Small animal positron emission tomography in food sciences

Summary. Positron emission tomography (PET) is a 3-dimensional imaging technique that has undergone tremendous developments during the last decade. Non-invasive tracing of molecular pathways in vivo is the key capability of PET. It has become an important tool in the diagnosis of human diseases as well as in biomedical and pharmaceutical research. In contrast to other imaging modalities, radiotracer concentrations can be determined quantitatively. By application of appropriate tracer kinetic models, the rate constants of numerous different biological processes can be determined. Rapid progress in PET radiochemistry has significantly increased the number of biologically important molecules labelled with PET nuclides to target a broader range of physiologic, metabolic, and molecular pathways. Progress in PET physics and technology strongly contributed to better scanners and image processing. In this context, dedicated high resolution scanners for dynamic PET studies in small laboratory animals are now available. These developments represent the driving force for the expansion of PET methodology into new areas of life sciences including food sciences. Small animal PET has a high potential to depict physiologic processes like absorption, distribution, metabolism, elimination and interactions of biologically significant substances, including nutrients, ‘nutriceuticals’, functional food ingredients, and foodborne toxicants. Based on present data, potential applications of small animal PET in food sciences are discussed.     

Visualizing protein-protein interactions in living animals

A variety of techniques have been developed to analyze protein-protein interactions in vitro and in cultured cells. However, these methods do not determine how protein interactions affect and are regulated by physiologic and pathophysiologic conditions in living animals. This article describes methodology for detecting and quantifying protein interactions in living mice, using an inducible two-hybrid system developed for positron emission tomography (PET) imaging. We discuss the methods to establish stably transfected cells with components of the imaging system, create tumor xenografts, synthesize PET radiopharmaceuticals used to visualize the imaging reporter, perform microPET imaging, and analyze data from imaging studies. Development and application of technologies for molecular imaging of protein-protein interactions in vivo should enable researchers to investigate intrinsic binding specificities of proteins during normal development and disease progression as well as aid drug development through direct interrogation of molecular targets within intact animals.     

<Emphasis Type="Italic">In vivo</Emphasis> molecular imaging of experimental joint inflammation by combined <Superscript>18</Superscript>F-FDG positron emission tomography and computed tomography

Introduction  

The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation.     

Application of Palladium-Mediated 18F-Fluorination to PET Radiotracer Development: Overcoming Hurdles to Translation

New chemistry methods for the synthesis of radiolabeled small molecules have the potential to impact clinical positron emission tomography (PET) imaging, if they can be successfully translated. However, progression of modern reactions from the stage of synthetic chemistry development to the preparation of radiotracer doses ready for use in human PET imaging is challenging and rare. Here we describe the process of and the successful translation of a modern palladium-mediated fluorination reaction to non-human primate (NHP) baboon PET imaging–an important milestone on the path to human PET imaging. The method, which transforms [¹⁸F]fluoride into an electrophilic fluorination reagent, provides access to aryl–¹⁸F bonds that would be challenging to synthesize via conventional radiochemistry methods.     

Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography

Summary. Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the best known ‘nutriceuticals’. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic, inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an ¹⁸F-labelled resveratrol derivative, 3,5-dihydroxy-4′-[¹⁸F]fluoro-trans-stilbene ([¹⁸F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional imaging of polyphenol metabolism in vivo. In a typical “three-step/one-pot” reaction, ¹⁸F-labelled resveratrol derivative [¹⁸F]-1 could be synthesized within 120–130 min including HPLC separation at a specific radioactivity of about 90 GBq/μmol. The radiochemical yield was about 9% (decay-corrected) related to [¹⁸F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [¹⁸F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake and metabolism in the liver and kidney, respectively, of [¹⁸F]-1. This study represents the first investigation of polyphenols in vivo by means of PET.     

Diversity of selected hop cultivars detected by fluorescent AFLPs

 The amplified fragment length polymorphism (AFLP) technique was used to assay eight hop cultivars. The application of fluorescent-labelled primers proved to be a valuable tool and substituted radiolabelling. Digestion with the enzymes EcoRI/ MseI and amplification with primers having three selective bases at the 3’end resulted in distinct banding patterns for imaging with a fluorescent scanner. A total of 523 AFLP fragments derived from eight primer combinations were analysed. On average, 18 polymorphisms per combination were displayed. The Saazer “noble” hop cultivars ‘Saazer’, ‘Tettnanger’ and ‘Spalter’ could not be discriminated. The lowest genetic similarity (GS) between lines was computed for the bitter hops ‘Hallertauer Magnum’ and ‘Wye Target’: GS value of 0.89. The high level of genetic similarity of the analysed hop cultivars is discussed. Received: 11 August 1997 / Accepted: 22 August 1997     

