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

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.

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Microbial antibiotic production aboard the International Space Station.

Previous studies examining metabolic characteristics of bacterial cultures have mostly suggested that reduced gravity is advantageous for microbial growth. As a consequence, the question of whether space flight would similarly enhance secondary metabolite production was raised. Results from three prior space shuttle experiments indicated that antibiotic production was stimulated in space for two different microbial systems, albeit under suboptimal growth conditions. The goal of this latest experiment was to determine whether the enhanced productivity would also occur with better growth conditions and over longer durations of weightlessness. Microbial antibiotic production was examined onboard the International Space Station during the 72-day 8A increment. Findings of increased productivity of actinomycin D by Streptomyces plicatus in space corroborated with previous findings for the early sample points (days 8 and 12); however, the flight production levels were lower than the matched ground control samples for the remainder of the mission. The overall goal of this research program is to elucidate the specific mechanisms responsible for the initial stimulation of productivity in space and translate this knowledge into methods for improving efficiency of commercial production facilities on Earth.

A novel combination of methods to assess sarcopenia and muscle performance in mice.

A novel combination of assays was developed to assess sarcopenia and muscle performance. Three techniques were tested to assess muscle function both during and upon termination of treatments designed to induce sarcopenia. In unsuspended (US) and hindlimb suspended (HS) mice, a Hindlimb Exertion Force Test (HEFT), cage wheel running, and in vitro muscle electrophysiology were performed. Twelve-week old, mature male C57BL/6J mice were HS (n = 24) for two weeks, or served as US controls (n = 26). Both groups were subjected to a HEFT on day 13; that is, the maximum force exerted against a beam force transducer (2 lb. linear range, Transducer Techniques, Temecula CA) following applied tail shock stimulus (0.15 mA, 300 msec). This test primarily evaluated the hindlimb muscles used for an escape response (i.e., hamstrings, quadriceps and calf muscles). Mice (n = 10-11/group) were given voluntary access to running wheels for 7 days post treatment to evaluate muscle endurance. On day 13, HS mice showed a mean 18.9% (p = 0.002) decrease in the maximum force exerted compared to US mice. After 7 days of wheel running, HS running distance tended to decrease (13.2%, p = 0.084). HS mice ran an average of 2.0 km/day less than US control mice, with similar running patterns: distance declined on day 2 following completion of HS but increased steadily thereafter. With in vitro testing, the maximum soleus tetanus response decreased by 31.8% (p = 0.01) with HS, in agreement with the changes observed by the other assays. These three assays, combined, appear to provide effective and complementary ways to measure muscle performance and functional differences.

Comparison of tail-suspension and sciatic nerve crush on the musculoskeletal system in young-adult mice.

Musculoskeletal unloading and disuse result in significant muscle and bone loss. These phenomena can be modeled using sciatic nerve crush or tail-suspension. Mature animals eliminate the complication of growth superimposed on bone and muscle loss. In the current study, young-adult (12-week old male) C57BL/6J mice were subjected to sciatic nerve crush (NC; n = 9) or tail-suspension (TS; n = 9) for 14 days, with a normal ambulatory control (n = 10). The soleus, gastrocnemius, and EDL muscles were collected and weighed at sacrifice. Femurs were analyzed in three-point bending for stiffness, elastic force and maximum force. Muscle masses in tail suspended mice were reduced by 41.9% (p < 0.001), 17.5% (p < 0.001), and 9.1% (N.S.) for the soleus, gastrocnemius, and EDL, respectively. In NC mice, muscle masses were reduced by 18.6% (p = 0.004), 37.2% (p < 0.001), and 22.5% (p = 0.003). Femur stiffness, elastic and maximum forces were reduced by 20.9% (p = 0.014), 14.7% (N.S.), and 11.6% (N.S.) in TS, compared to NC where masses were reduced by 15.5% (p = 0.022), 0.2% (N.S.) and 11.2% (N.S.) in the crushed leg compared to the contralateral control. NC resulted in a greater reduction of muscle mass in the gastrocnemius and EDL muscle; whereas tail-suspension had a greater effect on the soleus. Tail-suspension had the greatest effect on bone mechanical properties. When comparing these results to actual spaceflight data, it appears as though TS most closely models muscle loss, and NC most closely models changes in bone mechanical properties. These unloading models have tissue-specific effects that impact their applications for musculoskeletal research.

Exercise prevention of unloading-induced bone and muscle loss in adult mice.

