The regulation of Satellite Cells during skeletal muscle regeneration and neuromuscular disease.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

Assessment of vibratory stimulation on neuromuscular system

The thesis analyze a subject of renewed interest in bioengineering, the research and analysis of exercise parameters that maximize the neuromuscular and cardiovascular involvement in vibration treatment. The research activity was inspired by the increasing use of device able to provide localized or whole body vibration (WBV). In particular, the focus was placed on the vibrating platform and the effect that the vibrations have on the neuromuscular system and cardiovascular system. The aim of the thesis is to evaluate the effectiveness and efficiency of vibration applied to the entire body, in particular, it was investigated the effect of WBV on: 1) Oxygen consumption during static and dynamic squat; 2) Resonant frequency of the muscle groups of the lower limbs; 3) Oxygen consumption and electromyographic signals during static and dynamic squat. In the first three chapters are explained the state of the art concerning vibration treatments, the effects of vibration applied to the entire body, with the explanation of the basic mechanisms (Tonic Vibration Reflex, TVR) and the neuromuscular system, with particular attention to the skeletal muscles and the stretch reflex. In the fourth chapter is illustrated the set-up used for the experiments and the software, implemented in LabWindows in order to control the platform and acquire the electromyographic signal. In the fifth chapter were exposed experiments undertaken during the PhD years. In particular, the analysis of Whole Body Vibration effect on neurological and cardiovascular systems showed interesting results. The results indicate that the static squat with WBV produced higher neuromuscular and cardiorespiratory system activation for exercise duration <60 sec. Otherwise, if the single bout duration was higher than 60 sec, the greater cardiorespiratory system activation was achieved during the dynamic squat with WBV while higher neuromuscular activation was still obtained with the static exercise.

Techniques and Methods for a multi-scale analysis of neuromuscular fatigue

This thesis proposes an integrated holistic approach to the study of neuromuscular fatigue in order to encompass all the causes and all the consequences underlying the phenomenon. Starting from the metabolic processes occurring at the cellular level, the reader is guided toward the physiological changes at the motorneuron and motor unit level and from this to the more general biomechanical alterations. In Chapter 1 a list of the various definitions for fatigue spanning several contexts has been reported. In Chapter 2, the electrophysiological changes in terms of motor unit behavior and descending neural drive to the muscle have been studied extensively as well as the biomechanical adaptations induced. In Chapter 3 a study based on the observation of temporal features extracted from sEMG signals has been reported leading to the need of a more robust and reliable indicator during fatiguing tasks. Therefore, in Chapter 4, a novel bi-dimensional parameter is proposed. The study on sEMG-based indicators opened a scenario also on neurophysiological mechanisms underlying fatigue. For this purpose, in Chapter 5, a protocol designed for the analysis of motor unit-related parameters during prolonged fatiguing contractions is presented. In particular, two methodologies have been applied to multichannel sEMG recordings of isometric contractions of the Tibialis Anterior muscle: the state-of-the-art technique for sEMG decomposition and a coherence analysis on MU spike trains. The importance of a multi-scale approach has been finally highlighted in the context of the evaluation of cycling performance, where fatigue is one of the limiting factors. In particular, the last chapter of this thesis can be considered as a paradigm: physiological, metabolic, environmental, psychological and biomechanical factors influence the performance of a cyclist and only when all of these are kept together in a novel integrative way it is possible to derive a clear model and make correct assessments.

