Challenges to long-duration space exploration and colonization in microgravity and cosmic radiation environments by humans include poorly understood risks for gastrointestinal function and cancer. Nonetheless, constant remodeling of the intestinal microvasculature is critical for tissue viability, healthy wound healing, and successful prevention or recovery from vascular-mediated inflammatory or ischemic diseases such as cancer. Currently no automated image analysis programs provide quantitative assessments of the complex structure of the mucosal vascular system that are necessary for tracking disease development and tissue recovery. Increasing abnormalities to the microvascular network geometry were therefore mapped with VESsel GENeration Analysis (VESGEN) software from 3D tissue reconstructions of developing intestinal inflammation in a dextran sulfate sodium (DSS) mouse model. By several VESGEN parameters and a novel vascular network linking analysis, inflammation strongly disrupted the regular, lattice-like geometry that defines the normal microvascular network, correlating positively with the increased recruitment of dendritic cells during mucosal defense responses.
Exposure to ionizing radiation may negatively impact skeletal integrity during extended spaceflight missions to the moon, Mars, or near-Earth asteroids. However, our understanding of the effects of radiation on bone is limited when compared to the effects of weightlessness. In addition to microgravity, astronauts will be exposed to space radiation from solar and cosmic sources. Historically, radiation exposure has been shown to damage both osteoblast precursors and local vasculature within the irradiated volume. The resulting suppression of bone formation and a general state of low bone-turnover is thought to be the primary contributor to bone loss and eventual fracture. Recent investigations using mouse models have identified a rapid, but transient, increase in osteoclast activity immediately after irradiation with both spaceflight and clinically-relevant radiation qualities and doses. Together with a chronic suppression of bone formation after radiation exposure, this acute skeletal damage may contribute to long-term deterioration of bone quality, potentially increasing fracture risk. Direct evidence for the damaging effects of radiation on human bone are primarily demonstrated by the increased incidence of fractures at sites that absorb high doses of radiation during cancer therapy: exposures are considerably higher than what could be expected during spaceflight. However, both the rapidity of bone damage and the chronic nature of the changes appear similar between exposure scenarios. This review will outline our current knowledge of space and clinical exploration exposure to ionizing radiation on skeletal health.
One advantage of using cartilage to replace/repair bone is that the implant disappears as bone is formed by endochondral ossification. Previously, we showed that cartilage spheroids, grown in a rotating bioreactor (Synthecon, Inc.) and implanted into a 2 mm skull defect, contributed to healing of the defect. Skulls with or without implants were subjected to microCT scans. Mineralized regions from microCT sections correlated with regions of bone in histological sections of the defect region of demineralized skulls. Recently, sections from microCT scans of live mice were compared to histological sections from the same mice. The area of the defect staining for bone in histological sections of demineralized skulls was the same region shown as mineralized in microCT sections. Defects without implants were not healed. This study demonstrates that microCT scans are an important corollary to histological studies evaluating the use of implants in healing of bony defects.
Although it is well known that radiation causes mutational damage, little is known about the biological effects of long-term exposure to radiation in space. Exposure to radiation can result in serious heritable defects in experimental animals, and in humans, susceptibility to cancer, radiation-sickness, and death at high dosages. It is possible to do ground controlled studies of different types of radiation on experimental animals and to physically measure radiation on the space station or on space probes. However, the actual biological affects of long-term exposure to the full range of space radiation have not been studied, and little information is available about the biological consequences of solar flares. Biological systems are not simply passive recording instruments. They respond differently under different conditions, and thus it is important to be able to collect data from a living animal. There are technical difficulties that restrict the placement of an experimental organism in a space environment for long periods of time, in a manner that allows for the recovery of genetic data. Use of the self-fertilizing hermaphroditic nematode, Caenorhabditis elegans offers potential for the design of a biological dosimeter. In this paper, we describe the advantages of this model system and review the literature of C. elegans in space.
