Optimal force evaluation for isotonic fatigue characterization in mouse Tibialis Anterior muscle

Skeletal muscle fatigue is most often studied as a response to repeated stimulations in isometric conditions and it is usually quantified as the progressive loss of force generating capability over time. However, physical dynamic activity is based on the shortening of skeletal muscles. Therefore, the condition that best mimics body movements is the isotonic one, in which muscle is allowed to shorten against a constant load. In the literature, the isotonic fatigue test is performed allowing the muscle to lift a load corresponding to one-third of the maximal isometric force (reference optimal force), as best representative of the force at which the tissue develops its maximum power. The goal of this study was to devise a new testing protocol in which each muscle was tested for isotonic fatigue by shortening against its own optimal force, i.e. the force at which it really developed the maximum power. Our hypothesis was that testing all the muscle at a standard reference value would introduce significant errors in the parameters associated to muscle fatigue and in their variance. The proposed protocol was based on the real-time measurement of the maximum power a muscle was able to generate through the application of the after-load technique and a mathematical interpolation to the Hill's equation, that therefore allowed to determine the experimental optimal force to be applied during the fatigue test. Experimental results showed that the muscles tested with the experimental optimal force had a fatigue time significantly lower than the control muscles tested with the reference optimal force. A decrease, even if not statistically significant, was also measured for the power and work generated during the fatigue test. Of note, for all these parameters a huge decrease in the measurement variance was reported, confirming that a precise assessment of the muscle experimental optimal force was needed to increase the accuracy of the measurements. On the other hand, the application of the protocol proposed in this work required an increase in the test duration, due to the application of the after-load technique, and a real time measurement of the power generated by the tissue

Development of innovative techniques, experimental devices and testing protocols for the measurement of muscle and neuromuscular junction functionality in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) remains an invariably fatal disease, in which neuromuscular junction (NMJ) functionality is strongly impaired. To this, the aim of this research project was to develop a series of novel testing tools for a precise assessment of the altered communication between muscle and nerve in ALS progression. A novel technique for the in-situ measurement of murine Tibialis Anterior (TA) NMJ functionality in isotonic conditions was developed and validated. A novel parameter, named Isotonic Neurotransmission Failure (INF), was proposed. Results showed an increase in INF of SOD1G93A mouse TA muscles at the end-stage of the disease, highlighting, for the first time, an increased impairment of NMJ functionality in isotonic conditions. An embedded system for the measurement of 3D engineered skeletal muscle tissues’ contractility with a non-invasive technique was proposed. Results showed the capability of the system to not impair tissue's contractility during the entire growth, and to discriminate healthy and pathological conditions. Finally, a 3D microfluidic device was designed and realized to promote the formation of NMJ between spinal cord-derived neuronal cells and 3D engineered skeletal muscle. Results showed a good attraction between these two cells populations, paving the basis for the development of a more comprehensive 3D NMJ in-vitro model. On the other hand, since extracellular vesicles (EVs) are involved in ALS pathological proteins transportation, a series of preliminary experiments with muscle cells’ populations was carried out, with the final aim of evaluating the role of SOD1G93A mice-derived EVs on the novel experimental models here proposed. Results showed that SOD1G93A mice-derived EVs increased in number during the ALS progression, and impaired C2C12 cells’ differentiation. In conclusion, a series of novel testing tools have been developed for a precise assessment of the NMJ functionality in different models which, of note, can be also employed to unravel the mechanism behind muscle-nerve impairments in other neurodegenerative pathologies

Contribution of oxygen-dependent mechanisms to vascular responses of exercise in young and older men: the role of prostaglandins and adenosine

Previous work suggests vasodilating prostaglandins (PGs) are released during isometric handgrip exercise in an O$_2$-dependent manner in young men. This project investigates their contribution to the exercise hyperaemia of isometric and rhythmic handgrip contraction performed by healthy, recreationally-active young and older men. Hyperoxia (40% O$_2$), aspirin, and their combination equally attenuated exercise and post-exercise hyperaemia, and venous efflux of PGE$_2$ and PGI$_2$ in both age groups: efflux of these PGs was not attenuated with age, but their contribution to the hyperaemic response was. Further, the release of COX products evoked reflex vasoconstriction in an O$_2$-dependent manner. Moreover, 40% O$_2$, aspirin, and their combination equally inhibited the exercise-evoked vasoconstriction in both age groups. However, both the exercise-evoked attenuation in perfusion of resting skeletal muscles and the contribution of COX products were attenuated with age. Additional experiments showed that adenosine contributes to the hyperaemia of electrically evoked isometric twitch contractions in an O$_2$-dependent manner; adenosine may contribute to the increase in the concentrations of vasodilating PGs. Importantly, unlike 60% and 100% O$_2$, 40% O$_2$ did not attenuate acetylcholine-evoked endothelium-dependent dilatation in either age group, supporting the argument that the effect of 40% O$_2$ during exercise is independent of hyperoxia-related oxidative stress

Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years, and this has lead to the development of novel therapeutic strategies for these patients. In this book a review of the options available for the clinical management of patients with these tumors are outlined. In addition advances in radiology both for pre-operative diagnostic purposes along with surgical planning are described. Furthermore a review of newer developments in chemotherapy along with the evolving field of photodynamic therapy both for intra-operative management and subsequent therapy is provided. A discussion of certain surgical management issues along with tumor induced epilepsy is included. Finally a discussion of the management of certain unique problems including brain metastases, brainstem glioma, central nervous system lymphoma along with issues involving patients with a brain tumor and pregnancy is provided

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

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