Mouse Femoral Neck Architecture Determined by MicroCT Reflects Skeletal Architecture Observed at Other Bone Sites Robert Brommage, Sabrina Jeter-Jones, Wendy Xiong, Rose Champ and Jeff Liu Lexicon Pharmaceuticals, The Woodlands, Texas 77381 INTRODUCTION RESULTS (continued) RESULTS (continued) Effect of Age in WT and Sost KO Mice Sfrp4 KO FN: Large Diameter and Greater Trabecularization Mouse femoral neck architecture is seldom examined in genetic, physiology and pharmacology studies. We developed a microCT scanning protocol providing reproducible values for total area (diameter), bone area, BV/TV and BS/BV (trabecularization) FNs (Ns = 5 to 11) from female (16 weeks) and male (60, 68 and 105 weeks) KO mice have greater diameters (26% to 45%, P < 0.001) High bone volume (BV) at 16, 60 and 68 weeks (13% to 17%, P < 0.03) Low BV/TV at all ages, which declines dramatically in KO mice at 2 years Elevated trabecularization (BS/BV) at all ages, with dramatic increase in KO mice at 2 years Femoral neck architecture in Sfrp4 KO mimics phenotypes in LV5 (increased trabecular bone) and midshaft femur (high diameter with low cortical thickness) METHODS MicroCT Scanning (Scanco Medical µCT40) Scan parameters: 55 keV energy, 145 microamperes current, 200 milliseconds integration time, 240 threshold, with 6 micrometer isotropic voxel size. Careful positioning of the bones to obtain vertical orientation of the neck is critical. The entire neck (150 slices) is scanned. Using 2-D analysis, the slice having the lowest total area is identified. 3-D analysis is performed using 20 slices with the middle slice having the lowest total area in the 2-D analysis. No attempt is made to separate trabecular from cortical bone. Using the Direct, model-independent mode, total volume (TV), bone volume (BV) and BV/TV are determined. Since cross-sections are nearly circular, femoral neck diameters are calculated from total area, defined as total volume divided by the 120 micrometer length analyzed. BV/TV falls with age in males and females and is elevated in Sost KO mice FN diameter is higher in male than female and elevated in Sost KO mice Femoral Neck BV/TV Reduced Following OVX and Elevated with Teriparatide Treatment WT Mice Two-Factor ANOVA Two-Factor ANOVA P < 0.001 for OVX P = 0.29 for Lrp5 KO P < 0.001 for PTH P < 0.001 for PTH P = 0.08 for Interaction P = 0.64 for Interaction FN diameter is reduced in female Lrp5 KO mice above (16%, P < 0.001) and also male Lrp5 KO mice at 12 weeks of age (20%, P < 0.001) Sfrp4 KO Mice Post-Lactation Bone Recovery RESULTS C57BL/6J mice mated at 7 to 8 weeks of age. After parturition mice were fed standard purified diets containing 0.1% or 0.6% Ca. Nonlactating mice had their pups removed and were euthanized 3 weeks later. After 21 days of lactation, some mice fed both diets were euthanized, pups were weaned from the remaining mice and all post-lactation mice were fed the 0.6% Ca diet. After bone recovery of one week (mice fed 0.1% Ca diet during lactation only) or six weeks (mice fed both diets during lactation) mice were euthanized. MicroCT analyses of the LV5 vertebral body trabecular bone and midshaft femur cortical bone were performed in addition to femoral neck measurements. Bone loss during lactation was greatly enhanced in mice fed low Ca diet. Dramatic post-lactation bone recovery occurred in the FN, similarly to LV5 trabecular and midshaft femur cortical bone (data not shown). Skeletal Phenotypes of KO Mice SUMMARY AND CONCLUSIONS Gene KO LV5 Trabecular Bone Volume Midshaft Femur Cortical Thickness Midshaft Femur Diameter References Lrp5 Reduced Gong, 2001 Holman, 2004 Iwaniec, 2007 Sfrp4 Elevated Saito, 2008 Brommage, 2009 Kao, 2010 Sost Brommage, 2006 Li, 2008 Kramer, 2010 Bone mass and architecture can be determined in mouse femoral neck, a site at which bone strength measurements are performed by several groups Mouse femoral neck bone mass (BV/TV): Increased with KO of Sost and teriparatide treatment Decreased with KO of Sfrp4, ovariectomy and lactation Mouse femoral neck diameter: Greater in males than females Increased with KO of Sost and Sfrp4 Decreased with KO of Lrp5 Femoral neck architecture is consistent with trabecular (LV5) and cortical (midshaft femur) bone structure in all mice examined P Values in BLACK refer to effects of diet P Values in RED refer to effects of lactation References in RED provide information on KO mice examined at Lexicon Identical bone phenotypes are described by two independent laboratories Lexicon mice are F2 hybrids from C57BL/6J and 129 SvEv parental strains Presented at the ASBMR Annual Meeting – Baltimore, MD, USA, October 4 – 7, 2013 Disclosure: The authors of this poster are current employees of Lexicon Pharmaceuticals and own stock/stock options