Both vestibular and non-vestibular graviceptors contribute to spatial orientation [1]. Among non-vestibular influences, Vaitl et al. [2] shown that shifts in blood volume alter the perception of body posture. As postural stabilization depends on body spatial orientation perception, we hypothesized that factors affecting blood shift may alter postural control. The purpose of the present study was to investigate the effects of venous contention (a device known to decrease the downward blood shift induced by upright standing) on postural stability.

Twelve healthy volunteers were studied each in 3 randomized conditions: with no venous contention (C0), grade 1 (C1) or grade 3 venous contention (C3). In each condition the subject laid down supine on a bed with the legs up for 10 minutes before standing up on a balance plate covered with a small foam rubber mattress for 5 minutes with the eyes closed. To reproduce the natural standing, instruction was given to the subject to reach the force plate from the bed position as soon as possible after a starting signal and to close the eyes as soon as possible after a stable standing position was adopted.The time from starting signal to closing of the eyes (Te) was measured with a hand chronometer and the time from starting signal to detection of a force on the plate (Tf) was deduced from the force signal. The position of the subject (x, y coordinates) was recorded every 20 ms (50Hz). Standard deviation xy (square root of square x + square y; SDxy) position was calculated from the time a force was detected on the plate to the time closing of the eyes and for the next 10 s period.

No subject fell from the force plate after the eyes were closed. Te (mean ± SD) for C0, C1 and C3 contentions were respectively 6.54 ± 1.5 s, 6.41 ± 1.38 s and 6.54 ± 1.34 s (NS); Tf (mean ± SD) for C0, C1 and C3 contentions were respectively 3.31 ± 1.26 s, 3.29 ± 1.18 s and 3.48 ± 1.15 s (NS). Before closing of the eyes, SDxy (mean ± SD) for C0, C1 and C3 contentions were respectively 1.51 ± 0.36, 1.33 ± 0.35 and 1.49 ± 0.40 (NS), whereas during the first 10 seconds following closing of the eyes, SDxy was significantly increased (P = 0.014; ANOVA with Holm-Sidak method) with C3 contention (mean ± SD = 0.602 ± 0.185) as compared to C0 contention (mean ± SD = 0.494 ± 0.089). After this time, no significant difference was observed between the three conditions.

We found that strong venous contention increases postural instability at the beginning of standing with the eyes closed whereas contention has no effect at the beginning of standing when the eyes are open. As venous contention prevents blood shift induced by upright posture, this result is in line with the hypothesis that blood shifts influence body orientation perception and postural control among others factors as vision, vestibular inputs... In comparison with laying down, upright standing induces a decrease in splanchnic venous volume and an increase in leg venous volume. Thus it is not clear where the blood volume sensitive graviceptors are localized, in the profound blood vessels of legs, in the abdomen or both.

[1.Mittelstaedt H, Fricke E. The relative effect of saccular and somatosensory information on spatial orientation and control. Adv. Otol-Laryngol., 1988, 42:24-30 ; 2.Vaitl D, Mittelstaedt H, Saborowski R, Stark R, Baisch F. Shifts in blood volume alter the perception of posture: further evidence for somatic graviception, Int. J. Psychophysiol., 2002, 44:1-11]

Life-threatening cardiovascular disease is unique for humans and therefore should have human-specific mechanism. The most specific feature of human physiology is upright body position with significant gradient of gravitational potential across cardiovascular system GP=g*h(g-gravitational acceleration, h-posture height).

Objective: To explain human cardiovascular disease as human-specific gravitational damage of cardiovascular system associated with sitting lifestyle. Methods: Cross-disciplinary analysis of data from cardiovascular physiology, medicine and gravitational physics.

Results: In upright position high GP gradient across cardiovascular system pulls blood downward so that additional work must be performed by the system to maintain upper body circulation. This work may be defined as gravitational stress(GS) in the system and estimated as work to lift cardiac output(CO) in ascending limb of cardiovascular circuit GS=CO*d*g*hm*t (where hm=1/3h - mean height of blood lifting in circulation, d-blood density). Siphon principle does not work because descending and ascending limbs of the cardiovascular circuit are separated by autoregulated microcirculation. The only way to lift blood in ascending limb of the circuit is to create upward pressure gradient across it exceeding downward GP gradient. This is achieved by creating additional head-to-feet gradient of vasoconstriction opposite to the feet-to-head GP gradient at the price of increased power of heart work and excessive mechanical stress on vascular walls. For average h=1,75m and CO=5 l/min change from supine to standing increases mechanical power of heart output almost by 100% and total peripheral vascular resistance(TPR) almost by 50%. Built-in defense against GS is calf muscle pump(CMP) activated during walking that effectively returns blood upward making above compensatory response unnecessary. Then, actual GS depends on posture height and calf muscles activity being almost zero during walking/lying and maximal during prolonged sitting at desk that is high vertical body position with completely inactivated CMP. Modern lifestyle with reduced walking and prolonged upright sitting chronically increases gravitational stress in cardiovascular system. Resulting excessive compression of endothelium causes occlusive atherosclerosis while high tensile stress of media layer leads to wall rupture/dissection. Increased oxygen demand makes myocardium highly vulnerable to ischaemic damages. Cardiovascular morbidity/mortality was found to be significantly higher in workers spending work hours sitting upright (bus/train drivers, office clerks) than in those performing heavy manual work or general population.

Conclusion: Life-threatening human cardiovascular disease may be scientifically explained as human-specific gravitational damage of cardiovascular system caused by sitting lifestyle and thereby completely preventable by specific lifestyle interventions (decrease of sitting height/time, frequent walking/walking-like exercise).

The research goal of this investigation was to develop a mathematical model for simulation of the human cardiovascular responses to acute exposure and prolonged existence in the microgravity environment. We are particularly interested in the redistribution of transmural pressures, flows, blood volume, and the consequent alterations in local hemodynamics in different cardiovascular compartments during acute exposure and chronic adjustments. As a preliminary study, in this paper we first developed a multi-element, distributed hemodynamic model of human cardiovascular system, which is adaptive to gravitational alteration, and tested the ability of the model to simulate cardiovascular changes during head up/down tilt under various angles.

Development of the model: The distributed model was previously developed for the simulation study of cardiovascular responses to the hypergravity. To make it applicable to the microgravity environment, modifications has been made including the adjustment of parameters and inclusion of peripheral segments reflecting blood reservoir. In the model, gravitational stress is reflected by changes in hydrostatic pressure gradients and head up/down tilt is simulated by adjusting the gravitational component along the vessel segments at different angles. Computer simulations were performed to predict cardiovascular response to the head-up and head-down tilts at 10, 30, and 75 degrees for 5 min at each angle, respectively, as indicated by Katkov et alis work.

Simulation Results: In the head-up position, the mean pressure of the jugular vein and pulmonary artery decreased with the increase of the tilt angle; systolic pressure and diastolic pressure of the left ventricle decreased, but not significantly; the mean pressure of the femoral artery and vein increased almost the same. Correspondingly, in the head-down position, the mean pressure of the jugular vein and pulmonary artery increased with the angle, but that for the jugular vein became slow at 75 degree; in the left ventricle, while systolic pressure increased significantly with the angle, the increase of diastolic pressure was not remarkable; the mean pressure in the femoral artery decreased less than that in the femoral vein. Except for few results at a specific angle, most simulation data compare well with the published experimental data reported by Katkov et al[1]. Conclusion and perspectives: Comparison of simulation results with experimental data indicates that the model has the potential of simulating hemodynamic alteration during postural exposures and under microgravity. Further application on the mechanism study of orthostatic intolerance (OI) and anti-OI countermeasure is under investigation.
[1] Katkov, et al. Aviat. Space Environ. Med. 51(11):1234-1242, 1980

Aims: To study the reproducibility of presyncopal times across four graded orthostatic stress runs in healthy males. Procedures and methods: 10 healthy young males were subjected to graded orthostatic stress four times. Test runs were separated by two week interval. At the beginning and the end of every 14-day graded orthostatic stress (GOS), consisting of head-up tilt (HUT) combined with lower body negative pressure (LBNP), was used to achieve a pre-syncopal end-point. Orthostatic effects on cardiac and vascular function were continuously monitored and standing times noted. Results: In all the persons, mean arterial blood pressure dropped by 15%, pulse pressure by 36%, heart rate (HR) increased 112%, and stroke volume index decreased 51% (all n.s.) from supine control to presyncope. Thoracic impedance (TI) rose by 12 ± 1% (p < 0.0001), total peripheral resistance index (TPRI) decreased by 11 ± 5% (p = 0.03) and pulse pressure by 41 ± 8% (p = 0.0003). Repetitions of the orthostatic protocols did not influence standing times of test persons from the 1st to the 4th trial (15 ± 6 to 18 ± 7 min). Conclusion: Strategies for maintaining blood pressure in response to central hypovolemia in subjects induced by orthostatic stress are different between subjects. However, the standing times are highly reproducible when these subjects reach pre-syncope four times thus suggesting that LBNP tolerance does not develop across four runs, especially if these are separated by two weeks.

The purpose of this paper is to describe the main components and basic functioning principles of the antigravitational functional system (AFS). Methods: literary review and theoretical analysis of the neurogenic regulation functional system.

