A review of high-resolution X-ray computed tomography and other imaging modalities for small animal research

Dedicated high-resolution small animal systems have recently emerged as important new tools for laboratory animal research. These imaging systems permit researchers to noninvasively screen animal models for mutations or pathologies and to monitor disease progression and response to therapy. The authors survey various small animal imaging modalities, including MRI, PET, SPECT, and microCT, and discuss several representative microCT mouse imaging studies.     

Anesthesia and other considerations for in vivo imaging of small animals

The use of small animal imaging is increasing in biomedical research thanks to its ability to localize altered biochemical and physiological processes in the living animal and to follow these processes longitudinally and noninvasively. In contrast to human studies, however, imaging of small animals generally requires anesthesia, and anesthetic agents can have unintended effects on animal physiology that may confound the results of the imaging studies. In addition, repeated anesthesia, animal preparation for imaging, exposure to ionizing radiation, and the administration of contrast agents may affect the processes under study. We discuss this interplay of factors for small animal imaging in the context of four common imaging modalities for small animals: positron emission tomography (PET) and single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. We discuss animal preparation for imaging, including choice of animal strain and gender, the role of fasting and diet, and the circadian cycle. We review common anesthesias used in small animal imaging, such as pentobarbital, ketamine/xylazine, and isoflurane, and describe techniques for monitoring the respiration and circulation of anesthetized animals that are being imaged as well as developments for imaging conscious animals. We present current imaging literature exemplifying how anesthesia and animal handling can influence the biodistribution of PET tracers. Finally, we discuss how longitudinal imaging studies may affect animals due to repeated injections of radioactivity or other substrates and the general effect of stress on the animals. In conclusion, there are many animal handling issues to consider when designing an imaging experiment. Reproducible experimental conditions require clear, consistent reporting, in the study design and throughout the experiment, of the animal strain and gender, fasting, anesthesia, and how often individual animals were imaged.     

Imaging of experimental osteoarthritis in small animal models

Normally, tissue alterations in small animal models for osteoarthritis (OA) are assessed by time-consuming and destructive histology or biochemical assays. Some high resolution imaging modalities are used for longitudinal monitoring of the OA disease process in vivo. microCT is one of these imaging modalities, which is known for superb high-resolution imaging of bone architecture alterations. A major drawback of microCT is that it has low soft-tissue contrast, which makes direct imaging of cartilage impossible. The use of microCT in combination with negatively charged radiopaque contrast agents enables imaging of cartilage degeneration. We demonstrate the possibility of microCT to image cartilage degeneration as a consequence of experimental OA, by the use contrast enhanced microCT in vivo in a rat model for OA. Furthermore, for the assessment of alterations in molecular processes involved in OA we used the recently developed technique of multi pinhole SPECT. This enables us to assess molecular processes involved in experimental OA in a rat at sub-millimeter level. Here we show quantification of subchondral bone turnover in an OA rat knee. These new techniques demonstrate the possibilities of quantitative experimental OA assessment in small animal models such as mice and rats and might enable substitution of the conventional destructive methods.     

Three-dimensional in vivo imaging of the murine liver: a micro-computed tomography-based anatomical study

Various murine models are currently used to study acute and chronic pathological processes of the liver, and the efficacy of novel therapeutic regimens. The increasing availability of high-resolution small animal imaging modalities presents researchers with the opportunity to precisely identify and describe pathological processes of the liver. To meet the demands, the objective of this study was to provide a three-dimensional illustration of the macroscopic anatomical location of the murine liver lobes and hepatic vessels using small animal imaging modalities. We analysed micro-CT images of the murine liver by integrating additional information from the published literature to develop comprehensive illustrations of the macroscopic anatomical features of the murine liver and hepatic vasculature. As a result, we provide updated three-dimensional illustrations of the macroscopic anatomy of the murine liver and hepatic vessels using micro-CT. The information presented here provides researchers working in the field of experimental liver disease with a comprehensive, easily accessable overview of the macroscopic anatomy of the murine liver.     

