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Microgravity-induced apoptosis in cultured glial cells.

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.

Animals↗

Inhibitory effect of hypergravity on photosynthetic carbon dioxide fixation in Euglena gracilis.

Photosynthesis, the conversion of light energy into chemical energy, is a critical biological process, whereby plants synthesize carbohydrates from light, carbon dioxide (CO2) and water. The influence of gravity on this biological process, however, is not well understood. Thus, centrifugation was used to alter the gravity environment of Euglena gracilis grown on nutritive agar plates illuminated with red and blue light emitting diodes. The results showed that hypergravity (up to 10xg) had an inhibitory effect on photosynthetic CO2 fixation. Chlorophyll accumulation per cell was essentially unaffected by treatment; however, Chl a/Chl b ratios decreased in hypergravity when compared to 1xg controls. Photosynthesis in Euglena appears to have limited tolerance for even moderate changes in gravitational acceleration.

Animals↗

Impact of microgravity and hypergravity on free-running circadian rhythm of the desert beetle Trigonoscelis gigas Reitt.

Free-running circadian rhythms of locomotor activity of Tenebrionid beetles Trigonoscelis gigas Reitt., taken from the Turkmenian sand desert, were monitored in DD. The effects of microgravity --11 days in space flight aboard the Russian BION-10 "COSMOS" satellite, and of 2G hypergravity--seven days on a centrifuge, were determined. Two kinds of effects were found. In stable 2-peak records, there was a moderate decrease of tau in microgravity and an increase of tau in 2G, both of about 0.3 hr. In unstable records, alterations of gravity caused drastic deviations of tau and phi. Remarkably, two peaks of the activity rhythm, which are supposed to be controlled by separate oscillators, responded to gravity transitions in different ways. Gravity effects on the circadian system could be explained from a direct effect on the oscillator(s) itself or from a feed-back by altered locomotion to the pacemaker. Thus, for the first time the gravity dependence of a free-running circadian rhythm was proved in a combination of real space flight and centrifuge experiments.

Adaptation, Physiological↗

The influence of gravity on the process of development of animal systems.

The development of animal systems is described in terms of a series of overlapping phases: pattern specification; differentiation; growth; and aging. The extent to which altered (micro) gravity (g) affects those phases is briefly reviewed for several animal systems. As a model, amphibian egg/early embryo is described. Recent data derived from clinostat protocols indicates that microgravity simulation alters early pattern specification (dorsal/ventral polarity) but does not adversely influence subsequent morphogenesis. Possible explanations for the absence of catastrophic microgravity effects on amphibian embryogenesis are discussed.

Animals↗

Microgravity and bone cell mechanosensitivity.

Bone cells, in particular osteocytes, are extremely sensitive to mechanical stress, a quality that is probably linked to the process of mechanical adaptation (Wolff's law). The in vivo operating cell stress derived from bone loading is likely a flow of an interstitial fluid along the surface of the osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin synthesis and expression of inducible prostaglandin G/H synthase (PGHS-2 or inducible cyclooxygenase, COX-2), an enzyme that mediates the induction of bone formation by mechanical loading in vivo. Disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity has catabolic effects on the skeleton of astronauts, as well as on mineral metabolism in bone organ cultures. This might be explained simply as resulting from an exceptional form of disuse under weightlessness conditions. However, under microgravity conditions, the assembly of cytoskeletal elements may be altered, as gravity has been shown to determine the pattern of microtubular orientation assembled in vitro. Therefore, it is possible that the mechanosensitivity of bone cells is altered under microgravity conditions, and that this abnormal mechanosensation contributes to the disturbed bone metabolism observed in astronauts. In vitro experiments on the International Space Station should test this hypothesis experimentally.

Animals↗

A model for vestibular function in altered gravitational states.

During evolution, the vestibular organ was made to serve mainly two purposes: 1) to guide eye movements during sharp turns, so that the point of fixation in the visual field can be kept steady, a function accomplished by the semicircular canal system and 2) to indicate the terrestrial vertical, so that upright posture and gait can be maintained even in the dark. The otolith system serves the latter purpose. Since the function of the semicircular canal system does not depend on gravity, it is not grossly disturbed by gravitational levels different from 1 g. The proper function of the otolith system depends entirely on the presence of a gravitational force vector of 9.8/m/sec2 directed towards the center of the earth. This system therefore malfunctions when the amplitude of the combined gravito-inertial load is different from 1 g and also when the direction of the sensed gravitational pull is "contaminated" by additional inertial reactive forces as during horizontal acceleration. The effect of such inertial stimulations is probably even stronger in a weightless environment, in which case the background stimulation of terrestrial gravity is absent. Moreover, minor mass differences between the otolithic membranes of the left and right inner ear, even if well compensated on the ground, might lead to malcompensation in weightlessness as well as in hypergravity. A hypothetical model is developed to describe in the central nervous system compensating mechanisms in hypo- and hypergravitational states. The "space-sled" is introduced as a new research tool and recommendations are made for a prophylactic training regimen to reduce or prevent space sickness.

