The neutral posture of the cervical spine is not unique in human subjects

Cervical spine injuries often happen in dynamic environments (e.g., sports and motor vehicle crashes) where individuals may be moving their head and neck immediately prior to impact. This motion may reposition the cervical vertebrae in a way that is dissimilar to the upright resting posture that is often used as the initial position in cadaveric studies of catastrophic neck injury. Therefore our aim was to compare the “neutral” cervical alignment measured using fluoroscopy of 11 human subjects while resting in a neutral posture and as their neck passed through neutral during the four combinations of active flexion and extension movements in both an upright and inverted posture. Muscle activation patterns were also measured unilaterally using surface and indwelling electromyography in 8 muscles and then compared between the different conditions. Overall, the head posture, cervical spine alignment and muscle activation levels were significantly different while moving compared to resting upright. Compared to the resting upright condition, average head postures were 6–13° more extended, average vertebral angles varied from 11° more extended to 10° more flexed, and average muscle activation levels varied from unchanged to 10% MVC more active, although the exact differences varied with both direction of motion and orientation. These findings are important for ex vivo testing where the head and neck are statically positioned prior to impact – often in an upright neutral posture with negligible muscle forces – and suggest that current cadaveric head-first impact tests may not reflect many dynamic injury environments.     

Cervical and axioscapular muscle stiffness measured with shear wave elastography: A comparison between different levels of work-related neck disability

Assessing muscle mechanical properties in terms of stiffness may provide important insights into mechanisms underlying work-related neck pain. This study compared stiffness of cervical and axioscapular muscles between 92 participants (sonographers) with no (n = 31), mild (n = 43) or moderate/severe (n = 18) neck disability. It was hypothesized that participants with more severe neck pain and disability would present with altered distribution of stiffness in cervical and axioscapular muscles than those with no disability. Using shear wave elastography, the shear modulus (kPa) of five cervical and six axioscapular muscles or muscle segments were measured in a relaxed seated upright or side-lying position. Muscle activity was measured simultaneously using surface electromyography during the elastography measurements and scapular depression was measured using a measurement tape and inclinometer before the elastography measurements to evaluate their potential confounding influences on shear modulus. Increased shear modulus was found in deeper than superficial cervical muscles and more cranial than caudal axioscapular muscles. However, no differences in shear modulus of the cervical or axioscapular muscles were found between sonographers with varying levels of disability. This study suggests no alterations in stiffness of cervical and axioscapular muscles were associated with work-related neck pain and disability.     

Appearance of high-molecular-weight acetylcholinesterase in aneural muscle developing in vivo

Acetylcholinesterase was studied in the superior oblique muscle of the duck embryo during the course of in vivo development. Normally developing, paralyzed, and uninnervated muscles were studied using velocity sedimentation for separation of various forms and biochemical determination of enzyme activity, and light and electron microscopy for histochemical and cytochemical localization of enzyme. Results indicate that neither muscle activity nor contact by the motor neurons is essential for the appearance of high-molecular-weight form of acetylcholinesterase on muscle cells developing in vivo. Acetylcholinesterase activity per muscle was considerably lower in the paralyzed and aneural muscles than the normal muscle. The absolute loss of acetylcholinesterase parallels loss of muscle protein in paralyzed and aneural muscles and may be secondary. Paralysis or absence of innervation had no significant effect on the specific activity of acetylcholinesterase.     

Altered neuromuscular activity and postural stability during standing balance tasks in persons with non-specific neck pain

