NOTE: End date changed to 1/31/2011 (from 1/31/2010) per NSBRI (1/2010)

The spaceflight environment presents numerous challenges to astronauts' bone health, including the bone-debilitating effects of micro/reduced gravity. A reduction in bone volume and strength among astronauts represents a serious, mission-critical biomedical problem. Preliminary data and published accounts indicate that exposure to doses as low as 1 Gy of multiple types of ionizing radiation results in trabecular bone loss. However, it is unclear how bone already compromised by microgravity or partial gravity will be affected by exposure to space radiation.

Our intent is to enhance the understanding of the effects of radiation on bone health, particularly within a reduced gravity environment. Our ultimate goal is to develop appropriate countermeasures for bone loss in astronauts during exploratory missions. The proposed Aims will analyze the effects of modeled space radiation using scenarios applicable for Lunar Outpost missions.

Specific Aim 1: Examine the response of bone to a combination of spaceflight-associated challenges, including radiation exposure together with reduced limb bone loading.

Approach Aim 1: Skeletally mature female C57BL/6 mice, a strain sensitive to disuse-associated osteoporosis, were exposed to a 1 Gy dose or protons at Loma Linda Medical Center followed by a 4-week period of hindlimb suspension.

Main Findings Aim 1:

1) The combination of radiation and hindlimb suspension resulted in greater bone loss and deterioration of trabecular microarchitecture than the two challenges individually. Although both skeletal challenges induced atrophy, bone loss due to suspension alone was substantially greater than irradiation alone.

2) While reduced loading on the bone combined with microarchitecture resulted in excessive bone loss relative to either one challenge independently, the causal mechanisms for the stimuli may be independent. A thorough histological investigation was undertaken. Throughout the study, osteoclasts appear to be more active as an early response in both irradiated groups (irradiated only and irradiated + suspension) than non-irradiated groups.

Specific Aim 2: Examine the mechanistic causes of changes in bone at the cellular and tissue level following the individual and combined effects of radiation and unloading on mouse hindlimbs.

Approach Aim 2:

Year 1 of Funding: 13-week old C57BL/6 mice received whole body radiation with 2 Gy X-rays. X rays were chosen for these initial studies, as the relative biological effect of X-rays and protons are similar and bone loss from a 1 Gy dose has been shown to be similar (-13-15%) for both types at one month (preliminary data).

Year 2 of Funding: In an attempt to expand this NSBRI-funded developing technology to develop countermeasures for spaceflight and clinical applications, 16-week old mice were exposed to a 2 Gy dose of X rays. Some irradiated animals were given a commercially available osteoporosis medication (risedronate) to try to prevent any changes. Bone was examined at week 1, 2, and 3.

To apply this technology to clinical applications, the right hindlimbs of twenty-week old Sprague-Dawley rats received a dose of radiation modeling what the human hip receives during cancer radiotherapy. Animals were sacrificed at 2, 4, and 6 weeks to identify if modeled cancer therapy could induce bone loss. An antiresorptive agent (a commercially available bisphosphonate risedronate) was applied to try to prevent these changes.

Main Findings Aim 2:

1) Radiation appears to increase osteoclast number and activity as an early, acute response to radiation leading to early atrophy. Circulating markers of bone resorption are increased by 24 hours of exposure, with significantly increased osteoclast numbers present along trabeculae by 3 days. Trabecular bone loss is significant and substantial by Day 7 after exposure and occurs at multiple skeletal sites (proximal tibia; distal femur; fifth lumbar vertebrae). Bone quantity remains lower than control at Day 14 and 21.

2) Low dose irradiation of mice resulted in substantial deterioration of by one week after exposure. These changes occurred as a result of increased osteoclast activity and active bone resorption. An agent that blocks osteoclast activity (risedronate) completely prevented these changes. Modeling cancer radiotherapy resulted in severe deterioration of trabecular bone in rats at all time points investigated. Applying risedronate completely prevented deterioration of bone early after modeled cancer radiotherapy.

While Year 2 of funding was largely devoted to the development of a therapy for radiation-induced bone loss in both the spaceflight and clinical environment, identification of the causal mechanisms for bone loss must be identified. This past year we have clearly identified that radiation somehow turns on osteoclasts, and that this stimulation of active bone resorption (as opposed to suppressed bone formation) leads to bone loss. Our lab is identifying that markers of inflammation may be turning on these osteoclasts early after exposure. These include tumor necrosis factor-alpha (TNFa) and interleukin-1 (IL-1) activity. Therefore, for the coming year, we will investigate the role of these cytokines in radiation-induced bone loss. We will use a mutant mouse strain (reduced-inflammatory strain) developed in the lab of Dr. Albert Fornace, Jr. (Georgetown University) in collaboration with his NSBRI postdoctoral fellow, Dr. Daniella Trani. These mice will be exposed to a series of particle radiation exposures (both protons and iron nuclei; at low doses) at Brookhaven National Laboratory to identify if reducing inflammation can prevent bone loss.

