Morphogenesis of rat myotendinous junction

Myotendinous junction (MTJ) is the highly specialized complex which connects the skeletal muscle to the tendon for transmitting the contractile force between the two tissues. The purpose of this study was to investigate the MTJ development and rat EDL was chosen as a model. 1, 15, 30 day animals were considered and the junctions were analyzed by light and electron microscopy. The MTJ interface architecture increased during the development, extending the interaction between muscle and tendon. 1-day-old rats showed disorganized myofibril bundles, spread cytosol and incomplete rough endoplasmic reticulum, features partially improved in 15-day-old rats, and completely developed in 30-day-old animals. These findings indicate that muscle-tendon interface displays, during rat lifetime, numerically increased and longer tendon interdigitations, correlated with an improved organization of both tissues and with a progressive acquirement of full functionalit

Prevention of UVB radiation-induced cell death: “in vitro” studies

The ultraviolet component of sun light consists of UVA, UVB and UVC rays. UVB radiation represents an environmental hazard because of its role in skin aging, cancer and infection exacerbation. UVB stimulate the production of reactive oxygen species (ROS) in epidermal cells, resulting in skin lesions, accelerating aging and eliciting malignancies. At least 50% of UVB-induced damage is attributable to the formation of reactive ROS which cause cellular lesions if antioxidant defence mechanisms are down-regulated. Thus, exogenous supplementation of antioxidants may be an effective strategy to reduce or prevent skin damage. In the last years, we demonstrated the antioxidant effects of melatonin (Mel) (Luchetti et al., 2006) and, more recently of hydroxytyrosol (HyT) and its derivatives (Burattini et al., 2013) in hemopoietic human cells exposed to pro-oxidants. Therefore, in this project we propose to evaluate the antioxidant and/or anti-apoptotic effect of Mel and HyT in HaCaT human keratinocytes exposed to UVB. Keratinocytes in the non-irradiated condition are morphologically similar in Mel- and HyT-treated and untreated group. TUNEL reaction appears negative in both conditions, as well as in control. UVB radiation induces a significant decrease in cell confluence, with a diffuse cell detachment and the appearance of rounding and blebbed cells. TUNEL reaction evidences several nuclei with DNA fragmentation in UVB treated keratinocytes. In addition, cell viability evaluated by means of supravital propidium iodide (PI) evidences a diffuse staining positivity. Pre-treatment with Mel or HyT before UVB exposure is able to reduce cell death. In conclusion, HyT and Mel evidence an intringuing capability to prevent cell death in keratinocytes too. They could so represent a potential tool in skin protection from UVB radiation

Ultrastructural modifications of myotendinous junction with disuse

The skeletal muscle contraction is transmitted to the tendon through the myotendinous junction (MTJ), where the proximal extremity of tendon forms characteristic finger-like processes, penetrating into the muscle mass (Ciena et al., 2010). MTJ represents an intriguing and complex structure from both biological and functional point of view. We recently demonstrated that changes at MTJ level occur as an adaptation to exercise-induced tension increase. In particular, branching of finger-like processes increases, so enlarging the whole tendon-muscle surface area and, consequently, allowing a better tension resistance (Curzi et al., 2012). The aim of this study is to analyse MTJ behavior in disuse condition, furtherly investigating the strict morphofunctional correlation. Therefore, 4 hind-limb suspended and 4 control rats were studied. After sacrifice, MTJs of plantaris muscle were withdrawn, fixed in 1.4% glutaraldehyde in 0.2M sodium cacodylate and processed as previously described (Tidball et al., 1992; D’Emilio et al., 2010). After 5 days of suspension, skeletal muscle highlights signs of atrophy in response to decreased mechanical loading (Linderman et al., 1994). In fact, close to muscle-tendon interface, irregular misaligned sarcomeres, with absent Z-lines, were observed. Muscle intermyofibrillar component spreads, especially between terminal filaments and tendon finger-like processes. A lower number of muscle-tendon interdigitations, characteristically not bifurcated, also appears. In conclusion, along with muscle atrophy features, ultrastructural changes occur at the MTJ organization level, as an adaptation to muscle unloading

Cytoprotective effects of melatonin in C2C12 skeletal muscle cells: a multiple technical approach

