Animals
Ten crossbred (Landrace × large white) healthy female pigs (Sus scrofa domestica), 4 to 5 months old, with a mean body weight of 51.6 ± 2.8 kg, were used in the study. The animals were obtained from a local certified farm, inspected on arrival by an institutional veterinarian, and were not subjected to any other interventions before or after the study protocol. Prior to the study, the animals were housed in a standard, accredited, university animal facility. They were kept in metal cages with minimum dimensions of 1.8 × 2.2 m, with natural daylight and free access to water, and were fed twice daily with a mixture recommended for young swine. Ambient conditions (temperature, air) were regulated according to relevant standards. Animals were kept in groups of up to four. Anesthesia was provided during the whole study.
Anesthesia
After 24 h of fasting, sedation was induced by midazolam (0.3 mg/kg IM) followed by ketamine hydrochloride (15 to 20 mg/kg IM). Anesthesia was continued with initial boluses of propofol and morphine (2 mg/kg IV and 0.1 to 0.2 mg/kg IV, respectively), and animals were orotracheally intubated. A continuous IV infusion of propofol (8 to 10 mg/kg/h) combined with morphine (0.1 to 0.2 mg/kg/h) IV was used to maintain anesthesia, the depth of which was regularly assessed by photoreaction and corneal reflex.
Ventilation
Volume-controlled ventilation was delivered by the Hamilton G5 ventilator (Hamilton Medical, Bondauz, Switzerland) set at Vt 8 ml/kg, PEEP 5 cm H2O, FiO2 0.25, I:E 1:2, and MV adjusted to maintain pCO2 5.0–5.5 kPa (34–41 mmHg) and pO2 9.3–16 kPa (70–120 mmHg).
Invasive measurements
A triple-lumen central venous oximetry catheter (Multi-Med, Edwards Lifesciences) was inserted via the left external jugular vein to allow for (i) insertion of the Doppler Flow wire (0.014″) via its distal lumen, (ii) central venous pressure measurement (CVP), and (iii) administration of i.v. medications.
A Swan–Ganz continuous cardiac output (CCO) catheter (Edwards Lifesciences, USA) was inserted via the left femoral vein and advanced into the pulmonary artery to provide measurements of pulmonary arterial pressure (PAP) and pulmonary capillary wedge pressure (PCWP). CCO and mixed venous saturation (SVO2) were measured using the Vigilance monitor (Edwards Lifesciences, USA). In addition, a high-fidelity pressure–volume catheter (7F VSL, connected to ADV 500 console, Transonic Scisense Inc., London, ON, Canada) was placed retrograde into the left ventricular apex via a 7F sheath inserted percutaneously into the left external carotid artery. The correct position was confirmed by fluoroscopy. This catheter provided simultaneous measurements of left ventricle (LV) pressure and volume. LV pressure–volume loops were obtained from PV data using Powerlab Pro 7.0. software (ADInstruments, USA).
An introducer sheath (5F) was inserted into the femoral artery for invasive arterial blood pressure measurement. Another 6F introducer was placed in the contralateral femoral artery for blood removal during the hypovolemia protocol. A further 7F sheath was inserted into the right femoral vein for rapid blood and fluid replacement.
All blood pressure measurements (excluding LV pressure) were obtained via a fluid-filled pressure transducer (TruWave, Edwards Lifesciences, USA) connected to a patient monitor (Lifescope TR, Nihon Kohden, Japan). Peripheral saturation, capnometry, and temperature recordings were also performed via the patient monitor.
Doppler-based technique for superior vena cava blood flow assessment
The technical details of Doppler-based blood flow velocity measurement has been fully described previously, in a paper done by Doucette et al. [17]. A Flowire, connected to a Combomap console (Volcano Corp.), was inserted through the distal lumen of the central venous catheter, to the position in the SVC which had the best quality of laminar flow Doppler signal. The correct position was confirmed by fluoroscopy. The pulse Doppler of the SVC flow velocity was displayed on the console. The console automatically detected and plotted the envelope of the SVC flow velocity pattern. Data was read and recorded using the National Instruments LabView software. Doppler sampling frequency was 100 kHz.
The following parameters were recorded:
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SVC flow velocity as a velocity time integral (VTI) of the Doppler envelope.
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Venous return variation index (VRV), an analogue to the SVV as a new index for fluid responsiveness. We used airway pressure for respiration gating and ECG R-R intervals for heart beat detection. We analyzed flow variations during each individual respiratory cycle at 20-s intervals. Beats with maximal and minimal VTI values were used in the formula. Minimal flow occurred during mechanical inspiration; maximal flow was recorded in expiration. The VRV index was calculated as VRV = (VTImax − VTImin)/(VTImax + VTImin)/2. Individual respiratory cycle VRV values were averaged over a 20-s period.
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Systolic peak velocity (S peak) was also recorded. It is defined as peak velocity on a systolic wave using the final end-expiratory beat.
Data recording
Doppler envelope data were continuously recorded with a custom PC-based application specifically designed for the purposes of this trial. Other modalities recorded simultaneously by the same application were central venous pressure, arterial pressure, airway pressure, and ECG (Fig. 1). The application was designed to allow for both continuous manual and automatic parameter calculation (PPV, SVV, EDP, SVC flow with respiratory and ECG gating). Other physiological parameters were also recorded by the ADI LabChart Pro at 400 Hz (CCO, SvO2, PCWP, PAP, CVP, AP—arterial pressure, temperature).
Medication
Initial rapid IV infusion of 1000 ml of normal saline was given intravenously, followed by a continuous IV infusion to reach and maintain a central venous pressure of between 3 and 7 mmHg.
Unfractionated heparin (100 UI/kg IV) was given as a bolus after sheath placement, followed by 40 to 50 U/kg/h continuous IV drip to maintain activated clotting time of 180 to 250 s (values were checked every hour with Hemochron Junior+, International Technidyne Corporation, Edison, NJ, USA).
Study protocol
After sedation and ventilation set-up, the animals were kept in euvolemic status.
Euvolemia was defined as:
Euvolemia was then followed by bleeding periods.
The bleeding was induced by opening the 6F sheath introduced to the femoral artery. Blood was collected in transfusion sets, which were stored at room temperature. Estimated blood volume (EBV) was calculated as 65 ml/kg [18].
The first stage of bleeding removed 10% of EBV. This was followed by five subsequent stages, which each removed 5% of the initial EBV. A steady state of 10 min was kept after each bleeding period, after which measurements were performed.
The last bleeding was followed by two stages of 500 ml blood return (each lasting 30 min) separated by 10-min steady states. Fluid challenge (1000 ml saline over 30 min, given two times, separated by 10 min of steady state) administered after a non-responsive event was classified as fluid overload.
We were searching for a new predictor of fluid responsiveness, defined as an increase in the stroke volume by more than 10% (in comparison with the previous stage of the experiment).
Statistic analysis
The data presented is from 10 animals with a total of 44 events classifiable as responsive or non-responsive, in terms of changes in stroke volume variations. Fluid responsiveness was defined as a 10% change in stroke volume after all three study steps: bleeding, blood return, and fluid overload. During the trial, some events were not classifiable due to arrhythmias, PAC failure, or Doppler envelope detection failure. Five cases provided complete continuous trending data of all measured variables.