Small-Animal Research Imaging Devices Eugene J. Fine, MD,* Lawrence Herbst, DVM, PhD,† Linda A. Jelicks, PhD,‡,§ Wade Koba, BS, AS, CNMT,§,║ and Daniel Theele, DVM, PhD† The scientiﬁc study of living animals may be dated to Aristotle’s original dissections, but modern animal studies are perhaps a century in the making, and advanced animal imaging has emerged only during the past few decades. In vivo imaging now occupies a growing role in the scientiﬁc research paradigm. Imaging of small animals has been particularly useful to help understand human molecular biology and pathophysiology using rodents, especially using genetically engineered mice (GEM) with spontaneous diseases that closely mimic human diseases. Speciﬁc examples of GEM models of veterinary diseases exist, but in general, GEM for veterinary research has lagged behind human research applications. However, the development of spontaneous disease models from GEM may also hold potential for veterinary research. The imaging techniques most widely used in small-animal research are CT, PET, single-photon emission CT, MRI, and optical ﬂuorescent and luminescent imaging. Semin Nucl Med 44:57-65 C 2014 Elsevier Inc. All rights reserved.
n vivo imaging has become a mainstay in diagnostic veterinary medicine, employing instruments developed over the past several decades, in most cases originally designed for clinical diagnosis in humans. Plain radiographs are by far the most common imaging procedure performed in clinical veterinary practice, although some more recently developed devices may also be used on occasion, including PET, singlephoton emission CT (SPECT), x-ray CT, MRI, and ultrasonography. Meanwhile, research imaging of animals has evolved in different directions for humans and for veterinary purposes. In this respect, a major difference must be recognized in orientation between most veterinary-speciﬁc research and other areas of biomedical research. The species used for veterinary research is typically the species that we are interested in helping, and published imaging studies reﬂect this1-7. This is usually true because wide variation of disease manifestations across species and differences in physiology often make it impractical to develop small-animal models. This is quite
*Department of Radiology, Albert Einstein College of Medicine, Monteﬁore Medical Center, Nuclear Medicine Division, Bronx, NY. †Department of Pathology, Albert Einstein College of Medicine, Bronx, NY. ‡Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY. §Gruss Magnetic Resonance Research Center, Albert Einstein College of Medicine, Bronx, NY. ║ Department of Radiology, Nuclear Medicine Division, Albert Einstein College of Medicine, Bronx, NY. Address reprint requests to Eugene J. Fine, MD, Weiler Hospital-Monteﬁore Medical Center, Nuclear Medicine Division, 1st Floor 1825 Eastchester Rd, Bronx 10461, NY. E-mail: eugene.ﬁ[email protected]
0001-2998/14/$-see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.semnuclmed.2013.08.006
different from medical research for example, where research animals are used principally as surrogates for the human or more abstractly as models of vertebrate systems. The full genome of the mouse was determined, second only to the human genome, and this kind of knowledge advanced the small-animal mouse model to the status of principle human research surrogate. There are few large animals for which a mouse model can be said to have a comparable role. Therefore, although we assume that all veterinary research would beneﬁt from the advantages that imaging modalities provide, the above physiological limitations as well as logistical constraints (usually size) have shaped the uses of imaging devices in veterinary research. In vivo imaging instruments that have achieved wide distribution come in only a few basic sizes: small-animal devices suitable for mice, rats, and perhaps small cats and rabbits and human-scale instruments for any larger animal. There are also imaging devices that have been speciﬁcally modiﬁed for horses, but this represents a much smaller market than the other 2. In this article, the term small animal will be restricted to discussion of animals that ﬁt on small-animal imaging instruments. (This is different from the typical divide between small and large animals in veterinary medicine meaning dogs, cats, and pocket pets (rodents, etc.) vs horses, cattle, and swine, respectively.) The imaging of small animals as a research tool has developed into a distinct niche in human disease research just in the past decade. The use of small animal imaging devices and their relation to human research imaging was reviewed recently.8 In comparative medicine, by contrast, we understand that knowledge of a general vertebrate animal model 57
E.J. Fine et al.
