Gene expression changes induced by space flight in single-cells of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

This work describes a rare high-throughput evaluation of gene expression changes induced by space flight in a single plant cell. The cell evaluated is the spore of the fern Ceratopteris richardii, which exhibits both perception and response to gravity. cDNA microarray and Q RT-PCR analysis of spores germinating in microgravity onboard NASA space shuttle flight STS-93 revealed changes in the mRNA expression of roughly 5% of genes analyzed. These gene expression changes were compared with gene expression changes that occur during gravity perception and response in animal cells and multicellular plants. Our data contribute to a better understanding of the impact of space flight conditions, including microgravity, on cellular growth and development, and provide insights into the adaptive strategies of individual cells in response to these conditions.     

Whole blood leukocytes isolation with microfabricated filter for cell analysis

The flow cytometric analysis of leukocytes in whole blood usually requires isolation of leukocytes from other components of whole blood. Density gradient centrifugation and red blood cell lysis are the most commonly used methods to separate leukocytes but come with significant limitations. We report the results of the evaluation of a microfabricated filtration device for blood preparation that separates erythrocytes from leukocytes based on their size and mechanical properties. The microfabricated filter evaluated here requires a rapid and simple procedure and results in high leukocytes recovery without introducing bias among the leukocyte subpopulations. The filter removes erythrocytes, platelets, plasma proteins, and unbound staining reagent. This gentle filtration process produces very clean stained leukocytes for cytometric analysis without any apparent damage to leukocytes.     

Effect of simulated microgravity on human lymphocytes

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

Recording of blood pressure,heart rate and aortic nerve activity during parabolic flight in the rat via radio-telemetry

Exposure to microgravity induces cardiovascular deconditioning characterized by orthostatic hypotension when astronauts return to the earth. In order to understand the mechanism of cardiovascular deconditioning, it is necessary to clarify the changes in hemodynamics and the cardiovascular regulation system over the period of space flight. The telemetry system applied to freely moving animals will be a useful and appropriate technique for this kind of long term study of the cardiovascular system in the conscious animal during space flight. The purpose of the present study is twofold: firstly, to observe the detailed changes of arterial pressure and heart rate (HR) during microgravity elicited by the parabolic flight in order to study the acute effect of microgravity exposure on the cardiovascular system; and secondly, to test the feasibility of the telemetry system for recording blood pressure, HR and autonomic nervous activities continuously during space flight.     

Changes in hemodynamic and post-flights orthostatic tolerance of cosmonauts under application of the preventive device--thigh cuffs bracelets in short-term flights

Specific aims: to evaluate the influence of the use thigh cuffs "Bracelet" on the hemodynamic adaptation to microgravity during short-term (up to a month) space flights, in-flight tolerance to LBNP-tests and post-flight orthostatic tolerance. 6 cosmonauts applied and 7 others did not apply the occlusive cuffs when on flight. The "Bracelet" device notably relieved the cosmonauts from the subjective discomfort following by the blood redistribution at initial period of exposure to microgravity. It was established that "Bracelet" lessened shifts in central and peripheral hemodynamics typical for exposure to microgravity, venous stasis in the cervical-cephalic region in particular. There were no differences between the hemodynamic reaction on LBNP-test in cosmonauts who applied and not applied "Bracelet" during short-term flights. The objective data are received, that the application of the device during short-term space flight does not make negative effects on post-flight orthostatic tolerance.     

Shift in arm-pointing movements during gravity changes produced by aircraft parabolic flight.

It has been shown that target-pointing arm movements without visual feedback shift downward in space microgravity and upward in centrifuge hypergravity. Under gravity changes in aircraft parabolic flight, however, arm movements have been reported shifting upward in hypergravity as well, but a downward shift under microgravity is contradicted. In order to explain this discrepancy, we reexamined the pointing movements using an experimental design which was different from prior ones. Arm-pointing movements were measured by goniometry around the shoulder joint of subjects with and without eyes closed or with a weight in the hand, during hyper- and microgravity in parabolic flight. Subjects were fastened securely to the seat with the neck fixed and the elbow maintained in an extended position, and the eyes were kept closed for a period of time before each episode of parabolic flight. Under these new conditions, the arm consistently shifted downward during microgravity and mostly upward during hypergravity, as expected. We concluded that arm-pointing deviation induced by parabolic flight could be also be valid for studying the mechanism underlying disorientation under varying gravity conditions.     

