Thyroid cell growth: sphingomyelin metabolism as non-invasive marker for cell damage acquired during spaceflight

Prolonged spaceflights are known to elicit changes in human cardiovascular, musculoskeletal, and nervous systems, whose functions are regulated by the thyroid gland. It is known that sphingomyelin metabolism is involved in apoptosis (programmed cell death) of thyroid cells induced by UVC radiation, but at present no data exists with regard to this phenomenon, which occurs during space missions. The aim of this study was to analyze, for the first time, the effect of spaceflight on the enzymes of sphingomyelin metabolism, sphingomyelinase, and sphingomyelin synthase, and to determine whether the ratio between the two enzymes might be used as a possible marker for thyroid activity during space missions. Both quiescent thyroid cells and thyroid cells stimulated to proliferate with thyrotropin (TSH) were cultured during the Eneide and Esperia missions on the International Space Station. The results show that during space missions the cells treated with TSH grew only 1.5?±?0.65-fold and, thus, behave similarly to quiescent cells, while on the ground the same cells, maintained in experimental conditions that reproduced those of the flight, grew 7.71?±?0.67-fold. Comparison of the sphingomyelinase/sphingomyelin-synthase ratio and the levels of Bax, STAT3, and RNA polymerase II in proliferating, quiescent, pro-apoptotic, or apoptotic cells demonstrated that thyroid cells during space missions were induced into a pro-apoptotic state. Given its specificity and the small amount of cells needed for analysis, we propose the use of the sphingomyelinase/sphingomyelin-synthase ratio as a marker of functional status of thyroid cells during space missions. Further studies could lead to its use in real time during prolonged spaceflights.     

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

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

Response to hypogravity of normal in vitro cultured follicular cells from thyroid

Aim of this investigation is the study of molecular modifications occurring in differentiated mammalian cells exposed to gravitational changes. The test system chosen is a well characterized clone of differentiated, normal thyroid follicular cells (FRTL5) in long-term culture. As a follow-up to our recent experiment performed during the MASER-7 sounding rocket mission, flown for European Space Agency by Swedish Space Corporation in May 1996, we evaluated FRTL5 cells responses to Thyroid Stimulating Hormone dependent cAMP production under acute hypogravity conditions obtained in a fast rotating clinostat. Following this approach, we evaluated the FRTL5 cells response to TSH under microgravity conditions in order to optimize experimental tools and strategies in preparation to, and in between real flight missions.     

The properties of alamethicin incorporated into planar lipid bilayers under the influence of microgravity.

During evolution, life on earth had adapted to the gravity of 1g. Due to space flight, in the last decades the question arose what happens to the brain under microgravity on the molecular level. Ion channels among others are the molecular basis of brain function. Therefore, the investigation of ion channel function under microgravity seems to be a promising approach to gather knowledge on brain function during space flight. In a first step, the ion channel forming peptide Alamethicin was used as a model channel in an artificial membrane. It is well suitable for this kind of investigation, since its properties are well described under standard gravity. For that reason, changes due to microgravity can be detected easily. All experiments were performed in the German drop tower at ZARM-FAB, Bremen. A special set-up was constructed based on the bilayer technique introduced by Mueller and Rudin. All functions of this set-up can be observed and controlled remotely. In the first set of experiments, a dramatic change of electrical properties of Alamethicin under microgravity could be observed. Mainly, the pore frequency is significantly reduced.     

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity (microg) may modulate the proliferation and differentiation. We investigated the application of microg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated microg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated microg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.     

Early renal changes in 45 degrees HDT rats

Background: Both microgravity and simulated microgravity models, such as the 45HDT (45 degrees head-down tilt), cause a redistribution of body fluids indicating a possible adaptive process to the microgravity stressor. Understanding the physiological processes that occur in microgravity is a first step to developing countermeasures to stop its harmful effects, i.e., (edema, motion sickness) during long-term space flights. Hypothesis: Because of the kidneys' functional role in the regulation of fluid volume in the body, it plays a key role in the body's adaptation to microgravity. Methods: Rats were injected intramuscularly with a radioactive tracer and then lightly anesthetized in order to facilitate their placement in the 45HDT position. They were then placed in the 45HDT position using a specially designed ramp (45HDT group) or prone position (control group) for an experimental time period of 1 h. During this period, the 99mTc-DTPA (technetium-labeled diethylenepentaacetate, MW=492 amu, physical half-life of 6.02 h) radioactive tracer clearance rate was determined by measuring gamma counts per minute. The kidneys were then fixed and sectioned for electron microscopy. A point counting method was used to quantitate intracellular spaces of the kidney proximal tubules. Results: 45HDT animals show a significantly (p=0.0001) increased area in the interstitial space of the proximal tubules. Conclusions: There are significant changes in the kidneys during a 1 h exposure to a simulated microgravity environment that consist primarily of anatomical alterations in the kidney proximal tubules. The kidneys also appear to respond differently to the initial periods of head-down tilt.     

