Spaceflight Influences both Mucosal and Peripheral Cytokine Production in PTN-Tg and Wild Type Mice

Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-β1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice.     

Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS)

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.     

NASDA aquatic animal experiment facilities for Space Shuttle.

National Space Development Agency of Japan (NASDA) has been developed aquatic animal experiment facilities for space experiments using NASA Space Shuttle. Vestibular Function Experiment Unit (VFEU) has been firstly designed and developed for Spacelab-J mission (STS-47), and 8 days space experiment with carp has been performed. Following, the VFEU, Aquatic Animal Experiment Unit (AAEU) has been developed to accommodate small aquatic animals second International Microgravity Laboratory mission (IML-2, STS-65). Four kinds of space experiments with goldfish, medaka, newt, and newt eggs have been performed for 15 days mission duration. Then, VFEU has been improved to accommodate marine fish under low temperature condition for Neurolab (STS-90) and STS-95 missions. 17 days (STS-90) and 9 days (STS-95) experiments with oyster toadfish have been performed by using the VFEU. This report summarizes the outline of these aquatic animal experiment facilities.     

Evaluation of a Treadmill with Vibration Isolation and Stabilization (TVIS) for use on the International Space Station

A treadmill with vibration isolation and stabilization designed for the International Space Station (ISS) was evaluated during Shuttle mission STS-81. Three crew members ran and walked on the device, which floats freely in zero gravity. For the majority of the more than 2 hours of locomotion studied, the treadmill showed peak to peak linear and angular displacements of less than 2.5 cm and 2.5 degrees, respectively. Vibration transmitted to the vehicle was within the microgravity allocation limits that are defined for the ISS. Refinements to the treadmill and harness system are discussed. This approach to treadmill design offers the possibility of generating 1G-like loads on the lower extremities while preserving the microgravity environment of the ISS for structural safety and vibration free experimental conditions.     

STS-95 Space Experiments (plants and cell biology).

We report the outline of Space Experiments conducted on Space Shuttle (STS-95) launched in autumn of 1998. In this STS-95 mission, Japanese astronaut Dr. Chiaki Mukai achieved her 2nd space flight and conducted a part of 82 space experiments including Japanese experiments. US astronaut Senator John Glenn also achieved his second space flight, 36 years after his first space flight. Senator Glenn was a leader of the original (the first) 7 US astronauts and very famous in US because he succeeded US first orbital space flight around the earth. NASDA had started the project of space experiment using STS-95 at the summer of 1997, therefore we had only one year for the all preparation Yamashita, et al. Biological Sciences in Space, Vol.12 No.3(1998). Scientific results will be reported by investigators, therefore we report here how we had been developing the space experiment plan, on board operation procedure and ground operations including ground control experiments about four plant experiments and one cell biology experiment.     

Mice Drawer System: phase c/d development and perspective

MDS (Mice Drawer System) is the Facility that will support the research on board the International Space Station (ISS). Funded by ASI with Laben as industrial Prime Contractor, MDS will also permit its utilisation for other research programs with mice. The most attractive feature of MDS is that six mice can he kept separate inside dedicated and isolated cages. This permits to perform six experiments in parallel, one for each mouse. In the first utilisation flight of MDS facility the selected experiment is devoted to study human bone formation and specific countermeasures to prevent osteoporosis using mice under microgravity conditions as a model.     

BIOPACK: the ground controlled late access biological research facility

Future Space Shuttle flights shall be characterized by activities necessary to further build the International Space Station, ISS. During these missions limited resources are available to conduct biological experiments in space. The Shuttles' Middeck is a very suitable place to conduct science during the ISS assembly missions or dedicated science missions. The BIOPACK, which flew its first mission during the STS-107, provides a versatile Middeck Locker based research tool for gravitational biology studies. The core facility occupies the space of only two Middeck Lockers. Experiment temperatures are controlled for bacteria, plant, invertebrate and mammalian cultures. Gravity levels and profiles can be set ranging from 0 to 2.0 x g on three independent centrifuges. This provides the experimenter with a 1.0 x g on-board reference and intermediate hypogravity and hypergravity data points to investigate e.g. threshold levels in biological responses. Temperature sensitive items can be stored in the facilities' -10 degrees C and +4 degrees C stowage areas. During STS-107 the facility also included a small glovebox (GBX) and passive temperature controlled units (PTCU). The GBX provides the experimenter with two extra levels of containment for safe sample handling. This biological research facility is a late access (L-10 hrs) laboratory, which, when reaching orbit, could automatically be starting up reducing important experiment lag-time and valuable crew time. The system is completely telecommanded when needed. During flight system parameters like temperatures, centrifuge speeds, experiment commanding or sensor readouts can be monitored and changed when needed. Although ISS provides a wide range of research facilities there is still need for an STS-based late access facility such as the BIOPACK providing experimenters with a very versatile research cabinet for biological experiments under microgravity and in-flight control conditions.     

