Name: ultrasound based assessment of coronary artery flow and coronary flow reserve using the pressure overload model in mice
Uploaded: 2015-04-13T13:00:00
Description: Watch this Scientific Journal Video about Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice at JoVE.com

We thank Fred Roberts for exemplary technical support and also appreciate the help from the histology core in Beth Israel Hospital. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by a Department of Medicine Sundry Fund.

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
<ol>
	<li>Use 8-10 week old male C57BL/6 mice (BW~25 g) in the study.</li>
	<li>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.</li>
	<li>Prepare the animal fo...

<ol>
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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...

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 response1,2. 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 velocity4,6,7. 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 &#956;m, 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.

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ultrasound based assessment of coronary artery flow and coronary flow reserve using the pressure overload model in mice

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.

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 &#177; 110.9 mm/s, compared with 277.5 &#177; 10.51 mm/s in the sham mice on the day after the surgery (p&#60;0.05). The increase in velocity was the verification of the successful establishment of the pressure overload model. The SCA flow velocity, also referred t...

Watch this Scientific Journal Video about Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice at JoVE.com

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

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.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

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