Original Full Length ArticleIntravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo
Introduction
Osteocytes are the most abundant cellular component of bone, comprising around 90–95% of all bone cells. These cells are ‘entombed’ within bone tissue, but seem to survive for extended periods, up to 25 years in humans [1]. Osteocytes are terminally differentiated osteoblasts, considered dormant until recent evidence demonstrated their critical role in endocrine regulation and bone homeostasis [2], [3], [4], [5]. Osteocytes are connected to one another via a network of cytoplasmic projections [4], [5], consisting of disk-shaped osteocytic lacunae (OL) and numerous dendritic processes (canaliculi) radiating therefrom.
Since Baud reported electron micrographic observations of osteocytes' roughly bordered lacunar walls in 1962 [6], the concept of bone resorption by osteocytes, so-called ‘osteocytic osteolysis’, has been proposed and reviewed [7], [8], [9], [10], [11], although these initial histological studies provided little definite evidence. On the contrary, OL enlargement has also been attributed to an artifact of specimen preparation [12]; isolated avian osteocytes fail to resorb bone in vitro [13]. Since similar changes in OL can also be found in younger osteocytes, enlargement could result from insufficient mineralization of the periosteocytic matrix [12], [14]. In addition, the irregular, variable morphology of OL presents major challenges to examination of osteocytic osteolysis.
Nevertheless, recent circumstantial evidence supports the concept [15], [16]. OL enlargement was detected by lactation [17] in the presence of sclerostin [18] or microgravity [19], suggesting active regulation of the OL space and osteolysis. However, no direct evidence has yet been presented.
Over the past few years we have established a system for visualizing the bone tissues of living animals ‘intravitally’ under completely intact conditions [20], [21], [22], [23], [24], [25]. This novel system has unraveled mechanisms of migration and functions of bone-resorbing osteoclasts and their precursors in vivo. In this study, we exploited this new imaging technique to visualize and analyze the function of osteocytes in vivo.
Section snippets
Intravital in vivo bone tissue imaging
Intravital microscopy of mouse tibiae was performed using protocols modified from a previous study [20], [21]. Briefly, mice were anesthetized with isoflurane (2.0%, vaporized in 100% oxygen), and two-thirds of the length of the medial tibia was exposed by stripping the periosteum. Exposed cortical bone tissues were observed by two-photon excitation microscopy with a custom-made stereotactic holder. The imaging system was composed of a multiphoton microscope (SP5; Leica) driven by a laser
Intravital two-photon microscopy of the osteocytic lacuno-canalicular system
To visualize the osteocytic lacuno-canalicular system in bone, we systemically administered calcein, a commonly used fluorescent dye that attaches to the bone surface (20 mg/kg, s.c.) [29]. Histological examination of femur cortical bone specimens revealed that calcein was successfully incorporated in osteocytic lacunae (OL) (Fig. 1A–D). Intravital two-photon imaging of live tibia cortex also detected calcein-labeled OLs (shown in green), buried in bone matrices represented by second harmonic
Discussion
The function of osteocytes in bone metabolism has been discussed extensively, but numerous controversies remain. Among these, osteolysis is especially anecdotal, because most studies so far have employed histological analyses, providing no direct evidence. In this study, by using intravital bone imaging with fluorescent probes, we have found strong evidence for acidification in osteocytic lacunae in osteoporotic conditions such as sciatic neurectomy. Because acidification is a key step in
Disclosures
All authors declare that they have no conflicts of interest.
Acknowledgments
We thank Drs. K. Kikuchi, T. Kowada and H. Maeda (Osaka University Graduate School of Engineering) and H. E. Takahashi, N. Yamamoto and T. Shimakura (Niigata Bone Science Institute) for helpful discussions. This work was funded by Grants-in-Aid for Scientific Research on Innovative Areas (22113007), by Grant-in-Aid for Scientific Research (A) (25253070) from the Ministry of Education, Science, Sports and Culture of Japan, by grants from the International Human Frontier Science Program (
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