Melatonin has a wide range of physiological functions including protection against oxidative stress, which is carried out through its ability to act as a free radical scavenger and to stimulate antioxidant enzyme production (Allegra et al., 2003). Oxidative stress is a major player in initiating apoptosis in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in various myopathies (Loro et al., 2010). Several authors demonstrated that melatonin exerts antiapoptotic actions in various cell models (Hibaoui et al., 2009) and our previous studies evidenced that it prevents apoptosis induced by UV-B and H2O2 in U937 cells (Luchetti et al., 2006; Salucci et al., 2010). In this work, melatonin activity has been investigated in C2C12 cells, after apoptotic chemical treatments. Myoblasts and myotubes were pre-treated with melatonin and then exposed to H2O2, cisplatin, etoposide and staurosporine. Data, obtained by means of TEM and TUNEL-CLSM, show that melatonin prevents apoptosis induced by H2O2, cisplatin and etoposide. Differently, staurosporine-induced apoptosis is not inhibited, probably because this trigger has a mechanism of action different from free radical increase. These results confirm melatonin ability to act as an antioxidant and anti-apoptotic molecule, thus suggesting a possible therapeutic strategy for myophaties involving apoptosis misregulation

Melatonin effects in normal and tumoral skeletal muscle cells: a preliminary study

Melatonin (MEL), also chemically known as N-acetyl-5-methoxytryptamine, is a hormone found in animals, plants, and microbes. It exhibits strong antioxidant effects and thanks to its structure it is able to diffuse through all the biological membranes, also overcoming the blood-brain barrier and the placenta (Salucci et al., 2014). Numerous in vitro and in vivo studies have documented Mel ability to induce apoptosis in tumor cells while inhibiting it in the normal ones (Cristofanos et al, 2009; Lanoix et al., 2011). In this study MEL activity has been investigated in vitro both in murine skeletal muscle (C2C12) and in alveolar rhabdomyosarcoma (RH30) cell lines by means of morpho-functional approaches. If MEL low concentrations are well tolerated by normal skeletal muscle cells, its effect appears completely different in tumor cells, where MEL can be considered a powerful apoptotic trigger. In RH30 cells, blebbing, chromatin condensation and margination, apoptotic bodies occur as well as necrotic cell death features. The latter appeared after prolonged exposure to MEL. In conclusion, the neuro-hormone shows a strong dose and time dependent pro-apoptotic activity and it could represent a potential tool in association with the current chemotherapeutic compounds to resolve alveolar rhabdomyosarcoma, the most common pediatric skeletal muscle tissue malignancy

Physical Education on the Beach: An Alternative Way to Improve Primary School Children’s Skill- and Health-Related Outcomes during the COVID-19 Pandemic

: The COVID-19 restrictions could preclude children from participating in physical education (PE) interventions. This study aimed to evaluate the efficacy of a PE intervention conducted on the beach on children's skill- and health-related outcomes, as a possible alternative PE intervention that could be also applied during the COVID-19 pandemic. The study involved 106 primary school children, randomly assigned to the traditional indoor (TI) intervention or to the experimental outdoor (EO) intervention. The intervention period lasted 4 months and consisted of two 1-h sessions per week. Intervention was conducted just before the beginning of the COVID-19 pandemic. Children's anthropometric parameters (height, weight, BMI, body fat percentage, and abdominal circumference), fitness parameter (VO2peak), health parameters (resting heart rate, and systolic and diastolic blood pressure), gross motor coordination, and physical activity level were assessed before and after intervention. Both groups significantly improved fitness and motor coordination but worsened some anthropometric parameters (weight, abdominal circumference) after the intervention period. The EO group showed a higher increase of gross motor coordination than the TI group. Results of this study demonstrated that children benefited from a well-structured PE intervention conducted in the natural environment of the beach improving physical fitness and gross motor coordination. Therefore, planning outdoor PE interventions could be an alternative and safe way to encourage and implement physical activity at school during the particular period of COVID-19 pandemic

Tyrosol antioxidant effect in C2C12 cell line

Tyrosol (4-(2-hydroxyethyl) phenol) (Tyr) is a well-known phenolic compound that is mainly present in extra-virgin olive oil and white wine. It has many beneficial properties, such as antioxidant, antimicrobial and anti-inflammatory activity and has been reported to have scavenging effects on peroxynitrite and superoxide anion (1). Moreover, Tyr exhibits anticancer, anti-depressant, anti-stress, cardioprotective, antiosteoporosis, anti-inflammatory and neural protective effects (2). Here Tyr effect has been investigated in a skeletal muscle cell line exposed to known oxidative stress inducers (3, 4). To evaluate Tyr protection against oxidative stress and cell death, ultrastructural and functional analyses have been carried out. In particular, mitochondrial probes used and analysed at confocal microscopy. All techniques confirmed that Tyr is able to prevent skeletal muscle damage, to preserve mitochondrial membrane integrity and to down regulate oxidative stress levels. These findings demonstrated Tyr antioxidant property in skeletal muscle cell too, suggesting, for this molecule, an important potential role in all muscle diseases related to reactive oxygen species production

