Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.     

The effect of combined hypergravity and micro-grooved surface topography on the behaviour of fibroblasts

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and micro-grooved substrata (groove depth: 1 mum, width: 1, 2, 5, 10 microm), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and alpha(1)- and beta(1)-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces.     

The effect of simulated microgravity on seed germination and seedling anatomy of Phaseolus vulgaris L

Seed germination and root anatomy were investigated in seedlings of Phaseolus vulgaris L. developed on a slowly rotating bi-dimensional clinostat and in 1g. Germination time, percent germination, curvature and anatomy of developing root apexes were monitored on the clinostat and compared with the control. Interesting differences were found in germination and root features of the seeds developed on the clinostat compared with 1g ones: the main being germination time, root cap formation, the quantity and distribution of amyloplasts in statocytes. The use of a software to quantitatively analyse root cap anatomy allowed us to detect some differences otherwise unlikely to highlight. Our results showed that prolonged rotation on a bi-dimensional clinostat has an effect on some aspects of germination and on the statocytes that continuously perceives gravity from ever-changing directions.     

Morphological and morphometric study on the effect of simulated microgravity on rat testis

The present study was undertaken to determine the effect of simulated microgravity on the testis of the rats and to evaluate the possibility of spermatogenesis failure in space environment. Fifty-four adult male albino rats were used in this study. They were divided equally into intact control, stress control and experimental groups. The rats of the intact control group (Group Ia) were kept without intervention. The rats of both the stress control and experimental groups were subjected to inguinal canal ligation and tail-suspension. In the stress control group (Group Ib) the hindlimbs were not elevated above the floor of the housing units whereas in the experimental groups the hindlimbs were elevated for one week (Group II) and six weeks (Group III), respectively. In a third experimental group (Group IV) the rats were hindlimb-suspended for six weeks followed by another six weeks without suspension to allow recovery. Prior to sacrifice, the animals were weighed and anesthetized, and the testes were excised and weighed. Testicular specimens were processed for histological, histochemical and morphometric studies. The results of the present study revealed that only after six weeks of hindlimb-suspension, the rats showed a significant decline in testicular weight compared with the control groups. Histologically, few abnormalities were observed in some seminiferous tubules in one-week hindlimb-suspended group. Spermatogenesis was significantly reduced by six-week of hindlimb-suspension marked by atrophy of the testes and loss of all germ cells, except a few spermatogonia. Spermatogenesis was partially restored in the recovery group. In all groups the appearance of Sertoli cells remained the same. Proliferation of Leydig cells was observed in the experimental groups. It is concluded that spermatogenesis is severely inhibited by six weeks of hindlimb-suspension and that it is partially restored following six weeks of recovery. This study provides further insights regarding the serious effects of long-term exposure to microgravity on the testes of mammals, including human beings.     

模拟微重力对肺动脉和胸主动脉的影响

目的 :通过对模拟微重力 (SM)、肺动脉 (PA)和胸主动脉 (TA)局部调节机理研究 ,为大小循环动脉对SM适应机理和SM后立位耐力降低机理研究积累资料。方法 :XXH 2 0 0 0型小循环心功能检测仪检测人体头低位 6°卧床(HDT) 7d心肺循环功能变化。 - 30°尾部悬吊 (TS)大鼠模拟微重力 (microgravity ,M)的生理效应 ,测量 7d、1 4dPA和TA的反应性。结果 :人体HDT初期每搏PA(hz)、静脉 (hc)容量和左心前负荷 (hc/hz)均显著增加 ,96～ 1 4 4h大小循环均出现超调现象 ,前者出现时间早、幅度大。 7d尾部悬吊大鼠 (TS7)与对照组 (CON)比PA舒张反应显著增强 ,TS1 4有降低趋势。TS7TA与CON比舒张反应显著增强 ,TS1 4轻度升高。TS7PA收缩反应与CON比轻度降低 ,TS1 4显著降低。TS1 4TA收缩反应显著降低。去VECPA对KCl、苯肾上腺素和硝普钠的反应在所有组间无差异。结论 :SM对大小循环动脉影响不同 ,可能是SM时局部调节功能降低的重要表现 ,主要由于动脉血管内皮细胞功能变化 ,对立位耐力降低可能有贡献     

