December 16, 2003: TORONTO, ON - VisualSonics is pleased to share the featured article appearing in the Fall Release of the Academy of Molecular Imaging (AMI) News Release: Ultrasound: the less traveled path in molecular and pre-clinical imaging
By Dr. Stuart Foster
INTRODUCTION
The complexity of development, growth, and disease in mammalian models such as the mouse offer a major challenge to new microimaging technologies that have been developed over the past decade. Enormous strides have been made in the fields of optical imaging, positron emission tomography (PET), magnetic resonance (MR), ultrasound (US) and CT to refine the technologies for applications in the mouse. Yet each modality has its strengths and weaknesses for applications in molecular and pre-clinical imaging. A more complete picture of biological endpoints emerges when structure-function relationships determined by MR, CT and US are combined with the exquisite molecular sensitivity of PET and Optical techniques. In this “Mini Review” the potential of ultrasound to contribute to pre-clinical imaging is presented.
CLINICAL ULTRASOUND
Ultrasound in the 2 – 15 MHz range is a well established clinical imaging modality, accounting for more than 1/3 of all imaging procedures performed in north America. Ultrasound images are essentially maps of tissue echogenicity derived from the same principles as SONAR. The simplicity, ease of use, speed and safety of ultrasound have led to a significant role in diagnosis, treatment assessment, follow-up, and guidance of therapy. Conventional ultrasound images (B-scans) primarily report tissue structure. Ultrasound is also well established as a means of measuring blood flow or, more precisely, blood velocity using the Doppler principle. Scattering of ultrasound from moving red blood cells leads to a shift in the frequency of scattered ultrasound in comparison to the frequency of the insonifying pulse. This “Doppler” shift may be displayed in a variety of ways to contribute functional information on the flow of blood in the vascular system.
ULTRASOUND CONTRAST AGENTS AND MOLECULAR IMAGING
The field of ultrasound has recently been stimulated by the introduction of microbubble contrast agents for a wide variety of applications. Current generations of these agents consist of 1 ?m to 5 ?m microbubbles rendering them suitable for intravenous injection. A typical contrast agent consists of a thin flexible or rigid shell composed of albumin, lipid, or polymer confining a gas such as nitrogen, or a perfluorocarbon. The choice of shell and gas has an important influence on the properties of the agent. Examples of approved or soon to be approved contrast agents are Levovist (Schering Ag), Optison (Mallinckrodt Medical, https://www.mallinckrodt.com/), Sonovue (Bracco, https://www.bracco.com/) and Definity (DuPont Pharmaceutical). Due to their intrinsic compressibility (approximately 17,000 times more than water) microbubbles are very strong scatterers of ultrasound. The three key features of microbubble contrast agents are: 1) they are true intravascular agents unlike the diffusable agents commonly used in magnetic resonance imaging or computed tomography; 2) microbubbles have a strong nonlinear response to sound, creating a unique signature that permits echoes from the true microcirculation to be differentiated from those of surrounding tissues; and 3) microbubbles can be disrupted or “popped” with a controlled ultrasound pulse creating a quantitative means of probing perfusion of selected tissue regions or releasing a therapeutic payload. Exploitation of these properties has led to a number of potential applications in targeted chemotherapy[1], gene therapy[2, 3], thrombus detection/thrombolysis[4, 5], and monitoring of the microcirculation to follow anti and proagiogenic therapies[6, 7]. Nanoparticles composed of lipid shelled liquid perfluorocarbons have also shown promise as molecular targeted agents[8-10].
ULTRASOUND MICROIMAGING
The above molecular imaging approaches have been largely investigated in the diagnostic frequency range from 3 – 10 MHz and are suitable for human imaging. Instrumentation specifically designed for pre-clinical imaging has recently become commercially available in the 20 to 60MHz range (VisualSonics Inc., https://www.visualsonics.com/) [11, 12]. Ultrasound microimaging at high frequencies is often referred to as ultrasound biomicroscopy (UBM). Increasing the frequency of the scanner increases the resolution at the expense of penetration. Typical resolution ranges from 30 to 100 microns with 5 – 15 mm penetration. Such specifications are suitable for the mouse enabling visualization of embryo/organogenesis, neonatal, and adult development. Experimental protocols for mouse ultrasound studies have been described previously[12]. Briefly the mouse is anesthetized (isoflurane by face mask) and placed on a heated pad with temperature and ECG monitoring. The scanned region is cleared of hair by shaving or use of a chemical hair remover. The ability to perform rapid noninvasive longitudinal studies permits examination of normal development[12, 13], disease models[14, 15], and novel therapies[16].