Activation of nervous system development genes in bone marrow derived mesenchymal stem cells following spaceflight exposure

Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity‐induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA‐8 + Increment 13) from March 30 to April 8, 2006 (experiment “Stroma‐2”). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo‐inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell‐cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC. J. Cell. Biochem. 111: 442–452, 2010. © 2010 Wiley‐Liss, Inc.     

VFEU water quality control in STS-95 mission.

In STS-95 Space Shuttle mission, an aquatic animal research facility, Vestibular Function Experiment Unit (VFEU), was flown to perform neurobiological experiment with marine fish, oyster toadfish. For this purpose, we have developed a sea water purification system using highly active nitrifying bacteria at low temperature. With this system, the water quality in the VFEU was maintained in sufficient condition to keep the toadfish in healthy state for 9 days of the mission. This report summarizes the efficiency of the filter system based on the results from pre-flight bacterial preparation, water analysis of samples taken during flight, and the post-flight analysis of the bacterial filter.     

Embryogenesis and swimming behavior of Medaka fish in JUSTSAP STARS Program (STS-107).

JUSTSAP (Japan-US Science, Technology and Space Application Program) Medaka fish experiment was carried out as a part of STARS (Space Technology and Research for Student) experiment, a space shuttle mission, STS-107 in January 2003. Four eggs laid on earth under artificially controlled environment were put in a closed ecological system, AHAB (Aquatic Habitat), and launched by Space Shuttle Columbia. For the control experiment, four eggs were put in the AHAB and remained on the ground. There was no remarkable difference in the time course of the development. In ground experiment embryos were observed to rotate in the egg membrane, whereas in flight unit they did not rotate. One egg hatched out on L (Launch) +8 days in flight unit. Four eggs hatched out in ground unit. Fry in flight unit was observed to face its back usually to the camera with little swimming movement. Fry in ground unit were observed to move actively and also to control their posture with respect to gravity vector.     

Space shuttle flight (STS-45) of L8 myoblast cells results in the isolation of a nonfusing cell line variant

Myoblast cell cultures have been widely employed in conventional (1g) studies of biological processes because characteristics of intact muscle can be readily observed in these cultured cells. We decided to investigate the effects of spaceflight on muscle by utilizing a well characterized myoblast cell line (L8 rat myoblasts) as cultured in the recently designed Space Tissue Loss Flight Module “A” (STL-A). The STL-A is a “state of the art,” compact, fully contained, automated cell culture apparatus which replaces a single mid-deck locker on the Space Shuttle. The L8 cells were successfully flown in the STL-A on the Space Shuttle STS-45 mission. Upon return to earth, reculturing of these spaceflown L8 cells (L8SF) resulted in their unexpected failure to fuse and differentiate into myotubes. This inability of the L8SF cells to fuse was found to be a permanent phenotypic alteration. Scanning electron microscopic examination of L8SF cells growing at 1g on fibronectin-coated polypropylene fibers exhibited a strikingly different morphology as compared to control cells. In addition to their failure to fuse into myotubes, L8SF cells also piled up on top of each other. When assayed in fusion-promoting soft agar, L8SF cells gave rise to substantially more and larger colonies than did either preflight (L8AT) or ground control (L8GC) cells. All data to this point indicate that flying L8 rat myoblasts on the Space Shuttle for a duration of 7–10 d at subconfluent densities results in several permanent phenotypic alterations in these cells. © 1994 Wiley-Liss, Inc.     

Biological and metabolic response in STS-135 space-flown mouse skin

Abstract

There is evidence that space flight condition-induced biological damage is associated with increased oxidative stress and extracellular matrix (ECM) remodeling. To explore possible mechanisms, changes in gene expression profiles implicated in oxidative stress and in ECM remodeling in mouse skin were examined after space flight. The metabolic effects of space flight in skin tissues were also characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Female C57BL/6 mice were flown in the STS-135 using animal enclosure modules (AEMs). Within 3–5 h after landing, the mice were euthanized and skin samples were harvested for gene array analysis and metabolic biochemical assays. Many genes responsible for regulating production and metabolism of reactive oxygen species (ROS) were significantly (p < 0.05) altered in the flight group, with fold changes >1.5 compared to AEM control. For ECM profile, several genes encoding matrix and metalloproteinases involved in ECM remodeling were significantly up-/down-regulated following space flight. To characterize the metabolic effects of space flight, global biochemical profiles were evaluated. Of 332 named biochemicals, 19 differed significantly (p < 0.05) between space flight skin samples and AEM ground controls, with 12 up-regulated and 7 down-regulated including altered amino acid, carbohydrate metabolism, cell signaling, and transmethylation pathways. Collectively, the data demonstrated that space flight condition leads to a shift in biological and metabolic homeostasis as the consequence of increased regulation in cellular antioxidants, ROS production, and tissue remodeling. This indicates that astronauts may be at increased risk for pathophysiologic damage or carcinogenesis in cutaneous tissue.     

Lipid peroxidation rate and the antioxidant protection system during the readaptation period after long-term space flights on board the International Space Station

The content of lipid peroxidation (LPO) products (diene conjugates (DC), malondialdehyde (MDA), Schiff bases (SB), and tocopherol (TP, a main lipid antioxidant) were measured in blood serum of 17 astronauts taking part in long-term (125–217 days) missions on board the International Space Station (ISS) during the preflight period, on the day of the landing, and on the 7th and 14th days after landing (the rehabilitation period, RP). A decrease in the DC and MDA levels against a background of an increase in TP has been found in a group of eight astronauts after landing on board the Space Shuttle spacecraft and a group of eight astronauts after a space flight on board the Soyuz TM in the course of RP. The changes in measured indices were more pronounced in the group of astronauts after the space flight on board the Space Shuttle spacecraft. Inhibition of LPO during RP was regarded as an adequate response to readaptation stress to the conditions on earth. The possible mechanisms of differences in the efficiency of LPO inhibition between groups are discussed: the changes in the biomembrane phase state under the conditions of deceleration load during disorbiting and the stressful reaction to landing on board different spacecrafts.     

Shuttle radiation dose measurements in the International Space Station orbits.

The International Space Station (ISS) is now a reality with the start of a permanent human presence on board. Radiation presents a serious risk to the health and safety of the astronauts, and there is a clear requirement for estimating their exposures prior to and after flights. Predictions of the dose rate at times other than solar minimum or solar maximum have not been possible, because there has been no method to calculate the trapped-particle spectrum at intermediate times. Over the last few years, a tissue-equivalent proportional counter (TEPC) has been flown at a fixed mid-deck location on board the Space Shuttle in 51.65 degrees inclination flights. These flights have provided data that cover the expected changes in the dose rates due to changes in altitude and changes in solar activity from the solar minimum to the solar maximum of the current 23rd solar cycle. Based on these data, a simple function of the solar deceleration potential has been derived that can be used to predict the galactic cosmic radiation (GCR) dose rates to within +/-10%. For altitudes to be covered by the ISS, the dose rate due to the trapped particles is found to be a power-law function, rho(-2/3), of the atmospheric density, rho. This relationship can be used to predict trapped dose rates inside these spacecraft to +/-10% throughout the solar cycle. Thus, given the shielding distribution for a location inside the Space Shuttle or inside an ISS module, this approach can be used to predict the combined GCR + trapped dose rate to better than +/-15% for quiet solar conditions.     

Inertial shear forces and the use of centrifuges in gravity research. What is the proper control?