Changes in gene expression and signal transduction in microgravity

Studies from space flights over the past three decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. This laboratory has recently studied gene growth and activation of normal osteoblasts (MC3T3-El) during spaceflight. Osteoblast cells were grown on glass coverslips and loaded in the Biorack plunger boxes. The osteoblasts were launched in a serum deprived state, activated in microgravity and collected in microgravity. The osteoblasts were examined for changes in gene expression and signal transduction. Approximately one day after growth activation significant changes were observed in gene expression in 0-G flight samples. Immediate early growth genes/growth factors cox-2, c-myc, bcl2, TGF beta1, bFGF and PCNA showed a significant diminished mRNA induction in microgravity FCS activated cells when compared to ground and 1-G flight controls. Cox-1 was not detected in any of the samples. There were no significant differences in the expression of reference gene mRNA between the ground, 0-G and 1-G samples. The data suggest that quiescent osteoblasts are slower to enter the cell cycle in microgravity and that the lack of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-G. Here we examine ground-based and space flown data to help us understand the mechanism of bone loss in microgravity.     

Gravisensitivity of endothelial cells: the role of cytoskeleton and adhesion molecules

The review addresses the effect of microgravity on the endothelial cells, an important mechanosensory element of the cardiovascular system that is known to undergo functional changes in space flight. The chalanges that arise in performing space flight experiments are presented, as well as approaches used to simulate microgravity effects in vitro. The role of cytoskeletal elements as the putative gravity sensors in the cells is demonstrated. The changes in the expression of adhesion molecules that may underlie the mechanisms of gravity sensing by endothelial cells are described. The possible reasons for the discrepancies between the results obtained, such as the differences between the cell lines and experimental design, the variation in time of cultivation, and the specific spaceflight related factors, are analyzed.     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight

The "starch‐statolith" hypothesis has been used by plant physiologists to explain the gravity perception mechanism in higher plants. In order to help resolve some of the controversy associated with ground‐based research that has supported this theory, we performed a spaceflight experiment during the January 1997 mission of the Space Shuttle STS‐81. Seedlings of wild‐type (WT) Arabidopsis , two reduced‐starch strains, and a starchless mutant were grown in microgravity and then given a gravity stimulus on a centrifuge. In terms of development in space, germination was greater than 90% for seeds in microgravity, and flight seedlings were smaller (60% in total length) compared to control plants grown on the ground and to control plants on a rotating clinostat. Seedlings grown in space had two structural features that distinguished them from the controls: a greater density of root hairs and an anomalous hypocotyl hook structure. However, the slower growth and morphological changes observed in the flight seedlings may be due to the effects of ethylene present in the spacecraft. Nevertheless, during the flight, hypocotyls of WT seedlings responded to a unilateral 60‐min stimulus provided by a 1‐ g centrifuge while those of the starch‐deficient strains did not. Thus, the strain with the greatest amount of starch responded to the stimulus given in‐flight, and, therefore, these data support the starch‐statolith model for gravity sensing.     

"Critical periods" in vestibular development or adaptation of gravity sensory systems to altered gravitational conditions?