58 (primarily small rodents, rats, and mice) of a disease increases our understanding of the biology of all vertebrates. In other words, what we learn about mice may apply to our understanding and treatment of dogs and horses, as well as nonhuman and human primates, ie, one medicine concept. The value of studying an animal model system comes both from how well the model matches the subject species of interest and how easily ﬁndings can be extrapolated, but also from the ways in which the model does not ﬁt. There can be useful information in the differences, but this can only be discovered by knowing a system very well. Small-animal research, in general, has grown dramatically, particularly in rodents and teleost ﬁsh as well as other small vertebrate animal species. Another major contributory factor to small animal imaging research, especially for rodents, has been the development of genetically engineered mice (GEM) since the mid-1990s. In these animals, speciﬁc mutations can produce mice with diseases that closely mimic disorders of other animal species. GEM have principally found widespread application in the study of human diseases. The GEM, although relatively expensive to develop, can still realize substantial overall cost savings when compared with the expense of large-animal investigations. It should be noted, nevertheless, that housing GEM is quite costly, related primarily to providing environmental conditions where exposure to adventitious pathogens is prevented and which can be closely monitored and controlled. The costs of building and maintaining facilities and heating, ventilation, and air conditioning to meet these requirements are signiﬁcant. GEM husbandry is challenging because of unpredictable phenotypes, including immunologic deﬁciencies that may arise in tandem with the engineered genetic alterations. The GEM most susceptible to infections are naturally those with a deliberate impairment of their immune systems, such as nude athymic mice and severe combined immunodeﬁcient (SCID) mice. These extreme animal models are designed to blunt their ability to reject xenograft transplants, a feature that makes them attractive to oncology investigators, as tumors from a diverse number of species native to cats and dogs (as well as humans) can be transplanted into immunodeﬁcient animals to test chemotherapy agents without the confounding effect of host rejection of the xenograft. Such immunodeﬁcient rodents may also be used to establish chimeric animals having transplanted tissues and cell types from species of interest. For example, the humanized SCID mice have been transplanted with human bone marrow and therefore have circulating human blood cells. These can serve as surrogates for studying human viral infections, such as human immunodeﬁciency virus. Similar models have been constructed using feline tissues in immunodeﬁcient mouse hosts for the study of feline immunodeﬁciency virus. The other main application of GEM is to produce spontaneous diseases that mimic the features of speciﬁc veterinary and human diseases, ideal for research into these conditions. These types of GEM are also produced as genetic experiments themselves, each testing speciﬁc hypotheses about the possible role that a speciﬁc gene or combination of genes play in development or in the expression of disease. As mentioned,
this form of research is much more prevalent for study of human as opposed to veterinary diseases, in proportion to available funding. There are projects underway to speciﬁcally target and delete each of the known genes (“open reading frames”) identiﬁed in the mouse genome and evaluate the resulting effects on development, physiology, and behavior. Our further objectives are to review pertinent features of PET, SPECT CT, and MR imaging for research applications in small animals. Optical imaging is beyond the scope of this review. In addition to a brief outline of the technical aspects of the instruments, we highlight the general uses, advantages, and disadvantages of in vivo imaging as a research tool, illustrating with discussion of a few speciﬁc biological models of veterinary diseases. Finally, the husbandry challenges unique to a research environment are reviewed.
Overview of PET, SPECT, CT, and MR imaging CT, PET, and SPECT were all developed ﬁrst for use in humans and thereafter in large animals. All these techniques are 3-dimensional (3D) imaging tools that have grown to complement prior 2-dimensional (2D) devices. CT is a 3D x-ray, whereas PET and SPECT are 3D versions of planar (2D) nuclear medicine images. MRI, by contrast, was developed ﬁrst for use in small animals. Large-animal (ie, human scale) MRI came later in large part because of the expense and difﬁculty of making high-ﬁeld magnets of sufﬁcient size. Small-animal imaging with these instruments became achievable (for PET and SPECT) and desirable (for all instruments) only during the new millennium because of three major developments. First, there was a growing incentive to reduce animal sacriﬁce and animal suffering for several decades. Russell and Burch in the 1950s9 ﬁrst described the “3Rs,” which represent principles of reduction (of sacriﬁce), reﬁnement (of research methods), and replacement (of animals with other modalities, such as computer models, or animals of lower sentience). Imaging emerged as a desirable research tool as it permitted serial study using an animal as its own control. These features permitted reduced animal sacriﬁce with simultaneously improved statistical and scientiﬁc results. Second, the ongoing miniaturization of digital components made it possible to reduce the scale of many imaging procedures to permit small-animal imaging. Third, the increased use of small-animal genetic models (especially mice) emerged as a platform to study models of human and, to a lesser extent, veterinary animal disease.
CT CT is distributed worldwide and therefore is very familiar to clinical practitioners. In brief, the instrument produces 3D xray images. Essentially, information on tissue densities obtained from multiple 2D x-ray digital images that are acquired from different angles across the animal can be used to reconstruct an accurate 3D map (image) of the tissue densities. Current small-animal CT scanners can provide
Small-animal research imaging devices images with resolution from 100 m down to approximately 50 m. Images of even higher resolution are distinctly achievable but are also associated with higher radiation absorbed dose effects that may affect the biology of the research model being studied. Therefore, images at resolution below 50 m are best reserved for terminal animal study only.
