Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

Summary

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Abstract

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

Introduction

Aging is associated with progressive increases in cardiovascular calcification¹. Hemodynamically significant aortic valve stenosis affects 3% of the population over the age of 65², and patients with even moderate aortic valve stenosis (peak velocity of 3-4 m/s) have a 5 year event-free survival of less than 40%³. Presently, there are no effective treatments to slow the progression of aortic valve calcification, and surgical aortic valve replacement is the only available treatment for advanced aortic valve stenosis⁴.

Studies aimed at gaining a deeper understanding of the mechanisms that contribute to the initiation and progression of aortic valve calcification are a key first step in moving towards pharmacological and non-surgical methods to manage aortic valve stenosis^5,6. Genetically-altered mice have played a major role in developing our understanding of the mechanisms that contribute to a variety of diseases and are now coming to the forefront of mechanistic studies aimed at understanding the biology of aortic valve stenosis^6,7,8. Unlike other cardiovascular diseases such as atherosclerosis and heart failure-where standard protocols for evaluating vascular and ventricular function are for the most part well-established-there are unique challenges associated with in vivo phenotyping of heart valve function in mice. While recent reviews have provided thorough discussions regarding the advantages and disadvantages to numerous imaging and invasive modalities used to assess valve function in rodents^9,10,11, to date, we are not aware of a publication that provides a comprehensive, step-by-step protocol for phenotyping heart valve function in mice.

The purpose of this manuscript is to describe the methods and protocols to phenotype heart valve function in mice. All methods and procedures have been approved by the Mayo Clinic Institutional Animal Care and Use Committee. Key components of this protocol include the depth of anesthesia, the evaluation of cardiac function, and the evaluation of heart valve function. We hope this report will not only serve to guide investigators interested in pursuing research in the field of heart valve disease, but will also start a national and international dialogue related to protocol standardization to ensure data reproducibility and validity in this rapidly-growing field. Importantly, successful imaging using high-resolution ultrasound systems requires a working knowledge of the principles of sonography (and terminology commonly used in sonography), an understanding of the fundamental principles of cardiac physiology, and significant experience with sonography to allow for accurate and time-efficient assessment of cardiac function in rodents.

Protocol

1. Prepare the Materials and Equipment (Table 1 and Figure 1)

1. Turn on the ultrasound machine. Enter the animal ID, date, and time (for serial imaging experiments) and other relevant information.
2. Use a high-frequency ultrasound transducer, 40 MHz for imaging mice less than ~20 g or 30 MHz for mice greater than ~20 g.
3. Connect the platform to the electrocardiogram (ECG) monitor for ECG gating of imaging for certain modalities.
   NOTE: Critically, this also allows for the instantaneous calculation of the heart rate (HR), which can be used as one of several indices of an appropriate depth of anesthesia.
4. Pre-heat the platform to 37 °C.
   NOTE: All commercially-available ultrasound machines have a control panel that provides image acquisition controls and study management controls for B-mode, M-mode, and Doppler echocardiography. A cardiac measurement tool is embedded in the machine for the automatic measurement and computation of common echocardiographic parameters of cardiac and valvular functions.

