Published online before print August 20, 2007, doi: 10.1161/CIRCULATIONAHA.107.707737
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(Circulation. 2007;116:1250-1257.)
© 2007 American Heart Association, Inc.
Imaging |
In Vivo Characterization of Murine Myocardial Perfusion With Myocardial Contrast Echocardiography
Validation and Application in Nitric Oxide Synthase 3–Deficient Mice
From the Cardiac Ultrasound Laboratory in the Cardiology Division of the Department of Medicine (M.J.R., H.T., K.K.P., M.H.P., M.S.-C.), the Cardiovascular Research Center (M.J.R., H.T., K.D.B., M.S.-C.), the Department of Anesthesia and Critical Care (M.J.R., R.L., F.I., W.M.Z., K.D.B.), and the Institute for Technology Assessment (E.F.H.), Massachusetts General Hospital and Harvard Medical School, Boston, Mass; and INSERM E 0226 (H.T., G.D.), Université Claude Bernard Lyon I, Lyon, France.
Correspondence to Marielle Scherrer-Crosbie, MD, PhD, Cardiac Ultrasound Laboratory, Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114. E-mail marielle{at}crosbie.com
Received November 4, 2006; accepted June 29, 2007.
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Abstract |
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Background— The ability to noninvasively evaluate murine myocardial blood flow (MBF) in vivo would provide an important tool for cardiovascular research. Myocardial contrast echocardiography (MCE) has been used to measure MBF; however, it has not been validated in mice. This study assesses whether MCE can evaluate MBF at rest and after vasodilation and measure the maximal augmentation (coronary reserve) of MBF in mice. Wild-type (WT) and nitric oxide synthase 3 (NOS3)–deficient (NOS3–/–) mice were studied.
Methods and Results— MCE was performed at baseline and after intravenous infusion of acetylcholine or adenosine. Definity contrast agent was infused, and parasternal views were acquired in real-time mode. Replenishment curves of myocardial contrast were obtained, and rates of signal rise (ß) and plateau intensity (A) were calculated. MBF estimated by the product of A and ß (Aß) was compared with that measured with fluorescent microspheres. MCE analysis was feasible in 98% (52/53) of mice. MBF measured by microspheres increased with adenosine and correlated closely with Aß. There was no difference in MCE-derived MBF between WT and NOS3–/– mice at rest. Adenosine infusion increased MBF by 3.0±0.6-fold in NOS3–/– mice and 2.5±0.3-fold in WT (P=0.58 between genotypes). Acetylcholine induced an increase of 2.4±0.2-fold in MBF in WT mice but did not increase MBF in NOS3–/– mice (P<0.0005 versus WT).
Conclusions— MBF, coronary reserve, and vasodilator responses can be evaluated accurately in the intact mouse by MCE. This method demonstrated a preserved coronary response to adenosine but an impaired acetylcholine-induced vasodilation in NOS3–/– mice compared with WT mice.
Key Words: nitric oxide synthase • adenosine • acetylcholine • microspheres
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Introduction |
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Regulation of myocardial blood flow (MBF), a necessary process to meet the metabolic needs of the myocardium, is altered in diseases such as hypertension and diabetes mellitus and may have a role in the development of the cardiac complications seen in these pathologies.1,2 The regulation of MBF has been explored by studying the response of MBF to vasodilators such as adenosine and acetylcholine. The response to acetylcholine reflects endothelial function, and adenosine may be used to measure the maximal increase in blood flow available to the heart (coronary reserve).3
Clinical Perspective p 1257
Genetically modified mice are useful tools to study the pathways involved in the control of MBF. Whether adenosine-mediated coronary vasodilation depends on endothelial nitric oxide (NO) synthase (NOS3) and NO is controversial.4 Insights may be gained by comparing the response to adenosine of NOS3-deficient mice (NOS3–/–) with that of wild-type (WT) mice. Similarly, the importance of NOS3-derived NO in the endothelium-mediated coronary vasodilation induced by acetylcholine has been investigated with NOS3–/– mice, with conflicting results.5–10 In NOS3–/– mice, the coronary vasodilatory response to acetylcholine has been reported to be abolished,5 altered,8,10 or preserved.6,7,9 In these studies, coronary artery responses to vasodilators were evaluated in vitro with coronary artery rings5–7 or isolated hearts.8–10 Other methods to assess coronary flow, such as magnetic resonance imaging11 and Doppler echocardiography,12 have been proposed, but these technically challenging modalities remain unvalidated against a "gold standard" in mice. The ability to noninvasively evaluate MBF in vivo in mice both at rest and after administration of vasodilators would provide an important tool for cardiovascular disease research.