The effect of simulated microgravity on formation of the pigment apparatus in etiolated barley seedlings

The effect of horizontal clinorotation on the dynamics of the accumulation of the main photosynthetic pigments in the greening of 6-day-old etiolated barley seedlings has been studied. The content of protochlorophillide, the direct precursor of chlorophyll a, in clinorotated seedlings in the dark was 9–20% lower than in the control group. After exposure of barley seedlings to light for 12 h under clinorotation, chlorophyll accumulation lagged behind the control by 45% and reached the control value after 48–72 h. The total content of carotenoids increased many fold during greening; at the first stage the carotenoid level in clinorotated seedlings was less than in the control. The synthesis rates of δ-aminolevulinic acid and δ-aminolevulinate dehydratase activity in clinorotated seedlings were slower than in the control after 24 h of greening and after 72 h of greening reaching the control values. The activity of Mg-protoporphyrin IX chelatase catalyzing the incorporation of Mg ions in the structure of chlorophyll a, did not change when exposed to clinorotation. The results we obtained show inhibition of the initial stages of chlorophyll biosynthesis in the conditions of simulated microgravity. The light, to a certain extent, decreases the negative effect of microgravity on the formation of the photosynthetic apparatus in plants.     

The effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

The effects of simulated microgravity on characteristics of slow presaccadic potentials

The parameters of saccades and presaccadic slow potentials were studied in seven right-handed male volunteers with a dominant right eye before and after exposure to 6-day dry immersion. Visual stimuli were presented using three light diodes, which were located in the center of the visual field (the central fixation stimulus) and 10° to the right and left of it (peripheral stimuli (PSs)). The subjects performed a test with simple saccades to a PS and a test with antisaccades to the point located symmetrically in the opposite visual field. The EEG (19 monopolar leads) and electrooculogram were recorded. To isolate slow potentials, backward EEG averaging was performed, with the moment of switching on the PS serving as a trigger for the averaging. It was found that the characteristics of saccadic eye movements did not substantially change after exposure to immersion. However, both tests revealed a change in topography and a decrease in the amplitude of presaccadic slow negative potentials (PSNPs) during immersion. Characteristically, the focus of presaccadic negativity shifted to the right hemisphere so that the PSNP amplitude sharply decreased in the left and increased in the right hemisphere. A significant decrease in the PSNP amplitude on day 6 of immersion was found in the midline and left-hemispheric frontal and parietal leads. It may be suggested that, because of support unloading and a decrease in proprioceptive input, exposure to microgravity causes a decrease in the activity of the left hemisphere and prefrontal and parietal cortices, initially involved in preparation and realization of motor responses. The activation of the right hemisphere could be of compensatory character.     

Effects of simulated microgravity on embryonic stem cells

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.     

Salt-loading and simulated microgravity on baroreflex responsiveness in rats

Cardiovascular adaptations observed during exposure to microgravity results in impairment of baroreflex activity partially as a result of fluid and electrolyte shifts. The head-down tilt rat model mimics some of the physiological observations that have been made in astronauts. We examined the effects of salt-loading on baroreflex activity after 7 day simulated microgravity (30 degrees tail-suspension) and the subsequent 6 hr post-suspension in Sprague-Dawley (SD) rats, using low salt (0.3% NaCl) and high salt (8% NaCl) diets. In suspended animals on a low salt diet, the baroreflex response curve was shifted to the left, while the heart rate (HR) range and MAP50 values were reduced compared to their parallel tethered, non-suspended controls. For non-suspended animals, salt-loading shifted the curve to the right with a reduced HR range. In salt-loaded, suspended animals, the curve and its parameters resemble those of non-suspended animals on a low salt diet. In summary, these data have demonstrated that a short-term (seven days) simulated weightlessness may elicit cardiovascular deconditioning in rats after release from the simulation manifested as an altered responsiveness in baroreceptor-heart rate reflex and a lowered blood pressure while the rats are tethered and horizontal. Our results also suggest the counteracting effect of salt loading on cardiovascular deconditioning.     

Effect of simulated microgravity on potato minituber formation and structure

The effect of long-term clinorotation on potato minituber formation and the structural-functional organization of storage parenchyma cell in minitubers has been studied by using methods of organ culture in vitro, light- and electron microscopy, biochemistry as well as phenological observation. It was established some acceleration of growth, changes in the parenchyma cell ultrastructure and in the starch content as well as an intensification of phosphorylase activity in the storage tissue of minitubers under the influence of simulated microgravity.     

