INTRODUCTION

Cardiovascular molecular imaging is an emerging field that will have tremendous impact on the diagnosis and treatment of heart disease in the years ahead. While traditional cardiac imaging provides important information regarding myocardial perfusion, function, or heart structure and dimensions, cardiovascular molecular imaging utilizes tracers or contrast agents to measure physiologic and pathologic processes at the cellular and molecular level. These tracers are designed to target specific cell types, receptors or transporters, peptides, or even the expression of a particular gene product. Targeted tracers are currently being used or investigated for imaging cellular processes such as glucose transport or metabolism (i.e., FDG, BMIPP), apoptosis (annexin-V), sympathetic innervation (MIBG), as well as inflammation, hypoxia, angiogenesis, matrix metalloproteinase expression, and the transition of atherosclerotic plaques from the stable to the unstable state. In addition, cardiovascular molecular imaging approaches are being investigated as a tool for monitoring cell trafficking and tissue localization of injected therapeutic stem cells and the delivery and efficacy of targeted gene therapy.

Small animals such as mice, rats, and guinea pigs have long been utilized as models in most areas of molecular biology, toxicology, and drug discovery research, and well-characterized models of specific diseases have been developed using these animals. With the relatively recent development of transgenic mice, mouse models have gained even greater importance. The ability to manipulate the mouse genome to produce accurate models of many human diseases has resulted in significant progress in understanding these diseases. Historic methodologies for assessing molecular changes require euthanizing the animal and performing invasive and destructive assays such as autoradiography and in vitro histopathologic assays on specific tissue types. These techniques are not ideal, because they do not provide information in real time and in situ. As a result, large numbers of animals must often be euthanized to screen for the desired phenotypic changes, resulting in significantly higher cost as well as lost time. The full potential of these new transgenic mouse models can only be realized with the ready availability of accurate, noninvasive imaging methods to investigate disease progression and its response to therapeutic agents. For these reasons, there has been increasing interest in developing noninvasive imaging techniques that allow repeated in vivo imaging at the cellular and molecular level in small animals such as mice. Importantly, repeated imaging studies allow for the delineation of temporal changes of these functional processes.

While other imaging modalities like magnetic resonance imaging (MRI), myocardial contrast echocardiography (MCE), and the newer optical imaging modality are being utilized for small-animal imaging and have their particular strengths, imaging techniques based on radiolabeled probes and positron emission tomography (PET) and single-photon emission computed tomography (SPECT) instrumentation will remain at the forefront because of their unparalleled sensitivity. Unfortunately, however, the spatial resolution of commercially available SPECT and PET cameras designed for clinical imaging is on the order of 4 to 6 mm, which limits their usefulness in small animals such as mice in which the heart is typically 3 to 4 mm across the short axis and the left ventricular wall thickness is less than 1 mm (~0.8 to 0.9 mm). Accordingly, there has been a tremendous effort in recent years to develop high-resolution SPECT and PET cameras specifically designed for imaging small animals in biomedical research. At the present time, there are multiple commercial versions of both microSPECT and microPET cameras available, and intensive work is underway in both academic settings and industry to further develop and improve these cameras.

MICRO–POSITRON EMISSION TOMOGRAPHY

PET extends the investigative possibilities of radionuclide approaches that have historically focused mostly on the evaluation of myocardial blood flow. With PET tracers, it is now possible to explore and, more important, to noninvasively quantify functional processes such as substrate metabolism, oxygen consumption, receptor density and occupancy, and activation and expression of transfected genes. Because of PET’s quantitative and high temporal resolution capability, it is possible to accurately measure regional activity concentrations in the myocardium and blood as well as their changes over time. As described later, the radiotracer input function and the tissue response can be determined from serially acquired PET images. When combined with appropriate tracer kinetic models, absolute estimates of rates of functional processes can be derived (e.g., mL blood or mol substrate per minute per gram myocardium).

