Can ultrasound counteract bone loss? Effect of low-intensity ultrasound stimulation on a model of osteoclastic precursor

The aim of the present work is to determine whether mechanical stress caused by ultrasound (US) exposure affects osteoclastic precursor cells, thus addressing the hypothesis that mechanical strain-induced perturbation of preosteoclastic cell machinery can contribute to the occurrence of bone turnover alterations. Moreover, cell cytoskeleton was studied because of its supposed involvement in cell mechanotransduction.Our experimental model was the FLG 29.1 human cell line, previously characterized as an osteoclastic precursor model. Cell proliferation was quantified by trypan blue exclusion assay. Cell morpho-functional state was monitored by multispectral imaging autofluorescence microscopy. The expression of cytoskeletal components and markers of proliferation (Ki67) and osteoclastic differentiation (RANK) was analysed by immunocytochemistry.The findings demonstrated that US stimulation affects FLG 29.1 cell growth, depresses the expression of cytoskeletal components and markers of proliferation and differentiation, induces cell damage, thus supporting the hypothesis that US exposure inhibits osteoclastogenesis.These results have been compared with those obtained previously by exposure of FLG 29.1 cells to modelled hypogravity conditions. Finally, the possibility to utilize US stimulation for counteracting osteoporosis has been discussed.     

Cell morphological, ontogenic, and genetic reactions to 0-g simulation and hyper-g

Organisms use gravity for spatial orientation, and differentiation into species during evolution follows geological processes which are caused by gravity. On the other hand, the task of most organismic functions which have or may have a relation to gravity is to compensate gravity. Furthermore, today it is very obvious that organisms do not disintegrate under the conditions of weightlessness, at least for the currently tested durations. These previous statements indicate a large field of still unknown regulation and adaptation mechanisms. Experiments to simulate weightlessness on the fast clinostat and with hyper-g show a highly developed ability of the genetic chain and of differentiating cells in being autonomous against mechanical stresses caused by outer accelerations. Nevertheless, different strong and slight changes of different tested end points were found. The question remains if the cells react actively or only passively.     

Gravity perception and signal transduction in single cells

Cellular signal processing in multi-, as well as in unicellular organisms, has to rely on fundamentally similar mechanisms. Free-living single cells often use the gravity vector for their spatial orientation (gravitaxis) and show distinct gravisensitivities. In this investigation the gravisensitive giant ameboid cell Physarum polycephalum (Myxomycetes, acellular slime molds) is used. Its gravitaxis and the modulation of its intrinsic rhythmic contraction activity by gravity was demonstrated in 180 °turn experiments and in simulated, as well as in actual, near-weightlessness studies (fast-rotating clinostat; Spacelab D1, IML-1). The stimulus perception was addressed in an IML-2 experiment, which provided information on the gravireceptor itself by the determination of the cell's acceleration-sensitivity threshold. Ground-based experiments designed to elucidate the subsequent steps in signal transduction leading to a motor response, suggest that an acceleration stimulus induces changes in the level of second messenger, adenosine 3',5'-cyclic monophosphate (cAMP), indicating also that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.     

Cell-to-cell interactions in changed gravity: ground-based and flight experiments

Cell-to-cell interactions play an important role in all physiological processes and are mediated by humoral and mechanical factors. Mechanosensitive cells (e.g., osteocytes, chondrocytes, and fibroblasts) can be studied ex vivo to understand the effects of an altered gravity environment. In particular, cultured endothelial cells (EC) are very sensitive to a broad spectrum of mechanical and biochemical stimuli. Earlier, we demonstrated that clinorotation leads to cytoskeletal remodeling in cultured ECs. Long-term gravity vector changes also modulate the expression of surface adhesion molecules (ICAM-1, E-selectin, VCAM-1) on cultured ECs. To study the interactions of geterological cells, we cocultured endothelial monolayers and human lymphocytes, immune cells and myeloleucemic (K-560) cells. It was found that, although clinorotation did not alter the basal adhesion level of non-activated immune cells on endothelial monolayers, the adhesion of PMA-activated lymphocytes was increased. During flight experiments onboard the Russian segment of the International Space Station, we measured the cytotoxic activity of natural killer (NK) cells incubated with labeled target cells. It was found that immune cells in microgravity retained their ability to contact, recognize, and destroy oncogenic cells in vitro. Together, our data concerning the effects of simulated and real microgravity suggest that, despite changes in the cytoskeleton, cell motility, and expression of adhesion molecules, cell-cell interactions are not compromised, thus preserving the critical physiological functions of immune and endothelial cells.     

