Isoflurane and Carbon Dioxide Elicit Similar Behavioral Responses in Rats

Abstract

Euthanasia in rodents is an ongoing topic of debate due to concerns regarding the aversive nature of gases with anesthetic properties such as carbon dioxide (CO2) and isoflurane. The aim of this study was to expand upon previously published work evaluating the aversiveness of CO2 by introducing an isoflurane treatment group in parallel. Aversion was tested using a forced exposure setup and an aversion-avoidance setup. In the first part of the study, 12 naïve female Sprague–Dawley rats were exposed during four consecutive days, once to each of four treatments: isoflurane, fox urine, oxygen, and CO2. In the second part of the study, 24 naïve female Sprague–Dawley rats and 12 rats from the first experiment were exposed to CO2, isoflurane, or both gases. In the forced exposure study, there were no significant differences between CO2 and isoflurane treatments except in line crosses. Overall, rats were more active in the isoflurane and CO2 treatments compared to the control groups, suggesting that isoflurane and CO2 are similarly aversive. In the aversion-avoidance study, rats previously exposed to isoflurane left the dark chamber significantly earlier compared to naïve rats during exposure to isoflurane. We also show that learned aversion to isoflurane is sustained for at least 15 days after initial exposure. Given this result, we suggest that CO2 is superior to isoflurane when euthanizing rodents with prior exposure to isoflurane. Overall, these results confirm previous studies which suggest that care should be taken when considering the serial use of isoflurane as an anesthetic.

Thermal Response of Laboratory Rats (Rattus norvegicus) during the Application of Six Methods of Euthanasia Assessed by Infrared Thermography

“…This suggests that the anesthetic stress and physiological response triggered by isoflurane is more marked than that induced by other inhalant, injectable, and physical methods of euthanasia. The present results are in agreement with what other authors have stated regarding isoflurane as a refinement method for CO 2 [ 52 , 62 ] and affirm that precautions should be taken when deciding to use isoflurane as a sole method for the humane killing of research animals.…”

“…However, studies addressing the mechanism of action of CO 2 and isoflurane, as well as its overdose to induce euthanasia, have shown that both drugs trigger physiological stress and cardiovascular alterations in laboratory rodents [ 26 ]. Although both are considered safe, inexpensive, and effective methods to induce unconsciousness, the results obtained agree with previous findings suggesting that gases with anesthetic properties cause high levels of aversion and stress in mice and rats [ 15 , 52 ].…”

“…Before discussing the IRT results according to the thermal windows, the current literature regarding endocrine and behavioral changes after CO 2 and isoflurane euthanasia in rats and mice suggests that the thermal response due to stress/distress could be associated with both methods. CO 2 inhalation used in G 2 rats induces hypercapnia, acidosis, and suppression of the synaptic potentials [ 52 , 53 , 54 ] and activates the stress-mediated sympathetic HPA and SAM axes [ 55 ]. This was corroborated by Borovsky et al [ 56 ], who reported that exposure of rats to CO 2 increased their blood pressure (50–60 mmHg) and NE concentration up to ten times in response to hypoxia.…”

“…Isoflurane administered in G 4 rats is a volatile anesthetic that causes depression in the cardiorespiratory centers, leading to hypoxemia and death [ 26 , 60 ]. Although isoflurane has been considered as a method of CO 2 euthanasia refinement [ 14 , 15 ], in comparison with CO 2 and other volatile anesthetics, isoflurane has mild pungency [ 61 ], causing more aversion responses in laboratory rodents that is possibly due to airway irritation [ 52 , 62 ], air hunger, and dyspnea [ 52 ].…”

“…This pattern was expected because ear temperature assessed at the external ear canal is associated with the carotid artery and hypothalamic temperature, the main structure involved in central and peripheral thermoregulatory adaptations [ 79 ], particularly when exposed to stressors. Studies conclude that CO 2 and isoflurane exert acute stress in rodents [ 52 , 55 , 58 ].…”