Adaptation, Physiological↗

Gene expression in rat vestibular and reticular structures during and after space flight.

Space flight produces profound changes of neuronal activity in the mammalian vestibular and reticular systems, affecting postural and motor functions. These changes are compensated over time by plastic alterations in the brain. Immediate early genes (IEGs) are useful indicators of both activity changes and neuronal plasticity. We studied the expression of two IEG protein products [Fos and Fos-related antigens (FRAs)] with different cell persistence times (hours and days, respectively) to identify brainstem vestibular and reticular structures involved in adaptation to microgravity and readaptation to 1 G (gravity) during the NASA Neurolab Mission (STS-90). IEG protein expression in flight animals was compared to that of ground controls using Fisher 344 rats killed 1 and 12 days after launch and 1 and 14 days after landing. An increase in the number of Fos-protein-positive cells in vestibular (especially medial and spinal) regions was observed 1 day after launch and 1 day after landing. Fos-positive cell numbers were no different from controls 12 days after launch or 14 days after landing. No G-related changes in IEG expression were observed in the lateral vestibular nucleus. The pattern of FRA protein expression was generally similar to that of Fos, except at 1 day after landing, when FRA-expressing cells were observed throughout the whole spinal vestibular nucleus, but only in the caudal part of the medial vestibular nucleus. Fos expression was found throughout the entire medial vestibular nucleus at this time. While both Fos and FRA expression patterns may reflect the increased G force experienced during take-off and landing, the Fos pattern may additionally reflect recent rebound episodes of rapid eye movement (REM) sleep following forced wakefulness, especially after landing. Pontine activity sources producing rhythmic discharges of vestibulo-oculomotor neurons during REM sleep could substitute for labyrinthine signals after exposure to microgravity, contributing to activity-related plastic changes leading to G readaptation. Reticular structures exhibited a contrasting pattern of changes in the numbers of Fos- and FRA-positive cells suggestive of a major influence from proprioceptive inputs, and plastic re-weighting of inputs after landing. Asymmetric induction of Fos and FRAs observed in some vestibular nuclei 1 day after landing suggests that activity asymmetries between bilateral otolith organs, their primary labyrinthine afferents, and vestibular nuclei may become unmasked during flight.

Adaptation, Physiological↗

G-force induced alterations in rat EEG activity: a quantitative analysis.

A major physical limitation affecting pilots is G-force (+Gz, head-to-foot inertial load) induced loss of consciousness. Previous studies have shown that +Gz produces qualitatively similar effects on human and rat EEG activity. The present study sought to quantitatively correlate changes in rat EEG activity with increasing +Gz levels. A frontal-parietal differential electrode recorded rat EEG data during +Gz exposures (30 s) ranging from +0.5 to +25.0 Gz. Acceleration levels < or = +10 Gz had little effect on EEG activity. Acceleration levels of +15 to +20 Gz were associated with increased EEG slowing, depression and sharp waves. Acceleration levels > or = +17.5 Gz evoked burst suppression followed by isoelectric activity. Times to first onset of delta, depressed, and isoelectric EEG activity were approximately 12, 14 and 18 s, respectively. Acceleration effects on delta (1-4 Hz), theta (5-8 Hz), alpha (9-12 Hz), beta (13-30 Hz) and total (1-30 Hz) EEG powers were examined using Fourier transform analysis. EEG measures with the most predictive value at the following post-acceleration onset times (PAOT) were as follows (in s); increasing theta power: PAOT 0-2, decreasing delta power: PAOT 3-9, and decreasing beta power: PAOT > or = 12. This study provides a quantitative description of +Gz-induced alterations in EEG magnitude, time course and spectral content. Additionally, several EEG measures were identified which correlated with acceleration level at specific post-acceleration onset times.

Alpha Rhythm↗

Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia.

Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity.

Animals↗

Saccule contribution to immediate early gene induction in the gerbil brainstem with posterior canal galvanic or hypergravity stimulation.