ObjectivesTo compare neck, trunk, and lower extremity muscle activity in standing in persons with neck pain (NP) to healthy controls and determine associations with postural sway.MethodsParticipants included 25 persons with NP and 25 controls. Surface electromyography was recorded bilaterally from neck (sternocleidomastoid, SCM; splenius capitis, SC; upper trapezius, UT), trunk (erector spinae, ES), and lower extremity (rectus femoris, RF; biceps femoris, BF; tibialis anterior, TA; medial gastrocnemius, GN) muscles. Postural sway was measured using a force platform in narrow stance with eyes open/closed, on firm/soft surfaces.ResultsCompared to controls, the NP group demonstrated higher activity in all muscles, except UT and had higher amplitude ratios for neck muscles (SCM, SC) for all tasks (p < .05). No between-group difference was found in amplitude ratios for lower extremity muscles, except for GN. Lower extremity activity was moderately correlated with larger postural sway for both groups (r = 0.41–0.66, p < .05). There were no correlations between sway and neck and trunk muscle activity (p > .05).ConclusionIncreased muscle activity with NP is associated with increased postural sway. Both groups used similar postural control strategies, but the increased neck activity in the NP group is likely related to the NP disorder rather than postural instability.     

Strain and load thresholds for cervical muscle recruitment in response to quasi-static tensile stretch of the caprine C5–C6 facet joint capsule

The aim of this study was to investigate the response of cervical muscles to physiologic tensile stretch of cervical facet joint capsule (FJC) at a quasi-static displacement rate of 0.5 mm/s. In vivo caprine left C5–C6 FJC preparations were subjected to an incremental tensile displacement paradigm. EMG activity was recorded during FJC stretch from the right trapezius (TR) and multifidus (MF) muscle groups at the C5 and C6 levels and bilaterally from the sternomastoid (SM) and longus colli (LC) muscle groups at the C5–C6 level. Onset of muscular activity was later analyzed using visual and computer-based methods. Capsule load and strain at the time of onset were recorded and compared between the muscle groups. Results indicated capsule load was a better indicator of the tensile stretch thresholds for muscular recruitment than capsule strain. MF responded at significantly smaller capsule loads than TR and LC, while TR and LC activation loads were not significantly different. SM did not respond to physiologic FJC stretch. Muscle group recruitment order reflected the muscles’ fiber type compositions and functional roles in the spine. This study provides the first evidence that the cervical ligamento-muscular reflex pathways are activated via tensile FJC stretch and extend to superficial and deep musculature on the anterior and posterior aspects of the neck, ipsilateral and contralateral to the side of FJC stretch.     

The relationship between flexibility and EMG activity pattern of the erector spinae muscles during trunk flexion–extension

BackgroundMovements in the lumbar spine, including flexion and extension are governed by a complex neuromuscular system involving both active and passive units. Several biomechanical and clinical studies have shown the myoelectric activity reduction of the lumbar extensor muscles (flexion–relaxation phenomenon) during lumbar flexion from the upright standing posture. The relationship between flexibility and EMG activity pattern of the erector spinae during dynamic trunk flexion–extension task has not yet been completely discovered.ObjectiveThe purpose of this study was to investigate the relationship between general and lumbar spine flexibility and EMG activity pattern of the erector spinae during the trunk flexion–extension task.MethodsThirty healthy female college students were recruited in this study. General and lumbar spine flexibilities were measured by toe-touch and modified schober tests, respectively. During trunk flexion–extension, the surface electromyography (EMG) from the lumbar erector spinae muscles as well as flexion angles of the trunk, hip, lumbar spine and lumbar curvature were simultaneously recorded using a digital camera. The angle at which muscle activity diminished during flexion and initiated during extension was determined and subjected to linear regression analysis to detect the relationship between flexibility and EMG activity pattern of the erector spinae during trunk flexion–extension.ResultsDuring flexion, the erector spinae muscles in individuals with higher toe-touch scores were relaxed in larger trunk and hip angles and reactivated earlier during extension according to these angles (P < 0.001) while in individuals with higher modified schober scores this muscle group was relaxed later and reactivated sooner in accordance with lumbar angle and curvature (P < 0.05). Toe-touch test were significantly correlated with trunk and hip angles while modified schober test showed a significant correlation with lumbar angle and curvature variables.ConclusionThe findings of this study indicate that flexibility plays an important role in trunk muscular recruitment pattern and the strategy of the CNS to provide stability. The results reinforce the possible role of flexibility alterations as a contributing factor to the motor control impairments. This study also shows that flexibility changes behavior is not unique among different regions of the body.     

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.     

