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Session B5: Novel Methods for Science--Welfare ConcernsChairs: Christian Cook (New Zealand) and James Kirkwood (UK) B5: Reducing the Invasiveness of Obtaining Blood-borne Measures in Animals An important experimental design constraint in use of animals is to minimize invasive load and accompanying stress. This is particularly so in studies aimed to improve the welfare of animals. An often-found confounding problem is that the experimental techniques may themselves elicit stress responses in the animal. In particular, many changes of interest are reflected in analytes in the blood. Collection of blood is often stressful and mildly noxious and long-term catheterization imposes an invasive load on the animal. Many stress-related changes are transient and easily missed or diluted down by infrequent sampling, yet more frequent sampling and blood removal is also demanding on the animal. To overcome these inherent limitations, I am developing a new technique that combines low frequency ultrasound with a small electric current, termed electrosonophoresis. This technique involves the use of a portable instrumentation with a gun-like device that delivers an application at the skin surface of a brief (5-15 second) burst of ultrasound in combination with a low electric field. Although the mechanism is not fully understood, when this delivery is used in conjunction with an appropriate collecting head, positioned at the skin surface, a brief transdermal flux is created from underlying blood vessels, into extracellular fluid and to the skin. The technique appears to allow many of the analytes present in the blood to be collected and measured at the skin surface with a linear relationship to blood levels seen. In humans, the method is without sensation and innocuous. Actual blood removal is not required, and repeating the measures intensively in a short time period does not appear to cause any problems or discomfort. In animals accustomed to human handling, no behavioral disturbance has been seen in using the instrumentation. It appears applicable at most vascular surfaces and on cartilage. Successful readings have been made from sheep, cattle, cats, dogs, rodents, horses, birds, and sharks without any apparent ill effects. Large cellular substances, such as red blood cells, cannot be collected by this method, but a variety of analytes including hormones, amino acids, sugars, free fatty acids, immunoglobulins, lactate, and small proteins have been collected and measured with a high correlation (r > 0.89) to blood levels over a wide physiological range of change. Much remains to be assessed about this mechanism and its long-term uses and safety. We are also currently developing a small automated version that could be affixed onto an animal and allow collection while the animal was free roaming. The low invasiveness of use, the removal of the need to extract actual blood fluid, and the low stressfulness of the technique suggest that it may be a powerful future tool for welfare studies. B5: Use and Pitfalls of Allometry: An Essential Tool in Ethical Decisions About Validity of Extrapolating Between Species Where research on one species is justified on the grounds that it will provide benefits to another, the strength of the ethical case depends critically on whether findings can be extrapolated meaningfully. Valid extrapolation depends on the species being sufficiently similar in respects critical to the research and on knowledge of the bases and effects of salient differences. Many biological parameters vary with body mass (M) between species. When species of small size are used to model larger ones, the influence of size on physiological, immunological, and other processes must be taken into account. Between species, rates of physiological processes decrease with M-0.75, and durations of physiological events increase with M0.25. Providing potential pitfalls are understood, allometric scaling enables valid comparisons and extrapolations between species. Knowledge of these principles is crucial to making sound, ethical judgments about the justifiability of extrapolations between species on the very wide range of processes linked to rates of metabolism. B5: Non-invasive Imaging by Echocardiography to Study Mechanisms of Mitochondrial Cardiac Toxicity The noninvasive imaging technique of echocardiography has allowed us to evaluate cardiac function in awake rodents and characterize doxorubicin and 3-nitropropionic acid (3NPA) cardiac toxicity over multiple time points, reducing the number of mice needed by up to 70%. Conscious mouse echocardiography allows us to quantify left ventricular chamber diameter, wall thickness, aorta diameter and velocity time interval, ejection fraction, stroke volume, and cardiac output without the use of anesthesia and accompanying cardiac function depression. To reduce any stress involved in handling, rodents are habituated to the procedure before data collection. As examples of refinement and reduction applications to animal research, we will present results from two separate studies using echocardiography. B5: Better Data from Fewer Mice: High-Throughput Non-invasive Physiological Assays in Conscious Mice are Accelerating Functional Genomics Linking gene defect to disease in mouse models will continue to be a key step in drug development. Yet, most in vitro and in vivo assays entail anesthetic and surgery. We apply non-invasive conscious animal screens to provide clear evidence of gene defect with no harm to the mice. By application of a new device for recording electrocardiograms, we identified age, gender, and strain variations in heart function. Female C57BL6 mice have faster heart rate (HR) than C57BL6 males. Balb/C mice have slower HR than FVB mice. The remarkably steady HR in nursling neonates (6-days old) disappears at weaning. Importantly, this method has provided new evidence of heart defects in mouse models of muscular dystrophy and mucopolysaccharidoses reflective of human diseases. Application of a new system for monitoring gait dynamics has identified early markers of defects in models of amyotrophic lateral sclerosis and Down syndrome. Since these assays are non-invasive, the mice can be used for other tests. These conscious animal screens not only advance refinement and reduction efforts, but also provide the most comprehensive and physiologically relevant data for understanding drug and gene effects. B5: Refinement and Reduction of Animal Use by Application of Whole Animal Cell and Molecular Imaging Methods Increasingly, the subject of conservative use of laboratory animals is resonating with those who see the scientific and economic value of responsible animal use protocols. Imaging technologies, such as non-invasive Biophotonic Imaging (in vivo imaging of bioluminescent reporters in live animals), can yield more and higher quality experimental data per protocol by increasing the number of times that quantitative data can be collected and finally guiding appropriate endpoint tissue sampling for histology or biochemical analysis. By imaging the whole intact live animal at multiple time points, researchers can place biomolecular processes together with contextual influences in a live functioning animal. It is an added benefit that with these methods, fewer animals can deliver data with greater statistical significance. Lower stress, noninvasive methods are used to create more predictive animal models that share the characteristics of longitudinal study design, internal experimental control, molecular information, and quantitative data, and these methods will benefit both scientific inquiry and humane animal use. We will present data on three types of models: chronic infectious diseases, oncology, and genetically modified animals that are used in the pharmaceutical industry today and represent advances in the areas of refinement and reduction of animal use. B5: Suitability of Temperature-Sensitive Transponders to Measure Body Temperature During Animal Experiments Required for Regulatory Tests During development, production, and quality control of immunobiological medicines, such as vaccines, animal tests have to be done to prove safety, potency, and efficacy of the products. Body temperature is one of the important signs to detect systemic side effects (e.g. due to pyrogen contamination or to follow the course of artificial infection). Body temperature is also one of the key parameters to define humane endpoints in animal experiments. However, measuring body temperature via the rectal route is often troublesome, especially in animals of small body size or in species that are particularly prone to stress from handling, such as rabbits or pigs. Minimizing the duration of handling and restraint benefits the animals, improves reliability of data, and is also a human safety measure for staff performing the experiments. The aim of the study was to compare rectal body temperature of several animal species with temperature measured using subcutaneously implanted temperature-sensitive transponders (TST) under controlled conditions of regulatory tests. To test the accuracy of the devices, we calibrated TST at 38°C in a tempered bathwater and tested accuracy in vitro. We then implanted the transponders in animals. The temperature values obtained with the TST were compared with rectally measured body temperature of the same animal using commercially available calibrated digital thermometers. The results from experiments in mice, guinea pigs, rabbits, and pigs are presented and discussed.
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