Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.
Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR). In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.
Myocardial ischemia reperfusion (IR) is a condition where blood supply is restricted to the heart followed by the restoration of perfusion and simultaneous reoxygenation1. Occlusion of coronary arteries can be caused by an embolus or cholesterol plaque rupture, which results in a severe imbalance of metabolic supply and demand, causing tissue hypoxia. Salvage of jeopardized myocardium, improve left ventricular function, and enhance survival in patients with acute myocardial infarction have been observed by the reperfusion therapy. However, after recanalization of the coronary artery, functional abnormalities of small coronary vessels may occur2,3,4,5. A significant proportion of patients, perhaps as many as 40%, do not regain microvascular and myocardial perfusion despite the restoration of coronary flow. Visualization and evaluation of the coronary microvasculature can be difficult in vivo, but there are a number of invasive and noninvasive techniques that can be used to assess parameters directly depending on the coronary microvascular function6,7. Also, the endothelial function can be assessed at the level of the coronary circulation via the CFR5.
Transthoracic Doppler echocardiography is a noninvasive tool which allows us to study coronary artery flow velocity and CFR5. CFR represents the ratio of maximal coronary blood flow to the resting coronary blood flow8. During the stress challenge, a normal heart increases coronary blood flow up to four times above the resting value. Cardiovascular risk increases when CFR is diminished9. Ishihara et al showed that the CFR was severely impaired immediately after the coronary angioplasty5. In the absence of coronary artery stenosis, CFR decreases during the coronary microvascular dysfunction and is present in about half of the patients with stable coronary artery disease10.
The overall goal of this method is noninvasive visualization of left anterior descending coronary artery (LAD) function in rats via echocardiography, which may be used to calculate CFR. This offers an important assessment tool for diagnosing microvascular dysfunction and evaluating potential therapeutic treatments.
All procedures were performed in accordance with protocols approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC-approved protocol 18223) and the NIH Guide for the Care and Use of Laboratory Animals11.
1. Animals
2. Ultrasound Imaging before IR surgery
3. Ischemia Reperfusion Injury
4. Ultrasound imaging after IR surgery
For this study, we used 12 female Fisher 344 rats. We performed a stress test with Dobutamine and measured LAD coronary artery velocity before and 72 hours after the IR surgery. Before the IR surgery, resting LAD coronary artery velocity was measured as 423 ± 59 mm/s, which was increased after Dobutamine infusion (1005 ± 77mm/s) (Figure 3A). After 72 h of ischemia reperfusion, resting LAD coronary artery velocity was significantly higher compared to the resting LAD coronary artery velocity before the IR surgery (743 ± 40mm/s vs 423 ± 59 mm/s) (Figure 3A). Stress response to Dobutamine test after the IR surgery was significantly reduced compared to the before IR surgery responses (937 ± 67ms/s vs 1005 ± 77mm/s) (Figure 3A).
CFR is calculated as the ratio of the peak flow velocity during stress (Dobutamine) to the resting flow velocity (measured prior to Dobutamine infusion)8. CFR was 2.1 ± 0.35 in young rats before the IR surgery (Figure 3B) but significantly reduced (1.1 ± 0.25) after 72 h of the IR surgery, even though the resting LAD coronary artery velocity was higher in these rats compared to data obtained before IR surgery (Figure 3C). In addition, there were no significant changes in the systolic function of the left ventricle in rats after 72 h of the IR surgery (Figure 3C).
Figure 1: Coronary artery location. A. Probe position on the rat while obtaining LAD coronary artery velocity. B. Short axis anatomical representation of the pulmonary artery, aorta, and LAD coronary artery. C. Anatomical visualization of LAD coronary artery on echocardiography. Please click here to view a larger version of this figure.
Figure 2: The Pulse Wave Velocity imaging of the LAD coronary artery. A. Representation of the pulse wave velocity sensor placement on LAD coronary artery. B. LAD coronary artery pulse wave image during the rest condition. C. LAD coronary artery pulse wave image during the stress (Dobutamine) condition. Please click here to view a larger version of this figure.
Figure 3. Measurement of the coronary flow using Doppler echocardiography. A. Pulse wave velocity measured in Control during the rest (Cont B) and during Dobutamine infusion (Cont D) and in animals after 72 h of IR surgery during the rest (IR B) and during Dobutamine challenge (IR D), p < 0.05 Cont B vs. ischemia reperfusion B (*). B. CFR was calculated from pulse wave in the experimental animals (n=9), p < 0.05 Cont vs. IR (*). C. Fractional shortening assessment from the experimental groups (n=9). Data are presented as mean ± SD, analyzed with one-way ANOVA. Please click here to view a larger version of this figure.
The major findings from the present study are that IR increases the resting LAD coronary artery velocity and impairs CFR, even in the absence of any residual angiographic stenosis.
Understanding the coronary physiology is an essential part of the clinical decision-making for cardiologists to treat coronary artery disease. CFR is one of the important functional parameters in understanding the pathophysiology of coronary microcirculation7,13. CFR is a noninvasive method to assess both coronary artery stenosis and coronary microvascular circulation and is an indicator of myocardial blood supply, explicitly the ability of the coronaries to increase blood flow under stress conditions7. A normal CFR (>2.0) often reflects a good prognosis, while CFR less than 1.90 provides incremental diagnostic information for the identification of high-risk coronary artery disease14,15,16.
Our results show that, even though the systolic function was preserved after ischemia reperfusion (Figure 3C), CFR was significantly lower (Figure 3B). Thus, recanalization of stenotic coronary arteries does not improve microvascular perfusion. Decreased CFR enables detection of impaired microvascular vasodilation after ischemia reperfusion.
This study demonstrates the serial CFR evaluations to explore the effect of various pharmacological therapies using noninvasive transthoracic Doppler echocardiography. This method of coronary functional assessment can be used in small animal research as a feasible and viable clinical diagnostic tool. This will lead to minimizing the requirement of animal use, euthanasia, or necropsy in the small animal models. Critical steps in this protocol are visualizing the coronary artery and obtaining the PW velocity images of good quality. Another critical step is to maintain LAD visualization during the stress state. During Dobutamine challenge, the heart rate increases and the LAD may move from the field of view; researchers should be prepared to move the field in order to follow the coronary artery. Limitations in the current study include the relatively small sample size, the lack of correlation between CFR and the coronary artery lumen diameter in vivo in rats, due to the difficulty in obtaining accurate visualization for the size measurement of the coronary artery. However, the methods described here are reliable, reproducible, and offer insightful information on the damage inflicted on the cardiac microvasculature following ischemia reperfusion.
The authors have nothing to disclose.
We would like to thank the Helmsley foundation for providing ultrasound equipment to perform our experiments. This work was supported by NIA R01 053585 grant.
10 mL syringe | BD Syringe | 302995 | |
250S 13–24 MHz linear probe | FUJIFILM VisualSonics Inc | ||
Dobutamine hydrochloride | Sigma | D0676-10mg | |
Isoflurane | RRC | 27376 | |
Legato 100 Syringe pump | KD Scientific | 788100 | |
Vevo 3100 | FUJIFILM VisualSonics Inc | ||
Winged infusion set, 27G x 1/2", | Medline.com | TMOSV27ELZ |