Plos_one_SB_data

The age column is deleted

Additional file 2: of Neurocognitive sparing of desktop microbeam irradiation

Supplementary Method. (DOCX 16 kb

Additional file 1: of Neurocognitive sparing of desktop microbeam irradiation

Pretest results. (DOCX 754 kb

Rib Fractures and Death from Deletion of Osteoblast βcatenin in Adult Mice Is Rescued by Corticosteroids

<div><p>Ribs are primarily made of cortical bone and are necessary for chest expansion and ventilation. Rib fractures represent the most common type of non-traumatic fractures in the elderly yet few studies have focused on the biology of rib fragility. Here, we show that deletion of βcatenin in Col1a2 expressing osteoblasts of adult mice leads to aggressive osteoclastogenesis with increased serum levels of the osteoclastogenic cytokine RANKL, extensive rib resorption, multiple spontaneous rib fractures and chest wall deformities. Within days of osteoblast specific βcatenin deletion, animals die from respiratory failure with a vanishing rib cage that is unable to sustain ventilation. Increased bone resorption is also observed in the vertebrae and femur. Treatment with the bisphosphonate pamidronate delayed but did not prevent death or associated rib fractures. In contrast, administration of the glucocorticoid dexamethasone decreased serum RANKL and slowed osteoclastogenesis. Dexamethasone preserved rib structure, prevented respiratory compromise and strikingly increased survival. Our findings provide a novel model of accelerated osteoclastogenesis, where deletion of osteoblast βcatenin in adults leads to rapid development of destructive rib fractures. We demonstrate the role of βcatenin dependent mechanisms in rib fractures and suggest that glucocorticoids, by suppressing RANKL, may have a role in treating bone loss due to aggressive osteoclastogenesis.</p> </div

Changes in vertebral bone in βcatenin-CKO animals 10 days post tamoxifen.

<p>(<b>A,B</b>) High resolution micro CT of vertebrae of (<b>A</b>) control and (<b>B</b>) βcatenin-CKO animals (red arrowheads point to regions of bone loss) (<b>C</b>) mean BV/TV (<b>D,E</b>) Hematoxylin-eosin staining of sections of vertebrae of (<b>D</b>) control and (<b>E</b>) βcatenin-CKO animals (white arrowheads point to loss of trabecular bone) (mean±S.E.M.; n = 3 animals/group; *p<0.05, BV/TV: trabecular bone volume/total volume). (Scale bar: 100 µm).</p

βcatenin-CKO mice exhibit extensive rib resorption from osteoclastogenesis.

<p>(<b>A–C</b>) Micro CT of the thoracic cage of (<b>A</b>) control and (<b>B</b>) βcatenin-CKO animals. White arrowheads point to osteopenia and rib deformity. (<b>C</b>) Ribs shown in coronal plane with bone destruction (arrowheads) with (<b>D</b>) Rib bone volume/total volume, BV/TV) (mean±S.E.M.; n = 6 animals/group, *p<0.0001, BV/TV measurements were made on cortical rib) (<b>E,F</b>) Xgal staining <i>in-situ</i> of rib cage of (<b>E</b>) <i>Col1a2CreERT</i> and (<b>F</b>) βcatenin-CKO lineage reporter animals with callus and fracture deformities (black arrowheads). (<b>G,H</b>) Xgal staining of rib sections of (<b>G</b>) <i>Col1a2CreERT</i> and (<b>H</b>) βcatenin-CKO lineage reporter animals (arrowheads point to Cre expressing cells in blue) (<b>I–L</b>) Hematoxylin-eosin staining of rib sections of (<b>I</b>) control and (<b>J–L</b>) βcatenin-CKO animal showing (<b>J</b>) gross rib destruction with cellular infiltrate (<b>K</b>) involvement of consecutive ribs (<b>L</b>) complete rib resorption (<b>M,N</b>) TRAP staining of rib sections with TRAP positive cells (arrowheads) in (<b>M</b>) control and (<b>N</b>) βcatenin-CKO animals (<b>O</b>) Plasma alkaline phosphatase (mean±S.E.M., n = 10 animals/group, *p<0.05). All measurements and staining were done in animals 10 days post completion of tamoxifen. (Scale bar: 100 µm).</p

βcatenin CKO mice die within days of βcatenin deletion.

