Mouse isoflurane anesthesia using the drop method

Anesthesia with isoflurane prior to carbon dioxide euthanasia is recommended as a refinement, but vaporizer access can be limited. An alternative to vaporizers is the 'drop' method, introducing a fixed volume of isoflurane into the induction chamber. Previous work suggests that isoflurane administered at a concentration of 5% via the drop method is effective but aversive to mice; lower concentrations have not been tested. We assessed mouse behavior and insensibility with induction using the drop method for isoflurane concentrations below 5%. Male Crl:CD-1 (ICR) mice (n = 27) were randomly allocated to one of three isoflurane concentrations: 1.7%, 2.7%, and 3.7%. During induction, measures of insensibility and stress-related behaviors were recorded. All mice reached a surgical plane of anesthesia, and mice exposed to higher concentrations did so more quickly; as concentrations increased from 1.7 to 2.7 and 3.7%, the time to recumbency (Least squares means ± SE: 120.5 s ± 8.1, 97.9 s ± 8.1, and 82.8 s ± 8.1, respectively), loss of righting reflex (149.1 s ± 8.5, 127.7 s ± 8.5, and 100.7 s ± 8.5, respectively), and loss of pedal withdrawal reflex (214.5 s ± 8.3, 172.2 s ± 8.3, and 146.4 s ± 8.3, respectively) all declined. Rearing was the most frequently performed stress-related behavior, and was most pronounced immediately following isoflurane administration for all treatments. Our results indicate that the drop method can be used to effectively anesthetize mice with isoflurane concentrations as low as 1.7%; future work should assess mouse aversion.