The concept of a functional system was formulated at the beginning of the 20th century. Functional system was described as a dynamic, self-organizing, central-peripheral functional integration of structures of the nervous system whose activity was aimed at achieving adaptive useful results. This regulation pattern can be considered as the peak of the regulation systems evolution of the living matter. The main system forms on the basis of motivation for realizing adaptive action and achieving useful results. Muscular and vascular AFS are being formed for that. Their main goals are adaptive action realization and adequate blood distribution under different physical activity keeping.

The main difference between functional system and proposed regulating principles is the presence of the physiological mechanism of the prospective result prediction (action result acceptor). Action is programmed for defined result achievement. This is anticipatory regulation principle. Using this principle AFS timely provides cardiovascular system preparation for its impending functional conditions changes. It seems that gravity intolerance at the beginning and after space flight is related with AFS regulation peculiarities. Probably, during adaptation period, it precedes blood flow distribution adapted to "old" conditions. During this period change-over of the action result acceptor occurs and blood distribution disturbance is eliminated.

On earth all healthy individuals can be divided into three groups: with physiological cardiovascular response during orthostatic changes, hypo and hyper reactive response. We hypothesized that all non physiological orthostatic hemodynamic responses are results from AFS inadequate regulation. The same reactions observable over a long period can induce essential hypertension evolution that was confirmed by prospective studies.

Advanced studies of AFS are necessary because of short historical term of the gravity and microgravity influences on the cardiovascular system as well as rapidly increasing space flight duration. It is very important to create safe and comfort conditions for astronauts adaptation during gravitational loading changes as well as for certain diseases prophylaxis on earth.

Objective: We quantified the impact of a 60-day head-down tilt bed rest (HDBR) with countermeasures on the Cardiac function and arterial hemodynamics at rest.

Method: Twenty-one men (25-40y) divided into 3 groups [Control (Con), Foot Vibration (Vib) and Chinese Herb (Herb) were studied pre and at HDBR day 58. Post HDBR the subjects were identified as finisher at the orthostatic tolerance Tilt test (F) or non finishers at orthostatic tolerance Tilt test (NF). Cardiac and arterial size and flow velocity were investigated by Echography and Doppler.

Results: The following parameters at HDBR day 58 were significantly lower compare to pre HDBR in all group (Con, Vib, Herb, F, NF): Left ventricle diastolic volume and Myoc thickness, Mitral E velocity and E/A ratio, Pulmonary Systolic velocity, Portal Vein, Carotid and Femoral flow volume. Conversely Femoral and Tibial vascular resistance were significantly lower only in the Vib and NF group while it did not change in the others groups (Con, Herb, F).

Conclusion: The results will be discussed according to the countermeasure used and the fact that the subjects were finisher or non finisher at the post HDBR Tilt test.

The aim of the study was to get and analyse data on the changes in ECG/EKG, heart rate and blood pressure with the change in gravitational acceleration or g. Previous studies conducted in our laboratory showed that regular practice of Sheersasana (which is a Yogic posture) could help in tuning the cardiovascular system to adapt easily to the accumulation of blood in the upper part of the body in early days of spaceflight. In those studies, however, subjects were attached to a platform which could be rotated from 0° to 90°. Currently, studies have been done by attaching the subjects to a platform which could be rotated from 0° to 360°. Data viz., ECG/EKG, heart rate and blood pressure were first taken for the supine position (0°). The subjects were then rotated in steps of 15° upto 360°. Corresponding data were acquired for each position. All the subjects were healthy males in the age group of 18-25. The subjects were divided in three groups. One group consisted of those who regularly practiced Sheersasana and/or Sarvangasana atleast till the past two years while the second group consisted of those who regularly did physical exercise while the third group had never practiced Sheersasana or Sarvangasana or physical exercises and was taken as control group. This study was done as this is analogous to the pooling of blood in the head as in the case when in microgravity conditions. Moreover, it would help to estimate the role of gravity on the cardiovascular system. The results were plotted in the form of histograms for the differences in heart rate, systolic blood pressure, diastolic blood pressure, pulse pressure and PR, QT and RR intervals between two consecutive positions. The normal group showed a comparatively large spread than those in the Sheersasana group. Interestingly, the exercise group showed more spread than the other two groups. This suggests that regular practice of Sheersasana might possibly tune the cardiovascular system to the pooling of the blood in the head as in microgravity conditions.

We examined the effects of low and high dietetic NaCl (salt) intakes on plasma volume (PV) and cardiovascular variables [(heart rate (HR) stroke volume (SV), cardiac output (CO), mean arterial pressure (MAP) and total peripheral resistance (TPR)] of 8 young adult men in a randomized balanced cross-over, head-down bed rest (HDBR) study conducted at the metabolic ward of our Institute.

Methods: The study was conducted in two phases with 5 months wash-out in between. Each phase consisted of a 5-day baseline period, a 14-day HDBR [including low or high (0.7 or 7.7 mEq/kg body weight (BW) /d, respectively) dietetic salt intake, and a 4-day recovery period. Dietary NaCl intake was 2.8 mEq/kg BW during both, baseline and recovery. Plasma volume was measured by carbon monoxide rebreathing (CORB) on baseline day 4 and HDBR day 12; cardiovascular variables (finger blood pressure and pulse contour method) were determined non-invasively at rest supine and during 20' head up tilt (HUT) on baseline day 5, and on the first recovery day, just after termination of the HDBR-phase.

Results [mean (SD)]: HDBR caused a decrease in plasma volume (p<0.001) that was modulated by the salt intake regimes leading to a small though significant difference (low vs high salt intake on HDBR day 12 p < 0.05). After HDBR, supine HR was higher than before, but salt intake failed to modulate this effect, whereas MAP was higher on low than on high salt [89,7 (3,9) vs 85,9 (6,3) mmHg, respectively, p<0.05]. Neither HDBR nor salt intake affected supine SV, CO or TPR. After HDBR, HR increase during HUT was larger under low than under high salt intake [26,9 (9,1) vs 19,8 (10,8) beats.min-1, respectively, p<0.05], and the decrease in SV was, as expected, larger after HDBR than before, but there were no effects attributable to the salt regimen. The expected decrease in post bed rest orthostatic tolerance, measured as tolerance time to HUT, remained unaffected by dietetic salt intake.

Summary and conclusion: HDBR caused the expected effects on plasma volume, cardiovascular variables, and orthostatic tolerance. Different dietetic salt intake regimes modulated the decrease in plasma volume and the responses of MAP and HR to HDBR. Dietetic NaCl affects body fluids and modulates the activity of the RAAS and, likely, of the sympathetic nervous system, but it only exerts moderate influence upon the effects of HDBR, failing to ameliorate post bed rest orthostatic intolerance.

Previous studies, by our group and others, have reported an increased flow-mediated dilation (FMD) response in brachial, popliteal, and femoral arteries following long duration bed-rest protocols (14 to 56 days) and in spinal cord-injured individuals. We tested the hypothesis that FMD would be affected by 24 hours of head down tilt bed rest (HDBR). Six young, healthy, volunteers (3 women) completed FMD protocols before (BDC) and at the 4 and 24 hour mark of 6-degrees HDBR. Change in brachial artery diameter in response to reactive hyperemia initiated by 5 minutes of forearm circulatory occlusion was monitored by M-mode ultrasound throughout the protocols. We found that the maximum FMD response, at ~60 seconds after reperfusion, was not different between any of the time points (BDC: 4.45±0.83 to 4.80±0.81 mm, 7.6%; 4hr: 4.44±0.85 to 4.78±0.83 mm, 7.7%; 24hr: 4.44±0.80 to 4.80±0.80 mm, 8.1%).

These results are in contrast to the results of the longer term studies; suggesting that endothelial function does not follow the time-scale of the initial adaptation to HDBR but appears to take longer than the 24-hrs studied here. The conflicting results in endothelial function are paralleled by differences in vessel dimension with long vs. short term HDBR. The previous long term HDBR, as well as the spinal cord injury studies also showed a reduction in vessel cross-sectional area, not witnessed in this short term model, which may have a direct effect on endothelial function.