Skeletal unloading causes bone and muscle loss that may be reversed by post-unloading exercise. This study examines the effects of unloading and exercise, using tail-suspension for 14 days combined with a week of post-suspension cage wheel running in mice. Twenty-four adult, male, C57BL/6J mice were divided into four groups (n = 6 mice/group); unsuspended non-running, tail-suspended non-running, unsuspended running, and tail-suspended running. At sacrifice, the calf (soleus, gastrocnemeius and plantaris complex), heart, tibia and femur were collected and weighed. The femora and tibiae were cleaned of non-osseous tissue, subjected to 3-point bending (femurs only), and weighed for dry (105 degrees C; 24h) and ash mass (800 degrees C; 24h). The mean calf mass from the tail-suspended groups (157.13 +/- 2.83 mg) was significantly less than in the unsuspended groups (167.33 +/- 2.83 mg; p = 0.019), with no significant effect of cage wheel running. The mean heart mass in running groups (166.58 +/- 4.78 mg) was significantly greater than the non-running groups (148.17 +/- 4.78 mg; p = 0.013), with no effect of hindlimb suspension. The mean femur ash mass from tail-suspended groups (24.02 +/- 0.38 mg) were significantly less than the unsuspended groups (25.11 +/- 0.34 mg; p = 0.050), and the running groups (25.13 +/- 0.38 mg) were significantly greater than the non-running groups (24.00 +/- 0.34 mg; p = 0.043). No effect was observed for the femur dry mass or percent mineralization. Measurements of mechanical length tended to be lower in tail-suspension, with no significant affects do to cage wheel running. This study suggests that tail-suspension in adult mice significantly decreases skeletal muscle and bone mass, with no change in percent mineralization. Furthermore, one week of running does not reverse the effects on the skeletal muscle and bone mass.

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.

Skeletal muscle adaptations to microgravity exposure in the mouse.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to approximately 11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced (P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb (P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.

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 degrees 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.

Recent advances in technologies required for a "Salad Machine".

Future long duration, manned space flight missions will require life support systems that minimize resupply requirements and ultimately approach self-sufficiency in space. Bioregenerative life support systems are a promising approach, but they are far from mature. Early in the development of the NASA Controlled Ecological Life Support System Program, the idea of onboard cultivation of salad-type vegetables for crew consumption was proposed as a first step away from the total reliance on resupply for food in space. Since that time, significant advances in space-based plant growth hardware have occurred, and considerable flight experience has been gained. This paper revisits the "Salad Machine" concept and describes recent developments in subsystem technologies for both plant root and shoot environments that are directly relevant to the development of such a facility.

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

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

Cellular responses to gravity: extracellular, intracellular and in-between.

Our understanding of gravitational effects (inertial effects in the vicinity of 1 x g) on cells has matured to a stage at which it is possible to define, on the basis of experimental evidence, extracellular effects on small cells and intracellular effects on eukaryotic gravisensing cells. Yet undetermined is the nature of response, if any, of those classes of cells that are not governed solely by extracellular physical events (as are prokaryotes) and are devoid of obvious mechanical devices for sensing inertial forces (such as those possessed by certain plant cells and sensory cells of animals). This "in-between" class of cells needs to be understood on the basis of the combination of intracellular and extracellular gravity-dependent processes that govern experimentally-measurable variables that are relevant to the cell's responses to modified inertial forces. The forces that certain cell types generate or respond to are therefore compared to those imposed by approximately 1 x g in the context of cytoskeletal action and symmetry-breaking pathways.

Autonomous Biological System (ABS) experiments.

Three space flight experiments have been conducted to test and demonstrate the use of a passively controlled, materially closed, bioregenerative life support system in space. The Autonomous Biological System (ABS) provides an experimental environment for long term growth and breeding of aquatic plants and animals. The ABS is completely materially closed, isolated from human life support systems and cabin atmosphere contaminants, and requires little need for astronaut intervention. Testing of the ABS marked several firsts: the first aquatic angiosperms to be grown in space; the first higher organisms (aquatic invertebrate animals) to complete their life cycles in space; the first completely bioregenerative life support system in space; and, among the first gravitational ecology experiments. As an introduction this paper describes the ABS, its flight performance, advantages and disadvantages.

Plant-module for autonomous space support (P-MASS).

A wide variety of technical and science questions arise when attempting to envision the long-term support of plants, algae and bacteria in space. Currently, spaceflight data remain elusive since there are no U.S. carriers for investigating either the germane technical or scientific issues. The first flight of the Commercial Experiment Transporter (COMET) will provide a nominal 30 day orbital opportunity to evaluate such issues. The P-MASS is a small payload that is designed to meet the mass (40 lbs.), volume (1.5 cu.ft.), and power (120 W) constraints of one of several COMET payloads while enabling flight evaluations of plants, algae and bacteria. Various P-MASS subsystems have been subjected to extensive ground tests as well as KCl35 tests. Various biological sub-systems have been similarly evaluated. Through a variety of sensors coupled with color video, the P-MASS performance and the supported biological systems will be compared for terrestrial controls versus spaceflight materials. This small, low cost payload should return valuable information regarding the requirements for hardware and biological systems needed to move toward bioregenerative life support systems in space. In addition, it should be possible to accurately identify major unresolved difficulties that may arise in the long-term, spaceflight support of various biological systems. Finally, this generic spaceflight capability should enable a variety of plant research programs focused on the use of microgravity to modulate and exploit plant products for commercial applications ranging from new agricultural products to pharmacological feedstocks and new controlled agricultural strategies.

