Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations

• Published in: Bone (New York, N.Y.) N.Y.), 2012-05, Vol.50 (5), p.1115-1122
• Main Authors: Kennedy, Oran D, Herman, Brad C, Laudier, Damien M, Majeska, Robert J, Sun, Hui B, Schaffler, Mitchell B
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - genetics; Biological and medical sciences; Bone Resorption - genetics; Bone Resorption - pathology; Bone Resorption - physiopathology; Calcification, Physiologic; Caspase 3 - metabolism; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Immunohistochemistry; Microscopy, Fluorescence; Orthopedics; Osteoclasts - metabolism; Osteoclasts - pathology; Osteocytes - metabolism; Osteocytes - pathology; Osteoprotegerin - metabolism; RANK Ligand - metabolism; Rats; Signal Transduction - genetics; Stress, Mechanical; Ulna - metabolism; Ulna - pathology; Vertebrates: anatomy and physiology, studies on body, several organs or systems
• ISSN: 8756-3282; 1873-2763
• DOI: 10.1016/j.bone.2012.01.025

Abstract Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague–Dawley rats (16–18 weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7 days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6 mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300 μm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.