD after exercise (p < 0.05). Interestingly, miR-486 was upregulated ∼2-fold in the EVs of CP vs. TD both at baseline and after exercise. We then performed an in silico analysis of miR-486 targets and identified the satellite cell stemness factor Pax7 as a target of miR-486. C2C12 myoblasts were cultured with a miR-486 mimetic and RNA-sequencing was performed. Gene enrichment analysis revealed that several genes involved in sarcomerogenesis and extracellular matrix (ECM) were downregulated. Our data suggest that circulating miR-486 transported by EVs is elevated in individuals with CP and that miR-486 alters the transcriptome of myoblasts affecting both ECM- and sarcomerogenesis-related genes, providing a link to the skeletal muscle alterations observed in individuals with CP.

Epigenetic Marks at the Ribosomal DNA Promoter in Skeletal Muscle Are Negatively Associated With Degree of Impairment in Cerebral Palsy.

Introduction: Cerebral palsy (CP) is the most common motor impairment in children. Skeletal muscles in individuals with CP are typically weak, thin, and stiff. Whether epigenetic changes at the ribosomal DNA (rDNA) promoter are involved in this dysregulation remains unknown. Methods: Skeletal muscle samples were collected from 19 children with CP and 10 typically developed (TD) control children. Methylation of the rDNA promoter was analyzed using the Agena Epityper Mass array and gene expression by qRT-PCR. Results: Biceps brachii muscle ribosome biogenesis was suppressed in CP as compared to TD. Average methylation of the rDNA promoter was not different between CP and TD but negatively correlated to elbow flexor contracture in the CP group. Discussions: We observed a negative correlation between rDNA promoter methylation and degree of muscle contracture in the CP group. Children with CP with more severe motor impairment had less methylation of the rDNA promoter compared to less affected children. This finding suggests the importance of neural input and voluntary muscle movements for promoter methylation to occur in the biceps muscle.