Four educational programs in Space Life Sciences.

Four different educational programs impacting Space Life Sciences are described: the NASA/USRA Advanced Design Program, the NASA Specialized Center of Research and Training (NSCORT) Program, the Centers for the Commercial Development of Space (CCDS) Program, and the NASA Graduate Research Fellow Program. Each program makes somewhat different demands on the students engaged in them. Each program, at the University of Colorado, involves Space Life Sciences training. While the Graduate Student Research Fellow and NSCORT Programs are discipline oriented, the Advanced Design and CCDS Programs are focused on design, technologies and applications. Clearly, the "training paradigms" differ for these educational endeavors. But, these paradigms can be made to mutually facilitate enthusiasm and motivation. Discipline-oriented academic programs, ideally, must be flexible enough to accommodate the emergent cross-disciplinary needs of Space Life Sciences students. Models for such flexibility and resultant student performance levels are discussed based upon actual academic and professional records.

Postinjury changes in the biomechanics of nerves and roots in mice.

The biomechanical characteristics of sciatic nerve and associated spinal roots of mice were investigated. Both normal and postcrush nerve materials were tested in the same fashion using superimposed elongation, force and geometry data. The results show that nerve and roots differ considerably both in the force they sustain before failure and in the other biomechanics they exhibit. The nerves and associated structures transmit and absorb large amounts of force. The roots, in comparison, are mechanically frail but exhibit similar elongation before failure. The behavior of these elements of the nervous system are discussed with regard to implications for integrated nerve, root and spinal cord mechanical integrity.

Structural properties of spinal nerve roots: biomechanics.

The biomechanics of spinal nerve roots obtained from normal and nerve-crushed mice were evaluated. Photographs and longitudinal force measurements were taken as nerve roots were elongated through mechanical failure. Proportional limit stress and strain as well as the apparent modulus were calculated from photographic and force measurements to characterize nerve root strength, elasticity, and stiffness, respectively. Resulting mechanical data were indicative of an extremely weak material. Comparisons of nerve and nerve root mechanical properties revealed major differences. While nerve root elasticity was comparable to nerve, nerve root strength was only 10% that of nerve and root stiffness was only 20% of nerve values. Differences in nerve and root mechanics are attributed to the large discrepancies in relative amounts of connective tissue. Also in sharp contrast with peripheral nerve, unilateral nerve crush produced no significant alterations in root mechanics. Comparisons of nerve and nerve root strengths suggested possible pathways for dissipation of peripherally applied forces through epineurial and dural structures.

Structural properties of spinal nerve roots: protein composition.

The protein compositions of dorsal and ventral spinal nerve roots were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein readily soluble in sodium dodecyl sulfate (SDS) sample buffer made up 8.1% of the wet weight of dorsal and ventral roots. Spinal root protein samples consisted predominantly (60%) of myelin-associated proteins. Other major proteins including those tentatively identified as tubulin, actin, nuclear histones, and others accounted for the remainder of recovered protein. The protein constituents of nerve roots were similar to those of peripheral nerve but differed from those of spinal cord. Nerve roots and peripheral nerve were characterized by fewer major protein bands but greater concentrations of myelin proteins. Collagen which did not readily solubilize in SDS sample buffer was estimated by assaying for hydroxyproline. Nerve roots consisted of approximately 0.4% collagen by weight which was only one-fifth the amount estimated for nerve but six times more than spinal cord. It was apparent that the biomechanical frailty of roots compared with peripheral nerve might be explained by differences in the relative collagen contents of these tissues. The protein constituents of nerve roots after unilateral nerve crush were relatively stable compared with the profound changes seen in ipsilateral nerve and modest changes seen in contralateral uninjured nerve.

Relationships between the electrocardiogram and phonocardiogram: potential for improved heart monitoring.

Improvements in current heart monitoring techniques could reduce the number of heart attacks and resulting deaths. The potential for using time intervals measured between waveforms of the electrocardiogram (ECG), phonocardiogram (PCG), and peripheral blood flow pulse (PP) for heart monitoring was studied. The waveform locations identified in the simultaneously recorded signals included the R- and T-wave peaks of the ECG, the first (S1) and second (S2) sounds of the PCG, and the systolic peak of the PP. The signals were found to be highly consistent from one cardiac cycle to the next. Further, the time intervals measured between the different signals were stable with time. Strong relationships were found between the intervals R to T and R to S2 and the R to R interval. In contrast, R to PP and R to S1 correlated poorly with the R to R but strongly with each other. Additional differences between the measured intervals were revealed by studying changes due to exercise and different body positions. The relationships between the measured intervals were found to be independent of PCG recording location. This study demonstrates the feasibility and potential of using the electrical-contractile indices of heart function for monitoring heart patients. Design of a computer-based monitor using the techniques specified in this study is discussed along with relative strengths and weaknesses of such a system.