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The ultimate goal of the EU Directive 2010/63 is to phase out all animal testing. Wherever animal experimentation is necessary, the 3-R-principle of Russel and Burch is meant to be applied. The 3-R-principle stands for replacing 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 repeatedly anesthetized 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” . The aim of this study is to investigate if repeated anesthesia causes more stress in female mice than a single anesthesia.
Materials and methods
Female C57/BL6 J and the most common inhalation anesthetic isoflurane were used. All parameters observed were compared between controls and animals receiving a single or repeated anesthesia. Over a period of 3 weeks, the animals were anesthetized 6 times for 45 minutes, respectively . Vital parameters and reflexes were monitored when the animals were under anesthesia. The Mouse Grimace Scale was applied 30 and 150 minutes
after anesthesia. Besides pain, the Grimace Scale can also assess stress and discomfort. As the display of so-called luxury behaviors serves as an indicator of wellbeing, nest building and burrowing behavior were observed. Furthermore, activity, food and water intake were monitored for 24 hours. A behavioral test battery including the free exploratory paradigm and rotarod test was performed 2 and 9 days after the last anesthesia. Motor coordination and balance were assessed by the rotarod. The free exploratory paradigm estimated anxiety and exploratory behavior. Moreover, fecal corticosterone metabolites and steroid hormones in fur were measured in order to prove evidence of cumulative stress.
Mice being repeatedly anesthetized lost the righting reflex within a shorter time and showed more excitations during induction than mice which were anesthetized only once. The vital parameters did not differ between animals receiving a single or repeated anesthesia. After repeated anesthesia, the latency to first food intake decreased and the animals ingested less food over 24 hours than after a single anesthesia. This deficit was compensated for 8 days later. Both single and repeated anesthesia caused higher scores on the Mouse Grimace Scale versus control 30 minutes after anesthesia. Although repeated anesthesia reduced burrowing behavior, the nests of all mice were assigned equal scores. No effects were seen in the rotarod test and nocturnal activity. In the free exploratory paradigm, repeated anesthesia increased the latency to explore and decreased the total
duration of exploration one day after anesthesia. During the test period neither the body weight nor the preliminary analysis of fecal corticosterone metabolites differed between the groups.
Repeated anesthesia caused more stress in female mice during the immediate postanesthetic period only. Accordingly, we assume that the severity level of a single as well as of repeated anesthesia is mild.