The effects of long-term simulated microgravity on neuromuscular performance in men and women

The effects are reported of prolonged exposure to simulated microgravity (strict bed rest in an antiorthostatic position -6 degrees head-down tilt, HDT) on voluntary and electrically evoked contractions of the triceps surae muscle in men (n = 6) and women (n = 4). The subjects served as their own controls. Bed rest is a model that has commonly been used to simulate spaceflight. Measurements made in the control condition (10-8 days before the beginning of HDT) and after 120-days of HDT (on the 3rd day after it ended) included examination of the properties of isometric maximal voluntary contractions (MVC), isometric twitch contractions (Pt) and tetanic contractions (Po). After HDT, the MVC decreased by means of 44% and 33%, P, by means of 36% and 11%, Po by means of 34% and 24%, in the men and the women, respectively. The difference between Po and MVC, expressed as a percentage of Po and referred to as force deficiency (FD), has also been calculated. The FD increased by means of 60% and 28.8% in the men and the women, respectively. Time-to-peak tension of the triceps surae muscle increased by means of 12% and 14% in the men and the women, respectively, but half-relaxation time decreased by means of 9% and 19%. Total contraction time increased by a mean of 23% in the men and decreased by a mean of 17% in the women. Force-velocity of properties of the triceps surae muscle calculated according to a relative scale of voluntary contraction development significantly decreased more in the women than the men. The calculations of the same properties of electrically evoked contraction development did not differ substantially from the initial physiological state. It can be concluded that not only were the contractile properties of the triceps surae muscle significantly different in the men and the women, but that the effects of exposure to simulated microgravity on these properties were also different. These differences may be explained by sex differences in the muscle tissue itself and in its maximal neural activation.     

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.     

Influence of simulated microgravity on human skeletal muscle architecture and function

Ten male volunteers underwent a period of prolonged bed rest. Four subjects performed exercise countermeasures 2-3 times per week, while 6 subjects received no countermeasures. After bed rest plantarflexor force declined significantly (P < 0.001) in both exercise (-42%) and control (-55%) groups. The internal architecture of the gastrocnemius medialis (GM) muscle was significantly altered. This was associated with a reduction in fascicle shortening during isometric contraction. Exercise countermeasures partially mitigated the loss of muscle force and function following 90 days of bed rest.     

The collagen recognition sequence for fibroblasts depends on collagen topography

We found that the peptide Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) inhibited spreading of human fibroblasts inside collagen gels and markedly decreased gel contraction, but this peptide had no effect on cell spreading on collagen-coated surfaces. On the other hand, the peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), which inhibited cell spreading on collagen-coated surfaces, did not inhibit cell spreading within collagen gels and was a less effective inhibitor of collagen gel contraction than GRGESP. Based on these findings, we conclude that human fibroblasts can interact with different collagen cell recognition sequences depending upon topographical organization of the collagen.     

微重力环境下的细菌生物学效应

本文主要根据国内外微重力环境下细菌生物学效应的研究文献,从细菌生长繁殖、抗生素敏感性、毒力变化、新陈代谢等方面进行总结,并概述了微重力环境改变细菌生物学性状的作用机制,展望了微重力环境细菌生物学技术的未来发展。     

Influence of simulated microgravity on the longevity of insect-cell culture

Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.     

Effects of simulated microgravity conditions on carrageenin-induced oedema in rat

The effects of simulated microgravity conditions, using a three-dimensional clinostat (Random Positioning Machine, RPM), on carrageenin-induced paw oedema in rats as a model of local inflammation were evaluated. RPM-exposed animals showed a significant reduction of oedema and a more pronounced decrease in body weight with respect to control groups. Moreover, aspirine (ASA) treatment, an anti-inflammatory agent, on RPM-exposed rats did not exhibit any activity after carrageenin challenge with respect to RPM control animals on the ground. ASA activity on RPM could be prevented by RPM-induced anti-oedematous effect. RPM-induced anti-oedematous effect did not reversed by pre-treatment with the non-selective glucocorticoid receptor antagonist, mifepristone ruling out the supposed influence of an of cortisol release during the RPM treatment.