Online Issues

Journal Vol 49 (1)

<< All Back-issues
<< This Issue's Table of Contents

Introduction to Rodent Cardiac Imaging

Kennita Johnson

Kennita Johnson, PhD, is a biomedical engineering postdoctoral fellow in the Laboratory of Experimental Pathology at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina.

Address correspondence and reprint requests to Dr. Kennita Johnson, Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, 111 Alexander Drive, Research Triangle Park, NC 27709 or email johnso58@niehs.nih.gov.

Abstract

Imaging is a noninvasive complement to traditional methods (such as histology) in rodent cardiac studies. Assessments of structure and function are possible with ultrasound, microcomputed tomography (microCT), and magnetic resonance (MR) imaging. Cardiac imaging in the rodent poses a challenge because of the size of the animal and its rapid heart rate. Each aspect in the process of rodent cardiac imaging—animal preparation, choice of anesthetic, selection of gating method, image acquisition, and image interpretation and measurement—requires careful consideration to optimize image quality and to ensure accurate and reproducible data collection. Factors in animal preparation that can affect cardiac imaging are the choice of anesthesia regime (injected or inhaled), intubated or free-breathing animals, physiological monitoring (ECG, respiration, and temperature), and animal restraint. Each will vary depending on the method of imaging and the length of the study. Gating strategies, prospective or retrospective, reduce physiological motion artifacts and isolate specific time points in the cardiac cycle (i.e., end-diastole and end-systole) where measurements are taken. This article includes a simple explanation of the physics of ultrasound, microCT, and MR to describe how images are generated. Subsequent sections provide reviews of animal preparation, image acquisition, and measurement techniques in each modality specific to assessing cardiac functions such as ejection fraction, fractional shortening, stroke volume, cardiac output, and left ventricular mass. The discussion also includes the advantages and disadvantages of the different imaging modalities. With the use of ultrasound, microCT, and MR, it is possible to create 2-, 3-, and 4-dimensional views to characterize the structure and function of the rodent heart.

Key Words: animal preparation; cardiac imaging; gating; microcomputed tomography; magnetic resonance; rodent; ultrasound

Introduction

Imaging technologies such as ultrasound, microcomputed tomography (microCT¹), and magnetic resonance (MR¹) are noninvasive methods to determine cardiac function in murine models of heart disease. Ultrasound provides real-time assessment of cardiac function, and both MR and microCT can provide 3-dimensional (3D) and 4-dimensional (4D) views of the rodent heart (4D imaging is based on 3D images that change over time—for example, a 3D image of a beating heart).

In addition to anatomical views, each of the three modalities can assess function by measuring cardiac parameters. Fractional shortening (FS%) measures the percent change in the left ventricle (LV¹) dimension from end-diastole to end-systole. The ejection fraction (EF%), the fraction of the end-diastolic volume that is ejected per beat, measures contractility; a reduction in EF% suggests that the ventricle is excessively dilated (Wyngaarden and Smith 1982). The stroke volume is the difference between the end-diastolic and end-systolic volumes. Cardiac output, the amount of blood pumped by the left ventricle into the aorta each minute, is the product of the stroke volume and the heart rate (Guyton 1981). The heart rate is the number of beats per minute—approximately 300 in a healthy rat and 500 in a healthy mouse (Nahrendorf et al. 2003). Heart rate can be determined through electrocardiography or from the ultrasound image. Left ventricular mass is generally calculated as the difference between the epicardial and endocardial regions of the heart multiplied by an estimate of myocardial density (Kiatchoosakun et al. 2002). Left ventricular hypertrophy can be measured as an increased LV mass (Devereux et al. 2004) and may be physiological or pathological.

Each modality has its advantages and disadvantages, which warrant careful consideration when planning an imaging study. This article reviews the practical aspects of rodent cardiac imaging, including animal preparation considerations such as anesthesia, physiological monitoring, and image gating, which is necessary to eliminate motion artifacts. The sections on ultrasound, microCT, and MR include simplified explanations of the imaging physics for each, as well as the cardiac function measurements that are possible with each modality.

Animal Preparation

The preparation of the animal can increase—or compromise—image quality and throughput of small animal imaging studies (McConville et al. 2005). Anesthesia provides a consistent and controlled setting for rodent imaging studies. The ideal anesthesia for rodent imaging studies is easy to administer, reproducible, and rapid in onset and recovery; in addition, it causes minimal heart rate and cardiac depression and has a low toxicity (Roth et al. 2002). Two types of anesthesia commonly used in imaging studies are injectable (ketamine/xylazine) and inhaled (isoflurane or halothane) (Roth et al. 2002). Intraperitoneal (i.p.) injections of ketamine/xylazine require 5 to 20 minutes after injection to produce an adequate depth of anesthesia, which then lasts 30 to 60 minutes in healthy mice (Chaves et al. 2001). The depth of anesthesia with injectable drugs continuously decreases as the drug is metabolized (Lukasik and Gillies 2003). Ketamine/xylazine can cause bradycardia and hypotension in mice and has the greatest cardiac depression of the injectable anesthetics tested (Roth et al. 2002). Hypersalivation can occur with the use of ketamine (Lukasik and Gillies 2003).