The purpose of this study is to investigate that forced water drinking according to volume of urine output during -6�‹ head down bed rest for 20 days could alter the thermoregulatory function. Our hypothesis is that forced water drinking according to urine output for 20 days could increase sweat rate, blood flow during leg water immersion at 42�‹C for 45 min. To evaluate for this hypothesis, we measured the sweat rate and sweat expulsions which are able to evaluate the central drive for sweating, and also observe changes of fluid related hormones after -6�‹ head down bed rest. Twelve healthy men, 24 �}5 years old, participated in this study. Subjects were lying on the bed fixed at -6�‹ for 20 days under nursing care. During 20-day bed rest, water intake volume in subjects was forced to match up the urine volume from the previous day. The subjects underwent a heat exposure test at 2 days before the bed rest started (PRE) and 2 days after the 20-day head down bed rest (POST) in each. The heat exposure test consisted of the leg water immersion up to knee level at 42 �‹C for 45 min after a 10 min rest in the sitting position. Core temperature (tympanic), skin temperature (chest, arm, leg and thigh), blood flow (chest and forearm) and sweat rate (chest and forearm) were recorded continuously during rest and leg water immersion. Blood were sampled in PRE and POST from the left antecubital vein to analysed a plasma renin activity (PRA), angiotensin II (ANG II), aldsterone(ALD), antidiuretic hormone (ADH) and arterial natriuretic peptide (ANP). Blood were also sampled every morning before breakfast from the left antecubital vein to determine Hemoglobin (Hb) and Hematocrit (Hct). Plasma volume loss was calculated using the formula by Dill and Costill (1974). The results were 1) sweat rate in chest was significantly increased in POST during leg water immersion. 2) skin blood flow in chest was significantly increased in POST during leg water immersion. 3) the regression line between sweat rate and body temperature was not significantly different between PRE and POST. 4) PRA was significantly higher in POST than in PRE however ADH and ANG II were not different between PRE and POST. Our present study suggested that sweat rate and blood flow were increased under the forced water drinking with -6�‹ head down bed rest for 20 days, which partly contributes to the prevention of dehydration. We conclude that the forced water intake could prevent the impaired thermoregulatory responses, which was one of the impairments by the microgravity exposure (Michikami et al, 2004, Lee et al, 2002, Cui et al, 2000, Crandall et al, 1994), under microgravity condition such as during spaceflight.

Space flight induces a cardiovascular deconditioning syndrome. One of the most important symptoms of such state is a decrease in orthostatic tolerance, along with a significantly reduced exercise capacity and an increase of heart rate. Hypovolaemia, hormonal changes and vascular dysfunctions are associated with cardiovascular deconditioning. Dry immersion is a ground model of space environment inducing cardiovascular deconditioning.

We studied 8 healthy young men before, during and after 7 days of dry-immersion. Different aspects of cardiovascular deconditioning were studied. Microcirculatory response at the skin calf level was studied by laser Doppler flowmetry during a stand test before dry immersion (Day–1) and after dry immersion (recovery day+2). Plasma volume variations were evaluated by Dill and Costil method. Cardiovascular hormones were assessed in blood and plasma samples.

Our first results show a significant decrease in blood flow in standing position after 7 days of dry-immersion in favour of an enhancement of skin vasoconstriction. Plasma volume was decreased of 14,8+/-3,8 % (day 3 of immersion) and remained stable at day 5 of immersion (decrease of 16,4+/-3,2 %). Level of urinary catecholamines tended to decrease during the immersion phase whereas they raised immediately after dry immersion.

Increased vasoconstriction at the skin level in upright position after dry immersion might be related to the enhancement of sympathetic activity.

We examined that effectiveness of artificial gravity with ergometric exercise as countermeasure against orthostatic tolerance caused by simulated microgravity of -6° head-down bed rest (HDBR) for 20 days in human. Eight healthy male subjects underwent -6° HDBR for 20 days, and were randomly assigned to either a countermeasure group or a control group. The subjects in the countermeasure group performed artificial gravity with ergometric exercise training on alternate days (13 days) during HDBR.

Orthostatic tolerance test were performed before and after HDBR. The subjects lay on the tilt table at the 0° horizontal position for 15 min and was then declined to the -6° head-down position. Thereafter, it was inclined to the 0° horizontal and 30° head-up position and 60° head-up position. The tilt table remained in each position for 15 min. In control group, muscle sympathetic nerve activity increases during orthostatic tolerance test in post HDBR whereas countermeasure group was not exchange. Diastolic blood pressure in control group in post HDBR decreased though the muscle sympathetic nerve activity increases. Anti-G score and endurance time in G-tolerance test as the index of orthostatic tolerance has reduced both control and countermeasure group. These results suggest that artificial gravity with ergometric exercise of alternate days might not be effective as the prevention of the decrease orthostatic tolerance after HDBR.

Head-down bed rest of -6°(HDBR) was used as a model for studying the physiological changes during microgravity in spaceflight. Exposure of animals and humans to the space flight condition results in numerous immunological and hormonal changes similar to those observed during HDBR, but the mechanisms are not clear.

The aim of the present study was to investigate whether exposure to such an environment led to alterations in the synthesis of some acute-phase proteins and cytokines, and if countermeasures (artificial gravity with ergometric exercise) would prevent these changes.Twelve male volunteers were subjected to HDBR of -6° for 20 days; six were chosen to form the countermeasure group and were exposed to artificial gravity with ergometric exercise for 30 min per day, and the other 6 were used as controls. Plasma noradrenaline (NA), adrenaline (Ad), dopamine (DA), leukocyte count (WBC), interleukin 6 (IL-6), total serum protein (TP), C-reactive protein (CRP) and α1antichimotrypsin (ACT) were determined before and after HDBR, to assess the effects of HDBR and the countermeasure.In all subjects, Ad and NA concentrations increased significantly, and the concentration of CRP decreased, (ANOVA p<0.05) after HDBR. The concentration of CRP was significantly higher, and that of Ad was significantly lower, in the control group.(ANOVA p<0.05).

The results indicate that several neuroendocrine and immunological parameters are modulated by prolonged HDBR of -6° and that psychological stress and/or the increased sympathetic nervous system might play a role in inducing these changes and this alterations might be counteracted at least in a part by the artificial gravity with exercise.

In microgravity and the resulting absence of a hydrostatic component of local blood pressure the skin at the head shows an oedema whereas the skin at the legs undergoes dehydration. As a prerequisite to study the mechanisms behind this phenomena in more detail, new techniques are required for in vivo measurements of changes in volume and fluid content of different skin layers.

In this study the technology of the NMR-MOUSE® (Nuclear Magnetic Resonance-Mobile Universal Surface Explorer) was evaluated with regard to its capability to detect local differences in water content measured as relative changes in 1H signal intensity. In our study an instrument with 0.4 Tesla was used that recorded 1H spins at a resonance frequency of 17 MHz in a planar slice of 50 µm thickness at 4 mm distance from the magnet surface. This slice was oriented parallel to the plane of the skin. By moving the NMR-MOUSE in 80 equidistant 50 µm steps under computer control, the layer structure of the skin could be profiled up to a depth of 4 mm. The total measuring time for a profile was about 20 min. For 7 male subjects the skin at the outer side of the right lower arm was examined. The measurements were performed before and during venous occlusion (VO). During VO the NMR measurement started 5 min after induction of the occlusion.

NMR spin density profiles allow the identification of two skin layers which represent the dermis with rather homogeneous signal intensity and an average thickness of 1400 ± 250 µm; 1150 to 1900 µm (mean ± SD; range) and the subcutis with a more variable 1.5 to 2 times higher intensity and a thickness of 1500 ± 600 µm; 500 to 2150 µm, respectively. In the dermis of all subjects, the application of VO causes a significant increase in thickness by 180 ± 160 µm; 50 to 450 µm (P < 0.01). The thickness of subcutis increased only in 4 of 7 subjects with 100 ± 270 µm; -100 to 700 µm. The dermis signal intensity did not change at VO. In the subcutis a distinct drop in signal intensity was found in those subjects who also showed an increase in thickness. VO seemed to have different effects on dermis and subcutis in lean and fat skin. There were trends that a) subjects with a thin skin showed smaller changes in dermis with VO; b) in subjects with thin subcutis the thickness of subcutis increased during VO, whereas in subjects with thick subcutis a decrease or no change were observed; c) the water in lean skin probably shows a lower 1H signal intensity than the lipids stored in fat cells. However, all trends observed in this pilot study need further verification by examination of a larger number of subjects. The NMR-MOUSE has the potential to become a very useful tool studying local fluid volume regulation in human skin.

Introduction: Simulated weightlessness by bed rest is known to induce cardiovascular deconditioning with vascular impairment. Our objective was to evaluate the effects of a long term head-down bed rest on carotid properties evaluated by ultrasound.

Methods: We studied 14 volunteers (Age 30.2+/-1.2, weight 62.7+/-1.2, height 168.8+/-1.0) included in a 60-day bed rest study and randomly assigned to a control group (n=7) or to a group with daily Chinese Herb (n=7).

Ultrasound measurement of the left carotid was performed before (BDC - 6) and at the end of the bed rest (HDT + 52) in lying position with a Vivid-7 (GE) ultrasound operated by the same person. 10 sec clips were recorded in AVI file. Post analysis was performed via web transfer of images with the software "TELEVASC". Intima-media thickness (IMT) and carotid diameter (CDD) were measured in diastolic phase.

Results: IMT remains stable during head down bed rest in the control group (0,39mm +/-0,03 [BDC-6] vs 0,40 +/- 0,04[HDT +52]) and in the Chinese Herb group (0,38mm +/-0,03 [BDC-6] vs 0,36 +/- 0,03[HDT +52]). We observed no significant changes in basal carotid diameter in both groups (6,59+/-0,20 [BDC-6] vs 6,61 +/- 0,21[HDT +52] in control group / 6,54mm +/-0,19 [BDC-6] vs 6,72 +/- 0,18[HDT +52]) in Herb group).

Discussion, conclusion: "TELEVASC" software could measure accurately carotid morphological properties. Two months with enhanced physical inactivity are not sufficient to increase IMT at the carotid level. Two month head down position do not change significantly basal diastolic carotid diameter.

