Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Summary

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Abstract

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.

The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Introduction

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling¹ and is associated with pressure-overload conditions². Echocardiography (EchoCG) can be used for the characterization of RV diastolic dysfunction^3,4. Despite recent developments in small animal echocardiography, measurements of diastolic parameters are rarely reported. In contrast, measurements of the systolic function are widely used for the characterization of transgenic mice⁵, as well as for the evaluation of a treatment response⁶.

This can be partly explained by the difficulties in the measurement of diastolic parameters from the apical four-chamber view. Visualization of the heart in this position can be facilitated by tilting the fixation platform LeCa or RiCr. Even if these manipulations are used, echocardiographers do not report them in their manuscripts^4,7. Therefore, it remains unclear whether these manipulations provide comparable results. Moreover, this also precludes a development of standardized nomenclature of this position for mice.

The aim of this study was to describe two positions for apical four-chamber view visualization and compare their results. To determine the differences between the two positions, we have utilized the mouse pulmonary artery banding (PAB) model, in which a tantalum clip leads to a partial occlusion of the pulmonary artery. This occlusion results in right ventricle remodeling and dysfunction. Full details of the PAB operation can be found in previously published work³. Sham-operated mice, where the clip was placed next to the pulmonary artery, were used for comparison. EchoCG investigations were performed three weeks post-operation using the imaging system with a 30 MHz scan head (see Table of Materials for both). Nomenclature for the description of the positions and orientations between the mouse and the ultrasound beam is used as described by Zhou et al.⁷.

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Protocol

The study was performed according to national regulations for animal experimentation and EU Directive 2010/63. Prepare equipment as described previously by Brittain et al.⁸.

1. Mouse Preparation

1. Obtain 12 to 13 week-old male C57Bl6/J mice and house them with a 12 h light/dark cycle, at a constant room temperature, and with ad libitum access to standard laboratory chow and water, until the start of the experiment.
2. Anesthetize the mouse using general anesthesia approved by the Institute and check for the lack of response to the toe pinch. Under mild anesthesia with isoflurane 0.8%–1.2%, fix the mouse on a heated platform. Apply electrode gel to its extremities for the continuous monitoring of its heart rate and temperature.
3. Depilate the mouse's chest hair using depilation creme. To reduce pressure on its thorax, do not apply the ultrasound coupling gel directly on the thorax; rather, apply a layer of the gel to the tip of the transducer.

