The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.
The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.
The present methodology teaches the investigator how to measure and use the LAV, Aorta diameter and PA diameter with 2D echocardiography in mice under isoflurane. Heart Failure with preserved Ejection Fraction (HFpEF) in elderly people affects up to 10% in those 80 years and older1 and results in significant morbidity and mortality. Significant mortality also occur from Pulmonary Hypertension (PH), an insidious disease process presenting with similar symptoms as HFpEF, in which elevated pulmonary artery pressures lead to exertional dyspnea, progressive right heart failure, and often death.2 The increasing prevalence of both HFpEF and PH signify the need for developing a method that allows for accurate evaluation and monitoring of interventions in murine models in a non-invasive, non-terminal approach.
Aging leads to a deterioration of diastolic function via alterations in ventricular-arterial stiffening, vascular dysfunction, inflammation3, impaired calcium regulation4, decreased β-adrenergic responsiveness, and physical deconditioning producing slowed active relaxation and increased passive stiffness. Over time this leads to increased LV filling pressure and compensatory enlargement of the LA.5
Though other etiologies such as valvular dysfunction (mitral regurgitation or stenosis) and infiltrative processes cause elevations in pressure and volume in the LA1, the European Society of Cardiology supports the addition of LA size as a noninvasive reflection of LV function.6
Correlations between LA volume and invasive measures of diastolic function have been studied in mice. LA volume correlated with differences in function determined invasively within age groups for both 14- and 31-months-old mice. In the 14-months-old mice, LA volume correlated with three standard invasive measures of diastolic function −dP/dtmin (r2 = .5, p <0.05), Tau (time constant of relaxation, (r2 = .6, p <0.05), and left ventricular end diastolic pressure (r2 = .25, p <0.05). For the 31 months-old mice, the correlations between LA volume and -dP/ dtmin (r2 = .92, p <0.05) and LVEDP (r2 = .61, p <0.05) were apparent though the relationship with Tau was less clear. Therefore, LA volume increased with diastolic impairment not only across groups but within age groups.7
Studies of cardiac function in murine models using catheter techniques to evaluate cardiac performance, although rigorous and reliable, are limited due to their incompatibility with repeated assessments.8 Alternatives to invasive measures such as MRI and 3D echocardiography may also be more accurate than 2D echocardiographic techniques, but they are more expensive; 2D echocardiography is considered adequate for LA volume evaluation. 9,10
Assessment of the LA volume and PA diameter with echocardiography allows the discrimination between models that produce primary increases in pulmonary artery resistance resulting in an increase in PA diameter with no change in LA pressure or LAV from those where the Pulmonary Artery and Left Atrium both enlarge as a result of elevated filling pressures on the left side of the heart. This approach was taken by Scalia et al. who showed that in people, the echocardiographic Pulmonary Artery to Left Atrial Ratio (ePLAR) is a parameter to accurately differentiate between patients with pre-pulmonary capillary hypertension and post-capillaryhypertension.11
All animals were cared for in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals at Baylor College of Medicine.
1. Set-up
2. Image Acquisition
Figure 1: "Supine" Position. Representation of a mouse in the "Supine" Position. Approximately 30 – 45° angle along the X axis and 0° angle along the Y axis. This position is used to acquire all of the Left Atrium measurements. Please click here to view a larger version of this figure.
Figure 2: "Standing-up" Position. Representation of a mouse in the "Standing-up" Position. Approximately 5° angle along the X axis and a 60° angle along the Y axis. This position is used to acquire the Aorta and Pulmonary Artery diameters. Please click here to view a larger version of this figure.
Figure 3: Pulmonary Artery Doppler. Characteristic Pulmonary Artery Doppler imaging. The researcher may use Doppler imaging as a tool to ensure correct identification of the Pulmonary Artery. Please click here to view a larger version of this figure.
3. Post Procedure Animal Recovery
4. Image Analysis
Assessment of the LA volume and PA diameter with echocardiography allows the discrimination between models where increased pulmonary artery resistance results in increased PA diameter with no change in LAV from those where the Pulmonary Artery and Left Atrial enlargement are both a result of elevated filling pressures on the left side of the heart. Images displaying the differences in the two pathophysiologies are shown in Figure 4. Images of the LA and PA are from young, hypoxia induced pulmonary hypertension and age related LV diastolic dysfunction. The Pulmonary Hypertension models develop an increase in PA diameter without change in Left Atrium volume over a 4 weeks period. In comparison, the old animals develop an increase in both their Pulmonary Artery diameter and Left Atrium volume suggesting that backward propagation of pressure drive the increase in PA diameter in this group.
