Micro-CT of rodents: State-of-the-art and future perspectives

Micron-scale computed tomography (micro-CT) is an essential tool for phenotyping and for elucidating diseases and their therapies. This work is focused on preclinical micro-CT imaging, reviewing relevant principles, technologies, and applications. Commonly, micro-CT provides high-resolution anatomic information, either on its own or in conjunction with lower-resolution functional imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT). More recently, however, advanced applications of micro-CT produce functional information by translating clinical applications to model systems (e.g. measuring cardiac functional metrics) and by pioneering new ones (e.g. measuring tumor vascular permeability with nanoparticle contrast agents). The primary limitations of micro-CT imaging are the associated radiation dose and relatively poor soft tissue contrast. We review several image reconstruction strategies based on iterative, statistical, and gradient sparsity regularization, demonstrating that high image quality is achievable with low radiation dose given ever more powerful computational resources. We also review two contrast mechanisms under intense development. The first is spectral contrast for quantitative material discrimination in combination with passive or actively targeted nanoparticle contrast agents. The second is phase contrast which measures refraction in biological tissues for improved contrast and potentially reduced radiation dose relative to standard absorption imaging. These technological advancements promise to develop micro-CT into a commonplace, functional and even molecular imaging modality.     

Morphology of elastase-induced cerebral aneurysm model in rabbit and rapid prototyping of elastomeric transparent replicas

In this work, we describe a methodology to fabricate transparent elastomeric vascular replicas using rapid prototyping techniques. First, the three-dimensional morphology of an elastase-induced aneurysm model in rabbit is acquired. The morphology is reconstructed from in vivo rotational angiography and it is compared with three-dimensional reconstructions obtained by computerized tomography and magnetic resonance imaging of an intraluminal arterial cast that was obtained from the same animal at sacrifice. Results show that resolution of the imaging modality strongly influences the level of detail, such as small side branches, in the final reconstruction. We developed an average morphology model for elastase-induced aneurysms in rabbits including the surrounding vasculature and describe a method for rapid prototyping of vascular models from the three-dimensional morphology. Our replicas can be manufactured in a short period of time and the final product is optically clear. In addition, the elasticity of the models can be controlled to represent arterial elasticity, which makes them ideal for optical investigations of detailed flow dynamics using measurement tools such as particle image velocimetry.     

A realistic utilization of nanotechnology in molecular imaging and targeted radiotherapy of solid tumors

Precise dose delivery to malignant tissue in radiotherapy is of paramount importance for treatment efficacy while minimizing morbidity of surrounding normal tissues. Current conventional imaging techniques, such as magnetic resonance imaging (MRI) and computerized tomography (CT), are used to define the three-dimensional shape and volume of the tumor for radiation therapy. In many cases, these radiographic imaging (RI) techniques are ambiguous or provide limited information with regard to tumor margins and histopathology. Molecular imaging (MI) modalities, such as positron emission tomography (PET) and single photon-emission computed-tomography (SPECT) that can characterize tumor tissue, are rapidly becoming routine in radiation therapy. However, their inherent low spatial resolution impedes tumor delineation for the purposes of radiation treatment planning. This review will focus on applications of nanotechnology to synergize imaging modalities in order to accurately highlight, as well as subsequently target, tumor cells. Furthermore, using such nano-agents for imaging, simultaneous coupling of novel therapeutics including radiosensitizers can be delivered specifically to the tumor to maximize tumor cell killing while sparing normal tissue.     

New molecular mediators in tumor angiogenesis

Angiogenesis is essential for tumor growth and progression. It has been demonstrated that tumor growth beyond a size 1 to 2 mm³ requires the induction of new vessels. Angiogenesis is regulated by several endogenous stimulators and inhibitors of endothelial cell migration, proliferation and tube formation. Under physiological conditions these mediators of endothelial cell growth are in balance and vessel growth is limited. In fact, within the angiogenic balance endothelial cell turnover is sufficient to maintain a functional vascular wall but does not allow vessel growth. Tumor growth an progression has successfully been correlated to the serum concentration of angiogenic mediators. Furthermore, the vascular density of tumor tissues could be correlated to the clinical course of the disease in several tumor entities. Within the last years several new mediators of endothelial cell growth have been isolated e.g. angiopoietin 1, angiopoietin 2, midkine, pleiotropin, leptin and maspin. In this review we discuss the mechanisms leading to tumor angiogenesis and describe some of the newer mediators of endothelial cell stimulation and inhibition.     

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging,Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.For this purpose, we injected 10⁵ MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg¹. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula¹. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified^2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions^5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles⁷.In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.     

Imaging lung perfusion

From the first measurements of the distribution of pulmonary blood flow using radioactive tracers by West and colleagues (J Clin Invest 40: 1-12, 1961) allowing gravitational differences in pulmonary blood flow to be described, the imaging of pulmonary blood flow has made considerable progress. The researcher employing modern imaging techniques now has the choice of several techniques, including magnetic resonance imaging (MRI), computerized tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT). These techniques differ in several important ways: the resolution of the measurement, the type of contrast or tag used to image flow, and the amount of ionizing radiation associated with each measurement. In addition, the techniques vary in what is actually measured, whether it is capillary perfusion such as with PET and SPECT, or larger vessel information in addition to capillary perfusion such as with MRI and CT. Combined, these issues affect quantification and interpretation of data as well as the type of experiments possible using different techniques. The goal of this review is to give an overview of the techniques most commonly in use for physiological experiments along with the issues unique to each technique.     

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).     