Cells treated with RNAlaterTM have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4oC. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous_ kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlaterTM and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL_ Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant_ software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens. (Grant support to J. Becker: NAG9-1341)
Understanding the changes that occur in living organisms to bring about adaptation to the space environment is essential to support future plans for long-term missions to the Moon and Mars. The European Modular Cultivation System (EMCS), a life science research facility developed by the European Space Agency (ESA), will serve as a habitat for culturing multiple generations of Drosophila melanogaster. Based on the results obtained from previously tested prototype hardware, a new one was developed and designated as Prototype Containers III (PIII). The feasibility of culturing Drosophila specimens in the PIII hardware using the EMCS was tested at NASA Ames Research Center. The objective of the study presented here was to optimize the biocompatibility of the prototype hardware. Fly behavior, humidity levels, and hardware mechanics were examined. Modifications to the hardware were made as required to resolve any suboptimal performance issues. Flies were grown in containers using standard techniques for fly handling and videotaped using the video capabilities of the EMCS Experiment Reference Module (ERM). Offspring were counted and sorted to assess differences in population size and distribution. Preliminary results demonstrate that the ERM video system was successful in imaging the behavioral parameters of the flies. The total number of offspring and the percentage of males and females were similar in Prototype Containers as compared to standard lab containers which were used as controls. From these results, the Prototype hardware appears capable of supporting Drosophila growth. Further studies are underway to evaluate multigenerational growth and molecular indicators of stress.
To efficiently utilize available space in current launch vehicles, experiments that minimize mass and power consumptions, while still meeting NASA's space exploration initiative, are being developed. EMMYS-1, Effects of Microgravity on Model Yeast Specimen, is a yeast experiment designed to return samples grown on-orbit for morphological and molecular analysis. Due to the constraints set by the current payload scenarios, EMMYS—1 is being optimized for storage and operation at ambient for a period of up to 6 months. The purpose of this study was to determine which fixatives and conditions provided the best staining results as well as ability to fix yeast after storage at either 4oC or 25oC for various periods of time. The test parameters used are based on the current hardware configuration and how the fixatives interacted with the hardware. Formaldehyde and paraformaldehyde at different concentrations in either Phosphate Buffered Saline or Cacodylic Acid Buffer were tested. Stains used for the testing were DAPI, Mithramycin, Calcofluor, and Acridine Orange. Yeast were fixed at a ratio of 2 to 1, stained and compared to freshly fixed yeast. To determine effectiveness of the fixatives, viability post-fixation was tested. Yeast were exposed to freshly prepared fixatives for 1, 3 or 5 minutes, washed and allowed to recover for a minimum of 30 minutes prior to being plated on YPD. It was determined that formaldehyde fixed yeast more rapidly than paraformaldehyde. In addition to testing freshly prepared fixatives, fixatives stored at ambient for an extended period of time were also tested. Based on the viability and staining tests, 4% formaldehyde in PBS is the best fixative to use for the current EMMYS-1 scenario.
Lithium modifies thigmotropic responses through its effects on ethylene metabolism; lithium inhibits conversion of ACC ((1-aminocyclopropane-1-carboxylic acid) to ethylene. Lithium modifies second messenger systems in animal by inhibiting resynthesis of PIP2 and inhibiting inositol-1-phosphate phosphatase. Work in our laboratory has implicated auxin-induced ethylene production and intermediates in the second messenger system in the growth and gravitropic response of primary roots of maize. Li ions at concentrations greater than 0.1 mM promote the elongation rate of primary roots of maize; this may be due to inhibition of auxin induced ethylene. Further support for this hypothesis is provided by ssymmetric application of Li to graviresponding roots; application of Li ions to the lower surface of roots delays positive gravicurvature or results in negative gravicurvature. This may be due to inhibition of auxin-induced ethylene production during the motor response of the roots. Lithium ions enhance the phosphorylation of specific protein species while suppressing the phosphorylation of other protein species. The pattern of phosphorylation that is observed as a result of lithium treatment is similar to the pattern observed upon treatment of maize roots with promotive concentrations of indole-3-acetic acid (IAA) when ethylene biosynthesis has been inhibited using AVG (aminoethoxyvinyl glycine). Distinct phosphorylation patterns obtained from cytoplasmic and membrane fractions of roots which are exposed to inhibitory concentrations of indole-3-acetic acid can be suppressed by pretreatment of root tissue with 0.1 mM lithium; the resulting phosphorylation profile exhibited by these Li/inhibitory-IAA concentration treated roots are almost identical to the phosphorylation profile of IAA-stimulated root tissues.