Fund: ES-1 IBREP; CNES; Televasc project is funded by "Angers Technopole " and " Région des Pays de la Loire"

Previous reports indicate that the incidence of orthostatic intolerance (OI) among female astronauts is significantly higher than compared to their male counterparts. We have previously shown that astronauts who are orthostatically tolerant after spaceflight demonstrate an increase in arterial stiffness (Sa) during spaceflight. We hypothesized that the increase in Sa may represent a protective mechanism against OI by lowering the resistance to venous return and allowing for the maintenance of cardiac output during an orthostatic stress. In line with our previous work, we further hypothesize that the Sa of female astronauts does not increase during spaceflight which may explain, in part, the gender difference in the incidence of OI. We tested this hypothesis using previously collected astronaut hemodynamic data and two ground-based models of microgravity: -6° head-down tilt bedrest (HDTB) in humans and hindlimb unweighting (HLU) in rats. We estimated astronaut Sa as supine pulse pressure divided by stroke volume. We monitored arterial stiffness in humans during bedrest by measuring the central augmentation index before and after 90 days of HDTB. The Sa in rats was monitored by measuring aortic pulse wave velocity before and after 7 days of HLU. Female astronauts had a slight decrease in their Sa as compared to preflight values; however, male astronauts had increased Sa after spaceflight. Additionally, both human and rat female subjects demonstrated no statistical change in Sa after simulated microgravity while their male counterparts showed increases. Female astronauts had a non-significant decrease in Sa of 0.08 ± 0.066 mmHg/ml (N=11, P=0.2405) while male astronauts had a significant increase in Sa of 0.10 ± 0.04 mmHg/ml (N=46, P=0.0145). Female bedrest subjects had an increase of 1.33 ± 4.06% (N=3, p=0.6271) in their central augmentation index after 90 days of bedrest while male bedrest subjects had a statistically significant increase of 11.76 ± 2.67% (N=4, p=0.003). After HLU, the aortic pulse wave velocity of the female rat decreased by 0.05 ± .58 m/s (N=6, p=0.8509) while we have shown the aortic pulse wave velocity of male rats to increase by 3.77 ± 0.99 m/s (N=8, p<0.0001) after HLU. All the evidence indicates that while male Sa increased in response to microgravity (simulated), female vascular stiffness was unaltered. Together these data show that the female vasculature does not respond the same as the male vasculature to microgravity (simulated) and represents a possible underlying cause for the gender difference in the incidence of microgravity-induced orthostatic hypotension.

Reduced weight-bearing activity often occurs on Earth as a consequence of injury, disabling diseases or advanced age, but is also typically observed during long-duration spaceflight. Lower limbs of astronauts and bedridden or cast-immobilized patients show functional and structural adaptations mainly due to muscle unloading. Human data from spaceflight are scanty but fortunately bed rest is thought to be a reliable simulation model for at least part of the physiological effects of spaceflight, including bone and muscle changes and, maybe, endocrine system adaptations. Melatonin is a powerful immunomodulator and is the main synchronizer of daily light-dark and rest-activity cycles while cortisol maintains energy homeostasis and modulates immune function; they are out of phase with each other: cortisol approaches its nadir at bedtime, whereas melatonin peaks a few hours after  cortisol  drop.

Balance between the two is crucial for overall good health, so that Melatonin-Cortisol Index (MCI) has been proposed to assess cancer risk and immune function and to help diagnosing depression, heart disease, osteoporosis. Aim of this study was to verify whether these two hormones undergo any secretion pattern changes during bed rest. 10 healthy male volunteers participated in a 35 day bed-rest experiment, which was performed according to the Declaration of Helsinki and followed a protocol approved by the National Ethics Committee (Ministry of Health) of Slovenia. The subjects were hospitalized at the Orthopaedic Hospital Valdoltra (Slovenia). Saliva samples were collected three times (7.00, 10.00 a.m. and 5.00 p.m.) on days -3, -1, 0, 7, 14, 21, 28, 35 and +1 and frozen at -20°C until assayed for cortisol and melatonin by highly sensitive and accurate ELISA methods. Cortisol maintained its original circadian rhythm, but its concentrations decreased throughout bed rest (0.75±0.10, 0.21±0.09, 0.14±0.03 µg/dl on day -3; 0.42±0.12, 0.17±0.07, 0.12±0.08 on day 28, at7.00, 10.00 a.m. and 5.00 p.m. respectively, p<0.05). On day -3 melatonin displayed levels as high as 7.50±0.9 and 1.6±0.08 while on day 21 it reached down to 3.96±1.02 and 0.67±0.06 pg/ml at 7.00 a.m. and 5.00 p.m. respectively (p<0.05). The MCI got below 15 (from normal baseline levels ranging from 120 to 15, p< 0.05) on days 7 and 28. These preliminary data show that prolonged bed rest may affect cortisol and melatonin secretion pattern and the delicate equilibrium between these two hormones.

Introduction: Gastrointestinal (GI) motility plays a key role in the physiology and function of the GI tract. It directly affects absorption of medications and nutrients taken by mouth, in addition to indirectly altering GI physiology by way of changes in the microfloral composition and biochemistry of the GI tract. Astronauts have reported nausea, loss of appetite and constipation during space flight, all of which indicate a reduction in GI motility and function similar to the one seen in chronic bed rest patients. The purpose of this study is to determine GI motility and bacterial proliferation during -6° head-down-tilt bed rest (HDT).

Methods: Healthy male and female subjects between the ages of 25 and 40 y participated in a 60-day HDT study protocol. GI transit time (GITT) was determined using the lactulose breath hydrogen test and bacterial overgrowth was measured using the glucose breath hydrogen test. Helicobacter pylori colonization was determined using a C13-urea breath test (UBIT®). All three tests were conducted on 9 days before HDT, and repeated on HDT days 2, 28 and 58, and again on day 7 after HDT.

Results: GITT increased during HDT compared to the respective ambulatory control values; GITT was significantly lower on day 7 after HDT. A concomitant increase in bacterial colonization was also noticed during HDT, starting after approximately 28 days of HDT. However, H. pylori proliferation was not recorded during HDT as indicated by UBIT®.

Conclusion: GITT significantly increased during HDT with a concomitant increase in the proliferation of GI bacterial flora but not H. pylori.

Hypergravity-associated perturbations at launching and lending of spacecrafts augmented by the microgravity-induced deconditioning of the heart could be a serious health risk factor for space travelers. There are reasons to believe that the heart is especially susceptible to damages caused by reactive oxygen species, because higher rates of oxygen consumption of myocardium are usually coupled with relatively low antioxidant enzyme activities. Investigations carried out in this laboratory, as well as in other research institutions, have clearly shown exposure-dependent alterations in oxygen consumption rate and thermoregulation of hypergravity-treated animals. In an attempt to shed light on the heart antioxidant defense at hypergravity, we analyzed key antioxidant enzyme activities in hypergravity-treated laboratory mammals.

Methods. Hypergravity was modeled by 2 g centrifugation of adult male Wistar rats in an animal centrifuge (3 m in diameter) during 1 hour or 4 hours. Immediately after the centrifugation, the rats were sacrificed and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathine reductase were determined in the left ventricle of myocardium by the well-known spectrophotometric methods.

Results indicate that the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathine reductase were moderately increased after one-hour hypergravity. At 4 hours hypergravity, however, the activities of superoxide dismutase, catalase and glutathine reductase, but not glutathione peroxidase, declined and became lower the corresponding control values.

Conclusions: one-hour hypergravity increases activities of the antioxidant enzyme in myocardium, whereas longer exposures (4 hours) could result in opposite reactions of the heart antioxidant defense.

Isolated presynaptic nerve terminals (synaptosomes) can be identified and subjected to flow cytometric analysis without loss of physiological responsiveness. Flow cytometric studies (COULTER EPICS XL) demonstrated the insignificant decrease in cell size heterogeneity by forward scatter (FS) analysis of rat brain synaptosomes prepared from animals subjected to centrifuge-induced hypergravity as compared to control ones.

Cytoplasmic granularity measured by side scatter (SS) profiling also slightly decrease in hypergravity synaptosomes as compared to control ones. Membrane potential can be measured with flow cytometric methods using potentiometric optical dye rhodamine 6G. Populations of both control and hypergravity synaptosomes showed the ability to maintain a significant membrane potential that was demonstrated by the absence of negative peak of rhodamine 6G fluorescence.

The analysis of the main peak of rhodamine 6G fluorescence revealed the decrease in fluorescence intensity by 20% in synaptosomes after hypergravity as compared to control ones. High-KCl similarly reduced fluorescence signal inside of control and hypergravity synaptosomes reflecting rapid depolarization of the plasma membrane. A pH-sensitive fluorescent dye, acridine orange, selectively accumulated by synaptic vesicles (acid compartments of synaptosomes) was used for monitoring synaptic vesicle acidification (∆pH), an important component of electrochemical H+ gradient (∆μH+). ∆μH+ is a driving force for the accumulation of neurotransmitters into synaptic vesicles.

