The influence of therapeutic radiation on the patterns of bone remodeling in ovary-intact and ovariectomized mice

Abstract

Our purpose was to characterize changes in bone remodeling associated with localized radiation that models therapeutic cancer treatment in ovary-intact (I) and ovariectomized (OVX) mice and to evaluate the influence of radiation on the pattern of bone mineral remodeling. Young adult, female BALB/c mice, I and OVX, were used (n = 71). All mice were intravenously injected with 15 μCi (45)Ca. Thirty days post-(45)Ca administration, the hind limbs of 17 mice were exposed to a single dose of 16 Gy radiation (R). The time course of (45)Ca excretion, serum CTx and osteocalcin markers, and cancellous bone volume fraction (BV/TV) and cortical thickness (Ct.Th) of the distal femur were assayed. Cellular activity and dynamic histomorphometry were performed. Irradiation resulted in rapid increases in fecal (45)Ca excretion compared to control groups, indicating increased bone remodeling. CTx increased rapidly after irradiation, followed by an increase in osteocalcin concentration. BV/TV decreased in the I mice following irradiation. Ct.Th increased in the OVX groups following irradiation. I+R mice exhibited diminished osteoblast surface, osteoclast number, and mineral apposition. Our murine model showed the systemic effects (via (45)Ca excretion) and local effects (via bone microarchitecture and surface activity) of clinically relevant, therapeutic radiation exposure. The I and OVX murine models have similar (45)Ca excretion but different bone microarchitectural responses. The (45)Ca assay effectively indicates the onset and rate of systemic bone mineral remodeling, providing real-time assessment of changes in bone histomorphometric parameters. Monitoring bone health via a bone mineral marker may help to identify the appropriate time for clinical intervention to preserve skeletal integrity.

Figure 1

Schematic of the experimental design. Five groups of skeletally mature BALB/c female mice (16 weeks old) were used for this study. These groups were: 1) Biodistribution (BD); 2) Ovary-intact, non-irradiated (I−R); 3) Ovary-intact, irradiated (I+R); 4) Ovariectomized, non-irradiated (OVX−R); and, 5) Ovariectomized, irradiated (OVX+R). Ovariectomy was performed 27 days prior to ⁴⁵Ca injection by the vendor (Jackson Laboratory; Bar Harbor, ME) and confirmed by weighing uterii immediately after euthanization.

Figure 2

A. Shielding designed to deliver radiation to the hind limbs while protect the rest of the the animal. A specially designed lead shield (4 mm thickness) was placed over the body cranial to the femur, over the pelvis and caudal vertebrae, targeting the radiation exposure to only the hind limbs. Proper placement of shielding was confirmed using Kodak EDR-2 film placed beneath the animal and acrylic plate. Total dose delivered, including the shielding placement exposure, was 16 Gy. Total dose in the radiation field and under the block was verified using thermo-luminescent dosimeters (TLD). B. Hematoxylin and Eosin (H&E) stain (distal femur): (I) Demineralized distal femora vacuum embedded in paraffin were sectioned (5 um) and stained with H&E, then imaged at 100x. (II) the number of osteoclasts (Oc.N), and (III) surface occupied by functional osteoblasts (Ob.S). C. Dynamic labeling (proximal tibia). Twelve and twenty nine days post days post radiation, all mice (n=12) received tetracycline and calcein intravenous injections respectively. Mice were euthanized five days later (34d post-radiation). Fixed undemineralized tibiae were vacuum embedded in methyl methacrylate and sectioned at 5 um on an EXAKT system (Oklahoma City, OK) and imaged under UV light with a 40x objective on a Zeiss AxioPlan II (Oberkochen, Germany). Images were captured and stitched together by Image-Pro v7.0 using a Stage-Pro module (Media Cybernetics, Inc., Rockville, MD). A representative figure of collated images of a complete tibia with dual labeling is shown in C (I). A small region of tibia with dual labeling is shown for control mice (II) and for irradiated mice (III).

Figure 3

Biodistribution and pharmacokinetics of ⁴⁵Ca in various organs, bones and excreta. A. Biodistribution of ⁴⁵Ca in organs and bones; B: Biodistribution of ⁴⁵Ca in feces and urine; C. ⁴⁵Ca pharmacokinetics in feces of different mice groups, I-R and OVX-R. Values are mean ± SE, n=5. To examine isotope biodistribution, ⁴⁵Ca radioactivity is measured in cleaned and ashed femur, tibia, lumbar vertebrae, liver, kidneys, gut tissue (duodenum to anus, with contents), the quadriceps femoris muscle, urine and feces at days 1, 3, 5, 16. Particulars are detailed in the Methods section.

Figure 4

⁴⁵Ca excretion curves showing the temporal effect of 16 Gy radiation exposure on the hind limb of ovary-intact mice and ovariectomized (OVX) mice. There was a significant increase in ⁴⁵Ca excretions from the A). irradiation, ovary-intact group compared to the B). non-irradiated OVX controls (p<0.001). Effects of radiation on ⁴⁵Ca excretion in intact mice were apparent during the first week after treatment emerging transiently (i.e., approximately for two weeks). There appeared to be a smaller effect of radiation on mice ovariectomized 57 days prior.

Figure 5

MicroCT results of the distal femur. A. Representative microCT 3D images of trabecular bone in a non-irradiated (I-R) and an irradiated (I+R) mouse on day 30 after irradiation. The BV/TV of the I+R mouse shown was 36.4% less than that of the I-R mouse shown. B. Histomorphometric parameters for the I±R and OVX±R groups. Histomorphometric parameters shown are: relative cancellous bone volume fraction (BV/TV; %; i.e. the ratio of the segmented bone volume to the total volume of the region of interest) and cortical thickness (Ct.Th, mm). Data are presented as group means ± SEM, n=5–7 per group. Significant differences were observed in the I±R group for BV/TV at day 8 (p=0.051) and day 30 (p=0.016), but not in the OVX±R group at day 30. A significant difference in Ct.Th was observed in I±R group at day 8 (p=0.001), and the OVX±R group at day 30 (p<0.001). Time dependent changes in BV/TV were significant (adjusting for multiple comparisons) between the I±R day 3 cohort compared to the I±R day 30 group (p<0.001) and between day 8 compared to the day 30 set (p=0.002). There was no significant difference between days 3 and 8. Radiation induced reductions in BV/TV were greater in intact mice compared to OVX mice, as is shown by the excreta data (Figure 4).

Figure 6

Correlation of cumulative ⁴⁵Ca in feces with mean cancellous BV/TV for the I+R group on days 3, 8, and 30 after irradiation (p<0.001). Data are presented as means +/− standard error, n=5–7 per group.