Original contributionEffects of Isoflurane on Coronary Blood Flow Velocity in Young, Old and ApoE−/− Mice Measured by Doppler Ultrasound
Introduction
The noninvasive measurement of coronary blood flow by ultrasound has been notoriously difficult in both man and animals because coronary arteries are small, highly branched, lie deep within the chest and are in constant motion because of their attachment to the epicardial surface. Thus, measurements of coronary flow or velocity have required the use of invasive methods such as implantable flow probes (Hartley and Cole 1974) or coronary catheters (Cole and Hartley 1977, Sibley et al 1986, Wilson et al 1985). In addition, coronary blood flow (even if it could be measured accurately) is often normal at rest even in the presence of severe coronary artery disease (Gould et al 1974, Marcus et al 1981). This problem is commonly addressed by administering a coronary vasodilator such as adenosine (Hoffman 1984, Marcus 1983) to increase blood flow and then measuring the ratio of maximum hyperemic flow to resting baseline flow as an index of coronary vascular reserve (Gould et al. 1974). Coronary vascular reserve has been shown to be reduced in the presence of coronary lesions because of a reduction in hyperemic flow (Marcus et al. 1981) and by other cardiac pathologies because of an increase in baseline flow (Marcus et al 1982, Marcus 1983).
The apolipoprotein-E–deficient mouse (ApoE−/−) is a commonly used model of atherosclerosis because the arterial lesions resemble those found in humans at certain stages of the disease (Nakashima et al 1994, Paigen et al 1987). Although the atherosclerotic lesions are progressive and may become severe, their functional effect on blood flow and physiology is largely unknown. Cardiovascular reserve relevant to exercise performance is reduced (Niebauer et al. 1999) and we have previously shown a 55% increase in aortic and mitral flow velocities and a 59% increase in heart-weight to body-weight ratio at one year of age (Hartley et al. 2000) consistent with volume overload hypertrophy. However, the effect and significance of systemic and coronary arterial lesions and of cardiac hypertrophy on coronary blood flow and coronary flow reserve in ApoE−/− mice is unknown.
The administration of a specific and maximal coronary vasodilator such as adenosine (Wikstrom et al. 2005) is necessary to evaluate coronary flow reserve, and this is much more difficult and problematic in mice where the veins are more difficult to cannulate and the tolerated doses and volumes are much smaller. Fortunately, one of the most widely used anesthetic agents (isoflurane gas) is also a coronary vasodilator when administered at higher concentrations (Crystal 1996, Gamperi et al 2002, Reiz et al 1983, Zhou et al 1998). The use of an inhaled coronary vasodilator, if effective and well tolerated, would greatly simplify the estimation of coronary flow reserve in mice and make the procedure truly noninvasive and amenable to high throughput. Although it is known to lower systemic blood pressure (Zuurbier et al. 2002), isoflurane is now the preferred anesthetic for performing cardiovascular studies in mice because it has minimal effects on heart rate when compared with other nonvolatile agents (Jannsen et al. 2004). However, the ability of isoflurane gas to dilate coronary arteries and increase coronary blood flow is often unrecognized, and the required concentrations and responses are undocumented in mice.
During the last decade there have been several reports on the use of noninvasive Doppler ultrasound to estimate coronary flow reserve in man using adenosine (Neishi et al. 2005), dipyridamole (Galderisi et al 2004, Santagata et al 2005, Saraste et al 2001) or dobutamine (Cicala et al. 2004) to increase coronary flow. However, because it is difficult to locate and identify specific coronary lesions, these methods have not achieved wide acceptance and are not routinely used in assessing the relationship between anatomy and functional significance in patients with coronary artery disease (Rigo 2005, Voci et al 2004). Recently, there have also been reports showing coronary flow velocity signals recorded from mice using conventional echocardiography machines (Gan et al 2005, Wikstrom et al 2005) or a high-frequency scanner designed specifically for mice (Zhou et al. 2004). Although the signals shown were identifiable and quantifiable, they were often of suboptimal quality and fidelity. This encouraged us to test the feasibility of using a smaller and more focused Doppler probe to measure coronary flow velocity and to estimate reserve in a mouse model of atherosclerosis (Plump et al 1992, Wang 2005) where we hypothesized that the presence of lesions in the left main coronary artery (Wikstrom et al. 2005) coupled with volume overload hypertrophy (Hartley et al. 2000) would reduce coronary flow reserve. Thus, we report here a noninvasive method using Doppler ultrasound (Hartley et al. 2000) to measure left main coronary flow velocity and the response to low and high levels of inhaled isoflurane in young and old wild-type, and old (age-matched) ApoE−/− mice (Bernard et al 1992, Crystal 1996). We use the response to isoflurane to document systematic differences in baseline and hyperemic coronary flow velocity among the groups and to demonstrate large variations in baseline and hyperemic coronary artery velocities in ApoE−/− mice, likely because of the presence of stenotic coronary artery lesions.
Section snippets
Animal protocol
Three groups of mice (n = 40) were studied following a protocol approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. The groups consisted of 10 young C57BL6 wild-type (“Young”), 10 old ApoE+/+ wild-type (“Old”) and 20 old ApoE−/− (“ApoE−/−”) knockout mice. The 2-y-old ApoE−/− mice and their wild-type age-matched controls were derived from a C57BL6 background and were obtained from a colony at Berlex Biosciences (Richmond, CA, USA). All mice were
Results
Coronary flow velocity signals were obtained in all animals under baseline and hyperemic flow conditions. As illustrated in Fig. 2, the signal-to-noise ratio and fidelity of coronary velocity signals from mice were similar in quality to signals from other vessels such as the ascending aorta and carotid arteries. To generate this illustration two additional Doppler probes and modules were used to record all signals simultaneously (Hartley et al. 1978). The velocity signals from the left main
Discussion
In this report we introduced a simple and noninvasive method based on Doppler ultrasound to measure coronary flow velocity in mice under B and H conditions created by changing the concentration of isoflurane gas and documented differences because of age and atherosclerosis. The ratio of hyperemic to baseline velocity (H/B) is lower in Young mice than in Old mice, and it is lower in two-year old ApoE−/− mice than in two-year old wild-type mice. We were able to obtain adequate coronary velocity
Conclusions
We conclude that coronary flow velocity can be measured noninvasively in mice with Doppler ultrasound and that isoflurane gas can be used as a convenient and noninvasive coronary vasodilator. Our data suggest that the ratio of hyperemic to baseline coronary blood velocity can be a valuable index of coronary reserve despite the fact that isoflurane may not be a maximal coronary vasodilator. The differences noted in the ratio of hyperemic to baseline coronary blood velocity in Young, Old and ApoE
Acknowledgments
We acknowledge Dr. Y-X Wang of Berlex Biosciences for supplying the ApoE−/− and age-matched Old mice, Thuy Pham and Jennifer Pocius for technical assistance and James Brooks for editorial assistance.
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2013, Ultrasound in Medicine and BiologyCitation Excerpt :Second, heart rate was not controlled as well as desired, which could have influenced the flow values in the LMCA. Third, isoflurane can cause coronary artery vasodilation and increase coronary artery flow in mice (Hartley et al. 2007), which may influence the velocity level of the coronary artery. Fourth, an angle of 15° between the ultrasound beam and the blood flow results in underestimation of the true velocity by about 3.5% (Hartley et al. 2007).
This work was supported in part by National Institutes of Health Grants R01-HL22512, P01-HL42550, R01-AG17899, R41-HL76928 and K25-HL73041.