Studies on clonogenic hemopoietic cells of vertebrate in space: problems and perspectives.

Hemopoietic tissues were studied in vertebrates launched aboard the Soviet (Russian) biosatellites ("Cosmos-1129, 1514, 1667, 1887 and 2044"; "Bion-10 and 11") between 1980 and 1996. In the bone marrow of rats exposed to spaceflight conditions, a statistically significant decrease in cell number was revealed in the progenitor cell compartment accounting for the compensatory response of granulocyte-macrophage (CFU-gm) and erythrocyte lineages (BFU-e and CFU-e) and in the compartment of multipotent hemopoietic stem cells (CFU-s), which is responsible for the permanent renewal of hemopoietic tissue. The number of stromal fibroblastic progenitors (CFC-f) in the bone marrow of these rats was also reduced. Apparently, changes in the hemopoietic stroma damage the hemopoietic microenvironment and, hence, may be responsible for changes observed in the hemopoietic tissue proper. Attempts were made to develop methods for analyzing morphologically indiscernible clonogenic hemopoietic cells of newts, and studies on the effects of spaceflight factors on these cells were performed. The results showed that the numbers of clonogenic cells in newts of the flight group newts were significantly lower than in control newts. The data obtained are used as the basis for formulating the problems to be studied, drawing up a program for further research on the effects of spaceflight factors on stem and other clonogenic hemopoietic cells, and developing new experimental models for analyzing stem cells, the state of the hemopoietic stroma, etc.     

AstroNewt: early development of newt in space.

AstroNewt experiment explores the effects of earth gravity on the early development of Japanese red-bellied newt, Cynops pyrrhogaster. Since female newts keep spermatophore in cloaca, fertilized eggs could be obtained without mating. Fertilization of newt's egg occurs just prior to spawning, so that gonadotrophic cues applied to females in orbit leads to laying eggs fertilized just in space. A property of newt being kept in hibernation at low temperature may be of great help for the space experiment carried out with much limited resources. A general outline of the AstroNewt project is shown here in addition to some technical advances for the development of the project. Experimental schemes of two space experiments (IML-2 in summer 1994 and unmanned SFU at the beginning of 1995) are also shown.     

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.     

Development of human epithelial cell systems for radiation risk assessment.

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.     

Regeneration of organs and tissues in lower vertebrates during and after space flight.

In this paper most important data obtained in studies on the effect of space flight conditions on regeneration in the adult newt are summarized. We demonstrate a phenomenon of synchronization of limb and lens regeneration and increase in its rate during and after space flight. We also describe a peculiarities of cell proliferation in lens, limb and tail regenerates and of the process of minced muscle regeneration.     

Investigations onboard the biosatellite Cosmos-1667

The program of the 7-day flight of the biosatellite Cosmos-1667 launched in July 1985 included experiments on two rhesus monkeys, ten Wistar SPF rats, ten newts, Drosophila flies, maize seedlings, lettuce sprouts, and unicellular organisms - Tetrahymena. The primate study demonstrated that transition to orbital flight was accompanied by a greater excitability of the vestibular apparatus and an increased linear blood flow velocity in the common carotid artery. The rat studies showed that atrophy of antigravity muscles and osteoporosis of limb bones developed even during short-term exposure to microgravity. The experiments on other living systems revealed no microgravity effects on the cell division rate, proliferative activity of cells of regenerating tissues and organs, energy metabolism of developing insects, structure or chemical composition of higher plant seedlings.     

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.     

Effect of the space environment on the induction of DNA-repair related proteins and recovery from radiation damage.

Recovery of bacterial cells from radiation damage and the effects of microgravity were examined in an STS-79 Shuttle/Mir Mission-4 experiment using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space-Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity. No difference was observed for the survival of radiosensitive mutant rec30 cells whether incubated in space or on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, the product of a newly found gene related to the DNA repair mechanism of D. radiodurans, was enhanced under microgravity compared with ground controls.     

The Rhesus monkey as a model for testing the immunological effects of space flight.

