Experimental animals and cardiac arrest model

The Institutional Animal Care and Use Committee (IACUC) at the University of Colorado approved all experimental protocols in accordance with National Institutes of Health guidelines for the care and use of animals in research. Experiments were performed in accordance with ARRIVE guidelines.^31,32 Blinding was performed prior to electrophysiological recording. Adult (8–10-week-old) male C57Bl6 (Charles River, Wilmington, MA) were subjected to CA/CPR as previously described^12,15,33 during ON light cycle. A total of 119 surgeries were performed, including sham surgeries of which 14 mice did not survive to reach their final end point. There were an additional 10 animals that survived; however, we were unable to obtain high quality recordings.

Briefly, anesthesia was induced with 3% isoflurane and maintained with 1.5–2% isoflurane in oxygen-enriched air via facemask. Temperature probes were inserted in the left ear and rectum to monitor head and body temperature simultaneously. A PE-10 catheter was inserted into the right internal jugular vein for drug administration. Needle electrodes were placed subcutaneously on the chest for continuous EKG monitoring. Animals were endotracheally intubated, connected to a mouse ventilator (MiniVent Ventilator, Harvard Apparatus). Cardiac arrest was induced by injection of 50 µL KCl (0.5M) via the jugular catheter and confirmed by asystole on EKG. The endotracheal tube was disconnected and anesthesia stopped. During cardiac arrest, body temperature was allowed to spontaneously decrease to a minimum of 35.5℃, and head temperature was maintained at 37.5℃. Resuscitation was begun 8 min after induction of cardiac arrest by injection of 0.05–0.10 ml epinephrine solution (16 µg epinephrine/ml 0.9% saline), chest compressions, and ventilation with 100% oxygen at a respiratory rate of 200/min and 25% greater tidal volume. Chest compressions were stopped as soon as spontaneous circulation was restored. Resuscitation was abandoned if spontaneous circulation was not restored within 2 min. Mice were extubated after they recovered an adequate respiratory rate and effort. Sham controls underwent the same procedures as mice undergoing cardiac arrest including anesthesia intubation, placement of jugular catheter, EKG leads and temperature management. Sham controls did not receive KCl or epinephrine injections or chest compressions. The animals were placed in a single housed static recovery cage on a heated water blanket (35℃) for 24 h recovery and at room temp for long-term recovery (up to 30 days). Mice received soft food and subcutaneous saline for three days after surgery and had free access to water and regular chow.