PET and SPECT The principles of 3D PET10 and SPECT image reconstruction are completely analogous to CT. Instead of reconstructing 3D tissue densities from multiple 2D digital images, PET and SPECT reconstruct 3D images from multiple 2D digital radioactive emission proﬁles. But despite the same principles, practical instruments for radiotracer imaging did not become widely available until the mid-1990s, ﬁrst for human and large animals and then for small animals around the turn of the millennium. The delay was due to several factors, including (a) availability of radiotracers, which usually meant the proximity of a commercial cyclotron, (b) the demonstration of clinical utility of PET imaging, (c) hardware improvements in PET resolution (early instruments resolved no better than 2 cm), and (d) insurance coverage for PET procedures in humans. Insurance reimbursement had an indirect but powerful relevance for veterinary medicine. The ﬁnancial incentives for development and marketing of PET devices for human illness opened the door to make these instruments also available for large-animal clinical and research applications. PET and SPECT principles and technical details are discussed in the article by LeBlanc and Peremans (pages 47–56) in this issue. The capabilities of PET and SPECT speciﬁc to smallanimal imaging are unique, however, in several important ways. PET resolution of current commercially available instruments is better than 1.4 mm, comparing favorably to largescale instruments where only 4 mm is feasible. Interpretation of images from small animals, of course, requires this degree of improved resolution. State-of-the-art small-animal devices have been constructed with resolutions of 0.8 mm or better, approaching the physical limit for emissions of carbon-11 or ﬂuorine-18.
Radiotracers More important than spatial resolution of the instruments, however, are the biochemical properties of radioisotopes, which make them uniquely powerful probes of biological systems. In a simple case, radioiodine (as sodium iodide) behaves like ordinary iodide ion and has speciﬁc afﬁnity for thyroid cells and other tissues that express the sodium iodide symporter. The measurement of thyroid function in nuclear medicine, in fact, goes back to the late 1930s,11 almost immediately on the discovery of ﬁssion-derived radioisotopes such as 131I (nonradioactive iodine is 127I). Most radiotracers for general use however, unlike radioiodine, derive their function not from the intrinsic property of the radioactive moiety but from the biochemical properties of the
59 molecule to which the radioactive moiety is conjugated. Therefore, 18 F-labeled ﬂuorodeoxyglucose behaves like a glucose analog, at least up to the point of tissue uptake and phosphorylation. 18 F-ﬂuorothymidine, on the contrary, behaves more like a thymidine analog (again up to a point). Biologically useful radiotracers therefore depend on the imagination of the biologists who need them for speciﬁc purposes and the constraints imposed by the limits of radiotracer synthesis.12,13 Speciﬁc features of radiotracers include the following: (a) The ability to detect tracer quantities (picomolar sensitivity), which is unparalleled by any other technique.14 (b) The wide versatility of radiotracers, which permits functional assessment of disorders of metabolism, hypoxia, perfusion, proliferation of the heart, lung, brain, liver, kidney, bone, and disorders encompassing diabetes, hypertension, cancer, arthritis, infection, inﬂammation, and many others. (c) Radiotracer studies can be repeated, providing for a longitudinal research design that improves science and statistics by allowing animals to serve as their own controls while simultaneously reducing animal sacriﬁce. Longitudinal study therefore can potentially reduce costs of experimentation, particularly for multiple serial comparisons of the same animals (vs multiple groups of animals sacriﬁced at different time intervals). (d) Dynamic imaging of radiotracer distribution in early moments of the postinjection period permits measurement of the rates of tracer uptake into various tissues. Meanwhile late imaging of longer-lived tracers also provides unique information about delayed biodistribution, something that short-lived tracers explicitly cannot accomplish. (e) Radiotracers, by deﬁnition, are administered in quantities (picomolar) so small that they have no measureable physiological effects on the studied organism, unlike probes administered in macroscopic quantities that may be accompanied by various toxicities. (f) Finally, radiotracer use is directly translatable from small-animal models to large-animal study as the radiotracers used can be applied to any animal species. Including in vitro autoradiograph imaging of radiotracers, radioactive imaging is the only platform that is completely translatable from the cellular microscopic model to the large-animal scale.
A disadvantage of radiotracers is that radioactive animals must be housed in a way that satisﬁes safety requirements for human stafﬁng. The accumulated dose to the staff must be kept to a minimum, in accordance with safe practices, including the ALARA standards. For example, animals that receive radiotracers are not transportable through public spaces until their radioactivity has decayed ten half-lives in many university facilities. For tracers labeled with 18F (T ½ ¼ 110 minutes), this represents an overnight stay in the imaging laboratory, but for 124I (T ½ ¼ 4.2 days) animals would need
E.J. Fine et al.
