ALBERT

Description: Spaceflight has deleterious effects on skeletal structure and function, specifically causingprofound loss in bone mass, density, and strength, as well as changes in expression levels of genes related to oxidative stress [Hyeon et al., Smith et al.]. It is known that bone resorption remains elevated after spaceflight and that bone density and strength fail to recover completely even years following spaceflight [Smith et al., Carpenter et al.]. However, our current understanding of the signaling pathways and molecular mechanisms that control bone loss and that link oxidative stress, bone resorption, and mechanical unloading of skeletal tissue is incomplete. Here, we aim to examine skeletal responses to simulated long-duration spaceflight on bone loss using the ground-based hindlimb unloading (HU) model in adult (9 months old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative-defense genes and pro-osteoclastogenic factors, showing progression and eventual plateau during long-term unloading, and that transient changes at early timepoints in these pathways precede skeletal adaptations to long-duration unloading. We will identify oxidativestress and bone resorption-related changes using global gene expression analysis (Affymetrix arrays) for both acute (within 14 days) and long-term timepoints (90 days). We will also use quantitative PCR to examine changes in expression of genes related to oxidative metabolism (e.g. Nrf2, SOD-1), bone turnover (resorption and formation markers, e.g. TRAP, osteocalcin respectively, SOST), and osteoclastogenesis (e.g. RANKL, OPG) at both early and late timepoints. We will then use detailed microarchitectural and structural analysis through microcomputed tomography to relate gene expression changes with structural changes in bone, expecting that plateaus in gene expression correlate with long-term changes in bone microarchitecture.

Description: Space radiation and micro-gravity are the two major obstacles impeding human exploration of Mars and beyond. Long-duration space flights expose astronauts to high doses of high linear energy transfer (LET) radiation as well as prolonged periods of skeletal disuse due to weightlessness. One important consequence of both radiation exposure and micro-gravity is acute bone loss. However, biological responses to different radiation types and combined radiation and micro-gravity environments remain unknown. Thus, the purpose of this study is to compare the acute effects of different radiation species and simulated weightlessness on bone degeneration for the purpose of developing accurate risk assessments of prolonged space flight. Mouse models were used to simulate space flight-relevant doses of different radiation types as well as weightlessness via hind-limb unloading. Three groups of mice (n 9) were irradiated with 1 Gy (Gray) H+, 1 Gy 56Fe, and 1 Gy combined H+ and 56Fe (dual ion) respectively and compared to sham irradiated (n 9) and 2 Gy 56Fe irradiated positive controls (n 6). Two groups of mice (n 9) were hind-limb unloaded for three days and then either sham irradiated or dual ion irradiated respectively, followed by subsequent hind-limb unloading for 11 days. Cancellous tissue from tibiae metaphyses were harvested 11 days post-irradiation for ex vivo micro-computed tomography analysis. Microarchitecture parameters including bone volume to total volume ratio (BVTV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.S), and connectivity density (Conn.D) will be quantified using a novel automated segmentation procedure developed in our lab. The anticipated results will be instrumental in developing counter-measures against micro-gravity and radiation-induced bone loss. Moreover, possible synergistic effects may provide insight into underlying mechanisms mediating biological response.

Description: Damaging effects due to spaceflight and long-duration weightlessness are seen in the musculoskeletal system, specifically with regards to bone loss, bone resorption, and changes in overall bone structure. These adverse effects are all seen with indicators of oxidative stress and a variation in the levels of oxidative gene expression. Once gravity is restored, however, the recovery is slow and incomplete. Despite this, few reports have investigated the correlation between oxidative damage and general modifications within the bone. In this project, we will make use of a ground-based model of simulated weightlessness (hindlimb unloading, HU) in order to observe skeletal changes in response to induced microgravity due to changes in oxidative pressures. With this model we will analyze samples at 14-day and 90-day time points following HU for the determination of acute and chronic effects, each with corresponding controls. We hypothesize that simulated microgravity will lead to skeletal adaptations including time-dependent activation of pro-oxidative processes and pro-osteoclastogenic signals related to the progression, plateau, and recovery of the bone. Microcomputed tomography techniques will be utilized to measure skeletal changes in response to HU. With the results of this study, we hope to further the understanding of skeletal affects as a result of long-duration weightlessness and develop countermeasures to combat bone loss in spaceflight and osteoporosis on Earth.