The Rhesus monkey has been proposed as a model for the effects of space flight on immunity. In order to determine the feasibility of the use of the Rhesus monkey as a model, we studied the use of Rhesus monkey cells for immunological procedures that have been shown to be affected by space flight in both rodents and humans. We have shown that both lymph node cells and peripheral blood leukocytes can be stained with monoclonal antibodies to detect the following surface markers: CD4, CD-8, Ia and surface immunoglobulin. Also, the level of Ia antigen expression was increased by treatment of the cells with human interferon-gamma. In addition, cells were induced to produce interferons and interleukins. Isolated neutrophils also demonstrated increased oxidative burst. These data indicate that the Rhesus monkey will be a useful model for space flight studies of immunity.     

Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity

The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.     

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.     

The effects of proton radiation on UHMWPE material properties for space flight and medical applications

Ultra High Molecular Weight Polyethylene (UHMWPE) is a polymer widely used as a radiation shielding material in space flight applications and as a bearing material in total joint replacements. As a long chain hydrocarbon based polymer, UHMWPE’s material properties are influenced by radiation exposure, and prior studies show that gamma irradiation is effective for both medical sterilization and increased wear resistance in total joint replacement applications. However, the effects of space flight radiation types and doses on UHMWPE material properties are poorly understood. In this study, three clinically relevant grades of UHMWPE (GUR 1020, GUR 1050, and GUR 1020 blended with Vitamin E) were proton irradiated and tested for differences in material properties. Each of the three types of UHMWPE was irradiated at nominal doses of 0 Gy (control), 5 Gy, 10 Gy, 20 Gy, and 35 Gy. Following irradiation, uniaxial tensile testing and thermal testing using Differential Scanning Calorimetry (DSC) and Dynamic Mechanical Analysis (DMA) were performed. Results show small but significant changes in several material properties between the control (0 Gy) and 35 Gy samples, indicating that proton irradiation could have a effect on the long term performance of UHMWPE in both medical and space flight applications.     

Radiosensibility of higher plant seeds after space flight.

The influence of long-term storage of higher plant seeds under space flight conditions (49 to 827 days) on their radiosensibility was studied in the experiments on the orbital stations Salyut 6 and 7. Short-term storage has been proved to have no effect on radiosensitivity of Crepis capillaris seeds. Only in the case of maximal exposure duration the frequency of chromosome aberrations in post-flight irradiated seeds significantly exceeded the chromosome aberration frequency in the ground-based irradiated control. A statistically significant increase in the number of cells with multiple chromosome aberrations was also observed in this experiment. After gamma-irradiation of Arabidopsis thaliana seeds the germinating ability and survival rate of plants decreased depending on the duration of seed storage. Flight-exposed seeds were more sensitive to irradiations with respect to these parameters. A statistically significant increase in the frequency of recessive lethal mutations was observed only in two experiments of long exposure duration.     

Effect of microgravity on primordial germ cells (PGCs) in silk chicken offspring (Gallus gallus domesticus)

Primordial germ cells (PGCs), precursors of germline cells, display a variety of antigens during their migration to target gonads. Here, we used silk chicken offspring (Gallus gallus domesticus) embryos subjected to space microgravity to investigate the influence of microgravity on PGCs. The ShenZhou-3 unmanned spaceship carried nine fertilized silk chicken eggs, named the flight group, returned to Earth after 7 days space flight. And the control group has the same clan with the flight group. PGCs from flight and control group silk chicken offspring embryos were examined during migration by using two antibodies (2C9 and anti-SSEA-1), in combination with the horseradish peroxidase detection system, and using periodic acid-Schiff’s solution (PAS) reaction. After incubation for about 30 h, SSEA-1 and 2C9 positive cells were detected in the germinal crescent of flight and control group silk chicken offspring embryos. After incubation of eggs for 2–2.5 days, SSEA-1 and 2C9 positive cells were detected in embryonic blood vessels of flight and control group silk chicken offspring embryos. After incubation of eggs for 5.5 days, PGCs in the dorsal mesentery and gonad could also be identified in flight and control group silk chicken offspring embryos by using SSEA-1 and 2C9 antibodies. Based on location and PAS staining, these cells were identified as PGCs. Meanwhile, at the stage of PGCs migration and then becoming established in the germinal ridges, no difference in SSEA-1 or 2C9 staining was detected between female and male PGCs in flight and control group silk chicken offspring embryos. Although there were differences in the profiles of PGC concentration between male and female embryos during the special circulating stage, changing profile of PGCs concentration was similar in same sex between flight and control group offspring embryos. We concluded that there is little effect on PGCs in offspring embryos of microgravity-treated chicken and that PGC development appears to be normal.     