The imaging system is shown in figure 1a and an example of a 13.5 day mouse embryo is given in figure 1b. Clear high resolution visualization of the embryonic lateral ventricle of the brain (V), eye (E) and heart (H) is apparent. Because ultrasound is a real-time imaging modality its use in the guidance of injections of cells or genetic material for expression studies[17], is a natural application.
Figure 2 illustrates the use of UBM to inject material into the developing neural tube of a day 12.5 embryo in vivo. From left to right the needle is shown at the uterine wall, penetrating the embryonic brain, inserted into the neural tube, and after injection of micro-beads to confirm injection.
Doppler can add functional information on blood flow to complement the 2 dimensional B-scan images. Often this is done by windowing a particular location such as the mitral valve and entering pulsed Doppler mode. This creates a plot of blood velocity versus time in the sample volume assuming that the angle between the ultrasound beam and the vessel axis is known. Alternatively, the Doppler information can be processed to create two-dimensional (color flow) maps of average blood velocity toward or away from the transducer. This mode of operation has been demonstrated in prototype equipment[15, 18] and has been applied to cancer models as a means of monitoring antiangiogenic therapies[16].
CONCLUSIONS
Although ultrasound is the less traveled path in molecular and pre-clinical imaging, the seeds have been planted for a growing contribution. The versatility, speed, cost, and resolution of ultrasound will certainly complement more established tools. The further development of targeted blood pool contrast agents at both low and high frequencies has the potential to provide a range of new diagnostic and therapeutic opportunities. On the pre-clinical side, ultrasound should find its place as a potent means of rapid high throughput imaging with applications in disease modeling and guided interventions such as needle injection and biopsy. Technical advances such as multimodality imaging and 3 dimensional imaging will also contribute to future applications. Ultrasound's unique capabilities will provide a valuable resource both in animal labs and human clinical/interventional studies.
Figure Captions:
- Figure 1. a) Photograph of VisualSonics VEVO 660 system. A high frequency transducer is mechanically scanned over the mouse tissues of interest to generate 2 dimensional "B scan" images and duplex Doppler blood flow waveforms. b) Example image plane of a longitudinal section of a 13.5 day mouse embryo showing the developing eye (E), lateral ventricle (V), and heart (H). The large depth of field in this image is achieved using a method called zone focusing. Smallest scale divisions are 100 microns.
- Figure 2. Needle guided injection. Introduction of cells, DNA, retroviral particles etc. are possible under real-time continuous guidance. The needle tip (arrow) is shown, from left to right, at penetration of the uterine wall, penetration of the embryonic brain, entry into the neural tube, and after injection of contrast material to confirm delivery. Images courtesy of Slevin, J et al, Mt. Sinai Hospital, Toronto.
- Figure 3. Experimental treatment of MeWo melanoma with an antivascular agent (AstraZeneca ZD6126). a) Hoechst perfusion staining shows a marked decrease in perfusion at 4 hours post injection (20 mg/kg). b) This pattern of blood flow modification is also seen noninvasively with power Doppler ultrasound imaging. For details see Goertz et al [16].
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About VisualSonics
VisualSonics (www.visualsonics.com) is the leading manufacturer of high-resolution in vivo imaging systems for small animal research. VisualSonics' Vevo 660™ high-resolution imaging system provides real-time visualization and measurement of anatomical and hemodynamic function in small animals down to 30 microns. Because of the non-invasive nature of the technology, longitudinal studies of the same animal can be performed. This high-resolution imaging technology is being successfully applied to phenotypic-based research applications such as developmental biology, cardiovascular research, cancer biology, neurobiology and drug discovery.