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

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 5 days (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.

DI-5-Cuffs: Bone Remodelling and Associated Metabolism Markers in Humans After Five Days of Dry Immersion to Simulate Microgravity.

Background: The dry immersion (DI) model closely reproduces factors of spaceflight environment such as supportlessness, mechanical and axial unloading, physical inactivity, and induces early increased bone resorption activity and metabolic responses as well as fluid centralization. The main goal of this experiment was to assess the efficacity of venoconstrictive thigh cuffs, as countermeasure to limit cephalad fluidshift, on DI-induced deconditioning, in particular for body fluids and related ophthalmological disorders. Our specific goal was to deepen our knowledge on the DI effects on the musculoskeletal events and to test whether intermittent counteracting fluid transfer would affect DI-induced bone modifications. Methods: Eighteen males divided into Control (DI) or Cuffs (DI-TC) group underwent an unloading condition for 5 days. DI-TC group wore thigh cuffs 8-10 h/day during DI period. Key markers of bone turnover, phospho-calcic metabolism and associated metabolic factors were measured. Results: In the DI group, bone resorption increased as shown by higher level in Tartrate-resistant acid phosphatase isoform 5b at DI24h. C-terminal telopeptide levels were unchanged. Bone formation and mineralization were also affected at DI24h with a decreased in collagen type I synthesis and an increased bone-specific alkaline phosphatase. In addition, osteocalcin and periostin levels decreased at DI120h. Calcemia increased up to a peak at DI48h, inducing a trend to decrease in parathyroid hormone levels at DI120h. Phosphatemia remained unchanged. Insulin-like growth factor 1 and visfatin were very sensitive to DI conditions as evidenced by higher levels by 120% vs. baseline for visfatin at DI48h. Lipocalin-2, a potential regulator of bone homeostasis, and irisin were unchanged. The changes in bone turnover markers were similar in the two groups. Only periostin and visfatin changes were, at least partially, prevented by thigh cuffs. Conclusion: This study confirmed the rapid dissociation between bone formation and resorption under DI conditions. It revealed an adaptation peak at DI48h, then the maintenance of this new metabolic state during all DI. Notably, collagen synthesis and mineralisation markers evolved asynchronously. Thigh cuffs did not prevent significantly the DI-induced deleterious effects on bone cellular activities and/or energy metabolism.

The Skeletal Cellular and Molecular Underpinning of the Murine Hindlimb Unloading Model.

Bone adaptation to spaceflight results in bone loss at weight bearing sites following the absence of the stimulus represented by ground force. The rodent hindlimb unloading model was designed to mimic the loss of mechanical loading experienced by astronauts in spaceflight to better understand the mechanisms causing this disuse-induced bone loss. The model has also been largely adopted to study disuse osteopenia and therefore to test drugs for its treatment. Loss of trabecular and cortical bone is observed in long bones of hindlimbs in tail-suspended rodents. Over the years, osteocytes have been shown to play a key role in sensing mechanical stress/stimulus via the ECM-integrin-cytoskeletal axis and to respond to it by regulating different cytokines such as SOST and RANKL. Colder experimental environments (~20-22°C) below thermoneutral temperatures (~28-32°C) exacerbate bone loss. Hence, it is important to consider the role of environmental temperatures on the experimental outcomes. We provide insights into the cellular and molecular pathways that have been shown to play a role in the hindlimb unloading and recommendations to minimize the effects of conditions that we refer to as confounding factors.