Commercial Biomedical Test Module - 2 (CBTM-2) - 01.13.16
Commercial Biomedical Test Module - 2 (CBTM-2) uses a validated mouse model to examine the effectiveness of an experimental therapeutic as a possible countermeasure for muscle atrophy. Science Results for Everyone
Spaceflight doesn’t seem to agree with mice. Researchers analyzed mouse tissues after spaceflight and found a broad spectrum of effects: decreased expression of muscle cell calcium release channels; changes in amounts of some bone marrow cells and loss in body weight, suggesting chronic stress; modification in distribution, function, and gene expression of tumor-attacking cells; reductions in liver, spleen, and thymus masses; changes in mRNA expression of certain genes; adverse changes in lung tissue; and changes in eight of 67 liver proteins analyzed. No significant changes were seen in ovarian tissue exposed to space flight for 13 days or fewer. Experiment Details
H. Q. Han, Ph.D., M.D., Amgen Research, Thousand Oaks, CA, United States
Giovambattista Capasso, M.D., Secondi University of Napoli, Napoli, Italy
Michael D. Delp, Ph.D., Florida State University, Tallahassee, FL, United States
Allan D. Forsman, Ph.D., East Tennessee State University, Johnson City, TN, United States
Lora M. Green, Ph.D., Loma Linda University, Loma Linda, CA, United States
David L. Lacey, M.D., Amgen, Thousand Oaks, CA, United States
Gregory A. Nelson, Ph.D., Loma Linda University, Loma Linda, CA, United States
Bradley J. Behnke, University of Florida, Gainesville, FL, United States
David C. Zawieja, Ph.D., Texas A&M University, TX, United States
Brooke C. Harrison, Ph.D., University of Colorado, Boulder, CO, United States
Louis S. Stodieck, Ph.D., University of Colorado, BioServe Space Technologies, Boulder, CO, United States
David L. Allen, Ph.D., University of Colorado, Boulder, CO, United States
Virginia L. Ferguson, Ph.D., University of Colorado, Boulder, CO, United States
Timothy G. Hammond, M.B.B.S., Durham Veterans' Affairs Medical Center, Durham, NC, United States
Jean-Luc Morel, Physiologie Cellulaire et Pharmacologie Moleculaire, Bordeaux, France
Paolo Di Nardo, University of Rome Tor Vergata, Rome, Italy
Ted A. Bateman, Ph.D., University of North Carolina, Chapel Hill, NC, United States
Amgen Research, Thousand Oaks, CA, United States
BioServe Space Technologies, University of Colorado, Boulder, CO, United States
NASA Ames Research Center, Moffett Field, CA, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration 1
April 2007 - October 2007
Previous ISS Missions
A similar investigation, CBTM, flew round trip to the ISS on STS-108 during ISS Expedition 4. AEMs have flown on numerous Space Shuttle missions over the years.
- CBTM-2 research has the potential to contribute to the development of an effective countermeasure for the negative effects of space on human skeletal muscle. The muscle changes that occur as a result of space flight must be understood and addressed in order to send humans on long-duration missions to the Moon and Mars. Exercise is only partially effective in preventing muscle atrophy (wasting) during space flight. A complementary therapeutic countermeasure to muscle atrophy could make future missions to the Moon and Mars safer and more productive for space explorers.
- Mice will be flown in space where half will be treated with the muscle therapeutic and half given a placebo. Effects of treatment on muscle size and function will be compared between space flight and ground control mice.
- CBTM-2 could impact future long-duration missions by establishing a therapeutic approach to preventing or reducing the amount of muscle loss that occurs during long-duration space flight.
- CBTM-2 is also expected to contribute data to the current body of research on microgravity effects on organ systems through a tissue sharing program.
There are no approved pharmaceutical countermeasures to muscle loss for use in crewmembers or any currently in the NASA countermeasure "pipeline." To counter muscle atrophy currently, crewmembers engage in daily exercise while in space. Exercise routines last 1.5 - 3 hours per day and result in reduced crewmember productivity. These regimes also do not completely alleviate the muscle loss that occurs as a result of extended stays in space. Developing and approving a pharmaceutical countermeasure to this condition could be instrumental to long duration human exploration missions.
This investigation will examine the effectiveness of an experimental therapeutic in preventing muscle loss in mice exposed to microgravity. This will be the first time an experimental therapeutic for muscle loss will be investigated in space; an important and significant step in developing a more effective countermeasure to space induced muscle changes.
Physical deficits from muscle atrophy also affect millions of Americans and results in significant health care expenditures. This experimental therapeutic has the potential to benefit NASA crewmembers and could help inform the development of potential interventions for muscle wasting related to a range of diseases, including cancer, kidney failure and age-related frailty.
