Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Summary

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Abstract

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.

Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.

With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Introduction

Mouse models are increasingly employed for the investigation of cardiovascular disease (CVD) and particularly used in longitudinal studies which allow the characterization of different phases of disease development¹. Elastic properties of large arteries are related to different pathological conditions; from a technical point of view, arterial stiffness can be assessed by measuring pulse wave velocity (PWV), which represents the speed with which the pulse wave travels in a conduit vessel². Because of its clinical significance, it is increasingly measured even in preclinical small animal models³.

Protocol

Animal experiments were performed in accordance with the European Directive (2010/63/UE) and the Italian law (D.Lvo 26/2014), and it followed principles of laboratory animal care. The Local Ethical Approval Panel approved the study.

1. Imaging Procedure

1. Place the mouse in an anesthesia induction chamber filled with 2.5% isoflurane in 1 L/min pure oxygen. Verify the depth of anesthesia by unresponsiveness to toe pinch.
2. Place the animal on a temperature controlled board in a supine position. Moisten the animal's eyes with eye salve, in order to avoid them running dry. Supply anesthetic gas flow (1.5% isoflurane) by placing the mouse's nose i....

Results

The proposed approach has been applied to mice abdominal aorta in a previous study¹¹. The following figures show the results of the application of the described approach on real mice images. These data are from a single animal (wild type mice, 13 weeks old, strain: C57BL6, weight: 33 g) In particular, Figure 1 represents the result of the analysis of the US images. Edge detection and contour tracking techniques applied to B-mode images acquired wit.......

Discussion

In this study, an image processing algorithm based on the lnD-V loop for PWV assessment in mice has been described in detail. The proposed approach is based on the processing of US images only and, thus, could represent a valid alternative to existing techniques^6,13 for the evaluation of arterial stiffness in mouse models. In fact, conversely to invasive methods⁶ which are based on the acquisition of intra-arterial pressure signals and req.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
VEVO2100	FUJIFILM VisualSonics Inc, Toronto, Canada		micro-ultrasound equipment
MS250 Ultrasound Probe	FUJIFILM VisualSonics Inc, Toronto, Canada		micro-ultrasound probe
EKV Software	FUJIFILM VisualSonics Inc, Toronto, Canada		Software
Matlab R2015a	MathWorks Inc, Natick, MA, USA		Software
Conductive Paste	Chosen by the operator		Laboratory material
Petroleum Jelly	Chosen by the operator		Laboratory material
Depilatory Cream	Chosen by the operator		Laboratory material
Acoustic Coupling Gel	Chosen by the operator		Laboratory material
Developed Matlab Software			The authors are willing to collaborate with those researchers who are interested in the software and to make the software available under their supervision