Additionally, we will more appropriately model the skeletal effects of radiation during realistic spaceflight scenarios by irradiating mice with 0, 1, or 2 Gray dose of protons at a prolonged dose rate, modeling the maximum dose rate at the peak of a solar flare.

Work from this project has identified that by preventing osteoclast activity after radiation exposure, bone loss can be prevented. This work has helped lead to the development of several clinical trials within oncology departments in California and North Carolina. If these observations translate into the clinic, it is possible that the notion of preventing bone deterioration resulting from cancer therapy by targeting the osteoclast can improve patient quality of care.

Specific Aim 2: Examine the cause of radiation-induced bone loss and test preventative countermeasures (antiresorptive; anti-inflammatory) to prevent any early response that can lead to increased osteoclast activity.

Progress: 16-week old C57BL/6 mice received whole body radiation with 2 Gy X-rays. This low dose of radiation caused a very quick loss of bone volume and deterioration of architecture in mice: virtually all indices of bone volume and architectural integrity were lower than control by one week after exposure. Bone loss and deterioration of microarchitecture was present in the proximal tibia, distal femur, and lumbar vertebrae. This osteoporosis occurred as a result of increased osteoclast number and activity, which was elevated only during the first week (which was when the majority of bone loss occurred). Additionally, bone formation rates were unchanged during the first week, so that loss of bone cannot be attributed to suppressed bone formation. However, by applying an agent that decreased osteoclast activity (the bisphosphonate risedronate, an osteoporosis medication) we completely prevented deterioration of bone after irradiation. These results were accepted for publication by the journal Bone in September of 2009, and are currently in press.

Additionally, we attempted to determine if modeling the dose of radiation a human hip receives during radiation therapy for pelvic cancers (which increases the chance of hip fracture) in a rat model can induce bone loss Right hind limbs were exposed to 4 doses of 4 Gy at Wake Forest Comprehensive Cancer Center. Animals were sacrificed at 2, 4, and 6 weeks. Irradiation resulted in deterioration of the exposed bone relative to the non-irradiated contralateral limb. Reduced connectivity and a decreased number of more rod-like trabeculae were present . Deterioration was entirely prevented with risedronate administration. Thus, we have identified a potential target to prevent bone loss and fractures among cancer radiation therapy survivors.

International Bone and Mineral Society Sun Valley Workshop, Abstract Book, August 2009. , Aug-2009

Radiation Research Society 55th Annual Meeting, Abstract Book, October 2009. , Oct-2009

Radiation Research Society 55th Annual Meeting, Abstract Book, October 2009. , Oct-2009

The spaceflight environment presents numerous challenges to astronauts' bone health, including the bone-debilitating effects of micro/reduced gravity. A reduction in bone volume and strength among astronauts represents a serious, mission-critical biomedical problem. Preliminary data and published accounts indicate that exposure to doses as low as 1 Gy of multiple types of ionizing radiation results in trabecular bone loss. However, it is unclear how bone already compromised by microgravity or partial gravity will be affected by exposure to space radiation. Our intent is to enhance the understanding of the effects of radiation on bone health, particularly within a reduced gravity environment. Our ultimate goal is to develop appropriate countermeasures for bone loss in astronauts during exploratory missions. The proposed Aims will analyze the effects of modeled space radiation using scenarios applicable for Lunar Outpost missions.

Specific Aim 1: Examine the response of bone to a combination of spaceflight-associated challenges, including radiation exposure together with reduced limb bone loading. HYPOTHESIS: Proton or Fe radiation combined with unloading will induce more severe impacts on bone quantity and strength than unloading alone. Approach Aim 1: Skeletally mature female C57BL/6 mice, a strain sensitive to disuse-associated osteoporosis, were exposed to a 1 Gy dose or protons at Loma Linda Medical Center followed by a 4-week period of hindlimb suspension. Main Findings Aim 1: The combination of radiation and hindlimb suspension resulted in greater bone loss and deterioration of trabecular microarchitecture than the two challenges individually. Although both skeletal challenges induced atrophy, bone loss due to suspension alone was substantially greater than irradiation alone.