This research will utilize the animal enclosure modules (AEMs). Three AEMs will be flown; each containing eight mice for a total of twelve treated and twelve placebo control mice. Three AEMs in the same configuration as space will be operated on the ground as ground controls and run in parallel with the space flight modules.
This research is also expected to contribute data to the current body of research on microgravity effects on the skeletal, cardiovascular, and immune systems, liver and kidney function as well as other physiological systems through a tissue sharing program. Every effort will be made to harvest as many different samples and types of tissue from the mice as possible for other mission specific biomedical research. Positive results from this research may advance our understanding of mechanistic changes that occur in various physiological systems after exposure to microgravity and support overall efforts to reduce health risks to crewmembers. The investigations resulting from the CBTM-2 tissue sharing program are as follows:
- The effects of space flight on regulation of adipose (fat) tissues
David Allen, Ph.D.
The goal of this research is to determine whether expression of certain components of a recently discovered regulatory pathway for muscle atrophy are changed in the fat tissue of mice during space flight. After space flight, two types of mouse fat tissue will be taken from the ground control and space flight animals. A chemical found in the body of all mammals, called ribonucleic acid or RNA will be separated from the mouse fat tissue samples and quantified. RNA is responsible for the transfer of information from deoxyribonucleic acid (DNA), genetic material, and plays an important role in protein synthesis (formation) and other chemical activity that occurs within the cells of the body. This research could lead to a better understanding of how crewmembers bodies are negatively affected by the microgravity environment and lead to new or improved treatments to counter those affects.
- Relationship of microRNA expression and muscle atrophy in microgravity
Brooke Harrison, Ph.D.
To date, few studies have investigated the effects of altered muscle use on muscle-specific micro-ribonucleic acid, microRNA (single strand RNA molecules) expression. The overall goal of this investigation is to identify microRNAs that demonstrate altered expression as a result of changes in muscle activation, specifically, muscle changes that occur during exposure to microgravity. To quantify microgravity-induced alterations in skeletal muscle microRNA expression, total RNA will be isolated from both ground control and space flight animals. The expression of selected microRNAs of interest will then be determined using Northern blotting (technique to study gene expression) or quantitative RT-PCR (reverse transcription - polymerase chain reaction, technique for amplifying a defined piece of a RNA) methods. Microgravity-induced alterations will be compared to ground-based models of both muscle use/disuse. This research could lead to a more thorough understanding of the mechanism behind the muscle atrophy that occurs during space flight, which could lead to the development of a countermeasure for this condition.
- Space flight effects on mouse reproductive tissues
Allan Forsman, Ph.D.
The ovarian tissue of female mice is a rapidly changing tissue. Changes occur due to the rapid turnover of ovaries from the follicular phase (phase which ovary follicles mature) to the luteal phase (latter phase of the estrous cycle) of the four day estrous cycle in the mouse. Although follicular growth occurs over several estrous cycles the formation of the corpus luteum from the remnants of the follicle occurs within a relatively short time span, literally hours. Previous data has indicated decreased vascular endothelial growth factor (VEGF, signaling protein involved in formation of the embryonic circulatory system, and growth of blood vessels from pre-existing vasculature) levels in the ovaries of mice from ground-based models of space flight. These decreased levels of VEGF are believed to be due to decreased expression of the VEGF mRNA within the ovarian cells. This study will examine the gross morphology of the ovaries of the mice flown in space and the corresponding ground controls. Factors to be examined include ovarian/follicular/luteal size, shape, and overall tissue health. In the mouse, if the growth of the blood vessels that form within the corpus luteum is disturbed, the female mouse is unable to maintain a pregnancy. Understanding the effects of space flight on vessel growth in animal reproductive tissue may help scientists understand how the microgravity environment could affect the human reproductive system in space.
- Expression and localization of vascular myocyte calcium release channels
This investigation examines the expression of calcium ion signalling proteins in vascular smooth muscles. The calcium ion signals are implicated in vascular tone and contraction but also in cellular processes including growth and differentiation. This investigation is based, in part, on results from a ground-based model of space flight. With this model, vascular smooth muscle change of calcium ion signalling was due to a decrease of ryanodine receptor (RyR, calcium channels in muscle) expression. This family of sarcoplasmic calcium ion channels (RyR) is crucial because these calcium ion channels amplify all calcium ion signals in smooth muscle cells.
The investigation will concentrate on calcium channels (RyR and the L and T-type voltage-dependent calcium ion channels, TRP channels, InsP3R) and on enzymes implicated in InsP3 and cyclic ADP-ribose (second messengers activating InsP3R and RyR, respectively) and vasoconstrictor receptor (adrenaline, angiotensin II, endothelin-1) regulating the activity of calcium ion channels. The investigation will compare animals in-flight, ground control and baseline groups and will help further the understanding of the effects of space flight on smooth muscles. Smooth muscle is responsible for the contraction of hollow organs, such as blood vessels, the gastrointestinal tract, the bladder, and the uterus.