Figure 4: LA Superior-Inferior and PA Diameter on a Normal Mouse, a Pulmonary Hypertension Model Mouse and an Old Mouse with Age-associated LV Diastolic Dysfunction. Images (A) and (B) show the LA Superior-Inferior and PA diameter respectively of a 15 weeks old normal mouse. Images (C) and (D) show the LA Superior-Inferior and PA diameter respectively of a 15 weeks old Pulmonary Hypertension model mouse. Pulmonary hypertension was induced by exposure to 10% hypoxia for four weeks and the PA systolic pressure was increased 75% by right heart catheter studies. Please note the difference in PA diameter. Images (E) and (F) show the LA Superior-Inferior and PA diameter respectively of a 21 months old mouse with elevated LV end diastolic pressure. The filling pressure was increased almost 300% in the old mice. Please note that both the LA and PA diameter are larger compared to the 15 weeks old mice. Scale units in millimeters. The marker "Δ" denotes the point in the EKG at which the image shown was obtained. LA SI: Left Atrium Superior-Inferior, PA: Pulmonary Artery. Please click here to view a larger version of this figure.
To ensure accuracy in the results it is necessary that measurements are taken at their appropriate timing in the cardiac cycle. When measuring the LAV, the researcher wants to measure the atrium at its largest capacity for all three measures, this means before atrial contraction. If the LA appendage is seen in any mice in the Superior-Inferior view, the authors recommend to ignore it when doing the measurement. When measuring the great vessels the researcher wants to measure them at their largest capacity, meaning after the QRS or just after the ventricles have pumped blood into the vessels. Figures 5 through 9 show images corresponding to the same animal, measured at different time points on the EKG to illustrate the differences between a correct and incorrect measurement.
The researcher can now calculate the LAV using the formula of the prolate ellipse9,10 as in the following example:
(4 * π * Superior-Inferior Dimension * Anterior-Posterior Dimension * Medio-Lateral Dimension) / (3*2*2*2)
In Figure 5, a "Correct" measurement is that obtained using the EKG as a guide to ensure the researcher is measuring at the largest capacity of the cavity. In this case the atria is at its largest capacity when measured just at the P-wave. (A,C). An "Incorrect" measurement is that obtained at any point other than the P-wave. It tends to give smaller dimensions as the left atrium contracts or fills. (B,D). In Figure 6, a "Correct" measurement is that obtained using the EKG as a guide to ensure the researcher is measuring at the largest capacity of the cavity. In this case the atria is at its largest capacity when measured just at the P-wave (LA(1)). An "Incorrect" measurement is that obtained at any point other than the P-wave. It tends to give smaller dimensions as the left atrium contracts or fills. (LA(2)). In Figure 7, a "Correct" measurement is that obtained using the EKG as a guide to ensure the researcher is measuring at the largest capacity of the cavity. The atria is at its largest capacity when measured just at the P-wave. (A,C). An "Incorrect" measurement is that obtained at any point other than the P-wave. (B,D). *The authors think the cine is extremely helpful for determining the limits of the LA. The researcher should be able to see the LA move throughout the cardiac cycle. In Figure 8, a "Correct" measurement is that obtained using the EKG as a guide to ensure the researcher is measuring at the largest capacity of the vessel. In this case the aorta is at its largest capacity just after the QRS complex. (A,C). An "Incorrect" measurement is that obtained at any point other than that just after the QRS complex. It tends to give smaller dimensions as the aorta contracts or fills. (B,D). In Figure 9, a "Correct" measurement is that obtained using the EKG as a guide to ensure the researcher is measuring at the largest capacity of the vessel. In this case the pulmonary artery is at its largest capacity just after the QRS complex. (A,C). An "Incorrect" measurement is that obtained at any point other than just after the QRS complex. It tends to give smaller dimensions as the pulmonary artery contracts or fills. (B,D).
Figure 5: LA Superior-Inferior Measurements. Images (A) and (B) correspond to the same mouse specimen. Scale units in millimeters. Image (C) corresponds to the lower right fragment of EKG of Image (A). Not to scale. Image (D) corresponds to the lower right fragment of the EKG of Image (B). Not to scale. Note the marker "Δ" in images (C) and (D). The marker "Δ" denotes the point in the EKG at which the image shown was obtained. Please click here to view a larger version of this figure.
Figure 6: LA Anterior-Posterior Measurements. M-Mode view of Left Atrium. The image corresponds to an M-Mode view of Left Atrium in order to obtain the Anterior-Posterior Measurement. Please click here to view a larger version of this figure.
Figure 7: LA Medio-Lateral Measurements. Images (A) and (B) correspond to the same mouse specimen. Scale units in millimeters. (C) corresponds to a fragment of EKG of (A). Not to scale. (D) corresponds to a fragment of the EKG of (B). Not to scale. Note the marker "Δ" in images (C) and (D). The marker "Δ" denotes the point in the EKG at which the image shown was obtained. LV: Left ventricle, RA: Right atrium, LA: Left atrium. Please click here to view a larger version of this figure.
Figure 8: Aorta Measurements. Images (A) and (B) correspond to the same mouse specimen. Scale units in millimeters. (C) corresponds to a fragment of EKG of (A). Not to scale. (D) corresponds to a fragment of the EKG of (B). Not to scale. Note the marker "Δ" in images (C) and (D). The marker "Δ" denotes the point in the EKG at which the image shown was obtained. Please click here to view a larger version of this figure.
