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

Summary

The current article describes performing an intravital imaging approach to observe mechanically induced calcium signaling of embedded osteocytes in vivo in real-time in response to tissue-level mechanical loading of the mouse third metatarsal.

Abstract

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

Introduction

The bone matrix is organized according to mechanical demand^1,2 and can change dynamically to account for shifting mechanical requirements^3,4,5. Seminal work regarding the mechanosensory mechanism in bone was published as a modeling paper about 30 years ago^6,7,8, where it was proposed that osteocytes embedded within the bone sense tissue level mechanical deformation via fluid movement in their local environment. This ....

Protocol

All methods have been approved by the Institutional Animal Care and Use Committee (IACUC) at Cornell University.

1. Preparation of materials and equipment

1. Design and build the metatarsal loading device for use with an overhead multiphoton microscope.
   NOTE: Details for component specifications and device operation are described in the supplementary documentation of a previous study²⁵. Briefly, the major components consist of an actuato.......

Discussion

It is difficult to study osteocytes experimentally because of their embedded position in the hard tissue matrix, to which they rapidly deteriorate or dedifferentiate upon removal of this niche. Researching osteocytes has required immense ingenuity over the last 3 decades, such as the creation of osteocyte-like lines^29,30,31, the establishment of protocols to isolate primary osteocytes^32,

Materials

Name	Company	Catalog Number	Comments
#3 Handle scalpel	Electron Microscopy Sciences	72040-03	Surgical supplies
20x Water immersion objective	Olympus	XLUMPLFLN20XW	This is a 20x objective, but 40x can also be used for this protocol. We recommend water immersions because it needs to be dipped in the bath during imaging, so open-air objective may cause abberrations
A.M. Bickford Omnicon F/AIR	AM Bickford	80120	A sensible answer to anesthesia gas problems in the operating room, the F/AIR anesthesia gas filter was specifically designed to remove waste anesthesia gases such as Isoflurane, Halothane, Enflurane, etc. from the operating room environment.
A.M. Bickford Omnicon F/AIR Kit	AM Bickford	80000	An entire kit with tube and adaptors to connect F/AIR to setup
B6J.Cg-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/MwarJ	The Jackson Laboratory	28865	GCaMP6f Mice for cross-breeding with Dmp1-Cre mice
B6N.FVB-Tg(Dmp1-cre)1Jqfe/BwdJ	The Jackson Laboratory	23047	Dmp1-Cre Mice for cross-breeding with GCaMP6f  mice
Compression load cell	FUTEK Advanced Sensor Technology, Inc.	905898	LCM100 , 1000 g , Sub-miniature tension & compression load cell (Miniature/Inline Threaded) , RoHS lead free, Material - 17-4 PH S.S. , M3x0.5-thread , 34 Awg 4 conductor braided polyester cable , 5 ft Long
Corning 500 mL DPBS (Dulbecco's phosphate buffered saline), 1x [+] calcium, magnesium	VWR International	21-030-CV	Ionically balanced bath that contains calcium submerges the metatarsal during imaging
Dental pick tool	Electron Microscopy Sciences		Surgical supplies
Ethyl alcohol pure 200 proof ACS reagent >99.5%	Sigma Aldrich	SIAL-459844-500ML	Sterilization purposes
Fulcrum pin	Fathom	N/A	Fabricated by direct 3D laser sintering of stainless steel (PH1 alloy) from 3D SolidWorks STL files. Original vendor was GPI Prototype & Manufacturing Services, Inc, now acquired by FATHOM, specifications are provided in the previously published document
High resolution and speed USB220 output kit	FUTEK Advanced Sensor Technology, Inc.	717435	Used to connect load cell to laptop
ImageJ 1.53t with Java 1.8.0_172 (for Windows 64-bit)	NIH	N/A	Install here https://imagej.nih.gov/ij/
Isoflurane	Piramal Critical Care	66794-013-25	100% inhalation vapour liquid
Loading bracket	Fathom	N/A	Fabricated by direct 3D laser sintering of stainless steel (PH1 alloy) from 3D SolidWorks STL files. Original vendor was GPI Prototype & Manufacturing Services, Inc, now acquired by FATHOM, specifications are provided in the previously published document
MATLAB R2019a	MathWorks		For running the loading device
Matrx VIP 3000 vaporizer well fill isoflurane	Butler Schein Animal Health	14309	Vaporizer for anesthetic
Nonin pulse oximeter Model 2500A Vet		2500A Vet
Piezo servo controller	PI-USA	E-625	Electronic component recommended by the company to be used with the actuator
PiezoMove high-stiffness linear piezo actuator	PI-USA	P-602.5SL	Actuator for the loading device
Scalpel blades, No. 10 for handle No. 3, pack of 100	Electron Microscopy Sciences	72044-10	Surgical supplies
Stainless steel tweezers with sharp, fine tips. Length: 120 mm	Electron Microscopy Sciences	78326-42	Surgical supplies
Thorlabs Bergamo multiphoton microscope	ThorLabs	N/A	This is only the imaging system and does not have the laser included, although ThorLabs has laser options if desired
Titanium-Saphire Chameleon Discovery NX with Total Power Control (TPC)	Coherent	N/A	This system technically has two lasers, both a tunable and a fixed laser. However, for the protocol, only the tunable is needed.
Vannas spring scissors - 4 mm cutting edge	Fine Science Tools	15018-10	Surgical supplies
Water bath	Fathom	N/A	Fabricated by direct 3D laser sintering of stainless steel (PH1 alloy) from 3D SolidWorks STL files. Original vendor was GPI Prototype & Manufacturing Services, Inc, now acquired by FATHOM, specifications are provided in the previously published document