- Hypercalciuria during space flight: the role of calbindin D28K
Giovambattista Capasso, M.D.
This experiment investigates calcium metabolism, particularly as it relates to calcium excretion and re-absorption via the kidneys under space flight conditions. The importance of the membrane-transporter protein calbindin for calcium re-absorption is being investigated. In particular the focus is on the up and down regulation of the relevant gene, CaB, which is known to lead to effects on both sodium and calcium regulation in humans. Space flight and ground control mouse kidney tissue will be analyzed for this experiment. Disruption to the balance of calcium excreted and re-absorbed in humans during space flight is a significant complication that can lead to kidney stones during a mission. An improved understanding of how calcium loses its balance within the body may help to reduce or eliminate this complication for long-duration space missions.
- Muscle atrophy in microgravity: a case of imbalance between stem/progenitor cell activation and muscle cell death
Paolo Di Nardo, M.D.
Long duration exposure to microgravity induces skeletal and heart muscle deconditioning that can cause health concerns for crewmembers upon return to Earth. Several strategies are used to counteract muscle deconditioning, but the problem is still not completely solved. This is likely due to the fact that basic mechanisms behind gravitational atrophy (wasting) remain to be completely understood. The aim of this investigation is to evaluate whether there exists a possible imbalance between stem/progenitor cell recruitment and muscle cells in microgravity deconditioning and determine which biologically active factors are involved in the process. The results could be helpful in designing new strategies to avoid microgravity muscle deconditioning. This investigation will examine heart, diaphragm and skeletal muscle of the space flight and ground control mice.
- Effect of space flight on macrophase differentiation and activation
S. Keith Chapes, Ph.D.
Space flight suppresses the formation of both red and white blood cells. It also affects the numbers of cells that circulate in the blood that protect people from disease. This occurs not only in humans but also in rats, mice and rhesus monkeys. In fact, space flight offers a unique environment that impacts many physiological systems. Amongst these systems, bone metabolism is known to be significantly altered by the skeletal unloading of space flight. Hematopoiesis is the process which forms red and white blood cells. Therefore, it provides the body with mature, functioning cells necessary for oxygenation, clotting and immune functions. The bone, specifically, the bone marrow, is the site for hematopoiesis. Moreover, changes in bone impact all compartments, including the bone marrow. Valuable information will be obtained about what changes occur in bone marrow cells from the STS-118 space flight and ground control mice. This understanding will allow for more effective countermeasure planning. On Earth, skeletal unloading occurs to individuals that are bedridden for long periods of time, wheel-chair bound, or lose the ability to stand for long periods of time. These prolonged periods of skeletal unloading also induce changes in bone that parallel those seen in space flight. In addition, individuals with bone disorders, such as osteoporosis, are also subject to alterations in hematopoiesis. Therefore, this experiment also has direct relevance to individuals on Earth.
- The effects of space flight on stress and immunity
Michael Pecaut, Ph.D.
There is an indisputable link between stress and immunity that is particularly important when discussing space flight. Although crewmembers will be exposed to isotropic whole-body radiation fields, simultaneous exposure to other environmental factors, such as microgravity and other mission-related stressors, could act additively, or even synergistically. Previous microgravity experiments have shown that exposure to the space flight environment can lead to changes in virtually all of the immune parameters influenced by radiation. This study is particularly interested in three inflammatory cytokines. All three cytokines are released by activated macrophages in response to a pathogenic challenge. In small localized amounts, these cytokines contribute to the immune response by regulating activity, enhancing phagocytosis, promoting coagulation, and stimulating growth and repair in damaged tissues. Additionally, all three have been linked to "sickness behavior" and, therefore, CNS-immune communication. This study will characterize both the stress response, and its effect on functional immune parameters ex vivo (outside) utilizing spleen, liver, brain, adrenal and thymus tissues. Results from the study may increase the understanding of the link between stress and immune system functioning in crewmembers during space flight and help develop strategies to minimize any negative effects.
- Space radiation and microgravity effects on regulation of lung tissue
Daila Gridley, Ph.D.