60 to be housed for 42 days in the imaging laboratory unless an adjacent appropriate husbandry unit is constructed. Most small animal–imaging facilities possess a cyclotron, enabling them to produce radioactive 11C, 13N, and 15O, all positron emitters. The desirable physiology of these innately organic biochemical constituents coupled with their short halflives (20, 10, and 2 minutes, respectively) makes these radiotracers favorites for these institutions as 10 half-lives of decay permit return to distant housing facilities the same day. Nevertheless, the costs of purchase and installation of a cyclotron and annual maintenance of this complex machinery make a case for the use of radiotracers labeled with longer-lived isotopes such as 18F, 64Cu, and 124I as these can be purchased from commercial cyclotrons and shipped for use. The authors’ institution has no on-site cyclotron, for example, and has developed specialized housing near the imaging laboratory for longitudinal animal imaging studies of 18F, 64Cu, and 124I labeled tracers. The costs of the specialized housing, although not insigniﬁcant, are far lower than those of a cyclotron purchase and maintenance. In short, there are advantages and disadvantages to having a cyclotron to support an imaging facility.
MRI Even before the application to clinical imaging, MRI and nuclear magnetic resonance (NMR) spectroscopy, using vertical small-bore NMR spectrometers, were being used to study rodents.15,16 However, early animal studies were limited to anatomical imaging and crude spectroscopic measurements. Advances in technology and the recognition of the commercial potential for clinical MRI spurred development of human MRI systems. Subsequently, the need for noninvasive in vivo preclinical investigations in animal models of human diseases was recognized and high-ﬁeld small-animal MRI systems were designed and novel methods were developed speciﬁcally for small-animal studies. Advances in hardware and computing power continued to lead to higher magnetic ﬁeld strength, thus more powerful systems for both human and animal MRI. Today 3 T human and 9.4 T animal systems, with excellent magnetic ﬁeld homogeneity, are available for routine imaging. MRI is a high-resolution ( 50-100 μm for small-animal studies) imaging modality.17 It is noninvasive and nondestructive, using magnetic ﬁelds and nonionizing radiofrequency radiation to produce images, mainly of the abundant 1H in water, with soft tissue contrast depending on the local environment of the 1H (tissue, organ, ﬂuid, proximity to hemoglobin). Contrast can also be enhanced by exploiting relaxation properties of 1H in speciﬁc environments. A vast array of MR imaging sequences offers enormous versatility: experiments can be optimized for evaluating anatomical structure, tissue composition, perfusion, oxygenation, tissue elasticity, metabolism, and neuronal function. Furthermore, the development of targeted molecular probes18 using superparamagnetic iron particles, gadolinium, manganese, 19F, etc. permits the detection and tracking of cells,
antibodies, and therapeutics and improves diagnostic sensitivity. Technical details and discussion of the various applications of MRI to small-animal imaging (including diffusion tensor and functional imaging) have been previously reviewed in Seminars in Nuclear Medicine. MRI has been recognized as an outstanding diagnostic tool for humans and now is becoming common place in veterinary specialty practices. Veterinary applications include diagnosis of diseases of brain and spinal cord; evaluation of muscles, tendons, ligaments, and cartilage; detection of tumors, abscesses, and eye, ear, nose, and throat disorders; and evaluation of thoracic and abdominal disorders. Although the MRI examination itself is noninvasive and extremely safe, examinations of animals require anesthesia and, as such, carry a small risk. There are also potential complications, as would be expected in humans, such as hazards resulting from magnetic implants or adverse reaction to contrast agents. Although preclinical research instruments are most routinely used for studying small rodents or primates, clinical veterinary MRI systems capable of imaging cats, dogs, and other pets as well as larger animals (ie, horses) are available. A 2013 PubMed search (search terms: veterinary and MRI) detects almost 2000 publications, with recent studies including gas bubble disease in a California Sea Lion,19 bone marrow lesions in horses,20 and many studies on dogs and other small animals. In addition to imaging, MRI hardware permits acquisition of spectra that can be used to evaluate bioenergetics and cell metabolism. Although magnetic resonance spectroscopy is an insensitive technique, typically requiring submillimolar concentrations of metabolites, it is now routinely applied as 2D and 3D MR spectroscopic imaging in live animals. Although most tissue imaging and spectroscopic techniques measure signals derived from the 1H resonance (principally tissue water in vivo), there are a number of other nuclei that give rise to detectable NMR signals (such as 31P, 13C, 15N, 19F and 23Na) that can be exploited in spectroscopic studies for diagnostic purposes.
