1. 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 < for OVX P = 0.29 for Lrp5 KO
P < for PTH P < 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