exploring-effects-spaceflight-on-mouse-physiology-using-open-access

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

NASA Ames Research Center

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16 weeks) were exposed to high atomic number, high energy (HZE) iron ions ((56)Fe, 2 Gy, 600 MeV/ion) (IR, n=5) or sham-irradiated (Sham, n=5). In vivo axial loading was initiated 5 months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60 cycles/day, 3 day/wk for 4 weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation.

Mechanical loading causes site-specific anabolic effects on bone following exposure to ionizing radiation.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA's Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose Fe (200 cGy) increased  and . The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET (Cs γ) when irradiated in vitro, but had limited protective effects on high-LET Fe-exposed cells. In sum, either protons or Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System.

Segmental bone defects (SBDs) secondary to trauma invariably result in a prolonged recovery with an extended period of limited weight bearing on the affected limb. Soldiers sustaining blast injuries and civilians sustaining high energy trauma typify such a clinical scenario. These patients frequently sustain composite injuries with SBDs in concert with extensive soft tissue damage. For soft tissue injury resolution and skeletal reconstruction a patient may experience limited weight bearing for upwards of 6 months. Many small animal investigations have evaluated interventions for SBDs. While providing foundational information regarding the treatment of bone defects, these models do not simulate limited weight bearing conditions after injury. For example, mice ambulate immediately following anesthetic recovery, and in most cases are normally ambulating within 1-3 days post-surgery. Thus, investigations that combine disuse with bone healing may better test novel bone healing strategies. To remove weight bearing, we have designed a SBD rodent healing study in microgravity (µG) on the International Space Station (ISS) for the Rodent Research-4 (RR-4) Mission, which launched February 19, 2017 on SpaceX CRS-10 (Commercial Resupply Services). In preparation for this mission, we conducted an end-to-end mission simulation consisting of surgical infliction of SBD followed by launch simulation and hindlimb unloading (HLU) studies. In brief, a 2 mm defect was created in the femur of 10 week-old C57BL6/J male mice (n = 9-10/group). Three days after surgery, 6 groups of mice were treated as follows: 1) Vivarium Control (maintained continuously in standard cages); 2) Launch Negative Control (placed in the same spaceflight-like hardware as the Launch Positive Control group but were not subjected to launch simulation conditions); 3) Launch Positive Control (placed in spaceflight-like hardware and also subjected to vibration followed by centrifugation); 4) Launch Positive Experimental (identical to Launch Positive Control group, but placed in qualified spaceflight hardware); 5) Hindlimb Unloaded (HLU, were subjected to HLU immediately after launch simulation tests to simulate unloading in spaceflight); and 6) HLU Control (single housed in identical HLU cages but not suspended). Mice were euthanized 28 days after launch simulation and bone healing was examined via micro-Computed Tomography (µCT). These studies demonstrated that the mice post-surgery can tolerate launch conditions. Additionally, forces and vibrations associated with launch did not impact bone healing (p = .3). However, HLU resulted in a 52.5% reduction in total callus volume compared to HLU Controls (p = .0003). Taken together, these findings suggest that mice having a femoral SBD surgery tolerated the vibration and hypergravity associated with launch, and that launch simulation itself did not impact bone healing, but that the prolonged lack of weight bearing associated with HLU did impair bone healing. Based on these findings, we proceeded with testing the efficacy of FDA approved and novel SBD therapies using the unique spaceflight environment as a novel unloading model on SpaceX CRS-10.

Article	Year
Mechanical loading causes site-specific anabolic effects on bone following exposure to ionizing radiation. Bone\| PubMed ID: 26191778	2015
Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System. International journal of molecular sciences\| PubMed ID: 28994728	2017
Forces associated with launch into space do not impact bone fracture healing. Life sciences in space research\| PubMed ID: 29475520	2018

Yasaman Shirazi-Fard

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