Vascular functions in humans following cardiovascular adaptations to spaceflight

Purpose: Diminished vascular function is a primary cardiovascular risk of spaceflight identified in the 2004 NASA Bioastronautics Critical Path Roadmap based on: (1) structural and functional alterations in arterial vessels of animals undergoing hindlimb unloading and; (2) lower peripheral vascular resistance (PVR) in astronauts who became presyncopal after spaceflight.Methods: We conducted a critical review of published data obtained from spaceflight and relevant ground-based microgravity simulations in an effort to interpret the meaning of altered responses in PVR and their relationship to postflight presyncope.Results: Presyncope reported in astronauts on landing day was associated with lower peripheral resistance. However, non-presyncopal astronauts demonstrated significantly elevated vascular resistance in the upright posture after compared with before spaceflight. Results from both space and ground experiments suggest that preflight maximal vasoconstrictor capacity is inherently lower in presyncopal astronauts, but unaltered by spaceflight.Conclusions: Vasoconstrictor reserve is associated with lower blood volume adaptation to microgravity. Rather than reduced vascular function, low inherent maximal vasoconstrictor capacity and reduced vasoconstrictor reserve secondary to decreased circulating vascular volume explain lower peripheral vascular resistance in astronauts who experience presyncopal episodes on landing day.     

Application of GFP technique for cytoskeleton visualization onboard the International Space Station

Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity.     

Nemaline rod and degeneration of Z band of muscle cell in weightlessness at spaceflight

There are some studies demonstrating the skeletal muscle degeneration associated with the degeneration of Z band and appearance of nemaline rods in experimental animals of the simulation model for spaceflight but not in human heart tissues. In the present study, therefore, we investigated the pathological changes or degeneration in left auricular heart muscles obtained during operations of mitral valves replacement using both electron and light microscopies. The degeneration of Z band even in the myofibrils of comparatively little damaged cell was found. Furthermore, nemaline rods were detected in most of the heart muscle cells. These results suggest that the existence of nemaline rods is involved in the cell injury in the heart muscle of patients with heart disease without nemaline myopathy. Further study is necessary to know whether the similar pathological findings are observed not only in the skeletal muscle but also in the cardiac muscle in experimental animals of the simulation model for spaceflight or in a prolonged spaceflight.     

全球星摄动运动及摄动补偿运控策略研究

绕地卫星摄动运动是导致星座设计构型发散的主要因素,摄动补偿轨道控制是维持全     

Altered expression of mRNAs implicated in osteogenesis under conditions of simulated microgravity is regulated by CD200:CD200R

Mouse calvarial cells grown under simulated microgravity conditions (neutral buoyancy) show preferential differentiation towards the osteoclast lineage, as defined by surrogate mRNAs, bone nodule growth and TRAP⁺ cells, when compared with cells cultured under normal gravity conditions. This effect was suppressed in cultures which contained the immunoregulatory molecule CD200, and conversely enhanced by anti-CD200 mAb. Concomitant increases occur in expression of inflammatory cytokines, and their mRNAs, under simulated microgravity conditions. Again cultures containing exogenous CD200 showed suppressed cytokine and cytokine mRNA expression. Further alterations in osteoclastogenesis were seen using cells isolated from cytokine-receptor knockout mice. We conclude that, as assessed by altered expression of mRNAs associated with osteoblast differentiation, CD200:CD200R interactions play an important regulatory role in the enhanced osteoclastogenesis seen under simulated microgravity conditions, with changes in cytokine expression further modulating this effect.     

Choroidal responses in microgravity. (SLS-1, SLS-2 and hindlimb-suspension experiments)

Fluid and electrolyte shifts occuring during human spaceflight have been reported and investigated at the level of blood, cardio-vascular and renal responses. Very few data were available concerning the cerebral fluid and electrolyte adaptation to microgravity, even in animal models. It is the reason why we developed several studies focused on the effects of spaceflight (SLS-1 and SLS-2 programs, carried on NASA STS 40 and 56 missions, which were 9- and 14-day flights, respectively), on structural and functional features of choroid plexuses, organs which secrete 70–90 % of cerebrospinal fluid (CSF) and which are involved in brain homeostasis. Rats flown aboard space shuttles were sacrificed either in space (SLS-2 experiment, on flight day 13) or 4–8 hours after landing (SLS-1 and SLS-2 experiments). Quantitative autoradiography performed by microdensitometry and image analysis, showed that lateral and third ventricle choroid plexuses from rats flown for SLS-1 experiment demonstrated an increased number (about x 2) of binding sites to natriuretic peptides (which are known to be involved in mechanisms regulating CSF production). Using electron microscopy and immunocytochemistry, we studied the cellular response of choroid plexuses, which produce cerebrospinal fluid (CSF) in brain lateral, third and fourth ventricles. We demonstrated that spaceflight (SLS-2 experiment, inflight samples) induces changes in the choroidal cell structure (apical microvilli, kinocilia organization, vesicle accumulation) and protein distribution or expression (carbonic anhydrase II, water channels,…). These observations suggested a loss of choroidal cell polarity and a decrease in CSF secretion. Hindlimb-suspended rats displayed similar choroidal changes. All together, these results support the hypothesis of a modified CSF production in rats during long-term (9, 13 or 14 days) adaptations to microgravity.     

