Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone). Immunohistochemistry was used to identify apoptotic and RANKL-producing osteocytes in femoral diaphysis and distal trabecular bone, and μCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3- and 4-fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3-fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5-fold versus Ctrl, p <0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU-triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, μCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse.

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca responses of individual osteocytes by using a genetically encoded fluorescent Ca indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading.

Metabolic oxidative stress has been implicated as a cause of osteocyte apoptosis, an essential step in triggering bone remodeling. However, little is known about the oxidative behavior of osteocytes in vivo. We assessed the redox status and distribution of total and active mitochondria in osteocytes of mouse metatarsal cortical bone in situ. Multiphoton microscopy (MPM) was used to measure fluorescence of reduced pyridine nucleotides (NADH) under normoxic conditions and acutely following extreme (postmortem) hypoxic stress. Under non-hypoxic conditions, osteocytes exhibited no detectable fluorescence, indicating rapid NADH re-oxidation. With hypoxia, NADH levels peaked and returned to near baseline levels over 3 hours. Cells near the periosteal surface reached maximum NADH levels twice as rapidly as osteocytes near the mid-cortex, due to the time required to initiate NADH accumulation; once started, NADH accumulation followed a similar exponential relationship at all sites. Osteocytes near periosteal and endosteal bone surfaces also had higher mitochondrial content than those in mid-cortex based on immunohistochemical staining for mitochondrial ATPase-5A (Complex V ATPase). The content of active mitochondria, assessed in situ using the potentiometric dye TMRM, was also high in osteocytes near periosteum, but low in osteocytes near endocortical surfaces, similar to levels in mid-cortex. These results demonstrate that cortical osteocytes maintain normal oxidative status utilizing mainly aerobic (mitochondrial) pathways but respond to hypoxic stress differently depending on their location in the cortex, a difference linked to mitochondrial content. An apparently high proportion of poorly functional mitochondria in osteocytes near endocortical surfaces, where increased apoptosis mainly occurs in response to bone remodeling stimuli, further suggest that regional differences in oxidative function may in part determine osteocyte susceptibility to undergo apoptosis in response to stimuli that trigger bone remodeling.

Despite increased longevity and resistance to multiple stressors, growth hormone receptor null (GHRKO) mice exhibit severe skeletal impairment. The role of GHR in maintaining osteocyte mitochondrial function is unknown. We found that GHR ablation was detrimental to osteocyte mitochondrial function. In vivo multiphoton microscopy revealed significant reductions of >10% in mitochondrial membrane potential (MMP) in GHRKO osteocytes and reduced mitochondrial volumetric density. Reductions in MMP were accompanied by reductions in glucose transporter-1 levels, steady state ATP, NADH redox index, oxygen consumption rate, and mitochondrial reserve capacity in GHRKO osteocytes. Glycolytic capacity did not differ between control and GHRKO males' osteocytes. However, osteocytes from aged female GHRKO mice exhibited reductions in glycolytic parameters, indicating impairments in glucose metabolism, which may be sex dependent. GHRKO osteocytes exhibited increased levels of cytoplasmic reactive oxygen species (ROS) (both basal and in response to high glucose), insulin-like growth factor-1 (IGF-1), and insulin. Mitochondrial ROS levels were increased and correlated with reduced glutathione in GHRKO osteocytes. Overall, the compromised osteocyte mitochondrial function and responses to metabolic insults strongly correlated with skeletal impairments, suggesting that despite increased life span of the GHRKO mice, skeletal health span is decreased. © 2018 American Society for Bone and Mineral Research.