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Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

The Italian Space Agency, within the frame of the mission “Beyond” has promoted the "YiSS—Youth ISS Science" competition, to involve secondary school students in the process of conception and execution of a space experiment. The XENOGRISS experiment was selected by the Italian Space Agency to fly onboard the ISS. The project foresees an active involvement of students into a multi-disciplinary activity aimed at studying the effect of microgravity on growth and regeneration processes, using an animal model (Xenopus laevis) that allows both processes to be observed simultaneously. The project involves the preparation of a Xenopus laevis tadpole culture within a Xenopus Experiment Unit (XEU). The XEU has been integrated into a powered metallic sealed container called Biokon. The activation on the ISS ensured the feeding of the tadpoles, the recirculation of water and the acquisition of images. The control electronics, including the acquisition system, was designed and realized by the students under teacher supervision in collaboration with engineers from Kayser Italia. The integration process of the experiment onboard the ISS was provided by the UTISS Team (Argotec/Telespazio) that supported safety evaluation, requirements verification, payload manifesting, delivery, operations and recovery. The facility has been launched with Space-X CRS-19 and recovered after 30 days. The information derived from this experiment will help to understand the mechanisms underlying the effect of microgravity upon the processes of growth, repair and regeneration of tissues. A better knowledge of these processes is important in defining protocols for the management of traumatic injuries, wounds and chronic ulcers both in Space and on Earth.

Over the past three decades, physicians have used laser sources for the management of different pain conditions obtaining controversial results that call for further investigations. In order to evaluate the pain relieving possibilities of photobiomodulation therapy (PBMT), we tested two near infrared (NIR) laser systems, with different power, against various kinds of persistent hyperalgesia animal models. In rats, articular pain was reproduced by the intra-articular injection of sodium monoiodoacetate (MIA) and complete Freund's adjuvant (CFA), while compressive neuropathy was modelled by the chronic constriction injury of the sciatic nerve (CCI). In MIA and CFA models, (NIR) laser (MLS-Mphi, ASA S.r.l., Vicenza, Italy) application was started 14 days after injury and was performed once a day for a total of 13 applications. In MIA-treated animals, the anti-hyperalgesic effect of laser began 5 min after treatment and vanished after 60 min. The subsequent applications evoked similar effects. In CFA-treated rats, laser efficacy started 5 min after treatment and disappeared after 180 min. In rats that underwent CCI, two treatment protocols with similar fluence but different power output were tested using a new experimental device called Multiwave Locked System laser (MLS-HPP). Treatments began 7 days after injury and were performed during 3 weeks for a total of 10 applications. Both protocols reduced mechanical hyperalgesia and hindlimb weight bearing alterations until 60 min after treatment with a higher efficacy recorded for the animals treated using the higher power output. In conclusion, this study supports laser therapy as a potential treatment for immediate relief of chronic articular or neuropathic pain.

Microgravity-induced muscle atrophy is a problem of utmost importance for the impact it may have on the health and performance of astronauts. Therefore, appropriate countermeasures are needed to prevent disuse atrophy and favour muscle recov- ery. Muscle atrophy is characterized by loss of muscle mass and strength, and a shift in substrate utilization from fat to glucose, that leads to a reduced metabolic efficiency and enhanced fatigability. Laser therapy is already used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of the research we present was to get insights on possible benefits deriving from the application of an advanced infrared laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of the hypotrophic tissue. The source used was a Mul- tiwave Locked System (MLS) laser, which combines continuous and pulsed emissions at 808 nm and 905 nm, respectively. We studied the effect of MLS treatment on morphology and energy metabolism of C2C12 cells, a widely accepted myoblast model, previously exposed to microgravity conditions modelled by a Random Po- sitioning Machine. The MLS laser treatment was able to restore basal levels of serine/threonine protein phos- phatase activity and to counteract cytoskeletal alter- ations and increase in glycolytic enzymes activity that occurred following the exposure to modelled micro- gravity. In conclusion, the results provide interesting insights for the application of infrared laser in the treatment of muscle atrophy.

The number of astronauts involved in long-lasting missions and extra-vehicular activities is going to increase in the future. Consequently, the chance of injury due to traumatic events or unexpected emergency surgery will also increase and medical evacuation times to earth will be prolonged. Hence, the need to address requirements for surgery and trauma care in non terrestrial environments will be a priority. Tissue repair in weightlessness should therefore be regarded as a major issue not enough studied to date. Wound healing is a complex multi-step process, crucial to the survival of the organism. It starts with an inflammatory phase followed by a remodelling phase. During repair, the extracellular matrix (ECM) is sequentially remodelled by the concerted action of different cell types, in order to rebuild a functional tissue. The available literature concerning wound healing with mechanical unloading presents controversial results. However, many studies indicate impairment of the healing processes. Here we present a study on the behaviour of cells involved in the remodelling phase of repair, e.g. fibroblasts and endothelial cells, in response to microgravity (μg). In particular, their adhesion/migration, cytoskeleton organization, production of ECM molecules and receptors have been investigated. Cell response to pulsed Nd:YAG laser irradiation has also been investigated in order to evaluate the possibility to use laser irradiation for counteracting the effect of μg on wound healing. In μg, we observed alterations in production/assembling of ECM molecules. Increased fibronectin (FN) and laminin (LM) could be the cause for impaired ECM rebuilding and altered cell adhesion/migration. Treatment with Nd:YAG laser pulses induced organized fibrillogenesis and favoured endothelial cell spreading and monolayer formation. These findings open the way for a better understanding of tissue repair mechanisms in space and future clinical applications on earth.