1. A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Modification of environmental conditions during this specific period of life affects development in a long-term manner, or even irreversibly. Deprivation is the prefered approach to study the existence and duration of critical periods. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. 2. Studies in a fish (Oreochromis mossambicus) and an amphibian (Xenopus laevis) revealed a significant sensitivity of their roll-induced static vestibuloocular reflex (rVOR) to a 9- to 10-day gravity deprivation (microgravity) during a spaceflight. In some instances, the rVOR was augmented after the flight as demonstrated in young Oreochromis which were launched when their rVOR had not been developed, and in Xenopus tadpoles launched after their rVOR had developed. Fish which could perform the rVOR at launch were insensitive to microgravity exposure. A similar insensitivity to microgravity was observed in Xenopus tadpoles with normal body shape which had not yet developed their rVOR at launch. Some tadpoles, however, developed an upward bended tail during their space flight; their rVOR was significantly depressed after termination of microgravity independent of the age at onset of the flight. Hypergravity depressed the rVOR for all so far tested developmental stages in both Oreochromis and Xenopus. 3. Both adaptive processes during exposure to altered gravity as well as the existence of a critical period in vestibular development might be responsible for the modulation of the rVOR recorded after exposure to altered gravity. Deprivation studies have to be extended to older developmental stages to test the possibility of a critical period; however, this approach is limited due to the low number of space flights.     

Evidence of short-term adaptation to microgravity of neuromuscular synergy during a whole body movement

Human, like any other animal systems, moves in the terrestrial gravity field and must learn gravity-related motor strategies during his ontogenetic development. Considering continuous gravity action upon body segments, movement involves particular muscle activation patterns depending on body orientation to gravity. Gravitational-altered environments provided by parabolic flight or orbital space mission offer a great opportunity to investigate how gravity is taken into account in posture and movement planning. Indeed, in a context where the mechanical constraints are modified, movement execution involves that new muscular activity have to be produced. Almost, only few studies in microgravity environment are included electromyographic analysis and this parameter is generally used only to confirm modification of the muscular activation patterns. This study is focused to analyse the adaptation capacity of the brain to a modified gravitational environment. In this aim, EMG activity have been recorded during a whole body movement execution in both normo- and microgravity environment during parabolic flight. This procedure allowed us to analyse the EMG patterns recorded during the very first moments of weightlessness. In this study are reported the results of this analyse.     

Artificial gravity in space and in medical research

The history of manned space flight has repeatedly documented the fact that prolonged sojourn in space causes physiological deconditioning. Physiological deterioration has raised a legitimate concern about man's ability to adequately perform in the course of long missions and even the possibility of leading to circumstances threatening survival. One of the possible countermeasures of physiological deconditioning, theoretically more complete than others presently used since it affects all bodily systems, is artificial gravity. Space stations and spacecrafts can be equipped with artificial gravity, but is artificial gravity necessary? The term "necessary" must be qualified because a meaningful answer to the question depends entirely on further defining the purpose of space travel. If man intends to stay only temporarily in space, then he must keep himself in good physical condition so as to be able to return to earth or to land on any other planetary surface without undue exposure to major physiological problems resulting from transition through variable gravitational fields. Such a situation makes artificial gravity highly desirable, although perhaps not absolutely necessary in the case of relative short exposure to microgravity, but certainly necessary in interplanetary flight and planetary landings. If the intent is to remain indefinitely in space, to colonize space, then artificial gravity may not be necessary, but in this case the consequences of long term effects of adaptation to weightlessness will have to be weighed against the biological evolutionary outcomes that are to be expected. At the moment, plans for establishing permanent colonies in space seem still remote. More likely, the initial phase of exploration of the uncharted solar system will take place through successive, scope limited, research ventures ending with return to earth. This will require man to be ready to operate in gravitational fields of variable intensity. Equipping spacecrafts or space stations with some means of artificial gravity in this initial phase is, therefore, necessary without question. In a strict sense artificial gravity is conceived as a means of replacing natural gravity in space by the centripetal acceleration generated by some sort of rotating device. Rotating devices create an inertial force which has effects on bodies similar to those caused by terrestrial gravity, but artificial gravity by a rotation device is not the same as terrestrial gravity, as we shall see. Present research in artificial gravity for space exploration is projected in two main directions: artificial gravity for whole space stations and artificial gravity produced by short arm centrifuges designed for human use in space.     

Development of a noninvasive technique for the measurement of intracranial pressure.

