Further considerations on in vitro skeletal muscle cell death

The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death inductio

Ultrastructural Features of Apoptosis

Apoptosis is a gene-directed physiological and programmed process of cell deletion aimed at the regulation of tissue and organ development. It affects different cell types and is triggered by a variety of stimuli all inducing closely comparable structural changes. Despite the deeply different morphology and metabolism of the cell models and the various inducers and their initial effects, a convergence seems to take place in a common metabolic pathway that, in most cases, involves the activation of a Ca2+ dependent endonuclease. A growing body of data is now available on the molecular events that lead to DNA damage. DNA cleavage in nucleosomic or oligonucleosomic fragments is related to the appearance of unusual and very characteristic ultrastructural changes. The nucleus is especially affected, and shows chromatin rearrangements consisting of cup-shaped marginations, sharply separated from diffuse chromatin areas. Nuclear fragmentation subsequently appears, finally followed by the formation of numerous micronuclei. Cytoplasmic damage appears at a very late stage and the process takes place despite good preservation of plasma membrane and cytoplasm

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

Role of Bacterial Exopolymers and Host Factors on Adherence and Phagocytosis of Staphylococcus aureus in Foreign Body Infection

Using a previously developed guinea pig model of foreign body infection, we examined ultrastructural and functional surface alterations of Staphylococcus aureus strain Wood 46 during the early phase of infection. Exopolymer-free bacteria were prepared and inoculated into subcutaneously implanted tissue cages. After three hours, the bacteria showed abundant capsular and intercellular exopolymers, which were visualized by transmission electron microscopy. Exopolymers were also produced by S. aureus exposed in vitro to fluid from the tissue cage. In contrast, human serum albumin prevented exopolymer production by S. aureus. The influence of exopolymers on the susceptibility of S. aureus to ingestion and phagocytic killing by neutrophils was tested in vitro and found to be negligible. Furthermore, adherence of S. aureus to fibronectin-coated surfaces was unaffected by the presence or absence of exopolymers. Thus, in our experimental model, exopolymers are produced early during the onset of infection, but they have little impact on adherence and phagocytosi

Alfa-Tocopherol supplementation induces morphological changes in the hippocampus of adult offspring

none6noopenSalucci, Sara; Ambrogini, Patrizia; Lattanzi, Davide; Minelli, Andrea; Falcieri, Elisabetta; Gobbi, PietroSalucci, Sara; Ambrogini, Patrizia; Lattanzi, Davide; Minelli, Andrea; Falcieri, Elisabetta; Gobbi, Pietr

Nuclear pores in the apoptotic cell.

During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models, Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas, In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cistemae, A possible functional significance of this pore behaviour during apoptosis is discussed

Protective effect of different antioxidant agents in UVB-irradiated keratinocytes

Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, undergoing programmed cell death, when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases, including skin inflammation and cancer. Some polyphenols, present in virgin olive oil, well tolerated by organism after oral administration, show a variety of pharmacological and clinical benefits such as anti-oxidant, anti-cancer, anti-inflammatory, and neuro-protective activities. Here, the protective effects of antioxidant compounds against UV-induced apoptosis have been described in HaCat cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effects have been evaluated by means of ultrastructural analyses. After UVB radiation, a known cell death trigger, typical apoptotic features, absent in control condition and in antioxidant alone-treated cells, appear. An evident numerical decrease of ultrastructural apoptotic patterns and TUNEL positive nuclei can be observed when natural antioxidants were supplied before cell death induction. These data have been confirmed by molecular investigation of caspase activity. In conclusion, this paper highlights antioxidant compound ability to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, a potential role in preventing skin damage

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