Continuing data analyses on experiments supporting Specific Aim 2 [Determine impact of low-dose, high LET radiation exposure (modeling that expected on the Lunar surface) on musculoskeletal outcomes in modeled Lunar gravity conditions] allowed for more refinement of conclusions reached during year 3. In support of our original hypothesis, radiation exposure exacerbates the loss of cancellous bone (%BV/TV) seen with partial weightbearing; it took only 0.5 Gy of 28Si exposure to produce the same 14% decline in %BV/TV as observed with 1 Gy of reference x-ray radiation. The primary effect on cancellous bone microarchitecture was a decrease in trabecular number but not trabecular thickness, suggesting more of an effect on osteoclast resorptive activity than on osteoblast formation activity, consistent with published literature. One of our most intriguing results was that fractionating the 0.5 Gy 28Si dose into 3 doses spread over the period of PWB produced the same net bone loss as did one acute dose delivered on the first day of PWB. By contrast, fractionating our 1 Gy x-ray exposure into 3 exposures DID effectively mitigate loss of bone.

The major task accomplished in Year 4 of this project was to complete experiments supporting our final specific aim: would low dose, high LET radiation impair the ability of bone and muscle to respond to exercise during recovery from a period of simulated Lunar gravity conditions? Marshaling all available budgetary means, we returned to NSRL at Brookhaven to test impact of simulated GCR on the exercise response in bone and muscle, rather than relying on reference (x-ray) radiation. During NSRL's Fall 2011 run, we successfully exposed ~65 mice to acute dose (0.5 Gy) of 56-Fe, before they were shipped to Texas A&M, where the animal protocols were conducted, finishing in April 2012. We have generated some exciting and novel data on vertebral bone responses to PWB and recovery there from: the 0.5 Gy 56Fe exposure appears to significantly impair the ability of spinal bone to regain cancellous bone mass lost during the preceding period of PWB. In fact, further losses in trabecular thickness are observed during the recovery period (with and without exercise), implying a sustained suppression of osteoblast activity from the radiation exposure imposed 6 weeks earlier. Similar findings held for cancellous bone in the distal femur, but the effects are less dramatic than in vertebral bone. Preliminary assessment of bone formation rate (BFR) on the periosteal surface of the tibial shaft indicates a significant reduction in this marker of osteoblast activity in this separate bone compartment. On-going histomorphometric analyses of BFR in the distal femur will help verify if high LET radiation also suppresses osteoblast function some 6 weeks later in cancellous bone.

We have also produced some novel data regarding skeletal muscle outcomes from combined partial weightbearing and simulated galactic cosmic radiation exposure. Simulated Lunar gravity resulted in significant decreases in muscle mass and rates of muscle protein synthesis. However, contrary to the working hypothesis, both ion species used (0.5 Gy28Si, 300 MeV or 0.5 Gy 56Fe, 1GeV), contributed to an increase in muscle wet mass. In viable muscle, alterations of muscle mass are associated with alterations of protein content that are proportional with water content. Thus, in healthy muscle, changes in muscle mass do not lead to changes in protein concentration within the tissue. Our analyses reveal that protein concentration was constant among groups in the 28Si groups, but not 56Fe groups. Furthermore, it appears that alterations in fractional synthesis rates (assessed using novel deuterium-oxide methodologies) were consistent with changes in protein concentrations, where lower FSRs were documented in tissues with lower protein concentrations. In summary, our results verify that the increases in gastrocnemius wet mass exposed to 0.5 Gy of 300 MeV 28Si are reflective of muscle gains, with or without the presence of partial loading. Gains due to 50cGy exposure of 1 GeV 56Fe appear to indicate an accumulation of extraneous, non-protein substances. These experiments were designed to answer IRP Risk Degen 7 ["Are there significant synergistic effects from other spaceflight factors (microgravity, stress, altered circadian rhythms, changes in immune responses, etc.) that modify the degenerative risk from space radiation?"] in addition to IRP Risk B11 ["What are the effects of radiation on bone?"] Understanding the interaction of reduced weightbearing and radiation exposure is an important goal in support of human exploration of space. Although not designed to thoroughly explain mechanisms for observed effects on bone and skeletal muscle, we are generating supporting analyses to provide some insight to direct future investigations.

