Nancy D. Searby

Total-Body Irradiation of Postpubertal Mice with 137Cs Acutely Compromises the Microarchitecture of Cancellous Bone and Increases Osteoclasts

Abstract Kondo, H., Searby, N. D., Mojarrab, R., Phillips, J., Alwood, J., Yumoto, K., Almeida, E. A. C., Limoli, C. L. and Globus, R. K. Total-Body Irradiation of Postpubertal Mice with 137Cs Acutely Compromises the Microarchitecture of Cancellous Bone and Increases Osteoclasts. Radiat. Res. 171, 283–289 (2009). Ionizing radiation can cause substantial tissue degeneration, which may threaten the long-term health of astronauts and radiotherapy patients. To determine whether a single dose of radiation acutely compromises structural integrity in the postpubertal skeleton, 18-week-old male mice were exposed to 137Cs γ radiation (1 or 2 Gy). The structure of high-turnover, cancellous bone was analyzed by microcomputed tomography (microCT) 3 or 10 days after irradiation and in basal controls (tissues harvested at the time of irradiation) and age-matched controls. Irradiation (2 Gy) caused a 20% decline in tibial cancellous bone volume fraction (BV/TV) within 3 days and a 43% decline within 10 days, while 1 Gy caused a 28% reduction 10 days later. The BV/TV decrement was due to increased spacing and decreased thickness of trabeculae. Radiation also increased (∼150%) cancellous surfaces lined with tartrate-resistant, acid phosphatase-positive osteoclasts, an index of increased bone resorption. Radiation decreased lumbar vertebral BV/TV 1 month after irradiation, showing the persistence of cancellous bone loss, although mechanical properties in compression were unaffected. In sum, a single dose of γ radiation rapidly increased osteoclast surface in cancellous tissue and compromised cancellous microarchitecture in the remodeling appendicular and axial skeleton of postpubertal mice.

Oxidative stress and gamma radiation-induced cancellous bone loss with musculoskeletal disuse

Exposure of astronauts in space to radiation during weightlessness may contribute to subsequent bone loss. Gamma irradiation of postpubertal mice rapidly increases the number of bone-resorbing osteoclasts and causes bone loss in cancellous tissue; similar changes occur in skeletal diseases associated with oxidative stress. Therefore, we hypothesized that increased oxidative stress mediates radiation-induced bone loss and that musculoskeletal disuse changes the sensitivity of cancellous tissue to radiation exposure. Musculoskeletal disuse by hindlimb unloading (1 or 2 wk) or total body gamma irradiation (1 or 2 Gy of (137)Cs) of 4-mo-old, male C57BL/6 mice each decreased cancellous bone volume fraction in the proximal tibiae and lumbar vertebrae. The extent of radiation-induced acute cancellous bone loss in tibiae and lumbar vertebrae was similar in normally loaded and hindlimb-unloaded mice. Similarly, osteoclast surface in the tibiae increased 46% as a result of irradiation, 47% as a result of hindlimb unloading, and 64% as a result of irradiation + hindlimb unloading compared with normally loaded mice. Irradiation, but not hindlimb unloading, reduced viability and increased apoptosis of marrow cells and caused oxidative damage to lipids within mineralized tissue. Irradiation also stimulated generation of reactive oxygen species in marrow cells. Furthermore, injection of alpha-lipoic acid, an antioxidant, mitigated the acute bone loss caused by irradiation. Together, these results showed that disuse and gamma irradiation, alone or in combination, caused a similar degree of acute cancellous bone loss and shared a common cellular mechanism of increased bone resorption. Furthermore, irradiation, but not disuse, may increase the number of osteoclasts and the extent of acute bone loss via increased reactive oxygen species production and ensuing oxidative damage, implying different molecular mechanisms. The finding that alpha-lipoic acid protected cancellous tissue from the detrimental effects of irradiation has potential relevance to astronauts and radiotherapy patients.

Microgravity studies of cells and tissues.

