Animal preparation
We performed all procedures according to the previously described protocol [9]. Adult male Sprague-Dawley rats (450–550 g, Charles River Laboratories, Wilmington, MA, USA) were anesthetized with 4% isoflurane (Isosthesia, Butler-Schein AHS, Dublin, OH, USA) and intubated by a 14-gauge plastic catheter (Surflo, Terumo Medical Corporation, Somerset, NJ, USA). The rats underwent mechanical ventilation (Ventilator Model 683, Harvard Apparatus, Holliston, MA, USA). We fixed a minute ventilation volume at 180 mL/min at a respiratory rate of 45 breaths per minute (tidal volume: 8–9 ml/kg). The carbon dioxide (CO2) was continuously measured inline in the exhalation branch of the ventilator circuit by using an expiratory CO2 gas monitor (OLG-2800, Nihon Kohden Corp., Tokyo, Japan) with a CO2 sensor (TG-970P, Nihon Kohden Corp., Tokyo, Japan) and airway ETC adapter (YG-211T, Nihon Kohden Corp., Tokyo, Japan). The end-tidal CO2 (EtCO2) values were monitored within a range of 30–45 mm Hg during preparation. Under anesthesia with isoflurane 2% and a fraction of inspired O2 (FIO2) of 0.3, a core body temperature probe (T-type thermocouple probes, ADInstruments, Colorado Springs, CO, USA) was placed in the esophagus and the temperature was maintained at 36.5 ± 1.0 °C during the surgical procedure. We inserted a sterile polyethylene-50 catheter in the left femoral artery (FA) for continuous arterial pressure monitoring (MLT844, ADInstruments; Bridge Amplifier ML221, ADInstruments, Colorado Springs, CO, USA). Another polyethylene-50 catheter was cannulated from the left femoral vein and the tip was advanced 9 cm from the insertion site in order to monitor the pressure of the inferior vena cava nearby the right atrium as the central venous pressure (CVP) (MLT844, ADInstruments; Bridge Amplifier ML221, ADInstruments, Colorado Springs, CO, USA). 150 U of heparin (Heparin, SAGENT Pharmaceuticals, Schaumburg, IL, USA) was given through the CVP catheter during the surgical preparation and the basal data were obtained (preparation time, temperature, heart rate, femoral artery pressure, EtCO2, etc). At the end of preparation, blood samples were collected from the arterial line before asphyxia. The pH, the partial pressure of oxygen, the partial pressure of carbon dioxide, lactate, glucose, and hematocrit levels were measured (i-STAT, Abbott Laboratories, Abbott Park, IL, USA)
Survival study
Animals were assigned to 3 groups at the end of surgery: (1) 10-minute asphyxia arrest treated with 1-side chest compression (n=5), (2) 10-min asphyxia arrest treated with 2-side chest compression (n = 5), and (3) 10-min asphyxia arrest treated with 3-side chest compression (n = 5). After preparation, 2 mg/kg of vecuronium bromide (Hospira, Lake Forest, IL, USA) was slowly given to all animals. The ventilator was turned off to introduce asphyxia to rats. CA generally occurred within 3 to 4 min after asphyxia was initiated. All animals were positioned on the spine position. After the initial 10 min, we restarted mechanical ventilation at an FIO2 of 1.0 and performed 3 types of manual CPR. One-side was chest compression vertically performed with 2 fingers over the sternum at a rate of 240 to 300 per minute. The chest compression rate was obtained retrospectively from the record of pressure waveforms of the femoral arterial catheter. Two-side method was performed with 2 fingers horizontally squeezing the chest wall from both sides at the same rate, while 3-side chest compression was underwent with the right hand’s 2 fingers over the sternum in synchrony with left hand’s 2 fingers squeezing the chest wall (Additional file 1). Thirty seconds after the beginning of CPR, a 20-μg/kg bolus of adrenaline was administered through the venous catheter. We defined the return of spontaneous circulation (ROSC) as a systolic blood pressure over 60 mmHg, after which chest compression was discontinued. If we could not obtain ROSC by 5 min from the initiation of CPR, we terminated resuscitation. Mechanical ventilation was discontinued 2 h after CPR, and survival was monitored 10 min after extubation. The left FA pressure and CVP were monitored in all animals. After euthanizing the animals, the position of the central vein catheter tip was verified in all cases and, if not positioned appropriately, the CVP data was omitted accordingly. In an attempt to further elucidate the beneficial effects of 3-side chest compression in a rodent CPR model, we also tested ROSC rates of (1) 14-min asphyxia arrest treated with 3-side chest compression (n = 4) and (2) 15-min asphyxia arrest treated with 3-side chest compression (n = 4).
The comparison of arterial pressures and intrathoracic pressure
A set of experiments separated from the survival study was conducted for physiological measurements of three different chest compressions. In this study, we started chest compression 10 min after the induction of asphyxiation without giving adrenaline. No animals obtained ROSC in this experiment. The three sets of chest compressions were alternately tested every 60 s, and CPR was performed for up to 6 min. After the rats were intubated and mechanically ventilated with oxygen-containing isoflurane, a midline cervical incision was performed under local analgesics. The polyethylene-50 catheter was advanced into the right common carotid artery (CCA). The CCA and FA were recorded at the same time from the same animals (n = 6). In addition to these experiments, another set of experiments involving measurement of the intra-esophageal pressure, the endotracheal airway pressure, and the left ventricle pressure was conducted (n = 6). A pressure transducer catheter (MPC500, Millar Instruments, Houston, TX, USA) was placed in the esophagus and the intra-esophageal pressure was monitored. A pressure probe (MLT844, ADInstruments; Bridge Amplifier ML221, ADInstruments, Colorado Springs, CO, USA) was attached inline in the branch of the ventilator circuit and the endotracheal airway pressure was continuously recorded. The polyethylene-50 catheter was then advanced to the left ventricle from the right common carotid artery via ascending aortic arch. The catheter tip position was verified by monitoring the pressure waveforms.
Cardiac output
To determine the effect of different chest compressions on cardiac output, a 1.9 Fr pressure-volume (PV) conductance catheter (Model FTE-1912B-8018, Scisense Inc., London, ON, Canada) was inserted into the left ventricular from the right CCA via ascending aortic arch (n = 5). After a 10-min asphyxiation, we alternately performed 3 different chest compressions without mechanical ventilation or adrenaline injection. The 3 types of chest compression was applied every 20 s and CPR was continued for up to 5 min. We evaluated the stroke volume using the ADVantage™ system (Scisense Inc., London, ON, Canada).
Brain tissue oxygen
A quenching oxygen probe (AL300, Ocean Optics, Dunedin, FL, USA) and a fluorometer (NEOFOX-GT, Ocean Optics, Dunedin, FL, USA) were used to measure the oxygen level in the brain tissue during CPR (n = 4). The response time of this oxygen sensor is less than 1 s. After rats were intubated and mechanically ventilated with oxygen-containing isoflurane, the body was secured in a prone position. We made a 2-mm bur-hole on the right parietal cortex (3 mm posterior and 3 mm lateral to the bregma). A 20-gauge plastic catheter (Surflo, Terumo Medical Corporation, Somerset, NJ, USA) was inserted into the subdural space and the quenching oxygen probe was advanced through the catheter. After completing the surgical preparation, the animals were repositioned to supine position. After a 10-min asphyxiation, we alternately performed three different chest compressions without administering adrenaline. Each chest compression was performed and switched every 20 s and continued for up to 4 min. The average of 10 s at the last half of the compression cycle was recorded and analyzed as the oxygen level in the brain tissue.