Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress

Abstract

According to the EU Directive 2010/63, the severity of a procedure has to be classified as mild, moderate or severe. General anesthesia is thought to be mild, but the Directive does not differentiate between single and repeated anesthesia. Therefore, we investigated the impact of repeated administration of isoflurane, the most commonly used inhalation anesthetic, on the well-being of adult C57BL/6JRj mice, in comparison to single administrations and to untreated animals, when applied six times for 45 min at an interval of 3-4 days. For the animals anesthetized, excitations, phases of anesthesia, and vital parameters were monitored. Well-being after anesthesia was assessed using a behavioral test battery including luxury behavior like burrowing and nest building behavior, the Mouse Grimace Scale (MGS), the free exploratory paradigm for anxiety-related behavior, home cage activity and the rotarod test for activity, as well as food intake and body weight. Additionally, hair corticosterone and fecal corticosterone metabolites were measured. Our results show that nest building behavior, home cage activity, body weight, and corticosterone concentrations were not influenced by anesthesia, whereas changes in burrowing behavior, the MGS, food intake, and the free exploratory behavior indicated that the well-being of the mice was more affected by repeated than single isoflurane anesthesia. This effect depended on the sex of the animals, with female mice being more susceptible than male mice. However, repeated isoflurane anesthesia caused only short-term mild distress and impairment of well-being, mainly in the immediate postanesthetic period. Well-being stabilized at 8 days after the last anesthesia, at the latest. Therefore, we conclude that when using our anesthesia protocol, the severity of both single and repeated isoflurane anesthesia in C57BL/6JRj mice can be classified as mild. However, within the mild severity category, repeated isoflurane anesthesia ranks higher than single isoflurane anesthesia. Additionally, our results imply that male and female mice can differently perceive the severity of a procedure.

Fig 1. Flow chart of the test schedule.

⁺ Number [n] of rearings, number [n] of grooming episodes, duration of resting [s], duration of activity [s], duration of food intake [s], and the latency to first food intake [s] were observed during the recovery period.

Fig 2. Mouse Grimace Scale difference scores at 30 min and 150 min after last anesthesia.

MGS, Mouse Grimace Scale. Data are mean ± standard error. p values were calculated using Kruskal-Wallis-Test: ** p < 0.01. (A) Control ♀: n = 6, single anesthesia ♀: n = 9, repeated anesthesia ♀: n = 13; 4 mice of the single anesthesia group were excluded from statistics because of technical malfunction of the camera. (B) Control ♂: n = 6, single anesthesia ♂: n = 10, repeated anesthesia ♂: n = 13; 3 mice of the single anesthesia group and 1 control mouse were excluded from statistics because of technical malfunction of the camera.

Fig 3. Burrowing behavior in the immediate postanesthetic period.

Data are means ± standard error. p values were calculated using Kruskal-Wallis-Test: * p < 0.05. (A) Control ♀: n = 6, single anesthesia ♀: n = 9, repeated anesthesia ♀: n = 13; 4 mice of the single anesthesia group were excluded from statistics because of technical malfunctions of the scale. (B) Control ♂: n = 6, single anesthesia ♂: n = 10, repeated anesthesia ♂: n = 13; 3 mice of the single anesthesia group and 1 control mouse were excluded from statistics because of technical malfunction of the scale.

Fig 4. Latency to explore in the free exploratory paradigm for trait anxiety-related and exploratory behavior.

Latency to explore [s]; data are means ± standard error; p values were calculated using the Kruskal-Wallis-Test: ** p < 0.01 versus repeated anesthesia. (A) Control ♀: n = 6, single anesthesia ♀: n = 13, repeated anesthesia ♀: n = 13. (B) Control ♂: n = 7, single anesthesia ♂: n = 13, repeated anesthesia ♂: n = 13.

Fig 5. Course of body weight.

Data are means ± standard error. (A) Control ♀: n = 6, single anesthesia ♀: n = 13, repeated anesthesia ♀: n = 13. (B) Control ♂: n = 7, single anesthesia ♂: n = 13, repeated anesthesia ♂: n = 13. p values were calculated using repeated measures ANOVA with group as between-subject factor: * p < 0.05, ** p < 0.01, *** p < 0.001 versus 14 days after the last anesthesia; ^# p < 0.05 versus 9 days after the last anesthesia.