Our experiments focusing on effects of low-dose radiation on musculoskeletal structure and function provide unique and novel data about the potential degenerative effects to be expected by those humans living in areas with high natural background radiation (e.g., Ramsar, Iran); by individuals who accumulate high occupational exposures to ionizing radiation (e.g., commercial airplane crews); and by patients accumulating multiple medical irradiation exposures over time. A growing literature is documenting surprising and deleterious effects on bone with low-level radiation (as opposed to the high doses used in radiotherapy for cancer patients) and our results are consistent with those findings, especially in bone sites rich in cancellous bone, such as the femoral neck (site of hip fractures) and in vertebral bone. Very little is known at the present time about the impact on these low doses on maintenance of normal muscle protein synthesis and muscle mass. Our early results suggest a surprising (but small) gain in muscle mass with very low dose, high LET radiation exposure, even during a period of partial weightbearing. However, protein synthesis appears to be impaired, so these gains in mass may not reflect gains in functional tissue (perhaps connective tissue).

Specific Aim 2 (impact of low-dose, high energy radiation simulating galactic cosmic radiation): All experiments were completed earlier; during Year 4 of this project we focused on finalizing histomorphometry analyses, muscle protein synthesis determinations and started on manuscript preparation. Experiment 2 (pilot experiment testing various doses of x-ray radiation in weightbearing mice, with primary cell culture studies) manuscript is in preparation for submission to the journal Radiation Research; progress has been slowed by the departure of the post-doctoral fellow (F. Lima) in charge, who has committed to a February, 2013 deadline. A second manuscript targeted to Radiation Research is being submitted in March 2013 detailing the impact of x-ray and high LET (28-Si) radiation exposures on cortical bone (B.R. Macias, first author); some of these data were also presented at the International Congress of Radiation Research (Warsaw, Poland; August, 2011) and at the American Society for Bone and Mineral Research, Annual Meeting (San Diego, CA; September, 2011). At least one other bone-oriented manuscript will be submitted as soon as we finalize the more labor-intensive histomorphometry analyses on cancellous bone from these experiments. Muscle protein synthesis and related cell signaling data (by Western blot) collection is now complete; histological assays for BrdUincorporation and fiber-type specific cross-sectional areas are in progress and should provide compelling data for several more manuscripts.

Specific Aim 3 (impact of simulated GCR on bone/muscle response to exercise during recovery from a period of partial weightbearing(PWB): We completed an extensive pilot experiment in summer of 2011 comparing the efficacy of two exercise paradigms in mitigating bone loss during a PWB period: treadmill running (endurance training) vs. tower climbing (more resistance based). (A manuscript of these data will be ready for submission to Acta Astronautica in late February, 2013; R. Boudreaux, first author.) Marshaling all available budgetary means, we returned to NSRL at Brookhaven to test impact of simulated GCR on the exercise response in bone and muscle, rather than relying on reference (x-ray) radiation. During NSRL's Fall 2011 run, we successfully exposed ~65 mice to acute dose (0.5 Gy) of 56-Fe, before they were shipped to Texas A&M, where the animal protocols were conducted, finishing in April 2012. All micro-CT analyses were finished by December 2012, as well as some mechanical testing results. On-going are histomorphometry analyses and skeletal muscle histology. We anticipate 2-3 more manuscripts will result from these data within the next 6 months.

The major animal protocol work in Project Year 3 started with an intensive 5 ½ week stay at Brookhaven National Laboratory in June 2010 conducting a 3-week animal experiment testing responses to simulated galactic cosmic radiation in weightbearing (1 g) and partial (1/6) weightbearing mice, using 28Si, 300 MeV at the NASA Space Radiation Space Laboratory (NSRL). At experiment's end, we harvested multiple tissues beyond bone and muscle in hopes of tissue sharing with other PI's, and have found at least one laboratory at NASA-JSC and another at Texas A&M interested in these extra tissues. The rest of this third project year focused on processing of these tissues as well as those from parallel x-ray experiments performed at Texas A&M late in Project Year 2.