The effects of low-shear mechanical stress on Yersinia pestis virulence

Manned space exploration has created a need to evaluate the effects of spacelike stress on pathogenic and opportunistic microbes astronauts could carry with them to the International Space Station and beyond. Yersinia pestis (YP) causes bubonic, septicemic, and pneumonic plague and is capable of killing infected patients within 3-7 days. In this study, low-shear modeled microgravity (LSMMG), a spacelike stress, was used to physically stress YP; and its effects on proliferation, cold growth, and type III secretion system (T3SS) function were evaluated. YP was grown to saturation in either LSMMG or normal gravity (NG) conditions prior to being used for RAW 246.7 cell infections, HeLa cell infections, and Yop secretion assays. A mutant strain of YP (ΔyopB) that lacks the ability to inject Yersinia outer membrane proteins (Yops) into the host cell was used as a negative control in cell infection experiments. Our experimental results indicate that YP cultivated under LSMMG resulted in reduced YopM production and secretion compared to its NG-grown counterpart. Similarly, NG-grown YP induced more cell rounding in HeLa cells than did the LSMMG-grown YP, which suggests that LSMMG somehow impairs T3SS optimum function. Also, LSMMG-grown YP used to infect cultured RAW 246.7 cells showed a similar pattern of dysfunction in that it proliferated less than did its NG-grown counterpart during an 8-hour infection period. This study suggests that LSMMG can attenuate bacterial virulence contrary to previously published data that have demonstrated LSMMG-induced hypervirulence of other Gram-negative enterics.     

Responses of haloarchaea to simulated microgravity

Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites).     

Parabolic maneuvers of the Swiss Air Force fighter jet F-5E as a research platform for cell culture experiments in microgravity

Long-term sensitivity of human cells to reduced gravity has been supposed since the first Apollo missions and was demonstrated during several space missions in the past. However, little information is available on primary and rapid gravi-responsive elements in mammalian cells. In search of rapid-responsive molecular alterations in mammalian cells, short-term microgravity provided by parabolic flight maneuvers is an ideal way to elucidate such initial and primary effects. Modern biomedical research at the cellular and molecular level requires frequent repetition of experiments that are usually performed in sequences of experiments and analyses. Therefore, a research platform on Earth providing frequent, easy and repeated access to real microgravity for cell culture experiments is strongly desired. For this reason, we developed a research platform onboard the military fighter jet aircraft Northrop F-5E “Tiger II”. The experimental system consists of a programmable and automatically operated system composed of six individual experiment modules, placed in the front compartment, which work completely independent of the aircraft systems. Signal transduction pathways in cultured human cells can be investigated after the addition of an activator solution at the onset of microgravity and a fixative or lysis buffer after termination of microgravity. Before the beginning of a regular military training flight, a parabolic maneuver was executed. After a 1 g control phase, the parabolic maneuver starts at 13,000 ft and at Mach 0.99 airspeed, where a 22 s climb with an acceleration of 2.5g is initiated, following a free-fall ballistic Keplerian trajectory lasting 45 s with an apogee of 27,000 ft at Mach 0.4 airspeed. Temperature, pressure and acceleration are monitored constantly during the entire flight. Cells and activator solutions are kept at 37 °C during the entire experiment until the fixative has been added. The parabolic flight profile provides up to 45 s of microgravity at a quality of 0.05g in all axes. Access time is 30 min before take-off; retrieval time is 30 min after landing. We conclude that using military fighter jets for microgravity research is a valuable tool for frequent and repeated cell culture experiments and therefore for state-of-the art method of biomedical research.     

Sleep in space

Manned space flights have shown it is possible to sleep in microgravity. However, some sleep disturbances have been reported which influence performance of the crew and safety of space flight. This paper reviews the main studies of in-flight sleep in animal and man. Most disturbances are related to phase lags due to operational requirements. Factors which can disturb in-flight sleep are analysed: environmental factors. Some of them are secondary to space flight ergonomics. Conversely, effects of microgravity on light-dark alternance are less known and lead to interesting problems of fundamental research, psychological factors, especially during long duration flights.     

Short duration microgravity experiments in physical and life sciences during parabolic flights: the first 30 ESA campaigns

Aircraft parabolic flights provide repetitively up to 20 s of reduced gravity during ballistic flight manoeuvres. Parabolic flights are used to conduct short microgravity investigations in Physical and Life Sciences, to test instrumentation and to train astronauts before a space flight. The European Space Agency (ESA) has organized since 1984 thirty parabolic flight campaigns for microgravity research experiments utilizing six different airplanes. More than 360 experiments were successfully conducted during more than 2800 parabolas, representing a cumulated weightlessness time of 15 h 30 m. This paper presents the short duration microgravity research programme of ESA. The experiments conducted during these campaigns are summarized, and the different airplanes used by ESA are shortly presented. The technical capabilities of the Airbus A300 'Zero-G' are addressed. Some Physical Science, Technology and Life Science experiments performed during the last ESA campaigns with the Airbus A300 are presented to show the interest of this unique microgravity research tool to complement, support and prepare orbital microgravity investigations.     

