Abdominal aortic aneurysms (AAA) localize in the infrarenal aorta in humans, while they are found in the suprarenal aorta in mouse models. It has been shown previously that humans experience a reversal of flow during early diastole in the infrarenal aorta during each cardiac cycle. This flow reversal causes oscillatory wall shear stress (OWSS) to be present in the infrarenal aorta of humans. OWSS has been linked to a variety of proatherogenic and proinflammatory factors. The presence of reverse flow in the mouse aorta is unknown. In this study we investigated blood flow in mice, using phase-contrast magnetic resonance (PCMR) imaging. We measured blood flow in the suprarenal and infrarenal abdominal aorta of 18 wild-type C57BL/6J mice and 15 apolipoprotein E (apoE)−/− mice. Although OWSS was not directly evaluated, results indicate that, unlike humans, there is no reversal of flow in the infrarenal aorta of wild-type or apoE−/− mice. Distensibility of the mouse aortic wall in both the suprarenal and infrarenal segments is higher than reported values for the human aorta. We conclude that normal mice do not experience the reverse flow in the infrarenal aorta that is observed in humans.
- abdominal aortic aneurysms
- magnetic resonance imaging
- wall shear stress
differences exist between blood flow patterns in the suprarenal and infrarenal abdominal aorta of humans. A reversal of blood flow in early diastole is present in the infrarenal aorta of young, healthy humans, causing the flow waveform to be triphasic over the cardiac cycle (forward in systole—reverse in early diastole—forward in late diastole) (20). In contrast, flow in the suprarenal aorta is biphasic, with little or no reverse flow over the cardiac cycle (Fig. 1). The triphasic flow pattern has been attributed to the low resistance of the renal vasculature and the high capacitance and high resistance of the infrarenal vessels that serve the lower limbs (11). Although wall shear stress (WSS) and flow are not directly related, this triphasic flow pattern tends to cause a reduction of the mean WSS and an increase in the level of oscillatory wall shear stress (OWSS) in the infrarenal aorta. Low mean WSS as well as high levels of OWSS have been linked to a variety of proatherogenic conditions including increased intimal thickness (20, 22), endothelial dysfunction, increased expression of adhesion molecules, and increased levels of oxidative stress in cultured human endothelial cells (16, 20).
Both atherosclerosis and abdominal aortic aneurysm (AAA) exhibit a preference for developing in certain locations within the vascular tree. Although evidence is not conclusive, atherosclerosis tends to develop near bifurcations, branch points, and areas of curvature, where WSS is low or oscillatory (3, 16, 20). In humans, atherosclerotic lesions are more prevalent in the infrarenal aorta than the suprarenal aorta. Approximately 90% of all AAAs are associated with atherosclerosis, and ∼95% of AAAs develop in the infrarenal aorta, where mean WSS is low and WSS is oscillatory over the cardiac cycle. The suprarenal aorta is remarkably spared from AAA development (1, 17). Thus the localization of atherosclerosis and AAA to the infrarenal aorta may be partially explained by biomechanical effects of low or oscillatory WSS.
In contrast to humans, less is known about the flow patterns in the mouse aorta. Studies of the murine aorta have shown a biphasic flow pattern in the aortic arch and the suprarenal aorta (8, 27). Flow patterns in the infrarenal aorta have not been reported. Also, the location of AAA formation in the mouse differs markedly from that in the human. In all current mouse AAA models, murine AAA form exclusively in the suprarenal aorta (Fig. 2) (5).
The purpose of the present study was to characterize the hemodynamic environment of the suprarenal and infrarenal abdominal aorta of normal and apolipoprotein E (apoE)−/− mice with in vivo phase-contrast magnetic resonance (PCMR) velocity measurements. We hypothesized that there would be a biphasic flow pattern (i.e., absence of flow reversal) in the infrarenal aorta of mice, in contrast to what is seen in humans. We based our hypothesis on two factors. The first factor was the difference that is seen in anatomic localization of AAA development between mice and humans, and the second was the knowledge that oscillatory and low mean WSS create an environment prone to vascular pathology.
MATERIALS AND METHODS
The protocol used to obtain the human images was approved by Emory's Institutional Review Board, and informed consent was obtained from the research subject. The study protocol was reviewed and approved by Emory University's Institutional Animal Care and Use Committee.