2. Prepare the Mouse for Imaging and Induction of Anesthesia

1. Gently pick up the mouse by its tail and firmly hold the animal at the nape of its neck.
2. Guide the nose of the animal into the nose cone. Begin anesthesia flow at 1% isoflurane. Ensure that the animal is sedated within 3-5 s of exposure to the gas.
3. Quickly and accurately lay the animal on the platform in a supine position, making sure that the forefeet and hind feet lie on the ECG sensors of the platform.
4. Gently secure the animal with adhesive tape on all four limbs, lightly apply adhesive tape to stabilize the head in the nose cone apparatus, and apply adhesive tape to stabilize the tail. Both hind feet and forefeet should lie flat to ensure stable and clear ECG signal acquisition by the physiological imaging system.
5. Check the HR. Do this using an imaging platform with ECG capabilities or with external ECG devices. Ensure that the baseline HR is between 600 to 700 bpm. Ensure that the HR does not fall below 450 bpm under any circumstances.
   NOTE: During the procedure, the HR may decrease slightly due to anesthesia, but it should be above 500 bpm in most instances.
6. Adjust the anesthesia flow by small increments accordingly (~0.1% increments every 15 s until a stable state of anesthesia is reached).
   NOTE: A stable state of anesthesia is a condition in which the above-mentioned cardiac parameters are maintained (see step 2.5) and the animal does not overtly respond to stimuli from the placement of the probe on various imaging windows. Importantly, this is not a surgical plane of anesthesia, which results in marked cardiodepression in mice. For prolonged imaging sessions, the application of vet ointment to the eyes to prevent dryness is recommended.
7. Check the body temperature using a rectal thermometer. Keep the temperature between 36.5 °C and 38 °C.
   NOTE: In an appropriately environmentally-controlled room and on a heated platform, the body temperature (measured rectally) remains constant during the entire procedure and, consequently, is not a confounding factor influencing cardiovascular hemodynamics over time.
8. Shave off the hair from the chest using an electric clipper designed for use with fine hair. Wipe clean the chest with a damp paper towel. The animal is ready for imaging.
   NOTE: While chemical removal of hair can also be performed, avoid the use of such compounds, as they can cause significant skin irritation over time in long-term experiments. Furthermore, the appropriate application and removal of such chemically-based hair removal products can prolong the duration of anesthesia exposure by 2-3 min (~10-20%). The total time from induction of anesthesia to completion of skin preparation should take less than 3 min.

3. Follow Basic Principles and Guidelines in Acquiring Cardiac Ultrasound Images

NOTE: There are three ultrasound modalities used in acquiring the images: B-mode/2-D, M-mode, and Doppler (spectral pulsed-wave Doppler and color flow Doppler imaging). There are two basic transducer positions used to acquire images of the heart and heart valves: the parasternal and apical windows (Figure 2).

1. From each transducer position, obtain multiple tomographic images of the heart relative to its long and short axes by manually rotating and angulating the transducer.
   NOTE: Rotation refers to pivoting or twisting the transducer from a fixed position on the chest wall, while angulation refers to the side-to-side movement of the transducer from a fixed point on the chest wall. All ultrasound transducers have an image index marker in the form of a groove (notch), external ribbing, or button.
2. Ensure that the ultrasound signal is perpendicular to the target structure by adjusting the transducer position accordingly.
3. Optimize the color flow and peak velocity signals by aligning the transmitted ultrasound beam parallel to the flow. The angle between the ultrasound beam and flow should be less than 60°.
4. Optimize the image quality using the control panel controls. Only the area of interrogation should fill up the image display.
   NOTE: Fine adjustments in transducer and platform positions are almost always necessary to obtain clear images. Even during optimal conditions, respiratory movements, chest wall anatomy (e.g., small rib spacing), and variations in internal anatomy (both inherent and disease-induced) can limit the acoustic window and make image acquisition very challenging.
5. When measuring the left ventricular dimensions in M-mode and 2-D/B-mode, place the measurement caliper in the most continuous echo line.
6. Adjust the color Doppler sector and sample volume to the area of interrogation by adjusting the sector control, which is found on the panel.
   NOTE: The color-coding scheme in Doppler studies indicates the speed and directionality of blood flow. Doppler signals that are red indicate laminar blood flow towards the transducer. Doppler signals that are blue indicate laminar flow away from the transducer. A "mosaic" color pattern indicates regions of turbulent or non-laminar blood flow (which commonly occurs in valvular stenosis or valvular regurgitation).
7. Record a minimum of two 5 s strips (or 100 frames) of real-time B-mode/2D echo from each imaging window for offline analysis.
   NOTE: Commercially-available echo machines have image acquisition settings that capture a pre-set number of frames or cine-loop sizes. The image acquisition settings can be modified so that longer cine loops can be acquired. Acquisition of high-quality images requires extensive experience and experimentation. Investigators must find the right combination of transducer placement and platform angle to obtain images from many views and acoustic windows.

4. Evaluation of Aortic Valve (AV) Function

NOTE: Assessments of aortic valve function include qualitative evaluations of the valve (e.g., perceived cusp thickness, increased echogenicity due to valvular calcification, and the presence or absence of regurgitant jets using color Doppler) and quantitative measures of valve function (e.g., peak transvalvular velocity and cusp separation distance).

1. Begin to image the aortic valve by selecting B-mode image acquisition.
2. With the animal securely fastened on the platform and the head facing away from the investigator, tilt the table 15-20° to the left. This will bring the heart forward and leftward, closer to the chest wall. Apply a generous amount of ultrasound gel on the transducer or directly on the animal's chest.
3. Position the transducer parasternally, about 90° perpendicular with the long axis of the heart, with the image index marker of the transducer pointing posteriorly (Figure 2). While in 2D/B-mode, slide the transducer cephalad until the AV comes into view. This is the "short axis" view of the aortic valve.
   NOTE: A normal aortic valve has three thin cusps that open widely during systole and close adequately during diastole so that there is no regurgitation of blood back into the left ventricle. The cusps are very thin, move very rapidly, and can often be challenging to visualize.
4. Rotate the transducer clockwise until the image index marker points caudad. Observe the aortic root, aortic valve, left ventricular outflow tract, mitral valve, left atrium, and part of the right ventricular outflow tract on the image display.
   NOTE: This is the "parasternal long axis" view of the AV. The sonographer should ascertain that there are two aortic valve cusps visible throughout the cardiac cycle in the B-mode images, which will allow for subsequent M-Mode imaging and analysis (see below).
5. Evaluate the aortic root in this view. Carefully sweep back and forth so that the aortic root images contain the largest dimensions of the aortic root. Measure the largest antero-posterior dimension of the aorta using the electronic caliper associated with the measurement tool embedded in the machine.
6. Locate the aortic valve in the long axis. Reduce the image width so that only the aortic valve is on the image display by adjusting image width button in the control panel. Position the M-mode line of interrogation where it intersects the tips of the aortic valve to accurately assess aortic valve cusp separation.
7. In the M-mode display of the aortic valve, measure the cusp separation distance (box-like appearance in the systole) using the electronic caliper associated with the measurement tool embedded in the machine.
   NOTE: The greatest advantage of M-mode imaging is the very high temporal resolution, which is essential for the evaluation of aortic valve function. While M-mode images of the AV can be acquired in both the short- and long-axis views, the parasternal long-axis view is generally preferred because the imaging plane allows the sonographer to readily identify the orientation and location of the tips of the cusps during systole.
8. While still in the parasternal long-axis view of the aortic valve, press the color Doppler control key in the control panel. Apply color Doppler to the region of the aortic valve.
   NOTE: Normal flow from the left ventricle through the aortic valve during systole is toward the transducer and thus is encoded red.
9. Document the presence or absence of aortic valve regurgitation.
   NOTE: Aortic valve regurgitation is an abnormal flow that occurs during diastole and is directed away from the transducer; thus, it is encoded blue.
10. Press the pulsed-wave Doppler control key. Using the track ball located in the control panel, place the pulsed-wave sample volume in the proximal ascending aorta, just above the aortic valve, making sure that the angle between the ultrasound beam and the blood flow is less than 60° by tilting the platform and/or the transducer. If possible, obtain the peak velocity across the aortic valve from the suprasternal notch window.
11. Measure the peak velocity from the spectral display using the electronic calipers associated with the measurement tool embedded in the machine (Figure 3C and 3F).
    NOTE: A mosaic color denotes high flow velocity that is likely to contain non-laminar flow patterns.

5. Evaluation of Mitral Valve (MV) Function

NOTE: Assessment of mitral valve function includes qualitative evaluations of the valve (e.g., perceived cusp thickness, increased echogenicity due to valvular calcification, presence or absence of regurgitant jets using color Doppler) and quantitative measures of valve function.