Preliminary results derived from micro-CT analyses of distal femur cancellous bone from these two experiments indicate the highest doses of both x-ray and 28Si did exacerbate bone loss in our 1/6 g mice; these groups had 24% and 7% lower cancellous bone volume (%BV/TV) than sham exposed partial g mice. Interestingly, fractionating that highest dose protected against further loss of bone mass in the x-ray-exposed, but did not for silicon-exposed, mice on partial g. Micro-CT analyses of cancellous bone microarchitecture will soon be complemented by traditional histomorphometry measures of bone formation rate on both cancellous and cortical bone envelopes. Consistent suppression of femoral neck strength (load at failure) was observed in all partial g mice; this suppression was exacerbated in only one group exposed to low-dose radiation (17 cGy 28Si). Analyses in progress will provide some answers to potential mechanisms for these combined effects of reduced weightbearing and modeled space radiation (decreased bone formation? increased bone resorption? apoptosis of osteocytes/ osteoblasts?) which will address IRP Risk Degen 2 ["What are the mechanisms of degenerative tissue risks in the heart, circulatory, endocrine, digestive, lens and other tissue systems?"].

We are also examining skeletal muscle alterations with partial weightbearing and modeled space radiation exposure ; these analyses address IRP Risk M23 ["Do factors in addition to unloading contribute to muscle atrophy during space flight (e.g., radiation, inflammation, hydration, redox balance, energy balance)?"] Analyses on 1/6 partial g mice reveal gastrocnemius muscle atrophy consistently occurring; some of this muscle loss is due to a reduced protein synthesis rate. However, there was no detectable additional effect of co-exposure to low dose x-rays on either loss of muscle mass or the reductions in protein synthesis rate. Western blot assessment of proteins that regulate peptide chain initiation vs. elongation will reveal mechanisms for any alterations in protein synthesis rates. Early results suggest that impairment in muscle protein metabolism with partial gravity may occur at the level of peptide-chain elongation. Upcoming assays will quantify BrdU-positive nuclei to indicate radiation-induced damage to satellite cells and, secondly, fiber-type specific changes in fiber cross-sectional area.

In the end, we will have a rich data base of both bone and muscle outcomes from parallel experiments performed with reference x-ray radiation and the 300MeV 28Si, with the capability of providing some calculations of relative biological effectiveness (RBE) for a number of physiological/structural outcomes. These experiments were designed to answer IRP Risk Degen 7 ["Are there significant synergistic effects from other spaceflight factors (microgravity, stress, altered circadian rhythms, changes in immune responses, etc.) that modify the degenerative risk from space radiation?"] in addition to IRP Risk B11 ["What are the effects of radiation on bone?"]

In Year 4 (starting 6/1/2011) we will complete these analyses, prepare multiple manuscripts and conduct our final experiment to test whether acute high LET radiation exposure will impair the ability of bone and/or muscle to respond to a resistance-based training protocol administered during a 21-day period of partial weightbearing (at 1/6 g). To our knowledge, no other laboratories have tested this to date, so these data should be absolutely unique in the literature. NSRL beam time proposals were submitted to Brookhaven National Laboratory in February, 2010 and we await word of acceptance and scheduling, hopefully in the fall run, 2011. Mr. Brandon Macias, a senior doctoral student in Dr. Bloomfield's laboratory and NSBRI Pre-Doctoral Fellow at Texas A&M, will be attending NASA's Space Radiation Summer School in June 2011 at BNL, further enhancing our capabilities in radiation biology research.