For procedures that last longer than 30 to 45 minutes, inhaled anesthesia is preferable to injectable drugs (Lukasik and Gillies 2003). Inhaled anesthetics are easier to control and provide more stable and physiologically relevant heart rates (approximately 300 for a rat and 500 for a mouse) (Chaves et al. 2001); isoflurane, for example, produces minimal cardiac depression (Roth et al. 2002). A general rule to maximize recovery of the animal is to limit gas anesthesia to 3 hours or less. Longer image acquisition times under anesthesia risk compromising the animal's health (Chen 2006). In addition, sedated and anesthetized animals do not completely close their eyelids and so are at risk of corneal desiccation and ulceration. It is advisable to protect their eyes with sterile eye-lubricating ointment, especially in long-duration studies (Lukasik and Gillies 2003).

The effects of anesthesia may pose challenges in the image interpretation (Balaban and Hampshire 2001). For example, anesthetic choice has a profound effect on cardiovascular parameter estimation in mice; Chaves and colleagues (2001) found statistically significant differences in heart rate, cardiac output, and LV fractional shortening between injectable and inhaled anesthetics, despite a comparable depth of anesthesia. In transgenic animals, the phenotype might be expressed as an enhanced sensitivity to anesthesia, with minimal changes to the wild-type (Balaban and Hampshire 2001); and both the type of anesthesia and the timing of echocardiographic measurement after anesthesia can also have significant effects on cardiac parameters (Roth et al. 2002).

It is important to monitor the electrocardiogram (ECG¹), respiration, and body temperature during an experimental procedure on an anesthetized animal (Hartley et al. 2002). An ECG measurement in a rodent requires consideration of the animal's position, lead attachment(s), number of leads, noise suppression, and amplifier bandwidth (Hartley et al. 2002). ECG lead attachments can be made with copper tape, ECG pads, or silver needle electrodes, typically attached to the paws. The application of ECG gel improves the electrical contact between the leads and the animal (Chen 2006). The conventional method of cardiac gating, described in the next section, involves triggering image acquisition from the ECG signal (Heijman et al. 2007). But significant alterations occur in heart rate and cardiac function with changes in body temperature (Hartley et al. 2002). It is therefore useful to monitor the animal's body temperature, for example with a rectal probe (Chen 2006). Heat lamps or heating pads are commonly used to warm the anesthetized animal. However, heating devices can add noise to ECG signals (Hartley et al. 2002). When monitoring the animal's physiological functions during MR imaging, all connections and equipment near the magnet must be nonmagnetic.

After anesthesia and physiological monitoring, the animal should be restrained to reduce motion artifacts as well as to properly position it for the imaging examinations. Animal immobilization methods include bite bars, immobilizing splints, cradles, and medical tape (McConville et al. 2005). Cradles and immobilizing splints are effective for positioning the animal in the same location in the imaging field for repeated examinations in longitudinal studies. Schneider and colleagues (2003, 2004) used a cradle outfitted with a nose cone and tubing connections for delivering and scavenging the anesthetic gases, as well as equipment for controlling the animal's temperature and for ECG and respiratory monitoring. The animals were secured to the cradle with surgical tape, without compression of the abdomen or chest region (Schneider et al. 2004). The animal should be under anesthesia if restricted in this manner to reduce stress, which can confound the cardiac function under investigation.

Gating

Although the animal is anesthetized and immobilized during the examination, physiological (cardiac and respiratory) motion can produce artifacts in the images. "Gating" reduces these motion artifacts through the synchronization of the image acquisition with the cardiac cycle, and makes it possible to capture images at the same point in the cardiac cycle. This is a beneficial feature in cardiac imaging, where end-systolic and end-diastolic measurements are key.

Badea and colleagues (2005) described a prospective gating sequence that corresponds to the R peak in the ECG cycle. An acquisition window of 100 milliseconds is defined at end-expiration in the ventilation cycle and any QRS occurring within that window triggers an x-ray exposure. With this method, the animal is intubated and placed on a special-purpose mechanical ventilator that makes it possible to ventilate the animal at a controlled rate, provide the oxygen and anesthetic gas mixture, and measure the airway pressure with a pressure transducer in the breathing valve. This airway pressure, along with the ECG, informs the gating sequence (Badea et al. 2005).