Dedicated, high spatial resolution small-animal PET imaging devices have become available. Some are in-house-developed systems at several research institutions,^1–5 but more recently, dedicated small-animal systems have become available through several commercial suppliers. The spatial resolution of these imaging devices is typically in the range of 1.0 to 1.5 mm full-width at half-maximum (FWHM) and enables mapping of the distribution of positron-emitting radiotracer and its changes over time throughout the body of small animals. These imaging systems have been used quite extensively for measuring substrate metabolism and cell replication rates of tumors as indices of tumor growth rates and tumor aggressiveness, and for determining responses of tumor growth to investigational therapeutic agents.^6–10 Other research has utilized these small-animal imaging devices for the development and validation of novel noninvasive assays for identifying activation and expression of transgenes.^6,9–11

POSITRON EMISSION TOMOGRAPHY IMAGING OF THE RODENT HEART

The small dimensions of the rodent heart pose formidable challenges for PET studies of the cardiovascular system in rodents. In general, dedicated small-animal PET systems employ two different technical concepts: (1) crystal detector–based and (2) multiwire proportional chamber–based systems.^12,13 As shown in Figure 11-1, multiwire proportional chamber–based systems may offer superior spatial or volume resolution but generally are less sensitive and require longer image acquisition times. Conversely, crystal-based systems offer higher sensitivity (count rate per unit radioactivity) and offer superior temporal resolution but at the expense of somewhat lower spatial resolution (Fig. 11-2).

Figure 11-1 ¹⁸F-deoxyglucose uptake in a 29-g mouse imaged with a multiwire proportional chamber-based system (QuadHIDAC-PET, Oxford Positron Systems, Cambridge, England). The 15-minute whole body image was recorded 1 hour after injection of 10 MBq ¹⁸F-deoxyglucose. A, Coronal whole body slice. B, Reoriented myocardial slices.

(Courtesy of K. Schäfers, University of Münster, Münster, Germany.)

Figure 11-2 Sagittal, coronal, and transaxial images slices of ¹⁸F-deoxyglucose uptake in a mouse, obtained with a crystal-based, dedicated small-animal PET system (Concorde Microsystems, Knoxville, TN; Focus microPET system) and reconstructed using maximum a posteriori probability at 60 minutes after a tail-vein injection of ¹⁸F-deoxyglucose.

(Courtesy of David Stout, University of California, Los Angeles.)

Several commercial small-animal scanners are now available since the first prototype scanner was built at University of California, Los Angeles. Figure 11-3 shows one such small-animal PET scanner, the microPET Focus 120.¹⁴ This system consists of 96 scintillation detectors arranged in four rings with a 14.7-cm diameter and 7.6-cm axial extent. Each detector consists of a 12 × 12 array of 1.52 × 1.52 × 10 mm lutetium oxyorthosilicate (LSO) crystals coupled to a position-sensitive photomultiplier tube (PS-PMT) via optical fiber bundles. Data are acquired in three-dimensional (3D) mode. With standard filtered backprojection reconstruction algorithms, the system achieves an isotropic (in all three directions) spatial resolution of 1.5 mm in the center of the field of view (FOV) and remains less than 2.0 mm within 5 cm of the center of the FOV. Imaging of the myocardium in mice and rats with the flow tracer ¹³N-ammonia or the glucose analogue ¹⁸F-deoxyglucose typically requires activity doses of 0.5 to 1.5 mCi.¹⁵ Count rates at these radioactivity doses remain relatively low so that dead-time losses are small. Image reconstruction with the more recently developed maximum a posteriori probability (MAP) algorithm, now employed for cardiac studies in mice, provides images of myocardial perfusion and glucose uptake of good to excellent diagnostic quality (see Fig. 11-2).¹⁶ The MAP reconstruction algorithm improves the spatial resolution in the center of the FOV to 1.2 mm FWHM so that a volumetric resolution of less than 5 microliters is now possible.

Figure 11-3 MicroPET Focus 120 scanner.

(Image courtesy of Siemens Medical Solutions USA, Inc.)

The emission data are reconstructed initially into a set of contiguous transaxial slices with a 0.75-mm interplane spacing (Fig. 11-4).¹⁷ The transaxial images are typically reoriented into short-axis and vertical and horizontal long-axis cuts (Fig. 11-5)¹⁷ that can be assembled into polar maps analogous to those of myocardial perfusion studies in humans (Fig. 11-6).¹⁷ Images of glucose uptake obtained with ¹⁸F-deoxyglucose are of greater contrast and higher quality than images of perfusion obtained with ¹³N-ammonia. This is most likely because of the more complete clearance of ¹⁸F-deoxyglucose from blood and higher tracer activity concentrations in the myocardium. Another reason is the slower physical decay of ¹⁸F-deoxyglucose so that over the total image acquisition time, more counts are recorded. On the other hand, the shorter physical half-life of ¹³N-ammonia affords the ability to perform repeat examinations of myocardial blood flow during the same study session so that effects of interventions can be evaluated. ECG gated image acquisition is also possible with small PET systems so that left ventricular function can be assessed.¹⁸ For example, in normal 300-g rats, the left ventricular ejection fraction was about 60% to 70%, and the stroke volume was about 300 μL.¹⁸

Figure 11-4 Contiguous transaxial images of the chest in a mouse (inferior to superior from top left to bottom right, interplane spacing 0.75 mm) obtained 30 minutes after intravenous ¹⁸F-deoxyglucose.