Our experiments focusing on effects of low-dose radiation on musculoskeletal structure and function will provide unique and novel data about the potential degenerative effects to be expected by those humans living in areas with high natural background radiation (e.g., Ramsar, Iran); by individuals who accumulate high occupational exposures to ionizing radiation (e.g., commercial airplane crews); and by patients accumulating multiple medical irradiation exposures over time. A growing literature is documenting surprising and deleterious effects on bone with low-level radiation (as opposed to the high doses used in radiotherapy for cancer patients); very little is known at the present time about the impact on these low doses on maintenance of normal muscle protein synthesis and muscle mass. Our experiments directly address these issues.

A manuscript comparing is nearly ready for submission by 5/31/2011; target journal is Bone. Poster presentations including these data were made at the Oct 2010 meetings of the American Society of Bone & Mineral Research. A separate manuscript detailing muscle outcomes (muscle protein synthesis, satellite cell BrdU incorporation, histological assessment of fiber cross-sectional areas) is in preparation.

Specific Aim 2 [ impact of low-dose radiation simulating galactic cosmic radiation on the musculoskeletal response to partial weightbearing]:

Our Experiment 2 (performed 6/09-3/10) was a comprehensive pilot experiment testing the impact of low doses of reference x-ray radiation. These results have been reported in poster presentations at the Oct 2010 meetings of the American Society of Bone & Mineral Research (ASBMR) (Lima et al., JBMR 2010) and a manuscript is in preparation for submission to Radiation Research by July 1, 2011. Experiment 3, testing the combined effect of reference x-ray radiation (50 and 100 cGy) and partial weightbearing (1/6 g), was completed in Year 2; much of the current project year has been devoted to analysis of tissues collected (histomorphometry analyses, mechanical testing, immunostaining). Muscle protein synthesis assays are nearly complete; histological assays for BrdU incorporation and fiber-type specific cross-sectional areas are in progress.

Experiment 4 was performed entirely on site at Brookhaven's NSRL in June 2010 at start of Year 3 to test the combined impact of high LET radiation (17 and 50 cGy of 28Si, 300 MeV) and partial weightbearing (1/6 g), parallelling as closely as possible Experiment 3 described above. The balance of our year has been spent in processing tissue analyses from bone and muscle collected during this experiment and in planning for our final Year 4 experiments; beam time and animal use proposals submitted to BNL 2/11 for fall 2011 run). Preliminary results from Experiments 3 and 4 have been presented at the April 2011 IAA Humans in Space Symposium (Houston, TX) and May 2011 joint meetings of the European Calcified Tissue Society and the International Bone and Mineral Society in Athens, Greece. Additional abstracts were submitted for presentation at the September 2011 meetings of the American Society of Bone & Mineral Research (San Diego, CA). Muscle protein synthesis assays are nearly complete; histological assays for BrdU incorporation and fiber-type specific cross-sectional areas are in progress.

Experiments supporting Specific Aim 1 [does partial weightbearing (~1/6 g) mitigate losses observed with full unloading (~ 0 g)] are just concluding in early June 2009, hence we are not yet able to report on key findings. We have made a "course correction" in the design of those first experiments. To simulate the additional loadbearing incurred by crew members locomoting on the Lunar surface due to the weight of EVA spacesuits, we elected to add one additional group of animals designated "1/3 g" (since current spacesuit design is roughly equivalent to one body weight, hence doubling the load bearing in the 1/6 g environment). This is easily accommodated by our partial g mouse model, since weightbearing can be titrated to whatever fraction of 100% body weight is desired.

We are concurrently preparing to start Experiment 2 (supporting Specific Aim 2: does low-dose radiation exposure change the bone or muscle response to partial gravity). This is a dose-response study utilizing reference x-ray radiation to assess responses of bone and muscle at early (~3 days), "delayed" (~21 days) and "recovery" (~56 days) time points. To achieve the best sensitivity possible, we have added cell culture studies to this experiment at the earlier timepoints to assess the impact on osteoprogenitor cell populations in the marrow. Pilot experiments are in progress to verify our procedures with primary cell culture.