Myocardial contrast echocardiography (MCE) allows for the noninvasive quantitation of MBF with a continuous intravenous infusion of echogenic gas-filled microbubbles.13,14 After destruction of the microbubbles by high-energy ultrasound pulses, the increase of the myocardial signal intensity over time reflects the replenishment of the microbubbles in the myocardium. The increase of the myocardial signal intensity over time may be fitted to an exponential curve. Myocardial plateau signal intensity (A) and the rate constant of the rise of the signal intensity (ß) have been interpreted as the myocardial microvascular cross-sectional area and blood velocity, respectively. Aß, the product of A and ß, is an estimate of MBF.13 MCE has been applied in large animals13,15,16 and humans17,18 to measure coronary blood flow reserve. In mice, MCE with bolus injections of microbubbles allowed us to measure the extent of the nonperfused region in a model of coronary artery ligation.19
The present study investigated whether or not MCE with a continuous infusion of contrast is feasible in mice and would allow us to evaluate MBF, both at rest and after vasodilator infusion. Echocardiographically derived MBF was measured in C57BL6 WT mice before and after infusion of adenosine and acetylcholine. The measurement of MBF by MCE was validated with microspheres.20 To investigate in vivo the role of NOS3 in the regulation of MBF, MCE was also performed in NOS3–/– mice before and after infusion of adenosine and acetylcholine.
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Methods |
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MCE Technique
Mice were anesthetized with intraperitoneal injections of ketamine (0.1 mg/g), xylazine (0.01 mg/g), and pancuronium (0.002 mg/g). Heart rate and blood pressure were monitored with a carotid catheter. Venous lines were placed in both jugular veins for the separate infusion of microbubbles and vasodilators. A thoracotomy was performed to obtain unrestricted visualization of all myocardial regions. In 5 additional mice, the feasibility of closed-chest acquisition was tested by the performance of both closed- and open-chest imaging in the same animal.
MCE was performed with a linear transducer (14 MHz; Acuson Sequoia C512 system; Siemens, Mountain View, Calif) at a mechanical index of 0.24 (transmission power –7 dB) with contrast pulse sequencing. Gains were adjusted to obtain images with no signal from the myocardium and were then held constant. Perflutren lipid microspheres (Definity, Bristol-Myers Squibb Medical Imaging, Billerica, Mass) were diluted 1/10 in sterile saline. For the dose-response determination, contrast doses were infused intravenously for 30 seconds each. In all other mice, the contrast agent was infused intravenously at 20 µL/min.
Perfusion images were acquired in real time (frame rate of 30 Hz) after a sequence of 10 high-energy frames (mechanical index of 1.9). In 5 additional mice, real-time acquisition was compared with end-systolic acquisition. The acquisition time for 1 loop was 4±1 seconds. Parasternal long-axis views were acquired in all mice. To evaluate regional distribution of MBF, parasternal short-axis views at the papillary muscle level were also acquired in 7 mice.
After baseline images were acquired, adenosine (150 µg · kg–1min–1, 10 µL/min, Bedford Laboratories, Bedford, Ohio) or acetylcholine (2 µg · kg–1min–1, 10 µL/min, Sigma-Aldrich, St Louis, Mo) was infused intravenously. After 5 minutes of continuous infusion, MCE images were obtained. M-mode echocardiograms were also acquired before and after adenosine (n=5 WT) and acetylcholine (n=5 WT) to measure cardiac output.
MCE Analysis
Analysis of MCE was performed by readers blinded to the genotype and the treatment of the mice. Regions of interest were positioned within the anteroseptal and posterolateral walls in the long-axis view and within the septal and lateral walls in the short-axis view. To determine the relationship between the infused contrast dose and the left ventricular (LV) cavity signal intensity, an additional region of interest was positioned within the LV cavity. Average signal intensity within the region of interest was measured automatically on each frame (Syngo ACQ, Siemens). A curve of signal intensity over time was obtained in each region of interest and fitted to an exponential function: y=A(1–e–ßt), where y is signal intensity at any given time, ß is the initial slope of the curve, and A is the plateau intensity. MBF was estimated by the product Aß. Two to 3 curves were averaged for each animal under each condition. A goodness-of-fit coefficient (r2 of the fit) was obtained for each curve, and an arbitrary threshold of 0.8 was set. All curves with a goodness-of-fit of less than 0.8 were excluded.