Previous studies on NASA Spacelab missions have found that pulmonary function in astronauts is greatly altered by weightlessness including diffusing capacity and reduced residual volume. Furthermore, inhalation of bacteria and fungi with increased antibiotic resistance and more rapid proliferation may adversely affect pulmonary function as well. We have recently compared effects of acutely delivered photons (gamma-rays), protons and simulated solar particle event protons (sSPE) on expression of extracellular matrix (ECM) regulators in lung tissue of mice. Transforming growth factor-beta1 (TGF-beta), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 were assessed on days 4 and 21 after whole-body exposure to a total of 2-Gray (Gy). The data show dependence on radiation regimen in expression of these ECM regulators and suggest that lung function in crewmembers exposed to a large SPE may become compromised. This study proposes to examine the lungs' altered TGF-b1, MMP-2, TIMP-1 and TIMP-2 gene expression and for RT-Profiler PCR array, mouse oxidative stress and antioxidant defense. This research could lead to an increased understanding of overall pulmonary functioning during space flight.
- Effects of space flight on neurogenesis in the brain
Gregory A. Nelson, Ph.D.
Neurogenesis or the regeneration of new nerve cells is critical in maintaining cognitive function, learning and spatial memory in adult mammals. Neuronal stem cells are the most sensitive radiation targets in the central nervous system (CNS) and up to 50% loss of newly-born cells in the hippocampus (a critical structure for memory and learning) occurs at doses as low as 1-Gy. In mice and rats experimentally-induced reduction of neurogenesis has been shown to adversely affect learning and memory. The 1Gy level of exposure is anticipated for certain space operations such as extravehicular activities during a solar particle event (SPE). Therefore the potential for inhibition of neurogenesis by radiation with accompanying cognitive deficits is a significant risk factor for crewmembers. Neurogenesis is also highly dependent on stress levels (negative response) and environmental enrichment and activity (positive effect). Therefore space flight factors in general have the potential to influence neurogenesis and, by extension, memory and learning. This investigation will examine brain tissue for markers of neurogenesis and stress responses by a combination of immunohistochemistry on fixed tissue and Western blot analysis (method to detect protein) of key Wnt (regulate cell-to-cell interactions during embryogenesis) and IGF-1 (polypeptide protein hormone similar in molecular structure to insulin) pathway components from snap frozen samples.
- Effect of space flight on alterations of astrocyte-neuronal coupling
Lora Green, Ph.D.
Recent preliminary data suggests that astrocytes (a type of brain cell) and neurons have reciprocal functional responses to irradiation and very different recovery kinetics. This investigation will examine the mechanism for this uncoupling by measuring alterations in glutamate uptake and metabolism, and to determine how abnormal glutamate uptake affects intercellular signaling with increased radiation exposure during space flight. The investigation will also examine brain tissue for markers indicative of astrocyte-neuronal coupling by immunohistochemistry and western blot analysis. Additionally, this study will assess the effect of space flight stress on intercellular communication in brain tissues. Immunofluorescence will be used to measure the expressed levels of the connexins known to be important in neuronal and astrocytic communication. Additionally, the study will examine the inter-relationship between calcium and adenosine triphosphate (ATP) during glio-transmission (neural transmission within astrocytes), by measuring the levels of the purinergic receptors in brain tissues from ground control and flight mice. Changes in astrocyte-neuronal coupling due to the radiation and/or microgravity components of the space flight environment may ultimately, result in cognitive deficits in crewmembers, thus an important area of investigation to ensure health and safety during space flight missions.
- Tissue-specific expression of gravity-sensitive proteins in kidney and liver
Timothy Hammond, M.D.
This investigation will test the hypothesis that there are tissue specific changes in renal and liver protein expression in space flight. If ICAM and VCAM protein expression are elevated in the liver but not the kidney then it can be confirmed that there are tissue specificity of the responses to space flight at the protein level. If there are changes in both tissues or neither tissue then the other forces present in suspension culture, but not space flight, are important mediators of ICAM and VCAM protein expression. If angiotensin converting enzyme (ACE, peptides (substances smaller than proteins) that act as vasoconstricting agents (causing blood vessels to narrow). Narrowing the diameter of the blood vessels sends up the blood pressure.) activity increases in-flight, either there is more ACE protein or the activity of the ACE is increased. This is important as ACE is the major degradatory enzyme for the vasoactive peptide angiotensin II (acts as an endocrine, autocrine, paracrine, and intracrine hormone). This investigation will utilize kidney and liver tissue provided by the tissue sharing program of CBTM-2. The choice of proteins to study is based on the gene expression data from a previous space flight investigation. This investigation will help determine if the gene expression changes observed translate into protein changes as well.
- Examination of functional and molecular changes caused by spaceflight-induced bone loss in mice
Ted Bateman, Ph.D.