Taxane recovery from cells of Taxus in micro- and hypergravity

Cell suspension cultures of Taxus cuspidata produce taxanes that are released from the outer surface of cells into the culture medium as free and bound alkaloids. Paclitaxel (Taxol (TM)), is an anti-cancer drug in short supply. It has a taxane ring derived from baccatin III and a C-13 phenylisoserine side-chain. This drug is produced over a wide range of gravitational forces. Monoclonal and polyclonal antibodies to paclitaxel, baccatin III, and the C-13 phenylisoserine side chain were combined in multiple-labeling studies to localize taxanes and paclitaxel on cell surfaces or on particles released into the culture medium. Bioreactor vessel design altered the composition of taxanes recovered from cells in simulated microgravity. At 10(-2) and 2x10(-4)g, taxane recovery was reduced but biomass growth and percent paclitaxel was significantly increased. At 1 to 24g, growth was reduced with a significant recovery of total taxanes with low percent paclitaxel. Bound paclitaxel was also localized in endonuclease-rich fragmenting nuclei of individual apoptotic cells. A model is presented comprising TCH (touch) genes encoding enzymes that modify taxane-bearing xylan residues in cell walls, the calcium-sensing of gravitational forces by the cytoplasm, and the predisposition of nuclei to apoptosis. This integrates the adaptive physiological and biochemical responses of drug-producing genomes with gravitational forces.     

Medical considerations for extending human presence in space

The prospects for extending the length of time that humans can safely remain in space depend partly on resolution of a number of medical issues. Physiologic effects of weightlessness that may affect health during flight include loss of body fluid, functional alterations in the cardiovascular system, loss of red blood cells and bone mineral, compromised immune system function, and neurosensory disturbances. Some of the physiologic adaptations to weightlessness contribute to difficulties with readaptation to Earth's gravity. These include cardiovascular deconditioning and loss of body fluids and electrolytes; red blood cell mass; muscle mass, strength, and endurance; and bone mineral. Potentially harmful factors in space flight that are not related to weightlessness include radiation, altered circadian rhythms and rest/work cycles, and the closed, isolated environment of the spacecraft. There is no evidence that space flight has long-term effects on humans, except that bone mass lost during flight may not be replaced, and radiation damage is cumulative. However, the number of people who have spent several months or longer in space is still small. Only carefully-planned experiments in space preceded by thorough ground-based studies can provide the information needed to increase the amount of time humans can safely spend in space.     

Rapid rotations of a satellite with a cavity filled with viscous fluid under the action of moments of gravity and light pressure forces

Rapid rotational motion of a dynamically asymmetric satellite relative to the center of mass is studied. The satellite has a cavity filled with viscous fluid at low Reynolds numbers, and it moves under the action of moments of gravity and light pressure forces. Orbital motions with an arbitrary eccentricity are supposed to be specified. The system, obtained after averaging over the Euler-Poinsot motion and applying the modified averaging method, is analyzed. The numerical analysis in the general case is performed, and the analytical study in the axial rotation vicinity is carried out. The motion in the specific case of a dynamically symmetric satellite is considered.     

An endogenous growth pattern of roots is revealed in seedlings grown in microgravity

In plants, sensitive and selective mechanisms have evolved to perceive and respond to light and gravity. We investigated the effects of microgravity on the growth and development of Arabidopsis thaliana (ecotype Landsberg) in a spaceflight experiment. These studies were performed with the Biological Research in Canisters (BRIC) hardware system in the middeck region of the space shuttle during mission STS-131 in April 2010. Seedlings were grown on nutrient agar in Petri dishes in BRIC hardware under dark conditions and then fixed in flight with paraformaldehyde, glutaraldehyde, or RNAlater. Although the long-term objective was to study the role of the actin cytoskeleton in gravity perception, in this article we focus on the analysis of morphology of seedlings that developed in microgravity. While previous spaceflight studies noted deleterious morphological effects due to the accumulation of ethylene gas, no such effects were observed in seedlings grown with the BRIC system. Seed germination was 89% in the spaceflight experiment and 91% in the ground control, and seedlings grew equally well in both conditions. However, roots of space-grown seedlings exhibited a significant difference (compared to the ground controls) in overall growth patterns in that they skewed to one direction. In addition, a greater number of adventitious roots formed from the axis of the hypocotyls in the flight-grown plants. Our hypothesis is that an endogenous response in plants causes the roots to skew and that this default growth response is largely masked by the normal 1?g conditions on Earth.     