Intubating rodents is difficult because of the small size of the trachea and therefore requires skill to avoid tracheal damage, which may make future intubations more difficult (Ford et al. 2005). Ford and colleagues (2005) have described an approach for prospectively gating free-breathing rodents, which do not require intubation: an external pressure sensor on the animal's abdomen produces the respiratory signal. But this method is problematic when the respiration rate is unstable, for example if the animal is breathing shallowly or taking deep sighs.

In retrospective gating, image data collection occurs continuously with the corresponding physiological data and the projection data are retrospectively sorted based on where the images fell in the cardiac cycle (Drangova et al. 2007). Drangova and colleagues (2007) used retrospective gating with a microCT system to reconstruct 3D images that correspond to specific time points in the cardiac cycle. They performed this technique on free-breathing animals, thus eliminating the need for intubation.

The advantage of retrospective rather than prospective gating is that it enables the imaging of all time points in a cardiac cycle during one scan, analogous to human imaging (Drangova et al. 2007). This gating technique also makes possible the simultaneous imaging of multiple mice, which is difficult with prospective gating and triggering because the cardiac motion of different animals is completely asynchronous (Bishop et al. 2006; Drangova et al. 2007).

One disadvantage of retrospective gating is less motion control than in the prospective strategies (Bishop et al. 2006). Gating from the ECG can be difficult in the MR environment due to gradients, radiofrequency (RF¹) pulses, and magnetic fields, which can interfere with the ECG signal. Either fiber-optic or analog filters can help reduce interference from MR gradients (Chen 2006). Sabbah and colleagues (2007) made a real-time gating system that used digital low-pass filtering with automatic trigger-level adjustment to compensate for the MR effects on the ECG signal.

Ultrasound

Ultrasound refers to sound waves that are not detectable by human ears (frequencies greater than 20,000 cycles/second). The basic components of an ultrasound system are a transducer, a computer, and a monitor; the latter two can be placed on a cart, which makes the system portable. The ultrasound transducer generates sound waves that propagate through the body at a transmission velocity of 1540 m/s. The acoustic impedance is the product of the transmission velocity and the tissue density. At the boundary between differing tissue densities, there is an acoustic mismatch and some of the sound waves are reflected. The transducer detects these reflected sound waves, or "echoes," and the computer converts the signals to images for display on the monitor. The greater the acoustic mismatch, the more sound waves are reflected to the transducer and the brighter the image (Coatney 2001). To overcome the acoustic mismatch that results from the small amount of air between the transducer and the animal's skin, the application of gel on the skin "couples" the transducer to the body.

Ultrasound has three imaging modes. The first is brightness (b-mode), a gray-scaled, 2D cross-section view of the target organ moving in real time and with spatial resolution on the order of 50 μm². Figure 1A is a representative b-mode image of the mouse heart. The second imaging mode is motion (m-mode). For an m-mode echocardiogram, for example, a single ultrasound beam is transmitted through the heart and the resulting image is displayed over time, creating a strip chart of cardiac motion (Curry et al. 1990). Figure 1B is a representative m-mode image of the mouse heart. The third imaging mode is Doppler, which is used to determine the speed and direction of blood flow: as blood moves away from or toward the transducer, there is a frequency shift (the Doppler shift) in the reflected waves. This shift is affected by the alignment of the ultrasound beam and the flowing blood (Coatney 2001).

Figure 1 (A) A b-mode ultrasound image of a parasternal short-axis view of a mouse heart. The dotted line indicates where the ultrasound beam will create the m-mode image. (B) An m-mode image of a parasternal short-axis view of a mouse heart. The arrows show the corresponding locations bewtween b-mode and m-mode images.

The first step in preparing a rodent for ultrasound imaging is usually to remove the hair (with hair clippers or chemical hair remover) at the imaging site because it can interfere with the ultrasound image. The ultrasound coupling gel will moisturize the bare skin. The next step is to secure the animal (with medical tape) to a platform equipped with a warming pad, connections for a nose cone to deliver anesthetic gases, and ECG electrodes. The platform enables a fixed and reproducible position for imaging. Zhou and colleagues (2004) have described ultrasound imaging sections and transducer positions for cardiac structural, functional, and Doppler hemodynamic evaluation with a fixed-plane platform. These examination times are very short (e.g., 2 to 10 minutes), which is most productive for high-throughput studies. Experienced sonographers can hold anesthetized animals in one hand and position the transducer on the animal with the other.

