Elsevier

Bone

Volume 50, Issue 1, January 2012, Pages 390-399
Bone

Original Full Length Article
Structure and quantification of microvascularisation within mouse long bones: What and how should we measure?

https://doi.org/10.1016/j.bone.2011.09.051Get rights and content

Abstract

Bone marrow vascularisation is involved in both remodeling and hematopoïesis. Challenged mouse models often require imaging and quantitative assessment of blood vessels and bone cell activities for a better understanding of the role of the vascular system. In this study we compared images of mouse hind limb long bone vascularisation after infusion of either barium sulfate or lead chromate-loaded silicon. The images were then analyzed through histology as well as low-resolution and synchrotron-radiation microtomography. We show that barium sulfate infusion provides the best vessel images and furthermore, that it is compatible with staining procedures used in bone histomorphometry and CD31 immunohistochemistry. Bone marrow vascularisation displays large structural and spatial distribution heterogeneity, including large lobular clusters of sinusoids and an unexpectedly substantial amount of capillaries in the adipocytes-rich distal third of the tibia. For an unbiased assessment of bone vascular development/changes, these features must be taken into account. We describe the conditions under which the quantification of microvascularisation on histological sections of barium-infused long bones is reproducible, as applied to seven-month-old male C57/Bl6J and mixed CD1/129Sv/J mice, and we propose a nomenclature for the histological parameters measured. Finally, we validate our technique by studying the effect of ovariectomy on mouse tibial vascular density.

Highlights

► Barium sulfate provides better vascular images than silicon rubber in mouse bones. ► Vascular infusion with barium allows histomorphometry staining and IHC labeling. ► µCT imaging of microvascular network requires appropriate voxel size.

Introduction

Vascularisation plays a major role in bone metabolism. Blood vessels invade cartilage templates and trigger endochondral ossification during skeletal development. Later on, in the course of bone growth or repair, osteogenesis and angiogenesis are tightly coupled. Finally, at the adult stage, both hematopoiesis and bone remodeling take place in the bone marrow cavity. Both the vascular niche [1], where hematopoietic cells mature before moving towards the systemic circulation, and the bone remodeling compartments are built around blood vessels [2]. In reaction to physiological or pathological challenges, blood vessels may modulate the activity of bone cells while bone cells may influence the vascular bed through an intense molecular cross-talk. In a previous article we reported a technique to characterize the bone vessel network in rats, using barium sulfate infusion for quantitative assessment of both the microvascular bed and bone cell activities [3]. For a better understanding of the functional and spatial relationships between bone and vessels, however, we need studies in genetically modified mice, and therefore need to design techniques for this particular animal model. Histological evaluation of bone vascularisation relies on two approaches which could be complementary. The first one is to label the vessel wall, usually targeting endothelial cells or basal membrane components. Immunohistochemistry (IHC) is commonly used for quantitative assessment of the bone vascular network. While it does allow to measure areas and numbers of immunostained vessel walls [4], IHC does not permit the simultaneous assessment of histodynamic indices of bone formation after fluorochrome labeling. Either the acetylated low-density lipoprotein endothelial uptake through endocytosis or the overexpression of endothelial GFP-labeled specific genes are other blood vessel labeling methods [5] with the same limitations. The second approach consists in filling up blood vessels with infused contrasting products. These are either agents allowing the optical detection of vessels, such as India ink, light green or dextran-coupled fluorescent dies [6] or radio contrasting agents, like barium [7] or lead [8], [9], [10]. The latter can also allow histological imaging [3], and they offer the possibility of 2 or 3-D imaging of the vascular network with computed microtomography (μCT).

Given this context, the first aim of our study was to define the best radio contrasting agent to obtain optimal visualisation of vessels in infused bones. We show that barium sulfate provides the best imaging and preservation of the bone vascular network, and allows IHC as well as conventional bone histomorphometry of undecalcified bones. Using this technique, we first characterized the morphology of the bone vessels network, and then we developed and validated a reproducible procedure for the quantification of the bone vascular compartment on histological sections of mouse long bones. Finally, by showing a significant decrease in vascular density of tibial proximal metaphysis 15 days post ovariectomy, we confirmed that our technique discriminates biological effects.

Section snippets

Animals

The procedures for the care and killing of the animals were in compliance with the European Community standards on the care and use of laboratory animals (Ministère de l'Agriculture, France; Authorization 04827). Seven-month-old male mice from inbred C57/Bl6J (Charles River laboratories, L'arbresle, France) and mixed CD-1/Sv129J (CD-1/129) backgrounds were respectively used for synchrotron radiation μCT imaging (SR-μCT) and histological quantitative assessment of the vascular network. Two

Assessment of contrasting agent through histology and μCT

In histological sections, barium sulfate infusion proved to be an excellent optical contrasting agent, allowing us to locate unequivocally vascular structures whose diameters can be as small as 10 μm (Fig. 1A). Vessel content appeared homogenous when observed at magnifications of 10× or less, and became granular and birefringent at 25× (Fig. 1B). Although some rare vessel profiles were incompletely filled (Fig. 1B)—either because the contrasting agent was washed out or because the vessel had

Discussion

The functional relationship between bone and vasculature plays a pivotal role in the regulation of bone modeling and fracture repair. Indeed, animal models demonstrate that osteogenesis is accompanied by vessel growth with dramatic consequences on both bone and vessels networks when major signalling pathways such as that of HIF1α are challenged [17]. In contrast, the relationship between the two systems may differ during bone remodeling. We recently showed that osteo-anabolic intermittent

Acknowledgments

This study was supported by the Agence Nationale de la Recherche (ANR AdapHyG, Adaptation to hypergravity during mouse development, 2010–12), by the CNES (Centre National d'Etudes Spatiales) and by a grant from the Société Française de Rhumatologie.

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