ALBERT

Description: Future long duration missions outside the protection of the Earth's magnetosphere, or unshielded exposures to solar particle events, achieves total doses capable of causing cancellous bone loss. Cancellous bone loss caused by ionizing radiation occurs quite rapidly in rodents: Initially, radiation increases the number and activity of bone-resorbing osteoclasts, followed by decrease in bone forming osteoblast cells. Here we report that Dried Plum (DP) diet completely prevented cancellous bone loss caused by ionizing radiation (Figure 1). DP attenuated marrow expression of genes related to bone resorption (Figure 2), and protected the bone marrow-derived pre-osteoblasts ex vivo from total body irradiation (Figure 3). DP is known to inhibit resorption in models of aging and ovariectomy-induced osteopenia; this is the first report that dietary DP is radioprotective.

Description: Radiation-induced bone loss can occur with radiotherapy patients, accidental radiation exposure and during long-term spaceflight. Bone loss due to radiation is due to an early increase in oxidative stress, inflammation and bone resorption, resulting in an imbalance in bone remodeling. Furthermore, exposure to high-Linear Energy Transfer (LET) radiation will impair the bone forming progenitors and reduce bone formation. Radiation can be classified as high-LET or low-LET based on the amount of energy released. Dried Plum (DP) diet prevents bone loss in mice exposed to total body irradiation with both low-LET and high-LET radiation. DP prevents the early radiation-induced bone resorption, but furthermore, we show that DP protects the bone forming osteoblast progenitors from high-LET radiation. These results provide insight that DP re-balances the bone remodeling by preventing resorption and protecting the bone formation capacity. This data is important considering that most of the current osteoporosis treatments only block the bone resorption but do not protect bone formation. In addition, DP seems to act on both the oxidative stress and inflammation pathways. Finally, we have preliminary data showing the potential of DP to be radio-protective at a systemic effect and could possible protect other tissues at risk of total body-irradiation such as skin, brain and heart.

Description: Ionizing radiation-induced bone loss appears to be a two-stage process: first an early increase in pro-resorption cytokines and increased bone resorption by osteoclasts, followed by a decrease in bone formation by osteoblasts. This results in a net loss of mass in mineralized bone tissue. The molecular mechanisms underlying the imbalance in bone remodeling caused by exposure to radiation are not fully understood. We hypothesized that the radiation-induced rise in reactive oxygen species (ROS) damages osteoblast progenitors, leading to a decrease in number and activity of differentiated progeny. We have shown that a diet high in antioxidant capacity prevents radiation-induced bone loss in adult mice (Schreurs et al. 2016) by reducing the early increase in pro-resotption cytokines. Here, we investigated the damaging effects of radiation exposure on cells in the osteoblast lineage, testing if addition of the exogenous antioxidant enzyme, superoxide dismutase (SOD) can mitigate radiation damage. Osteoprogenitors were grown in vitro from the marrow of 16wk old, male C57Bl/6 mice. Cells were irradiated 3 days after plating (day 0) with either gamma (Cs-137, 0.1-5Gy) or iron (Fe-56, 600 MeV/n, 0.5-2Gy), and then grown until day 10. SOD or vehicle was added 2 hours before irradiation (SOD at 200U/ml), twice a day and up to day 5, for a total of 2 days treatment. Cell behavior was assessed by: (a) colony number (counted on day 7), (b) DNA content (surrogate for cell number) to assess cell growth (percent change between day 3 and day 10) and (c) alkaline phosphatase activity (osteoblast differentiation marker). Results show that SOD protected cells from the adverse effects of low-LET ionizing radiation, but not high-LET radiation. These novel results provide an interesting platform to explore further diverse effects and damages caused by low-LET and high-LET, pointing toward different mechanisms and possible intervention strategies for radiation-induced bone loss.

Description: NASA has flown animals to space as part of trailblazing missions and to understand the biological responses to spaceflight. Mice traveled in the Lunar Module with the Apollo 17 astronauts and now mice are frequent research subjects in LEO on the ISS. The ISS rodent missions have focused on unravelling biological mechanisms, better understanding risks to astronaut health, and testing candidate countermeasures. A critical barrier for longer-duration animal missions is the need for humans-in-the-loop to perform animal husbandry and perform routine tasks during a mission. Using autonomous or telerobotic systems to alleviate some of these tasks would enable longer-duration missions to be performed at the Deep Space Gateway. Rodent missions performed using the Gateway as a platform could address a number of critical risks identified by the Human Research Program (HRP), as well as Space Biology Program questions identified by NRC Decadal Survey on Biological and Physical Sciences in Space, (2011). HRP risk areas of potentially greatest relevance that the Gateway rodent missions can address include those related to visual impairment (VIIP) and radiation risks to central nervous system, cardiovascular disease, as well as countermeasure testing. Space Biology focus areas addressed by the Gateway rodent missions include mechanisms and combinatorial effects of microgravity and radiation. The objectives of the work proposed here are to 1) develop capability for semi-autonomous rodent research in cis-lunar orbit, 2) conduct key experiments for testing countermeasures against low gravity and space radiation. The hardware and operations system developed will enable experiments at least one month in duration, which potentially could be extended to one year in duration. To gain novel insights into the health risks to crew of deep space travel (i.e., exposure to space radiation), results obtained from Gateway flight rodents can be compared to ground control groups and separate groups of mice exposed to simulated Galactic Cosmic Radiation (at the NASA Space Radiation Lab). Results can then be compared to identical experiments conducted on the ISS. Together results from Gateway, ground-based, and ISS rodent experiments will provide novel insight into the effects of space radiation.

Description: Astronauts using high-force resistance training while weightless show a high-turnover remodeling state within the skeletal system, with resorption and formation biomarkers being elevated. One countermeasure for the skeletal health of astronauts includes an antiresorptive of the bisphosphonate (BP) drug class. We asked, does the combination of an anti-resorptive and high-force exercise during weightlessness have negative effects on bone remodeling and strength? In this study, we developed an integrated model to mimic the mechanical strain of exercise via cyclical loading (CL) in mice treated with the BP Zoledronate (ZOL) combined with hind limb unloading (HU) to simulate weightlessness. We hypothesized that ZOL prevents structural degradation from simulated weightlessness and that CL and ZOL interact to render CL less effective. Thirty-two C57BL/6 mice (male, 16 weeks old, n=8/group) were exposed to 3 weeks of either HU or normal ambulation (NA). Cohorts of mice received one subcutaneous injection of ZOL (45g/kg), or saline vehicle (VEH), prior to the start of HU. The right tibia was axially compressed in vivo 60x/day to 9N (+1200strain on the periosteal surface) and repeated 3x/week during HU. Left tibiae served as a within subject, non-compressed control. Ex vivo CT was performed on all subjects to determine cancellous and cortical architectural parameters. Static and dynamic histomorphometry were carried out for the left and right tibiae to determine osteoclast- and osteoblast relevant surfaces. Further, micro damage was assessed in select groups by basic-fuchsin staining to test whether CL had an effect. For all assays, a multivariate (2x2x2) ANCOVA model was used to account for body weight changes. Additionally, for the tibiae, we incorporated a random effect for the subject (hence, a mixed model) to account for observations of both left and right tibiae within each subject. P 〈 0.05 was considered significant. In the cancellous compartment of the proximal tibial metaphysis, we observed a main effect from each independent variable, as determined by structural and histomorphometric assays. Specifically, as expected, ZOL showed an increase in the cancellous bone volume to total volume fraction (BV/TV, +32%) and trabecular number (+18%) compared to the VEH. As expected, ZOL decreased osteoclast surface (OC/BS) by -45% compared to VEH. Surprisingly, ZOL reduced mineralizing surface (MS/BS) and bone formation rate (BFR), indicators of osteoblast activity, by -40% and -54%, respectively, compared to VEH. Altogether, ZOL-treated mice displayed a low turnover state of remodeling in the metaphysis. In the context of skeletal aging, we speculate that ZOL prevented age-related cancellous strut loss during the experiment. As a main effect, as expected, HU decreased BV/TV by - 31% via reductions in both trabecular thickness (-11%) and number (-22%) compared to NA controls. Additionally, HU decreased MS/BS by -38% and bone formation rate (BFR) by -50% compared to NA controls. Altogether, these data are consistent with structural degradation resulting from imbalanced remodeling that favors resorption. As a main effect, CL increased BV/TV by +15% via increased trabecular thickness (+12%) compared to the noncompressed limb. As expected, CL increased MS/BS (+20%) and BFR (+24%), indicating osteoblast mineralization contributed to bone gains. These data show that CL provided an anabolic stimulus to the cancellous tissue. We observed unique interactions in ZOL*CL and HU*CL. First, ZOL prevented CL-induced increases in BV/TV and trabecular number, as compared to VEH. In the context of skeletal aging, these data suggest no added benefit from CL in the ZOL-treated mice. Interestingly, no microdamage was observed in mice that were unloaded and treated with ZOL (independent of CL). Secondly, HU prevented CL-induced increases in BFR, as compared to NA controls. These data suggest that either exercise is less effective or the kinetics of formation are slower during simulated weightlessness. Osteoclast surface was unchanged by either treatment. Thus, in contrast to exercising astronauts, these data do not suggest a high-turnover state in the metaphysis. To assess mechanical properties as a function of HU or ZOL, we tested the left femur in three-point bending ex vivo. As expected, HU decreased stiffness (-30%) compared to NA, and ZOL increased stiffness compared to VEH (+28%). Interestingly, HU increased the post-yield displacement, related to collagenous tensile loading, compared to NA (+20%). ZOL increased yield force (+11%) and ultimate force (+17%), which seems to explain the significant effect of ZOL increasing total energy (work-to-fracture, +15%), while not affecting the post yield displacement. Taken together, ZOL did not have detrimental affect on mechanical properties. Our integrated model simulates the combination of weightlessness, exercise-induced mechanical strain, and anti-resorptive treatment that astronauts experience during space missions. We conclude that Zoledronate was an effective countermeasure against weightlessness-induced bone loss, though zoledronate, as well as weightlessness, rendered exercise-related mechanical loading less effective.