2. Image Acquisition

1. Apical four-chamber view with a left and caudal tilt of the platform
   1. After the mouse preparation, angulate the platform to the left at 10°–15° and then caudally at 10°–15°.
   2. Position the transducer above the apex with the imaging plane ~45° to the coronal plane and the central axis of the ultrasound beam directed cranially, posterior, and to the left to obtain the apical four-chamber view. Press the B-Mode button to activate the B-mode/2-D image.
      NOTE: The transducer can be held manually or fixed by a stage. The term "B-mode" comes from the imaging system that was used instead of the more familiar term "two-dimensional" (2-D) and is used throughout the protocol.
   3. Look for the appearance of the following structures in the acoustic window: the left ventricle (LV), the left atrium (LA), the RV, the right atrium (RA), the mitral valve (MV), and the tricuspid valve (TV).
   4. Manipulate the imaging plane in the coronal plane and rotate clock- and counterclockwise around the central axis until both ventricles are visualized at their longest dimension and both atria are visible. This is the four-chamber view (Figure 1).
   5. Press the Cine store button to save the recording.
   6. Press the Scan/Freeze button to pause the system.
2. Measurement of transtricuspid blood flow velocities
   1. Press the Scan/Freeze button to activate the system.
   2. Press the Overlay button several times to activate the sample volume for PW (pulsed wave) mode.
   3. While keeping the obtained four-chamber view, use the trackball to position the sample volume at the opening of the tricuspid valves for the measurement of inflow velocities (E and A peak velocities).
   4. Press the PW mode button for the measurement of inflow velocities (E and A peak velocities).
      NOTE: Because tricuspid valves are difficult to visualize in this position, performing several measurements helps to align correctly the sample volume with the blood flow. Perform the Doppler sampling with the smallest incidence angle between the Doppler beam and the blood flow direction. The obtained blood flow profile should correspond to the following criteria: 1) an inflow profile similar to an M-shape with the first peak lower than the second; 2) a respiratory modulation with an increased amplitude at inspiration; 3) a maximal amplitude of velocities in several measurements (Figure 2).
   5. Press the Cine store button to save the optimized recording.
   6. Press the Scan/Freeze button to pause the system.
3. Measurement of the tricuspid annular plane systolic excursion (TAPSE)
   1. Press the Scan/Freeze button to activate the system.
   2. Switch to B-mode by pressing the B-Mode button. Some manipulations on the image might be necessary to regain the correct four-chamber view.
   3. Press the Overlay button several times to activate the sample volume of the M-mode. Using the trackball, align the sample volume with the lateral part of the tricuspid annulus. By pulling the edges of the sample volume using the trackball, align the length of the sample volume to cover the entire amplitude of the cardiac movement during the cardiac cycle.
   4. Press the M-Mode button to activate M-mode. Tricuspid annulus' movements should appear as a wave (Figure 2).
   5. Press the Cine store button to save the recording.
   6. Press the Scan/Freeze button to pause the system.
4. Measurement of tissue Doppler parameters
   1. Press the Scan/Freeze button to activate the system.
   2. Press the B-Mode button to activate B-mode.
      NOTE: Some manipulations by angulation in the coronal plane and rotation clock- and counterclockwise around the central axis of the image might be necessary to regain the correct four-chamber view.
   3. Press the Overlay button several times to activate the sample volume for TDI (tissue Doppler imaging). Using the trackball, align the sample volume with the lateral part of the tricuspid annulus, where the RV free wall creates an angle with the tricuspid valve. By pulling the edges of the sample volume using the trackball, adjust the sample volume to include both the systolic and the diastolic extreme positions of the annulus.
   4. Press the Tissue button to activate TDI mode.
      NOTE: Yellow tracing of the TDI recording appears corresponding to the following criteria:1) a recording similar to an inverted M-shape; 2) clearly distinguishable E' and A' peaks during diastole and S' peak during systole; 3) a maximal amplitude of velocities in several measurements (Figure 2).
   5. Press the Cine store button to record an optimized image.
   6. Press the Scan/Freeze button to pause the system.
5. Apical four-chamber view with right and cranial tilt of the platform
   1. Angulate the platform to the right at 10°–15° and then cranial at 10°–15°. Perform the measurements as described in the previous sections for the LeCa steps (steps 2.1, 2.2, 2.3, and 2.4).
      NOTE: During the investigation, isoflurane should be titrated between 0.–1.2 to keep the mouse's heart rate at 400–440 bpm. In this range, separate peaks of transtricuspid blood flow and tissue Doppler (DTI) velocities are measurable. To avoid the effects of the heat loss on hemodynamics, the data are recorded, and the analysis is performed off-line. Only signals obtained at the end-expiration are used for analysis. Measurements of 3 - 5 heartbeats are averaged.

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Results

The apical four-chamber view is difficult to obtain in mice. Therefore, manipulations of the platform position can help to visualize the heart by changing its position in the thorax. The tilting of the platform to the left and to the right improved the acoustic window and provided images of comparable quality in B-mode (Figure 1). After obtaining the correct positions, measurements in PW-, M-, and TDI-modes provided images of comparable quality (

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Discussion

The echocardiographic RV function and dimension assessment from parasternal positions have been well described. In contrast, the apical position in mouse echocardiography has been neglected partly due to technical difficulties. Using a horizontal platform position, it is difficult to obtain a sufficient acoustic window for four-chamber view imaging. To facilitate the imaging of this position, the platform can be tilted to the left, a manipulation similar to the left-sided positioning of patients. This should result in a ...

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Materials

Name	Company	Catalog Number	Comments
RMV-707B scan head 30 MHz	Visual Sonics	P/N 11459	mouse scan head
VisualSonics Vevo 770® High-Resolution Imaging System	Visual Sonics	770-230	ultrasound machine
Veet depilation creme for sensitive skin	Veet	07768307
Surgical tape Durapore 3M	3M Deutschland GmbH	1538-1	for fixation
Askina Brauncel cellulose swabs	B.Braun	9051015
Aquasonic ultrasound gel	Parker Laboratories Inc.	BT025-0037L
Electrode Gel	GE medical systems information technologies Inc.	2034731-002	apply to extremities for countinous ECG and heart rate monitoring
Thermasonic gel warmer	Parker Laboratories Inc.	82-04-20	to reduce heat loss warm up the ultrasound gel before use