Description: As humans venture further into space and beyond low Earth orbit, space radiation is one of the main challenges for astronauts' health. Radiation-induced bone loss is a potential health problem for long duration habitation in space. We showed that a dietary countermeasure prevents bone loss in mice exposed to total body irradiation (TBI). We used a range of ionizing radiation, gamma (137Cs), proton (1H), iron (56Fe), and a combination of sequential proton and iron beam (1H/56Fe/1H) to evaluate skeletal responses. These TBI cover a range of linear energy transfer (LET), from low-LET such as proton, to high-LET such as 56Fe (HZE: high Z- high energy) at doses between 1-2 Gy. The countermeasure diet, composed of 25% Dried Plum (DP) was effective at preventing radiation-induced cancellous bone loss in appendicular bone (tibia). Furthermore, exposing mice to HZE radiation, such as 56Fe (1Gy), impaired ex vivo growth of marrow-derived, bone-forming osteoblasts, which led to reduced mineralization capacity (-77%). In contrast, mice fed the DP diet did not display these deficits, showing the diet's capacity to protect marrow-derived osteoprogenitors. Dietary DP prevented the increase of bone resorbing osteoclast cells, inflammation and oxidative stress, while protecting the osteoprogenitors and mesenchymal stem cells, which few drugs against osteoporosis may achieve. Spaceflight is a combination of multiple factors including microgravity, in addition to space radiation. Therefore, we conducted additional studies to determine if the DP diet could prevent simulated spaceflight (simulated microgravity and radiation combined) bone loss. Mice were exposed to gamma (TBI, 137Cs, 2 Gy), simulated microgravity (using the hindlimb unloading system, HU) or TBI+HU. While we observed bone loss in mice fed the control diet (CD) due to both treatments (TBI=14%, HU=20%), and a worse effect with combined treatments (TBI+HU=25%), mice fed the DP diet did not sustain significant bone loss relative to untreated controls. The DP diet prevented microarchitectural decrements in both appendicular bone (tibia) and axial bone (vertebrae). In addition, the DP diet mitigated HU-induced deficits in osteoblastogenesis. Interestingly, lower doses of DP diet (5%, 10%) did not appear to prevent cancellous bone loss, which shows the importance of identifying the active component(s) of DP. Finally, we have preliminary data showing the potential of DP to prevent radiation-induced damage at a systematic level.. In summary, this novel dietary countermeasure is a promising candidate nutritional countermeasure for spaceflight-induced bone loss and tissue damage.

Description: Future long-duration space exploration beyond low earth orbit will increase human exposure to space radiation and microgravity conditions as well as associated risks to skeletal health. In animal studies, radiation exposure (greater than 1 Gy) is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility. Definitive measurements and detection of bone loss typically require large and specialized equipment which can make their application to long duration space missions logistically challenging. Towards the goal of developing non-invasive and less complicated monitoring methods to predict astronauts' health during spaceflight, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (IR). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and IR (0.5 Gy 56Fe 600 MeV/n and 0.5 Gy 1H 150 MeV/n), euthanized at one and 11 days post-irradiation (IR). To determine the extent of bone loss, tibiae were harvested and cancellous microarchitecture in the proximal tibia quantified ex vivo using microcomputed tomography (microCT). Statistical analysis was performed using Student's t-test. At one day post-IR, expression of FGF18 in skin was significantly greater (3.8X) than sham-irradiated controls, but did not differ at 11 days post IR. Expression levels of other genes associated with antioxidant response (Nfe2l2, FoxO3 and Sod1) and the cell cycle (Trp53, Cdkn1a, Gadd45g) did not significantly differ between the control and IR groups at either time point. Radiation exposure resulted in a 27.0% increase in FGF18-positive hair follicles at one day post-IR and returned to basal levels at 11 days post-IR. A similar trend was observed from FGF18 gene expression analysis of skin. In bone (femora), there was an increase in the expression of the pro-osteoclastogenic cytokine, MCP-1, one day after IR compared to non-irradiated controls. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibia from these animals showed reduced cancellous bone volume (-9.9%) at 11 days post- IR. These results suggest that measurements of early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass. Further research may lead to the development of a relatively simple diagnostic tool for bone loss, with the advantage that hair follicles and skin are relatively easy to acquire from human subjects.

Description: In this study, we aim to examine skeletal responses to simulated long-duration spaceflight (90 days) and weight-bearing recovery on bone loss using the ground-based hindlimb unloading (HU) model in adolescent (3-month old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative defense genes and pro-bone resorption factors, where there is a progression and eventual plateau; furthermore, early transient changes in these pathways precede skeletal adaptations.

Description: Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The potential for exposure to space radiation to contribute to lasting decrements in bone mass is not yet understood. Sustained changes to bone mass have a relatively long latency for development, however skin is a radiation sensitive organ and changes in skin gene expression may serve as an early radiation biomarker of exposures and may correlate with adverse effects on skeletal tissue. Previous studies have shown that FGF18 gene expression levels of hair follicles collected from astronauts on the ISS rose over time. In the hair follicle, FGF18 signaling mediates radioresistance in the telogen by arresting the cell cycle, and FGF18 has the potential to function as a radioprotector. In bone, FGF18 appears to regulate cell proliferation and differentiation positively during osteogenesis and negatively during chondrogenesis. Cellular defense responses to radiation are shared by a variety of organs, hence in this study, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We have examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (TBI). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and TBI (0.5 Gy Fe-56 600 MeV/n and 0.5 Gy H-1 150 MeV/n). Animals were euthanized one and 11 days post-IR. Statistical analysis was performed via a Student's ttest. In skin samples one day after IR, skin expression of FGF18 was significantly greater (3.8X) than sham-irradiated controls (3.8X), but did not differ 11 days post TBI. Expression levels of other radiation related genes (Nfe2l2, Trp53, Cdkn1a, FoxO3, Gadd45g, SOD1), was not different due to TBI at either time point. In bone (femora) TBI significantly increased (3.8X) expression of the pro-bone resorption cytokine, MCP-1, one day after TBI. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibae from these animals showed reduced fractional cancellous bone volume (-21.7%) at 11 days post exposure. These results suggest that early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass.