The development of spaceflight experiments withArabidopsis as a model system in gravitropism studies

Experiments withArabidopsis have been developed for spaceflight studies in the European Space Agency's Blorack module. The Biorack is a multiuser facility that is flown on the United States Space Shuttle and serves as a small laboratory for studying cell and developmental biology in unicells, plants, and small invertebrates. The purpose of our spaceflight research was to investigate the starch-statolith model for gravity perception by studying wild-type (WT) and three starch-deficient mutants ofArabidopsis. Since spaceflight opportunities for biological experimentation are scarce, the extensive ground-based testing described in this paper is needed to ensure the success of a flight project. Therefore, the specific aims of our ground-based research were: (1) to modify the internal configuration of the flight hardware, which originally was designed for large lentil seeds, to accommodate smallArabidopsis seeds; (2) to maximize seed germination in the hardware; and (3) to develop favorable conditions in flight hardware for the growth and gravitropism of seedlings. The hardware has been modified, and growth conditions forArabidopsis have been optimized. These experiments were successfully flown on two Space Shuttle missions in 1997.     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

Spaceflight and development of immune responses

The NIH.R1 Space Shuttle experiment wasdesigned to study the effects of spaceflight on rodent development.Pregnant rats were flown on the Space Shuttle for 11 days, and pregnantcontrol rats were maintained in animal enclosure modules in aground-based chamber under conditions approximating those in flight.Additional controls were in standard housing. The effects of the flighton immunological parameters of dams, fetuses, and pups were determined. Blastogenesis of spleen cells in response to mitogen was inhibited inflown dams but was not inhibited in cells from their pups. Interferon- production by spleen cells showed a trend toward inhibition in flown dams but not in their pups. The response of bonemarrow cells to colony-stimulating factor showed a trend towardinhibition after spaceflight in dams, but the response of fetus and pupliver cells was not inhibited. Total serum IgG was not affected byspaceflight. None of the examined immune parameters that were alteredin rat dams after spaceflight was found to be altered in theiroffspring.

     

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.     

Spaceflight induces changes in splenocyte subpopulations: effectiveness of ground-based models

Spaceflight produces changes in the immune system. The mechanisms for the alterations in immune function after spaceflight remain unclear due in part to the difficulties associated with conducting spaceflight research. The purpose of the following studies, therefore, was to create a ground-based protocol that can reproduce the immunological changes found after spaceflight, i.e., changes in splenic lymphocyte populations. Rats were exposed to either flight aboard the Space Shuttle Endeavor (STS-77) or ground-based simulations of various components of the spaceflight experience. The ground-based mock spaceflight was comprised of exposure to launch and landing loads and unloading of the hindlimbs. In addition, each component of this ground-based mock spaceflight was tested separately. The results were that spaceflight reduced splenic CD4(+) T (helper/inducer) cells and CD11b(+) (neutrophils/macrophages) cells. The ground-based simulations of spaceflight did not reproduce the same pattern of splenocyte changes. In fact, exposure to landing loads alone increased splenic CD4(+) T (helper/inducer) cells. These findings support the conclusion that the ground models tested did not induce similar changes in the immune system as did spaceflight. It is possible, therefore, that stressors/factors unique to the spaceflight experience impact the immune system in ways that cannot be currently, fully modeled on the ground.     

The influence of microgravity and spaceflight on columella cell ultrastructure in starch-deficient mutants of Arabidopsis.

The ultrastructure of root cap columella cells was studied by morphometric analysis in wild-type, a reduced-starch mutant, and a starchless mutant of Arabidopsis grown in microgravity (F-microgravity) and compared to ground 1g (G-1g) and flight 1g (F-1g) controls. Seedlings of the wild-type and reduced-starch mutant that developed during an experiment on the Space Shuttle (both the F-microgravity samples and the F-lg control) exhibited a decreased starch content in comparison to the G-1g control. These results suggest that some factor associated with spaceflight (and not microgravity per se) affects starch metabolism. Elevated levels of ethylene were found during the experiments on the Space Shuttle, and analysis of ground controls with added ethylene demonstrated that this gas was responsible for decreased starch levels in the columella cells. This is the first study to use an on-board centrifuge as a control when quantifying starch in spaceflight-grown plants. Furthermore, our results show that ethylene levels must be carefully considered and controlled when designing experiments with plants for the International Space Station.     