This research will help develop a better understanding of weightlessness and its impact on the skeletal system. Currently crewmembers lose 1.5 - 2% of their skeletal mass each month during ISS missions, with bone strength declining ~2.5% per month. Due to improved analysis techniques over the last few years, a more in-depth analysis can occur. These improved techniques include microcomputed tomography (microCT), histology and immuno-histochemistry (IHC) all of which will be performed on the tibia of space flight and ground control samples. The humerus diaphysis (shaft of humerus) may also be used to examine levels of messenger RNA (mRNA) expression of specific genes including RANKL (Receptor Activator for Nuclear Factor Kappa B Ligand - a molecule important in bone metabolism; overproduction of RANKL is implicated in a variety of degenerative bone diseases, such as rheumatoid arthritis and osteomyelitis), OPG (osteoprotegerin, a cytokine that specifically acts on bone, increasing bone mineral density and bone volume) and osteocalcin (noncollagenous protein found in bone and dentin. It is secreted by osteoblasts and thought to play a role in mineralization and calcium ion homeostasis). This skeletal work will be performed in collaboration with BioServe Space Technologies and will include mechanical testing, compositional analysis and quantitative histomorphometry (identification of cellular and tissue components for the measurement of lengths) at the femur middiaphysis, middle shaft region of the femur.
The results of this research could be applied towards minimizing the risk of fracture in crewmembers during future exploratory missions to the moon and Mars. It will also help scientists better understand the molecular causes of bone loss in space and could have far reaching impact in furthering research in overall bone health.
Testing the effects of a potential countermeasure to the muscle loss that occurs during long-duration space flight will potentially provide NASA with a non-exercise, therapeutic countermeasure that helps ensure astronaut health and well being.
Muscle atrophy resulting from disuse and reduced physical activity affects millions of Americans particularly in the aging population. This condition contributes to increased bone fractures, negative metabolic changes and decreased levels of physical activity. Results from this investigation could help inform the development of potential interventions for muscle wasting related to a range of disease, including cancer, kidney failure and age-related frailty.
Once the mice are in space, the flight hardware and experiment are relatively self-sufficient. The AEMs contain enough food and water to house the mice safely and effectively for the mission duration. An astronaut will check the health status of the mice on a daily basis, by assessing them through the viewing window on each AEM.
For this study nine week old female C57/B6 mice will be launched on the space shuttle. The mice will spend a total of 11-12 days in microgravity. Flight mice will be treated once with a placebo vehicle or therapeutic agent approximately 24 hours before launch. Ground control mice will be treated in the same manner but with a 48 hour offset. Ground control mice will be housed under the same environmental conditions (temperature, light/dark cycle, humidity, oxygen levels and carbon dioxide levels) as the flight mice. All mice will receive the same full access to food and water. Upon return to Earth, the AEMs will be returned to the research team for analysis. Body weight will also be measured pre and post flight. Statistical comparisons will be made between the treated and control mice.
Decadal Survey Recommendations
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During ISS Expedition 15, 24 mice were flown to ISS on shuttle flight STS-118 in three AEMs. The AEMs remained on STS-118 throughout the mission. Utilizing a tissue sharing program CBTM-2 was able to support several additional investigations which have yielded the results below.
Expression and localization of vascular myocyte calcium release channels
Researchers found that exposure to microgravity during 8 days in the International Space Station decreases the expression of the ryanodine receptor 1 (RyR1) which is a calcium release channels inside cells in primary cultured myocytes (muscle cells) from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the effects of microgravity, were compared with those obtained in smooth muscle cells from rats injected with chemical agent directed against the RyR1 protein. In both conditions calcium release were significantly decreased. In contrast, in spontaneous hypertensive rats, an increase in RyR1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, these results show that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to vascular pressure by the regulation of the RyR1 expression (Dabertrand 2011).
Effect of space flight on macrophase differentiation and activation
This study involved the analysis of bone marrow cells from the CBTM-2 payload following a 13-day flight on the Space Shuttle to determine how space flight affected differentiation of cells in the granulocytic lineage (important white blood cells produced in bone marrow). The bone marrow cells were isolated from the humerii (long bones of the upper limb or forelimb) of mice. A cell counting method was utilized to assess the expression of several molecules (Ly6C, CD11b, CD31 (PECAM-1), Ly6G (Gr-1), F4/80, CD44 and c-Fos) that defines the maturation state of cells in the granulocytic lineage on three bone marrow cell subpopulations ( R1, R2 and R3) defined by their size and light-scattering properties. There were no observable characteristic differences between total bone marrow cells isolated from flight and ground-control mice. Nevertheless, there were subpopulation differences observed which suggests neutrophil activation in response to landing. Decreases were noticed in Ly6C, c-Fos, CD44high and Ly6G. An increase in F4/80 suggested that the cells in the bone marrow R3 subpopulation of the mice flown on Shuttle were more differentiated compared to the ground controls. A loss in body weight was also noticed in the mice that flew in space which suggest that they were subjected to chronic stress beyond that what was endured during landing. Therefore, it is not unreasonable to suggest that there are significant changes in bone marrow phenotype in response to the stress of the space flight experience (Ortega 2008).