Imaging modalities to assess structural birth defects in mutant mouse models

Assessment of structural birth defects (SBDs) in animal models usually entails conducting detailed necropsy for anatomical defects followed by histological analysis for tissue defects. Recent advances in new imaging technologies have provided the means for rapid phenotyping of SBDs, such as using ultra‐high frequency ultrasound biomicroscopy, optical coherence tomography, micro‐CT, and micro‐MRI. These imaging modalities allow the detailed assessment of organ/tissue structure, and with ultrasound biomicroscopy, structure and function of the cardiovascular system also can be assessed noninvasively, allowing the longitudinal tracking of the fetus in utero. In this review, we briefly discuss the application of these state‐of‐the‐art imaging technologies for phenotyping of SBDs in rodent embryos and fetuses, showing how these imaging modalities may be used for the detection of a wide variety of SBDs. Birth Defects Research (Part C) 90:176–184, 2010. © 2010 Wiley‐Liss, Inc.     

Inside out: modern imaging techniques to reveal animal anatomy

Animal anatomy has traditionally relied on detailed dissections to produce anatomical illustrations, but modern imaging modalities, such as MRI and CT, now represent an enormous resource that allows for fast non-invasive visualizations of animal anatomy in living animals. These modalities also allow for creation of three-dimensional representations that can be of considerable value in the dissemination of anatomical studies. In this methodological review, we present our experiences using MRI, CT and μCT to create advanced representation of animal anatomy, including bones, inner organs and blood vessels in a variety of animals, including fish, amphibians, reptiles, mammals, and spiders. The images have a similar quality to most traditional anatomical drawings and are presented together with interactive movies of the anatomical structures, where the object can be viewed from different angles. Given that clinical scanners found in the majority of larger hospitals are fully suitable for these purposes, we encourage biologists to take advantage of these imaging techniques in creation of three-dimensional graphical representations of internal structures.     

Clinical considerations in rodent bioimaging

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution magnetic resonance imaging (MRI), optical imaging, and high-resolution ultrasound are rapidly becoming invaluable research tools. These advanced imaging technologies are now commonly used to investigate rodent biology, metabolism, pharmacokinetics, and disease in vivo. Choosing an appropriate anesthetic regimen as well as monitoring and supporting the animal's physiologic balance is key to obtaining images that truly represent the biologic process or disease state of interest. However, there are many challenges in rodent bioimaging such as limited animal access, small sample volumes, anesthetic complications, strain and gender variability, and the introduction of image artifacts. Because each imaging study presents unique challenges, a thorough understanding of the imaging modality used, the animal's health status, and the research data desired is required. This article addresses these issues along with other common laboratory animal clinical considerations such as biosecurity and radiation safety in in vivo rodent bioimaging.     

Aptamer-based molecular imaging

Molecular imaging has greatly advanced basic biology and translational medicine through visualization and quantification of single/multiple molecular events temporally and spatially in a cellular context and in living organisms. Aptamers, short single-stranded nucleic acids selected in vitro to bind a broad range of target molecules avidly and specifically, are ideal molecular recognition elements for probe development in molecular imaging. This review summarizes the current state of aptamer-based biosensor development (probe design and imaging modalities) and their application in imaging small molecules, nucleic acids and proteins mostly in a cellular context with some animal studies. The article is concluded with a brief discussion on the perspective of aptamer-based molecular imaging.     

Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent

BACKGROUND: Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities. METHODS: A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye. RESULTS: Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging. CONCLUSION: A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.     

The usefulness of radiotracers to make the body biochemically transparent

Summary. Radioactive isotopes are uniquely applicable to observe reactions or circuits of reactions at the molecular level without disturbing the system being studied. The advent of molecular imaging modalities, particularly positron emission tomography (PET), is a major breakthrough for the visualisation and quantitative assessment of cellular and molecular processes occurring in living tissues. The recent development of animal PET scanners that offers 2-mm resolution and is tailored to laboratory rodent models, has made a further great impact on in vivo biochemistry. With these live-imaging modalities at hand, radiotracer-based technologies allow to look directly at biochemical distribution and interaction processes. Tremendous progress made in radiotracer chemistry, primarily in carbon-11 and fluorine-18 radiochemistry, and in the design of imaging devices strengthens the usefulness of radiotracers in nuclear medicine and drug research and development and opens exciting opportunities for new applications, e.g., in food science.     