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.     

The BIORACK facility and its performance during the IML-2 Spacelab mission

The configuration and performance of the Biorack facility during the Second International Microgravity Laboratory mission (IML-2; 8-23 July 1995) is described in detail. During this Spacelab mission, Biorack flew with two incubators (22 degrees C and 37 degrees C), glovebox, cooler (5 degrees C) and four passive thermal conditioning units (PTCU; 5 degrees C and 10 degrees C) in the stowage. The crew worked more than 40 h to perform 19 Biorack experiments originating from seven European countries. Almost 200 Biorack experiment containers had to be translocated in about 1500 predetermined steps before the Space Shuttle Columbia returned after nearly 14 days: 18 h or 236 orbits in space to Kennedy Space Center, Florida.     

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.     

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.     

Developmental testing of the Advanced Animal Habitat to determine compatibility with rats and mice

The Research Animal Holding Facility (RAHF) and the Animal Enclosure Module (AEM) have housed rats during Space Shuttle flights since the 1980s, but the operational constraints of the hardware have limited the scientific return from these Shuttle flights. The RAHF provides environmental control and monitoring for 24 rats with in-flight animal access, but it must be flown in the Spacelab. Due to the infrequent availability of Spacelab flights, rodent experiments rely heavily on the AEM. Unfortunately, the AEM supports only six rats, has no environmental control and provides no animal access in flight. The Advanced Animal Habitat (AAH) is being developed to support up to 12 adult rats or 30 adult mice for up to 30 days, provide active temperature control, animal telemetry and on-orbit video, record environmental parameters in the animal cage, and provide in-flight animal access in the Middeck, the Spacelab or the Space Station. To ensure the AAH can meet these requirements, animal testing is being conducted with rats and mice in every step of development. Testing began with the cage configuration.     

Preventing annoyance from odors in spaceflight: a method for evaluating the sensory impact of rodent housing.

For the scientific community, the ability to fly mice under weightless conditions in space offers several advantages over the use of rats. These advantages include the option of testing a range of transgenic animals, the ability to increase the number of animals that can be flown, and reduced demands on shuttle resources (food, water, animal mass) and crew time (for water refill). Mice have been flown in animal enclosure module (AEM) hardware only once [Space Shuttle Transport System (STS)-90] and were dissected early in the mission, whereas rats have been flown in the AEM on >20 missions. This has been due, in part, to concerns that strong and annoying odors from mouse urine (vs. rat urine) will interfere with crew performance in the shuttle middeck. To screen and approve mice for flight, a method was developed to evaluate the odor containment performance of AEMs housing female C57BL/6J mice compared with AEMs housing Sprague-Dawley rats across a 21-day test period. Based on the results of this test, consensus was reached that mice could fly in the AEM hardware for up to 17 days (including prelaunch and contingency) and that the AEM hardware would likely contain odors beyond this duration. Human sensory and electronic nose analysis of the AEMs postflight demonstrated their success in containing odors from mice for the mission duration of STS-108 (13 days). Although this paper focuses specifically on odor evaluations for the space shuttle, the concern is applicable to any confined, closed-system environment for human habitation.     

Plastid Distribution in Columella Cells of a Starchless Arabidopsis Mutant Grown in Microgravity

Wild-type and starchless Arabidopsis thaliana mutant seedlings(TC7) were grown and fixed in the microgravity environment ofa U.S. Space Shuttle spaceflight. Computer image analysis oflongitudinal sections from columella cells suggest a differentplastid positioning mechanism for mutant and wild-type in theabsence of gravity. (Received September 24, 1996; Accepted January 21, 1997)     

The Second Annual Symposium of the NASA Specialized Center of Research and Training (NSCORT) in Gravitational Biology

The second annual meeting of the NSCORT in Gravitational Biology was held at Kansas State University on September 29-October 1, 1992. Symposium presentations at the meeting included ones on basic gravitational cellular and developmental biology, spaceflight hardware for biological studies, studies on Space Shuttle, and special talks on Space Station Freedom and on life support systems.     