Year 2 of this project will hence start with Experiment 2 (beginning July 2009) utilizing 108 mice (12 groups of 9 each) to test responses at the 3 time points listed above at 3 different radiation exposures. We will choose the lowest radiation dose producing detectable change in muscle protein synthesis and bone formation rate to use in the subsequent Experiment 3, commencing in February 2010, which will test the effects of x-ray exposure on the response to simulated partial gravity; in this experiment, mice subjected to 1/3 g (and weightbearing control mice) will be exposed to one acute dose of x-ray (low LET) radiation as well as 3 fractionated doses. Results of Experiment 2 will also be critical to constructing a beam time proposal for a repetition of Experiment 3 at Brookhaven National Lab during Year 3 of this project.

The project PI (S. Bloomfield) is attending NASA's Space Radiation Summer School at Brookhaven National Laboratory in June 2009. Both the conceptual and practical knowledge gained will prove invaluable for the later preparation of a beam time proposal as well as preparing for the logistics of conducting 3-week long experiments with live animals at the NASA Space Radiation Laboratory facility.

Our experiments focusing on effects of low-dose radiation on musculoskeletal structure and function will provide unique and novel data about the potential degenerative effects to be expected by those humans living in areas with high natural background radiation (e.g., Ramsar, Iran), by individuals that accumulate high occupational exposures to ionizing radiation (e.g., commercial airplane crews), and by patients accumulating multiple medical irradiation exposures over time. A growing literature is documenting surprising and deleterious effects on bone with low-level radiation (as opposed to the high doses used in radiotherapy for cancer patients); very little is known at the present time about the impact on muscle.

Specific Aim 2 [impact of low-dose radiation simulating galactic cosmic radiation on the musculoskeletal response to partial weightbearing]: We first completed a dose-response experiment to determine the best dose of x-ray radiation to use in Experiment 3, as well as timing of animal sacrifice after the radiation exposure. In the summer of 2009, mice were exposed to 0 (sham), 17, or 50 or 100 cGy of reference x-ray radiation; one group was sacrificed 3 days after exposure and a second group at 21 days. Micro-CT analyses of distal femurs, histological assessment of bone formation rate, were completed. A significant addition to this experiment was performance of primary cell culture studies of bone marrow stromal cells (BMSC) to assess radiation effects on BMSC proliferative and differentiation capabilities. An additional round of mice were irradiated in spring of 2010 to repeat cell culture experiments. Most analyses have been completed; preliminary results will be presented at the 2010 Skeletal Tissue Biology Workshop (Sun Valley, August, 2010) and at the American Society for Bone & Mineral Research (Toronto, October 2010).

The main experiment for Specific Aim 2 (Experiment 3) was completed by April 2010; both weightbearing (1 g) and partial (1/6) g mice were exposed to sham irradiation or 1 dose of 17 or 50 cGy of x-ray radiation on Day 0. A fourth group was exposed to 3 fractionated doses (0.17 Gy at 1 week intervals, on Day 0, 7 and 14). Animals were sacrificed after 21 days; analysis of bone and muscle collected at sacrifice are on-going. April and May of 2010 has been dominated by planning for experiments at the NASA Space Radiation Laboratory at Brookhaven National Laboratory, which will expose weightbearing and partial (1/6) g mice to similar doses of a higher LET ionizing radiation (silicon). These experiments are scheduled for the Summer 2010 run, starting on May 31.

Experiments supporting Specific Aim 1 [does partial weightbearing (~1/6 g) mitigate losses observed with full unloading (~ 0 g)] are just concluding in early June 2009, hence we are not yet able to report on key findings. We have made a "course correction" in the design of those first experiments. To simulate the additional loadbearing incurred by crew members locomoting on the Lunar surface due to the weight of EVA spacesuits, we elected to add one additional group of animals designated "1/3 g" (since current spacesuit design is roughly equivalent to one body weight, hence doubling the load bearing in the 1/6 g environment). This is easily accommodated by our partial g mouse model, since weightbearing can be titrated to whatever fraction of 100% body weight is desired.

