1,720,967 research outputs found
Uranyl-free staining as a suitable contrasting technique for nuclear structures at transmission electron microscopy
No full textUranyl acetate solution has widely been used as staining reagent for samples processed for ultrastructural morphology, cytochemistry, and immunocytochemistry. Although uranyl acetate guarantees high performance as a staining reagent, the radioactive uranyl salts make its use and purchase severely restricted. In this view, we used a non-radioactive lanthanide mix solution as contrasting dye for both nucleoplasmic and nucleolar ribonucleoprotein-containing components. This method guarantees a good contrast without masking the probe-antigen immunoreaction, thus proving to be a suitable tool for high-resolution studies of both cyto- and immunocytochemistry on acrylic resin-embedded samples
Ultrastructural immunocytochemistry shows impairment of RNA pathways in skeletal muscle nuclei of old mice: A link to sarcopenia?
Full text linkDuring aging, skeletal muscle is affected by sarcopenia, a progressive decline in muscle mass, strength and endurance that leads to loss of function and disability. Cell nucleus dysfunction is a possible factor contributing to sarcopenia because aging-associated alterations in mRNA and rRNA transcription/maturation machinery have been shown in several cell types including muscle cells. In this study, the distribution and density of key molecular factors involved in RNA pathways namely, nuclear actin (a motor protein and regulator of RNA transcription), 5-methyl cytosine (an epigenetic regulator of gene transcription), and ribonuclease A (an RNA degrading enzyme) were compared in different nuclear compartments of late adult and old mice myonuclei by means of ultrastructural immunocytochemistry. In all nuclear compartments, an age-related decrease of nuclear actin suggested altered chromatin structuring and impaired nucleus-to-cytoplasm transport of both mRNA and ribosomal subunits, while a decrease of 5-methyl cytosine and ribonuclease A in the nucleoli of old mice indicated an age-dependent loss of rRNA genes. These findings provide novel experimental evidence that, in the aging skeletal muscle, nuclear RNA pathways undergo impairment, likely hindering protein synthesis and contributing to the onset and progression of sarcopenia
A Fluid Dynamic In Vitro System to Study the Effect of Hyaluronic Acid Administration on Collagen Organization in Human Skin Explants
Full text linkHyaluronic acid (HA) is an unbranched polysaccharide particularly abundant in the extracellular matrix (ECM) of soft connective tissues. In humans, about 50% of the total HA in the organism is localized in the skin. HA plays an essential role in the hydration of the ECM, in the regulation of tissue homeostasis, in the resistance to mechanical stimuli/forces, and in the modulation of tissue regeneration. For these reasons, HA is widely used in regenerative medicine and cosmetics. In this study we used an innovative fluid dynamic system to investigate the effects of a cross-linked macrostructural HA formulation on dermal collagen of healthy human skin explants. The good preservation of skin explants provided by the bioreactor allowed applying refined high-resolution microscopy techniques to analyze in situ the HA-induced modifications on the ECM collagen fibrils up to 48 h from the application on the skin surface. Results demonstrated that this HA formulation, commercially proposed for subcutaneous injection, may act on dermal ECM also when applied transcutaneously, improving ECM hydration and modifying the organization of the collagen fibrils. These findings, obtained by the original combination of explanted human skin use with an advanced culture system and multiscale imaging techniques, are consistent with the volumizing and anti-aging effect of HA
Physical Training Chronically Stimulates the Motor Neuron Cell Nucleus in the Ts65Dn Mouse, a Model of Down Syndrome
Full text linkDown syndrome (DS) is a genetically-based disease based on the trisomy of chromosome 21 (Hsa21). DS is characterized by intellectual disability in association with several pathological traits among which early aging and altered motor coordination are prominent. Physical training or passive exercise were found to be useful in counteracting motor impairment in DS subjects. In this study we used the Ts65Dn mouse, a widely accepted animal model of DS, to investigate the ultrastructural architecture of the medullary motor neuron cell nucleus taken as marker of the cell functional state. Using transmission electron microscopy, ultrastructural morphometry, and immunocytochemistry we carried out a detailed investigation of possible trisomy-related alteration(s) of nuclear constituents, which are known to vary their amount and distribution as a function of nuclear activity, as well as the effect of adapted physical training upon them. Results demonstrated that trisomy per se affects nuclear constituents to a limited extent; however, adapted physical training is able to chronically stimulate pre-mRNA transcription and processing activity in motor neuron nuclei of trisomic mice, although to a lesser extent than in their euploid mates. These findings are a step towards understanding the mechanisms underlying the positive effect of physical activity in DS
Low ozone concentrations differentially affect the structural and functional features of non-activated and activated fibroblasts in vitro
Full text linkOxygen-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 and procaine increase secretion of platelet-derived factors in platelet-rich plasma
Full text linkPlatelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways
Physical training promotes remodeling of the skeletal muscle extracellular matrix: An ultrastructural study in a murine model of Down syndrome
Full text linkDown syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by multi-systemic premature aging associated with deficit in motor coordination, balance, and postural control. Using a morphological, morphometrical, and immunocytochemical ultrastructural approach, this study investigated in vastus lateralis muscle of Ts65Dn mouse, a murine model of DS, the effect of an adapted physical training on the extracellular matrix (ECM) characteristics and whether the forecasted exercise-induced ECM remodeling impacts on sarcomere organization. Morphometry demonstrated thicker basement membrane and larger collagen bundles with larger interfibrillar spacing as well as irregularly arrayed myofibrils and lower telethonin density on Z-lines in trisomic versus euploid sedentary mice. In agreement with the multi-systemic premature aging described in DS, these ECM alterations were similar to those previously observed in skeletal muscle of aged mice. Adapted physical training induced remodeling of ECM in both trisomic and euploid mice, that is, enlargement of the collagen bundles associated with hypertrophy of collagen fibrils and reduction of the interfibrillar spacing. A re-alignment of the myofibrils and a higher telethonin density on Z-line was found in trisomic mice. Altogether, our findings suggest that physical training is an effective tool in limiting/counteracting the trisomy-associated musculoskeletal structural anomalies. The current findings constitute a solid experimental background for further study investigating the possible positive effect of physical training on skeletal muscle performance. RESEARCH HIGHLIGHTS: Vastus lateralis muscle of trisomic mice shows aging-like alterations of extracellular matrix. Training promotes extracellular matrix remodeling. Training may be an effective tool to counteract trisomy-associated alterations of skeletal muscle
The aging of skeletal muscle: morphological experimental approaches
No full textThe research project of my doctorate was to further investigate on myonuclear involvement in skeletal muscle aging as well as age-related changes in muscle ECM. In this purpose, two different methodological approaches were used. First, ultrastructural immunocytochemistry revealed age-related differences in the distribution and density of some molecular factors involved in RNA pathways between old (28 months) mice and their late adult counterpart (19 months). Second, morphological and morphometrical evaluation (performed by fluorescent and transmission electron microscopy) combined with proteomic approach showed higher amount of several muscle ECM components in old (24 months) compared to adult mice (12 months), suggesting that muscle ECM remodelling may hinder muscle cell-matrix interplay as well as its structural organization. Once age-related muscular dysfunctions had been identified, we investigated two strategies potentially able to counteract sarcopenia: physical exercise (as a non-pharmacological approach) and nanotechnology (considered as a suitable tool for pharmacological intervention). Preliminary data based on morphometrical and morphological evaluations performed at transmission and fluorescence microscopy showed that in old (24 months) trained mice physical activity limit/prevent age-related modifications of myofiber (at cellular and sub-cellular level) as well as muscle ECM organization. Further, a pilot study tested the suitability of phospholipid-based nanoparticles (NPs: ethosomes, transethosomes) as nanocarriers to deliver vitamin D3, based on evidence suggesting that vitamin D supplementation may improve muscle functionality in elderly. We have evaluated NPs biocompatibility in three different cell line representative of epithelial, connective and muscle tissue. Then, observation at bright-field, fluorescence and transmission electron microscopy were performed to assess NPs internalization, intracellular fate and the potentially associated subcellular alteration. Our preliminary results set the stage for in vivo investigations aimed at testing vitamin D transdermal administration as a novel strategy to address age-related muscular disease. Finally, during my research period abroad I collaborated in setting up an in vitro co-culture system containing primary macrophages and murine myoblast cells which were previously exposed to etoposide (a drug able to induce DNA damage). Muscle cells were investigated by real-time PCR, to determine whether myoblast gain or lose their intrinsic function in presence of DNA damage and in presence or absence of macrophages. Lastly, immunofluorescence investigations were carried out in muscle cells to investigate the ability of macrophages to counteract DNA damage. The purpose of this preliminary investigation was to describe in vitro the crosstalk between macrophages and muscle cells under DNA damage condition
Aged gastrocnemius muscle of mice positively responds to a late onset adapted physical training
Full text linkIntroduction: A regular physical training is known to contribute to preserve muscle mass and strength, maintaining structure and function of neural and vascular compartments and preventing muscle insulin resistance and inflammation. However, physical activity is progressively reduced during aging causing mobility limitations and poor quality of life. Although physical exercise for rehabilitation purposes (e.g., after fractures or cardiovascular events) or simply aiming to counteract the development of sarcopenia is frequently advised by physicians, nevertheless few data are available on the targets and the global effects on the muscle organ of adapted exercise especially if started at old age.Methods: To contribute answering this question for medical translational purposes, the proteomic profile of the gastrocnemius muscle was analyzed in 24-month-old mice undergoing adapted physical training on a treadmill for 12 weeks or kept under a sedentary lifestyle condition. Proteomic data were implemented by morphological and morphometrical ultrastructural evaluations.Results and Discussion: Data demonstrate that muscles can respond to adapted physical training started at old age, positively modulating their morphology and the proteomic profile fostering protective and saving mechanisms either involving the extracellular compartment as well as muscle cell components and pathways (i.e., mitochondrial processes, cytoplasmic translation pathways, chaperone-dependent protein refolding, regulation of skeletal muscle contraction). Therefore, this study provides important insights on the targets of adapted physical training, which can be regarded as suitable benchmarks for future in vivo studies further exploring the effects of this type of physical activity by functional/metabolic approaches
L-Carnitine Functionalization to Increase Skeletal Muscle Tropism of PLGA Nanoparticles
Full text linkMuscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na+-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells
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