Hindlimb unloading in C57BL/6J mice induces bone loss at thermoneutrality without change in osteocyte and lacuno-canalicular network.

Abstract

Impaired mechanical stimuli during hindlimb unloading (HLU) are believed to exacerbate osteocyte paracrine regulation of osteoclasts. We hypothesized that bone loss and deterioration of the osteocyte lacuno-canalicular network are attenuated in HLU mice housed at thermoneutrality (28°C) compared with those housed at ambient temperature (22°C). Following acclimatization, 20-week-old male C57BL/6J mice were submitted to HLU or kept in pair-fed control cages (CONT), for 5days (5d) or 14d, at 22°C or 28°C. In the femur distal metaphysis, thermoneutral CONT mice had higher bone volume (p=0.0007, BV/TV, in vivo μCT, vs. 14dCONT22) whilst osteoclastic surfaces of CONT and HLU were greater at 22°C (5dCONT22+53%, 5dHLU22+50%, 14dCONT22+186%, 14dHLU22+104%, vs matching 28°C group). In the femur diaphysis and at both temperatures, 14dHLU exhibited thinner cortices distally or proximally compared to controls; the mid-diaphysis being thicker at 28°C than at 22°C in all groups. Expression of cortical genes for proteolytic enzyme (Mmp13), markers for osteoclastogenic differentiation (MCSF, RANKL), and activity (TRAP, Ctsk) were increased following 22°C HLU, whereas only Ctsk expression was increased following 28°C HLU. Expression of cortical genes for apoptosis, senescence, and autophagy were not elevated following HLU at any temperature. Osteocyte density at the posterior mid-diaphysis was similar between groups, as was the proportion of empty lacunae (<0.5%). However, analysis of the lacuno-canalicular network (LCN, fluorescein staining) revealed unstained areas in the 14dHLU22 group only, suggesting disrupted LCN flow in this group alone. In conclusion, 28°C housing influences the HLU bone response but does not prevent bone loss. Furthermore, our results do not show osteocyte senescence or death, and at thermoneutrality, HLU-induced bone resorption is not triggered by osteoclastic activators RANKL and MCSF.