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Background: The EU Directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. Wherever animal experimentation is necessary, the 3-Rprinciple of Russel and Burch (replace, reduce, refine) has to be observed. The primary goal of the 3-R-principle is to replace animal testing with alternative methods. If no alternative method can be applied, the total number of animals is supposed be reduced. Consequently, some animals are used multiple times in the course of an experiment. For example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. However, the Directive claims that “the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal”.
Objective: We are looking into whether multiple exposures to anesthesia cause more stress than a single exposure.
Methods: The most common mouse strain C57/BL6 J and anesthetics isoflurane and the combination of ketamine/xylazine are used. With regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. All parameters observed are compared between controls, animals with a single and repeated anesthesia. The interval between the administration of the anesthesias is three to four days. When the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. The grimace scale is scored 30 and 150 minutes after anesthesia. Besides pain, the grimace scale can also assess anxiety, stress and malaise. The display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. Furthermore, activity, food and water intake are monitored for 24 hours. A behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. Motor coordination and balance are assessed by the balance beam and rota rod. The open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. Moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress.
Results: The first results of our study will be presented at the 82nd meeting of DGPT. Conclusion: We are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. BB3R is funded by BMBF.