Viral nanoparticles as tools for intravital vascular imaging

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.     

Retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample. One method which circumvents these issues is micro-Computed Tomography (μCT). μCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry). However, imaging vessels by ex vivo μCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by μCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for μCT imaging. Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch), or ex vivo via a Langendorff perfusion system. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network--arteries, capillaries, and veins alike.     

VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype

Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype.     

In vivo characterization of tumor and tumor vascular network using multi‐modal imaging approach

We present a multi‐modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vivo micro-CT imaging of liver lesions in small animal models

Three-dimensional micro computed tomography (microCT) offers the opportunity to capture images liver structures and lesions in mice with a high spatial resolution. Non-invasive microCT allows for accurate calculation of vessel tortuosity and density, as well as liver lesion volume and distribution. Longitudinal monitoring of liver lesions is also possible. However, distinguishing liver lesions from variations within a normal liver is impossible by microCT without the use of liver- or tumor-specific contrast-enhancing agents. The combination of microCT for morphologic imaging with functional imaging, such as positron emission tomography (PET) or single photon emission tomography (SPECT), offers the opportunity for better abdominal imaging and assessment of structure discrepancies visible by functional imaging.This paper describes methods of current microCT imaging options for imaging of liver lesions compared to other imaging techniques in small animals.     

Multimodality imaging of nitric oxide and nitric oxide synthases

Nitric oxide (NO) and NO synthases (NOSs) are crucial factors in many pathophysiological processes such as inflammation, vascular/neurological function, and many types of cancer. Noninvasive imaging of NO or NOS can provide new insights in understanding these diseases and facilitate the development of novel therapeutic strategies. In this review, we will summarize the current state-of-the-art multimodality imaging in detecting NO and NOSs, including optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET). With continued effort over the last several years, these noninvasive imaging techniques can now reveal the biodistribution of NO or NOS in living subjects with high fidelity which will greatly facilitate scientists/clinicians in the development of new drugs and/or patient management. Lastly, we will also discuss future directions/applications of NO/NOS imaging. Successful development of novel NO/NOS imaging agents with optimal in vivo stability and desirable pharmacokinetics for clinical translation will enable the maximum benefit in patient management.     

Unsupervised Deconvolution of Dynamic Imaging Reveals Intratumor Vascular Heterogeneity and Repopulation Dynamics

With the existence of biologically distinctive malignant cells originated within the same tumor, intratumor functional heterogeneity is present in many cancers and is often manifested by the intermingled vascular compartments with distinct pharmacokinetics. However, intratumor vascular heterogeneity cannot be resolved directly by most in vivo dynamic imaging. We developed multi-tissue compartment modeling (MTCM), a completely unsupervised method of deconvoluting dynamic imaging series from heterogeneous tumors that can improve vascular characterization in many biological contexts. Applying MTCM to dynamic contrast-enhanced magnetic resonance imaging of breast cancers revealed characteristic intratumor vascular heterogeneity and therapeutic responses that were otherwise undetectable. MTCM is readily applicable to other dynamic imaging modalities for studying intratumor functional and phenotypic heterogeneity, together with a variety of foreseeable applications in the clinic.     

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.     

Current and future anatomical and functional imaging approaches to pheochromocytoma and paraganglioma

After establishing a biochemical diagnosis, pheochromocytomas and extra-adrenal paragangliomas (PPGLs) can be localized using different anatomical and functional imaging modalities. These include computed tomography, magnetic resonance imaging, single-photon emission computed tomography (SPECT) using 123I-metaiodobenzylguanidine or 111In-DTPA-pentetreotide, and positron emission tomography (PET) using 6-[18F]-fluorodopamine (18F-FDA), 6-[18F]-fluoro-l-3,4-dihydroxyphenylalanine (18F-DOPA), and 2-[18F]-fluoro-2-deoxy-d-glucose. We review the currently available data on the performance of anatomical imaging, SPECT, and PET for the detection of (metastatic) PPGL as well as parasympathetic head and neck paragangliomas. We show that there appears to be no 'gold-standard' imaging technique for all patients with (suspected) PPGL. A tailor-made approach is warranted, guided by clinical, biochemical, and genetic characteristics. In the current era of a growing number of PET tracers, PPGL imaging has moved beyond tumor localization towards functional characterization of tumors.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

Three-dimensional imaging of the mouse neurovasculature with magnetic resonance microscopy

Knowledge of the three-dimensional (3D) architecture of blood vessels in the brain is crucial because the progression of various neuropathologies ranging from Alzheimer's disease to brain tumors involves anomalous blood vessels. The challenges in obtaining such data from patients, in conjunction with development of mouse models of neuropathology, have made the murine brain indispensable for investigating disease induced neurovascular changes. Here we describe a novel method for "whole brain" 3D mapping of murine neurovasculature using magnetic resonance microscopy (μMRI). This approach preserves the vascular and white matter tract architecture, and can be combined with complementary MRI contrast mechanisms such as diffusion tensor imaging (DTI) to examine the interplay between the vasculature and white matter reorganization that often characterizes neuropathologies. Following validation with micro computed tomography (μCT) and optical microscopy, we demonstrate the utility of this method by: (i) combined 3D imaging of angiogenesis and white matter reorganization in both, invasive and non-invasive brain tumor models; (ii) characterizing the morphological heterogeneity of the vascular phenotype in the murine brain; and (iii) conducting "multi-scale" imaging of brain tumor angiogenesis, wherein we directly compared in vivo MRI blood volume measurements with ex vivo vasculature data.