We developed a PCMR imaging sequence and processing software to acquire cine velocity maps of blood flow in the mouse aorta. In vivo mouse imaging was performed with a 4.7-T Varian INOVA MRI scanner (Varian, Palo Alto, CA) with a 37-mm-diameter, 16-element birdcage quadrature coil. ECG gating was used to acquire 10 frames equally spaced over the cardiac cycle with the R wave as the trigger for image acquisition. This sequence provided cine images that allowed both measurement of blood flow velocity and assessment of aortic wall motion. Temporal resolution was 12–15 ms depending on heart rate. In-plane resolution of the sequence was 68 × 68 μm, and the slice thickness was 1 mm (Fig. 3). This resolution provided for 12–18 pixels across the mouse aorta, depending on the size of the aorta.
The PCMR imaging sequence was first validated on a set of tubes in which the flow rate was known. A gravity-fed reservoir flow system was used to generate steady flow. Timed collections in a graduated cylinder were acquired to compare actual flow and velocity values to those measured from the PC-MRI scans. Tubes ranged in diameter from 1 to 4 mm, and flow velocities ranged from 60 to 120 cm/s.
Mouse imaging protocol.
The PCMR sequence described above was used to obtain velocity measurements in the suprarenal and infrarenal aorta of 18 normal, wild-type C57BL/6J mice and 15 apoE−/− mice. The mice were anesthetized with 1.8% isoflurane inhaled anesthetic, and the ECG leads were placed on the arms and left leg. First, a transverse slice positioned in the superior abdominal cavity was obtained with an ECG-gated gradient-echo sequence. A stack of coronal and sagittal slices passing through the long axis of the aorta was planned from this axial image. Coronal slices in which the kidneys were visible were used for the positioning of the suprarenal and infrarenal velocity measurements (Fig. 4). The superior pole of the right kidney and the inferior pole of the left kidney were used as landmarks for the positioning of the suprarenal and infrarenal velocity images. The imaging plane was positioned perpendicular to the aorta on the sagittal and coronal images.
Postprocessing and analysis.
Average cross-sectional velocity values were obtained by averaging the velocity values over the lumen for each frame imaged during the cardiac cycle with Image J (National Institutes of Health, Bethesda, MD). User-defined contour lines on the magnitude and phase images were used to identify vessel boundaries. An average velocity value as well as an instantaneous flow rate was obtained in this manner for each of the 10 frames during the cardiac cycle in both the suprarenal and infrarenal aorta. By integrating the velocity values over the blood vessel lumen, a flow for each time point in the cardiac cycle was obtained. Integrating the instantaneous flow values over the cardiac cycle provided volumetric flow value over the cardiac cycle.
Background phase offsets from field inhomogeneities or eddy currents can introduce errors in velocity measurements. To ensure we did not have such an offset in our measurements, we obtained values of the velocity of the background stationary tissue in the paraspinal muscle tissue and verified that these were zero.
We used the magnitude images from our PC-MRI to segment the luminal cross-sectional area of the aorta and obtain estimates of its distensibility expressed as the percent change in area over the cardiac cycle due to blood pressure changes. The distensibility of the aorta was calculated as [(Dmax − Dmin)/Dmin] × 100, where Dmax and Dmin are the maximum and minimum diameters of the aorta, respectively, during the cardiac cycle.
Results of the validation experiments indicated excellent correlation between PCMR flow measurements and timed collections (r2 = 0.97).
Mouse velocity and flow measurements.
Suprarenal and infrarenal velocity measurements were successfully obtained in 18 of 20 C57BL/6J mice and 15 of 15 apoE−/− mice. Two of the 20 C57BL/6J mice died before the scan could be completed. Table 1 shows the results of our morphometric and hemodynamic measurements in the mice.
Both velocity and flow in the infrarenal aorta were higher in C57BL/6J mice than in apoE−/− mice. Higher velocity and flow were also seen in the suprarenal aorta of C57BL/6J mice compared with apoE−/− mice. The higher flow may be due to the larger size of the aorta in C57BL/6J mice compared with apoE−/− mice (Table 1). Cardiac output was not measured in these mice, so it is unknown whether the velocity and flow differences are due to higher cardiac output in C57BL/6J mice.