Figure 9: Pulmonary Artery Measurements. Images (A) and (B) correspond to the same mouse specimen. Scale units in millimeters. (C) corresponds to a fragment of EKG of (A). Not to scale. (D) corresponds to a fragment of the EKG of (B). Not to scale. Note the marker "Δ" in images (C) and (D). The marker "Δ" denotes the point in the EKG at which the image shown was obtained. Please click here to view a larger version of this figure.
To demonstrate the reproducibility and consistency of this technique, the authors offer two examples derived from their own work. The first one presents a group of 19 months old mice that underwent baseline echocardiography and subsequent echocardiography one week later. The authors collected the measurements for Left Atrium and Great Vessels as previously described and obtained the following results. The left side graph shows each individual mouse's Left Atrium Volume calculated using the prolate ellipse formula9,10. The right side graph shows each individual mouse's Pulmonary Artery Diameter. The data showed variation of only a few percent.
Figure 10: Left Atrium Volume and Pulmonary Artery Diameter on two subsequent assessments. (A) shows the Left Atrium Volume in mm³ of six 19 months old mice on 2 subsequent assessments. (B) shows the Pulmonary Artery diameter in mm of the same 19 months old mice group. Please click here to view a larger version of this figure.
As a second example, the authors present two images of the same 21 months old mouse acquired on the same day by two different researchers. The first two panels correspond to the images acquired by Researcher #1 and the second two panels correspond to the images acquired by Researcher #2. Please note that the difference in "Time Gain Compensation" does not affect the measurements. This example demonstrate the great importance of selecting the right point in QRS while measuring the cavity size, as previously described.
Figure 11: LA Superior-Inferior and PA diameter of the same 19 months old mouse acquired by two different researchers. (A) shows the Left Atrium Superior-Inferior diameter. (B) shows the PA diameter of the same mouse. Both images were acquired by the same researcher on the same day. (C) shows the Left Atrium Superior-Inferior diameter. (D) shows the PA diameter of the same mouse. Both images were acquired by a second researcher on the same day. Scale units in millimeters. The marker "Δ" denotes the point in the EKG at which the image shown was obtained. LA SI: Left Atrium Superior-Inferior. Please click here to view a larger version of this figure.
There are three critical steps to successfully measure the LAV, Aorta and PA diameters. During the Set-up it is important to completely remove the fur on the chest; failure to do so will result in interference with image quality. During the Image Acquisition steps it is important to obtain each image with their corresponding probe and filter as the frames per second vary from probe to probe: that is 25 MHz and Cardiac Filter for the assessment of the LAV and 30 MHz and Abdominal Filter for assessment of the Aorta and PA diameters. The researcher may be limited sometimes by poor image acquisition if the mouse has a great amount of fat or if a rib makes it difficult to get a good window. During the Image Analysis steps it is important to measure the cavities at their maximum capacities as described in the Representative Results section. Use the EKG as guidance.
Echocardiography is known to be operator dependent. This technique requires familiarization with the cardiac anatomy and echocardiographic windows for best image acquisition as well as to reduce the time of the study. Familiarization becomes relevant to prevent complications derived from the prolonged use of isoflurane such as slowing of the heart rate and hemodynamic compromise. To prevent this, close monitoring of the animal must be ensured by maintaining the heart rate above 400 beats/min throughout the study.
This technique offers many advantages compared to other available techniques to evaluate cardiac function in mice. Compared to invasive catheterization it is a non-invasive, non-terminal technique that allows serial measurements; compared to MRI, it is cheaper and faster8. This new technique for measuring LAV and PA diameter through 2D echocardiography opens doors to monitor interventions and further research in cardiopulmonary sciences.
The authors have nothing to disclose.
This work was supported by the Huffington Center on Aging at Baylor College of Medicine, Geriatrics, CVS DeBakey Heart Center at Houston Methodist Hospital and BCM, NIH RO-1 HL 13870 to ML Entman, as well as a Career Development Award # IK2BX002410 from the United States Department of Veterans Affairs (LMP), Biomedical Laboratory Research and Development Program.
We are immensely grateful to Mark Entman M.D. for sharing his wisdom and supporting this work.
Vevo 770 high-resolution in vivo micro-imaging system | Visual Sonics | Vevo 770-120 | Echocardiographic Equipment |
707B RMV (Real time MicroVisualization) 30 MHz Scanhead with encapsulated transducer | Visual Sonics | 707B-256 | Real Time Microvisualization Scanhead |
710B RMV (Real time MicroVisualization) 25 MHz Scanhead with encapsulated transducer | Visual Sonics | 710B-159 | Real Time Microvisualization Scanhead |
Vevo integrated rail system including physiological monitoring unit | Visual Sonics | ||
Inhalation Anesthesia System | VetEquip | VE2627 | Anesthesia System |
Isofluorane | Henry Schein | 50033 | |
Electric razor | Wahl | General supply | |
Hair removal cream | Nair | General supply | |
Transductor cream | Parker | ||
Transductor gel | Parker | ||
Standard Gauze pads | McKeeson | General supply | |
Tape | Durapore | General supply | |
Nose cone | For anesthesia delivery | ||
Water | |||
Vet eye ointment | Puralube | General supply | To prevent dryness |
Cotton tipped applicators | General supply | ||
USB Flash Drive | General supply |