Description: Space flight modulates bone remodeling to favor bone resorption. Current countermeasures include an anti-resorptive drug class, bisphosphonates (BP), and high-force loading regimens. Does the combination of anti-resorptives and high-force exercise during weightlessness have negative effects on the mechanical and structural properties of bone? In this study, we implemented an integrated model to mimic mechanical strain of exercise via cyclical loading (CL) in mice treated with the BP Zoledronate (ZOL) combined with hindlimb unloading (HU). Our working hypothesis is that CL combined with ZOL in the HU model induces additive structural and mechanical changes. Thirty-two C57BL6 mice (male,16 weeks old, n8group) were exposed to 3 weeks of either HU or normal ambulation (NA). Cohorts of mice received one subcutaneous injection of ZOL (45gkg), or saline vehicle, prior to experiment. The right tibia was axially loaded in vivo, 60xday to 9N in compression, repeated 3xweek during HU. During the application of compression, secant stiffness (SEC), a linear estimate of slope of the force displacement curve from rest (0.5N) to max load (9.0N), was calculated for each cycle once per week. Ex vivo CT was conducted on all subjects. For ex vivo mechanical properties, non-CL left femurs underwent 3-point bending. In the proximal tibial metaphysis, HU decreased, CL increased, and ZOL increased the cancellous bone volume to total volume ratio by -26, +21, and +33, respectively. Similar trends held for trabecular thickness and number. Ex vivo left femur mechanical properties revealed HU decreased stiffness (-37),and ZOL mitigated the HU stiffness losses (+78). Data on the ex vivo Ultimate Force followed similar trends. After 3 weeks, HU decreased in vivo SEC (-16). The combination of CL+HU appeared additive in bone structure and mechanical properties. However, when HU + CL + ZOL were combined, ZOL had no additional effect (p0.05) on in vivo SEC. Structural data followed this trend with ZOL not modulating trabecular thickness in CL + NAHU mice. In summary, our integrated model simulates the combination of weightlessness, exercise-induced mechanical strain, and anti-resorptive treatment that astronauts experience during space missions. Based on these results, we conclude that, at the structural and stiffness level, zoledronate treatment during simulated spaceflight does not impede the skeletal response to axial compression. In contrast to our hypothesis, our data show that zoledronate confers no additional mechanical or structural benefit beyond those gained from cyclical loading.