The small G proteins of the Rho family (Rho GTPases) are key operators in the signaling arising from integrins and cell-matrix focal adhesions. They function as binary molecular switches that cycle between an inactive GDPbound and an active GTP-bound form that in turn activates downstream effectors and a variety of signaling pathways. The best known Rho GTPases, RhoA, Rac1 and Cdc42 control the organization of the cytoskeleton and the focal adhesions-mediated transduction of exogenous and endogenous mechanical signals. Their localization at the cross-road between signaling, mechanical forces and the cytoskeleton along with reported effects of microgravity on cell shape and cytoskeletal arrangement led us to hypothesize that Rho GTPases might be involved in the reception and reaction of cells to gravity. To explore this hypothesis we created stable cell lines, SV40-transformed fibroblasts (WI-26), expressing a constitutively active form (QL) of RhoA, Rac1 or Cdc42 (manuscript in preparation). The reverse situation, i.e. suppression of the GTPases expression was obtained by transfecting small interfering RNA (siRNA) targeting each of these GTPases (Deroanne et al., 2003,2005).
Decreased interstitial flow under microgravity has been thought to be a cause of bone mineral loss during space flight. Although mechanical stimulations such as flow significantly affect cellular activities, the molecular nature of putative mechanoreceptor has been unclear. While studying effects of mechanical stimuli on the expression of an immediate early gene c-fos by an osteoblast-like line MC3T3-E1, we noticed that mere manual handling of culture flasks affected the response far greater than intended mechanical stimulation such as centrifugation. Carrying culture flasks in conventional laboratory practices may induce culture fluid flow even with extremely gentle handling. Moreover, such procedures bring with cooling down of culture fluid to the room temperature, which may also affect the response. We then developed a simple technique to minimize the effects of such problems. Temperature change and fluid flow were independently applied to the cells in culture flasks. It was shown that culture fluid flow, generated by manual rocking of the flask at stable temperature, induced transient c-fos expression. Temperature change at standing still also induced similar response. It has been reported that kidney cells could sense fluid flow by molecules named polycystins -1 and -2 located on primary cilia. MC3T2-E1 cells also had primary cilia and expressed polycystin-2, suggesting analogous events in bone cells. Polycystins are TRP channel super family proteins. Some TRP channel proteins are known to be either hot or cold temperature receptors. The finding that fluid flow and temperature shift separately induced similar response, suggested possible involvement of receptors molecules related both mechanical and thermal perceptions in osteoblast activation.
The International Space Station ISS offers the opportunity for physiological long-term observations in microgravity in awake animals. The 3-month period of experimentation favours heredity, development and physiological adaptation as scientific fields that can profit from those longterm exposures to microgravity. However, the most complicating problems are (i) the lack of proper animal habitats, and (ii) the available crew time. These facts dramatically decrease the types of experiments and, in particular, the animal species that can be used for long-term studies. From the methodological point of view, automatic working recording devices are the most suitable techniques. Experience with neurophysiological long-term recordings based on ground studies, in particular in insects (cf. Miller, 1979), favours the study of central integration of neuronal, sensory and muscular activity of the organisms and, in particular, its adaptation to the microgravity environment. The lack of proper animal habitats on ISS favours the use of animals that are adapted to a life in extreme environmental conditions with rare food supply. Only those animals can be used that are adapted to a life in extreme environmental conditions and that are able to starve for long periods of their life due to limited access to food and water. Desert animals such as beetles and scorpions are species of first choice. In 2001, an experiment with scorpions titled SCORPI was selected for flight on ISS mounted in the European research facility BIOLAB. Its main purpose is to analyse the adaptation of coordinating mechanisms between vegetative and sensorimotor activities to microgravity by means of neurophysiological recordings.
Plant roots direct their growth in response to gravity, light, and mechanical stimuli. Spatial changes in the rates of cell elongation and division are responsible for the directional growth. Local changes in hormone concentration and/or sensitivity have been shown to be part of the signal transduction and response mechanisms that result in those tropic growth responses. Part of most hormone regulated mechanisms are regulation of transcription and transcript stability. We have focused our analysis of whole genome microarrays on the differential regulation of transcript abundance changes during the first hour after gravity and mechanical stimulation with respect to the involvement of hormones – especially auxins and brassinosteroids.