One advantage of ultrasound is the possibility of imaging unanesthetized mice. Yang et al. (1999) and Rottman et al. (2007) describe methods of training mice before the start of a study in order to image conscious, unanesthetized mice. Training includes holding the mouse in the position required for imaging and touching its chest to simulate the probe. Initially, the mice developed bradycardia, but after several training sessions over 1 to 3 days, they remained calm and the bradycardia disappeared (Yang et al. 1999). During the examination, the operator held the mouse by the nape of the neck in the prone position in one hand with the tail held between the last two fingers.

Two-dimensional m-mode echocardiograms can be obtained at the level of the papillary muscle from the parasternal short-axis view (Rottman et al. 2007). Echocardiography is a readily interpreted tool for evaluating ventricular function in mice, using techniques and indices familiar from human echocardiography (Rottman et al. 2007). M-mode images enable the measurement of wall thickness, LV dimensions, and cardiac mass, and b- and m-mode images combined reveal endpoints such as contractility, LV dilation, and fractional shortening (Madamanchi 2004).

Collins and colleagues (2001) compared LV mass estimates using 2D (b-mode) and echocardiographic (m-mode) images. To estimate LV mass, they used two different algorithms with b-mode images (the area-length method and the truncated ellipsoid method), and for the m-mode they used a cubic formula. They found that the area-length measurements (a formula that includes the parasternal short-axis area multiplied by parasternal long-axis length) of LV mass did not differ significantly from the necropsy weights, whereas the truncated ellipsoid formula underestimated the mass. Collins and colleagues (2001) also used a linear regression analysis to compare the LV mass estimates to the true LV weights found at necropsy. Using a formula that included the internal diameter of the LV cubed (cubic formula) with m-mode images, Collins and colleagues (2001) performed left-ventricular mass calculations that overestimated the mass compared to necropsy values. The findings of Kiatchoosakun and colleagues (2002) agreed with those of Collins and colleagues (2001). Watson and colleagues (2004) determined baseline echocardiographic values for anesthetized male Sprague-Dawley rats and obtained 2D, m-mode, and Doppler images both before and 2 weeks after animals underwent a sham operation. Their report provides a comprehensive listing (b-mode, m-mode, and Doppler) of measurements that are possible with small animal ultrasound (Watson et al. 2004).

In addition to the possibility of imaging unanesthetized mice, another advantage of ultrasound is short imaging times—on the order of minutes—whether holding the animal in the hand or using the platform. One of the limitations of ultrasound is that it requires the knowledge and expertise of a trained operator to position the transducer to obtain accurate, repeatable, high-quality images. Using a single operator to image all animals in a given study minimizes this limitation (Coatney 2001). It is also necessary to have a good interpreter of the images to ensure reliable measurements from the ultrasound images, which have lower image contrast than microCT or MR images. Some geometric assumptions are necessary in ultrasound (but not in microCT and MR, the 3D modalities). Nonetheless, ultrasound can be effective for measures of functional parameters of a 3D object (e.g., the heart) from 2D images.

Microcomputed Tomography

A projection image is produced from radiation, transmitted from an x-ray source, that is attenuated by the tissue as it passes through the body and then picked up by a detector that converts the signal to an image (Acharya et al. 1995). A computed tomography (CT) scanner produces 3D images by taking x-ray projection views, acquired at hundreds of equally spaced angular positions around the animal, and reconstructing them by means of a back-projection algorithm (Holdsworth and Thornton 2002). The resulting CT dataset is a series of 2D cross-sectional images that can be stacked to form a 3D representation. The data can be sliced in different orientations to reveal different views of the anatomy. MicroCT produces the same type of images as CT with a spatial resolution better than 100 μm³. Figure 2 shows representative microCT images of a mouse heart.

Figure 2 A microCT image of a normal mouse heart at (A) axial, (B) dorsal-ventral, and (C) sagittal views. Image courtesy of NIH, NINDS, Mouse Imaging Facility.

The 3D image is composed of voxels, small volume elements that are the 3D equivalent of a pixel. The x-ray attenuation is proportional to the density of the tissue, making different tissue types (e.g., bone, lung, soft tissue) appear with different grayscale values in the microCT image (Acharya et al. 1995). These grayscale values, called the CT number, serve as quantitative measurements of the density of a specific tissue relative to the density of water. Hounsfield units (HU), the CT number multiplied by a magnification constant of 1,000 (Curry et al. 1990), are also a measure of tissue density.

MicroCT offers limited inherent image contrast between different soft tissue types (e.g., blood and myocardial tissue) because there is not a significant change in tissue density among soft tissues. Investigations of individual organs require the introduction of a contrast agent (through a tail-vein injection or catheterization; Chen 2006) to help define the boundaries of the target organ. Blood-pool contrast agents can increase the difference between blood and muscle in the microCT image by approximately 500 HU (Badea et al. 2006). A contrast agent can be useful for tracing vessels and determining whether there are blockages, leaks, or other abnormalities (Ford et al. 2006).

