Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Summary

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Abstract

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy.

During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS).

The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography.

The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Introduction

Clinical aortic stenosis (AS) is well known to promote a progressive increase in left ventricular (LV) afterload. To compensate for this chronically rising hemodynamic load, LV hypertrophy (LVH) ensues as an adaptive response^1,2. The development of LVH is often associated with abnormalities in coronary microcirculation. It is thought that microvascular dysfunction contributes to chronic ischemia in these patients ⁵. In addition to coronary flow ^3,4, coronary flow reserve (CFR) represents functional change of coronary arteries ^1,3 and is defined as the ratio of maximal flow velocity in hyperemia to baseline flow velocity or resting flow velocity^4,6,7. CFR is decreased during LV remodeling ^1-3,5-9 and is used as an index of the extent of functional severity of coronary dysfunction ^1,10,17. It is known to be impaired in many forms of dilated cardiomyopathy ¹⁰ and also coronary stenosis⁶. CFR is also a prognostic marker for poor clinical outcomes ¹².

LV remodeling in the setting of cardiac dysfunction such as ischemia or LVH is also accompanied by extensive fibrosis, changes in coronary microcirculation and thickening of coronary arteries ^1,2. As a result of these changes in coronary physiology, there is likely remodeling of the coronary arteries. This helps mitigate the effects of low oxygen diffusion and LV diastolic dysfunction that could result in susceptibility to myocardial ischemia ^1,2,13.

Genetically modified mice are now a widely prevalent investigational tool for mimicking human disease conditions such as coronary atherosclerosis ^5,7,10,12,17. Particularly the pressure overload model in mice has been widely studied ^14,17. The trans-aortic constriction model (TAC) has been shown to be associated with extensive fibrosis, and coronary stenosis resulting, in part, from medial thickening of coronary arteries and with accompanying changes in coronary flow patterns ^1,11,17,19 similar to what is seen in the setting of LVH in humans. While it is known that prolonged pressure overload leads to decompensated heart failure in about 4-8 weeks, the effects on coronary flow dynamics and flow reserve in these models, early in the process of disease progression, and at different stages after banding, are yet to be clearly delineated.

Numerous strains of mice are currently available for research use, including well-characterized LDLR-/- or ApoE-/- mice ^10-12, and these have prompted development of sensitive techniques for assessing cardiovascular function and morphology in living mice ^11-15. Such techniques include MRI, PET, contrast CT, high frequency ultrasound, and electron beam tomography ^2,9,17,19, all of which provide promising alternatives to invasive methods such as cardiac catheterizations and coronary angiography ¹². However, in mice with very small size of the coronary arteries and high heart rates (HR), imaging of coronary circulation still constitutes a technical challenge for many currently available techniques ^4,12. Interestingly, there has been an exponential rise in technical advances in the field of transthoracic Doppler echocardiography (TTDE), including the development of high-frequency array scan heads with center frequencies from 15 to 50 MHz allowing axial resolutions of approximately 30-100 μm, at depths of 8-40 mm, and frame rates greater than 400 frames-captured/sec. In turn, TTDE-based techniques have emerged as a potentially powerful tool for imaging larger ² or even smaller vessels such as coronary arteries ^5,12.

Another critical advance that has allowed investigators to conduct diagnostic imaging studies of the vasculature in small animals is the carefully controlled use of anesthetics that maintain the heart and respiratory rate of the animals during imaging ¹¹. Controlled anesthesia maintenance is particularly important for studies related to vasodilation in mice, and the effect of anesthesia also needs to be further explored in this context ^10,11. In humans, on the other hand, TTDE-derived CFR measurements have become a more commonly used tool for evaluation of stenosed and non-obstructed epicardial coronary arteries, predominantly in left anterior descending (LAD) coronary artery ^5,16. However, the prognostic role of CFR and coronary flow changes in asymptomatic patients or mice with preserved LV systolic function at rest has been much less explored ¹⁶. Therefore, the aim of the study was to first establish a clear step-by-step protocol, to evaluate changes in coronary flow using TTDE in a pressure overload mouse model; second, this study examined the prognostic significance of CFR and coronary flow changes in response to pressure overload stress in these mice. We hypothesized that TTDE based assessment of CFR and coronary flow may be useful in the early detection of coronary dysfunction that may precede LV dysfunction.

Protocol

NOTE: All procedures were performed in mice in accordance with American Veterinary Medical Association (AVMA) guidelines and approved Institutional Animal Care and Use Committees (IACUC) protocols.

1. Study Design

1. Use 8-10 week old male C57BL/6 mice (BW~25 g) in the study.
2. Randomize the mice (n = 11) into two groups, the study group selected for aortic banding (n = 8), and the control group (n = 3) to undergo sham operation via thoracotomy.
3. Prepare the animal for imaging by removing hair from the chest using depilatory cream that is medical grade.
4. Perform a first ultrasound (section 2) 24 hr prior to aortic banding to determine baseline parameters at Day -1, between a range of 1% and 2.5% isoflurane (mixed with 100% O₂ via nosecone) induced anesthesia.
5. Choose a medically approved anesthetic agent (i.e. isoflurane) and monitor the degree of anesthesia (2-3% to induce, and 1.0% to maintain).
   NOTE: Proper anesthesia is crucial in the maintenance of heart beat at normal physiological rates (about 500 beats/min).
6. Confirm the depth of anesthesia by loss of motion from the animal in response to a pedal-withdrawal reflex. Use paralube vet ointment on the eyes to prevent dryness while under anesthesia.
7. Perform surgery at Day 0 ^20,21.
8. For aortic banding, ligate the aorta using a 7-0 silk suture around a tapered 26 G needle placed on the arch.
   NOTE: Details regarding the experimental protocol, including the surgical aortic banding procedures, have been described previously ^20,21.
9. Perform post-surgery ultrasound imaging (section 2) at Day(s) 2, 6 and 13.
10. Euthanize the mice on Day 14 and harvest the hearts for histological assessment. Euthanize the animals using an overdose of pentobarbital followed by removal of a vital organ such as the heart. Arrest the hearts in diastole and fix with formalin. Use the procedure of the heart harvesting that has been described previously ²².
11. Fix all heart tissues with buffered 10% formalin solution. For trichrome staining, embed tissues in paraffin before sectioning. Use the details of trichrome staining that have been well illustrated previously ^14,23.
12. Analyze the data using offline software (section 3).