Immunolabeling patterns of the immediate early gene-related protein Fos in the gerbil brainstem were studied following stimulation of the sacculus by both hypergravity and galvanic stimulation. Head-restrained, alert animals were exposed to a prolonged (1 h) inertial vector of 2 G (19.6 m/s2) head acceleration directed in a dorso-ventral head axis to maximally stimulate the sacculus. Fos-defined immunoreactivity was quantified, and the results compared to a control group. The hypergravity stimulus produced Fos immunolabeling in the dorsomedial cell column (dmcc) of the inferior olive independently of other subnuclei. Similar dmcc labeling was induced by a 30 min galvanic stimulus of up to -100 microA applied through a stimulating electrode placed unilaterally on the bony labyrinth overlying the posterior canal (PC). The pattern of vestibular afferent firing activity induced by this galvanic stimulus was quantified in anesthetized gerbils by simultaneously recording from Scarpa's ganglion. Only saccular and PC afferent neurons exhibited increases in average firing rates of 200-300%, suggesting a pattern of current spread involving only PC and saccular afferent neurons at this level of stimulation. These results suggest that alteration in saccular afferent firing rates are sufficient to induce Fos-defined genomic activation of the dmcc, and lend further evidence to the existence of a functional vestibulo-olivary-cerebellar pathway of adaptation to novel gravito-inertial environments.

Animals↗

A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation.

Callus cells of Arabidopsis thaliana (cv. Landsberg erecta) were exposed for 8 h to a horizontal clinostat rotation (H, simulated weightlessness), a vertical clinostat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and fructose apparently decreased, while starch content increased in the H compared with the V- and S-treated cells. In order to investigate the influences of clinostat rotation on the cellular proteome further, the proteome alterations induced by horizontal and vertical clinostat rotation have been comparatively analysed by high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 80 protein spots showed quantitative and qualitative variations that were significantly (P <0.01) and reproducibly different between the clinorotated (H or V) and the stationary control samples. Protein spots excised from 2-D gels were analysed by microbe high performance liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. 18 protein spots, which showed significant expression alteration only under the H condition compared with those under V and S conditions, were identified. Of these proteins, seven were involved in stress responses, and four protein spots were identified as key enzymes in carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated in the H samples. Other proteins such as protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins also exhibited altered expression under the H condition. The data presented in this study illustrate that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins involved in general stress responses, metabolic pathways, gene activation/transcription, protein synthesis, and cell wall biosynthesis.

Arabidopsis↗

[Mineral metabolism in man under altered gravitation].

The book contains data about the functions and the metabolism of mineralized tissues, and the results of investigations of bone tissue and calcium metabolism during manned space missions and ground-based simulations. Consideration is given to the bone strength characteristics under experimental hypo- and hypergravity. The role of hormonal and physical/chemical factors in the mechanisms of iron control alterations in man during space missions of various lengths is analyzed. Evaluated is the efficiency of different countermeasures opposing the shifts in electrolyte exchange and bone mineral losses in these conditions. A concept is proposed for hypogravity-induced changes in the mineral metabolism and bone tissue status. The book is addressed to biologists, physiologists, biochemists, specialists in space biology and aerospace medicine.

Aerospace Medicine↗

Developmental study of rat vestibular neuronal circuits during a spaceflight of 17 days.

The aim of this study was to investigate the potential plasticity of the vestibular system, in structural and biochemical terms, at the level of the gravity receptors (the sensory hair cells), the primary neurons relaying the sensory signals (the vestibular ganglion neurons) and their projections into the vestibular nuclei. We studied the biochemical differentiation of the sensory cells and of the vestibular ganglion by investigating which calcium-binding proteins were present. We studied the development of peripheral synaptic connections of the efferent system by investigating the distribution of CGRP (calcitonin-gene related-peptide) and we also studied the cerebellar synaptic connections in the vestibular nuclei, as identified by the presence of calbindin. Putative changes were studied after a 17-day episode of microgravity (Neurolab STS-90), in developing rats between postnatal days 8 and 25. The extent to which these changes could be caused by alterations in gravity was determined by examining sensory and nervous structures not involved in gravity detection, the cochlea and the cochlear nuclei.

Acoustic Maculae↗

Readaptation of the vestibuloocular reflex to 1g-condition in immature lower vertebrates (Xenopus laevis) after micro- or hypergravity exposure.