Average cross-sectional blood velocities are similar in the suprarenal and infrarenal aortic segments in both C57BL/6J mice and apoE−/− mice, but flow is lower in the infrarenal aorta since the area of the vessel is smaller (P < 0.01) (Table 1).
Figures 5 and 6 show the flow of the suprarenal and infrarenal aorta averaged over 18 wild-type C57BL/6J mice and 15 apoE−/− mice, respectively. There was no evidence of triphasic blood flow in either suprarenal or the infrarenal aorta. No flow reversal was seen at any time during the cardiac cycle in any of the mice. Unlike the triphasic (forward-reverse-forward) waveform seen in the infrarenal aorta of humans (Fig. 1), the infrarenal blood flow of mice is biphasic. The suprarenal biphasic aortic flow pattern is similar to what is seen in humans.
Distensibility of the aortic wall in the suprarenal and infrarenal segments, 72 ± 26% (mean ± SD) and 63 ± 18%, respectively, was similar in the normal mice (P = 0.08). These percent changes in area are significantly greater then their analogous values in humans (12), indicating that a relatively high amount of elasticity exists in these vessels compared with humans.
In this study we found that there was no evidence of triphasic flow reversal in the infrarenal aorta of either normal C57BL/6J or apoE−/− mice. Mice have biphasic flow in both the suprarenal and infrarenal aorta. In addition, the distensibility of the mouse aorta was greater than that of the human aorta.
Human AAA develop almost exclusively inferior to the renal arteries. Two of the most widely used mouse models of AAA are apoE−/− or LDL receptor−/− mice infused with angiotensin II (ANG II) (5). Both of these models exhibit AAA formation superior to the renal and celiac arteries, a distinctly different location from those seen in humans. Except for an elastase-induced aneurysm model (which shows aneurysmal dilatation at the site of injection), there are no other mouse models of aneurysms that form AAA below the renal arteries (5). The lack of reverse flow in the infrarenal aorta during early diastole in the mouse aorta will provide a hemodynamic environment that will tend to reduce or eliminate OWSS in that vascular segment. A reduction in OWSS may help explain why AAA do not form in the murine infrarenal aorta. The reasons why AAA may form in the suprarenal aorta of mice is not clear. This is an area that requires further investigation and likely will require a complete computational fluid dynamic (CFD) simulation. Although a plethora of studies have linked low and oscillatory WSS to proatherogenic events in the cellular constructs and model studies, there have been in vivo studies in which this link has not been shown to exist (14, 26, 28).
The mouse has become a commonly studied animal model for many cardiovascular diseases such as atherosclerosis and aneurysm development. However, differences exist between many of these human diseases and their murine counterparts. Investigating these differences is a crucial part of determining the initiating factors in the pathogenesis of these diseases. One of these differences is the large disparity in mean WSS values that is seen between humans and mice. Aortic blood velocities in mice and humans are close in value (21). The smaller diameter of the mouse aorta causes WSS to be higher. Humans tend to exhibit a mean WSS value of 5–10 dyn/cm2 (21), while values in mice have been found to be much higher, ∼45–50 dyn/cm2 (8, 9, 27). The flow values found in the present study support the finding of high WSS values in mice compared with humans.
Five percent of human AAA form in the suprarenal aorta. Although this is a relatively small number, this is a vascular segment that does not exhibit a triphasic flow pattern. Therefore, clearly there are other mitigating factors that contribute to AAA development. In this small minority of cases it is also possible that there exist circumstances that allow for the reversal of flow to extend into the suprarenal arteries. For example, pathological events causing a patient's renal vasculature pressure to increase to a higher level or changes in vascular distensibility may affect suprarenal flow patterns.
To our knowledge, this is the first time evidence has been presented on the absence of reversal of blood flow in the infrarenal aorta of mice. The triphasic blood flow in the infrarenal aorta of humans has been explained by both the low resistance of the renal circulatory system as well as the high capacitance and resistance of the blood vessels that supply the lower limbs (11). However, similar reasoning may not be sufficient to explain the lack of a triphasic flow pattern in mice. First, it is unlikely that the relative resistance of the murine renal vasculature is much higher than that of the human renal vasculature because they are both governed by similar physiological principles and serve similar functions in both mice and humans. One may argue that the overall capacitance of the lower limb vessels in mice is lower than that in humans because the relative total vascular volume in the lower limbs of mice is low. This reduced lower limb vascular volume may prevent the backflow of blood from the lower limbs into the infrarenal aorta during diastole. Although the results noted above show high elasticity of the abdominal aorta during the cardiac cycle, capacitance of the lower limb vessels is determined by the total volume of blood held within the vessels from systole to diastole. Therefore, capacitance will depend on the relative volume of the vessels below the renal arteries and the elasticity of these vessels, which we have not explicitly measured here. If the volume of blood in the vessels below the renal arteries is small, the capacitance of these vessels may also be small despite high elasticity.