Multimodal imaging for tumour characterization from micro‐ to macroscopic level using a newly developed dorsal chamber designed for long‐term follow‐up

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One‐shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow‐up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long‐term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long‐term biological processes, as in cancer research.     

Illuminating the Regenerative Properties of Stem Cells In Vivo with Bioluminescence Imaging

Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real-time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research.     

Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Introduction to rodent cardiac imaging

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.     

Quantitative in vivo imaging of embryonic development: opportunities and challenges

Animal models are critically important for a mechanistic understanding of embryonic morphogenesis. For decades, visualizing these rapid and complex multidimensional events has relied on projection images and thin section reconstructions. While much insight has been gained, fixed tissue specimens offer limited information on dynamic processes that are essential for tissue assembly and organ patterning. Quantitative imaging is required to unlock the important basic science and clinically relevant secrets that remain hidden. Recent advances in live imaging technology have enabled quantitative longitudinal analysis of embryonic morphogenesis at multiple length and time scales. Four different imaging modalities are currently being used to monitor embryonic morphogenesis: optical, ultrasound, magnetic resonance imaging (MRI), and micro-computed tomography (micro-CT). Each has its advantages and limitations with respect to spatial resolution, depth of field, scanning speed, and tissue contrast. In addition, new processing tools have been developed to enhance live imaging capabilities. In this review, we analyze each type of imaging source and its use in quantitative study of embryonic morphogenesis in small animal models. We describe the physics behind their function, identify some examples in which the modality has revealed new quantitative insights, and then conclude with a discussion of new research directions with live imaging.     

Design of a multimodal fibers optic system for small animal optical imaging

Small animals optical imaging systems are widely used in pre-clinical research to image in vivo the bio-distribution of light emitting probes using fluorescence or bioluminescence modalities. In this work we presented a set of simulated results of a novel small animal optical imaging module based on a fibers optics matrix, coupled with a position sensitive detector, devoted to acquire bioluminescence and Cerenkov images. Simulations were performed using GEANT 4 code with the GAMOS architecture using the tissue optics plugin. Results showed that it is possible to image a 30 × 30 mm region of interest using a fiber optics array containing 100 optical fibers without compromising the quality of the reconstruction. The number of fibers necessary to cover an adequate portion of a small animal is thus quite modest. This design allows integrating the module with magnetic resonance (MR) in order to acquire optical and MR images at the same time. A detailed model of the mouse anatomy, obtained by segmentation of 3D MRI images, will improve the quality of optical 3D reconstruction.     

Cerebral Glioma Grading Using Bayesian Network with Features Extracted from Multiple Modalities of Magnetic Resonance Imaging

Many modalities of magnetic resonance imaging (MRI) have been confirmed to be of great diagnostic value in glioma grading. Contrast enhanced T1-weighted imaging allows the recognition of blood-brain barrier breakdown. Perfusion weighted imaging and MR spectroscopic imaging enable the quantitative measurement of perfusion parameters and metabolic alterations respectively. These modalities can potentially improve the grading process in glioma if combined properly. In this study, Bayesian Network, which is a powerful and flexible method for probabilistic analysis under uncertainty, is used to combine features extracted from contrast enhanced T1-weighted imaging, perfusion weighted imaging and MR spectroscopic imaging. The networks were constructed using K2 algorithm along with manual determination and distribution parameters learned using maximum likelihood estimation. The grading performance was evaluated in a leave-one-out analysis, achieving an overall grading accuracy of 92.86% and an area under the curve of 0.9577 in the receiver operating characteristic analysis given all available features observed in the total 56 patients. Results and discussions show that Bayesian Network is promising in combining features from multiple modalities of MRI for improved grading performance.     

Molecular-genetic imaging: a nuclear medicine-based perspective

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.