2. Imaging Protocol

1. Long and short axis images of septal coronary artery (SCA) (B- Mode)
   1. Using MS550D probe with center frequency of 40 MHz connected to the active-port, set the application preset to “cardiac imaging”.
   2. With the animal supine on the heated platform, and under anesthesia controlled via nose cone, position the probe using the rail system to obtain the parasternal long axis view (PSLAX) (Figure 1A). Always ensure that the animal is kept warm on the prewarmed platform and body temperature is maintained at physiologic levels.
   3. Rotate the probe (with notch pointing caudally) clockwise such that the probe angle is 15° to the left parasternal line (long-axis view) (Figure 1B).
   4. Adjust the probe angle by tilting slightly along y axis of the probe to obtain a full-length longitudinal view of the SCA in the center of the screen (Figure 1B).
   5. Once the proper landmarks (aortic valve and pulmonary artery) are viewed, cine store the image using the highest frame-rate possible.
   6. By using the “xy” axes micro-manipulators (Figure 1D), adjust the probe position to obtain the clearest image of the SCA.
   7. Rotate the probe 90° (with notch pointing caudally) clockwise such that notched end of the probe is to the left of midline (short-axis) (Figure 1C).
2. Long and short axis images of SCA (Color-Doppler Mode)
   1. Once a B Mode image is captured or cine-stored, click the color Doppler key on the keyboard to turn on color Doppler acoustic window (Figure 2).
      NOTE: This helps to isolate coronary artery (white arrow indicates SCA) either in the long (Figure 2A) or in the short axis (Figure 2C). Red color is as seen in real time and is indicative of the direction of flow (away from the aortic valve).
   2. Ensure that the focus depth (indicated by a yellow arrowhead on right of the image screen), lies in the center of coronary artery.
   3. Ensure that the data is recorded, using the cine-store key, at the highest possible frame rate (>100 frames/sec).
3. PW Doppler Imaging of SCA (Pulsed-Wave or PW Mode)
   1. While in color-Doppler mode, click on the PW key to bring up a yellow-indicator line on the coronary artery (Figure 2, shown in red).
   2. Place the yellow PW line in the middle of the coronary artery in view, at an angle that parallels the directionality of the flow. Note that velocity measurements are highly dependent on the angle of image acquisition.
   3. Adjust the angle of flow (PW angle key) and sample volume (SV key) such that the PW angle key is 60° or less and sample volume captures flow right in the center of the SCA.
   4. Use cine store to capture the wave forms that indicate the velocity of the coronary flow at peak systole (S) and diastole (D) (Figures 3A and 3B), using 1% and 2.5% isoflurane.

3. Data Calculation and Analysis

1. Select the velocity time integral (VTI) tool to obtain the peak systolic and diastolic velocities from the images shown in Figures 3A and 3B.
2. Calculate the coronary flow reserve index (CFR) as the ratio of hyperemic (2.5% isoflurane) peak diastolic flow velocity to baseline (1% isoflurane) peak diastolic flow velocity.
3. Calculate the S/D ratio as the peak systolic coronary flow velocity/ peak diastolic coronary flow velocity. Determine the ratio at baseline (1% isoflurane) and at hyperemia (2.5% isoflurane).
4. For standard cardiac function parameters such as FS, FAC, LVM, refer to the manuals from the manufacturer to perform data analysis using proprietary software or refer to Cheng’s JoVE paper ².

Results

Of the 11 mice that were studied (banded, n=8 and sham, n = 3), adequate and reproducible images were obtained by a single observer at several time-points: at baseline (D-1), D2, D6 and D13. Also, the flow velocity at the constrictive site was measured as 2225 ± 110.9 mm/s, compared with 277.5 ± 10.51 mm/s in the sham mice on the day after the surgery (p<0.05). The increase in velocity was the verification of the successful establishment of the pressure overload model. The SCA flow velocity, also referred to here as t...

Discussion

In this ultrasound based study, non-invasive assessment of coronary flow was reproducibly performed in real time, over days, in live experimental mice; furthermore, the protocol demonstrated the potential to detect coronary artery dysfunction that was present at an early stage and was associated with deficiency in myocardial perfusion. This method could ultimately be leveraged as a clinical tool for cardiovascular risk stratification and/or assessing response to therapeutic intervention.

First...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Depilatory cream	Miltex, Inc.	Surgi-Prep	Apply 24 hours prior to imaging
Isoflurane	Baxter International Inc.	NDC 10019-773-40	2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute
High Frequency Ultrasound	FUJIFILM VisualSonics, Inc.	Vevo 2100
High-frequency Mechanical Transducer	FUJIFILM VisualSonics, Inc.	MS250, MS550D, MS400