Physiologic and Behavioral Effects in Mice Anesthetized with Isoflurane in a Red-tinted or a Traditional Translucent Chamber

Abstract

Isoflurane has been characterized as a distressing agent for rodents, causing both physiologic and behavioral effects. Using a "darkened home cage" has been recommended during CO₂ administration for rodent euthanasia; this is arguably a similar animal experience to anesthetic induction with isoflurane. Based on the premise that rodents perceive red light as darkness via the primary optic tract, we compared physiologic and behavioral markers of stress in 2 inbred strains of mice (C57BL/6J and BALB/cJ) anesthetized with isoflurane in either a red-tinted (dark) induction chamber or a traditional translucent induction chamber. Physiologic stress was assessed based on plasma levels of norepinephrine, epinephrine, and corticosterone. Stress-related behaviors (rearing, face wiping, and jumping) were recorded on video and scored from initiation of induction to loss of consciousness. No significant correlations were found between chamber type and physiologic stress hormones. As compared with the translucent chamber, stress-related behaviors were more frequent in the red-tinted chamber, including: 1) significantly higher rearing frequencies in BALB/cJ mice; 2) higher behavioral stress scores in BALB/cJ and male C57BL/6J mice; and 3) more face wiping behavior when considering all mice combined. These findings suggest that mice do not experience significant alleviation of physiologic indices of stress when anesthetized in a red-tinted induction chamber. Furthermore, isoflurane induction in the red-tinted chamber appeared to increase the expression of stress-related behaviors, particularly in BALB/cJ mice. Based on our findings and a growing body of literature on the unintended effects of red light, we do not recommend using red-tinted chambers for induction of anesthesia in mice.

Figure 1.

(A) Mouse behavioral scoring sheet used for assessment during video recordings. Rearing scores were adjusted posthoc to better capture the range of rearing frequency. (B) Original behavioral scoring for readers’ reference.

Figure 2.

Transmission spectrum of both the red-tinted and traditional chambers used. Values plotted include the transmittance (%) and wavelength (nm).

Figure 3.

Radiometric and photometric values for the traditional and red-tinted chambers. Calculations made using the Rodent Toolbox spreadsheet.

Figure 4.

Comparison of mean plasma norepinephrine level across all groups. No significant correlations were found based on chamber type, sex, or strain.

Figure 5.

(A) Comparison of mean plasma epinephrine level across all groups. No significant correlation was found based on chamber type or sex. (B) Comparison of mean plasma corticosterone level across all groups. No significant correlation was found based on chamber type. (C) Comparison of epinephrine levels between strains indicated significantly higher epinephrine levels in BALB/cJ mice (P < 0.0001). Comparison of corticosterone levels by (D) strain and (E) sex found significantly higher corticosterone levels in both BALB/cJ (P < 0.0001) and female (P ≤ 0.05) groups, respectively.

Figure 6.

Comparison of average rearing frequency (per minute) across all groups. Asterisks indicate highly significant differences (P < 0.0001) between the red-tinted chamber compared with the traditional chamber.

Figure 7.

Comparison of adjusted behavioral stress scores across all groups. Asterisks indicate highly significant differences (P < 0.0001) between the red-tinted chamber compared with the traditional chamber.

Figure 8.

Comparison between chamber types of the proportion of mice displaying face wipe behavior. Asterisks indicate significant differences (P < 0.05) between chambers.