Measurements were made on five pregnant sheep (gestational age, 122–130 days; term, 145 days) that were part of a research protocol investigating the effect of antihypertensive therapy on maternal and fetal circulation. All experiments were performed in accordance with the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (1986) and in compliance with the European Union Directive 86/609/EEC (1997). The research protocol was approved by the Animal Care and Use Committee of the University of Oulu, Finland.
Surgical procedure and instrumentation
Before surgery, food was withheld for 18 h. Premedication consisted of intramuscular ketamine (2 mg/kg) and midazolam (0.2 mg/kg). General anesthesia was induced with intravenous propofol (4–7 mg/kg). The anesthesia was maintained with isoflurane (1–2.5%) in an oxygen–air mixture delivered via an endotracheal tube, combined with intravenous boluses of fentanyl as required. Mechanical ventilation was maintained throughout the surgical procedure with a Siemens 730 ventilator (Siemens-Elema AB, Solna, Sweden). An auricular artery was cannulated to measure the arterial blood pressure (BP) and heart rate. An 8.5-F (2.8-mm) Introflex polyurethane sheath with bonded Touhy-Borst valve and a sideport (Edwards Lifesciences LLC, Irvine, CA, USA) was inserted into the left external jugular vein for intravenous access and for the insertion of a thermodilution catheter on the experimental day.
A midline laparotomy was performed and a 6-mm transit-time ultrasonic flow probe (Transonic Systems Inc., Ithaca, NY, USA) was placed around the uterine artery supplying the pregnant uterine horn proximal to its bifurcation. The flow probe was secured in a fixed position with sutures, tunneled subcutaneously, and exteriorized through a small incision in the ewe's flank. The laparotomy incision was closed. For postoperative analgesia, a fentanyl patch was attached to the ewe's tail, and intramuscular fentanyl (2 µg/kg) was given in the first 48 h and buprenorphine (0.01 mg/kg) thereafter. The ewes were given 1 g ampicillin daily via the jugular venous catheter, which was flushed daily with heparinized saline. The animals were allowed to recover for 4 days before experiments started.
Data acquisition was carried out on anesthetized animals on the 5th postoperative day. General anesthesia was induced with intravenous propofol (4–7 mg/kg) and maintained throughout the experiments with isoflurane (1–1.5%) in an oxygen–air mixture delivered via an endotracheal tube.
A 16-gauge polyurethane catheter was inserted into the descending aorta via a femoral artery for monitoring the aortic BP and acid–base status. A thermodilution catheter (Criticath SP5107H, Becton Dickinson, Sandy, UT, USA) was inserted into the pulmonary artery through the external jugular vein access. In addition, a 19-gauge epidural catheter was placed into the epidural space just above the lumbosacral junction. Ringer's lactate solution was infused freely until pulmonary capillary wedge pressure (measured by filling the balloon located close to the tip of thermodilution catheter with 1.5 mL air to occlude a pulmonary capillary artery) reached a value of 6 mmHg, and thereafter at a fixed rate of 200 mL/h. The sheep was then positioned supine with a right lateral tilt.
The animals were allowed to stabilize for 30 min before the baseline (Phase 0) measurements were made. In Phase I, noradrenaline infusion was started at an initial rate of 0.2 µg/kg/min and adjusted to achieve a systolic BP of at least 30% above the baseline, or over 140 mmHg. The maternal hemodynamic and uterine artery Doppler measurements were obtained 15 min after achieving the targeted increase in maternal systolic BP. In Phase II, the ewe received an infusion of a beta-blocker (labetalol 1mg/kg) over 30 min in order to decrease the systolic BP to baseline level. In Phase III, hypoxemia was induced by replacing oxygen with medical air (3 L/min) in the rebreathing circuit until the maternal oxyhemoglobin saturation level reached 80–90%. In Phase IV, the ewe was allowed to recover from hypoxemia by returning the inhaled oxygen concentration to baseline. In Phase V, 0.5% bupivacaine was administered through the epidural catheter to a total dose of 0.3 mL/kg body weight 2 min after an initial test dose of 5 mL. Hypotension was allowed to develop to at least a 20% decrease in the maternal systolic BP. In Phase VI, 5-mg boluses of ephedrine were administered intravenously to raise the maternal systolic BP to at least 90% of the baseline level. The total experiment lasted for an average of 3 h.
The aortic BP of the ewe was monitored continuously using a disposable pressure transducer (DT-XX, Ohmeda, Hatfield, UK). The mean arterial pressure (MAP) was calculated as: MAP = diastolic BP + 1/3(systolic BP − diastolic BP). The central venous pressure (CVP) was measured continuously from the superior vena cava just above the right atrium with the thermodilution catheter, which has an extra lumen for CVP measurement approximately 30 cm proximal to the catheter tip. The mQUtA was measured directly with a perivascular transit-time ultrasonic flow probe (T206, Transonic Systems Inc., Ithaca, NY, USA). All these variables were recorded continuously at a sampling rate of 100 Hz using a polygraph (UIM100A, Biopac Systems Inc., Santa Barbara, CA, USA) and computerized data acquisition software (Acqknowledge v. 3.5.7 for Windows, Biopac Systems Inc., Santa Barbara, CA, USA).
Ultrasonography was performed, using a Vivid 7 Dimension ultrasound system (GE Vingmed Ultrasound, Horten, Norway) with a 4-MHz curvilinear probe, during each phase of the experiment. The high-pass filter (low velocity reject) was set at minimum (1 cm/s). Color Doppler was used to identify the uterine artery and to optimize the insonation angle (kept < 20°) for pulsed-wave Doppler interrogation. Blood flow velocity waveforms were obtained from the proximal portion (before bifurcation) of the same uterine artery around which the perivascular flow probe was attached (Figure 1a). Online measurements were performed using the software supplied with the ultrasound machine and the following parameters were obtained: peak systolic velocity (PSV), end-diastolic velocity (EDV), time-averaged maximum velocity (TAMXV), time-averaged intensity-weighted mean velocity (TAMEANV) and heart rate (HR). An average value of three consecutive cardiac cycles was used for statistical analysis.
Figure 1. (a) Uterine artery blood velocity waveforms obtained by Doppler ultrasonography and (b) measurement of uterine artery diameter in a pregnant sheep using power Doppler angiography.
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The diameter of the uterine artery was measured during systole using power Doppler angiography (Figure 1b) as described by Konje et al.5. Measurements were repeated three times and the average value was used for the calculation of uterine artery CSA, assuming the vessel to have a circular lumen: CSA = π(diameter/2)2. cQUtA was calculated according to the following formula: cQUtA(mL/min) = TAMEANV (cm/s) × CSA (cm2) × 60.
During each phase of the experiment, maternal hemodynamic and uterine artery Doppler parameters were obtained simultaneously and maternal arterial blood samples were taken and analyzed for acid–base status (corrected for a body temperature of 39 °C) using an i-Stat® 1 Analyser (i-Stat Corporation, East Windsor, NJ, USA). At the end of the experiment the animals were euthanized using a lethal dose of pentobarbital.
Data were analyzed using Statistical Software for Social Sciences for Windows version 13.0 (SPSS Inc. Chicago, IL, USA). Linear regression analysis was used to examine the association between variables. Correlations were tested using Pearson's correlation coefficient. Agreement between the two methods of QUtA measurement was assessed for bias and precision using Bland–Altman analysis11, 12 on log-transformed data. Bias was defined as the geometric mean of all volume flow measurement ratios (cQUtA/mQUtA) and precision as 95% confidence limits of agreement. The statistical significance level was set at a P-value of < 0.05.