Comparison With Microsphere-Estimated Blood Flow
Mice underwent MCE either at baseline (n=10) or after adenosine-induced vasodilation (n=8). Immediately after MCE acquisition, 180 000 10-µm (diameter) fluorescent microspheres (Molecular Probes, Eugene, Ore) diluted in 200 µL of saline were injected directly into the LV over 5 seconds. A reference blood sample was withdrawn from the carotid artery at a rate of 200 µL/min, starting 5 seconds before microsphere injection and lasting for a total of 60 seconds. Mice were euthanized, and the blood, LV, and kidneys were digested with 3 mL of 8N ethanolic KOH with 2% Tween-80 for 72 hours. Microspheres were isolated with Spin-X centrifuge tube membrane filters (Corning Costar, Acton, Mass). The filters were washed with saline and 2% Tween-80 and incubated for 6 hours with 2-ethoxyethyl acetate to elute fluorescent dyes. Fluorescence in each sample was measured with a 96-well plate reader (VICTOR3, PerkinElmer, Wellesley, Mass). Blood flow value in a tissue sample, i, was calculated with the formula:
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where Qi is the blood flow in the sample i, Qref is the withdrawal rate of the reference blood sample, and Inti and Intref are the fluorescence intensity in sample i and the reference blood sample, respectively.
Statistical Analysis
Statistical analysis was performed with the JMP statistical software package (SAS Institute, Cary, NC). All values are expressed as mean±SEM, except interobserver and intraobserver variabilities, which are expressed as mean±SD. Normality of the distribution was checked with the Shapiro-Wilk test for each group of mice. The comparisons involving nonnormally distributed groups (perfusion parameters of NOS3-deficient mice after adenosine and blood pressure of WT mice after acetylcholine) were done with ordinal logistic and the Wilcoxon sign test. For normally distributed groups, results were analyzed by means of 1-way ANOVAs for repeated measurements. The effects of adenosine and acetylcholine on both genotypes were analyzed separately with 1-factor ANOVAs for repeated measurements. The effects of interest were treatment and genotype. If the ANOVA showed a difference in the response between genotypes, paired t tests were used to compare the postdilation state with the baseline within each genotype. To correct for the multiple comparison error (effects of interest A, ß, Aß, blood pressure, and heart rate), these effects were considered to be significant if P<0.01. Two to 3 replenishment curves were obtained for each mouse at each stage and the values of the perfusion parameters averaged. The interobserver variability was measured on these averaged values by 2 independent observers (KKP and HT) in identical loops in 5 mice. To measure intraobserver variability, a single observer (HT) repeated the measurements on the same loops several weeks after the first measurement set. Intraobserver and interobserver variabilities were calculated as the difference between the 2 observations divided by the means of the observations and were expressed as both absolute numbers and percentages. The intraobserver and interobserver correlation coefficients were also measured. To estimate the variability between the curves obtained in each mouse, a pooled SD for each of the measurements is also reported.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Relationship of Contrast Dose and LV Signal
Infusion of Definity at a rate of 2 µL/min ensured maximal detection of contrast in the posterolateral myocardial wall while still being on the linear portion of the dose-signal curve (Figure 1). The signal of the LV cavity, however, was on the saturated portion of the dose-signal curve.
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Baseline Hemodynamic Parameters in Mice Undergoing MCE
At baseline, systolic arterial pressure was higher in NOS3–/– mice than in WT mice (102±2 versus 93±2 mm Hg, respectively, P<0.01) and heart rate was lower (514±8 versus 545±7 bpm, respectively, P<0.01). MCE did not affect blood pressure or heart rate (data not shown).
Feasibility, Baseline Values, and Variability of Murine MCE
A total of 206 curves were analyzed. Twenty-one curves (10%) had a goodness-of fit <0.8 (insufficient visualization of myocardial contrast despite the routinely administered contrast dose). This led to the exclusion of 1 mouse from 53 mice analyzed (2%).