Badea and colleagues (2004) have described an experimental in vivo microCT system in which the x-ray remains stationary and the animal rotates vertically in the x-ray beam. They reduced motion artifacts with cardiac gating and synchronization of the image acquisition with the ventilatory cycle. They used this technique to produce a 4D view of the mouse heart by displaying 3D images over six time points (0, 15, 20, 45, 60, and 90 msec after the R peak) of the cardiac cycle; from this dataset they were able to calculate LV volume, ejection fraction, stroke volume, and cardiac output (Badea et al. 2005). This microCT system has resolution in the 50- to 100-μm range and an acquisition time of approximately 10 to 15 minutes (Badea et al. 2004).

In commercial in vivo microCT systems, the animal is stationary in the horizontal position and the x-ray system rotates around the animal. The latest commercially available systems use a clinical x-ray tube and a 1,024 x 1,024–pixel flat-panel detector mounted on a slip-ring gantry, which allows continuous rotation around the animal with imaging times of less than 1 minute (Drangova et al. 2007; Ford et al. 2006). Drangova and colleagues (2007) used a commercial system along with retrospective gating in a free-breathing mouse to calculate LV function.

Because the voxels in 3D microCT images are isotropic, it is possible to measure ventricular volumes at different phases of the cardiac cycle (Badea et al. 2005). Analysis of the left ventricle, for example, may entail segmenting the 3D image; a region-growing algorithm can automatically segment the LV chamber when a contrast agent is used. The volume is calculated by taking the number of voxels in the segmented chamber and multiplying it by the physical voxel dimension (μm³). From that volume it is then possible to calculate the stroke volume, ejection fraction, and cardiac output (Drangova et al. 2007). In addition, microCT images enable evaluations of the wall dynamics, which Badea and colleagues (2005) analyzed by measuring changes in wall thickness as an index of myocardial ischemia.

The disadvantage to using microCT is the ionizing radiation. Although the microCT scanner is shielded for the operator, the animal's exposure is unavoidable. The lethal dose to small rodents is ~6 Gy and should be taken into consideration in a longitudinal study. In addition, soft tissue contrast is inherently poor with x-ray CT and contrast agent may be necessary to discern some anatomical features in vivo (Holdsworth and Thornton 2002).

Magnetic Resonance

Magnetic resonance imaging does not rely on ionizing radiation but rather the generation of magnetic resonance (MR) from spinning protons in the animal's body. The rodent is made up of mostly hydrogen atoms whose nuclei are spinning positively charged protons that induce a magnetic field along the axis of each proton. In a zero-magnetic field, the orientation of the spins is random. Placement of the rat or mouse in an external magnetic field causes the hydrogen proton spins to align with that field (Pautler 2004).

With the application of a radiofrequency (RF) pulse, the nuclei of the hydrogen atoms are promoted to a higher energy state and their nuclei change orientation as they absorb the RF energy. This change of energy state is called "excitation" (Pautler 2004). A coil that surrounds the animal generates the RF pulse; once the pulse ceases, the nuclei return to their original orientation, releasing the absorbed energy. This released energy constitutes the MR signal, which is detected by the coil and made into an image. A representative MR image of a mouse heart is shown in Figure 3. (A discussion of the selection criteria for an appropriate RF coil is beyond the scope of this article but should be considered in plans to use MR in cardiac studies; Doty and colleagues (2007) discuss RF coil technology for small animal MR.)

Figure 3 An MR image of a normal mouse heart taken at 9.4 T. Image courtesy of University of North Carolina, Chapel Hill.

When the RF pulse ceases and the nuclei "relax" back to their equilibrium position, the MR signal indicates two different relaxation times, T1 (recovery) and T2 (decay). T1 is due to the nuclei giving up energy to the surrounding environment, and T2 is due to the nuclei exchanging energy with each other. Different tissues relax at different rates and therefore have different T1 and T2, which appear as different grayscale values in the MR image. Although MR imaging produces an inherent tissue contrast (Pautler 2004), the characteristics of the signal depend on the specific molecular environment of the nucleus. Through the proper RF stimulation—i.e., in the timing of the pulse sequences—it is possible to gain information about the different tissue characteristics (Acharya et al. 1995). The time between RF pulses is repetition time (TR) and the timing from the excitation RF pulse to the acquisition of the signal induced in the coils is echo time (TE). The TR and TE settings can optimize T1 and T2 contrast to highlight pathology in the animal (Pautler 2004).