Morphological study of cartilage cell death in patients affected by osteoarthritis and chondrocalcinosis

The role of chondrocyte death in the pathogenesis of osteoarthritis (OA) has been largely discussed in literature, but its relative contribution is difficult to assess (1). Chondrocyte death, be it apoptotic, necrotic or chondroptotic, has been clearly documented in OA and a certain correlation between the degree of cartilage damage and chondrocyte apoptosis has been demonstrated (2;3). Conversely, the relationship between the different types of cell death and chondrocalcinosis (CC) is still little known, as well as the presence and role of chondroptotic cells. The aim of this research was to compare chondrocyte behavior in the cartilage of osteoarthritic and chondrocalcinotic knees, evaluating the different types of cell death by means of optical and electron microscopy. During total knee replacement surgeries, cartilage specimens of femoral condyle have been withdrawn and their transversal semithin sections, stained with toluidine blue and alizarin solutions, have been investigated by optical microscopy. From the same samples, thin sections were obtained for transmission electron microscopy to evaluate, at high magnification, the specific ultrastructural features of different types of cell death. Cartilage specimens from both conditions revealed a thickness reduction of superficial layer and a high number of empty lacunae in the middle layer. Calcium pyrophosphate crystals appeared in the samples of patients affected by CC. In osteoarthritic cartilage, numerous chondrocytes revealed necrotic features, whereas, in chondrocalcinotic tissue, the middle zone was characterized by morphological patterns suggestive of chondroptosis, such as chromatin condensation mostly localized at the nuclear periphery, mitochondria alterations, a marked increase in endoplasmic reticulum, the presence of a diffuse autophagic component and the extrusion of cellular material into the lacunae. In conclusion, a different distribution of cell death types seems to characterize the intermediate layers of cartilage specimens from patients affected by CC compared to OA

Growth hormone and resistance exercise effects on myotendinous junction in hind-limb unloaded rats: an ultrastructural study

Myotendinous junction (MTJ), is the site at which the contractile forces are transmitted from myofibrils to extracellular matrix, and, when observed at ultrastructural level, it displays an interdigitated profile (Kojima et al., 2008). The aim of this study is to investigate the junctional behaviour in the atrophic condition and during particular prevention protocols. The MTJs of plantaris muscles from twenty hypophysectomized rats were processed for electron microscopy (Curzi et al., 2012). The animals were assigned to one of five groups: control (CTRL), hind-limb suspended (HS), hind-limb suspended and exercised (EX), hind-limb suspended and growth hormone injected (GH) and hind-limb suspended, GH injected and exercised (GH+EX). After unloading, the tendon finger-like processes appeared small and irregular and the contact between tissues is reduced to 61.3%. The prevention treatments increased the interface area up to 6.2% in GH, 25.3% in EX and 46.4 % in GH+EX respectively. The resistance exercise protocol, as well as GH treatment, was not capable of maintaining the contact surface between tissues, but in both exercised groups the number of bifurcated interdigitations was higher than in the CTRL. In conclusion, ultrastructural changes occur at MTJ in HS, as an adaptation to muscle unloading. Differently, MTJ structure is partially maintained by resistance training or GH treatment, while the exercise with simultaneous somatropin administration showed a greater effect

Melatonin effects in normal and tumoral skeletal muscle cells: a preliminary study

Melatonin (MEL), also chemically known as N-acetyl-5-methoxytryptamine, is a hormone found in animals, plants, and microbes. It exhibits strong antioxidant effects and thanks to its structure it is able to diffuse through all the biological membranes, also overcoming the blood-brain barrier and the placenta (Salucci et al., 2014). Numerous in vitro and in vivo studies have documented Mel ability to induce apoptosis in tumor cells while inhibiting it in the normal ones (Cristofanos et al, 2009; Lanoix et al., 2011). In this study MEL activity has been investigated in vitro both in murine skeletal muscle (C2C12) and in alveolar rhabdomyosarcoma (RH30) cell lines by means of morpho-functional approaches. If MEL low concentrations are well tolerated by normal skeletal muscle cells, its effect appears completely different in tumor cells, where MEL can be considered a powerful apoptotic trigger. In RH30 cells, blebbing, chromatin condensation and margination, apoptotic bodies occur as well as necrotic cell death features. The latter appeared after prolonged exposure to MEL. In conclusion, the neuro-hormone shows a strong dose and time dependent pro-apoptotic activity and it could represent a potential tool in association with the current chemotherapeutic compounds to resolve alveolar rhabdomyosarcoma, the most common pediatric skeletal muscle tissue malignancy