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Theme B Posters: Refinement and Reduction AlternativesB1: Fish as an Alternative Model to Mammals for Genotoxicity Research Ever since the Three R's concept of Russell and Burch (1959) came into vogue, scientists all over the world have been working to develop models alternative to animal use for biomedical research. We have been able to standardize a fish in vivo model to screen environmental pollutants for genotoxicity. A number of commonly used pesticides have been tested, employing micronucleus test (MNT) in the peripheral blood erythrocytes of two live fishes, Heteropneustes fossilis and Anabas testudenius. In general, treated results differed significantly from respective controls. In addition, the fish system has certain advantages: 1) fish are easy to procure, handle, and rear in the laboratory; 2) unlike mammals, erythrocytes are nucleated, facilitating easy scoring of any abnormality; and 3) it is cost-effective. Considering all these features and the present results, it is suggested that the fish model can be used as an alternative to mammals to screen the genotoxicity of environmental pollutants. Being cost-effective, the use of the fish model would be particularly helpful to the researchers of the developing countries. B2: Optimizing Resources via Reduction: The Frame Reduction Committee The FRAME Reduction Committee, formed in 1998, comprises experienced professionals in the fields of statistics, experimental design, animal welfare, and alternatives. Literature surveys suggest that often the number of laboratory animals used in experiments could have been reduced, while still resulting in the generation of statistically valid data. The FRAME Reduction Committee undertakes projects designed to contribute to reduction by addressing the implementation of recommendations made at the ECVAM workshop, "The Three R's: The Way Forward." The projects include ongoing reviews of computer statistical packages and the creation of a directory of training material on experimental design and statistical analysis. The poster highlights one practical example of a reduction strategy to address the logistical problem posed by an anti-cancer drug development screening system. B3: Environmental Enrichment: Beneficial Effects in a Rodent Model of Lead Neurotoxicity Long-term deficits in cognitive function are the principle effects of lead exposure in children and can be modeled in experimental animals. Using a rodent model of lead-induced neurotoxicity, we show that environmental enrichment reverses cognitive and molecular deficits induced by this developmental neurotoxicant. Long-Evans female rats were exposed to 0 or 1500 ppm lead acetate 10 days prior to mating and during gestation and lactation until weaning. At weaning, pups were removed from the lead exposure and housed in isolation (one per cage) or environmental enrichment cages (8 per cage). Enrichment cages were larger and contained toys, mazes, a running wheel, and a hammock. Rats exposed to lead and reared in an isolated environment were significantly impaired in acquiring a spatial learning task. On the other hand, lead-exposed rats reared in an enriched environment performed as well as control enriched rats and better than control isolated rats. Recovery of learning performance in lead-exposed animals reared in enriched cages was associated with recovery of NMDA receptor deficits. These findings demonstrate the beneficial effects of an enriched environment on an animal model of developmental lead neurotoxicity. Further, the findings suggest that the living environment is an important modifier on the effect of toxins on the central nervous system. B4: Murine Models of Tobacco Smoke-Induced Pulmonary Inflammation and Emphysema We have investigated the acute inflammatory response following a single exposure to tobacco smoke (TS) and the effects of chronic TS exposure in female A/J mice. The acute inflammatory response was measured from 0.5 to 24 hours post-exposure (groups of five unrestrained mice exposed to TS from six cigarettes for approximately 42 minutes). The peak cell influx occurred at six hours, making this acute model suitable for evaluating new anti-inflammatory concepts. In the chronic model, mice were exposed to TS for five days/week for fourteen weeks, exposure over the first two weeks of the study being gradually increased from two to six cigarettes per day. The responses of the mice to exposure were closely monitored visually and are reported at this meeting (Thornton et al). Morphometric analysis of lungs showed that mice chronically exposed to TS had a 14.7% (p < 0.01) greater linear mean intercept (LMI, a measurement of emphysema) compared to lungs from air-exposed controls. In conclusion, this chronic model, in which emphysema develops relatively rapidly, offers the opportunity to further study the pathogenesis of emphysema. B5: Experimental Design: Computer Simulation for Improving the Precision
of an Experiment Poorly designed animal experiments cannot be ethically justified. Understanding variation in order to control variability and to minimize experimental error should be a first priority for designing experiments that are going to have reliable, efficient results. Realistic experimental conditions can be simulated with computer simulations. A computer aided learning program has been developed that allows formal design such as completely randomized, randomized block, crossover, and sequential design and their statistical analysis. Unwanted variability will increase the number of animals used in subsequent experiments. This can be simulated in examples. Accurate data can then be collected. With visualized mathematical methods, the appropriate sample size can be determined and a final choice of an optimal experimental design can be selected. By paying careful attention to the factors that affect variability and efficient experimental design, the information provided by real experiments will increase, resulting in a reduction in animal use.
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