Intracranial pressure (ICP) dynamics are important for understanding adjustments to altered gravity. Previous flight observations document significant facial edema during exposure to microgravity, which suggests that ICP is elevated during microgravity. However, there are no experimental results obtained during space flight, primarily due to the invasiveness of currently available techniques. We have developed and refined a noninvasive technique to measure intracranial pressure noninvasively. The technique is based upon detecting skull movements of a few micrometers in association with altered intracranial pressure. We reported that the PPLL technique has enough sensitivity to detect changes in cranial distance associated with the pulsation of ICP in cadavera. In normal operations, however, we place a transducer on the scalp. Thus, we cannot rule out the possibility that the PPLL technique picks up cutaneous pulsation. The purpose of the present study was therefore to show that the PPLL technique has enough sensitivity to detect changes in cranial distance associated with cardiac cycles in vivo.     

Behavioral reactions of the bat Carollia perspicillata to abrupt changes in gravity.

As part of an ongoing survey of the behavioral responses of vertebrates to abrupt changes in gravity, we report here on the reactions of bats (Carollia perspicillata) exposed to altered gravity during parabolic aircraft flight. In microgravity, mammals typically behave as if they were upside-down and exhibit repetitive righting reflexes, which often lead to long axis rolling. Since bats, however, normally rest upside-down, we hypothesized that they would not roll in microgravity. Only one of three specimens attempted to fly during microgravity. None rolled or performed any righting maneuvers. During periods of microgravity the bats partially extended their forearms but kept their wings folded and parallel to the body. Between parabolas and occasionally during microgravity the bats groomed themselves. Both the extended limbs and autogrooming may be stress responses to the novel stimulus of altered gravity. This is the first behavioral record of Chiroptera in microgravity.     

Antibody binding in altered gravity: implications for immunosorbent assay during space flight

A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.     

Characteristics of adaptation and maladaptation of human cardiovascular system under space flight conditions

The goal of this study was to analyze and generalize hemodynamic data collected over 20 years from 26 cosmonauts, who had flown from 8 to 438 days aboard orbital stations Salut-7 and Mir. This paper describes the results of ultrasonographic studies of the heart and arterial and venous peripheral vessels in different parts of human body as well as the study of venous capacity by occlusion aeroplethysmography. It was established that, at rest, the key hemodynamic parameters (the pumping function of the heart and blood supply of the brain) and integral parameters (blood pressure and heart rate) were best “protected” and remained stable throughout long exposure in microgravity. In the absence of gravitational stimulation, arterial resistance decreased in almost all vascular regions below the heart level; i.e., the antigravity distribution of the vascular tone was gradually lost as unneeded in microgravity. Venous hemodynamics was found to be most sensitive to microgravity: changes in it were expressed earlier and were more pronounced than in the arterial part of the vasculature. The changes included deceleration of venous return, a decrease in the vascular resistance in the lower body, and an increase in the leg’s venous network capacity. The functional test with the lower body’s negative pressure revealed a deterioration of gravity-dependent responses, which increased with an increase in the duration of the space flight. Cardiovascular deconditioning clearly manifested itself after the return to the Earth’s gravity as a decreased g-tolerance during reentry and orthostatic instability in the post-flight period. The results of this study confirmed the multifactorial genesis of orthostatic instability during space flights including blood redistribution, changes in the regulation of vascular tone of arterial and venous vessels in legs, and hypovolemia.     

Noninvasive beat-to-beat stroke volume computation during acute hydrostatic pressure changes in parabolic flight

A three-element model of the cardiovascular system was used to monitor stroke volume (SV) changes during parabolic flight. Aortic blood flow was estimated from continuous arterial finger pressure and SV computed by integrating simulated aortic flow during each systole. SV was significantly higher in microgravity (microgravity) compared to 1 G whereas in hypergravity (hG), SV was significantly lower. Exponential SV transients were observed after the transitions to and from microgravity and the succeeding or preceeding hG phases. These SV transients present different time constants, which reflect two different mechanisms of cardiovascular adaptation to sudden gravitational changes. These results show that beat-to-beat computation of SV provides noninvasive information on circulatory adaptation to acute hydrostatic pressure changes.     

Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions.

Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype.