Establishment of dosing conditions for etomidate anesthesia

While animals were in a tubular restrainer, the lateral tail vein was cannulated with either a hypodermic or a butterfly needle which was connected via a catheter to an infusion pump (Harvard Apparatus, Boston, MA, USA). Anesthesia was induced with isoflurane (rats: 4%; mice: 2.5–3%; Abbot, Baar, Switzerland) delivered by a calibrated vaporizer in a carrier gas consisting of oxygen and air (1:5 v/v). Subsequently, etomidate (Etomidate-®Lipuro; B. Braun Melsungen AG, Melsungen, Germany) was administered as a primed continuous intravenous infusion. Isoflurane supply was discontinued after 8 min whereas the supply of oxygen-enriched breathing gas was maintained throughout the duration of the anesthesia. Species- and strain-specific dose-finding for etomidate anesthesia was carried out. The priming dose of etomidate was set to a rate of 4 mg/kg/min (mice) or 5 mg/kg/min (rats) for the initial 3 min. Thereafter, anesthesia was sustained with rates of 0.17, 0.25, 0.33 or 0.50 mg/kg/min for rats and 0.5 or 0.75 mg/kg/min for C57BL/6J mice, 0.75 or 1.0 mg/kg/min for BTBR T+tf/J mice or 0.75, 1.0, 1.2 or 1.5 mg/kg/min for CD1 mice (each n = 2 per dose). Anesthesia was continued for 2 h to reflect the maximum duration of a typical MRI assessment.

Body temperature was continuously monitored with a rectal probe and maintained at 37.3 ± 0.3 °C with a feedback-regulated electric heating blanket (Prang + Partner AG, Pfungen, Switzerland). Induction of anesthesia was assessed by the loss of the righting reflex⁴². The depth of anesthesia was further characterized by standardized tests for nociceptive (tail pinch), withdrawal (toe, ear pinch) or palpebral (medial eye canthus) reflexes and the response to vibrissal (mechanical deflection) or loud sound stimuli (hand clapping) carried out every 10 min. In addition, the color of the mucous membranes was evaluated. After discontinuation of etomidate infusion, animals were further monitored until they were fully ambulatory. Table 1 lists the species- and strain-specific infusion rates of etomidate as well as doses of other anesthetics tested in this study that produced adequate anesthetic depth and good recovery.