<p>(<b>A</b>) Strategy for deleting βcatenin in <i>Col1a2</i> expressing cells of 8 week old <i>Col1a2CreERT</i>/βcatenin^fl/fl mice. (<b>B</b>) Survival of mice following tamoxifen administration (n = 45 animals for βcatenin-CKO and15 animals for each of the control groups). (<b>C</b>) Peripheral blood oxygen saturation of mice pre and post pulmonary challenge with inhaled isoflurane (n = 10 animals in βcatenin-CKO group and 5–6 animals for control groups, *p<0.001) (<b>D</b>) Tidal volume (mean±S.E.M.; n = 6 animals/group, *p<0.05 versus control groups) and (<b>E</b>) lung histology, 10 days post tamoxifen (<b>F</b>) CT scan and (<b>G</b>) 3-dimensional lung reconstruction at Day 0 and Day 10 post tamoxifen (n = 6 animals/group). (White arrowheads indicate chest wall deformity and black arrowheads loss of lung volume. Oil injection refers to Cre+/βcatenin^fl/fl animals injected with oil and Cre neg control refers to tamoxifen injected βcatenin^fl/fl animals). (Scale bar: 100 µm).</p

Changes in femur of βcatenin-CKO animals 10 days post tamoxifen.

<p>(<b>A</b>) X gal staining <i>in situ</i> of femur of <i>Col1a2Cre:R26R^lacZ</i> mice (arrowheads point to lacZ expressing regions, arrow shows femoral head) (<b>B, C</b>) X gal staining of section of (<b>B</b>) metaphyseal end of femur and (<b>C</b>) femoral diaphysis (arrowheads point to lacZ expressing cells, with very few cells expressing lacZ in the diaphysis). (<b>D</b>) Change in femoral BV/TV in βcatenin-CKO animal (mean±S.E.M, n = 3 animals/group, p>0.05, bone volume measured at metaphyseal ends, BV/TV: trabecular bone volume/total volume) (<b>E</b>) Micro CT of femurs and (<b>F, G</b>) TRAP staining of metaphyseal region of femurs of (<b>F</b>) control and (<b>G</b>) βcatenin-CKO animals (arrowheads show TRAP positive cells) and (<b>H</b>) number of osteoclasts/mm² of femoral metaphysis (n = 3 femurs/group, n.s = not significant) (<b>I, J</b>) Hematoxylin-eosin staining of femoral diaphysis sections of (<b>I</b>) control and (<b>J</b>) βcatenin-CKO animals and (<b>K</b>) histomorphometric assessment of cortical bone area of femoral diaphysis (n = 9 femurs/group, n.s = not significant) (Scale bar: 100 µm).</p

Dexamethasone improves survival and preserves rib structure by decreasing RANKL and osteoclastogenesis in βcatenin-CKO mice.

<p>(<b>A</b>) Kaplan-Meier survival curve of βcatenin-CKO animals treated with dexamethasone or pamidronate (p<0.005 between dexamethasone and βcatenin-CKO groups, p<0.05 between dexamethasone and pamidronate groups, n = 16 animals in dexamethasone and pamidronate groups) (<b>B,C</b>) Hematoxylin-eosin staining of βcatenin-CKO animals 10 days post tamoxifen (<b>B</b>) untreated or (<b>C</b>) treated with dexamethasone (<b>D–F</b>) High resolution micro CT of ribs from (D) Cre negative control animal (<b>E</b>) βcatenin-CKO and (<b>F</b>) dexamethasone treated βcatenin-CKO 10 days post tamoxifen (<b>G</b>) Rib BV/TV 10 days post tamoxifen (mean±S.E.M.; *p<0.05, n = 4 ribs/animal with 3 animals/group) (<b>H</b>) Micro CT of ribs of dexamethasone treated βcatenin-CKO 65 days post tamoxifen (<b>I,J</b>) Hematoxylin-eosin staining of ribs 65 days post tamoxifen in dexamethasone treated βcatenin-CKO animal showing (<b>I</b>) cartilage formation and (<b>J</b>) restoration of bone circumference. (<b>K</b>) CT scan 65 days post tamoxifen of dexamethasone treated animal showing bilaterally inflated lungs. (<b>L–N</b>) TRAP staining 10 days post tamoxifen showing osteoclasts (arrowheads) in ribs of (<b>L</b>) untreated and (<b>M</b>) dexamethasone treated βcatenin-CKO animals with (<b>N</b>) number of TRAP positive cells normalized to rib surface area (mean±S.E.M.; *p<0.05, n = 16 ribs/animal with 3 animals/group) (<b>O</b>) Serum RANKL levels 8 days post tamoxifen in control and βcatenin-CKO mice with/without dexamethasone treatment (mean±S.E.M.; *p<0.05 versus Tam Inj Cre-, **p<0.05 versus βcatenin-CKO, n = 10 animals/group). (Scale bar: 100 µm).</p