Mouse models in neurological disorders: Applications of non-invasive imaging

Neuroimaging techniques represent powerful tools to assess disease-specific cellular, biochemical and molecular processes non-invasively in vivo. Besides providing precise anatomical localisation and quantification, the most exciting advantage of non-invasive imaging techniques is the opportunity to investigate the spatial and temporal dynamics of disease-specific functional and molecular events longitudinally in intact living organisms, so called molecular imaging (MI). Combining neuroimaging technologies with in vivo models of neurological disorders provides unique opportunities to understand the aetiology and pathophysiology of human neurological disorders. In this way, neuroimaging in mouse models of neurological disorders not only can be used for phenotyping specific diseases and monitoring disease progression but also plays an essential role in the development and evaluation of disease-specific treatment approaches. In this way MI is a key technology in translational research, helping to design improved disease models as well as experimental treatment protocols that may afterwards be implemented into clinical routine. The most widely used imaging modalities in animal models to assess in vivo anatomical, functional and molecular events are positron emission tomography (PET), magnetic resonance imaging (MRI) and optical imaging (OI). Here, we review the application of neuroimaging in mouse models of neurodegeneration (Parkinson's disease, PD, and Alzheimer's disease, AD) and brain cancer (glioma).     

Delectable Creatures and the Fundamental Reality of Metaphor: Biosemiotics and Animal Mind

This article argues that organisms, defined by a semi-permeable membrane or skin separating organism from environment, are (must be) semiotically alert responders to environments (both Innenwelt and Umwelt). As organisms and environments complexify over time, so, necessarily, does semiotic responsiveness, or ‘semiotic freedom’. In complex environments, semiotic responsiveness necessitates increasing plasticity of discernment, or discrimination. Such judgements, in other words, involve interpretations. The latter, in effect, consist of translations of a range of sign relations which, like metaphor, are based on transfers (carryings over) of meanings or expressions from one semiotic ‘site’ to another. The article argues that what humans describe as ‘metaphor’ (and believe is something which only pertains to human speech and mind and, in essence, is ‘not real’) is, in fact, fundamental to all semiotic and biosemiotic sign processes in all living things. The article first argues that metaphor and mind are immanent in all life, and are evolutionary, and, thus, that animals certainly do have minds. Following Heidegger and then Agamben, the article continues by asking about the place of animal mind in humans, and concludes that, as a kind of ‘night science’, ‘humananimal’ mind is central to the semiotics of Peircean abduction.     

Real time non-invasive imaging of receptor-ligand interactions in vivo

Non-invasive longitudinal detection and evaluation of gene expression in living animals can provide investigators with an understanding of the ontogeny of a gene's biological function(s). Currently, mouse model systems are used to optimize magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging modalities to detect gene expression and protein function. These molecular imaging strategies are being developed to assess tumor growth and the tumor microenvironment. In addition, pre-labeling of progenitor cells can provide invaluable information about the developmental lineage of stem cells both in organogenesis and tumorigenesis. The feasibility of this approach has been extensively tested by targeting of endogenous tumor cell receptors with labeled ligand (or ligand analog) reporters and targeting enzymes with labeled substrate (or substrate analog). We will primarily discuss MRI, PET, and SPECT imaging of cell surface receptors and the feasibility of non-invasive imaging of gene expression using the tumor microenvironment (e.g., hypoxia) as a conditional regulator of gene expression.     

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.     

Synthesis and evaluation of a novel near-infrared fluorescent probe based on succinimidyl-Cys-C(O)-Glu that targets prostate-specific membrane antigen for optical imaging

Prostate-specific membrane antigen (PSMA), which is highly expressed in both localized and metastatic prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported radiolabeled asymmetric urea derivatives as a PSMA-targeting radiotracer for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging. Here, based on these radiopharmaceutical probes, we designed a novel near-infrared (NIR) fluorescent imaging probe (800CW-SCE) by chemical conjugation between IRDye 800CW-Maleimide and an asymmetric urea compound, known as PSMA inhibitor, for optical imaging. In the in vitro cellular uptake study, 800CW-SCE was internalized into PSMA-positive PCa cells (LNCaP cells) but not into PSMA-negative PCa cells (PC-3 cells). Moreover, in the in vivo imaging study, the probe was highly accumulated in LNCaP tumors but not in PC-3 tumors, and remained in LNCaP tumors until 24 h after intravenous administration. These results suggest that the potent NIR conjugate may contribute to clinical intraoperative optical imaging.     