Regional baseline values for A, ß, and Aß in WT mice are reported in Table 1. There was no significant difference in A, ß, and Aß between the different myocardial walls. Consequently, only the values obtained for the posterolateral wall are reported. In the posterolateral wall, the pooled SDs of A, ß, and Aß were 1.3, 0.1, and 1.6, respectively. The intraobserver variability as assessed by the SD of the error was 1.2 dB (10%) for A, 0.05 s–1 (7%) for ß, and 0.6 dB/s (8%) for Aß. The intraobserver correlation coefficients were 0.85, 0.99, and 0.99 for A, ß, and Aß, respectively. The interobserver variability was 1.5 dB (13%) for A, 0.09 s–1 (14%) for ß, and 1 dB/s (11%) for Aß. The interobserver correlation coefficients were 0.73, 0.96, and 0.97 for A, ß, and Aß, respectively.
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In additional studies, the feasibility of closed-chest MCE was tested. Imaging artifacts such as rib shadowing were more prevalent in closed-chest than in open-chest conditions, and an increased number of loops4–5 were acquired and analyzed for each animal to more accurately evaluate myocardial perfusion. No mice were excluded from the analysis. A, ß, and Aß were not different between closed-chest and open-chest mice acquisitions (A: 10±2 versus 11±1 dB; ß: 0.6±0.1 versus 0.6±0.1 s–1; and Aß: 6±1 versus 7±1 dB/s, respectively). The feasibility of triggered end-systolic imaging was also tested. A, ß, and Aß were not different between real-time and triggered end-systolic imaging (data not shown).
Hemodynamic Responses to Vasodilators
Adenosine infusion lowered blood pressure and heart rate in NOS3–/– but not in WT mice (Table 2). Infusion of acetylcholine did not alter blood pressure in either genotype, decreased the heart rate in WT mice by 7±1%, but had no effect on the heart rate in NOS3–/– mice (P<0.01 versus heart rate in WT). Neither adenosine nor acetylcholine modified the cardiac output in WT mice (12±2 mL/min after adenosine versus 10±1 mL/min at baseline and 12±1 mL/min after acetylcholine compared with 11±1 mL/min at baseline).
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Comparison of MCE-Estimated MBF Measurements With Microspheres
Injection of microspheres was accompanied by a transient increase in blood pressure (11±9%, P<0.05) with no change in heart rate. This increase peaked 15±1 seconds after the start of the injection. All tissue and blood samples contained >7000 microspheres. MBF measured by microspheres was 6±0.5 mL · min–1 · g–1 at baseline. Blood flow in both kidneys was similar (9±1 and 8±1 mL · min–1 · g–1 in the right and left kidneys, respectively). WT mice treated with adenosine had a higher MBF (14±2 mL · min–1 · g–1) than did untreated WT mice (P<0.0001). Adenosine did not alter blood flow in the kidneys (6±1 and 7±1 mL · min–1 · g–1 in the right and left kidneys, respectively). MBF estimated by MCE (as the product of Aß) was 6±1 dB/s in WT mice at baseline and 14±2 dB/s after adenosine (P<0.001 versus control). There was a close correlation between MBF as measured by fluorescent microspheres and that derived from MCE both from the anteroseptal wall (microsphere-measured MBF=1.16 Aß+0.6, r2=0.73, P<0.0001; Figure 2, upper panel) and the posterolateral wall (microsphere-measured MBF=0.92 Aß+0.6, r2=0.7, P<0.0001; Figure 2, lower panel).
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Coronary Reserve and Coronary Vasodilatory Response to Acetylcholine Measured by MCE
In WT mice, adenosine increased MBF by 2.5±0.3-fold (P<0.005), primarily by increasing blood velocity (estimated by ß) without modification of myocardial blood volume as estimated by A (Table 3). Increasing the dose of adenosine did not further increase the MBF (data not shown). Acetylcholine also increased MBF in WT mice by 2.4±0.2-fold by augmenting blood velocity and blood volume (Table 3; Figure 3).
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At baseline, there was no difference in MCE-estimated MBF between WT and NOS3–/– mice (Table 3). Adenosine infusion increased MBF in both genotypes by increasing myocardial blood velocity. No change in myocardial blood volume was noted in either genotype. The increase in MBF elicited by adenosine was similar in NOS3–/– and WT mice (P=0.58; Table 3). Acetylcholine induced an increase in MBF in WT, but no response was noted in NOS3–/– mice (2.4±0.2-fold increase in WT compared with 1.0±0.1-fold in NOS3–/– mice, P<0.0005; Figure 3).
