Thirty-two C57BL/6 mice (13 weeks old; Charles River Breeding Laboratories, Wilmington, MA) either were irradiated or served as nonirradiated controls (n = 16 per group). Subsets of these mice were used for one of three assays: histological analysis, microcomputed tomography (microCT), and serum chemistry analysis. All animals were allowed a 1-week acclimation period prior to the start of the study, with food and water provided ad libitum. The Institutional Animal Care and Use Committee at Clemson University approved of all procedures. All animals were killed humanely 3 days after irradiation to collect data on body mass, tibial length and morphology.
While under anesthesia (1.5% isoflourane), mice were irradiated in the prone position with a single field of 140 kVp X rays to a single-fraction mid-plane dose of 2 Gy. Irradiation was performed at a nominal dose rate of 1.37 Gy/min with an exposure time of 1.46 min. A 150 kV industrial portable X-ray unit was used (Philips Medical Systems, Bothell, WA). Anesthetized control mice were placed inside the inactive X-ray unit for a similar amount of time as the treated animals for sham irradiation.
To examine the postirradiation activity of osteoclasts, a set of 12 mice (n = 6/group) were used for histological analyses. Three days after whole-body irradiation, tibiae were isolated, cleaned of soft tissue, fixed in formalin for 48 h, and stored in 70% ethanol. The right tibia was decalcified in a weak formic acid solution (Immunocal™, Decal Chemical Corporation, Tallman, NY). Radiographs assessed the earliest time of complete decalcification. Then tibiae were embedded in a glycol methacrylate resin (Immunobed™, Polysciences, Warrington, PA) and cut into sagittal sections with a thickness of 1.5 μm. The presence of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP) staining of the slides using a commercial kit (Sigma, St. Louis, MO) and then counterstaining with hematoxylin (Sigma).
Histomorphometric evaluation was performed from captured micro-graphs (400×) throughout the metaphysis, starting approximately 0.25 mm distal from the growth plate (to exclude the primary spongiosa) and extending a further 0.5 mm. Bone histomorphometric parameters for the proximal metaphysis of the tibia were measured as described in the report of the American Society of Bone and Mineral Research (ASBMR) Histomophometry Nomenclature Committee (22
). Surface measurements were quantified relative to total bone surface (BS). These measurements included osteoblast surface (Ob.S/BS; %), osteoclast surface (Oc.S/BS; %), eroded surface with the inclusion of osteoclast surface (surface covered by Howship's lacunae plus osteoclasts, [ES(Oc+)/BS], %), and eroded surface with the exclusion of osteoclast surface (surface covered by Howship's lacunae, [ES(Oc−)/BS], %). The number of osteoclasts (N.Oc) within the region of interest along trabeculae of the secondary spongiosa was also determined (N.Oc/BS, mm−1
Twenty mice (n = 10/group) were treated identically for examination of trabecular microarchitecture and serum chemistry markers of bone resorption. The right tibiae were scanned with microCT (Scanco Medical AG, Bassersdorf, Switzerland), with isotropic voxels of 9 μm/side. A 3-mm section of bone immediately distal to the proximal growth plate was scanned. Evaluation of the scanned region began immediately adjacent to the primary spongiosa and extended 1 mm. Trabecular bone parameters including bone volume fraction (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were quantified.
To confirm osteoclast activation with a systemic analysis technique, serum was collected from these mice after they were killed by cardiac puncture and exsanguination. Sufficient blood for further serum analysis was collected from eight of the ten mice in each group. ELISAs were used to determine the blood serum concentration of tartrate-resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity that is associated with bone resorption (ImmunoDiagnostic Systems Inc., Fountain Hills, AZ), and osteocalcin, a marker of osteoblast activity that is associated with bone formation (Biomedical Technologies Inc., Stoughton, MA).
Serum and other histomorphometric analyses were tested for statistical significance using SigmaStat Version 3.5 (Systat Software Inc., Richmond, CA). A Student's t test was performed to reveal significant differences between the control and irradiated groups. A P value of less than 0.05 was considered a significant difference. All data are presented as means ± SEM.