The effects of space flight on stress and immunity
Understanding lymphocyte activity associated with spaceflight stressors is important in determining the impact on associated cancer risk. This study examined the T-lymphocytes in C57BL/6 mice (a traditional inbred strain of lab mice) after the return from a 13-day Space Shuttle mission. Flight mice (FLT) and ground controls similarly housed in Animal Enclosure Modules (AEMs) were evaluated within 3 to 5 hours after landing. Muscle strength testing and nuclear magnetic resonance body composition measurements were performed on the mice. After euthanasia (painlessly put to death), spleen and thymus samples were analyzed. DNA synthesis in splenocytes (any one of the different white blood cell types situated in the spleen) from FLT mice was low in response to phytohemagglutinin (PHA-plant chemical used to stimulate the multiplication of white blood cells, specifically T cells) compared to AEM controls. There was a lower percentage of T cells and higher percentage of natural killer (NK) cells, (both of which are involved in attacking tumor cells) in the FLT mice, but the percentage of B cells (involved in producing antibodies) was similar to AEM controls. The secretion capacity of four cytokines (small secreted proteins that serve to regulate the immune system) in response to activation via signaling molecules, similarly to what occurs in the body, was significantly different in the FLT mice compared with the AEM controls. Cancer-related gene expression profiles in the thymus differed greatly between the FLT and AEM groups. The data obtained from this study collectively exhibits that T cell distribution, function and gene expression are significantly modified shortly after return from the spaceflight environment. However, it remains to be determined whether the quantified changes are brief and primarily due to the tremendous physiological stress of landing and readaptation or have an enduring effect on risk for infection and/or cancer (Gridley 2009).
Studies have shown that the spaceflight environment can impact several physiological systems potentially resulting in serious consequences for immunity. The primary aim of this study was to investigate changes in immune parameters concerning the spleen, liver and thymus in response to flight. C57BL/6 mice (a common inbred strain of lab mice) were flown on a 13-day Space Shuttle mission. In response to flight, the mice exhibited reductions in liver, spleen and thymus masses in comparison to ground controls. The changes in organ masses suggest that the mice were subject to psychological and/or physiological stress inflight or during landing. Splenic (pertaining to the spleen) white blood cells (WBCs) and numbers of leukocyte (cells in the blood that destroy disease-causing microorganisms) subpopulations were significantly reduced after flight. To determine the recovery and proliferative capacity of lymphocytes, this study characterized spontaneous blastogenesis (unstimulated DNA synthesis). The observed increase in [3H]-TdR incorporation into DNA (tritiated thymidine-method for estimating capacity for cell regeneration) by splenic lymphocytes demonstrated that ex vivo (outside of the body) DNA synthesis was increased after flight and suggests that the cells were capable of shifting to a proliferative (or recovery) state once removed from any stress-induced inhibition in vivo (inside of the body). In contrast, LPS (lipopolysaccharides-large molecules found in the outer membrane of many common bacteria) induced proliferation was decreased in the flight mice, indicating that the ability to respond to a potent B cell mitogen (substance that induces cell division) may be compromised. The flight mice demonstrated an increased capacity to produce biological responses, interleukin-6 and interleukin-10 (IL-6 and IL-10 - chemical messengers secreted by cells of the immune system), but not TNF-α (Tumor Necrosis Factor-alpha - protein that can cause tumor cell death when injected into tumor-bearing mice). The genes responsible for scavenging ROS (Reactive Oxygen Species - play vital roles in normal cell functions, but are also sources of tissue and DNA damage) were shown to be up-regulated after flight. The data confirm that immune parameters are influenced by the space flight environment. Furthermore, these data also suggest that exposure to the space flight environment can increase anti-inflammatory mechanisms and change the ex vivo response to LPS, which is a bacterial component that typically induces a strong response from the immune system (Farnaz 2009).
Genetic analysis in young adult mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle for a period of 13 days demonstrate that spaceflight induces significant changes in mRNA expression of genes in the thymus that regulate stress, hormone receptor metabolism, and T white blood cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure microgravity. The results of this study provide insight into how spaceflight affects stress-related gene expression in addition to influencing genes associated with specific immunological processes in the thymus itself. The results also show the connection between many of the altered genes via their relation to a wide range of physiologic processes, including stress and immune response (Lebsack 2010).