We have demonstrated the decrease in acridine orange fluorescence by 10% after hypergravity. Thus, exposure to hypergravity resulted in depolarization of the synaptosomal plasma membrane and diminution in synaptic vesicle acidification. Recently, we have shown the decrease in the uptake of glutamate, major excitatory neurotransmitter in the CNS, the attenuation of the exocytotic and increase in transporter-mediated release of glutamate in rat brain synaptosomes under hypergravity conditions. The latest changes might result from the alterations of membrane potential and synaptic vesicles functional state we have shown in the present study.

The objective was to evaluate the calf vein response to hypergravity, and check the efficiency of elastic compression stocking (ECS) in preventing their distension.

Method: Tibial (Tib csa) and Gasrocnemius (Gast csa) vein cross section area were investigated continuously during the experiment by echography using a flat skin probe stick on the calf. While instrumented the subject performed (a) a 10 min stand test (ST10), (b) then he sited inside the long arm centrifuge and was submitted to 2G gravity for 2 min, (c) at last he performed again a 10 min stand test. The whole session was performed without ECS and repeated with ECS.

Results: Without ECS the Tib and Gast max csa, and percent increase at ST10 were similar at pre and post 2G centrifugation. Conversely with ECS Tib and Gast max csa and percent increase at ST10 were lower than without ECS but similar at pre and post 2G centrifuge ST10 .During the 2G period the Tib and Gast maximal csa without ECS were significantly higher than at ST10 without ECS. With ECS the vein maximal csa at 2G was similar to the maximal csa at ST10 without ECS. The minimal csa supine with ECS was lower than without ECS, thus during the 2G period the percent increase in vein csa was similar with or without ECS.

Conclusion: Centrifugation at 2G induced a higher vein distension for both the muscular (Gast) and non muscular (Tib) veins compare to Stand test. At 2G centrifugation, ECS reduced the amplitude of the vein csa increase and limited the max vein csa to the Stand test value without ECS.

The original method of our LBNP (lower body negative pressure) examination was developed for the pilots’ pre-selection with low level of orthostatic tolerance. Flights with alternating ±Gz acceleration in agile aircraft invoke a cardiovascular response of the organism, named a Push-Pull effect. This phenomenon is characterized by the rapid and progressive decrease of blood pressure (BP) accompanied by a slower return to normality during +Gz load following previous –Gz load. Just the slower return of BP to the standard value after its previous substantial decrease is very dangerous reality during flights. At this period pilot’s performance is reduced and an impairment of the vision and G-LOC can occur.

Our LBNP device was reconstructed in order to enable not only to estimate the level of probable +Gz tolerance but also to reveal pilots with low tolerance to the Push-Pull effect. The examination consists of two stages continuing upon one another. The first stage represents the Push-Pull simulation the second stage corresponds to the standard or so-called isolated LBNP exposure. The Push-Pull effect is simulated so that the LBNP device is tilted backwards to the -43 HDT (head down tilt) position. There is atmospheric pressure in the LBNP chamber. This first step corresponds to the plateau of microgravity. Pilots remain in this position for duration of two minutes. Plus Gz load is created by the rapid LBNP exposition with a rapid simultaneous elevation of the LBNP device to the vertical position. Negative pressure level has the value of –70 mmHg and this value is achieved in one second. The return of the device to the vertical position is accomplished also very rapidly, namely in two seconds. The exposition is finished at the end of the two minutes interval in case of sufficient tolerance. Otherwise, it is finished naturally sooner. The second stage is launched after two minutes pause. The LBNP examination is accomplished again with one step exposure at the negative pressure level of -70 mmHg with achievement of this value again in one second. Pilots are furthermore examined in the sitting position. As well as in the first stage the exposition is finished at the end of the two minutes interval in case of sufficient tolerance.

LBNP exposition is also used as a method of drill in anti-g manoeuvres. This new LBNP examination method is an important training stage before centrifuge training.

We compared BP responses between the isolated LBNP load and Push-Pull load. BP responds very sensitively to the LBNP exposure, which stands for a significant parameter reflecting the cardiovascular regulation level in both type of the load. Push-Pull effect causes the progressive BP decrease during approximately first ten seconds. Only then BP increases but, in contrast to its course during the isolated exposure, very slowly. Relative changes of the BP decrease (related to the values before the exposure beginning) were compared at fifteen-second intervals and again relative changes of the BP increase at fifty-second intervals in both stages of examinations. The statistical test proved that the BP changes during exposition with Push-Pull effect is much more significant than during the isolated LBNP examination and very good correspond to Push-Pull phenomenon. BP behaviour indicates that the designed examination method is suitable for a pilots’ testing in conditions of Push-Pull effect.

The study of the hypergravity influence on plant growth and development is one of the main tasks of space biology in connection with planned of long-term space flight. Ca2+ is known to play an essential role in realization of a gravitropic reaction of plant and gravity signal transduction. However, the participation of Ca2+, as secondary messenger involved in hypergravity signal transduction is not clearly understood. The effect of hypergravity on the Ca2+ distribution and on the relative amount of Ca2+ in walls, cytoplasm and chloroplasts has been analyzed in Nicotiana tabacum callus cells.

Callus was grown in a Murashige and Scoog medium. 15-day-old N. tabacum callus was exposed to hypergravity at 7 g and 14 g for 15 and 60 min at +20oC in the dispersed white light using the centrifuge. The cells of callus were loaded with Fluo-4 with and then studied a confocal laser-scanning microscopy (LSM5 Pascal). For analysis of cell readaptation after centrifugation callus was growing at +24oC for 1-24 h. The study localization of Ca2+ by confocal laser microscopy showed the green fluorescence of calcium ions in cell walls, cytoplasm, and chloroplasts. The relative content of calcium ions in cell walls was 3.7 times more than that in cytoplasm, and 2.8 times more than in chloroplasts of control cells. Fluorescence of calcium ions is also revealed in cell walls, cytoplasm and chloroplasts of all cells, which were centrifuged. The effect of hypergravity on cells was high. The relative content of Ca2+ in centrifuged cells differed from that of control cells. The decrease of the relative content of Ca2+ in cell walls and the increase of calcium ions in the cytoplasm and chloroplasts is revealed after 15 min action of hypergravity at 7 and 14 g. The formation of the structures, which were like calcium microdomains of animal cells, was the peculiarity of callus cells after action of hypergravity. The relative content of Ca2+ in such structures was very large. Readaptation of Ca2+ distribution in the cells occurred in 2 h after hypergravity ending. It is suggested that under the influence of hypergravity an increase of Ca2+ in cytoplasm occur in consequence of the disturbance of calcium channels functioning of cytoplasmic membrane.

This investigation aims to determine whether the growth response of tomato seedlings on reorientation to the horizontal induces alterations in distribution of electrolytes in cells of the main root elongation zone, the site where induction of the gravitropic curvature takes place. Tomato (Lycopersicon esculentum, Rio Grande) seedlings were grown on agar surface in 9 cm Petri dishes. The gravitropic response of seedlings was evaluated by the angle of gravitropic curvature after the roots were reoriented 90° from the vertical. Root tips and segments of several mm basipetal to the root tip were fixed in liquid nitrogen, freeze-substituted with Lowicril K11M at -35° C. 100 and 1000 nm thick root tip longitudinal sections and transversal sections of root elongation zone curvature were cut using LKB Ultrotome V, collected by dry method and analyzed in the 6060 LA SEM at accelerating voltage 15 kV. Using different modes of X-ray microanalysis (X-ray map, - line and - point analysis), distribution of the physiologically relevant ions (Ca, K, Na, P) along growth axis of root tip and in the direction between upper and lower sides of the root curvature were investigated. The peculiarities in localization of the electrolytes in the different subcellular compartments as well as distribution of the electrolytes between surface layers of upper and lower sides of the root curvature are discussed.

Comparison of gravisensing in negatively and positively gravitropic organs was studied by the analysis of location and motion of amyloplasts in hypocotyl endodermal and root columella cells of cress (Lepidium sativum L.) seedlings during a subsequent 6-min period of lateral (reoriented 90°) gravitropic stimulation. The amyloplast positioning has been evaluated taking linear measurements of each plastid position with respect to the longitudinal wall and original bottom of the gravity sensing cells. After growth for 30 h at 1 g, the amyloplasts are distributed symmetrically across the cells and located closer to the bottom cell wall in endodermis, than in root columella (15.2% and 27.7%, respectively). A more rapid plastid displacement (approximately 3 µm/min) towards the gravity and simultaneous sliding along the cells was determined within the first minute of gravitropic stimulation in hypocotyl endodermal cells as compared with that (1 µm/min) in root statocytes. During the second minute, the amyloplast location remained almost unchanged in endodermal cells, while they continued intensively to slide along the columella cells shifting slightly downwards. After the 6-min period, the final relative statolith distances from the lower longitudinal wall and bottom of the statocytes equalled to 31.1% and 28.9% in hypocotyls and to 36.5% and 38.8% - in roots. The data allow that in common with the gravity force the elastic forces of the cytoskeleton act pulling actively the amyloplasts along the statocytes in the both organs within the 6-min gravitropic stimulation. However, during the first treatment minutes, the relation of these forces differs significantly in gravisensing cells of hypocotyls and roots.

