Measuring neuromuscular junction functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle. Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1)

A DIC based technique to measure the contraction of a skeletal muscle engineered tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response.The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based systemallows for an accurate and noninvasive measurement of biological tissues’ spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

Development and validation of a device for in vitro uniaxial cell substrate deformation with real-time strain control

Substrate deformation affects the behavior of many cell types, as for example bone, skeletal muscle and endothelial cells. Nowadays, in vitro tests are widely employed to study the mechanotransduction induced by substrate deformation. The aim of in vitro systems is to properly reproduce the mechanical stimuli sensed by the tissue in the cellular microenvironment. An accurate strain measurement and control is therefore necessary to ensure the cell sensing the proper strain for the entire treatment. Different types of in vitro systems are commercially available or custom made designed; however, none of these devices performs a real-time measurement of the induced strains. In this study, we proposed a uniaxial strain device for in vitro cell stimulation with an innovative real-time strain control. The system was designed to induce sinusoidal waveform stimulation in a huge range of amplitude and frequency, to three silicone chambers stretched by a linear actuator. The real-time strain measurement and control algorithm is based on an optical tracking method implemented in LabView 2015, and it is able adapting the input amplitude to the linear motor, if necessary, hanging the stimulation signal for about 120 ms. A validation of the strain values measured during the real-time tracking algorithm was carried out through a comparison with digital image correlation (DIC) technique. We investigated the influence of number of reference points and image size on the algorithm accuracy. Experimental results showed that the tracking algorithm allowed for a real-time measurement of the membrane longitudinal strains with a relative error of 0.3%, on average, in comparison to the strains measured with DIC in post-processing analysis. We showed a high homogeneity of the strain pattern on the entire chamber base for different stimulation conditions. Finally, as proof of concept, we employed the uniaxial strain device to induce substrate deformation on human Osteosarcoma cell line (SaOS-2). Experimental results showed a consistent cells’ change in shape in response to the mechanical strain

Measuring the maximum power of an ex vivo engineered muscle tissue with isovelocity shortening technique

The final aim of muscle tissue engineering (TE) is to create a new tissue able to restore the functionality of impaired muscles once transplanted in the site of injury. Therefore, functional contractile properties close to that of healthy muscles are desirable to allow for a good compatibility and a proper functional contribution. Since skeletal muscles deal with locomotion during their normal activity, an accurate measurement of ex vivo muscle engineered tissues' isotonic properties is crucial. In this paper, we devised an experimental system to measure the mechanical power generated by an ex vivo muscle engineered tissue, the X-MET, based on the isovelocity contraction technique. The X-MET is developed without the use of any scaffolds, so that its mechanical properties are not affected by endogenous components. Our experiments allowed for delimiting the ranges of shortening and shortening velocity for which the tissue is able to generate and maintain power for the entire stimulation, which is the condition that better reproduces muscle physiological activity. Then, we measured the power generated by the X-MET and fit the experimental results to the Hill's equation usually employed for modeling the force-velocity relationship of skeletal muscles. The use of this model yielded to the measurement of maximum power and maximum shortening velocity. Results revealed that most of the isotonic properties were consistent with that proposed in the literature for slow-twitch muscles; in particular, the X-METs were able to generate a maximum power of 2.08± 0.78 W/kg and had a maximum shortening velocity of 1.84 ± 0.57 L₀/s, on average

Reactive postural responses to continuous yaw perturbations in healthy humans: the effect of aging

Maintaining balance stability while turning in a quasi-static stance and/or in dynamic motion requires proper recovery mechanisms to manage sudden center-of-mass displacement. Furthermore, falls during turning are among the main concerns of community-dwelling elderly population. This study investigates the effect of aging on reactive postural responses to continuous yaw perturbations on a cohort of 10 young adults (mean age 28 ± 3 years old) and 10 older adults (mean age 61 ± 4 years old). Subjects underwent external continuous yaw perturbations provided by the RotoBit1D platform. Different conditions of visual feedback (eyes opened and eyes closed) and perturbation intensity, i.e., sinusoidal rotations on the horizontal plane at different frequencies (0.2 Hz and 0.3 Hz), were applied. Kinematics of axial body segments was gathered using three inertial measurement units. In order to measure reactive postural responses, we measured body-absolute and joint absolute rotations, center-of-mass displacement, body sway, and inter-joint coordination. Older adults showed significant reduction in horizontal rotations of body segments and joints, as well as in center-of-mass displacement. Furthermore, older adults manifested a greater variability in reactive postural responses than younger adults. The abnormal reactive postural responses observed in older adults might contribute to the well-known age-related difficulty in dealing with balance control during turning

A DIC Based Technique to Measure the Contraction of a Skeletal Muscle Engineered Tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response. The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based system allows for an accurate and noninvasive measurement of biological tissues' spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

Skeletal muscle is a primary target of SOD1G93A-mediated toxicity

The antioxidant enzyme superoxide dismutase 1 (SOD1) is a critical player of the antioxidative defense whose activity is altered in several chronic diseases, including amyotrophic lateral sclerosis. However, how oxidative insult affects muscle homeostasis remains unclear. This study addresses the role of oxidative stress on muscle homeostasis and function by the generation of a transgenic mouse model expressing a mutant SOD1 gene (SOD1(G93A)) selectively in skeletal muscle. Transgenic mice developed progressive muscle atrophy, associated with a significant reduction in muscle strength, alterations in the contractile apparatus, and mitochondrial dysfunction. The analysis of molecular pathways associated with muscle atrophy revealed that accumulation of oxidative stress served as signaling molecules to initiate autophagy, one of the major intracellular degradation mechanisms. These data demonstrate that skeletal muscle is a primary target of SOD1(G93A) -mediated toxicity and disclose the molecular mechanism whereby oxidative stress triggers muscle atrophy

Remodeled eX vivo muscle engineered tissue improves heart function after chronic myocardial ischemia

: The adult heart displays poor reparative capacities after injury. Cell transplantation and tissue engineering approaches have emerged as possible therapeutic options. Several stem cell populations have been largely used to treat the infarcted myocardium. Nevertheless, transplanted cells displayed limited ability to establish functional connections with the host cardiomyocytes. In this study, we provide a new experimental tool, named 3D eX vivo muscle engineered tissue (X-MET), to define the contribution of mechanical stimuli in triggering functional remodeling and to rescue cardiac ischemia. We revealed that mechanical stimuli trigger a functional remodeling of the 3D skeletal muscle system toward a cardiac muscle-like structure. This was supported by molecular and functional analyses, demonstrating that remodeled X-MET expresses relevant markers of functional cardiomyocytes, compared to unstimulated and to 2D- skeletal muscle culture system. Interestingly, transplanted remodeled X-MET preserved heart function in a murine model of chronic myocardial ischemia and increased survival of transplanted injured mice. X-MET implantation resulted in repression of pro-inflammatory cytokines, induction of anti-inflammatory cytokines, and reduction in collagen deposition. Altogether, our findings indicate that biomechanical stimulation induced a cardiac functional remodeling of X-MET, which showed promising seminal results as a therapeutic product for the development of novel strategies for regenerative medicine