204 research outputs found

    Satellite cells in skeletal muscle of the hibernating dormouse, a natural model of quiescence and re-activation: focus on the cell nucleus

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    Satellite cells (SCs) participate in skeletal muscle plasticity/regeneration. Activation of SCs implies that nuclear changes underpin a new functional status. In hibernating mammals, periods of reduced metabolic activity alternate with arousals and resumption of bodily functions, thereby leading to repeated cell deactivation and reactivation. In hibernation, muscle fibers are preserved despite long periods of immobilization. The structural and functional characteristics of SC nuclei during hibernation have not been investigated yet. Using ultrastructural and immunocytochemical analysis, we found that the SCs of the hibernating edible dormouse, Glis glis, did not show apoptosis or necrosis. Moreover, their nuclei were typical of quiescent cells, showing similar amounts and distributions of heterochromatin, pre-mRNA transcription and processing factors, as well as paired box protein 7 (Pax7) and the myogenic differentiation transcription factor D (MyoD), as in euthermia. However, the finding of accumulated perichromatin granules (i.e., sites of storage/transport of spliced pre-mRNA) in SC nuclei of hibernating dormice suggested slowing down of the nucleus-to-cytoplasm transport. We conclude that during hibernation, SC nuclei maintain similar transcription and splicing activity as in euthermia, indicating an unmodified status during immobilization and hypometabolism. Skeletal muscle preservation during hibernation is presumably not due to SC activation, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting

    Embedding cell monolayers to investigate nanoparticle-plasmalemma interactions at transmission electron microscopy

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    Transmission electron microscopy is the technique of choice to visualize the spatial relationships between nanoconstructs and cells and especially to monitor the uptake process of nanomaterials. It is therefore crucial that the cell surface be preserved in its integrity, to obtain reliable ultrastructural evidence: the plasmalemma represents the biological barrier the nanomaterials have to cross, and the mode of membrane-nanoconstruct interaction is responsible for the intracellular fate of the nanomaterials. In this paper, we describe a simple and inexpensive method to process cell monolayers for ultrastructural morphology and immunocytochemistry, ensuring consistent preservation of the cell surface and of the occurring interactions with nanoparticles of different chemical composition

    Quantitative magnetic resonance characterization of the effect of physical training on skeletal muscle of the Ts65Dn mice, a model of Down syndrome

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    Down syndrome (DS) is characterized by muscle hypotonia and low muscle strength associated with motor dysfunction. Elucidation of the determinants of muscle weakness in DS would be relevant for therapeutic approaches aimed at treating/mitigating a physical disability with a strong impact on the quality of life in persons with DS. The Ts65Dn mice is a recognized mouse model of DS, with trisomic mice presenting gross motor and muscle phenotypes. The aim of this work was to assess the effect of physical exercise, a well-known tool to improve skeletal muscle condition, in the hindlimbs of trisomic and euploid male mice using quantitative magnetic resonance imaging (MRI). Magnetic resonance spectroscopy (MRS) metabolomics and histological fiber typing were used to further characterize the post-exercise muscle. Quantitative MRI showed not significantly different amounts of skeletal muscle in proximal hindlimbs in trisomic and euploid mice both at baseline and after physical exercise (P>0.05). Similar results were obtained for hindlimbs subfascia adipose tissue, and subcutaneous adipose tissue (P>0.05). MRS showed lower amounts of exercise-related metabolites (valine, isoleucine, leucine) in euploid vs. trisomic mice after exercise (P <=.0.05). The percentage of slow-twitch fibers was similar in the two genotypes (P>0.05). We conclude that in DS adapted physical exercise (one month of training) does not induce quantitative changes in skeletal muscle or fiber type composition therein; however, the metabolic response of skeletal muscle to exercise may be affected by trisomy. These findings prompt further research investigating the role of physical exercise as a cue to clarify the mechanisms of the muscular deficit found in DS

    Fluorescence and electron microscopy to visualize the intracellular fate of nanoparticles for drug delivery

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    In order to design valid protocols for drug release via nanocarriers, it is essential to know the mechanisms of cell internalization, the interactions with organelles, and the intracellular permanence and degradation of nanoparticles (NPs) as well as the possible cell alteration or damage induced. In the present study, the intracellular fate of liposomes, polymeric NPs and mesoporous silica NPs (MSN) has been investigated in an in vitro cell system by fluorescence and transmission electron microscopy. The tested nanocarriers proved to be characterized by specific interactions with the cell: liposomes enter the cells probably by fusion with the plasma membrane and undergo rapid cytoplasmic degradation; polymeric NPs are internalized by endocytosis, occur in the cytoplasm both enclosed in endosomes and free in the cytosol, and then undergo massive degradation by lysosome action; MSN are internalized by both endocytosis and phagocytosis, and persist in the cytoplasm enclosed in vacuoles. No one of the tested nanocarriers was found to enter the nucleus. The exposure to the different nanocarriers did not increase cell death; only liposomes induced a reduction of cell population after long incubation times, probably due to cell overloading. No subcellular damage was observed to be induced by polymeric NPs and MSN, whereas transmission electron microscopy revealed cytoplasm alterations in liposome-treated cells. This important information on the structural and functional relationships between nanocarriers designed for drug delivery and cultured cells further proves the crucial role of microscopy techniques in nanotechnology

