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

Pharmacological inhibition of PKCθ counteracts muscle disease in a mouse model of duchenne muscular dystrophy

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease

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

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

Identification of the best stimulation parameters to measure in situ the comunication between muscle and nerve in mouse tibialis muscle

Investigating the path functionality of the nerve stimulation signal and the muscle contraction is of primary importance in the study of a wide variety of pathologic conditions: neuromuscular diseases like Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy, as well as acute denervation and aging. Alterations of coupling between motor neuron conduction and muscle contraction can be studied in mice, comparing the muscle contraction elicited by two alternating stimulation paradigms: direct stimulation on the membrane and indirect stimulation through the nerve. The fundamental assumption behind this approach is that in a healthy model the two stimulations should lead to the same contractile response of the muscle. In this work we have searched for the pulse stimulation parameters that better resemble the physiological action potential. Applying these optimized stimulations it is then possible to design new final protocols to evaluate all the contractile parameters of muscle tissue in a wide variety of pathological models

Measuring neuromuscular junction functionality in the SOD1G93A animal model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to motor neuron degeneration, alteration in neuromuscular junctions (NMJs), muscle atrophy, and paralysis. To investigate the NMJ functionality in ALS we tested, in vitro, two innervated muscle types excised from SOD1G93A transgenic mice at the end-stage of the disease: the Soleus, a postural muscle almost completely paralyzed at that stage, and the diaphragm, which, on the contrary, is functional until death. To this aim we employed an experimental protocol that combined two types of electrical stimulation: the direct stimulation and the stimulation through the nerve. The technique we applied allowed us to determine the relevance of NMJ functionality separately from muscle contractile properties in SOD1G93A animal model. Functional measurements revealed that the muscle contractility of transgenic diaphragms is almost unaltered in comparison to control muscles, while transgenic Soleus muscles were severely compromised. In contrast, when stimulated via the nerve, both transgenic muscle types showed a strong decrease of the contraction force, a slowing down of the kinetic parameters, as well as alterations in the neurotransmission failure parameter. All together, these results confirm a severely impaired functionality in the SOD1G93A neuromuscular junctions

Identification of the best stimulation parameters to measure in situ the comunication between muscle and nerve in mouse tibialis muscle

Investigating the path functionality of the nerve stimulation signal and the muscle contraction is of primary importance in the study of a wide variety of pathologic conditions: neuromuscular diseases like Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy, as well as acute denervation and aging. Alterations of coupling between motor neuron conduction and muscle contraction can be studied in mice, comparing the muscle contraction elicited by two alternating stimulation paradigms: direct stimulation on the membrane and indirect stimulation through the nerve. The fundamental assumption behind this approach is that in a healthy model the two stimulations should lead to the same contractile response of the muscle. In this work we have searched for the pulse stimulation parameters that better resemble the physiological action potential. Applying these optimized stimulations it is then possible to design new final protocols to evaluate all the contractile parameters of muscle tissue in a wide variety of pathological models