Human and experimental animals in space develop the anemia of space flight (Tavassoli, M., 1982; Udden et al., 1995). Studies have shown that erythropoietin (Epo) is implicated in this microgravity-induced abnormality (Udden et al., 1995). The status of the Epo receptor (EpoR) in microgravity, however, is unknown. The Rotary Cell Culture System (RCCS), based on NASA rotating wall vessel (RWV) technology and manufactured by Synthecon Inc., is an in vitro culture system. Within this system individual cells, to a certain extent, experience an environment with similarities to true microgravity (Gao et al., 1997; Battle et al., 1999). BaF3 cells stably expressing the transfected human Epo receptor (BaF3- EpoR cells) were cultured in RPMI-1640 supplemented with 5% fetal bovine serum and either 1 unit/mL of recombinant human Epo (rhEpo) or 5 ng of recombinant mouse interleukin-3/mL (rmIL-3) in either the RCCS or in 175 cm2 standard tissue culture flasks (control) at 37 oC in a humidified atmosphere of 95% air/5% CO2, 37 C for 48 hours. Cells were then harvested by centrifugation, incubated with 125I-labeled rhEpo or 125I-labeled rmIL-3, and the bound/free ligands were separated by centrifuging the cells through a serum cushion according to published methods (Yonekura, 1991). The radioactivity of the cell pellet was quantified by gamma scintillation spectrometry.
There is little evidence obtained from space flight to support the notion that occurrence of cardiac dysrhythmias, impaired cardiac and vascular function, and manifestation of asymptomatic cardiovascular disease represent serious risks during space flight. Therefore, the development of orthostatic hypotension and instability immediately after return from spaceflight probably reflect the most significant operational risks associated with the cardiovascular system of astronauts. Significant reductions in stroke volume and lower reserve for increasing peripheral vascular resistance contribute to ineffective maintenance of systemic arterial blood pressure during standing after spaceflight despite compensatory elevations in heart rate. The primary mechanism underlying reduced stroke volume appears to be a reduction in preload associated with less circulating blood volume while inadequate peripheral vasoconstriction may be caused partly by hyporeactivity of receptors that control arterial smooth muscle function. A focus for development of future countermeasures for hemodynamic responses to central hypovolemia includes the potential application of pharmacological agents that specifically target and restore blood volume (e.g., fludrocortisone, electrolyte-containing beverages) and reserve for vasoconstriction (e.g., midodrine, vasopressin). Based on systematic evaluations, acute physical exercise designed to elicit maximal effort or inspiratory resistance have shown promise as successful countermeasures that provide protection against development of orthostatic hypotension and intolerance without potential risks and side effects associated with specific pharmacological interventions.
As humans explore further into space, biocontamination of other planets remains a concern. Another issue is the protection of crew health and the prevention of microbe-induced sickness. A silane quaternary ammonium salt (QAS; AEGIS Microbe Shield TM) was applied to aluminum coupons and tested against Bacillus subtilis, Escherichia coli, and Staphylococcus epidermidis to determine its value as an antimicrobial surface shield. The bacteria tested are commonly associated with spacecraft assembly, space travel and the human body. The QAS was initially effective as an antimicrobial agent but gradually lost its activity through repeated use, a result contrary to company claims.
Animal models have been used to study the effects of space flight on physiological systems. The animal models have been used because of the limited availability of human subjects for studies to be carried out in space as well as because of the need to carry out experiments requiring samples and experimental conditions that cannot be performed using humans. Experiments have been carried out in space using a variety of species, and included developmental biology studies. These species included rats, mice, non-human primates, fish, invertebrates, amphibians and insects. The species were chosen because they best fit the experimental conditions required for the experiments. Experiments with animals have also been carried out utilizing ground-based models that simulate some of the effects of exposure to space flight conditions. Most of the animal studies have generated results that parallel the effects of space flight on human physiological systems. Systems studied have included the neurovestibular system, the musculoskeletal system, the immune system, the neurological system, the hematological system, and the cardiovascular system. Hindlimb unloading, a ground-based model of some of the effects of space flight on the immune system, has been used to study the effects of space flight conditions on physiological parameters. For the immune system, exposure to hindlimb unloading has been shown to results in alterations of the immune system similar to those observed after space flight. This has permitted the development of experiments that demonstrated compromised resistance to infection in rodents maintained in the hindlimb unloading model as well as the beginning of studies to develop countermeasures to ameliorate or prevent such occurrences. Although there are limitations to the use of animal models for the effects of space flight on physiological systems, the animal models should prove very valuable in designing countermeasures for exploration class missions of the future.
