Tracking Mouse Bone Marrow Monocytes In Vivo

Summary

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Abstract

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter ¹, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Introduction

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span ^2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells ⁴. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen ^{5, 6, 7} and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow ^{8, 5, ....}

Protocol

NOTE: All experiment protocols were approved by the French Animal Experimentation and Ethics Committee and validated by the “Service Protection et Santé Animales, Environnement” with number A-75-2065. Sample sizes are chosen to ensure reproducibility of the experiments, and according to the 3R of animal ethic regulation.

1. Preparation of the Mouse

1. Anaesthesia
   1. For short period of imaging (less than 1 hr), anaesthetize the mouse with an intraperitoneal injection of 200 µl of a saline solution containing Ketamine (100 mg/kg) and Xylazine (10 mg/kg).
   2. Alternatively, for longer per....

Results

Mouse skull structure offers a good opportunity to study the bone marrow physiology by intravital imaging. The bone being thin around the front-parietal area, it is possible to get access to medullar niches without abrasion of the bone. Figure 1 represents a wide 2D field of the skull of a MacBlue transgenic mouse. The bone matrix is mainly composed of collagen I easily detectable by SHG ¹⁹. Injection of rhodamin-dextran stains the vascular network of the bone marrow and allows for the identif.......

Discussion

The critical points of in vivo imaging methodology are to ensure stability of the focus in order to maximize the duration of imaging and to minimize the risk of bacterial contamination and inflammation, which might impact the dynamics of the inflammatory cells. Imaging of the skull bone marrow follows these aims as the surgery performed to get access to the bone marrow is minimal. The use of sterile material and antiseptics is essential to limit the risk of infection that might induce perturbation in cel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamin	Merial		100 mg/ml, anesthetic
Xylazin	Bayer HealthCare		10 mg/ml, anesthetic
Isofluran	Baxter		2.5%, anesthetic
O2/NO2			70/30 mixture, anesthetic
Rhodamin-Dextran	Invitrogen		2 MDa, 10 mg/ml, Vascular staining
Ly6G-PE	Becton-Dickinson		clone 1A8, neutrophils staining
Stereotactic holder	Home made		surgery
Ethanol 70%			surgery
Sterile scissors and nippers			surgery
Rubber ring			18 mm diameter, surgery
Glubran 2	Queryo Medical		Surgical Glue, rubber ring fixation
Small gauge needles	Terumo		surgery
Zeiss LSM 710 NLO multiphoton microscope	Carl Zeiss		Microscope
Ti:Sapphire crystal laser	Coherent Chameleon Ultra		140 fsec pulses of NIR light
Zen 2010	Carl Zeiss		Acquistion Software
Imaris Bitplane	Bitplane		Analysis Software, 3D automatic tracking
PBS 1X	D. Dutscher		surgery
Thermostated chamber	Carl Zeiss		intravital imaging