Description: Microgravity and ionizing radiation may contribute to cellular stress; resulting in increased generation of reactive oxygen species (ROS), DNA damage, cell cycle arrest, and cell death. We hypothesized that suppression of excess ROS in osteoblasts and osteoclasts will improve bone microarchitecture. To test our hypothesis, we used irradiated transgenic mCAT mice overexpressing human anti-oxidant catalase gene targeted to the mitochondria (main site for ROS production). mCAT mice expressed the transgene and displayed elevated catalase activity in bone and ex vivo osteoblast and osteoclast cultures. Treated bone from wildtype mice showed elevated levels of oxidative damage whereas mCAT mice did not. Also, increased catalase activity correlated with decreased MDA levels and that increased oxidative damage correlated with decreased % bone volume. Ex-vivo osteoblast colony growth positively correlated with osteoblast catalase activity. mCAT mice displayed reduced % bone volume. Treatment caused significant bone loss in wildtype mice. Treatment also caused slight deficits in microarchitecture of mCAT mice. In conclusion, ROS signaling in both osteoblast and osteoclast lineage cells contribute to skeletal development and remodeling and quenching oxidative damage could play a role in bone loss prevention.

Description: Long-term spaceflight leads to extensive changes in the musculoskeletal system attributable, in part, to unloading during microgravity exposure. Additionally, irradiation at doses similar to that of a solar flare or a round-trip sojourn to Mars may cause significant depletion of stem/progenitor cell pools throughout the body as well as inflammation associated with prompt skeletal-tissue degradation. Previously, we demonstrated that irradiation leads to rapid bone loss, which can be mitigated in the short term by injection of a potent antioxidant (-lipoic acid). Furthermore, simulated weightlessness in adult mice adversely affects skeletal responses to low linear energy transfer (LET) radiation (137Cs). Here, we hypothesized that simulated weightlessness exacerbates the adverse effects of simulated space radiation (including both protons and 56Fe ions) by adversely affecting skeletal structure and functions as well as associated vasculature. Furthermore, we hypothesized that an antioxidant cocktail, which has been shown to be protective in other tissues, mitigates space radiation induced bone loss.

Description: Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even after return to Earth. We hypothesize that heavy-ion irradiation, a component of Galactic Cosmic Radiation, damages osteoblast progenitors and may contribute to bone loss during long duration space travel beyond the protection of the Earth's magnetosphere. Male, 16 week-old C57BL6/J mice were exposed to high-LET (56-Fe, 600MeV) radiation using either low (5 or 10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 3-4, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 m pixel size) and marrow cell redox assessed using membrane permeable, free radical-sensitive fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo, then mineralized nodule formation quantified by imaging and gene expression analyzed by RT-PCR. Interestingly, 3-4 days post-exposure, fluorogenic dyes that reflect cytoplasmic generation of reactive nitrogen/oxygen species (DAF-FM Diacetate or CM-H2DCFDA) revealed irradiation (50cGy) reduced free radical generation (20-45%) compared to sham-irradiated controls. Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation (MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive oxygen/nitrogen levels remained lower (24%) relative to sham-irradiated controls. After one month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy) compared to controls. High dose irradiation (200cGy) up-regulated expression of a late osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure or ex vivo nodule formation, but did down-regulate iNOS by 0.54-0.58 fold. Thus, both low- and high-doses of heavy-ion irradiation cause time-dependent, adaptive changes in redox state within marrow cells but only high doses (50, 200cGy) inhibit osteoblastogenesis and cause cancellous bone loss. We conclude space radiation has the potential to cause persistent damage to bone marrow-derived stem and progenitor cells for osteoblasts despite adaptive changes in cellular redox state.