Coregistration The functional and physiological images obtained with PET and SPECT must be compared with anatomical images to derive maximum usefulness, whether for clinical or research applications. Side-by-side comparisons of separately obtained PET and CT scans were originally performed, but by the year 2000, it became possible to digitally superimpose the images, a process now called coregistration.21 This process had originally been achieved by imaging patients in identical positions on separate instruments followed by applying specialized software to permit the image superposition, or coregistration. The challenges included the inconvenience of maintaining precise patient position between studies as well as the requirement that image ﬁles from separate instruments were both compatible with the coregistration software. Both difﬁculties were overcome with the development of instruments such as PET/CT,21,22 SPECT/CT,23 and PET/SPECT/CT. In these
Small-animal research imaging devices
Figure 1 Coregistered PET/MRI of the thorax. The MRI is in high-deﬁnition grayscale, whereas the lower resolution of the PET image demonstrates speciﬁc uptake in the myocardium (a glucose-avid organ) of 18F-ﬂuorodeoxyglucose (FDG) on the superimposed PET scan. (Courtesy: Linda Jelicks, PhD, and Min-Hui Cui, PhD, Gruss Magnetic Resonance Research Center, AECOM).
instruments, multiple imaging modalities were combined in a single unit sharing an imaging gantry, permitting immediate sequential studies with minimal patient motion and more accurate coregistration than was possible using software-only methods. More recently, novel hardware and software developments now permit PET/MRI.24,25 An overlaid PET/MRI of rat heart (Figure 1) performed at our institution required separate imaging sessions and moving an anesthetized animal between imaging stations, followed by software alignment. However, true simultaneous image acquisition is now possible with specialized new hardware cameras, a combination which promises to be particularly useful for brain imaging.
Husbandry Challenges of Small Research Animals The use of animals as research subjects for the study of human and animal physiology and disease can be dated back to Aristotle, who anticipated some aspects of the scientiﬁc method 24 centuries ago, notably that scientiﬁc knowledge is based on experimental observation. His greatest contribution to science was, arguably, his use of dissection to understand the inner workings of numerous animals. However, the modern sciences of anatomy, physiology, and pathophysiology however may be said to have begun with the Renaissance. Leonardo da Vinci and Michelangelo both performed dissections of animals (including human cadavers) principally to understand their proper anatomy and form for better artistic rendering. Leonardo’s works also include careful drawings from the dissection of insects, ﬁsh, birds, amphibians, reptiles, as well as mammals. In a similar fashion, the modern study of physiology from experiment is often dated to the studies of William Harvey’s dissections of human cadavers to understand the human circulatory system.26 Harvey’s work was anticipated, in many ways, by the animal dissection studies of Ibn Al Naﬁs, an Arab
physician in 12th century Egypt and Syria who gave the ﬁrst description of the mammalian pulmonary circulatory system. But the performance of animal research speciﬁcally to better understand animal diseases and conditions is more recent. It was not until 1762 that the ﬁrst veterinary school in the world was established by Claude Bourgelat in Lyons, France. John Hunter in England was a veterinary practitioner in the 18th century who also performed research speciﬁcally to improve his ability to treat animals (most particularly horses). Much more recently, the use of dogs by Pavlov became a classic model applied to help understand human as well as animal behavior. By the mid-1950s the use of mice and other rodents became a common standard in biomedicine as the “lab rat” entered common parlance. However, genetic engineering of animals allowed a quantum leap in the use of laboratory mice, in particular. Speciﬁc genetic traits due to inbreeding of laboratory animals had long been cultivated for research study. But genetic engineering took that capability much further, as speciﬁc gene deletions and additions became possible. These GEM were rapidly adopted by the research community for medical as well as veterinary disease investigations. However, it became clear by the late 1990s that inbreeding, and the difﬁcult conditions imposed by close quarters, caused many of these animals to acquire infections. The incidence of new, unexpected opportunistic infections had 2 major effects on research of small animals. First, some animals died of these infections, an obvious liability for any research program. Second, however, was that nonfatal infections could result in unexpected (and unwanted) responses to the planned study interventions. The close quartering of research animals in general and GEM in particular, introduced yet another physiological variable to the planned investigation that could invalidate the study. It has become increasingly clear to academic research centers that small-animal GEM investigation is necessary, but that protection against opportunistic infection is essential for valid study interpretation. This has led to the development of the “barrier” facility, built and maintained separately and
E.J. Fine et al.
