Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

Bone is a highly dynamic organ in which several cell types function cooperatively. Among these, osteocytes have recently emerged as an important regulator of bone homeostasis, although their mechanism of regulation is unclear. Here, intravital bone imaging by two-photon excitation microscopy allowed us to directly visualize 'osteocytic osteolysis', or resorption of bone in the lacuno-canalicular system. Osteocyte lacunae and the canalicular network in the cortex of murine tibiae were imaged by in vivo calcein staining, and local acidification in these structures was monitored using a topically applied pH sensor. We also demonstrated that sciatic neurectomy causes significant acidification around osteocytic lacunae and enlargement of lacuno-canalicular areas. These results provide strong evidence for osteocytic osteolysis, and demonstrate that two-photon intravital microscopy is useful for analysis of this phenomenon.