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.     

Sleep restriction does not affect orthostatic tolerance in the simulated microgravity environment.

Orthostatic intolerance (OI) is a major problem following spaceflight, and, during flight, astronauts also experience sleep restriction. We hypothesized that sleep restriction will compound the risk and severity of OI following simulated microgravity and exaggerate the renal, cardioendocrine, and cardiovascular adaptive responses to it. Nineteen healthy men were equilibrated on a constant diet, after which they underwent a tilt-stand test. They then completed 14-16 days of simulated microgravity [head-down tilt bed rest (HDTB)], followed by repeat tilt-stand test. During HDTB, 11 subjects were assigned to an 8-h sleep protocol (non-sleep restricted), and 8 were assigned to a sleep-restricted protocol with 6 h of sleep per night. During various phases, the following were performed: 24-h urine collections, hormonal measurements, and cardiovascular system identification. Development of presyncope or syncope defined OI. There was a significant decrease in time free of OI (P = 0.02) and an increase in OI occurrence (P = 0.06) after HDTB among all subjects. However, the increase in OI occurrence did not differ significantly between the two groups (P = 0.60). The two groups also experienced similar physiological changes with HDTB (initial increase in sodium excretion; increased excretion of potassium at the end of HDTB; increase in plasma renin activity secretion without a change in serum or urine aldosterone). No significant change in autonomic function or catecholamines was noted. Simulated microgravity leads to increased OI, and sleep restriction does not additively worsen OI in simulated microgravity. Furthermore, conditions of sleep restriction and nonsleep restriction are similar with respect to renal, cardioendocrine, and cardiovascular responses to simulated microgravity.     

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     

Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling.

Bone loss occurs as a consequence of exposure to microgravity. Using the hindlimb-unloaded rat to model spaceflight, this study had as its purpose to determine whether skeletal unloading and cephalic fluid shifts alter bone blood flow. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal structures of the forelimbs and head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7 days, and 28 days of hindlimb unloading (HU). Femoral and tibial perfusion were reduced with 10 min of HU, and blood flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of femora (-11%, P < 0. 05) and tibiae (-6%, P < 0.1) was lowered with 28 days of HU. In contrast, blood flow to the skull, mandible, clavicle, and humerus was increased with 10 min HU but returned to control levels with 7 days HU. Mandibular (+10%, P < 0.05), clavicular (+18%, P < 0.05), and humeral (+8%, P < 0.1) mass was increased with chronic HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.     

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.     

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.     

Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

Effect of simulated microgravity on vascular contractility

Microgravity was simulated inSprague-Dawley (SD) and Wistar (W) rats by using a tail harness toelevate the hindquarters, producing hindlimb unweighting (HU). After 20 days of HU treatment, blood vessels from both HU and control rats werecut into 3-mm rings and mounted in tissue baths for the measurement ofisometric contraction. HU treatment decreased the contractile responseto 68 mM K⁺ in abdominal aortafrom W rats. HU treatment also decreased the contraction to 68 mMK⁺ in carotid arteries from bothrat strains and in femoral arteries from W but not SD rats. HUtreatment reduced the maximal response to norepinephrine in allarteries except the femoral from SD rats. HU treatment reduced themaximal response of jugular vein from W rats to 68 mMK⁺ but had no effect on thatresponse in femoral vein from either rat strain. HU treatment also hadno significant effect on the maximal response to norepinephrine inveins. These results demonstrate that HU treatment caused a nearlyuniversal reduction of contractility in arteries, but generally had noeffect in veins.

     

Regulation of leptin secretion: effects of aging on daily patterns of serum leptin and food consumption

We have investigated the effects of age on the daily rise in serum leptin levels during the dark-phase of the light-dark cycle. The results show that in young 7-week-old rats, serum leptin levels increase significantly at 2300 h from the levels at 1500 h in association with increased food consumption. However, in middle-aged rats 25 weeks old, the dark-phase increase in serum leptin is absent despite retention of the daily dark-phase increase in food consumption. When compared to our earlier published results, these finding show that the loss of dark-phase rise in serum leptin occurred despite the daily increase in adipocyte leptin gene expression. These results are in accord with the view that the daily pattern in serum leptin is unlikely to be a contributor to the daily patterning of food consumption.     

Psychophysiological monitoring under conditions of real and simulated microgravity.

Dynamics of indices characterizing the process of adaptation of operators to complicated conditions of vital activity was studied with psychophysiological monitoring. This technique is based on the complex analysis of subjective and objective estimations of the current psychoemotional state and physiological and biochemical indices. We analyzed the data obtained during the examination of three healthy volunteers, who participated in the HUBES experiment (135-day isolation in the ground-based model of the orbital station "Mir"), and during a biorhythmological study conducted by six Russian cosmonauts of the main expeditions on the station "Mir." It was shown that psychophysiological monitoring is an informative method for studying different parameters of the operator's state and regulatory processes, the dynamics of biological rhythms, and individual characteristics of adaptation. By means of the analysis of a population of the variation in the characteristics of the parameters under study the experimental period of isolation was divided into three stages. The first and third stages were characterized by a relatively high variability, which was minimized at the second stage. The phasic character of the adaptation processes with a phase duration of about 60 days was revealed and a correlation was shown between the dynamics of parameters obtained by means of psychophysiological monitoring and specific features of the system regulation in different phases of the space flight and its simulation. It is suggested that the phases of the minimal variability of the studied characteristics are related to the periods of the minimal reliability of an operator's work. [Translated from Fiziologiya Cheloveka, vol. 25, no. 5, 1999 Sep-Oct, p. 86-91]     

Effects of simulated microgravity (HDT) on blood fluidity.