Figure 2 Breast tumors in mouse PET scan. Approximately 1 hour after the injection of approximately 300 μCi of FDG via tail vein, PET images reveal the distribution of the trapped radiotracer. Normal uptake is seen in glucose-utilizing organs such as heart, brain (cerebellum best seen in this image), and eyes, which are quite intense. Kidney uptake and bladder excretion is apparent. But breast uptake is not normally seen. These are PyMT (Polyoma middle T) mice that have a very high rate of spontaneous breast tumor and breast cancer development. The cancers are usually glycolytically dependant, glucose-avid tumors. The location of the 10 mammary glands is seen by marked oval regions indicated, and it is apparent that tumor development, size, and glycolytic behavior are quite heterogeneous. (Courtesy: Wade Koba and the M.Donald Blaufox, Laboratory of Molecular Imaging, and Pamela Stanley, PhD, and Hazuki Miwa, PhD, whose animal model was studied; all of the Albert Einstein College of Medicine).
speciﬁcally for GEM. “Conventional” mice and other conventional animals are kept in a separate, “nonbarrier” facility to prevent transmission of infections to the barrier GEM animals. The reaction of universities depended on their circumstances. GEM were particularly useful in the development of cancer models of disease, both in SCID or nude mice and in GEM with spontaneous tumor development. The Memorial Sloan Kettering Hospital Center in New York proceeded by building a “barrier-only” institution, eliminating their conventional animal facilities. The additional expense of 2 distinct animal facilities with different requirements was, on balance, not justiﬁed for the research goals of the institution. The debate at the Albert Einstein College of Medicine, the parent institution of this review’s authors, continues over these issues as well. The advantages of a “barrier-only” facility are not just ﬁnancial. The barrier’s sterility can be expected to be easier to maintain without the potential for staff to track in microorganisms carried inadvertently from the conventional animal facility. However, there remain many investigators at Einstein who wish to continue studies on conventional animals. In speciﬁc animal models of human disease, pertinent to our institution, genetically altered animals are not always needed; conversely, conventional animals are less expensive to purchase, breed, and maintain. The types of animal facilities required for veterinary research inevitably harbor these same conﬂicts.
Speciﬁc Models of Animal Disease The authors of the present article work at a medical university whose animal enterprise concerns research relevant to human physiology and disease rather than veterinary, mostly in rodents. However, the kinds of animal models that are used include spontaneous diseases in rodents which can apply to analogous diseases in other animal species. Notable examples include spontaneous breast tumor models, which besides human application would have relevance to canine breast
Figure 3 Lung cancers in aging mice on PET/CT. The CT image is displayed in grayscale, while the superimposed and coregistered FDG PET image demonstrates, in hot iron colors, 2 glycolytically dependant tumors at the base of the right lung. (Courtesy: Wade Koba and the M.Donald Blaufox Laboratory of Molecular Imaging, AECOM, and Claudia Gravekamp, PhD whose animal model was studied).
Small-animal research imaging devices
Figure 4 Tibial fracture on PET scan and healing: In both images, a PET bone scan was performed approximately 1 hour after the injection of 500 μCi of 18F sodium ﬂuoride. On the left, the image was obtained about 1 week after an experimental fracture of the tibia. Increased uptake in the fracture area is apparent, owing to increased osteoblastic activity and resultant radioﬂuoride ion substituting for hydroxyl groups in hydroxyapatite crystal of repairing bone. On the right, a follow-up image shows complete resolution of the bone. (Courtesy: Wade Koba and the M.Donald Blaufox Laboratory of Molecular Imaging, AECOM, and Berish Strauch, MD, and whose animal model was studied).
cancers, as well as models of cancers resulting from aging (also a signiﬁcant risk factor for cancer in dogs). The most common spontaneous breast tumor model is the polyoma mixed T genetically modiﬁed mouse that Einstein investigators have studied extensively (Figure 2). Spontaneous cancers in aging mice could potentially be a useful model for canine cancers (Figure 3). Bone fractures are a serious veterinary problem in thoroughbred horses, particularly. It is problematic for any rodent
model to recapitulate the physiology and species differences in long-bone stresses that lead to these kinds of fractures, but speciﬁc strategies to speed the healing process such as electromagnetic stimulation may have applications across species (Figure 4). Thyroid diseases, common in cats as well as dogs, include hyperthyroidism and more rarely thyroid cancer.27 Innovative conditional mouse thyroid gene modiﬁcations28,29 include knockout of the PTEN gene, which results in thyroid
Figure 5 Thyroid tumors in mice. PET images are obtained approximately 45 minutes after the injection of 300 μCi of FDG via tail vein. A mouse with conditional knockouts of thyroidal PTEN and p53 genes ﬁrst develops thyroid hyperplasia, as seen on the left coronal slice image, reﬂecting thyroid tissue proliferation due to lack of PTEN protein expression. Within just 2 weeks, the suppression of apoptosis (caused by the additional p53 knockout) has already led to anaplastic thyroid cancer as seen by the chaotic appearing glycolytic thyroid tumor in the same mouse on the right image. (Courtesy: Wade Koba and the M.Donald Blaufox Laboratory of Molecular Imaging, AECOM, and Antonio Di Cristofano, PhD whose animal model was studied).