Retinal degenerations can be promoted by many factors including ageing, ischemia, fluctuation in oxygen tension, oxidative stress, and increased intraocular pressure. We present new evidence that the environment encountered in space shuttle flight can also disrupt normal retinal development and mimic stimuli that induce retinal degenerations on earth. There is experimental evidence linking anomalies in visual perception with space flights since the Apollo missions (Phillpot et al., 1978; Newberg and Alavi, 1998). There is also strong evidence that pathological stimuli that disrupt retinal structure and function on earth are encountered in the space shuttle environment as well. Orbital space flights cause physiological disturbances in humans including cephalad fluid shift (Hoffler et al., 1977; Drummer, 2000), increased intraocular pressure (Mader et al., 1990; Draeger, 2000) disruption of cardiovascular function (Wang et al., 1996) and stress on the musculoskeletal system (Lane and Feedback, 2002; LeBlanc, 2000).
Microgravity and its environment have adverse effects on the immune system. Abnormal immune responses observed in microgravity may pose serious consequences, especially for the recent directions of NASA for long-term space missions to Moon, Mars and deep Space exploration. The study of space flight immunology is limited due to relative inaccessibility, difficulty of performing experiments in space, and inadequate provisions in this area in the United States and Russian space programs (Taylor 1993). Microgravity and stress experienced during space flights results in immune system aberration (Taylor 1993). In ground-based mouse models for some of the microgravity effects on the human body, hindlimb unloading (HU) has been reported to cause abnormal cell proliferation and cytokine production (Armstrong et al., 1993, Chapes et al. 1993). In this report, we document that a nutritional nucleotide supplementation as studied in ground-based microgravity analogs, has potential to serve as a countermeasure for the immune dysfunction observed in space travel.
In previous studies, we determined that cells of Pleurochrysis cartarae do not start to move in a measurably oriented manner until a certain cell density is reached, concomitant with the start of bioconvection (con-vection due to movement of microorganisms.) The process begins with the accumulation of cells at the surface of the medium, which in turn requires that the cells swim up. The objective of the current study was to determine if other taxes, specifically photo- and aerotaxis, were the cause of surface accumulation of cells. Because our experiment using this alga was deselected from spaceflight on ISS due to lack of videomicroscopy, macroscopic methods were used in these experiments. Cells from a stock cell culture of P.carterae, Plymouth strain 136, were grown overnight in F/2 medium (18oC), then divided into 4 t-flasks. After 24 hours of culture, a green halo of cells surrounded the air bubble in each flask, demonstrating aerotaxis. For phototaxis, box covers, with only a small opening at the bottom of one side, were placed over the t-flasks as an obstruction of the light source. Cells in these cultures did not accumulate in any region of the culture, and cultures did not grow due to the lack of light. The t-flasks were then placed horizontally in the incubator with the boxes covering all but the top 25% of the flask. The air bubble was located in the dark region of the flask. When the covers were removed, cells were found in the uncovered region of the t-flasks, demonstrating positive phototaxis. No cells accumulated around the air bubble, showing that phototaxis was stronger than aerotaxis. Upswimming both in the lab and in nature is likely due to phototaxis and /or aerotaxis, not negative geo-or gyro-taxis. This knowledge can be used to design spaceflight experiments, and the development of a macromethod obviates the need for videomicroscopy to see overall cell accumulations.
Light and gravity give clues about time and space which plants use to direct their growth. Roots grow towards the vector of gravity (positive gravitropism) to access water and nutrients in the soil and to provide stability. Their response to light depends on its wavelength and intensity. Roots grow towards red light (positive phototropism) and away from blue and white light (negative phototropism) (Kiss et al. 2003). Plants respond to these signals via differential cell elongation which results in directional growth. Light and gravity are simultaneous stimuli so plants must respond to both at the same time and integrate them into a single response. Our hypothesis is that integration of light and gravity responses in roots can be observed at the level of gene expression. One of the fastest known signal transduction elements described for gravity and light responses is inositol- 1,4,5 triphosphate (InsP3). Cellular levels of InsP3 increase within 15s to 30s in response to stimulation by gravity or light, respectively (Morse et al. 1987; Perera et al. 1999). We therefore analyzed light induced changes in transcript abundances of genes known to respond specifically to gravity in roots of wild type and transgenic plants dampened in their InsP3-signaling (Kimbrough et al. 2004; Perera et al., in preparation.).
