Real-time intravital imaging of pH variation associated with osteoclast activity.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Sphingosine-1-phosphate-mediated osteoclast precursor monocyte migration is a critical point of control in antibone-resorptive action of active vitamin D.

The migration and positioning of osteoclast precursor monocytes are controlled by the blood-enriched lipid mediator sphingosine-1-phosphate (S1P) and have recently been shown to be critical points of control in osteoclastogenesis and bone homeostasis. Here, we show that calcitriol, which is the hormonally active form of vitamin D, and its therapeutically used analog, eldecalcitol, inhibit bone resorption by modulating this mechanism. Vitamin D analogs have been used clinically for treating osteoporosis, although the mode of its pharmacologic action remains to be fully elucidated. In this study, we found that active vitamin D reduced the expression of S1PR2, a chemorepulsive receptor for blood S1P, on circulating osteoclast precursor monocytes both in vitro and in vivo. Calcitriol- or eldecalcitol-treated monocytoid RAW264.7 cells, which display osteoclast precursor-like properties, migrated readily to S1P. Concordantly, the mobility of circulating CX3CR1(+) osteoclast precursor monocytes was significantly increased on systemic administration of active vitamin D. These results show a mechanism for active vitamin D in controlling the migratory behavior of circulating osteoclast precursors, and this action should be conducive to limiting osteoclastic bone resorption in vivo.

Molecular mechanism of cerebral edema improvement via IL-1RA released from the stroke-unaffected hindlimb by treadmill exercise after cerebral infarction in rats.

Cerebral edema following cerebral infarction can be severe and directly affect mortality and mobility. Exercise therapy after cerebral infarction is an effective therapeutic approach; however, the molecular mechanism remains unclear. Myokines such as interleukin-1 receptor antagonist (IL-1RA) are released during skeletal muscle contraction with effects on other organs. We hypothesized that myokine release during exercise might improve brain edema and confirmed the hypothesis using transient middle cerebral artery occlusion (tMCAO) model rats. Rats subjected to tMCAO were divided according to the severity of illness and further assigned to exercise and non-exercise groups. Treadmill exercises were performed at a speed of 2-8 m/min for 10 min from 1-6 days post-reperfusion after tMCAO. Exercise significantly reduced edema and neurological deficits in severely ill rats, with a reduction in aquaporin-4 (AQP4) expression in the ischemic core and increased blood IL-1RA release from the stroke-unaffected hindlimb muscle after tMCAO. Administration of IL-1RA into the lateral ventricles significantly reduced edema and AQP4 expression in the ischemic core. In conclusion, treadmill exercise performed in the early phase of stroke onset alleviated the decrease in blood IL-1RA following ischemic stroke. IL-1RA administration decreased astrocytic AQP4 expression in the ischemic core, suppressing brain edema.

Dynamic Analyses of the Short-Term Effects of Different Bisphosphonates Using Intravital Two-Photon Microscopy.

Bisphosphonates are commonly used for the treatment of bone disorders such as osteoporosis; however, the mechanism by which they affect the dynamics of living mature osteoclasts in vivo remains unknown. Here, we describe the short-term effects of different bisphosphonates on controlling the bone resorptive activity of mature osteoclasts in living bone tissues of mice using intravital two-photon microscopy with a pH-sensing chemical fluorescent probe. Three types of nitrogen-containing bisphosphonates, risedronate, alendronate, and minodronate, inhibited osteoclastic acidification during osteoporotic conditions just 12 hours after i.v. injection. Among the three types of drugs, risedronate was the most effective at increasing osteoclast motility and changing the localization of proton pumps, which led to an inhibition of bone resorption. Together, these results demonstrate that the intravital imaging system is a useful tool for evaluating the similarities and differences in currently used antibone resorptive drugs. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Direct cell-cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo.

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial-temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell-cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell-cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell-cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.