Space radiation and microgravity effects on regulation of lung tissue
NASA has reported pulmonary abnormalities in astronauts on space missions, but changes in lung tissue have not been fully documented. CBTM-2 evaluated the health effects on the lungs resulting from increased levels of radiation, inhalation of possible pathogen, and low oxygen levels. Tissue examination showed profibrosis-like (excess growth of fibrous connective tissue) changes occurred in flown mice, more abundant collagen accumulation around blood vessels, and thicker walls compared with lung samples from ground mice. However, no marked abnormality was found in bronchiolar and alveolar lining. The findings suggest that the Flight mice may have experienced some degree of lung remodeling. Taken together, the data demonstrate that significant changes can be readily detected shortly after return from space flight in the expression of factors that can adversely influence lung function. The study concludes that compromised lung function due to space flight may result from disturbance of the balance between deposition and breakdown connective tissue of the lungs. In the future, investigations should be performed on samples taken during flight, after long-term missions, and at later time points after landing to fully document the take-off, residence in space, landing, and oxygen availability effects on lung morphology (Tian 2009).
Ovarian follicular and luteal development in the spaceflight mouse
This study aimed to assess changes in the gross morphology of the ovarian tissue from mice flown on shuttle mission STS-118 and tissues from corresponding control animals. The experiment consisted of three groups of animals: two sets of control animals and one set of flight animals (each set contained 12 mice). The results showed that there was a presence of developing follicles (structures which contain immature eggs) at all stages as well as the presence of corpora lutea (structure formed after the release of the egg) in all three treatment groups indicating no significant gross morphological changes occur within ovarian tissue when exposed to space flight for 13 days or less. Flight tissue was morphologically indistinguishable from both ground control and baseline tissue.
Future studies of this nature should consider the stages of the estrous cycle for each mouse both pre and postflight. This should be confirmed with hormonal data as well as by vaginal smears. Mice should be housed in as small groups as possible. The scent of male excreta could be introduced to the environment to circumvent the Lee-Boot effect, where the estrous cycle of female mice is prolonged or halted in crowded environments or those which lack a male. Analysis of the ovarian tissue should go beyond gross morphology and should look at indicators such as capillary density and kit ligand, a signaling molecule important in the formation of eggs, expression (Smith 2012).
Effects of space flight on the expression of liver proteins in the mouse
The aim was to investigate changes in the profile of major liver proteins of the mouse that are introduced by conditions associated with space flight. Raw data was derived from mass spectroscopic analyses of formalin-fixed paraffin-embedded tissue sections of liver from mice. The raw data was analyzed by two different search engines, using shotgun proteomics, a process by which the proteins are digested and the smaller components separated and read.
Statistically significant (p<0.05) changes of eight proteins were achieved in the mouse liver. Of the 8 proteins, only carbamoyl-phosohate synthetase and 60kDa heat shock protein, a chaperonin, gave significant positive log2 fold change values, a statistical approach for detecting differential expression. Carbomoyl-phosphate synthetase was the protein present in the highest concentration and up-regulation is consistent with increased amino acid breakdown resulting from gravitational changes and/or stress associated with missions in space. Glycine N-methyltransferase, a methyl group transferring enzyme which participates in detoxification chemistry in the liver cells was down-regulated. Fructose-bisphosphate aldolase B levels were also down-regulated.
Results reveal eight of the sixty-seven proteins showed statistical differences between flight and ground. Some of these are linked to detoxification pathways within the liver (carbamoyl-phosohate synthetase, glycine N-methyltransferase, S-adenosylmethionine synthetase) and some to carbohydrate metabolism (fructose-bisphosphate aldolase B, alpha-enolase). 60kDa heat shock protein was up-regulated, probably because of its relation to stress. Regucalcin was highly down-regulated possibly limiting osteoporosis. Ribonuclease UK114, also known as heat responsive protein 12, was down regulated also possibly due to the stress of space flight (Gridley 2012).^ back to top
Tian J, Delp MD, Slater JM, Zawieja DC. Spaceflight Modulates Expression of Extracellular Matrix, Adhesion, and Profibrotic Molecules in Mouse Lung. Journal of Applied Physiology. 2010 January; 108(1): 162-171. DOI: 10.1152/japplphysiol.00730.2009. PMID: 19850731.
Ortega MT, Delp MD, Zawieja DC, Stodieck LS, Ferguson VL, Behnke BJ. Shifts in Bone Marrow Cell Phenotypes Caused by Space Flight. Journal of Applied Physiology. 2009 Feb; 106(2): 548-555. DOI: 10.1152/japplphysiol.91138.2008. PMID: 19056998.
Baqai FP, Zawieja DC, Slater JM, Luo-Owen X, Stodieck LS, Ferguson VL, Behnke BJ, Delp MD. Effects of spaceflight on innate immune function and antioxidant gene expression. Journal of Applied Physiology. 2009; 106: 1935-1942. DOI: 10.1152/japplphysiol.91361.2008.