Specific Aim 2: Examine the mechanistic causes of changes in bone at the cellular and tissue level following the individual and combined effects of radiation and unloading on mouse hindlimbs. HYPOTHESIS: An early, inflammatory-type response following irradiation results in increased resorption via activation of osteoclasts. Approach Aim 2: Thirteen-week old C57BL/6 mice received whole body radiation with 2 Gy X-rays. X rays were chosen for these initial studies, as the relative biological effect of X-rays and protons are similar and bone loss from a 1 Gy dose has been shown to be similar (-13-15%) for both types at one month (preliminary data). Main Findings Aim 2: Radiation appears to increase osteoclast number and activity as an early, acute response to irradiation leading to early atrophy. Circulating markers of bone resorption are increased by 24 hours of exposure, with significantly increased osteoclast numbers present along trabeculae by 3 days. Trabecular bone loss is significant and substantial by Day 7 after exposure and occurs at multiple skeletal sites (proximal tibia; distal femur; fifth lumbar vertebrae). Bone quantity remains lower than control at Day 14 and 21. However, these changes in bone can completely be prevented by application of a commercially available osteoporosis medication (bisphosphonate) that prevents osteoclast activity.

These findings largely support the two hypotheses of the original proposal. Bone loss after exposure to multiple skeletal challenges appears additive relative to the two challenges individually. From Aim 2, radiation exposure resulted in a very early increase in osteoclast activity and number, with appreciable bone loss occurring by week 1. A large component of Aim 2 was to describe if the inflammatory response can lead to increased osteoclast activity early, as inflammation can induce bone loss and can occur within irradiated tissue. Thus, the findings from the first year have driven this research to focus on the following aspect of Aim 2: describe what cellular and molecular mechanism can lead to an increase in osteoclast activity early after exposure. Additionally, countermeasures (e.g., antiinflammatory and antiresorptive agents) will be employed in an attempt to both prevent radiation induced bone loss as well as to provide more information as to the nature of the increase in osteoclast activity. The research for the second year of NSBRI support will describe any role that early inflammation will play in this acute activation of osteoclasts. We will use primarily single limb irradiation to reduce the role of systemic confounding factors, such as changes in circulating estrogen if the gonads are irradiated. We will also focus on factors associated with inflammation. In particular, we will examine the response in bone after proinflammatory cytokine knock-out mice are exposed to radiation (e.g., tumor necrosis factor-alpha (TNFa), interleukin (IL)-1, and IL-6). These cytokines can activate osteoclasts. We will examine the role for transforming growth factor-beta (TGFb) in radiation-induced bone loss: this cytokine is often produced within irradiated tissues. This research will also examine if TNFa binding protein therapy or TGFb blockade with a type 1 receptor kinase inhibitor will block radiation-induced bone loss in this setting. Finally, we will continue to explore bisphosphonates as a countermeasure for radiation-induced bone loss.

Radiation-induced bone damage is a concern for radiation oncologists. Improvements in cancer treatment and diagnosis have led to increased long-term cancer survivorship. For example, of the more than 200,000 men who are diagnosed with prostate cancer each year, the 10 year survival rate approaches 90%. However, this growing population of long-term survivors are at risk of developing deleterious effects in normal tissues resulting from the use of therapeutic radiation. Skeletal complications, particularly fractures, are a recognized late-radiation induced effect. Fractures of the hip, ribs, clavicle, and humerus have documented in patients receiving targeted radiotherapy for pelvic and breast tumors. These bones absorb radiation during cancer treatment. The increased incidence of hip (particularly the femoral neck) fractures in postmenopausal women after receiving pelvic cancer treatment is substantial and can have severe negative impacts on a patient's mobility, independence, and survival.

Bone atrophy and fractures following direct exposure to radiation is thought to occur as a late response resulting from damage to osteoblasts and vascularity within marrow and throughout osteons. Data suggest radiation can impair bone formation after exposure by killing or damaging osteoblasts. The contribution of late vascular damage to skeletal complications is less clear. However, late radiation-induced bone atrophy has been observed in clinical and animal studies.

The effects of ionizing radiation on osteoclast activity are poorly documented, yet some evidence suggests an early stimulation of active bone resorption after exposure (as opposed to just reduced formation) could contribute to the etiology of radiation-induced bone damage. The possibility exists that an early, radiation-induced increase in osteoclast activity can contribute to late-observed atrophy. If this is true, atrophy of bone that is irradiated during the treatment of tumors as well as subsequent fractures of these structures may be reduced or prevented by early blockage of osteoclast activity using antiresorptive countermeasures, such as a bisphosphonate like risedronate.

Specific Aim 1: Examine the response of bone to a combination of spaceflight-associated challenges, specifically radiation exposure with reduced limb bone loading. Progress: Skeletally mature female C57BL/6 mice, a strain sensitive to disuse-associated osteoporosis, were exposed to a 1 Gy dose or protons at Loma Linda Medical Center, followed by a 4-week period of hindlimb suspension. Both of the skeletal challenges resulted in loss of bone volume fraction and deterioration of skeletal elements. The combination of radiation and hindlimb suspension resulted in greater bone loss and deterioration of trabecular microarchitecture than the two challenges individually. These changes appeared additive: relative reduction in bone volume fraction within the tibia after irradiation in the normally loaded mice (-15%) was very similar as the suspended mice (-17%) when compared with non-irradiated normally loaded and suspended mice, respectively.