E.J. Fine et al.
Figure 6 Goldﬁsh PET bone scan and FDG uptake images. Goldﬁsh were injected with approximately 300 μCi of 18F-NaF (left) or FDG (right), and in vivo PET images were obtained approximately 45 minutes later in a small research aquarium designed for the purpose. The left image demonstrates the bony distribution of radioactive ﬂuoride, whereas the metabolic distribution of FDG is seen on the right. (Courtesy: Wade Koba and Eugene Fine of the M.Donald Blaufox Laboratory of Molecular Imaging, AECOM).
hyperplasia, while an additional knockout of the p53 gene leads to anaplastic thyroid cancer (Figure 5).
In Vivo Small Fish and Aquatic Species Imaging Small-animal models used for preclinical imaging include a variety of mammals, although mice are preferred, owing to lower costs associated with purchase, housing, and animal care. Until recently, ﬁsh were not considered a useful imaging option as most ﬁsh diseases are not shared with humans, and in vivo imaging remained challenging for aquatic species in general. The zebraﬁsh (Danio rerio) has been studied since the 1970s as a research model whose genome is now sequenced. giant danios, at 6 times the length of zebraﬁsh, are more comparable to mouse size and could prove suitable for the resolution capabilities of small-animal imaging. Survivability of most ﬁsh species in air is poor for durations exceeding a few minutes. In the past, Carassius auratus (goldﬁsh) have been imaged by both MRI and CT, but physiological in vivo study of ﬁsh using molecular imaging techniques has proven challenging. Hydrogel30-32 has been used to sustain a ﬁsh for a few minutes out of water for research purposes but cannot be considered physiological. Hydrogel is also unrealistic for PET or SPECT imaging which may require the living animal to remain motionless for up to 120 minutes or longer, depending on the application. An immobilizing imaging aquarium, designed by 2 of the authors (W.K. and E.J.F.), sustains a conscious aquatic animal by supplying oxygenated water through either direct bubble aeration or continuous water ﬂow. The vessel provides a volume of water adequate for oxygen delivery, but small
enough to minimize attenuation of γ radiation that would be detected by a PET or SPECT imaging system. Although PET, SPECT, and CT can use bubbler or continuous ﬂow oxygen delivery systems, MRI is corrupted by air pocket artifacts from air bubbles so aeration must be achieved using a continuous water ventilation system with intake tubing and a sink drain for the return. Although more cumbersome, continuous water replacement permits removal of waste contaminants therefore allowing sustained studies, beneﬁcial for imaging or nonimaging studies. The aquarium’s restraints minimize head and ﬁn motion while permitting movement of the operculum. The animal remains conscious during this process without harm. However, acclimatization may still be helpful during an experiment. An interventional port within the aquarium offers the potential for physiological interventions or animal monitoring or both in applications such as (a) gavage (controlled feeding); (b) electroencephalography and electrocardiogram monitoring, or gating or both; (c) water temperature monitoring or modulating; and (d) introduction of pharmaceuticals. Hibernation studies are also possible. The goldﬁsh model has proven that ﬁsh can be imaged in vivo (Figure 6), but at present has limited research applications. On the contrary, the zebraﬁsh or perhaps giant danios would provide a broader variety of applications and may also be cost-effective. The giant danio may be the only animal that can be completely studied from the embryonic stage by light microscopy to molecular in vivo imaging of the adult.