Latchney SE, Rivera PD, Mao XW, Ferguson VL, Bateman TA, Stodieck LS, Nelson GA, Eisch AJ. The effect of spaceflight on mouse olfactory bulb volume, neurogenesis, and cell death indicates the protective effect of novel environment. Journal of Applied Physiology. 2014 June 15; 116(12): 1593-1604. DOI: 10.1152/japplphysiol.01174.2013.
Zawieja DC, Slater JM, Luo-Owen X, Rizvi A, Behnke BJ, Stodieck LS, Ferguson VL, Delp MD. Spaceflight Effects on T-lymphocyte distrubution, function and gene expression. Journal of Applied Physiology. 2009 January; 106(1): 194-202. DOI: 10.1152/japplphysiol.91126.2008. PMID: 18988762.
Mao XW, Delp MD, Stodieck LS, Ferguson VL, Bateman TA, Bouxsein ML, Jones TA, Moldovan M, Cunningham C, Chieu J, Zawieja DC. Spaceflight environment induces mitochondrial oxidative damage in ocular tissue. Radiation Research. 2013 September 13; epub. DOI: 10.1667/RR3309.1.
Zawieja DC, Delp MD, Green LM, Herrmann EC, Bianski B, Slater JM, Stodieck LS, Ferguson VL, Sandberg LB. Effects of Space Flight on the Expression of Liver Proteins in the Mouse. Journal of Proteomics and Bioinformatics. 2012; 5(10): 256-261. DOI: 10.4172/jpb.1000246.
Dabertrand F, Porte Y, Macrez N, Morel J. Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension.. Journal of Applied Physiology. 2012 February 1; 112(3): 471-480. DOI: 10.1152/japplphysiol.00733.2011.
Sung M, Li J, Spieker AJ, Spatz JM, Ellman R, Ferguson VL, Bateman TA, Rosen GD, Bouxsein ML, Rutkove SB. Spaceflight and hind limb unloading induce similar changes in electrical impedance characteristics of mouse gastrocnemius muscle. Journal of Musculoskeletal and Neuronal Interactions. 2013 December; 13(4): 405-411. PMID: 24292610.
Zawieja DC, Mao XW, Stodieck LS, Ferguson VL, Bateman TA, Moldovan M, Cunningham C, Jones TA, Slater JM, Delp MD. Changes in Mouse Thymus and Spleen after Return from the STS-135 Mission in Space. PLOS ONE. 2013 September 19; 8(9): e75097. DOI: 10.1371/journal.pone.0075097.
Romer C, Forsman AD. The effects of the spaceflight environment on the vaginal mucin layer of the mouse. Gravitational and Space Research. 2015 July; 3(1): 20-28.
Lebsack , Fa , Woods , Gruener , Manziello , Delp MD, Zawieja DC, Stodieck LS, Ferguson VL, DeLuca . Microarray Analysis of Spaceflown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors. Journal of Cellular Biochemistry. 2010 May 15; 110(2): 372-381. DOI: 10.1002/jcb.22547. PMID: 20213684.
Smith IM, Forsman AD. Ovarian Follicular and Luteal Development in the Spaceflight Mouse. Gravitational and Space Biology. 2012; 26(2): 30-37.
Ground Based Results Publications
Cannon CM, Dieter-Seelig H, Stodieck LS, Hanson AM, Simske SJ, Ferguson VL. A novel combination of methods to assess sarcopenia and muscle performance in mice. Biomedical Sciences Instrumentation. 2005; 41: 116-121.
Harrison BC, Allen DL, Girten BE, Stodieck LS, Kostenuik PJ, Bateman TA, Morony SE, Lacey DL, Leinwand LL. Skeletal muscle adaptations to microgravity exposure in the mouse. Journal of Applied Physiology. 2003; 95(6): 2462-2470. DOI: 10.1152/japplphysiol.00603.2003. PMID: 12882990.
Lloyd SA, Ferguson VL, Simske SJ, Dunlap AW, Livingston EW, Bateman TA. Housing in the Animal Enclosure Module Spaceflight Hardware increases trabecular bone mass in ground-control mice. Gravitational and Space Research. 2013 October; 1(1): 2-19.
Bar-Shai M, Carmeli E, Coleman R, Reznick AZ. Mechanisms in muscle atrophy in immobilization and aging. Annals of the New York Academy of Sciences. 2004; 1019: 475-478.
NASA Image S118E09308: The Commercial Biomedical Test Module - 2 (CBTM-2) hardware seen in this image flew onboard STS-118/13A.1 in August 2007. CBTM-2 will test the effectiveness of an experimental therapeutic as a possible countermeasure for muscle atrophy.
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