Vibration as a method for muscular stimulation

Human reactions to vibration have been extensively investigated in the past. Vibration, as well as whole-body vibration (WBV), has been commonly considered as an occupational hazard for its detrimental effects on human condition and comfort. Although long term exposure to vibrations may produce undesirable side-effects, a great part of the literature is dedicated to the positive effects of WBV when used as method for muscular stimulation and as an exercise intervention. Whole body vibration training (WBVT) aims to mechanically activate muscles by eliciting neuromuscular activity (muscle reflexes) via the use of vibrations delivered to the whole body. The most mentioned mechanism to explain the neuromuscular outcomes of vibration is the elicited neuromuscular activation. Local tendon vibrations induce activity of the muscle spindle Ia fibers, mediated by monosynaptic and polysynaptic pathways: a reflex muscle contraction known as the Tonic Vibration Reflex (TVR) arises in response to such vibratory stimulus. In WBVT mechanical vibrations, in a range from 10 to 80 Hz and peak to peak displacements from 1 to 10 mm, are usually transmitted to the patient body by the use of oscillating platforms. Vibrations are then transferred from the platform to a specific muscle group through the subject body. To customize WBV treatments, surface electromyography (SEMG) signals are often used to reveal the best stimulation frequency for each subject. Use of SEMG concise parameters, such as root mean square values of the recordings, is also a common practice; frequently a preliminary session can take place in order to discover the more appropriate stimulation frequency. Soft tissues act as wobbling masses vibrating in a damped manner in response to mechanical excitation; Muscle Tuning hypothesis suggest that neuromuscular system works to damp the soft tissue oscillation that occurs in response to vibrations; muscles alters their activity to dampen the vibrations, preventing any resonance phenomenon. Muscle response to vibration is however a complex phenomenon as it depends on different parameters, like muscle-tension, muscle or segment-stiffness, amplitude and frequency of the mechanical vibration. Additionally, while in the TVR study the applied vibratory stimulus and the muscle conditions are completely characterised (a known vibration source is applied directly to a stretched/shortened muscle or tendon), in WBV study only the stimulus applied to a distal part of the body is known. Moreover, mechanical response changes in relation to the posture. The transmissibility of vibratory stimulus along the body segment strongly depends on the position held by the subject. The aim of this work was the investigation on the effects that the use of vibrations, in particular the effects of whole body vibrations, may have on muscular activity. A new approach to discover the more appropriate stimulus frequency, by the use of accelerometers, was also explored. Different subjects, not affected by any known neurological or musculoskeletal disorders, were voluntarily involved in the study and gave their informed, written consent to participate. The device used to deliver vibration to the subjects was a vibrating platform. Vibrations impressed by the platform were exclusively vertical; platform displacement was sinusoidal with an intensity (peak-to-peak displacement) set to 1.2 mm and with a frequency ranging from 10 to 80 Hz. All the subjects familiarized with the device and the proper positioning. Two different posture were explored in this study: position 1 - hack squat; position 2 - subject standing on toes with heels raised. SEMG signals from the Rectus Femoris (RF), Vastus Lateralis (VL) and Vastus medialis (VM) were recorded. SEMG signals were amplified using a multi-channel, isolated biomedical signal amplifier The gain was set to 1000 V/V and a band pass filter (-3dB frequency 10 - 500 Hz) was applied; no notch filters were used to suppress line interference. Tiny and lightweight (less than 10 g) three-axial MEMS accelerometers (Freescale semiconductors) were used to measure accelerations of onto patient’s skin, at EMG electrodes level. Accelerations signals provided information related to individuals’ RF, Biceps Femoris (BF) and Gastrocnemius Lateralis (GL) muscle belly oscillation; they were pre-processed in order to exclude influence of gravity. As demonstrated by our results, vibrations generate peculiar, not negligible motion artifact on skin electrodes. Artifact amplitude is generally unpredictable; it appeared in all the quadriceps muscles analysed, but in different amounts. Artifact harmonics extend throughout the EMG spectrum, making classic high-pass filters ineffective; however, their contribution was easy to filter out from the raw EMG signal with a series of sharp notch filters centred at the vibration frequency and its superior harmonics (1.5 Hz wide). However, use of these simple filters prevents the revelation of EMG power potential variation in the mentioned filtered bands. Moreover our experience suggests that the possibility of reducing motion artefact, by using particular electrodes and by accurately preparing the subject’s skin, is not easily viable; even though some small improvements were obtained, it was not possible to substantially decrease the artifact. Anyway, getting rid of those artifacts lead to some true EMG signal loss. Nevertheless, our preliminary results suggest that the use of notch filters at vibration frequency and its harmonics is suitable for motion artifacts filtering. In RF SEMG recordings during vibratory stimulation only a little EMG power increment should be contained in the mentioned filtered bands due to synchronous electromyographic activity of the muscle. Moreover, it is better to remove the artifact that, in our experience, was found to be more than 40% of the total signal power. In summary, many variables have to be taken into account: in addition to amplitude, frequency and duration of vibration treatment, other fundamental variables were found to be subject anatomy, individual physiological condition and subject’s positioning on the platform. Studies on WBV treatments that include surface EMG analysis to asses muscular activity during vibratory stimulation should take into account the presence of motion artifacts. Appropriate filtering of artifacts, to reveal the actual effect on muscle contraction elicited by vibration stimulus, is mandatory. However as a result of our preliminary study, a simple multi-band notch filtering may help to reduce randomness of the results. Muscle tuning hypothesis seemed to be confirmed. Our results suggested that the effects of WBV are linked to the actual muscle motion (displacement). The greater was the muscle belly displacement the higher was found the muscle activity. The maximum muscle activity has been found in correspondence with the local mechanical resonance, suggesting a more effective stimulation at the specific system resonance frequency. Holding the hypothesis that muscle activation is proportional to muscle displacement, treatment optimization could be obtained by simply monitoring local acceleration (resonance). However, our study revealed some short term effects of vibratory stimulus; prolonged studies should be assembled in order to consider the long term effectiveness of these results. Since local stimulus depends on the kinematic chain involved, WBV muscle stimulation has to take into account the transmissibility of the stimulus along the body segment in order to ensure that vibratory stimulation effectively reaches the target muscle. Combination of local resonance and muscle response should also be further investigated to prevent hazards to individuals undergoing WBV treatments.