Effects of Isoflurane on Coronary Blood Flow Velocity in Young, Old and ApoE^−/− Mice Measured by Doppler Ultrasound

Abstract

The commonly used anesthetic agent isoflurane (ISO) is a potent coronary vasodilator that could potentially be used in the assessment of coronary reserve, but its effects on coronary blood flow in mice are unknown. Coronary reserve is reduced by age, coronary artery disease and other cardiac pathologies in man, and some of these conditions can now be modeled in mice. Accordingly, we used Doppler ultrasound to measure coronary flow velocity in mice anesthetized with low (1%) and high (2.5%) levels of ISO to generate baseline (B) and elevated hyperemic (H) coronary flows, respectively. A 20-MHz Doppler probe was mounted in a micromanipulator and pointed transthoracically toward the origin of the left main coronary arteries of 10 6-wk (Young [Y]), 10 2-y (Old [O]) and 20 2-y apolipoprotein-E null (ApoE^−/−) atherosclerotic (A) mice. In each mouse, we measured (B) and (H) peak diastolic velocities. B was 35.4 ± 1.4 cm/s (Y), 24.8 ± 1.6 (O) and 51.7 ± 6.4 (A); H was 83.5 ± 1.3 (Y), 86.5 ± 1.9 (O) and 120 ± 16.9 (A) and H/B was 2.4 ± 0.1 (Y), 3.6 ± 0.2 (O) and 2.5 ± 0.2 (A). The differences in baseline velocities and H/B between O and Y and between A and O were significant (p < 0.01), whereas the differences in hyperemic velocities were not (p > 0.05). H/B was higher in old mice as a result of decreased baseline flow rather than increased hyperemic flow velocity. In contrast, ApoE^−/− mice have increased baseline and hyperemic velocities, perhaps because of coronary lesions. The differences in baseline velocities between young and old mice could be the result of age-related changes in basal metabolism or to differential sensitivity to isoflurane. We conclude that Doppler ultrasound combined with coronary vasodilation via isoflurane could provide a convenient and noninvasive method to estimate coronary reserve in mice, but also that care must be taken when assessing coronary flow in mice under isoflurane anesthesia because of its potent coronary vasodilator properties. (E-mail: [email protected])

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.