The Combined Effects Of Disuse And Chronic Low-dose Gamma Radiation Exposure On Trabecular And Cortical Bone In Mice Hindlimb

[Background and Objective] During spaceflight,crewmembers are subjected to biomechanical and biological challenges including microgravity and radiation.In the skeleton,spaceflight leads to bone loss,increasing the risk of fracture.Studies utilizing hindlimb suspension(HLS)as a ground-based model of spaceflight often neglect the concomitant effects of radiation exposure,and even when radiation is accounted for,it is often delivered at a high dose rate over a short period of time,which does not faithfully mimic spaceflight conditions.This study was to design and test a animal model which is similar with the cosmic environment and to investigate the skeletal effects of low-dose gamma irradiationwhen administered simultaneously to disuse from HLS.The goal was to determine the internal mechanism of bone loss happened in astronaut in a space flight mission and was to set up a basic model for other cosmic related projects.[Methods] Four groups of 14 weeks old female C57BL/6 mice were studied: weight bearing + no radiation(WB+NR),HLS + NR,WB + radiation exposure(WB+RAD),and HLS+RAD.Hindlimb suspension was performed using a modification of theMorey-Holton method which led mice hind limbs disused.Swivel clips were attached to the tail harness and secured to an adjustable-height pulley system that allowed mobility about the cage in the x-and y-directions.Whole-body irradiation was performed using a 137 Cs gamma-ray irradiator in a vivarium facility.The dose rate to the mice was 8.5 cGy/day over a 20 day period,a total(23-25)dose for each mouse of1.7 Gy.At the end of the 20 day experimental period,hindlimbs were removed and femurs and tibias were cleaned of soft tissue.Right femurs were tested on a MTS mechanical testing system.Ultimate force(maximum load during testing,N)and failure energy(area under force-deformation curve to fracture point,J)were calculated from the loading data.Bone architecture of left femur and tibia was analyzed using micro-computed tomography(microCT).The left femur from each mouse was embedded in paraffin and prepared by H&E staining and TRAP straining for histological analysis.Static bone histomorphometric parameters,normalized to bone perimeter(B.Pm,mm)were quantified according to specifications of the American Society for Bone and Mineral Research(30)including osteoblast number(N.Ob/B.Pm,#/mm),osteoblast surface(Ob.Pm/B.Pm,%),osteoclast number(N.Oc/B.Pm,#/mm),osteoclast surface(Oc.Pm/B.Pm,%)and the ratio of osteoblast to osteoclast surfaces(Ob.Pm/Oc.Pm,%).[Results]1.Body mass was not different between groups upon animal arrival(p>0.05).However,at the conclusion of the study,body masses were significantly(p<0.05)different between groups,with the lowest body masses found in the HLS-NR group.HLS(p = 0.059),but not radiation(p>0.05),tended to decrease 4.8% bodymass in 2-factor ANOVA analyses.2.In the distal femoral metaphysis,trabecular bone volume fraction was significantly different(P<0.05)between the four groups,with the lowest values found in the HLS+RAD mice.The HLS+NR group demonstrated a 21% decrease in BV/TV as compared to WB+NR mice,and the HLS+RAD group demonstrated a 32%decrease in BV/TV as compared to WB+RAD mice.Indeed,HLS(P <0.05),but not radiation(P >0.05)decreased bone volume fraction in 2-factor ANOVA analyses.Similar trends in trabecular bone architecture were seen in the proximal tibia,where bone volume fraction was significantly(P <0.05)different between the four groups,with the lowest values found in the HLS+RAD mice.HLS(P <0.05)but not radiation(P >0.05)decreased bone volume fraction in 2-factor ANOVA analyses,and there were no interaction effects(P >0.05).3.Mechanical testing by MTS system revealed that HLS tended(P =0.051)to reduce fracture load,but did not affect failure energy(P >0.05).Radiation did not affect either property(P >0.0.05)in 2-factor ANOVA analyses.However,microCT of mid-diaphyseal cortical bone revealed that cortical bone area(P <0.05)and cortical bone volume(P <0.05)were significantly different between the four groups,and maximum section modulus(P >0.05)and cortical bone tissue mineral density(P >005)approached significance.HLS(P <0.05),but not radiation(P >0.05)had detrimental impacts on cortical bone area,volume,and maximum section modulus,and there were no interaction effects between loading and radiation exposure(P >0.05).4.In the histomorphometry analyses of trabecular bones we find a highvariability between groups which cause unremarkable differences.No differences were detected between the four groups in either osteoclast(P >0.05)or osteoblast(P >0.05)metrics,nor in the ratio of osteoblast to osteoclast surface(P >0.05).Neither HLS(P >0.168)nor radiation exposure(P >0.150)affected outcome measures for either osteoblast or osteoclast metrics,But we found a 54% decrease in osteoblast to osteoclast ratio,but radiation did not affect this property(P >0.05),nor was there a significant interaction effect(P >0.05).[Conclusion]In the model combining hindlimb suspension with chronic low-dose gamma radiatin,it is HLS caused bone loss in mice skeleton system,but not radiation and there was no significant interaction effect between these two factors.