Abstract: Controlled in vitro studies of cells and tissues under the conditions of microgravity (simulated on Earth, or actual in space) can improve our understanding of gravity sensing, transduction, and responses in living cells and tissues. This paper discusses the scientific results and practical implications of three NASA‐related biotechnology projects: ground and space studies of microgravity tissue engineering (JSC‐Houston), and the development of the cell culture unit for use aboard the International Space Station (ARC‐Ames).

Heavy ion irradiation and unloading effects on mouse lumbar vertebral microarchitecture, mechanical properties and tissue stresses

Astronauts are exposed to both musculoskeletal disuse and heavy ion radiation in space. Disuse alters the magnitude and direction of forces placed upon the skeleton causing bone remodeling, while energy deposited by ionizing radiation causes free radical formation and can lead to DNA strand breaks and oxidative damage to tissues. Radiation and disuse each result in a net loss of mineralized tissue in the adult, although the combined effects, subsequent consequences for mechanical properties and potential for recovery may differ. First, we examined how a high dose (2 Gy) of heavy ion radiation ((56)Fe) causes loss of mineralized tissue in the lumbar vertebrae of skeletally mature (4 months old), male, C57BL/6 mice using microcomputed tomography and determined the influence of structural changes on mechanical properties using whole bone compression tests and finite element analyses. Next, we tested if a low dose (0.5 Gy) of heavy particle radiation prevents skeletal recovery from a 14-day period of hindlimb unloading. Irradiation with a high dose of (56)Fe (2 Gy) caused bone loss (-14%) in the cancellous-rich centrum of the fourth lumbar vertebra (L4) 1 month later, increased trabecular stresses (+27%), increased the propensity for trabecular buckling and shifted stresses to the cortex. As expected, hindlimb unloading (14 days) alone adversely affected microarchitectural and mechanical stiffness of lumbar vertebrae, although the reduction in yield force was not statistically significant (-17%). Irradiation with a low dose of (56)Fe (0.5 Gy) did not affect vertebrae in normally loaded mice, but significantly reduced compressive yield force in vertebrae of unloaded mice relative to sham-irradiated controls (-24%). Irradiation did not impair the recovery of trabecular bone volume fraction that occurs after hindlimb unloaded mice are released to ambulate normally, although microarchitectural differences persisted 28 days later (96% increase in ratio of rod- to plate-like trabeculae). In summary, (56)Fe irradiation (0.5 Gy) of unloaded mice contributed to a reduction in compressive strength and partially prevented recovery of cancellous microarchitecture from adaptive responses of lumbar vertebrae to skeletal unloading. Thus, irradiation with heavy ions may accelerate or worsen the loss of skeletal integrity triggered by musculoskeletal disuse.

Short-Term Effects of Whole-Body Exposure to 56Fe Ions in Combination with Musculoskeletal Disuse on Bone Cells

Abstract Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to 56Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to 56Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP+ osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.

Effects of chair‐restraint on gastrointestinal transit time and colonic fermentation in male rhesus monkey (Macaca mulatta)

Abstract: The incidence of an 18 day chair‐restraint on the digestive physiology of male rhesus monkey was investigated for space research purposes, comparing four trained restraint subjects with two vivarium controls. Chair‐restraint induced a 2.5‐fold acceleration of the gastrointestinal transit time, which persisted throughout the 7 day postrestraint period, and an increase of the fecal dry matter content, which mean value rose from 40.7% to 69.6%. Fecal pH remained unaltered throughout the experiment. Modifications of fermentative metabolites produced by the colonic microflora and excreted through the breath (hydrogen and methane) or in the feces (short chain fatty acids and ammonia) could not be reliably related to chair‐restraint and probably involved side‐stress factors. On the whole, alterations due to chair‐restraint are shown to be different from those reported in the literature, following a modification of the dietary composition. These data may help to predict the alterations of digestive physiology likely to occur in immobilized human patients.

Flow Field Measurements in the Cell Culture Unit

Abstract: The cell culture unit (CCU) is being designed to support cell growth for long‐duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity (μg), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground‐based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the CSC flow environment are presented and discussed. The final configuration of the CSC employs magnetic stir bars with angled paddles to achieve the necessary flow requirements within the CSC.