Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model
ABSTRACTOsteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl) under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs.
[…] For live fluorescence imaging, larvae were anesthetized with 0.01% ethyl 3-aminobenzoate methanesulfonate (tricaine; Sigma A5040) and pictures were taken using a Nikon SMZ1000 stereomicroscope equipped with NIS-Elements BR 3.0 software (Nikon, Japan). For live confocal imaging, larvae were anesthetized with 0.005% tricaine and embedded in 1.5% low-melting-point agarose in a glass-bottomed Petri dish. Confocal pictures were taken with a Zeiss LSM 510 Meta using 405, 488 and 543 nm laser lines for DAPI, GFP and mCherry analysis, respectively. Imaging data were processed using Zeiss LSM Image Browser Version 184.108.40.206, Imaris 7.1.1 (bitplane), Image-Pro Plus 6.0, ImageJ (220.127.116.11) and Adobe Photoshop CS6 (18.104.22.168) software. […]