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Discussion |
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This study describes and validates the use of MCE for the evaluation of MBF in mice. Both resting MBF and the response of MBF to vasodilators were accurately estimated, and the technique was applied to NOS3–/– mice. In the WT mouse heart, adenosine and acetylcholine infusions were accompanied by a similar increase in MBF: adenosine increased MBF velocity without altering myocardial blood volume, whereas acetylcholine increased both MBF velocity and volume. MBF was similar in NOS3–/– and WT mice at rest. Adenosine augmented MBF in both genotypes, but the vasodilatory MBF response to acetylcholine was markedly blunted in NOS3–/– mice.
Murine MCE presents several challenges. The high-frequency probe necessary to permit sufficient spatial resolution precludes the use of harmonic imaging. Harmonic imaging relies on ultrasound excitation of the bubbles at their resonant frequency rather than the fundamental frequency reflected by both tissue and bubbles. The resonant frequency depends on bubble size and is 
3 MHz for a 3-µm-diameter bubble. Such a low frequency would not provide the spatial resolution necessary to image the murine heart. Although fundamental frequencies were used, we were still able to detect intramyocardial bubbles. The ability to detect low concentrations of microbubbles may be due to the fact that bubble destruction at a high probe frequency is minimal.21 Furthermore, the contrast pulse sequencing modality enhances the microbubble signal by detecting the nonlinear energy that exists at the same frequency band as the transmitted signal.22
MCE can be acquired with either a real-time mode or a mode in which imaging is intermittent and triggered to the ECG.13,23 Both modes allow the quantification of perfusion. In the present study, real-time acquisition was used and produced an adequate signal from the myocardial walls. In additional mice, the acquisition was triggered to end systole only. No difference was found between the MCE parameters obtained with the 2 techniques.
In this validation study, a thoracotomy provided unrestricted views that enabled analysis of all myocardial segments, without the artifacts produced by the sternum or the lungs. To extend the applicability of the present study to chronic models, the feasibility of closed-chest MCE was tested. There were no differences in the estimation of myocardial blood volume, blood flow velocity, or myocardial perfusion between closed-chest and open-chest conditions.
Fluorescent microspheres were injected into the LV to validate the MCE measurement of MBF. In a small sample such as the mouse heart or kidney, it is necessary to ensure that the sample contains >400 microspheres to yield meaningful blood flow.24 This was the case in the present study, in which all samples contained >7000 microspheres. Blood flow was similar in both kidneys, which suggests adequate mixing of the microspheres in the LV and aorta. Myocardial and renal blood flow values in the present study are similar to those reported previously.25 A small increase in blood pressure was noted after microsphere injection; however, this increase was within the variability of the blood pressure in awake mice. Adenosine-treated mice had an increased MBF, both when measured by microspheres and by MCE (2.5±0.3- and 2.3±0.2-fold, respectively). There was a close correlation between MCE- and microsphere-derived MBF. Unlike the microsphere technique, repeat assessment of MBF with MCE is possible. Although several injections of microspheres can be administered in larger animals, hemodynamic conditions in the mouse could not be easily maintained after the first injection of microspheres and subsequent withdrawal of blood. In contrast to microspheres, MCE was able to estimate MBF both at rest and after infusion of a vasodilator in the same mouse.
This ability to obtain both baseline and stimulated MBF allows the measurement of coronary reserve and the detection of differences between genotypes in the MBF response to interventions. In the present study, the maximum increase in MCE-derived MBF elicited by adenosine was less than that observed in normal humans, a finding confirmed by use of the fluorescent microsphere technique. Interestingly, Wikstrom et al12 reported that the adenosine-induced increase in coronary velocity measured by the Doppler effect was less in mice than in humans. There are several potential explanations for the lower coronary reserve in mice than in humans. Adenosine could decrease cardiac output in mice by decreasing heart rate and blood pressure, thereby reducing myocardial perfusion. In the present study, cardiac output was preserved after adenosine infusion. It is also recognized that an increase in myocardial oxygen consumption is a major trigger that increases MBF.26 Mice do not increase their oxygen consumption by more than 2-fold, even at peak exercise27 or after cage-switch stress.28 Murine heart rate is predominantly under sympathetic control29 and does not increase by more than 50% at maximal exercise.30 Mice, therefore, may need less coronary reserve than humans to respond to increased myocardial oxygen requirements. In the present study, acetylcholine infusion produced vasodilation and an increase in MBF similar to that induced by adenosine. This 2-fold increase in MBF observed in the present study with acetylcholine was similar to that reported in isolated mouse hearts.31
The increase in MBF observed after adenosine infusion was due to an increase in blood flow velocity, without any change in the myocardial blood volume. A similar observation has been made with MCE in dogs13,32 and has been attributed to the fact that myocardial blood volume is mainly contained in the capillaries, which do not appear to be recruited by intracoronary adenosine administration.33
At baseline, NOS3–/– mice had a higher blood pressure and a lower heart rate than WT mice, as previously reported in both awake and anesthetized animals.34,35 Whether or not the vasodilatory properties of adenosine are independent of the endothelium or mediated in part by NOS3-derived NO is controversial. In previous studies, chemical inhibition of NO synthesis either blunted36,37 or had no effect38,39 on adenosine-induced coronary vasodilation. In contrast to pharmacological NOS inhibitors, assessment of the response to adenosine in intact NOS3–/– mice ensures a specific and complete absence of the endothelial NOS isoform. Adenosine infusion decreased blood pressure only in NOS3–/– mice. This decrease in blood pressure was accompanied by a preserved increase of MBF, which suggests that adenosine does not require the presence of NOS3 to vasodilate both the systemic and coronary circulations of mice.