Navigating in a Virtual 3D Maze: Body and Gravity,Two Possible Reference Frames for Perceiving and Memorizing

Although recent studies have brought new insight into the mechanisms of spatial memory and cognitive strategies during navigation, most of these studies have concerned two-dimensional navigation and little is known regarding the problem of three-dimensional (3D) spatial memory. We found previously that memorizing complex 3D-structured corridors was easier with natural self-motion that included only yaw turns, and vertical translations facing the walls at vertical sections. This suggests that when only sideways (yaw) mental rotations had to be performed in order to shift from the experienced egocentric to the allocentric reference frame where recognition was tested, memorization of such corridors was improved. In the present investigation we studied the effect of tilting separately subject's body axis and self-motion's rotation axis relative to gravity. With a computerized 3D reconstruction task of the maze, we examined whether having any single rotation axis was enough to facilitate this reference shift or, if not, what aspect of the terrestrial condition-where visual displacement rotation, gravity and body axes were aligned-led to better performance. Field dependent (FD) and independent (FI) subjects, as determined by the rod and frame test, showed distinct effects of the navigation conditions. The FD group performance was markedly impaired when gravity and body axis were in conflict, independently of the rotation axis, whereas FI performance only slightly worsened when the body was tilted and the rotation axis remained aligned with gravity. Moreover, tilting the body in the control condition only worsened performance for the FD group.     

Study of physiological effects of weightlessness and artificial gravity in the flight of the biosatellite Cosmos-936.

In the 18.5-day flight of the Soviet biosatellite Cosmos-936 (3-22, August 1977) com-parative investigations of the physiological effects of prolonged weightlessness (20 rats) and artificial gravity of 1 g (10 rats) were carried out. Throughout the flight artificial gravity was generated by means of animal rotation in two centrifuges with a radius of 320mm. Postflight examination of animals and treatment of the flight data were performed by Soviet scientists in collaboration with the specialists from Bulgaria, Czechoslovakia, the German Democratic Republic, Hungary, Poland, Rumania, France and the U.S.A. During the flight the total motor activity of the weightless rats was higher and their body temperature was lower than those of the centrifuged animals. Postflight examination of the weightless rats showed a greater percentage of errors during maze an increase in water intake and a decrease in diuresis; a fall of the resistance of peripheral red cells; an increase in the conditionally pathogenic microflora in the mouth; a decrease of oxygen consumption, carbon dioxide production and energy expenditures; a drop in the static physical endurance; a decline in the capacity to keep balance on the rail; an increase in the latent period of the lifting reflex, etc. The centrifugal animals displayed lesser or no change of the above type. These findings together with the biochemical and morphological data give evidence that during and after flight adaptive processes in the centrifuged rats developed better.     

T cell regulation in microgravity – The current knowledge from in vitro experiments conducted in space,parabolic flights and ground-based facilities

Dating back to the Apollo and Skylab missions, it has been reported that astronauts suffered from bacterial and viral infections during space flight or after returning to Earth. Blood analyses revealed strongly reduced capability of human lymphocytes to become active upon mitogenic stimulation. Since then, a large number of in vitro studies on human immune cells have been conducted in space, in parabolic flights, and in ground-based facilities. It became obvious that microgravity affects cell morphology and important cellular functions. Observed changes include cell proliferation, the cytoskeleton, signal transduction and gene expression. This review gives an overview of the current knowledge of T cell regulation under altered gravity conditions obtained by in vitro studies with special emphasis on the cell culture conditions used. We propose that future in vitro experiments should follow rigorous standardized cell culture conditions, which allows better comparison of the results obtained in different flight- and ground-based experiment platforms.     

Role of O.G. Gazenko in formation and advancement of space biology and medicine

Oleg G. Gazenko belongs to the noble cohort of pupils of well-known Russian physiologist L.A. Orbeli. He was one of the fathers of space biology and medicine, discipline in which he displayed his brilliant talents of experimenter and thinker. He was acknowledged for the investigations of spaceflight effects on living systems, the concept of medical operations system to support long-term piloted missions and implementation of biological researches that fostered the advance of space and gravitational biology. The analytical works of Oleg G. Gazenko are of imperishable significance for future researches to the benefit of space biomedicine.     

The natural cytotoxicity in cosmonauts on board space stations

The nature of the changes of resistance to infection seems to be very important. Our studies indicate that different functions of natural killers could be depressed after the spaceflight. The decrease of the percentage of the lymphocytes that can bind target cells lead to the lowering of the “active” NK level and this can be resulted in the depression of total NK activity and lowering of resistance to viral and tumor antigens. The investigation of natural killer cells in cosmonauts before and after short and long-term spaceflights also revealed the important role of spaceflight duration, stress and individual immune reactivity.