Powerlab

The aorta was catheterized via the left carotid artery for continuous measurement of mean arterial pressure (MAP, Gould-Statham pressure transducers P23ID, Elk Grove, IL) and intermittent arterial blood gas samples adjusted for temperature (Rapidlab 865, Siemens, Eschborn, Germany) from appropriate syringes (PICO50, Radiometer Medical, Brønshøj, Denmark). Cardiac output (CO) was determined via transpulmonary thermodilution (PiCCO 4.2 non US, PULSION Medical Systems, Munich, Germany) at the end of each intervention, at least every 30 min. Heart rate (HR) was continuously measured by electrocardiography (Powerlab, ADInstruments, Castle Hill, Australia). All hemodynamic and respiratory variables were recorded on a personal computer after analog to digital conversion (Powerlab, ADInstruments, Castle Hill, Australia) for later analysis.

The autonomic function testing was done in a dedicated Patient Observation Room operated by the Autonomic Medicine Section at the NIH Clinical Center. After urinating to empty the bladder, the participant lay supine with head on pillow on a motorized tilt table. An intravenous (IV) catheter was placed in an arm vein, usually the left antecubital vein, and attached by a 3-way stopcock and connection tubing to a plastic bag containing normal saline that was infused continuously at a slow rate to keep the vein open.
Physiological data were recorded using LabChart Pro 8 running a 16-channel PowerLab electronic physiological recorder (ADInstruments, Colorado Springs, CO). Electrocardiographic leads were attached to the skin and connected by a harness to the PowerLab. A noninvasive automated finger cuff system was used for continuous BP recording [BMEYE Nexfin, BMEYE B.V., Amsterdam, The Netherlands) or Finapres Nova (Finapres B.V., Amsterdam, the Netherlands)]. Respiration was monitored using a Respitrace plethysmography device placed around the upper abdomen or lower chest and connected to the PowerLab (ADInstruments, Colorado Springs, CO).

The methods were previously described (Zheng et al., 2014 (link); Liu et al., 2018 (link)). Briefly, after fasting for 24 h, rats were anesthetized. After laparotomy, a strain gauge force transducer (WS100; Xinhang Xingye 349 Tech. Co., Beijing, China) was sutured onto the serosal surface of the gastric antrum (0.5 cm caudal from the pyloric ring) to measure the contractile activity of the longitudinal muscle. The abdomen was then closed, and the wires from the transducer were connected to the recording system (ML112, PowerLab, AD Instruments, Australia). After stabilizing for 2 h, the contraction data were collected by the “Chart & Scope” software (PowerLab, AD Instruments, Australia). The normal migrating motor complexes (MMCs), which include a 4-part cycle comprising phases I, II, III, and IV, were identified in the stomach. The contraction amplitude of phase III was evaluated.

BP, ECG, heart rate, and respiratory frequency were continuously recorded (PowerLab, ADInstruments, Colorado Springs, CO, USA) and acquired, amplified, and filtered at 1 kHz (Neurology, Digitimer, Welwyn Garden City, UK; PowerLab, ADInstruments, Colorado Springs, CO, USA). For basal autonomic evaluation, a baseline recording of 10 min was obtained. There was an interval of at least 3 min between each stimulation.

Rats were anesthetized and hearts were dissected carefully to isolate the left ventricular papillary muscles to the physiological salt solution of NaCl: 112 mM, MgCl₂: 1 mM, KCl: 5 mM, NaH₂PO₄: 0.5 mM, NaHCO₃: 25 mM, glucose: 10 mM, CaCl₂: 1.8 mM (pH: 7.4), KH₂PO₄: 0.5 mM and EDTA: 0.004 mM which carbogenated with 5% CO₂ and 95% O₂. Then the papillary muscles were attached vertically to an isometric force transducer (MLT 1030/D, ADInstruments, PowerLab, Spain) under a tension of 0.5 g in a 25 ml chamber of an organ bath (ADInstruments, PowerLab, Spain). The temperature of the bathing buffer was 33 °C. Papillary muscles were equilibrated in the organ bath for 90 min. For threshold, the muscles were exposed to an electrical-field stimulation at 1 Hz and then at 20% higher than the threshold to record the contractile force [20 (link)]. The papillary muscle excitation and contraction forces in all four groups were recorded and analyzed.

Heart rate and electrocardiogram (ECG) were recorded by PowerLab (PowerLab, ADinstruments, Oxfordshire, United Kingdom). Mean arterial pressure (MAP) and cardiac output (CO) were determined by pulse index continuous cardiac output (PiCCO, PiCCO Plus, Pulsion Medical Systems) (21 (link), 22 (link)).

Flow-controlled ventilation (FCV) was performed by Evone^® ventilator (Ventinova Medical, Eindhoven, Netherlands). Regarding to the manufacturer’s instructions FCV can be applied through a special ultra-thin endotracheal tube (Tritube^®) or a standard endotracheal tube (ETT) supplemented with an adapter which measures the airway pressure in the trachea, at the distal end of the setup. To standardize FOT measurements and avoid the risk of derecruitment due to reintubation, we used a standard ETT during the whole protocol. Tracheal pressure was continuously recorded also at the proximal end of the ETT by PowerLab (PowerLab, ADinstruments, Oxfordshire, United Kingdom). Peak inspiratory pressure (PIP), positive end-expiratory pressure (PEEP), flow and inspiratory to expiratory ratio can be set manually on the Evone ventilator. The setting of the respiratory rate on the ventilator is determined by different parameters such as flow, ventilatory pressures, minute volume. As the ventilator registers the mechanical parameters of the respiratory system (resistance, compliance) periodically, a 2-s-long inspiratory hold was interposed after every 10–12 breathing cycle. This mechanism resulted an elevated respiratory rate between the inspiratory holds to ensure the minute volume.