The underlying biochemical triggers in the formation of atherosclerotic plaque and aneurysms have been hypothesized to be very similar. Shimizu et al. (25) have proposed that in persistent inflammatory conditions one of the first triggers in both atherosclerosis and aneurysm development is increased T-helper cell type 1 (Th1) cytokine signaling, mainly IFN-γ. These increased levels of inflammatory cytokines activate chemokines and adhesion molecules and further recruit more inflammatory cells, leading to an atheroocclusive aorta. From this point forward, a set of second triggers that include both biochemical and environmental factors (e.g., smoking) act to develop AAA.
Another similarity in biochemical factors in AAA and atherosclerosis is the renin-angiotensin system, which is now known to be an important therapeutic target of atherosclerotic vascular disease (2, 7). ANG II induces the production of reactive oxygen species and stimulates the expression of adhesion molecules [vascular cell adhesion molecule-1 (VCAM-1)] and chemokines [monocyte chemoattractant protein-1 (MCP-1)] (2, 7, 10). Infusion of ANG II in hypercholesterolemic mice dramatically accelerates the atherosclerotic process, leading to the development of extensive atherosclerotic plaques and AAA (6, 29). Furthermore, by blocking the pathway that is thought to mediate the effects of ANG II aortic inflammatory changes as well as lipid accumulation were markedly attenuated (13). Therefore it is not surprising that almost 90% of AAA are associated with atherosclerosis. However, the fact that most plaques do not lead to the development of AAA argues that there are other mitigating factors. We believe biomechanics, specifically low and or oscillatory WSS, may be one of these mitigating factors.
Murine velocity and flow values measured in our study agree well with previously published values. Mean flow in the ascending aorta was measured at ∼20 ml/min (8), which matches well with our value of 15.3 ml/min in the suprarenal aorta. As expected, our values should be lower since we measured flow more distally in the vascular tree. The mean infrarenal flow that we obtained in C57BL/6J mice (7.5 ml/min) was greater than previously published data from Greve et al. (9), who measured mean infrarenal flow at 2.8 ml/min. We believe this can be accounted for by differences in the positioning of the PCMR imaging slice either above or below branching vessels in the abdomen.
There are limitations to our study. First, the absence of reverse flow does not necessarily imply that there is an absence of WSS oscillation. WSS is determined from the gradient of the velocity profile at the wall. Therefore, the bulk flow (determined by integrating the velocity over the entire vessel cross section) could be going forward but at specific points near the wall velocity could be reversed, causing negative WSS. Such a velocity profile is predicted under certain conditions by the Wormersley solution to unsteady flow in a circular tube (18, 19). We examined the velocity profiles and did not see this type of velocity profile in any mice. However, because of the fairly poor spatial resolution of the velocity profile (10 pixels across vessel), we cannot exclude this effect. A complete fluid-solid interaction (FSI)-based CFD model based on the MR geometry, vessel motion, and PCMR flow images would be required to fully explore this issue (4, 15, 23, 24).
Another limitation is that we made measurements in only two locations in the aorta and have extrapolated the measurements from the suprarenal aortic location to the entire thoracic aorta and the measurements from the infrarenal aortic location to the entire abdominal aorta.
In conclusion, we found no evidence of reversal of blood flow in the infrarenal aorta of wild-type C57BL/6J or apoE−/− mice, and this is an important difference in blood flow patterns between humans and mice. This reversal of flow occurs during each cardiac cycle and may increase oscillatory shear stress in the human infrarenal aorta, the segment where AAA are prominent in humans. In mice this same segment is spared from AAA formation, and here we have presented evidence that a similar reversal of flow does not exist in mice. Further investigations into the effect of blood flow reversal on the localization of AAA and/or plaque are warranted.
- Copyright © 2009 American Physiological Society