Commonly used magnetic field strengths are 4.7 tesla (T) and 7 T, but there are also reports of experiences at 11 T or above for cardiac applications (Vallee et al. 2004). Higher-powered small animal MR magnets may have a vertical instead of a horizontal bore (i.e., the animal hangs vertically during the examination). The advantage of the higher-powered fields is the accompanying increase in signal that produces higher spatial resolution in small animal models. In studies of less than 1 hour, measurements estimated from vertically imaged animals agreed with those of horizontally imaged animals (Schneider et al. 2003; Vallee et al. 2004). However, when Schneider and colleagues (2004) used a vertical bore magnet to image normal and infarcted mice with examination times of up to 3 hours, they found significant changes over time in the LV volumes, which were more pronounced in the failing mouse heart. Their theory was that the animal's vertical position over an extended period of time reduced venous return and end-diastolic pressure. They concluded that, to compensate for these effects, there was an increase in contractility and stroke volume as well as an elevation of cardiac output to maintain peripheral blood pressure (Schneider et al. 2004).

The advantages of MR imaging are its high tissue contrast, the ability to acquire 3D images, and the capacity to study many physiological parameters. Because the inherent tissue contrast is so good, there may be no need for contrast agents. In cardiac imaging, data are collected from multiple levels of the heart to provide more details about the overall cardiac chamber's function and morphology (James et al. 1998). Because the entire heart is imaged, there is no need for geometric assumptions when calculating ventricular mass and function, as with echocardiography (Hoit 2001; Weiss 2001). Cardiac mass estimates are highly accurate, reproducible, and observer- and technician-independent (Hoit 2001). Furthermore, through the use of gating, MR is able to generate high-resolution images at multiple time points in the cardiac cycle (Madamanchi 2004). Another advantage of MR imaging is the potential for multiple mouse imaging, an option not available with ultrasound or microCT. Bishop and colleagues (2006) were able to image three mice simultaneously at four temporal phases of the cardiac cycle using a retrospective gating strategy.

In order to calculate the functional parameters of the left ventricle, the cavity must be segmented out of the image. Nahrendorf and colleagues (2003) used a series of 1-mm-thick slices (in-plane resolution of 230 to 310 μm²) through the heart to determine the LV parameters. They calculated myocardial and ventricular volumes from end-diastolic and end-systolic images by multiplying the compartment area and slice thickness. Schneider and colleagues (2003) measured left ventricular parameters by using software to automatically segment the LV cavity at end-diastolic and end-systolic frames. They multiplied the number of voxels in each compartment by the physical voxel dimension to yield the volume of the cavity, and determined the mass by multiplying the volume by the specific gravity (1.05 g/cm3). They were also able to calculate stroke volume, ejection fraction, and cardiac output (Schneider et al. 2003).

MR imaging is the leading noninvasive technique for characterizing the structure and function of the irregularly shaped right ventricle in mice (Weiss 2001). The right ventricle (RV) is hard to image because of its complex geometry, with a wide angulation between inflow and outflow tract and relatively coarse trabecularization. Furthermore, geometric models are difficult to apply because of the complexity of RV contraction as well as the unpredictability of changes in its dimensions under pathophysiological conditions. In addition, the position of the normal RV is immediately beneath the sternum, which makes imaging the RV difficult for echocardiography (Weismann et al. 2002). MR surmounts all these obstacles to produce clear, useful images of the mouse RV.

The primary disadvantages of MR imaging include the very costly equipment, limited availability of MR systems for small animals, and the fact that operating a small animal MR facility is resource-intensive. In addition, examination times can range up to 90 minutes per animal (Hoit 2001), which can prohibit high-throughput studies.

Summary

In vivo imaging is nondestructive and therefore can be an advantageous tool in investigations of cardiac disease with animal models. Ultrasound, microCT, and MR are each capable of measuring classic cardiac function parameters such as ejection fraction, fractional shortening, stroke volume, cardiac output, and LV mass. All three modalities have their strengths and weaknesses, which are summarized in Table 1. Ultrasound uses high-frequency sound waves to produce images and is the least expensive and fastest (1 to 5 minutes) method of cardiac imaging. It permits images in real time on conscious or anesthetized animals, which is not possible in the other two modalities. Ultrasound has its limitations, though, as the calculations of ventricular mass rely on geometric assumptions, which can cause errors in the estimations.

MicroCT uses x-radiation to produce images and can provide 3D and 4D images of the rodent heart in relatively short time periods (10 to 15 minutes). Because of the low tissue contrast, contrast agent is necessary to enhance the visual differences between the blood and myocardial tissue. The ionizing radiation used in microCT may limit its use in longitudinal studies.