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thaliana under Spaceflight Conditions

Successful development of seeds under spaceflight conditionshas been an elusive goal of numerous long-duration experimentswith plants on orbital spacecraft. Because carbohydrate metabolismundergoes changes when plants are grown in microgravity, developingseed storage reserves might be detrimentally affected duringspaceflight. Seed development in Arabidopsis thaliana plantsthat flowered during 11 d in space on shuttle mission STS-68has been investigated in this study. Plants were grown to therosette stage (13 d) on a nutrient agar medium on the groundand loaded into the Plant Growth Unit flight hardware 18 h priorto lift-off. Plants were retrieved 3 h after landing and siliqueswere immediately removed from plants. Young seeds were fixedand processed for microscopic observation. Seeds in both theground control and flight plants are similar in their morphologyand size. The oldest seeds from these plants contain completelydeveloped embryos and seed coats. These embryos developed radicle,hypocotyl, meristematic apical tissue, and differentiated cotyledons.Protoderm, procambium, and primary ground tissue had differentiated.Reserves such as starch and protein were deposited in the embryosduring tissue differentiation. The aleurone layer contains alarge quantity of storage protein and starch grains. A seedcoat developed from integuments of the ovule with gradual changein cell composition and cell material deposition. Carbohydrateswere deposited in outer integument cells especially in the outsidecell walls. Starch grains decreased in number per cell in theintegument during seed coat development. All these characteristicsduring seed development represent normal features in the groundcontrol plants and show that the spaceflight environment doesnot prevent normal development of seeds in Arabidopsis. Arabidopsis ; spaceflight; embryo; endosperm; seed coat; storage reserves     

The Fluid Processing Apparatus: from flight hardware to electron micrographs

Sweet clover seeds were grown in the Fluid Processing Apparatus (FPA) flown on STS-54 (January 1993) and STS-60 (February 1994). After germination and seedling growth for 40 hours, seedlings were fixed in space. Electron microscopy was used to examine the seedling ultrastructure to verify that fixation occurred and preserved the samples. Micrographs revealed well-preserved cell structure and the presence of calcium precipitates indicating complete fixation. FPA is a useful piece of hardware for preservation/fixation during spaceflight.     

Lessons from Immune 1-3: what did we learn and what do we need to do in the future?

Sprague-Dawley rats were subjected to three 8-to-10 day space flights on the Space Shuttle. Housed in NASA's Animal Enclosure Modules, rats were flown to test the hypotheses that therapy with pegylated interleukin-2 or insulin-like growth factor-1 would ameliorate some of the effects of space flight on the immune system. As part of these experiments, we measured body and organ weights, blood cell differentials, plasma corticosterone, macrophage colony forming units, lymphocyte mitogenic, super-antigenic and interferon-gamma responses, bone marrow cell and peritoneal macrophage cytokine secretion and bone strength and mass. This paper compares some of the immunophysiological parameters of the control animals used in the Immune1-3 flight series and presents data from an animal infection model for use during space flight.     

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.     

Changes in Growth,Developmment,and Physiological and Biochemical Activity of Asparagus Seedlings Grown from Space Flown Seeds

Asparagus seeds were sent on board retrievable satellites and they were flown in space for 8 days. Experiments were conducted in fields and laboratory after the seeds returned to the earth. Comparative studies were made on the growth and development patterns of plants growing from space flown seeds and controls kept on the earth. Changes in physiological and biochemical characteristic of the seedlings were also studied. The results obtained are summarized as follows: (1) Space flight markedly raised the germination rate of seeds as compared with the controls kept on the earth. After 5 days of imbibition, 40% of the space flown seeds germinated while the germination rate for ground controls was only 22.5%. After 6 days of imbibition the germination rate of space flown seeds was 65 % and that of ground controls 40%. After 10 days of imbibition, the rates rose to 87. 5% and 72. 5% respectively. The seedlings from space flown seeds grew in fields much faster than ground controls. The yield of tender stems of the former was 34% higher than the latter. (2) An assay on respiration showed that the respiratory intensity of space seedlings was 61% higher than that of ground controls. This indicated that the vigor of seeds enhanced under space conditions, accelerating the germination of seeds and growth of seelings. (3) The proline content of space seedlings was 33% higher than that of the ground controls. At the same time, the permeability of the plasma membrane of the space seedlings was markedly lower than that of the ground controls. The content of aspartic acid in plants grown from space seeds was slightly higher than in ground controls while the content of asparagus was markedly lower.