Effects of space flight on surface marker expression.

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.     

Animal models for the study of the effects of spaceflight on the immune system.

Animal models have been used to determine the effects of spaceflight on the immune system. Rats and rhesus monkeys have been the primary animals used for actual space flight studies, but mice have also been utilized for studies in ground-based models. The primary ground based model used has been hindlimb unloading of rodents, which is similar to the chronic bed-rest model for humans. A variety of immune responses have been shown to be modified when animals are hindlimb unloaded. These results parallel those observed when animals are flown in space. In general, immune responses are depressed in animals maintained in the hindlimb unloading model or flown in space. These results raise the possibility that spaceflight could result in decreased resistance to infection in animals.     

Cellular changes in microgravity and the design of space radiation experiments.

Cell metabolism, secretion and cell-cell interactions can be altered during space flight. Early radiobiology experiments have demonstrated synergistic effects of radiation and microgravity as indicated by increased mutagenesis, increased chromosome aberrations, inhibited development, and retarded growth. Microgravity-induced changes in immune cell functions include reduced blastogenesis and cell-mediated, delayed-type hypersensitivity responses, increased cytokine secretions, but inhibited cytotoxic effects and macrophage differentiation. These effects are important because of the high radiosensitivity of immune cells. It is difficult to compare ground studies with space radiation biology experiments because of the complexity of the space radiation environment, types of radiation damage and repair mechanisms. Altered intracellular functions and molecular mechanisms must be considered in the design and interpretation of space radiation experiments. Critical steps in radiocarcinogenesis could be affected. New cell systems and hardware are needed to determine the biological effectiveness of the low dose rate, isotropic, multispectral space radiation and the potential usefulness of radioprotectants during space flight.     

Influence of different natural physical fields on biological processes.

In space flight conditions gravity, magnetic, and electrical fields as well as ionizing radiation change both in size, and in direction. This causes disruptions in the conduct of some physical processes, chemical reactions, and metabolism in living organisms. In these conditions organisms of different phylogenetic level change their metabolic reactions undergo changes such as disturbances in ionic exchange both in lower and in higher plants, changes in cell morphology for example, gyrosity in Proteus (Proteus vulgaris), spatial disorientation in coleoptiles of Wheat (Triticum aestivum) and Pea (Pisum sativum) seedlings, mutational changes in Crepis (Crepis capillaris) and Arabidopsis (Arabidopsis thaliana) seedling. It has been found that even in the absence of gravity, gravireceptors determining spatial orientation in higher plants under terrestrial conditions are formed in the course of ontogenesis. Under weightlessness this system does not function and spatial orientation is determined by the light flux gradient or by the action of some other factors. Peculiarities of the formation of the gravireceptor apparatus in higher plants, amphibians, fish, and birds under space flight conditions have been observed. It has been found that the system in which responses were accompanied by phase transition have proven to be gravity-sensitive under microgravity conditions. Such reactions include also the process of photosynthesis which is the main energy production process in plants. In view of the established effects of microgravity and different natural physical fields on biological processes, it has been shown that these processes change due to the absence of initially rigid determination. The established biological effect of physical fields influence on biological processes in organisms is the starting point for elucidating the role of gravity and evolutionary development of various organisms on Earth.     

Comparison of hindlimb unloading and partial weight suspension models for spaceflight-type condition induced effects on white blood cells

Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems.     

Alkylating agent (MNU)-induced mutation in space environment.

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.     

The mechanics of air-breathing in anuran larvae: implications to the development of amphibians in microgravity.

Because of their rapid development, amphibians have been important model organisms in studies of how microgravity affects vertebrate growth and differentiation. Both urodele (salamanders) and anuran (frogs and toads) embryos have been raised in orbital flight, the latter several times. The most commonly reported and striking effects of microgravity on tadpoles are not in the vestibular system, as one might suppose, but in their lungs and tails. Pathological changes in these organs disrupt behavior and retard larval growth. What causes malformed (typically lordotic) tadpoles in microgravity is not known, nor have axial pathologies been reported in every flight experiment. Lung pathology, however, has been consistently observed and is understood to result from the failure of the animals to inflate their lungs in a timely and adequate fashion. We suggest that malformities in the axial skeleton of tadpoles raised in microgravity are secondary to problems in respiratory function. We have used high speed videography to investigate how tadpoles breathe air in the 1G environment. The video images reveal alternative species-specific mechanisms, that allow tadpoles to separate air from water in less that 150 ms. We observed nothing in the biomechanics of air-breathing in 1G that would preclude these same mechanisms from working in microgravity. Thus our kinematic results suggest that the failure of tadpoles to inflate their lungs properly in microgravity is due to the tadpoles' inability to locate the air-water interface and not a problem with the inhalation mechanism per se.     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

Effects of gravity perturbation on developing animal systems

Developing systems provide unique opportunities for analyzing the effects of microgravity on animals. Several unusual types of cells as well as various extraordinary cellular behavior patterns characterize the embryos of most animals. Those features have been exploited as test systems for space flight. The data from previous experiments are reviewed, and considerations for the design of future experiments are presented.     