In the body, connective tissues have a major function in sustaining mechanical stresses. On the other hand, mechanical forces are important factors for connective tissue homeostasis. Connective tissues dynamically interact with mechanical and gravitational stimuli, changing their mechanical properties through the continuous modification of their composition, and thus improving their function. In connective tissues, mechanical forces are major regulators of extracellular matrix turnover, strongly affecting the production of extracellular matrix proteins. On the contrary, unloading conditions, such as bed rest or space flight, have a negative effect on these tissues, with loss of mass and impairment of mechanical properties. Here we describe the effect of photomechanical stress, supplied by a pulsed Nd:YAG laser, on extracellular matrix production by fibroblasts and chondrocytes, and compare it with the effect produced by hypergravity conditions. Cell morphology and structure, extracellular matrix production, cell adhesion, cell energy metabolism have been studied in treated human fibroblasts and chondrocytes by using immunocytochemistry, fluorescence and autofluorescence microscopy. The results show that photomechanical stress induce cytoskeleton remodelling, redistribution of membrane integrins, increase in production of ECM molecules, changes in cell energy metabolism. The effects are similar to those observed in the same cells exposed to cyclic hypergravitational stress (10×g).

Dendritic cells (DCs), the most potent antigen-presenting cells inducing specific immune responses, are involved in the pathogenesis of atherosclerosis. In this inflammatory disease, DCs increase in number, being particularly abundant in the shoulder regions of plaques. Since the exposure to altered gravitational conditions results in a significant impairment of the immune function, the aim of this study was to investigate the effects of hypergravity on both the function of DCs and their interactions with the vascular wall cells. Monocytes from peripheral blood mononuclear cells of healthy volunteers were sorted by CD14+ magnetic beads selection, cultured for 6 days in medium supplemented with GM-CSF and IL-4, followed by a further maturation stimulus. DC phenotype, assessed by flow cytometry, showed a high expression of the specific DC markers CD80, CD86, HLA-DR and CD83. The DCs obtained were then exposed to hypergravitational stimuli and their phenotype, cytoskeleton, ability to activate lymphocytes and interaction with vascular wall cells were investigated. The findings showed that the exposure to hypergravity conditions resulted in a significant impairment of DC cytoskeletal organization, without affecting the expression of DC markers. Moreover, an increase in DC adhesion to human vascular smooth muscle cells and in their ability to activate lymphocytes was observed.

It is well known that endothelial cells (ECs), which play a major role in cardiovascular system functioning, are very sensitive to mechanical stimuli. It has been demonstrated that changes in inertial conditions (i.e. microgravity and hypergravity) can affect both phenotypic and genotypic expression in ECs. In this report we describe the effects of hypergravity on ECs isolated from bovine aorta (BAECs). ECs were repeatedly exposed to discontinuous hypergravity conditions (5 × 10 min at 10×g with 10 min at 1×g between sets), simulated in a hyperfuge. Then, cell morphology and metabolism were analyzed by autofluorescence techniques. The phenotypic expression of cytoskeleton constituents (β-actin, vimentin, tubulin), adhesion and survival signals (integrins), mediators of inflammation and angiogenesis was evaluated by immunocytofluorescence. Quantitative PCR (Q-PCR) with Low Density Arrays (LDAs) was used to evaluate modifications in gene expression. After hypergravity exposure, no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substratum, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. On the whole, the results documented that hypergravity exposure maintained EC survival and function by activation of adaptive mechanisms.

The exposure to microgravity conditions results in a significant impairment of the immune function. Many reports describe morphological and functional changes in T-lymphocytes, monocytes and neutrophil granulocytes cultured in microgravity, both real and modeled, but very few studies have been made on the effect of microgravity on dendritic cells (DCs) and DC differentiation. DCs are able to process antigens and are the most efficient cells in presenting them to T-lymphocytes, thus giving a crucial contribution to the rise of an effective immune response. The aim of this study was to investigate whether the maturation of DCs from monocytes of astronauts was altered postflight. Blood samples from a crew-member of the Eneide mission were collected before the flight, soon after the return to earth and one year after the mission. In order to generate DCs, monocytes were used as precursors. They were separated, cultured for 6 days in medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4), then furtherly stimulated for 24 hours with a cocktail of cytokines. Differentiation was assessed by flow cytometry and immunofluorescence, assaying the expression of typical DC markers. Gene expression was analysed by RT-PCR. Morphological and functional characteristics were studied by autofluorescence microscopy. The findings showed that the maturation of DCs from monocytes collected from the astronaut immediately postflight was altered. In comparison with controls, significant differences were found in expression of DC markers, expression of genes involved in DC maturation, morphological and functional characteristics.

Cardiovascular diseases are major health problems in astronauts and pilots. The basic problem in cardiovascular diseases is the loss of function by vascular endothelium. It has been demonstrated that changes in inertial conditions (i.e. hypo- and hypergravity) can affect both phenotypic and genotypic expression in endothelial cells. This report describes the effects observed in endothelial cells from coronary post-capillary venules after repeated exposures to hypergravity conditions, alternating with recovery periods. The results showed changes in gene expression, cell energy metabolism, morphology and cytoskeleton organization.

The characterisation of leukemic cell autofluorescence during differentiation, induced by 12-O-tetradecanoylphorbol 13-acetate and all-trans retinoic acid, was performed by autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy. We have found that a dependence exists between the cell autofluorescence pattern and the degree of cell differentiation. When cells differentiate, their autofluorescence emission changes, following the morphological and functional rearrangement of cell structures. A decrease in emission intensity and a different distribution of endogenous fluorophores are observed. Thus, autofluorescence monitoring on living cells is a potentially useful tool for in vitro study of the differentiation processes. Furthermore, different maturation steps can be distinguished on the basis of the cell fluorescence pattern, leading the way to future application of the technique in diagnostics.