1. Place the transducer in the apical position in B-mode. Position the transducer so that it is angled towards the head of the mouse (Figure 2C). Observe the right ventricle (RV), left ventricle (LV), right atrium (RA), and left atrium (LA) on the image display. Manually tilt the platform slightly so that the animal is in a "head-down" position to visualize the mitral valve as it opens into the LV.
   NOTE: The apical 4-chamber view is the optimal view for examining blood velocity across the mitral and tricuspid valves, as well as the tissue velocity of the mitral annulus. This is also a good view to assess the motion and size of the RV and interventricular septum.
2. From the apical 4-chamber view, bring the mitral valve in focus by reducing the image width. Observe that the mitral valve leaflets appear as two thin, mobile filaments opening and closing during each cardiac cycle.
   NOTE: Mitral leaflets of a "normal" mouse can be difficult to visualize if imaging is done at physiological HR (i.e., >450 bpm).
3. Place the M-mode cursor across the mitral valve to assess the thickness of the leaflets.
   NOTE: The anterior leaflet is best visualized in systole when it is perpendicular to the ultrasound beam (Figure 4).
4. Using the apical 4-chamber view, apply color Doppler to image the flow from the left atrium through the mitral valve during diastole. Observe for mitral valve regurgitation.
   NOTE: Flow is directed toward the transducer and is therefore encoded red. Regurgitant flow will be encoded blue and occurs during systole (Figure 5).
5. Using the apical long-axis view, switch to pulsed-wave mode. Move the Doppler sample volume to the tips of the mitral valve leaflet. Note the two peaks of the mitral inflow spectral display. If the leaflets are not well-visualized, use color Doppler to identify regions with bright red or mosaic color patterns and place the sample volume at that point.
   NOTE: The spectral display of mitral flow has two peaks in slow HRs (<450 bpm). In normal HRs (>450 bpm), the early- (E) and late-filling (A) flows are fused. The spectral Doppler display of flow across the mitral valve is used in the assessment of left ventricular diastolic function (see step 7.5).

6. Evaluation of Right-sided Heart Valve Function

NOTE: The tricuspid and pulmonic valves comprise the right-sided heart valves. The tricuspid valve can be readily visualized in the apical long-axis view, while the pulmonic valve can be visualized in both the parasternal long- and short-axis views.

1. From the apical long-axis view, tilt or point the transducer tip using a rocking motion so that the right ventricle is at the center of the image display. Reduce the image width so that only the right ventricle is visible in the image display.
2. In the same image plane, visualize the tricuspid valve leaflets, which appear as thin, mobile filaments between the right atrium and right ventricle and that open and close over the course of each cardiac cycle.
3. Apply color Doppler in the region of the tricuspid valve. Note for tricuspid valve regurgitation. 
   NOTE: Normal flow occurs during diastole, is directed toward the transducer, and therefore is encoded red. Abnormal regurgitant flow occurs during systole, is directed away from the transducer, and therefore is encoded blue. The peak velocity of the regurgitant jet is used to estimate right ventricular systolic pressure.
4. Move the transducer to the parasternal short-axis position at the level of the aortic valve. Above the aortic valve are the right ventricular outflow tract, the pulmonic valve, the proximal main pulmonary artery, and the right and left pulmonary arteries (Figure 6).
5. Rotate the transducer clockwise to a modified parasternal long-axis position. Then, tilt the transducer slightly upward to obtain a short-axis view of the pulmonic valve.
6. In this view, apply M-mode imaging to evaluate the separation distance of the pulmonic valve cusps (Figure 7).
7. Apply color Doppler in the region of the pulmonic valve to assess for valvular regurgitation (a mosaic-patterned, high-velocity jet during diastole) and stenosis (a mosaic-patterned, high-velocity jet during systole).
8. Press the pulsed-wave control key and place the sample volume just after the pulmonic valve.
   NOTE: Analysis of the spectral Doppler display of flow is used to estimate pulmonary artery pressure (Figure 8).

7. Evaluation of Cardiac Function

NOTE: The assessment of cardiac function includes qualitative evaluations of left ventricular contractility (e.g., visual estimation of the ejection fraction, the regional wall motion abnormality, and the perceived thickness of the walls) and quantitative measures of left ventricular function (e.g., the ejection fraction, left ventricular mass, left ventricular diastolic function, and indices of myocardial performance).