The effects of altered gravitational conditions (AGC) on the development of the static vestibulo-ocular reflex (VOR) and readaptation to 1g were investigated in the amphibian Xenopus laevis. Tadpoles were exposed to microgravity during the German Space Mission D-2 for 10 days, using the STATEX closed survival system, or to 3g for 9 days during earth-bound experiments. At the beginning of AGC, the tadpoles had not yet developed the static VOR. The main results were: (i) Tadpoles with microgravity- or 3g-experience had a lower gain of the static VOR than the 1g-controls during the 2nd and 5th post-AGC days. (ii) Readaptation to response levels of 1g-reared controls usually occurred during the following weeks, except in slowly developing tadpoles with 3g-experience. Readaptation was less pronounced if, during the acute VOR test, tadpoles were rolled from the inclined to the normal posture than in the opposite test situation. It is postulated that (i) gravity is necessarily involved in the development of the static VOR, but only during a period including the time before onset of the first behavioural response; and (ii) readaptation which is superimposed by the processes of VOR development depends on many factors including the velocity of development, the actual excitation level of the vestibular systems and the neuroplastic properties of its specific pathways.

Adaptation, Physiological↗

Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity.

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered

Animals↗

Short-term (Fos) and long-term (FRA) protein expression in rat locus coeruleus neurons during the neurolab mission: contribution of altered gravitational fields, stress, and other factors.

Changes in immediate-early gene (IEG) expression during and after space flight were studied in the rat locus coeruleus (LC) during the NASA Neurolab mission. The LC sends widespread projections throughout the brain and releases the neuromodulator norepinephrine. LC neurons respond to natural vestibular stimulation; their firing rate also increases during waking and decreases or ceases during sleep. LC neurons express IEGs such as c-fos after activation. Adult male albino Fisher 344 rats were killed at four mission time points, and the number of Fos- and Fos-related antigen (FRA)-positive LC cells were counted in flight and ground-based control rats. Half of the subjects at each time point were exposed to light for 60 min prior to killing to standardize their sleep-waking state. FRA-expressing cells were more numerous than Fos-expressing cells in both flight- and ground-based subjects. The difference between FRA- and Fos-expressing cells within individuals was significantly larger 24 h after landing in subjects exposed to both space flight and light pulse than in all other subjects at any mission time point. Fos and FRA responses scaled in proportion to the maximum response observed in any single individual showed similar patterns of variation. Analysis of the scaled and combined responses showed that LC IEG levels responded to both gravity changes and light pulses. Subjects exposed to either single stimulus had equivalent responses, significantly greater than those of control subjects maintained in dim light. The combination of gravity change and light pulse gave significantly higher LC responses than either stimulus alone 24 h after takeoff, and to a lesser extent after 12 days in space; the highest responses were obtained 24 h after landing. By 14 days after landing, animals exposed to space flight and light pulse responded no differently than ground-based subjects. No difference in LC IEG expression was clearly attributable to changes in the sleep-waking state of subjects. Activity of noradrenergic LC neurons has been previously shown to modulate IEG expression in target structures. The increased IEG LC activity (seen most especially 24 h after landing) may reflect large-scale activation of noradrenergic neurons that may serve as a trigger for molecular changes in target structures, and be critical for adaptation to gravity changes.

Adaptation, Physiological↗

Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles (Xenopus laevis).

In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1)the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) microgravity induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.

Adaptation, Physiological↗

Time-frequency analysis of the surface EMG during maximum height jumps under altered-G conditions.

Surface electromyographic (SEMG) signals are often used to study motor control during human movement. Typically, the SEMG signal is used to determine when the muscle was on or off during the movement. However, determining the time-based frequency content of the SEMG signal in conjunction with the time-history of the movement may provide us with more insight into how the motion is organized. We collected SEMG from 4 muscles, (soleus, vasti, gluteus maximus, hamstrings) while subjects performed jumping and sit-to-stand tasks under altered g-environments. The experiment was performed on the Dynamic Environmental Simulator at Wright Patterson AFB, OH. The subjects performed the tasks at 1.0, 1.2, 1.4, 1.6, and 1.8 g. The SEMG signals were then analyzed using a continuous discrete wavelet transform based on a 4-coefficient Daubechie wavelet. Our initial results show that although the time-history of muscle activation patterns during movements do not always vary significantly at different g levels, the frequency content does change, with more high frequency activation at low g levels, and more low frequency activation at higher g levels. One possible explanation for the difference in frequency activation is fatigue of fast twitch fibers during the experiment. These differences were not apparent from the time-history activation patterns, and are difficult to interpret from standard FFT manipulations.

Electromyography↗