Exposures to microgravity and head-down tilt (HDT) produce similar changes in body fluid. This causes an increase in hematocrit that significantly affects hemorheological values. Lack of physical stimulation under bed rest conditions and the relative immobility of the crew during spaceflight also affects the blood fluidity. A group of six healthy male subjects participated as volunteers, and blood samples were collected 10 days before, on day 2 and day 9, and 2 days after the HDT phase. Blood rheology was quantified by plasma viscometry, red cell aggregability, and red cell deformability. A reduced red cell deformability, an indication of the diminished quality of the red blood cells, was measured under HDT conditions that finally led to the so-called "space flight anemia." Enhanced red cell membrane fragility induced by diminished physical activity and an increase in hemoglobin concentration are responsible for this effect. Plasma viscosity is reduced as a result of diminished plasma proteins. However, despite the reduction in plasma proteins, including fibrinogen, alpha 2-macroglobulin, and immunoglobulin M, red cell aggregation was enhanced, principally because of the increase in hematocrit. Our results of hemorheological alterations under HDT conditions may help to elucidate the formerly documented hematologic changes during spaceflight.     

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.     

The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells

Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72 h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.     

Vascular hyporesponsiveness in simulated microgravity: role of nitric oxide-dependent mechanisms.

Simulated microgravity depresses the ability of arteries to constrict to norepinephrine (NE). In the present study the role of nitric oxide-dependent mechanisms on the vascular hyporesponsiveness to NE was investigated in peripheral arteries of the rat after 20 days of hindlimb unweighting (HU). Blood vessels from control rats and rats subjected to HU (HU rats) were cut into 3-mm rings and mounted in tissue baths for the measurement of isometric contraction. Mechanical removal of the endothelium from carotid artery rings, but not from aorta or femoral artery rings, of HU rats restored the contractile response to NE toward control. A 10-fold increase in sensitivity to ACh was observed in phenylephrine-precontracted carotid artery rings from HU rats. In the presence of the nitric oxide synthase (NOS) substrate L-arginine, the inducible NOS inhibitor aminoguanidine (AG) restored the contractile responses to NE to control levels in the femoral, but not carotid, artery rings from HU rats. In vivo blood pressure measurements revealed that the peak blood pressure increase to NE was significantly greater in the control than in the HU rats, but that to AG was less than one-half in control compared with HU rats. These results indicate that the endothelial vasodilator mechanisms may be upregulated in the carotid artery, whereas the inducible NOS expression/activity may be increased in the femoral artery from HU rats. These HU-mediated changes could produce a sustained elevation of vascular nitric oxide levels that, in turn, could contribute to the vascular hyporesponsiveness to NE.     

Effects of simulated microgravity on thyroid carcinoma cells

We aimed to investigate whether simulated microgravity on thyroid carcinoma cells could help to perform in vitro cancer studies such as antitumor drug tests more reliable and to spare animal experiments. We cultured cancer cells at 0 g to enable formation of three-dimensional multicellular tumor spheroids (MCTS), which will resemble the originating tumors. Under microgravity human follicular cells (ML-1 cell line) keep floating with-out stirring so that initial cell-cell interactions required for spheroid formation will be induced by forces due to biochemical components actually expressed on surfaces of cells, whereas gravity related push- or shear events will not influence MCTS formation. Within 12 hours of clinorotation the monolayer turned spontaneously into MCTS with remarkable features: An increase of extracellular matrix proteins and TGF-beta 1. Thyroglobulin, ft3 and ft4 secretion were markedly reduced. These data are in agreement with the observation that astronauts show low thyroid hormone levels after spaceflight.     

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.     

Effects of insulin and exercise on rat hindlimb muscles after simulated microgravity.

This study was designed to examine insulin- and exercise-stimulated glucose uptake and metabolism in the hindlimb muscles of rats after conditions of simulated microgravity. To simulate microgravity, male Sprague-Dawley rats were suspended in a head-down (45 degrees) position with their hindlimbs non-weight bearing (SUS) for 14 days. In addition, rats were assigned to suspension followed by exercise (SUS-E), to cage control (CC), or to exercising control (CC-E) groups. Exercise consisted of five 10-min bouts of treadmill running at the same relative intensity for the CC-E and SUS-E rats (80-90% of maximum O2 consumption). Hindlimb perfusion results indicated that glucose uptake for the entire hindquarter at 24,000 microU/ml insulin (maximum stimulation) was significantly higher in the SUS (8.9 +/- 0.5 mumol.g-1.h-1) than in the CC (7.6 +/- 0.4 mumol.g-1.h-1) rats, signifying an increased insulin responsiveness. Glucose uptake at 90 microU/ml insulin was also significantly higher in the SUS (48 +/- 4; % of maximum stimulation over basal) than in the CC (21 +/- 4%) rats. In addition, exercise-induced increases in glucose uptake for the hindlimbs (133%) and glucose incorporation into glycogen for the plantaris (8.4-fold), extensor digitorum longus (5.4-fold), and white gastrocnemius (4.8-fold) muscles were greater for the SUS-E rats than for the CC-E rats (39% and 1.9-, 1.9-, and 3.0-fold, respectively). Therefore, suspension of the rat with hindlimbs non-weight bearing leads to enhanced muscle responses to insulin and exercise when they were applied separately. However, insulin action appeared to be impaired after exercise for the SUS-E rats, especially for the soleus muscle.