(Reproduced with permission from Schelbert HR, Inubushi M, Ross RS: PET imaging in small animals, J Nucl Cardiol 10:513–520, 2003.)

Figure 11-5 Reoriented images of the myocardial ¹⁸F-deoxyglucose uptake in the same mouse as depicted in Figure 11-4. Selected short-axis (upper row) and vertical and horizontal long-axis cuts are shown.

(Reproduced with permission from Schelbert HR, Inubushi M, Ross RS: PET imaging in small animals. J Nucl Cardiol 10:513–520, 2003.)

Figure 11-6 Polar map display of the ¹⁸F-deoxyglucose uptake in the left ventricular myocardium in the mouse depicted in Figures 11-4 and 11-5.

(Reproduced with permission from Schelbert HR, Inubushi M, Ross RS: PET imaging in small animals. J Nucl Cardiol 10:513–520, 2003.)

The image reconstruction algorithm corrects for radioactive decay and for dead-time losses. It also includes routines for photon attenuation correction. These routines utilize transmission images that are typically acquired prior to radiotracer administration and are performed using a Co57 point source or, for hybrid microPET/CT systems, an x-ray attenuation map. Despite the small body size of rats, photon attenuation causes about 30% systematic underestimation of the true myocardial radioactivity concentration.¹⁹ Albeit somewhat less in mice, photon attenuation still amounts to about 17% underestimation of the true activity. Accordingly, even in small animals, proper attenuation correction is important for the measurement of the true absolute intensities in cardiac imaging. Chow et al.¹⁹ have shown that CT-based attenuation correction methods result in less noise correction to emission images compared to the transmission-based attenuation corrections.

The stationary imaging gantry of microPET permits rapid serial (or “dynamic”) acquisition of images. Theoretically, images can be recorded at sampling rates of less than 1 second. For practical purposes, however, image acquisition rates depend on the number of counts required for statistically adequate image frames. Framing rates in the range of 2 to 10 seconds per image following an intravenous bolus of ¹³N-ammonia, ¹⁸F-deoxyglucose, or ¹¹C-palmitate produce statistically adequate images for generating tissue time-activity curves and thus are appropriate for radiotracer kinetic studies.²⁰ Figure 11-7 depicts selected frames acquired during and shortly after an intravenous bolus injection of ¹³N-ammonia in a rat. High temporal-resolution tissue time-activity curves for the right and left ventricular blood pools and the left ventricular myocardium in a rat following an intravenous bolus of ¹⁸F-deoxyglucose into a tail vein are shown in Figure 11-8.

Figure 11-7 Selected serially acquired images during and following a bolus administration of ¹³N-ammonia into the tail vein of a rat. The initial two images depict the tracer transit through the inferior vena cava through the right and then to the left ventricle, and the third image depicts the arrival and retention of tracer in both kidneys. Delayed (13.5 and 17.5 minutes) images demonstrate declining tracer activities in the kidneys and continued accumulation of tracer in the bladder. Tracer activity is also noted in the myocardium and throughout the liver.

(Reproduced with permission from Schelbert HR, Inubushi M, Ross RS: PET imaging in small animals, J Nucl Cardiol 10:513–520, 2003.)

Figure 11-8 Time-activity curves derived from the right and the left ventricular (RV, LV) blood pool and the left ventricular myocardium in a mouse. Serial images were recorded beginning with a tail vein injection of ¹⁸F-deoxyglucose. Note the high temporal resolution of the time-activity curves, with clearly separated activity peaks in the right and left ventricular blood pool.

(Courtesy of Michael Kreissl.)

IMAGING THE CARDIOVASCULAR SYSTEM IN MICE AND RATS

Experimental Coronary Occlusions in the Rat

The extent and severity of regional myocardial perfusion defects that are experimentally induced by transient coronary occlusions in rats can be accurately determined with microPET.¹⁵ In Figure 11-9, myocardial perfusion was imaged with intravenous ¹³N-ammonia at baseline, 45 minutes later during a 20-minute coronary occlusion, and again 45 minutes following reperfusion. Figure 11-10 shows the corresponding polar map displays. The location and extent of the image perfusion defects corresponded well with the actual regions observed on postmortem stained tissue. Using a threshold of 50% of the maximum myocardial activity concentration as the lower limit of normal, the size of the flow defect derived from polar maps of myocardial perfusion correlated linearly with the defect sizes determined by postmortem examination (Fig. 11-11).