How human sleep in space--investigations during space flights.

Sleep problems have been observed during many of the space flights. The existence of poor quality of sleep, fatigue, insomnia or different alterations in sleep structure, organization and sleep cyclicity have been established. Nevertheless results obtained from investigations of human sleep on board manned space vehicles show that it is possible to keep sleep patterns related to the restorative and adaptive processes. For the first time in the frame of the "Intercosmos" program a multi-channel system for recording and analysis of sleep in space was constructed by scientists of the Bulgarian Academy of Sciences and was installed on board the manned Mir orbiting station. In 1988 during the joint Bulgarian-Russian space flight continues recording of electro-physiological parameters necessary to estimate the sleep stages and sleep organization was made. These investigations were continued in next space flights of different prolongation. The results were compared with the findings obtained under the conditions during the pre- and post-flight periods.     

The biological clock of Neurospora in a microgravity environment.

The circadian rhythm of conidiation in Neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). An experiment was conducted on space shuttle flight STS-9 in order to test this hypothesis; during the first 7-8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscillation, an increase in the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of flight, the tubes were exposed to light while their growth fronts were marked. Some aspect of the marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. These results from the last 86 hours of flight demonstrated that the rhythm can persist in space. Since the aberrant rhythmicity occurred prior to the marking procedure, but not after, it was hypothesized that the damping on STS-9 may have resulted from the hypergravity pulse of launch. To test this hypothesis, we conducted investigations into the effects of altered gravitational forces on conidiation. Exposure to hypergravity (via centrifugation), simulated microgravity (via the use of a clinostat) and altered orientations (via alterations in the vector of a 1 g force) were used to examine the effects of gravity upon the circadian rhythm of conidiation.     

微重力下胶原蛋白纤维化及羟基磷灰石结晶的初步研究

在长期空间飞行过程中, 骨质丢失是一个严重问题. 羟基磷灰石(HAP)晶体是骨骼的主要成分, 骨骼中的胶原蛋白纤维在HAP生长结晶过程中起到关键作用. 研究了胶原蛋白纤维化过程在模拟微重力和常重力条件下的变化, 对以胶原 蛋白纤维作为模板生长出的HAP晶体形貌进行了观察. 结果表明, 不同浓度胶原蛋白溶液中形成的胶原蛋白纤维, 其内部孔隙数量和尺寸在模拟微重力条件下要明显大于常重力条件下, 胶原蛋白纤维内部孔隙的分布也不同于常重力条 件下的结果. 以模拟微重力条件下形成的胶原蛋白纤维为模板生长出的HAP 晶体主要为立方体状, 而以常重力条件下形成的胶原蛋白纤维为模板生长出的 HAP晶体形貌主要为板状. 该结果有助于未来进一步阐明空间骨质丢失的机理.      

First flight of the ASTROCULTURE (TM) experiment as a part of the U.S. Shuttle/MIR program.

A number of space-based experiments have been conducted to assess the impact of microgravity on plant growth and development. In general, these experiments did not identify any profound impact of microgravity on plant growth and development, though investigations to study seed development have indicated difficulty in plants completing their reproductive cycle. However, it was not clear whether the lack of seed production was due to gravity effects or some other environmental condition prevailing in the unit used for conducting the experiment. The ASTROCULTURE (TM) flight unit contains a totally enclosed plant chamber in which all the critically important environmental conditions are controlled. Normal wheat (Triticum aestivum L.) growth and development in the ASTROCULTURE (TM) flight unit was observed during a ground experiment conducted prior to the space experiment. Subsequent to the ground experiment, the flight unit was transported to MIR by STS-89, as part of the U.S. Shuttle/MIR program, in an attempt to determine if super dwarf wheat plants that were germinated in microgravity would grow normally and produce seeds. The experiment was initiated on-orbit after the flight unit was transferred from the Space Shuttle to MIR. The ASTROCULTURE (TM) flight unit performed nominally for the first 24 hours after the flight unit was activated, and then the unit stopped functioning abruptly. Since it was not possible to return the unit to nominal operation it was decided to terminate the experiment. On return of the flight unit, it was confirmed that the control computer of the ASTROCULTURE (TM) flight unit sustained a radiation hit that affected the control software embedded in the computer. This experience points out that at high orbital inclinations, such as that of MIR and that projected for the International Space Station, the danger of encountering harmful radiation effects are likely unless the electronic components of the flight hardware are resistant to such impacts.     

