A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes

This protocol provides a way to observe embedded osteocytes in vivo representing an important advancement in the study of bone mechanobiology. The main advantage of this technique is the ability to observe cellular and molecular level events in osteocytes in vivo, maintaining their native 3D spatial arrangement, blood flow, and endocrine conditions, and overcoming the limitations typically imposed by in vitro cellular experimentation. This technique is designed for basic science investigation in mouse models. They do not extend to therapy or diagnosis just yet, but do provide the opportunity for insights that define the next generation of bone treatments.

[Narrator] To begin, make a scalpel incision through the dermis between the second and third metatarsals, starting at the distal end and progressing proximally along the length of the bones. Using forceps, open the surgical site by pulling the skin toward the medial or lateral edges. Resect the extensor tendons using straight Vannas scissors to expose the third metatarsal. Insert the center fulcrum pin of the metatarsal loading device into the small space at the distal end where the third metatarsal meets the proximal phalange. Then move the pin proximally until it rests at the mid shaft of the third metatarsal. Take special care to guarantee a direct interface between the bone and the pin, excluding any muscle and fascia. Hold the ankle with forceps and place the foot into the DPBS filled water bath. Secure the paw in place with the loading bracket and connect the bracket to the actuator load cell arrangement. Carefully pick up the loading device setup with the mouse and place it onto the microscope platform. Start with the low magnification objective in epifluorescence mode to locate and center the mid diaphysis of the bone. After a region of interest is located, switch to a higher water immersion objective magnification and switch the optics to two photon. Set the pixel density by using the Galvo/Resonance area control settings. To avoid photo bleaching, reduce the power using the power control option and increase the PMT amplification in the Galvo/Resonance scanner control settings. Identify the surface of the bone in two photon mode by looking for the fibrous periosteum autofluorescence in the green channel that uses a band pass filter centered at 525 nanometers. If there is a visible movement of the bone during loading, select a different region of interest along Y or Z directions to ensure it is directly over the center fulcrum pin. If movement persists, adjust the surgical position of the pin manually underneath the third metatarsal. Conduct Z stack imaging with loading according to the experimental design to record the morphometric signaling events, and then T series imaging with loading to record the dynamic load induced signaling events. A single plane two photon image of metatarsal osteocytes expressing GCaMp6f in vivo is shown here. Endogenous expression of calcium reporter constructs provides a fluorescent target without the need for incubation. 3D Z projection of osteocytes in vivo captured with two photon microscopy is presented in this figure. The transverse cut in the XY plane and a sagittal cut show the depth of the Z stack. The representative image shows an example time course of GCaMp6f fluorescent signal from an embedded osteocyte. At rest, low level noisy fluctuations are exhibited. With loading, the calcium fluctuations increase in magnitude and regularity, often occurring in step with applied loading.

This method paves the way for researchers to answer functional based questions regarding osteocyte mechanobiology in vivo that were previously only possible in vitro or ex vivo.