We are concurrently preparing to start Experiment 2 (supporting Specific Aim 2: does low-dose radiation exposure change the bone or muscle response to partial gravity). This is a dose-response study utilizing reference x-ray radiation to assess responses of bone and muscle at early (~3 days), "delayed" (~21 days) and "recovery" (~56 days) time points. To achieve the best sensitivity possible, we have added cell culture studies to this experiment at the earlier timepoints to assess the impact on osteoprogenitor cell populations in the marrow. Pilot experiments are in progress to verify our procedures with primary cell culture.

Year 2 of this project will hence start with Experiment 2 (beginning July 2009) utilizing 108 mice (12 groups of 9 each) to test responses at the 3 time points listed above at 3 different radiation exposures. We will choose the lowest radiation dose producing detectable change in muscle protein synthesis and bone formation rate to use in the subsequent Experiment 3, commencing in February 2010, which will test the effects of x-ray exposure on the response to simulated partial gravity; in this experiment, mice subjected to 1/3 g (and weightbearing control mice) will be exposed to one acute dose of x-ray (low LET) radiation as well as 3 fractionated doses. Results of Experiment 2 will also be critical to constructing a beam time proposal for a repetition of Experiment 3 at Brookhaven National Lab during Year 3 of this project.

The project PI (S. Bloomfield) is attending NASA's Space Radiation Summer School at Brookhaven National Laboratory in June 2009. Both the conceptual and practical knowledge gained will prove invaluable for the later preparation of a beam time proposal as well as preparing for the logistics of conducting 3-week long experiments with live animals at the NASA Space Radiation Laboratory facility.

Our experiments focusing on the effect of chronic exposure to low-dose radiation on musculoskeletal structure and function will provide unique and novel data about the potential degenerative effects experienced by those humans living in areas with high natural background radiation (e.g. Ramsar, Iran), DOE/nuclear industry workers accumulating occupational exposures, and patients accumulating large exposures with multiple medical procedures.

While these experiments were still running, work has begun on Specific Aim 2 (determine impact of low-dose radiation exposure on muscle/bone integrity in modeled Lunar gravity); in particular, we have been planning for Experiment 2, the preliminary dose-response experiment with reference x-ray radiation Multiple consultations with other team PIs and with Dr. Marcelo Vasquez (NSBRI HQ) helped us refine our specific exposure doses and time of animal sacrifice relative to exposure day. Our post-doctoral fellow, skilled in primary cell culture work, has added osteoprogenitor cell differentiation and proliferation assays to our outcomes from these experiments. Pilot studies started in May 2008 to confirm viability and lack of contamination of primary cultures derived from mouse bone marrow samples are thus far successful. We plan to begin Experiment 2, testing 3 x-ray exposure doses and three times of sacrifice after exposure, no later than July of 2009.

We will use a novel, partial-gravity mouse model to first determine the independent impact of the moons 1/6 gravity on multiple bone and muscle outcomes, including direct determinations of bone breaking strength and other mechanical properties as well as muscle function in the live animal. We will then test the additional impact of low-dose radiation modeling galactic cosmic radiation during partial gravity conditions by exposing these mice to one acute dose, or four, fractionated doses on a weekly basis, of ionizing radiation.

Data from these experiments will be used to justify expanded experiments at the Brookhaven NASA Space Radiation Laboratory utilizing heavy iron ions to simulate galactic cosmic radiation. Finally, we will assess the impact of the lunar environment (partial gravity plus modeled space radiation) on the musculoskeletal response to exercise countermeasures.

Using an animal model to pursue these objectives provides for these advantages:

1. Direct determinations of bone quality with mechanical testing of bone at the end of each experiment;

2. Discovering the effect of radiation on the response to partial gravity and to exercise countermeasures, which cannot be ethically tested in human subjects; and

3. More rapid turn-around and much reduced cost for experiments than those involving long-duration human bed-rest studies.

These experiments will provide unique and valuable data about bone loss and impaired muscle function and determine efficacy of two different exercise countermeasures in a modeled lunar environment.