MR uses magnetic fields to "excite" the tissue in order to produce images. The inherent tissue contrast easily separates blood from heart muscle tissue. Right ventricular measurement, which is not easily assessed in ultrasound, is possible with MR. But MR examinations (0.5 to 3 hours) take longer than microCT and ultrasound. Studies with MR may also be limited by availability and expense.

It is important to carefully consider each aspect in the process of rodent cardiac imaging—from animal preparation to image interpretation—in order to optimize image quality and ensure accurate and reproducible data collection. Factors to consider in animal preparation are anesthesia regime (injectable or inhaled), intubated or free-breathing animals, physiological monitoring (ECG, respiration, and temperature), and animal immobilization. In vivo imaging also requires technical support to monitor the animal throughout the procedure and recovery (Balaban and Hampshire 2001). Gating strategies, prospective or retrospective, are an important component to reduce physiological motion artifacts and to isolate specific points in the cardiac cycle (i.e., end-diastole and end-systole) for the measurements.

With careful consideration and use, these imaging modalities allow characterization of both structure and function in the investigation of murine cardiac models.

Acknowledgments

The author expresses her appreciation to Page Myers for assistance with ultrasound imaging; Daniel Schimel, RLATG, and Brenda Klaunberg, VMD, of NIH, NINDS, Mouse Imaging Facility for the microCT images; and Weili Lin, PhD, and Hongyu An, DSc, of the University of North Carolina—Chapel Hill for the MR images. The author thanks Drs. Robert R. Maronpot and Jill Marcus for editorial assistance. This research was supported in part by the Intramural Research Program of the NIH National Institute of Environmental Health Sciences.

Abbreviations used in this article: ECG, electrocardiogram; LV, left ventricle; microCT, microcomputed tomography; MR, magnetic resonance; RF, radiofrequency

References

Acharya R, Wasserman R, Stevens J, Hinojosa C. 1995. Biomedical imaging modalities: A tutorial. Comput Med Imaging Graph 19:3-25.

Badea CT, Hedlund LW, Johnson GA. 2004. MicroCT with respiratory and cardiac gating. Med Phys 31:3324-3329.

Badea CT, Fubara B, Hedlund LW, Johnson GA. 2005. 4-D microCT of the mouse heart. Mol Imaging 4:110-116.

Badea CT, Bucholz E, Hedlund LW, Rockman HA, Johnson GA. 2006. Imaging methods for morphological and functional phenotyping of the rodent heart. Toxicol Pathol 34:111-117.

Balaban RS, Hampshire VA. 2001. Challenges in small animal noninvasive imaging. ILAR J 42:248-262.

Bishop J, Feintuch A, Dazai J, Davidson L, Henkelman RM. 2006. Retrospective gating for mouse cardiac MRI. Magn Reson Med 55:472-477.

Chaves AA, Weinstein DM, Bauer JA. 2001. Non-invasive echocardiographic studies in mice: Influence of anesthetic regimen. Life Sci 69:213-222.

Chen XJ. 2006. Mouse morphological phenotyping with magnetic resonance imaging. Methods Mol Med 124:103-127.

Chilton HM, Ekstrand KE. 1984. Principles and applications of nuclear magnetic resonance imaging. Am J Hosp Pharm 41:763-768.

Coatney RW. 2001. Ultrasound imaging: Principles and applications in rodent research. ILAR J 42:233-247.

Collins KA, Korcarz CE, Shroff SG, Bednarz JE, Fentzke RC, Lin H, Leiden JM, Lang RM. 2001. Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J Physiol Heart Circ Physiol 280:H1954-H1962.

Curry TS, Dowdey JE, Murry RC. 1990. Christensen's Physics of Diagnostic Radiology, 4th ed. Media, PA: Lea and Febiger.

Devereux RB, Wachtell K, Gerdts E, Boman K, Nieminen MS, Papademetriou V, Rokkedal J, Harris K, Aurup P, Dahlof B. 2004. Prognostic significance of left ventricular mass change during treatment of hypertension. JAMA 17:2350-2356.

Doty FD, Entzminger G, Kulkarni J, Kranti P, Staab JP. 2007. Radio frequency coil technology for small-animal MRI. NMR Biomed 20:304-325.

Drangova M, Ford NL, Detombe SA, Wheatley AR, Holdsworth DW. 2007. Fast retrospectively gated quantitative four-dimensional (4D) cardiac microcomputed tomography imaging of free-breathing mice. Invest Radiol 42:85-94.

Ford NL, Nikolov HN, Norley CJ, Thornton MM, Foster PJ, Drangova M, Holdsworth DW. 2005. Prospective respiratory-gated microCT of free breathing rodents. Med Phys 32:2888-2898.