References 1. Head LL, Daniel GB, Becker TJ, et al: Use of computed tomography and radiolabeled leukocytes in a cat with pancreatitis. Vet Radiol Ultrasound: 2005;46(3):263-266
Small-animal research imaging devices 2. Hecht S, Daniel GB, Mitchell SK: Diuretic renal scintigraphy in normal dogs. Vet Radiol Ultrasound: 2006;47(6):602-608 3. Lenard ZM, Zuber RM, Nicoll RG, et al: Evaluation of lymphoma in a cat using 99m Tc-sestamibi. Vet Radiol Ultrasound: 2005;46(6):533-535 4. Morandi F, Cole RC, Echandi RL, et al: Transsplenic portal scintigraphy using 99m Tc-mebrofenin in normal dogs. Vet Radiol Ultrasound: 2007;48(3):286-291 5. Steyn PF, Uhrig J: The role of protective lead clothing in reducing radiation exposure rates to personnel during equine bone scintigraphy. Vet Radiol Ultrasound: 2005;46(6):529-532 6. Sykes JM, Ramsay EC, Schumacher J, et al: Evaluation of an implanted osmotic pump for delivery of amikacin to corn snakes (Elaphe guttata guttata). J Zoo Wildl Med: 2006;37(3):373-380 7. Sykes JM, Schumacher J, Avenell J, et al: Preliminary evaluation of 99m Technetium diethylenetriamine pentaacetic acid, 99m Technetium dimercaptosuccinic acid, and 99m Technetium mercaptoacetyltriglycine for renal scintigraphy in corn snakes (Elaphe guttata guttata). Vet Radiol Ultrasound: 2006;47(2):222-227 8. Koba W, Lipton ML, Jelicks L, et al: Imaging devices for use in small animals. Semin Nucl Med: 2011;41(3):151-165 9. Russell WMS, Burch RL: The Principles of Humane Experimental Technique. London, Methuen, 1959. Reprinted: Universities Federation for Animal Welfare (UFAW), England, 1992 10. Wagner HN Jr: A brief history of positron emission tomography (PET). Semin Nucl Med: 1998;28(3):213-220 11. Hamilton JG, Soley MH: Studies in iodine metabolism by the use of a new radioactive isotope of iodine. Am J Physiol: 1939;127(3):557-572 12. Hwang DW, Ko HY, Kim SK, et al: Development of a quadruple imaging modality by using nanoparticles. Chemistry: 2009;15(37):9387-9393 13. Louie A: Multimodality imaging probes: Design and challenges. Chem Rev: 2010;5(3):146-3195 14. Gambhir SS: Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer: 2002;2(9):683-693 15. DeLuca F, DeSimone BC, Maraviglia B: Role of 3D imaging in small scale NMR tomography. Magn Reson Imaging: 1982;1(4):205-208 16. Buonanno FS, Pykett IL, Kistler JP, et al: Cranial anatomy and detection of ischemic stroke in the cat by nuclear magnetic resonance imaging. Radiology: 1982;143(1):187-193 17. Stafford RJ: High ﬁeld MRI—Technology, applications, safety and limitations. Medical Phys: 2005;32(6):2077
65 18. Hasegawa S, Furukawa T, Saga T: Molecular MR imaging of cancer gene therapy: Ferritin transgene reporter takes the stage. Magn Reson Med Sci: 2010;9(2):37-47 19. VanBonn W, Dennison S, Cook P, et al: Gas bubble disease in the brain of a living California sea lion (Zalophus californianus). Front Physiol 2013;4:5 20. Olive J, Vila T, Serraud N: Comparison of inversion recovery gradient echo with inversion recovery fast spin echo techniques for magnetic resonance imaging detection of navicular bone marrow lesions in horses. Am J Vet Res: 2013;74(2):232-238 21. Beyer T, Townsend DW, Brun T, et al: A combined PET/CT scanner for clinical oncology. J Nucl Med: 2000;41(8):1369-1379 22. Townsend DW: Dual-modality imaging: Combining anatomy and function. J Nucl Med: 2008;49(6):938-955 23. Cherry SR: Multimodality imaging: Beyond PET/CT and SPECT/CT. Semin Nucl Med: 2009;39(5):348-353 24. Catana C, Wu Y, Judenhofer MS, et al: Simultaneous acquisition of multislice PET and MR images: Initial results with a MR-compatible PET scanner. J Nucl Med: 2006;47(12):1968-1976 25. Judenhofer MS, Wehrl HF, Newport DF, et al: Simultaneous PET-MRI: A new approach for functional and morphological imaging. Nat Med: 2008;14(4):459-465 26. Harvey W.: On the motion of the heart and blood in animals, 1628; translated by Wills R1847; revised Bowie A (ed) 1989; Google ebook 2012 〈http://ebooks.adelaide.edu.au/h/harvey/william/motion/contents.html〉 27. Hibbert AS, Gruffydd-Jones T, Barrett EL, et al: Feline thyroid carcinoma: Diagnosis and response to high-dose radioactive iodine treatment. J Feline Med Surg: 2009;11(2):116-124 28. Antico Arciuch VG, Russo MA, Dima M, et al: Thyrocyte-speciﬁc inactivation of p53 and Pten results in anaplastic thyroid carcinomas faithfully recapitulating human tumors. Oncotarget: 2011;2(12): 1109-1126 29. Russo MA, Antico Arciuch VG, DiCristofano A: Mouse models of follicular and papillary thyroid cancer progression. Front Endocrinol (Lausanne) 2011;2:119 30. Hwang DC, Damodaran S: Synthesis and properties of ﬁsh protein-based hydrogel. J Am Oil Chem Soc: 1997;74(9):1165-1171 31. Rathna GVN, Damodaran S: Effect of nonprotein polymers on wateruptake properties of ﬁsh protein-based hydrogel. J Appl Polym Sci: 2002;85(1):45-51 32. Zhang SG: Hydrogels—Wet of let die. Nat Mater: 2004;3(1):7-8