Figure 1: Quantitative MicroCT Bone Recovery in Ovariectomized Mice Following Lactation  Andrea Y. Thompson, Jeff Liu, Sabrina Jeter-Jones, and Robert Brommage Department of Metabolism, Lexicon Pharmaceuticals, The Woodlands, Texas 77381 INTRODUCTION METHODS RESULTS RESULTS (continued) CONCLUSIONS Bone is lost during lactation in all species examined and this loss is not prevented by consuming high calcium (Ca) diets. Feeding rats low Ca diets greatly increases the amount of bone loss during lactation. Following lactation the bone lost is restored, and women undergoing several pregnancies and lactations are not at increased risk for osteoporotic fractures. We examined post-lactation cortical and trabecular bone gain in mice previously fed normal or low Ca diets during lactation. To evaluate the possible role of estrogens, some mice were ovariectomized at the end of lactation. Bone loss and formation were evaluated by microCT and histomorphometric methods. Figure 1: Quantitative MicroCT Figure 3: Midshaft Femur Histology Figure 5: Endocortical Bone Labeling A steady decrease in endocortical surface labeling is observed from 1 to 5 weeks post-lactation. Feeding LCD during lactation causes marked resorption of cortical bone resulting in a thin cortex, irregular bone surfaces, and foci of intracortical porosity. Post-lactation fluorescent labeling indicates that endocortical and periosteal bone formation occur during recovery, with intracortical bone formation also evident in LCD mice. Table 2: Dynamic Histomorphometry Midshaft Femur Endocortical Surface C57BL/6 mice at 7 to 8 weeks of age were mated and pregnant mice were assigned to study groups as shown in Table 1. Non-lactating mice had pups removed after birth and lactating mice had pups weaned at 3 weeks. Mice were fed purified diets with normal Ca (0.6%; Control Diet) or low Ca (0.1%; LCD) starting at parturition. All mice received Control Diet post-lactation. For designated mice, ovariectomy (OVX) was performed at the end of lactation. Non-lactating mice were euthanized at 3 weeks post- parturition, and lactating mice were euthanized at 0, 1 (LCD only), 3, or 6 weeks post-lactation. Mice euthanized at 6 weeks were given fluorescent labels by subcutaneous injections at 1 week (10 mg/kg calcein), 3 weeks (20 mg/kg alizarin complexone), and 5 weeks (30 mg/kg demeclocycline) during the post-lactation recovery period. Standard microCT (midshaft femur and LV5 vertebral body) and histomorphometry (midshaft femur) analyses were performed. MicroCT measurements were made with a voxel size of 8 µm and high resolution images were scanned at 6 µm. Femurs embedded in methacrylate were sectioned (80 µm thickness) using a Leica SP1600 Saw Microtome. Histomorphometric measurements were made using OsteoMeasure (v.2.31; OsteoMetrics, Inc.). Means ± SEMs are shown. During lactation, moderate bone loss (21% cortical and 25% trabecular) occurs in mice fed Control Diet while feeding LCD results in marked bone loss (63% cortical and 81% trabecular). Dramatic post-lactation bone recovery of femoral cortical bone and vertebral trabecular bone occurs after 3 weeks, with robust restoration of bone during the first week post-lactation in mice fed LCD. OVX after lactation results in lower trabecular bone volume at 6 weeks post-lactation, primarily due to reduced trabecular thickness. Figure 4: Fluorescent Labeling of Cortical Bone Brightfield (left) and Epifluorescence (right) Control Diet + OVX LCD Mice fed LCD during lactation show lower Ec.MAR and Ec.BFR/BS during the first 3 weeks of post-lactation recovery in comparison to mice fed a Control Diet. OVX after lactation results in decreased Ec.MS/BS and increased Ec.MAR, but does not significantly affect endocortical bone formation rates. Figure 2: MicroCT Images of LV5 Trabecular Bone Table 1: Study Groups Low Calcium Diet During Lactation and Post-Lactation Ovariectomy Bone loss during lactation in mice is greatly increased by feeding low Ca diet. There is rapid bone gain post-lactation, with normal bone mass reached in 6 weeks. Post-lactation bone gain is minimally affected by ovariectomy at the end of lactation. The bone mechanostat appears to be largely independent of estrogen. Understanding pathways involved in this physiological bone recovery might provide insights that could lead to novel anabolic therapies for osteoporosis. Mice fed Control Diet show moderate trabecular bone loss while mice fed LCD show severe bone loss and thin trabeculae. Remarkable bone restoration is observed at 1 week post- lactation in mice fed LCD during lactation. Trabecular structure is mostly restored in all mice by 3 weeks post-lactation. Midshaft femur cortical bone shows labeling of endocortical and periosteal surfaces during post-lactation recovery: calcein at week 1 (green), alizarin at week 3 (red), and demeclocycline at week 5 (yellow). Restored bone in mice fed Control Diet has normal lamellar structure, while cortical bone from LCD mice contains regions of woven bone and prominent intracortical calcein labeling. Presented at the XIIth Congress of the International Society of Bone Morphometry, October 16-19, 2012 Disclosure: Authors are employees of Lexicon Pharmaceuticals, Inc., and may own stock or may have been given stock options.