1. Obtain a short-axis view of the LV in 2D/B-mode, with the transducer in the parasternal short-axis position at the level of the papillary muscles. Move the transducer up and down to scan the LV from the base to the apex. Observe for wall motion abnormalities.
2. From a parasternal short-axis view of the left ventricle, press the M-mode button, located in the control panel. Using the track ball, position the M-mode cursor at the center of the left ventricular cavity at the level of the papillary muscles and obtain M-mode images.
3. Measure the left ventricular cavity dimension at end-diastole, where the distance between the anterior wall and posterior wall is the largest, and in end-systole, where the inward motion of both anterior and posterior walls is maximal (Figure 9).
4. Measure the anterior and posterior wall thickness at end-diastole and end-systole.
   NOTE: While the papillary muscles are an essential landmark to ensure the correct imaging plane, be careful not to include them in any measurements.
5. Move the transducer to the apical window. See step 5.1. Assess the left ventricular diastolic function using pulsed-wave Doppler of blood flow across the mitral valve in the apical long-axis view.
6. Place the sample volume at the tips of the mitral valve leaflets. Measure the peak mitral inflow velocity from the spectral display of pulsed-wave Doppler velocities across the mitral valve.
7. Position the sample volume between LV inflow and outflow. Note the mitral and aortic valve closing and opening signals. Measure the isovolumic relaxation time, isovolumic contraction time, and left ventricular ejection time (Figure 10).
8. Perform tissue Doppler imaging (TDI) of the mitral annulus in the apical long-axis view. Press the TDI control key and place the sample volume at the medial aspect of the mitral annulus. Make sure that the sample volume does not encroach on the mitral leaflets. Keep the Doppler sample volume size between 0.21 mm and 0.27 mm. Measure the early diastolic velocity (e') of the mitral annulus (Figure 11).

8. Final Steps

1. Review the acquired images. Ascertain that all required images were obtained. 
2. Remove any excess ultrasound gel from the chest of the mouse and gently remove the tape securing the animal in place. Turn off the anesthesia.
3. Place the animal on an absorbent paper towel (not bedding, which can be aspirated or can block airways during recovery). Observe the animal until sternal recumbency is attained. If the anesthesia is administered appropriately, recovery should occur within 30 to 60 s.

Results

Examples of images that are routinely obtained from animal cardiac ultrasound imaging are included in this manuscript. An illustration of transducer placement on the animal's chest is provided to give the reader a clear understanding of where the transducer is positioned to obtain the images as described. A photograph of the ultrasound laboratory set-up is also included to emphasize the importance of the proper equipment, particularly the ultrasound transducer to be used and the metho...

Discussion

Induction of anesthesia

Proper induction and maintenance of anesthesia is critical for the accurate assessment of changes in heart valve and cardiac function in mice. Given the rapid induction of anesthesia elicited by isoflurane and the relatively long wash-out time of this anesthetic following deep anesthesia, we do not use a stand-alone anesthesia chamber for induction. Instead, as noted in detail above, animals are guided directly to the anesthesia cone, which allows for rapi...

Materials

Name	Company	Catalog Number	Comments
High resolution ultrasound machine	VisualSonics, Fujifilm	Vevo 2100
Isoflurane diffuser (capable of delivering 1 % to 1.5 % isoflurane mixed with 1 L/min 100% O2	VisualSonics, Fujifilm	N/A
Transducers for small mice (550D) or larger mice (400)	MicroScan, VisualSonics, Fujifilm	MS 550D, MS 400
Animal platform	VisualSonics, Fujifilm	11503
Advanced physiological monitoring unit	VisualSonics, Fujifilm	N/A
Isoflurane	Terrell	NDC 66794-019-10
Nose cone and tubing connected to isoflurane diffuser and 100% O2	Custom Engineered in-house	--
Hair razor	Andis Super AGR+ vet pack clipper	AD65340
Ultrasound gel	Parker Laboratories	REF 01-08
Electrode gel	Parker Laboratories	REF 15-25
Adhesive tapes	Fisher Laboratories	1590120B
Paper towels