A novel muscle biopsy clamp yields accurate in vivo sarcomere length values

The measurement of in vivo muscle sarcomere length facilitates the definition of in vivo muscle functional properties and comparison of muscle designs amongst functional muscle groups. In vivo sarcomere lengths are available for just a handful of human muscles, largely due to the technical challenges associated with their measurement. The purpose of this report was to develop and test a muscle biopsy clamp that can quickly and accurately measure in vivo muscle sarcomere length. To test the device, muscle biopsies (n=23) were removed from the tibialis anterior muscles of New Zealand White rabbits immediately after sarcomere length measurements were made using laser diffraction. The muscle biopsy contained within the clamp was immediately fixed in Formalin for subsequent sarcomere length measurement. Comparisons of clamp-based and diffraction-based sarcomere lengths demonstrated excellent agreement between the two techniques, especially when the biopsy was obtained at relatively long lengths (above 2.6 μm). Given the intraoperative speed and simplicity of this technique and the relatively low-cost of the biopsy clamp, this method of measuring muscle sarcomere length should help investigators generate much-needed in vivo muscle structural and functional data.     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

Thick Filaments in Unstretched Mammalian Smooth Muscle

THE controversy concerning the organization of myosin in mammalian smooth muscle was reviewed (Nature New Biology, 231, 225; 1971) at a time when the studies of Rice's laboratory and our own demonstrated a regular, quasi-rectangular array of thick filaments in guinea-pig taenia coli (TC) and rabbit portal-anterior mesenteric vein (MV), and, further, that, by excessive stretch and by the use of hypertonic incubation solutions, the thick filaments in this lattice could be aggregated into ribbon-like structures^1,2. These observations were made on muscles stretched to approximately 1.5 times their excised length. Both the TC³ and the rabbit MV^2,4 are spontaneously active smooth muscles, which shorten to less than their in vivo length when excised from the body: stretching by approximately 1.5 times brings these muscles close to their in vivo length. Nevertheless, recent reports^5,6, indicating that thick filaments were more readily visualized (but see Figs. 2 and 3 in ref. 5) in stretched smooth muscles, prompted the editorial writer of Nature (231, 423; 1971) to consider it a debatable question whether thick filaments are present in unstretched muscle. Thick filaments have been observed in relaxed muscles^1,5,6 and we now show that an array of thick filaments can also be observed in completely unstretched guinea-pig and rabbit MV smooth muscle (excised and dropped into the fixative) and that such arrays are present after two different modes of fixation.     

Effects of load inversion in cockroach walking

To examine how walking patterns are adapted to changes in load, we recorded leg movements and muscle activities when cockroaches (Periplaneta americana) walked upright and on an inverted surface. Animals were videotaped to measure the hindleg femoro-tibial joint angle while myograms were taken from the tibial extensor and flexor muscles. The joint is rapidly flexed during swing and extended in stance in upright and inverted walking. When inverted, however, swing is shorter in duration and the joint traverses a range of angles further in extension. In slow upright walking, slow flexor motoneurons fire during swing and the slow extensor in stance, although a period of co-contraction occurs early in stance. In inverted walking, patterns of muscle activities are altered. Fast flexor motoneurons fire both in the swing phase and early in stance to support the body by pulling the animal toward the substrate. Extensor firing occurs late in stance to propel the animal forward. These findings are discussed within the context of a model in which stance is divided into an early support and subsequent propulsion phase. We also discuss how these changes in use of the hindleg may represent adaptations to the reversal of the effects of gravity.     

Rho family GTPases: Making it to the third dimension

The role of Rho family GTPases in controlling the actin cytoskeleton and thereby regulating cell migration has been well studied for cells migrating on 2D surfaces. In vivo, cell migration occurs within three-dimensional matrices and along aligned collagen fibers with rather different spatial requirements. Recently, a handful of studies coupled with new approaches have demonstrated that Rho GTPases have unique regulation and roles during cell migration within 3D matrices, along collagen fibers, and in vivo. Here we propose that migration on aligned matrices facilitates spatial organization of Rho family GTPases to restrict and stabilize protrusions in the principle direction of alignment, thereby maintaining persistent migration. The result is coordinated cell movement that ultimately leads to higher rates of metastasis in vivo.     