Ford NL, Graham KC, Groom AC, Macdonald IC, Chambers AF, Holdsworth DW. 2006. Time-course characterization of the computed tomography contrast enhancement of an iodinated blood-pool contrast agent in mice using a volumetric flat-panel equipped computed tomography scanner. Invest Radiol 41:384-390.

Guyton AG. 1981. Textbook of Medical Physiology, 6th ed. Philadelphia: WB Saunders.

Hartley CJ, Taffet GE, Reddy AK, Entman ML, Michael LH. 2002. Noninvasive cardiovascular phenotyping in mice. ILAR J 43:147-158.

Heijman E, de Graaf W, Niessen P, Nauerth A, van Eys G, de Graaf L, Nicolay K, Strijkers GJ. 2007. Comparison between prospective and retrospective triggering for mouse cardiac MRI. NMR Biomed. 20:439-447.

Hoit BD. 2001. New approaches to phenotypic analysis in adult mice. J Mol Cell Cardiol 33:27-35.

Holdsworth DW, Thornton MM. 2002. MicroCT in small animal and specimen imaging. Trends Biotech 20:S34-S39.

James JF, Hewett TE, Robbins J. 1998. Cardiac physiology in transgenic mice. Circ Res 82:407-415.

Kiatchoosakun S, Restivo J, Kirkpatrick D, Hoit BD. 2002. Assessment of left ventricular mass in mice: Comparison between two-dimensional and m-mode echocardiography. Echocardiography 19:199-205.

Lukasik VM, Gillies RJ. 2003. Animal anaesthesia for in vivo magnetic resonance. NMR Biomed 16:459-467.

Madamanchi A. 2004. Mouse models in cardiography research. McGill Journal of Medicine 8:34-39.

McConville P, Moody JB, Moffat BA. 2005. High-throughput magnetic resonance imaging in mice for phenotyping and therapeutic evaluation. Curr Opin Chem Biol 9:413-420.

Nahrendorf M, Hiller KH, Hu K, Ertl G, Haase A, Bauer WR. 2003. Cardiac magnetic resonance imaging in small animal models of human heart failure. Med Image Anal 7:369-375.

Pautler RG. 2004. Mouse MRI: Concepts and applications in physiology. Physiology 19:168-175.

Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross J. 2002. Impact of anesthesia on cardiac function during echocardiography in mice. Am J Physiol Heart Circ Physiol 282:H2134-H2140.

Rottman JN, Ni G, Brown M. 2007. Echocardiographic evaluation of ventricular function in mice. Echocardiography 24:83-89.

Sabbah M, Alsaid H, Fakri-Bouchet L, Pasquier C, Briguet A, Canet-Soulas E, Fokapu O. 2007. Real-time gating system for mouse cardiovascular MR imaging. Magn Reson Med 57:29-39.

Schneider JE, Cassidy PJ, Lygate C, Tyler DJ, Wiesmann F, Grieve SM, Hulbert K, Clarke K, Neubauer S. 2003. Fast high-resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on a vertical 11.7 T system. J Magn Reson Imaging 18:691-701.

Schneider JE, Hulbert KJ, Lygate CA, Ten Hove M, Cassidy PJ, Clarke K, Neubauer S. 2004. Long-term stability of cardiac function in normal and chronically failing mouse hearts in a vertical-bore MR system. MAGMA 17:162-169.

Vallee JP, Ivancevic MK, Nguyen D, Morel DR, Jaconi M. 2004. Current status of cardiac MRI in small animals. MAGMA 17:149-156.

Watson LE, Sheth M, Denyer RF, Dostal DE. 2004. Baseline echocardiographic values for adult male rats. J Am Soc Echocardiogr 17:161-167.

Weiss RG. 2001. Imaging the murine cardiovascular system with magnetic resonance. Circ Res 88:550-551.

Wiesmann F, Frydrychowicz A, Ratenberg J, Illinger R, Rommel E, Haase A, Neubauer S. 2002. Analysis of right ventricular function in healthy mice and a murine model of heart failure by in vivo MRI. Am J Physiol Heart Circ Physiol 283:H1065-H1071.

Wyngaarden JB, Smith LH. 1982. Cecil Textbook of Medicine, 16th ed. Philadelphia: WB Saunders.

Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, Carretero OA. 1999. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am J Physiol 277:H1967-H1974.

Zhou YQ, Foster FS, Nieman BJ, Davidson L, Chen XJ, Henkelman RM. 2004. Comprehensive transthoracic cardiac imaging in mice using ultrasound biomicroscopy with anatomical confirmation by magnetic resonance imaging. Physiol Genomics 18:232-244.