Specific Aim 2: Examine the mechanistic causes of changes in bone at the cellular and tissue level following irradiation and identify if bone loss can occur as an acute, early response. Test preventative countermeasures (antiresorptive; anti-inflammatory; antifibrotic) to prevent any early response that can lead to increased osteoclast activity. Progress: Radiation appears to increase osteoclast number and activity as an early, acute response to irradiation leading to early atrophy. C57BL/6 mice received whole body radiation with 2 Gy X-rays, as the relative biological effect of X-rays and protons are similar and bone loss from a 1 Gy dose has been shown to be similar (-13-15%) for both types at one month (preliminary data). Circulating serum markers of resorption (TRAP5b; +50%) are increased by 24 hours, which remains elevated by Day 3 (+14%) and Day 7 (+21%). Increased osteoclast numbers were present along trabeculae by Day 3 (+80%) and Day 7 (+218%). Surface covered by osteoclasts was also greater (+210%) at Day 3 after exposure. Trabecular bone loss is significant and substantial by Day 7 at multiple skeletal sites (proximal tibia; distal femur; fifth lumbar vertebrae). Deterioration bone was completely prevented by administering an antiresorptive osteoporosis countermeasure (risedronate).

American Society for Bone and Mineral Research 30th Annual Meeting, Abstract Book, September 2008. , Sep-2008

American Society for Bone and Mineral Research 30th Annual Meeting, Abstract Book, September 2008. , Sep-2008

American Society for Bone and Mineral Research 30th Annual Meeting, Abstract Book, September 2008. , Sep-2008

Radiation Research Society 54th Annual Meeting, Abstract Book, September 2008. , Sep-2008

Crews on exploration missions will experience reduced gravity as well as radiation from cosmic and solar sources, such as during solar flares. The character of space radiation is complex; components ranging from protons to iron particles are accompanied by secondary radiation (e.g., Bremmstrahlung x-rays and neutrons) produced as heavy ions that strike shielding materials and generate fragments. We identified loss of cancellous (trabecular or spongy) bone in mice after exposure to 1 gray (Gy) doses of multiple radiation types, several of which are present in the space environment and at doses and rates modeling a solar flare. The health status of bone is already compromised by altered gravity, thus any radiation-induced challenge may compound this problem.

Our intent is to enhance the understanding of the effects of radiation on bone health, particularly within a reduced-gravity environment. The project will address the following goal: reduce the risk of accelerated bone loss leading to osteoporosis. Ultimately, we intend to use this information to develop an appropriate countermeasure to prevent astronaut bone loss during exploration missions.

To define the risk associated with space radiation-induced bone loss, the project's objectives will examine the effects of modeled space radiation using scenarios applicable for Lunar Outpost missions.

Objectives:

1. Examine the response of bone to a combination of challenges involved with exploration missions, specifically radiation that models a solar particle event with hindlimb unloading.

PURPOSE: The stated challenges have been documented as inducing osteoporosis in astronauts and/or animal models. However, these conditions have been examined independent of each other. Therefore, the combined effects of these challenges on the skeletal system (i.e. synergistic, antagonistic, or neither) are unknown.

APPROACH: Right hindlimbs from rats will be exposed to one Gy proton, iron and x-ray radiation. Proton exposures will mimic the doses and dose rates of a solar particle event. The treated mice will then be divided into two groups, a hindlimb unloaded (tail suspension) and a fully loaded group, with non-irradiated mice serving as controls. After one month of suspension, the skeletal response of all groups will be evaluated. Bone volume and microarchitectural effects within a long bone of the hindlimb (tibia) will be examined via microCT. Mechanical testing via three-point bending will document any functional changes in bone strength after combined treatments. This study will therefore provide insight into how these challenges together might influence bone quantity, quality and ultimately mission-critical fractures in astronauts.

2. Examine the mechanistic causes of any changes in bone at the cellular and tissue level following the individual and combined effects of radiation and unloading on rat hindlimbs.

PURPOSE: The cause of bone loss can vary from an increase in bone resorption by cells called osteoclasts, decreased formation due to effects on bone-generating osteoblasts, or a combination of these factors. Understanding the causal nature of the bone loss will ultimately help develop an appropriate countermeasure. Our preliminary data suggests an early, radiation-induced inflammatory response results in increased resorption. This may enhance bone loss caused by the effects of unloading on osteoblasts and osteoclasts, as well as the radiation-induced death of osteoblasts.

APPROACH: We will examine the cells present within the tibiae of the irradiated and non-irradated hindlimbs from all previously mentioned groups via histological techniques to document effects on osteoclast and osteoblast number and activity. We will also examine proteins within the marrow of limb bones (femur) to identify any factors (i.e. cytokines such as TNFa) that can induce bone loss after such challenges.