Figure 11-9 Serial images of the regional myocardial perfusion with intravenous ¹³N-ammonia in a rat. Selected short axis slices through the left ventricular myocardium are shown. The initial image obtained at baseline depicts the homogenously distributed myocardial blood flow. The images in the center were recorded after a second ¹³N-ammonia injection during a transient coronary occlusion. The severe perfusion defect is seen in the base portion of the anterior wall (arrows). The defect has resolved completely at 45 minutes after release of the coronary occlusion, as seen on the third image set (right panel) obtained after another intravenous dose of ¹³N-ammonia.

(Reproduced with permission from Kudo T, Fukuchi K, Annala AJ, et al: Noninvasive measurement of myocardial activity concentrations and perfusion defect sizes in rats with a new small-animal positron emission tomograph, Circulation 106:118–123, 2002.)

Figure 11-10 Polar map display of the distribution of myocardial blood flow recorded serially before, during, and following a transient coronary occlusion in the rat depicted in Figure 11-7. Note the perfusion defect on the polar map during the occlusion, as indicated in blue.

(Reproduced with permission from Kudo T, Fukuchi K, Annala AJ, et al: Noninvasive measurement of myocardial activity concentrations and perfusion defect sizes in rats with a new small-animal positron emission tomograph, Circulation 106:118–123, 2002.)

Figure 11-11 Extent of perfusion defects induced by coronary occlusions in nine rats. Note the close linear correlation between the PET image–derived defect extents and the perfusion defect sizes determined following blue dye staining and postmortem measurements.

(Reproduced with permission from Kudo T, Fukuchi K, Annala AJ, et al: Noninvasive measurement of myocardial activity concentrations and perfusion defect sizes in rats with a new small-animal positron emission tomograph, Circulation 106:118–123, 2002.)

Studies in Genetically Modified Mice

Initial studies have demonstrated the possibility of phenotyping genetically modified mice. For example, cardiac myocyte-specific excision of the murine β-1 integrin gene results in extensive myocardial fibrosis.²¹ In adulthood, the mice develop a form of dilated cardiomyopathy and symptoms of congestive heart failure. MicroPET imaging with ¹⁸F-deoxyglucose in β-1-integrin knockout mice revealed heterogeneously distributed tracer uptake consistent with either replacement of myocytes by fibrotic tissue or as a consequence of regional alterations in substrate metabolism (Fig. 11-12). Assessment of myocardial perfusion with ¹³N-ammonia in the β-1-integrin knockout mouse further revealed heterogeneously distributed myocardial blood flow in addition to patchy regions of reduced tracer uptake.

Figure 11-12 Transaxial images of myocardial perfusion and of exogenous glucose utilization, obtained with intravenous ¹³N-ammonia and ¹⁸F-deoxyglucose in a β-1 integrin knockout mouse. Note the left ventricular enlargement and the heterogeneous tracer retentions.

(Reproduced with permission from Schelbert HR, Inubushi M, Ross RS: PET imaging in small animals, J Nucl Cardiol 10:513–520, 2003.)

Development of Novel Radiotracer Assay Approaches

Noninvasive, image-based probe systems for studying location, extent, magnitude, and duration of gene expression have also been explored and are now being used in small animals. One approach used in oncologic studies in mice has been the PET reporter gene/reporter probe system.⁹–^11,22 In one of these systems, a mutant herpes simplex virus thymidine kinase gene is delivered by an adenoviral vector and represents the PET reporter gene. If expressed in tissue, thymidine kinase phosphorylates the radiolabeled 9-(4-[¹⁸F]-fluoro-3 hydroxymethylbutyl) (guanine) (¹⁸FHBG) that serves as the PET reporter probe. Because the phosphorylated product of ¹⁸FHBG is relatively impermeable to the cell membrane, it becomes effectively trapped in tissue and thus can be imaged with PET. Details on this in vivo assay approach and on other probe systems are described in several recent reviews.^6,9,10,23

Pilot studies with in vitro imaging approaches had explored the feasibility of adapting these probe systems to the heart.²⁴

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