Animals were exposed to isoflurane as previously described3 (link),5 (link). Briefly, isoflurane was administered in a custom-built chamber in a step-down protocol over six hours starting at 2%, decreasing to 1.4% and then 0.8% every two hours. The carrier gas was a mixture of humidified air and oxygen (approximately 45% oxygen). A carbon dioxide absorber (Litholyme, Allied Healthcare, St. Louis, MO) was present during exposure. Isoflurane, oxygen, and carbon dioxide were monitored with a Datex-Ohmeda gas analyzer (West Bloomfield, MI). Temperature was regulated with a heat pad (Thermo Haake, Waltham MA), and monitored every 15 minutes by an infrared thermometer. Recorded parameters (isoflurane, oxygen and carbon dioxide concentrations and average animal temperature) during exposure are reported in the supplementary material (Supplemental Digital content 1: Figures showing physiologic variables averaged across all anesthetic exposures). There was a single mortality during isoflurane exposure of 33 animals in total exposed.