Production and action of cytokines in space.

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.     

Effects of long duration space flight on rice seed (or embryo) radiation sensitivity and element microlocalizations.

In long duration space experiments Rice caryopses and embryos, which are able to remain alive 10 years (or more) and tolerate extreme physical conditions (temperature, few water content) during irradiation and post-irradiation storage, were used (8, 40, 201 and 457 days on board of Salyut 7, 2107 days on LDEF). In certain experiments (Salyut 7), samples were irradiated either before or after the flight. Effects of the flight and radiosensitivity were observed in Rice seedlings cultivated in in vitro conditions. Statistical results indicate an increase in radiosensitivity when irradiations occur before the flight. Microanalyses were made in different parts of one caryopsis and of one embryo, and the results compared with those of control samples. With caryopses and embryos of the same Rice varieties, but from LDEF, we made the same kinds of experiments to compare results.     

The response of endocrine system to stress loads during space flight in human subject.

The responses of endocrine system to the exposure to stress-work load and hormonal changes during oral glucose tolerance tests were studied in the Slovak astronaut before (three weeks before flight), during (on the 4th and the 6th days of space flight), and after space flight (1-3 days and 15-17 days after space flight) on board of space station MIR. Blood samples during the tests were collected via cannula inserted into cubital vein, centrifuged in the special appliance Plasma-03, frozen in Kryogem-03, and at the end of the 8-day space flight transferred to Earth in special container for hormonal analysis. Preflight workload produced an increase of plasma norepinephrine and a moderate elevation of epinephrine levels. Plasma levels of insulin, growth hormone, prolactin and cortisol were not markedly changed immediately or 10 min after the end of work load. The higher increases of plasma growth hormone, prolactin and catecholamine levels were noted after workload during space flight as compared to preflight response. The higher plasma glucose and insulin levels were noted during the oral glucose tolerance test in space flight and also in the post flight period. Plasma epinephrine levels were slightly decreasing during glucose tolerance test; however, plasma norepinephrine levels were not changed. The similar patterns of catecholamine levels during glucose tolerance test were found when compared the preflight, in-flight and post flight values. These data demonstrate the changes of the dynamic responses of endocrine system to stress-work and metabolic loads during space flight in human subject.     

Solar cosmic rays as a specific source of radiation risk during piloted space flight.

Solar cosmic rays present one of several radiation sources that are unique to space flight. Under ground conditions the exposure to individuals has a controlled form and radiation risk occurs as stochastic radiobiological effects. Existence of solar cosmic rays in space leads to a stochastic mode of radiation environment as a result of which any radiobiological consequences of exposure to solar cosmic rays during the flight will be probabilistic values. In this case, the hazard of deterministic effects should also be expressed in radiation risk values. The main deterministic effect under space conditions is radiation sickness. The best dosimetric functional for its analysis is the blood forming organs dose equivalent but not an effective dose. In addition, the repair processes in red bone marrow affect strongly on the manifestation of this pathology and they must be taken into account for radiation risk assessment. A method for taking into account the mentioned above peculiarities for the solar cosmic rays radiation risk assessment during the interplanetary flights is given in the report. It is shown that radiation risk of deterministic effects defined, as the death probability caused by radiation sickness due to acute solar cosmic rays exposure, can be comparable to risk of stochastic effects. Its value decreases strongly because of the fractional mode of exposure during the orbital movement of the spacecraft. On the contrary, during the interplanetary flight, radiation risk of deterministic effects increases significantly because of the residual component of the blood forming organs dose from previous solar proton events. The noted quality of radiation responses must be taken into account for estimating radiation hazard in space.     

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.