Study protocol
After approval from the regional animal study ethics committee, 7 adult pigs were prepared. The animals (70 kg) were anesthetized and mechanically ventilated. When the animals showed circulatory stability, a 10 minute resting period without any activity or stimulation was instituted. After this period, a shed blood surrogate containing lipid micro-emboli was infused according to the protocol illustrated in Figure 1. The animals were monitored for the following three hours, after which cardiac output and pulmonary pressure were increased by infusing 200 μg of epinephrine (Adrenaline Merck NM, Merck NM, Stockholm, Sweden) together with 500 ml Ringer's lactate (Fresenius Kabi, Uppsala, Sweden).
Monitoring
Catheters for monitoring, drug delivery and blood sampling were inserted into a vein in one of the ears, the jugular internal vein, the left and right atrium, the pulmonary artery and the femoral and carotid arteries. During the experiment, arterial and pulmonary blood pressure, central venous and left atrium pressure, pulse, nasopharyngeal temperature, ventilator settings and pulse oximetry were monitored continuously with a SpaceLabs Medical monitoring system (SpaceLabs, Issaquah, WA, USA). Cardiac output was recorded with a Transonic HT207 ultrasonic flow meter (Transonic System Inc., Ithaca, New York, USA) or a Cardiomed CM 4000 transit time ultrasound flow meter (Cardiomed, Toronto, Canada). A 14 mm or 16 mm probe was used to measure the cardiac output in the pulmonary artery. Complete monitoring data were obtained from all animals except one. The recordings of pulmonary artery pressure in one animal were unstable due to deficient contact between the ultrasonic transducer and the pulmonary artery, and this pressure was excluded from the analysis.
Anesthesia
Premedication was performed with an intramuscular injection of 15 mg/kg ketamine chloride (Ketalar®, Pfizer Inc., New York, NY, USA) and 0.2 mg/kg xylasine (Rompun®, Bayer, Gothenburg, Sweden). Induction and maintenance of anesthesia were achieved using an infusion of 0.15 mg/kg/min ketamine chloride and 0.01 mg/kg/min pancuronium bromide (Pavulon®, N.V. Organon, Oss, the Netherlands), or an infusion of 0.1-0.2 mg/kg/min propofol (Diprivan®, Astra-Zeneca, Sweden), together with intermittent injections of fentanyl (Leptanal®, Lilly, France) and atracrium besylate (Tracrium®, Glaxo, Täby, Sweden). The different anaesthetic protocols were due to a change in laboratory practice for other reasons, and not related to this study.
The animals underwent tracheostomy and were connected to a ventilator (Siemens Servo 900C, Solna, Sweden). Volume controlled ventilation was applied with 50% FiO2. Arterial blood was drawn for blood gas sampling at the start of the study, immediately before administration of radiolabelled triolein, 5, 15, 30 and 45 minutes thereafter, and immediately before the period of increased pressure, and 5, 15, 30 and 45 minutes thereafter. Exhaled gases were monitored continuously with a NiCO or CO2SMO Plus respiratory profile meter (Novametrix Medical Systems Inc., Norwell, MA, USA). The dead space was calculated using the Bohr equation principle from blood gases and exhaled CO2 [18].
Experimental procedure
A sternotomy was performed to expose the heart and 400 IU/kg heparin (LEO Pharma A/S, Copenhagen, Denmark) was administered before starting the experiment. At the end of the experiment the animals were sacrificed using potassium chloride (B. Braun, Melsungen, Germany) and thiopental sodium (Pentothal®, Abbot, Chicago, IL, USA).
Administration of radiolabelled triolein
Radioactive triolein (Amersham BioSciences, Little Chalfont, UK) was mixed with 65% non-radioactive triolein solution (Carl Roth GmbH., Karlsruhe, Germany). The proportions used were such that 5 ml of the final solution contained 1 mCi of radioactivity.
A shed blood surrogate was then produced by mixing 200 ml arterial blood with 200 ml saline and 5 ml of the 1 mCi radioactive triolein solution. This will yield approximately 1% lipid content, and has been used in similar studies [6, 19]. The surrogate was gently agitated for approximately five minutes and retransfused from a pressurized transfusion bag with a 40-micron filter in the infusion aggregate, into the animal via the venous line during a 2-minute period.
Blood sampling
Blood samples for the determination of radioactivity were drawn from a separate catheter in the carotid artery at baseline, at the start of infusion of the shed blood surrogate, every minute for fifteen minutes and then every ten minutes up to 3 hours after infusion.
When the infusion of adrenaline and 500 ml Ringer's lactate was started, blood was sampled at the start of infusion, every minute for fifteen minutes and then every ten minutes up to one hour after the infusion. On each occasion 0.2 ml blood was collected for the determination of radioactivity.
Sample preparation
To each sample of blood, 2 ml Soulene-350® (Packard Bioscience, Groningen, the Netherlands) was added to dissolve the cells. The sample was left in an air heater at 37°C overnight. To decolorize the samples, 0.2 ml hydrogen peroxide was added twice, with overnight incubation at 37°C between. One ml 95% ethanol was added, followed by 15 ml scintillation fluid (Hionic Fluor, Packard Bioscience, Groningen, the Netherlands) [6]. The samples were then left to rest for 4-6 days in order for the chemoluminescence to decrease.
Scintillation counting was performed with a liquid scintillation counter (14814 Win Spectral Guardian, Wallac Oy, Turku, Finland). The specific activity of tritium was calculated for each sample. Two separate measurements were performed, and the mean value of the two measurements was used. Radioactivity is reported as the number of disintegrations per minute per ml (DPM/ml).
Statistics
All values are expressed as the mean ± 1 standard deviation (SD). Comparisons between groups were made with a two-tailed student's t-test, unless otherwise stated. To quantify the physiological response in terms of a decrease or increase in blood pressure, the area under the curve (AUC) was calculated from the period of blood pressure change (Figure 1). The end of the change was defined as the point of time when the blood pressure had returned to the pre-event value.