<Emphasis Type="Italic">Ex vivo</Emphasis> expansion of hematopoietic cells from CD34<Superscript>+</Superscript> cord blood cells in various culture conditions

This study compared the cell expansion and colony-forming ability of human cord blood stem cells cultured ex vivo with 2 types of cytokine combinations, and 2 types of media characterized by the presence or absence of fetal bovine serum (FBS) in 2 or 3 dimensional (2D or 3D) culture environments. Purified CD34⁺ cells derived from different donors were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) and Ultraculture serum-free medium (SFM) containing the cytokine cocktail-I (coc-I) (EPO, GM-CSF, SCF, and IL-3) or cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher CFU-GM values were observed in the IMDM compared to the SFM. In the coc-I conditions, the ‘IMDM + coc-I’ and ‘IMDM + coc-I + FBS’ conditions gave the greatest cell (1,667 ± 274 and 1,600 ± 140-fold, respectively) and colony-forming units (CFU) expansions (BFU-E: 21 ± 3, 36 ± 5; CFU-GM: 95 ± 19, 81 ± 17; and CFU-GEMM: 2 ± 1, 3 ± 1-fold, respectively) in 26 day culture, respectively. In the coc-II conditions, the ‘SFM + coc-II’ condition gave the greatest cell expansion (2,143 ± 134-fold), but the ‘IMDM + coc-II’ condition gave the best CFU-GM expansion (924 ± 110-fold) in 26 day culture. In conclusion, ‘IMDM + coc-I’ and ‘IMDM + coc-II’ were the most accessible conditions for CFU expansion for all culture cases. The 2D stationary culture had affirmative effect on CFU expansion compared to the 3D culture using semisolid Methocult™. These results are believed to be significant in the ex vivo expansion of hematopoietic stem cells.     

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.     

A dynamic model of motor basal ganglia functions

 Fast aiming movements were measured in a choice reaction paradigm in a healthy control group and in Parkinsonian patients. The patients were tested without (‘off ’) and with 3,4-dihydroxyphenylalanine (‘on’) (L-dopa) medication. The movement trajectories were used to estimate the parameters of a dynamic linear model. The model is based on the functional structure of the basal ganglia-thalamocortical circuit with direct and indirect pathways linking the putamen to the basal ganglia output nuclei (Albin et al. 1989). The output of the circuit is connected to a model for the motor neuron-musculo-skeletal system. The gain k _d for the direct pathway and the gain k _i for the indirect pathway were estimated. They were found to be significantly decreased for Parkinsonian patients in ‘off ’ compared with the control group. L-dopa therapy in Parkinsonian patients increased the gains of the direct and the indirect pathway almost to normal values which implies that the long-term dopamine level in the striatum was excitatory for the direct and for the indirect pathway. This result is restricted to movements of correct size. For movements of diminished size, which are typical for Parkinsonian patients, the model predicts that the dopamine level in the striatum is excitatory for the direct pathway but inhibitory for the indirect pathway. The simulated values for neuronal activities are in agreement with expected values according to the experimental data. The proposed model of the ‘motor’ basal ganglia thalamocortical circuit implies that information about biomechanical properties of the musculo-skeletal system is stored in the ‘motor’ basal ganglia-thalamocortical circuit, and that the basal ganglia are involved in computation of the desired movement amplitude. Received: 24 April 1996/Accepted in revised form: 25 February 1997     

Use of positron emission tomography to study tumors in vivo

Positron emission tomography (PET) is a non-invasive imaging technique. The ability of PET to visualize biochemistry and physiology in vivo distinguishes this technique from other imaging modalities and renders it of particular interest for oncological studies. PET studies can of en differentiate between normal and neoplastic tissue, as well as identify early signs of malignant degeneration through biochemical or physiological changes. Over the past several years, PET studies have been useful in the early diagnosis and the selection of treatment, as well as in following the progression or regression of malignant disease processes. Of particular significance, PET findings can be quantified by using mathematical modeling and computerized data analysis, which makes it possible to produce quantitative images of human pathophysiology in vivo.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies

Summary. The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer’s disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Radiolabelling of both nLDL and oxLDL using [¹⁸F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [¹⁸F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [¹⁸F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [¹⁸F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997