Response and adaptation of bone cells to simulated microgravity

Bone loss induced by microgravity during space flight is one of the most deleterious factors on astronaut’s health and is mainly attributed to an unbalance in the process of bone remodeling. Studies from the space microgravity have demonstrated that the disruption of bone remodeling is associated with the changes of four main functional bone cells, including osteoblast, osteoclast, osteocyte, and mesenchymal stem cells. For the limited availability, expensive costs and confined experiment conditions for conducting space microgravity studies, the mechanism of bone cells response and adaptation to microgravity is still unclear. Therefore, some ground-based simulated microgravity methods have been developed to investigate the bioeffects of microgravity and the mechanisms. Here, based on our studies and others, we review how bone cells (osteoblasts, osteoclasts, osteocytes and mesenchymal stem cells) respond and adapt to simulated microgravity.     

Effects of microgravity and hypergravity on the cell: investigations on Paramecium tetraurelia.

Previous space CYTOS experiments have shown that space flights resulted in an increase in growth of Paramecia cultures. Microgravity is the major factor responsible of this response: indeed the stimulatory effect disappeared in inflight cultures placed on a 1 g centrifuge aboard the Spacelab. On the other hand, exposure to different levels of hypergravity on Earth resulted in an opposite response, i.e. to a reduced cell growth rate. A possible mechanism of microgravity on paramecia is discussed.     

Body orientation and center of mass control in microgravity

The control of the body orientation and the center of mass position with respect to the feet was investigated under normo- and microgravity (space flight Altair), during erect posture and at the end of a forward or backward upper trunk movement.

It was observed that during erect posture, the trunk orientation with respect to the vertical was inclined some 6 ° forward in both subjects under microgravity, whereas it was vertical or slightly backward oriented under normogravity. Under microgravity, on the contrary, the initial position CM changed either backwards or forwards. This result suggests that the inclined trunk posture might be due to misevaluating the vertically under microgravity and that different control mechanisms are involved in orienting the trunk and placing the CM.

It was also noted that the final position of the CM at the end of the movement did not differ markedly between microgravity and normogravity. This result suggests that the kinematic synergies which stabilize the CM during uppertrunk movements may result from an automatic central control which is independent from the gravity constraints.     

Experimental Study of the Structures of Macroparticles in the Dust Plasma under Microgravity Conditions onboard the MirOrbital Complex

The dynamics of formation of the ordered structures of charged macroparticles under microgravity conditions is investigated. The experimental observations of the behavior of an ensemble of macroparticles were carried out onboard the Mirspace station. The analysis and comparison of results of experimental and theoretical investigations allow us to conclude that under microgravity conditions the formation of elongated, ordered structures of macroparticles, charged by solar radiation, is possible.     

Mental representation of spatial cues in microgravity: Writing and drawing tests

Humans have mental representation of their environment based on sensory information and experience. A series of experiments has been designed to allow the identification of disturbances in the mental representation of three-dimensional space during space flight as a consequence of the absence of the gravitational frame of reference. This NASA/ESA-funded research effort includes motor tests complemented by psychophysics measurements, designed to distinguish the effects of cognitive versus perceptual-motor changes due to microgravity exposure. Preliminary results have been obtained during the microgravity phase of parabolic flight. These results indicate that the vertical height of handwritten characters and drawn objects is reduced in microgravity compared to normal gravity, suggesting that the mental representation of the height of objects and the environment change during short-term microgravity. Identifying lasting abnormalities in the mental representation of spatial cues will establish the scientific and technical foundation for development of preflight and in-flight training and rehabilitative schemes, enhancing astronaut performance of perceptual-motor tasks, for example, interaction with robotic systems during exploration-class missions.     

Voluntary head stabilization in space during trunk movements in weightlessness

The ability to voluntarily stabilize the head in space during lateral rhythmic oscillations of the trunk has been investigated during parabolic flights. Five healthy young subjects, who gave informed consent, were examined. The movements were performed with eyes open or eyes closed, either during phases of microgravity or phases of normal gravity. The main result to emerge from this study is that the head may be stabilized in space about the roll axis under microgravity conditions with, as well as without vision, despite the reduction of the vestibular afferent and the muscle proprioceptive inputs. Moreover, the absence of head stabilization about the yaw axis confirms that the degrees of freedom of the neck can be independently controlled, as it was previously shown [1]. These results seem to indicate that voluntary head stabilization does not depend crucially upon static vestibular afferents. Head stabilization in space may be in fact organized on the basis of either dynamic vestibular afferents or a postural body scheme.     

A review of muscle atrophy in microgravity and during prolonged bed rest

With the prospect of long duration space missions in Earth orbit or to Mars, there is a need for adequate information on the physiological adaptations that will occur. One consequence of prolonged exposure to microgravity is muscle atrophy (loss of muscle mass). After a long duration space flight, muscle atrophy along with skeletal calcium loss would affect the capacity of astronauts to re-adapt to gravity on return to Earth. Of importance are any countermeasures which can attenuate the adaptive responses to microgravity. Experimentation is difficult in space with small subject numbers and mission constraints. Prolonged bed rest using healthy volunteers is used as an Earth-based model to simulate the muscle atrophy which occurs in the microgravity environment.     

Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells.

The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985.