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE*

In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.     

Development of a finite element musculoskeletal model with the ability to predict contractions of three-dimensional muscles

Representation of realistic muscle geometries is needed for systematic biomechanical simulation of musculoskeletal systems. Most of the previous musculoskeletal models are based on multibody dynamics simulation with muscles simplified as one-dimensional (1D) line-segments without accounting for the large muscle attachment areas, spatial fibre alignment within muscles and contact and wrapping between muscles and surrounding tissues. In previous musculoskeletal models with three-dimensional (3D) muscles, contractions of muscles were among the inputs rather than calculated, which hampers the predictive capability of these models. To address these issues, a finite element musculoskeletal model with the ability to predict contractions of 3D muscles was developed. Muscles with realistic 3D geometry, spatial muscle fibre alignment and muscle-muscle and muscle-bone interactions were accounted for. Active contractile stresses of the 3D muscles were determined through an efficient optimization approach based on the measured kinematics of the lower extremity and ground force during gait. This model also provided stresses and strains of muscles and contact mechanics of the muscle-muscle and muscle-bone interactions. The total contact force of the knee predicted by the model corresponded well to the in vivo measurement. Contact and wrapping between muscles and surrounding tissues were evident, demonstrating the need to consider 3D contact models of muscles. This modelling framework serves as the methodological basis for developing musculoskeletal modelling systems in finite element method incorporating 3D deformable contact models of muscles, joints, ligaments and bones.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Validation of indicators used to assess unconsciousness in veal calves at slaughter

European legislation states that after stunning regular checks should be performed to guarantee animals are unconscious between the end of the stunning process and death. When animals are killed without prior stunning these checks should be performed before the animal is released from restraint. The validity of certain indicators used to assess unconsciousness under different stunning and slaughter conditions is under debate. The aim of this study was to validate the absence of threat-, withdrawal-, corneal- and eyelid reflex as indicators to assess unconsciousness in calves subjected to different stunning and slaughter methods. Calves (201±22 kg) were randomly assigned to one of the following four treatments: (1) Captive bolt stunning followed by neck cut in an inverted position (n=25); (2) Non-stunned slaughter in an upright position (n=7); (3) Non-stunned slaughter in an inverted position (180° rotation) (n=25); (4) Non-stunned slaughter in an upright position followed by captive bolt stunning 40 s after the neck cut (n=25). Each calf was equipped with non-invasive electroencephalogram (EEG) electrodes before the slaughter procedure. All reflexes were verified once before the slaughter procedure. At the beginning of the procedure (T=0 s) calves were stunned (treatment 1) or neck cut in an upright position (treatment 2, 4) or inverted position (treatment 3). Calves of treatment 4 were captive bolt stunned 34±8 s after the neck cut. Reflexes were assessed every 20 s from T=15 s for all treatments until all reflex tests resulted in a negative response three times in a row and a flat line EEG was observed. In addition, reflexes were assessed 5 s after captive bolt stunning in calves of treatments 1 and 4. Visual assessment of changes in the amplitude and frequency of EEG traces was used to determine loss of consciousness. Timing of loss of consciousness was related to timing of loss of reflexes. After captive bolt stunning, absence of threat-, withdrawal-, corneal- and eyelid reflex indicated unconsciousness as determined by EEG recordings. After non-stunned slaughter, both threat- and withdrawal reflex were on average lost before calves were unconscious based on EEG recordings. The eyelid- and corneal reflex were on average lost after calves had lost consciousness based on EEG recordings and appeared to be distinctly conservative indicators of unconsciousness in non-stunned slaughtered calves since they were observed until 76±50 and 85±45 s (mean±SD), respectively, after EEG-based loss of consciousness.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

Heparan sulfate controls skeletal muscle differentiation and motor functions

BackgroundHeparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear.MethodsWe examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system.ResultsCRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl₂ injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells.SignificanceThese results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.