The importance of the endothelium in the arterial vasodilatory response to acetylcholine is well-known.40 The role of NOS3 in the coronary response to acetylcholine has been assessed with pharmacological inhibition39,41 and more recently in NOS3–/– mice.5–8,10 Pharmacological inhibition of NO synthesis reduces the coronary vasodilatory response to acetylcholine.39,41 Studies of NOS3–/– mice, however, have produced conflicting results. Chataigneau et al5 reported that acetylcholine did not vasodilate coronary vascular rings of NOS3–/– mice. Conversely, using coronary rings or isolated hearts, other investigators found partially impaired8,10 or preserved6,7 responses to acetylcholine. In the present study, acetylcholine infusion did not increase MBF in NOS3–/– mice, which underlines the major role of NOS3-derived NO in the vasodilator response to acetylcholine infusion in intact mice.
There are several limitations to the present study. The anesthetics used in our mouse studies may have decreased the coronary reserve.42 The high-energy frame-destruction sequence used in this experiment may decrease the replenishment rate by transiently decreasing microbubble delivery in the coronary vasculature.43 However, the microbubble destruction was the same in all of our studies, because an identical protocol was used in all mice at all times. We cannot exclude that the microsphere injection by itself may have had some effect on MBF. Neither closed-chest MCE nor end-systolic triggering acquisition was validated with the established technique of microspheres; however, real-time acquisition is an accepted technique for MBF estimation.44,45 Finally, we did not quantify absolute MBF. Recently, Vogel et al14 demonstrated that normalizing Aß obtained in the myocardial walls by the LV cavity signal intensity provides absolute measurements of MBF in humans. In the mouse, because of the small number of microbubbles within the myocardium, a high concentration of microbubbles relative to body weight must be infused. In the present study, the dose of contrast that was chosen ensured maximal detection of contrast in the myocardial walls within the linear portion of the dose-signal curve. However, the contrast signal intensity in the LV cavity did not reflect the amount of microbubbles therein, because the LV cavity signal was on the saturated portion of the dose-signal curve. The saturation of the signal in the LV cavity precluded normalization of the blood flow estimate.
In conclusion, MCE is feasible in mice and can reproducibly provide an accurate index of myocardial blood perfusion. MCE-estimated MBF correlates closely with the standard method of microsphere-derived MBF and can be used to measure murine coronary reserve and coronary responses to infused vasodilators. MCE showed that NOS3–/– mice have a preserved adenosine response, which suggests that NOS3 is not required for adenosine-mediated coronary vasodilation. NOS3–/– mice had an impaired MBF response to acetylcholine, which underscores the crucial role of NOS3 in acetylcholine-induced coronary vasodilation. Murine MCE can be used in future studies with other genetically modified mice to further our understanding of resting and stimulated MBF responses.
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Acknowledgments |
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We thank Dr A.E. Weyman for his thoughtful review of the paper and Hélène Houle, Barbara Conwell, Dr Edward Gardner, and Larry Brennan from Siemens for their technical support.
Sources of Funding
The present study was supported by an American Heart Association Scientist Development Grant (Dr Scherrer-Crosbie), the Fédération Française de Cardiologie (Dr Thibault), the National University Hospital, National Healthcare Group, Singapore and a fellowship award from the National Medical Research Council, Singapore (Dr Poh), as well as grants from the National Heart, Lung, and Blood Institute Public Health Service (HL-42397, HL-70896, HL-71987).
Disclosures
None.
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*The first 2 authors contributed equally to this work.
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