Physical effects at the cellular level under altered gravity conditions.

Several modifications of differentiated functions of animal cells cultivated in vitro have been reported when cultures have been exposed to increased or decreased inertial acceleration fields by centrifugation, clinorotation, and orbital space flight. Variables modified by clinorotation conditions include inertial acceleration, convection, hydrostatic pressure, sedimentation, and shear stress, which also affect transport processes in the extracellular chemical environment. Autocrine, paracrine and endocrine substances, to which cells are responsive via specific receptors, are usually transported in vitro (and possibly in certain embryos) by convection and in vivo by a circulatory system or ciliary action. Increased inertial acceleration increases convective flow, while microgravity nearly abolishes it. In the latter case the extracellular transport of macromolecules is governed by diffusion. By making certain assumptions it is possible to calculate the Peclet number, the ratio of convective transport to diffusive transport. Some, but not all, responses of cells in vitro to modified inertial environments could be manifestations of modified extracellular convective flow.     

微重力环境中哺乳动物细胞分子生物学效应研究进展

随着载人航天事业的不断发展,空间失重环境引起的航天员健康问题（心血管疾病、免疫抑制、肌肉萎缩、骨质疏松等）日益突出,这已成为人类探索空间的一大阻碍.越来越多的研究关注到微重力条件下机体及细胞的变化.近期的研究表明,在细胞水平上,微重力会引起细胞降解,改变细胞骨架,并造成细胞在分子水平（如细胞增殖、分化、迁移、粘附、信号转导等过程）的一系列改变.本文对微重力条件下免疫细胞、内皮细胞、骨细胞、癌细胞的相关研究进行了归纳总结,研究结果可为微重力条件下机体及相关细胞的研究提供指导,为治疗或缓解微重力条件造成的疾病提供方法和思路.      

Human reproductive issues in space.

Animal studies in space or analogous environments have suggested that there may be problems in the reproductive sphere; such factors might limit mankind's ability to live and work for extended periods of time in microgravity or on non-terrestrial planetary surfaces. A review of reproductive functioning in animal species studied during space flight demonstrated that most species were affected significantly by the absence of gravity and/or the presence of radiation. These two factors induced alterations in normal reproductive functioning independently of, as well as in combination with, each other. Based on animal models, we have identified several potential problem areas regarding human reproductive physiology and functioning in the space environment. While there are no current space flight investigations, the animal studies suggest priorities for future research in human reproduction. Such studies will be critical for the successful colonization of the space frontier.     

Approaches in the determination of plant nutrient uptake and distribution in space flight conditions.

The effective growth and development of vascular plants rely on the adequate availability of water and nutrients. Inefficiency in either the initial absorption, transportation, or distribution of these elements are factors which impinge on plant structure and metabolic integrity. The potential effect of space flight and microgravity conditions on the efficiency of these processes is unclear. Limitations in the available quantity of space-grown plant material and the sensitivity of routine analytical techniques have made an evaluation of these processes impractical. However, the recent introduction of new plant cultivating methodologies supporting the application of radionuclide elements and subsequent autoradiography techniques provides a highly sensitive investigative approach amenable to space flight studies. Experiments involving the use of gel based 'nutrient packs' and the radionuclides calcium-45 and iron-59 were conducted on the Shuttle mission STS-94. Uptake rates of the radionuclides between ground and flight plant material appeared comparable.     

An experimental system for determining the influence of microgravity on B lymphocyte activation and cell fusion.

The influence of microgravity on lymphocyte activation is central to the understanding of immunological function in space. Moreover, the adaptation of groundbased technologies to microgravity conditions presents opportunities for biotechnological applications including high efficiency production of antibody forming hybridomas. Because the emerging technology of microgravity hybridoma generation is dependent upon activation and cultivation of B lymphocytes during flight, we have adapted mitogen-driven B lymphocyte stimulation and culture that allows for the in vitro generation of large numbers of antibody forming cells suitable for cell fusion over a period of 1-2 weeks. We believe that this activation and cultivation system can be flown on near-term space flights to test fundamental hypotheses about mammalian cell activation, cell fusion, metabolism, secretion, growth, and bio-separation.     

Metabolic energy required for flight.

This paper reviews data available from U.S. and U.S.S.R. studies on energy metabolism in the microgravity of space flight. Energy utilization and energy availability in space seem to be similar to those on Earth. However, negative nitrogen balances in space in the presence of adequate energy and protein intakes and in-flight exercise, suggest that lean body mass decreases in space. Metabolic studies during simulated (bed rest) and actual microgravity have shown changes in blood glucose, fatty acids, and insulin levels, suggesting that energy metabolism may be altered during flight. Future research should focus on the interactions of lean body mass, diet, and exercise in space and their roles in energy metabolism during space flight.