To investigate effects of hypergravity on plant growth and development, we conducted a series of experiments on large-diameter centrifuges at NASA Ames Research Center in Moffett Field, CA, during summer and fall 2006. Brassica rapa L. cv. Astroplant was grown for 16 days inside plant growth chambers within the Plant Growth Facility hardware. Responses of the plants grown in the hypergravity treatments (2-g and 4-g) were compared with the 1-g controls. For purposes of growth form analysis, Brassica plants were photographed daily, during short-duration rotor stops, and stem angles were subsequently measured using Image-J software. At both 2-g and 4-g, Brassica plants assumed a more horizontal growth form as their exposure to hypergravity progressed, with a ≈45° plant angle after 16 days at 4-g. Stem diameter increased in both hypergravity treatments. Previous work found that lignin content increases in hypergravity as mechanical support challenges increase, while the response of other small molecular secondary metabolites to hypergravity varies. Immediately following the 16-d growth period at 1-g, 2-g or 4-g, plants were harvested, immediately frozen in liquid nitrogen, and lyophilized prior to analysis for glucosinolates and lignin. In general, glucosinolate concentration was the highest in stems, followed by leaves, then roots. Glucosinolate concentration was significantly lower in stems of the 2-g and 4-g plants - averaging 4.6 and 2.5 ng/g DW, respectively - compared with the stationary control plants, which averaged 7.9 ng/g DW. Combining these results with our previous findings from an experiment with Brassica rapa on the ISS confirms a negative correlation between glucosinolate and gravity, as well as between plant erectness and gravity. Plant secondary compounds are important as flavor and nutraceutical components, so understanding gravity’s affect on them contributes to the program’s exploration mission. This project was supported by NASA grants NAG10-329 and NNX07AT77G to MEM, and by NWO-ALW-SRON grant MG-057 to JvL.

A new method for quantifying environmental impact on plant growth will be presented. The method utilizes germinating fern spores as a bioassay to indicate the degree of environmental impact. The bioassay is easily conducted on a microscope slide and once initiated requires only observation for a short time (3 to 5 days). Development of the technique was focused on characterizing the dose response to phytotoxic materials in the environment. In the process, a rapid screening tool for presence of a phytotoxin also resulted. This new bioassay effectively provides quantification of dose response characteristics such as unit toxicity and chronic and acute toxicity points. A critically important finding is that unit response and toxicity points determined using the bioassay are identical to whole plant responses. Further testing of the method indicates similar responsiveness to a range environmental signals and gradients. Given the small size and short duration of the assay, as well as stability and containment of the germinating spores, we expect the method can be utilized to quantify the impact of the space environment on plant growth and development.

Inositol 1,4,5-triphosphate (InsP3) is a soluble second messenger in eukaryotic organisms. Changes in inositol phosphate metabolism correlate positively with plant responses to gravity and light stimulation (Perera et al., 2006; Salinas-Mondragon et al., 2005). We have shown that expression of the human inositolpolyphosphate 5-phosphatase in tomato plants reduced InsP3 concentrations. The transgenic lines are more tolerant to drought, showed a 2-4 fold increase in total vegetative biomass, and lycopene concentrations in the red ripe fruits was 5 fold increased compared to control plants. The roots of these transgenic tomato lines showed a delayed and reduced gravitropic response to reorientation. Increased nutritional value and improved drought tolerance are important traits in crop plants for human space exploration. In collaboration with BioServe Space Technologies (Colorado, USA), we are currently investigating growth and development of these transgenic and control tomato lines under microgravity conditions on the International Space Station.

Perera, I.Y., Hung, C.Y., Brady, S., Muday, G.K. and Boss, W.F. (2006) A universal role for inositol 1,4,5-trisphosphate-mediated signaling in plant gravitropism. Plant Physiol, 140, 746-760.

Salinas-Mondragon, R., Brogan, A., Ward, N., Perera, I., Boss, W., Brown, C.S. and Sederoff, H.W. (2005) Gravity and light: integrating transcriptional regulation in roots. Gravit Space Biol Bull, 18, 121-122.

Nitric oxide (NO), a ubiquitous signaling molecule in animals, has been implicated in numerous plant signaling systems. Our work has focused on understanding the role of NO and cGMP in two model plant gametophyte systems, Lilium longiflorum pollen tubes, and Ceratopteris fern spores. Both of these haploid, single cell systems are known to be developmentally dependent on localized calcium gradients, which actively drive cellular polarity. Lily pollen tubes were grown in vitro and showed a negative tropism when challenged with an artificially generated NO point source (1-3 microns). We imaged NO localization in pollen, using the fluorophore DAF-2 and confocal microscopy, and were able to determine that the distribution pattern was consistent with peroxisomes as the NO source. cGMP is suggested as the putative downstream messenger of NO in experiments that show the application of the phosphodiesterase inhibitor sildenfil citrate (Viagra) to pollen tubes could induce growth responses in the presence of a weak suboptimal NO source that previously failed to induce reorientation. In the Ceratopteris fern gametophyte, the haploid spores cells undergo a process of gravity-directed cell polarity development that, like pollen tubes, is driven by polar calcium currents. Application of specific NO donors and scavengers disrupts the calcium-dependent gravity response that controls cell polarity development in a dose dependent manner. Furthermore the effects of NO donor (Spermine nonoate) exposure were antagonized by application of NO scavenger compounds. Similarly drugs that modulate NO-dependent cGMP levels in the cells (guanylate cyclase inhibitors LY83583 and ODQ; and PDE5 inhibitor Viagra) disrupt gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers suggesting a connection between NO and cGMP in the signaling pathway. We used microarray analysis, to determine how gene expression changes in response to Viagra® treatment. This identified approximately 65 genes that could mediate signaling, nuclear regulation, and protein synthesis/folding/degradation in higher plants. We subsequently analyzed gene expression changes using quantitative real-time RT-PCR (Q RT-PCR) for four of the identified genes, which have strong sequence similarity to known signal transduction or stress response genes. In spores treated with Viagra or dibutryly cGMP these four genes have significant changes in abundance throughout the first 24 h of development not seen in control samples. These results demonstrate NO/cGMP signaling cascades are active components in calcium dependent signaling associated with cellular polarity and gravisensing in plant systems.

Several generations of healthy plant are essential part of spacemen's life support in long-term space missions. Many problems in areas of discovery of plant growth and fructification peculiarities, physiological state and productivity as well as diseases, especially virus infection, development, which can emerge under microgravity conditions in space, remain unresolved up to now. Cycle-2 and horizontal KG-8 clinostats have been developed by researchers of Taras Shevchenko Kyiv national university; mutual relations between viruses and plants under simulating microgravity are investigated.

The first one realizes several variants of plants growth longitudinal axis relatively acceleration vector of terrestrial gravity. The clinostat ensures acceleration from 0.001 g, consequently it sets off the effect of gravity by horizontal axial rotation of plants. Wheat is one of the main objects for space green-house. In conditions of Ukraine, Russia, USA and Europe countries Wheat steak mosaic virus (WSMV) is the most widespread and harmful plant pathogen. Consequently, our work was directed on investigation WSMV reproduction in wheat plants under clinorotation, also we searched for substances, which possess antistress effect.

Wheat cultivars of different ecological-geographical origin such as Chayka, Albatros odeskyy, Donska semidwarf, Saratovskaya-29 (recommended for zone of Steppe in Ukraine and Russia), Kolektyvna-3, Lada, Myronivska-65 and Myronivska-67 (recommended for zone of Forrest-steppe of Ukraine), Apogee (the cultivar was designed especially for spaceflights by selectionist B.Bugbee from University of Utah, USA) and medicinal plants (Panax ginseng C. A. Meyer) were investigated in clinostats. Physiological responds of these cultivars on viral infection under simulating microgravity were studied. Virus reproduction was investigated by das-ELISA. The investigations, carried out, showed that WSMV reproduction were depressed and its elimination from artificially infected wheat cv. Apogee took place when plants were clinorotated. Apogee cultivar is promising for cultivation in space green-house since other spring cultivars (Saratovskaya-29 and Kolektyvna-3) don't form ear for the period of cultivation.

Reduction of WSMV reproduction leads to forming of yield due to different physiological-biochemical mechanisms of adaptation establishing. Substances of phytohormonal nature, which possess the prominent antistress effect, actively assist adaptation process. We have found out bioactive substances, obtained from natural raw-material, which increase wheat root length and other biometrical indicators, including productivity wheat plants from (8 to 35 %), under simulating microgravity. They are Garant, Peat oxidate, Dublín, Shokolin and Maltanin. Thereby, alternations of virus-host-plant system in wheat plants take place and reactions of adaptation are formed under microgravity conditions. Just these reactions lead to reduction of reproduction of wheat streak mosaic virus. Process of adaptation depends both on plant genotype and on application of bioactive preparations, containing phenolic substances. Such preparations raise wheat plants resistance for stress conditions of microgravity.

The Phenomenon of the reducing of the WSMV reproduction in Apogee wheat in simulated microgravity may be used for the treatment of medicinal plants against viral infections.

During space flight, plant organisms growing in microgravity may undergo variations of different environmental factors, such as radiation, temperature, humidity etc. It has been shown microgravity influences plant cell homeostasis that seems to affect stress tolerance. The objective of this study was to examine whether simulated microgravity may change thermotolerance of pea seedlings. It is known that up-regulation of heat shock proteins (Hsps) is a common cellular response to heat stress (HS) as well as other kinds of stress. Electrolyte leakage increases under HS indicating deterioration of cell membranes. Therefore in order to characterize thermotolerance of the seedlings, Western analysis of Hsp70 and Hsp90 content, a conductometric test, as well as a hypocotyl- and root-elongation assay were carried out. Pea seedlings (cv. Intensive) were grown in the stationary conditions or on a horizontal clinostat from onset of germination for 5 days at 21±1°C in the dark and then exposed to different HS treatments (mild, severe and severe after pretreatment with mild HS). Seedlings grown in the stationary conditions or under clinorotation without HS served as controls. It was shown the mild HS had little negative effect on seedling elongation - less on the clinorotated seedlings comparatively to the stationary grown ones, while the severe HS significantly blocked elongation in both variants. At the same time, up-regulation of both Hsp70 and Hsp90 was higher in the clinorotated seedlings under all of the HS regimes. On the other hand, increased electrolyte leakage was determined in the clinorotated seedlings under normal temperature conditions as well as after mild HS and severe HS with pretreatment. After severe HS without pretreatment, electrolyte leakage had close high values in both variants. These data suggest that clinorotation may influence distinct functions, including Hsps synthesis and protection of membrane integrity, that affect plant growth activity and thermotolerance as a result.

We have analysed the morphogenesis of the root system of Arabidopsis thaliana seedlings grown on a clinostat at 1 rpm. During the early stages of the root development, it appeared that the length of the primary root was similar in clinorotated and control plants, and that the formation of secondary roots was similar in both conditions. After 8 days, the primary roots of the clinostated plants grew faster and showed a greater production of lateral roots than those of the controls. As auxin and abscisic acid play an important role in tropisms and apical dominance, we have studied the distributions of these phyhormons in the root system by using transgenic Arabidopsis expressing DR5::uidA or Rd29::uidA constructions, respectively.

DR5::uidA seedlings displayed a maximum of GUS activity in the columella of the root cap, the quiescent center, and part of the root meristem, showing that auxin was present in the root apex. Auxin was also present in the pericycle cells and in secondary roots. Although patterns of GUS expression was similar in clinorotated and control roots, it must be noted that the clinorotated roots presented a higher GUS expression levels than control roots. The impact of auxin transport have also be studied.

Our data also indicated that the clinostat induced an early decrease of abscisic acid content in Rd29::uidA roots. At the 8 days of culture, a slight GUS expression was observed in the elongation zone of the control root, while no GUS staining was detected in clinostated roots.

These results demonstated that clinoration induced some modifications in auxin and abscisic acid distributions, which could be correlated with the morphological changes in root development observed on the clinostat.

A robust, flexible protocol was developed for the cultivation of Arabidopsis thaliana for spaceflight experimentation. Seeds were germinated on rock-wool plugs contained within microtiter dishes and subsequently thinned out after 5 days. The rock-wool was easy to work with, showed consistent germination and good early seedling growth. The seedling-plugs were transferred into modified 15 mL polypropylene centrifuge tubes (5 cm lengths) at 7-10 days after planting. Each tube was cut with four side-situated slits through which capillary mat strips were woven in order to provide greater access to water and nutrients. An additional capillary mat wick extended from outside of the tubes up through a hole in the bottom to the middle of the tubes.

The incorporation of Fibrous Ion Exchange Resin Substrate (FIERS) was found to greatly promote vigorous seedling development, and resulted in thicker primary stems. The tubes could be maintained within culture chambers or transferred to flight-suitable substrate compartments containing a root-cube foam matrix that displayed advantageous properties of water retention, ease of use, and biocompatibility. The plants grew vigorously and produced numerous siliques whose seeds frequently adhered to the foam surfaces and germinated, yielding a second generation of seedlings produced without a vernalization treatment. The methods used herein for Arabidopsis could be easily applied to many plant species of interest to the Bioregenerative Life Support System community.

The present research examines responses of garden cress (Lepidium sativum L.) seedlings to separate and combined spectral components of light under fast clino-rotation (50 rpm) and vertically at 1g. The seedlings were illuminated from above using custom-built illumination modules with blue (peak wavelength 450 nm, photon flux density 5 µmol m^-2s^-1), red (660 nm, 13 µmol m^-2s^-1) and far-red (735 nm, 0.8 - 1 µmol m^-2s^-1) light emitting diodes (LEDs). The plants were grown on a solid MS medium with one-half-strength salts and 0.2% (w/v) gelrite in special containers fitted to the stationary control and centrifuge-clinostat devices, for 1g control and for the rotation on a horizontal clinostat, respectively. Biometric parameters of hypocotyls and leaves of Lepidium seedlings were measured after 5-day cultivation in both 12 h/d light photoperiod and without illumination. Histological and cytomorphometrical studies of the central part of the first leaf lamina were performed on transverse sections using light microscopy.

It was established that blue lighting reduced the elongation of 1-g and clino-rotated hypocotyls by 16% and 29%, red lighting - by 29% and 43%, and far-red lighting - by 57% and 64%, respectively. Illumination by both red and blue LEDs combined with far-red light suppressed the growth of hypocotyls more effectively in altered gravity. Leafstalks of leaves responded to gravity alteration and in the dark were significantly longer on the clinostat than at 1g. However, in light this difference was insignificant with the exception of effects provoked by red and far-red illumination. Red and far-red light inhibited the elongation of clino-rotated leafstalks as compared with 1-g control ones by 17 and 22%, respectively. The leaf area increased in red and far-red light due to the radial expansion; however, no difference between the leaf area and lamina length in 1-g and clino-rotated seedlings was determined. On the basis of histological and cytological analysis of Lepidium leaves, we can assert that the applied illumination promoted differentiation of leaf tissues more effectively when the induction of gravitropic reaction was suppressed by clino-rotation. Without illumination, the impact of clino-rotation on leaf tissue cells was negligible. The obtained data support the view that the rate of growth responses in altered gravity can be modulated by separate components of spectrum and by precise photon flux density of light.

Acknowledgements. This work is supported by the Lithuanian State Science and Studies Foundation, grant T-84/08.

Experiments recently performed during parabolic plane flights have provided clear evidence that gravireceptor molecules in characean rhizoids are not activated by pressure that is exerted by sedimenting statoliths upon gravistimulation. Even weightless statoliths trigger gravity perception as long as there are in contact with the gravireceptor, which suggests that direct (electrical/chemical) interactions between statoliths and gravireceptor initiate the graviperception process. To further investigate the statolith-gravireceptor interactions membrane-potential measurements were performed in the apex of graviresponding cells by using DiBAC4(3), a potent membrane potential sensitive dye.

A modular Nikon laser scanning microscope was rearranged such that the rhizoids could be examined in their normal vertical orientation; and, by rotating the stage, the cells could also be observed throughout the different phases of gravitropism. A dense population of depolarized vesicles was found in the apex of growing Chara rhizoids, its distribution reflecting the steep tip-high gradient of cytoplasmic free calcium. When polar growth of rhizoids was stopped by applying Latrunculin B, the fluorescence faded which indicates that the vesicles were no longer depolarized. Throughout gravitropic bending, the DiBAC4(3) fluorescence strongly faded in the area where statoliths sedimented onto the plasma membrane of the lower cell flank. We suggest that the statolith-induced local reduction of cytoplasmic free calcium impairs vesicle depolarization, inhibits exocytosis of vesicles and eventually causes gravicurvature due to differential growth of the opposite cell flanks.

Depolarised vesicles have also been found in other tip growing cell types like pollen tubes and root hairs. It is concluded that increasing concentrations of calcium cause the depolarization of vesicles which are delivered to the apex for exocytosis. Vesicle depolarization seems to be a prerequisite for gravitropic tip growth in characean rhizoids and for polar growth in general.

The mechanism by which plants sense gravity is not fully understood. The hydrostatic model was proposed to explain gravitropic sensing in systems which lack statoliths. These experiments are designed to provide further understanding about the underlying mechanism of the gravitropic sensing. The hydrostatic model states that the buoyancy of the cell is affected by the density of its external environment. When buoyancy of the cell is altered, stretch channels respond and control the amount of calcium ions external to the cell membrane; calcium ions are known to play a major role in control of cell growth and gravitropic response in plant tissue.

Primary roots of maize (Federal Hybrid RK112-1, Elgin, IA) with a length of about 1 cm were used. The roots were placed in environments of various density and buoyancy using air, water, sucrose/polyethylene glycol (PEG), and Ficoll PM 400 (Amersham Biosciences, Piscataway, NJ). The rates of growth and gravitropic curvature were monitored using time-lapse video and digital recordings. The time lapse images were analyzed using digital analysis software. Comparison of roots in air to roots in oxygenated water indicate that there is no significant difference in growth rates but the higher density of water significantly slows the gravitropic response. Ten percent sucrose solution with 5% PEG inhibits elongation of roots for approximately 2 hrs after the root is submerged in the solution. After 2 hr the elongation rate recovers to within 10% of the control rate; horizontally-oriented roots in 10% sucrose/5% PEG solutions exhibit no significant curvature after 8 hrs. Ficol PM 400, a polymer of sucrose with greatly reduced osmotic activity when compared to sucrose/PEG, does not significantly inhibit elongation of roots. Altering the environmental density and buoyancy of the solution surrounding the root does not appear to alter sedimentation of statoliths within the root tip. Data will be presented comparing environmental density and buoyancy on growth and parameters of the gravitropic response in primary roots of maize.

Experiments with plants carried out in microgravity have shown that plant roots experienced extreme variations in moisture, oxygen and nutrients availability. This is due to microgravity specific fluid distribution and problems with control of water supply systems in the plant growth media (substrates). Changes in substrate moisture are connected with corresponding changes in nutrient and oxygen availability to plant roots. The higher water content leads to oxygen deficiency and the lower water content leads to nutrient deficiency. Many plants grown in different space plant growth facilities have shown signs of stress connected with moisture availability.

Ground experiments were carried out in the laboratory prototype of SVET-2 Space Greenhouse to study the effect of variation of root-zone moisture conditions on growth of lettuce and pea plants. The effect of transient increase (for 1 day) and drastic increase (waterlogging for 10 days) of substrate moisture was studied with pea and lettuce plants respectively.

Moisture dynamics in the upper substrate layers during substrate moisture variations with or without root module components – hydro-accumulators and wicks was investigated. Substrate particles rearrangement in root module volume was observed.

Transient rise of substrate moisture initiated leaf wither and senescence in pea plants. Net photosynthetic rate (Pn) of pea leaves showed fast response to moisture changes while chlorophyll content of pea leaves and growth parameters did not change. Drastic change of substrate moisture (waterlogging) suppress lettuce Pn, chlorophyll biosynthesis and plant growth. These parameters slowly recovered after termination of waterlogging treatment but lettuce yield was greatly affected.

The results showed that the most sensitive physiological parameter to substrate moisture variations is photosynthesis. Assessment of plant physiological parameters could help adaptive substrate moisture control both during flight and ground-based experiments ensuring optimal plant production.

One of the first fundamental questions in early space biology research was whether plants could complete a full life cycle (from seed to seed) under spaceflight conditions. Once it was established that spaceflight environment was not a significant impediment to plant growth, the question that followed was whether this environment could cause changes in subsequent generations, even those grown in a normal Earth environment. In this study we used a genomic approach to answer this question. We tested whether changes in gene expression patterns can be detected in wheat plants that are several generations removed from growth in space, compared to wheat plants with no spaceflight exposure in their lineage. Triticum aestivum L. cv. Super Dwarf, flown on MIR in 1991, formed viable seeds back on Earth. These seeds were grown on the ground for three additional generations. Leaf material was harvested from two fourth generation MIR flight plants and two Triticum aestivum L. cv. USU-Apogee control plants.

Gene expression of fourth generation MIR flight leaves was compared to that of the Apogee control leaves by using custom-made wheat microarrays and a reference design. The median signal intensities detected for the MIR flight, Apogee control and reference samples were statistically analyzed using a two-step strategy including ANOVA. After correcting for multiple testing, expression levels of wheat genes represented by the 10263 probes on the arrays showed no statistically significant differences between the two sets of plants. This suggests that exposure to the spaceflight environment in low earth orbit space stations does not cause significant, heritable changes in gene expression patterns in plants. Follow-up studies could test the effects of exposure to spaceflight for several contiguous generations on gene expression in plants and their offspring. Furthermore, diploid or tetraploid species could be analyzed and compared to each other or to hexaploid species like the wheat used in this study.

Long-duration future habitation of space will require a controlled ecological life support system (CELSS) to simultaneously revitalize atmosphere (liberate oxygen and fix carbon dioxide), purify water (via transpiration), and generate food. Effects of microgravity on living organisms during space flight have been a topic of interest for some time, and a substantial body of knowledge has accumulated. Comparatively little information is available regarding the influence of microgravity on algae, even though it has been suggested for long duration flight or occupancy in space that plant growth systems, including both higher plants and algae, are likely to be necessary for bioregenerative life support systems. The Hydrodynamic Focusing Bioreactor (HFB) developed by NASA at Johnson Space Center provides a unique hydrofocusing capability that simultaneously enables a low-shear culture environment and unique hydrofocusing-based suspension of cells and tissue aggregates. The HFB provides a laboratory-based approach to investigating the effects of microgravity on cellular reactions.

In this study, the HFB was used to examine the influence of simulated microgravity on the growth and metabolism of Chlorella pyrenoidosa. After 3 days of culture, cell numbers increased more slowly in simulated microgravity than in the gravity control; after 7 days, growth in simulated microgravity was just over half (50%) that of the gravity control and at 14 days it was less than half (40%). Chlorella grown in simulated microgravity formed 3-D aggregates within 2 days of culture. Chlorophyll and protein were followed as indices of cell competence and function; after 2-3 days, protein and chlorophyll levels were slightly reduced in simulated microgravity compared to gravity controls. Chlorella exhibited an overall decrease in steady-state proteins, but an overall increase in various proteins throughout the 7 day growth cycle. Photosynthesis is a sensitive biochemical index of the fitness of photosynthetic organisms; thus, CO2-dependent 02 evolution was tested as a measure of photosynthetic capacity of cells grown in simulated microgravity. When data were expressed with respect to cell number, simulated microgravity appeared to have little effect on C02 fixation. Thus, even though overall growth rate was lower in microgravity, photosynthetic capacity appears unaffected.

This study suggests that cell growth and morphological characteristics of green algae may be altered by culture in simulated microgravity. Data obtained to date should provide a solid basis for additional experimentation regarding the influence of simulated microgravity on cell morphology, physiological activity, protein production and possibly gene expression in algal and plant cell systems.

Plants are subjected to various environmental changes during their life cycle. To protect themselves against unfavorable influences, plant cells synthesize several classes of heat shock proteins (Hsp/chaperones). Hsps are responsible for protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize proteins and membranes, and can assist in protein refolding under stress conditions. They can play a crucial role in protecting plants against stress by re-establishing normal protein conformation and thus cellular homeostasis. The aim of our study was to evaluate stress gene expression in Pisum sativum seedlings exposed to centrifugation and temperature elevation. We studied small heat shock proteins (sHsp), which are able to enhance the refolding of chemically denatured proteins in an ATP-independent manner, in other words they can function as molecular chaperones. Applying the RT-PCR and immunoblot analysis, we investigate sHsps expression in pea 5 day old seedlings subjected to centrifugation under 3, 7, 10 and 14g (during 15 minute and one hour) and temperature elevation (42°C). Temperature elevation, as the positive control, significantly increased sHsps expression. Expression of the housekeeping gene, actin was a constant and comparable to unstressed controls for all treatments. A question whether sHsp genes are under the same regulation after the thermal and hypergravity stress is discussed.

Potato occupies the fourth place as a food staple of the world’s humans after wheat, maize, and rice. Potato production allows to fight hunger in the most poverty-stricken regions of the globe, but the increase in its total production is very much dependent on the practices of plant protection from the diseases and the ways of pest and weed control. Up-to-date losses of potato yields destroyed by pests and diseases may be up to one-third of their amount and viral diseases may be a priority destroyer. It is important to understand the behavior of the system "virus-host plant" in man-made ecological systems. The problem becomes more acute with the creation of life-supply systems for prolonged space flights which become very significant nowadays in connection with the planning’s of piloted in remote outer space like a visit to Mars. As there exits a possibility of viral infection in outer space (flying vehicles) it is important to study the behavior of viruses and host plants in simulated microgravity.

Potato plant is being infected by 37 viruses. The most dominant of them in Ukraine are PMV, PSV, PYV and PXV. Some research activities (Mishchenko & Taran 2006, 2007) have found that in tolerant potato plants the content of viruses becomes lower under the influence of simulated microgravity. Clinostating behaves like an inducer of plant resistance and appears to be an alternative, non – standard, and environmentally safe technique of plant protection.

The use of the mechanisms of induced resistance in the practice of crop production allows to minimize the use of chemical preparations, which favors the development of sustainable agricultural production.

Vesta variety of potato plants, under conditions of simulated microgravity, showed a positive tendency in weight and quantity of tubers. Tuber weight increased two times with clinostating, compared with control test variant. The number of tubers per plant also increased. Positive tendencies in the development of tubers show that Vesta variety plants succeeded in adapting to the stress conditions of cultivation and microgravity became a stimulating factor for the formation of tubers.

Research of potato crop productivity under simulated microgravity allows to identify the plants which can become potentially productive under such stress conditions and that might allow to identify the technological parameters of potato production in other space expeditions.

One of the traditional practices of planting material treatment against the viruses are the species in vitro.
Researchers of the Institute of Agricultural Microbiology, UAAS (Zaritzky, et al, 2007) focal out that 1 infected plant in vitro will produce 14-16 000 infected species in a leaf-year!

Clinostating conditions affected the growth and development of the in vitro regenerants. The weight of the above-ground parts of Krymska Rosa variety potato plant increased 1,9 times compared with stationary controls, whereas the height of plants increased 1.4 times. As it is possible to obtain the in vitro plants free from some pathological viruses, by clinostating, there is no doubt that the employment of such biotechnological practices will prove effective and feasible in agricultural production.