Centrifuges are used for 1 x g controls in space flight microgravity experiments and in ground based research. Using centrifugation as a tool to generate an Earth like acceleration introduces unwanted inertial shear forces to the sample. Depending on the centrifuge and the geometry of the experiment hardware used these shear forces contribute significantly to the total force acting on the cells or tissues. The inertial shear force artifact should be dealt with for future experiment hardware development for Shuttle and the International Space Station (ISS) as well as for the interpretation of previous space-flight and on-ground research data.     

The effect of space flight on the production of actinomycin D by Streptomyces plicatus

The effect of space flight on production of the antibiotic actinomycin D by Streptomyces plicatus WC56452 was examined onboard the US Space Shuttle mission STS-80. Paired space flight and ground control samples were similarly prepared using identical hardware, media, and inoculum. The cultures were grown in defined and complex media under dark, anaerobic, thermally controlled (20°C) conditions with samples fixed after 7 and 12 days in orbit, and viable residuals maintained through landing at 17 days, 15 h. Postflight analyses indicated that space flight had reduced the colony-forming unit (CFU) per milliliter count of S. plicatus and increased the specific productivity (pg CFU⁻¹) of actinomycin D. The antibiotic compound itself was not affected, but its production time course was altered in space. Viable flight samples also maintained their sporulation ability when plated on agar medium postflight, while the residual ground controls did not sporulate. Received 21 August 2001/ Accepted in revised form 30 July 2002     

Centrifuges and inertial shear forces

Centrifuges are often used in biological studies for 1 x g control samples in space flight microgravity experiments as well as in ground based research. Using centrifugation as a tool to generate an Earth like acceleration introduces unwanted inertial shear forces to the sample. Depending on the centrifuge and the geometry of the experiment hardware used these shear forces may contribute as much as 99% to the total force acting on the cells or tissues. The inertial shear force artifact should be dealt with for future experiment hardware development for Shuttle and the International Space Station (ISS) as well as for the interpretation of previous spaceflight and on-ground research data.     

Development of the brine shrimp Artemia is accelerated during spaceflight

Developmentally arrested brine shrimp cysts have been reactivated during orbital spaceflight on two different Space Shuttle missions (STS-50 and STS-54), and their subsequent development has been compared with that of simultaneously reactivated ground controls. Flight and control brine shrimp do not significantly differ with respect to hatching rates or larval morphology at the scanning and transmission EM levels. A small percentage of the flight larvae had defective nauplier eye development, but the observation was not statistically significant. However, in three different experiments on two different flights, involving a total of 232 larvae that developed in space, a highly significant difference in degree of flight to control development was found. By as early as 2.25 days after reactivation of development, spaceflight brine shrimp were accelerated, by a full instar, over ground control brine shrimp. Although developing more rapidly, flight shrimp grew as long as control shrimp at each developmental instar or stage.     

Mini ecosystem in space--preliminary experiment on board STS-77.

An enclosed ecosystem which is stable on Earth will behave differently when brought into space. Micro-gravity and radiation will affect the dynamics of material circulation or the activities of small creatures of the ecosystem. One series of space experiments aiming to address such issues was planned in the United States (It is termed as ABS--Autonomous Biological System) and Japanese group has been involved with cooperating with the analysis of the flight samples. Before the ecosystem will be on board Russian Space Station "Mir" later 1996 for 3 months, a preliminary flight was carried out in May 1996 on Space Shuttle (STS-77) for 10 days flight. It was the first of such experiments to fly one whole ecosystem in space.     

Crystallization of biological macromolecules from flash frozen samples on the Russian Space Station Mir

One hundred eighty-three flash frozen, liquid-liquid diffusion and batch method protein and virus crystallization samples were launched aboard the Space Shuttle Discovery on June 27 (STS-71) and transferred to the Russian Space Station Mir on July 1, 1995. They were returned to earth November 20, 1995 (STS-74). Subsequent examination showed that of the 19 types of proteins and viruses investigated, 17 were crystallized during the period on Mir. The experiment demonstrates the utility of this very simple and inexpensive approach for the crystallization of biological macromolecules in space over extended time periods. The distribution of crystals among the three types of containers used indicated small samples yielded results equal or better than larger samples and that long diffusion path lengths were clearly better. Distribution of crystals within the container tubes showed a striking gradient of quality and size that indicated long, narrow tubes yield superior crystals, as predicted from other work based on crystallization in capillaries.