    Low ozone concentrations differentially affect the structural and functional features of non-activated and activated fibroblasts in vitro

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    Oxygen-ozone (O2-O3) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O3 concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O2-O3 gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O3 stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-β1 secretion, while in LPS-activated fibroblasts, O3 stimulated only antioxidant response and cytokines secretion. Therefore, the low O3 concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated

    Ozone at low concentrations does not affect motility and proliferation of cancer cells in vitro

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    Exposure to low ozone concentrations is used in medicine as an adjuvant/complementary treatment for a variety of diseases. The therapeutic potential of low ozone concentrations relies on their capability to increase the nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing the transcription of Antioxidant Response Elements (ARE)-driven genes and, through a cascade of events, a general cytoprotective response. However, based on the controversial role of Nrf2 in cancer initiation, progression and resistance to therapies, possible negative effects of ozone therapy may be hypothesised in oncological patients. With the aim to elucidate the possible changes in morphology, migration capability and proliferation of cancer cells following mild ozone exposure, we performed wound healing experiments in vitro on HeLa cells treated with low ozone concentrations currently used in the clinical practice. By combining a multimodal microscopy approach (light and fluorescence microscopy, scanning electron microscopy, atomic force microscopy) with morphometric analyses, we demonstrated that, under our experimental conditions, exposure to low ozone concentrations does not alter cytomorphology, motility and proliferation features, thus supporting the notion that ozone therapy should not positively affect tumour cell growth and metastasis

    Formulative Study and Intracellular Fate Evaluation of Ethosomes and Transethosomes for Vitamin D3 Delivery

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    In this pilot study, ethosomes and transethosomes were investigated as potential delivery systems for cholecalciferol (vitamin D3), whose deficiency has been correlated to many disorders such as dermatological diseases, systemic infections, cancer and sarcopenia. A formulative study on the influence of pharmaceutically acceptable ionic and non-ionic surfactants allowed the preparation of different transethosomes. In vitro cytotoxicity was evaluated in different cell types representative of epithelial, connective and muscle tissue. Then, the selected nanocarriers were further investigated at light and transmission electron microscopy to evaluate their uptake and intracellular fate. Both ethosomes and transethosomes proven to have physicochemical properties optimal for transdermal penetration and efficient vitamin D3 loading; moreover, nanocarriers were easily internalized by all cell types, although they followed distinct intracellular fates: ethosomes persisted for long times inside the cytoplasm, without inducing subcellular alteration, while transethosomes underwent rapid degradation giving rise to an intracellular accumulation of lipids. These basic results provide a solid scientific background to in vivo investigations aimed at exploring the efficacy of vitamin D3 transdermal administration in different experimental and pathological conditions

    Magnetic resonance imaging investigations in a murine model of Down syndrome: the Ts65Dn mouse

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    Trisomy 21, or Down’s syndrome, is the most common genetic cause of intellectual disability; moreover, DS patients suffer from muscle hypotonia and low muscle strength whose mechanisms are still unknown and only partially explained by intellectual disability. Genetic mouse models (e.g., the Ts65Dn mouse) may offer insights into the responsible mechanisms . Moreover, the Ts65Dn mouse show muscle weakness and share morphological similarities with sarcopenia of aging [1]. To characterize skeletal muscle in theTs65Dn model, we explored the hindlimb of young adult (6-month-old) mice in euploid (n=9, mean weight: 43.8±4.10 g) and trisomic (n=9, mean weight: 38.2±4.0 g ) individuals by magnetic resonance imaging. Mice were imaged in a Bruker Tomograph at 4.7 Tesla using a RARE T2-weighed sequence (TR: 5000ms, TE: 56ms; spatial resolution 0.0182cm2 per pixel). Muscle cross sectional area was measured in a section taken at mid-femur in either hindlimbs and values averaged for further analysis. Results showed that mean hindlimbs muscle cross sectional area was larger in trisomic than in euploid mice (0.29±0.05 vs. 0.23±0.05 cm2, p=0.03 [t test]). Because of significant difference in body weight between euploid and trisomic mice (p=0.009), data were adjusted per body weight. Using adjusted data, a significant difference was still found between groups (euploid: 0.005±0.001; trisomic: 0.007±0.001 cm2/g body weight). It is concluded that young adult trisomic mice has increased apparent muscle mass in hindlimbs. Since trisomic Ts65Dn mice show reduced grip strength as well as running and swimming speed in the presence of roughly normal muscle biochemistry [2], possible structural and fibertype changes in hindlimb muscles of these mice are under investigation to explain such a discrepancy

    Ozone activates the Nrf2 pathway and improves preservation of explanted adipose tissue in vitro

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    In clinical practice, administration of low ozone (O3) dosages is a complementary therapy for many diseases, due to the capability of O3 to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O3 concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O3 treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O3 treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O3 concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O3 as an adjuvant treatment to improve fat engraftment

    Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study

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    Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells
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