This study was performed in accordance with the National Institutes of Health (Bethesda, MD, USA) guidelines for the care and use of experimental animals. The protocol was approved by the animal ethics committee of Canton Bern, Switzerland. Twenty-seven domestic pigs (weight 28 to 32 kg) were fasted overnight but had free access to water. The pigs were sedated with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg). Then a peripheral intravenous catheter was inserted in an ear vein for initial administration of fluids and medications. Anesthesia was induced with midazolam 0.4 mg/kg and atropine 1 mg. After induction, the pigs were orally intubated and ventilated with oxygen in air (fraction of inspired oxygen = 0.3). Anesthesia was maintained with midazolam 0.5 mg/kg per hour, fentanyl 15 μg/kg per hour, pancuronium 0.3 mg/kg per hour, and low-dose propofol 0.15 mg/kg per hour. The animals were ventilated with a volume-controlled ventilator with a positive end-expiratory pressure of 5 cm H2O (Servo 900C; Siemens, Solna, Sweden). Tidal volume was kept at 8 to 10 mL/kg, and the respiratory rate was adjusted (22 to 26 breaths per minute) to maintain end-tidal carbon dioxide tension (PaCO2) at 5.3 ± 0.5 kPa. Immediately after induction, all animals received 1.5 g of Cefuroxim intravenously as an antibiotic prophylaxis. The stomach was emptied with a large-bore orogastric tube.
Surgical preparation
Through a left cervical cut-down, indwelling catheters were inserted into the left carotid artery and superior vena cava. A balloon-tipped catheter was inserted into the pulmonary artery through the right external jugular vein. Location of the catheter tip was determined by observing the characteristic pressure trace on the monitor as the catheter was advanced through the right heart into the pulmonary artery. Similarly, a fiberoptic hepatic vein catheter was inserted through the right jugular vein. Correct positioning was verified by a 15% to 20% decrease in the continuously measured hepatic vein saturation versus the mixed venous saturation and by a significant decrease in lactate concentration compared with mixed venous blood. The right carotid artery was dissected free and a 4-mm ultrasound transit time flow probe was placed around the vessel to measure carotid artery blood flow.
With the pig in the supine position, a midline laparotomy was performed. A catheter was inserted into the urinary bladder for drainage of urine. A second catheter was inserted into the mesenteric vein for blood sampling. The superior mesenteric artery (SMA), the celiac trunk, and the hepatic artery were identified close to their origin. After dissection to free these vessels from the surrounding tissues, precalibrated ultrasonic transit time flow probes (Transonic Systems, Ithaca, NY, USA) were placed around the vessels and connected to an ultrasound blood flowmeter (T 207; Transonic Systems).
Through a small incision in the jejunum, a custom-made laser Doppler flowmetry (LDF) probe (Oxford Optronix, Oxford, UK) was sutured to the jejunum mucosa for measurements of microcirculatory blood flow in the mucosa. A second LDF probe was sutured to the adjacent jejunum muscularis. Both LDF probes were attached with six microsutures to ensure continuous and steady contact with the tissue under investigation, preventing motion disturbance from respiration and gastrointestinal movements throughout the experiment. The signals of the LDF probes were visualized on a computer monitor. If the signal quality of a probe was poor, the position of the probe was corrected immediately. The incision in the jejunum also allowed controlled positioning of an air tonometer tube (TRIP Sigmoid catheter; Datex-Ohmeda, GE Healthcare, Helsinki, Finland). The bowel incision was then closed with continuous sutures.
For intramural intestinal tissue oxygen tension measurement, a polarographic tissue oxygen tension sensor was inserted into a section of healthy jejunum between the serosal and the mucosal tissue planes. The method has been described previously [15, 16]. Care was taken to minimize handling of the small intestine and to return the bowel to a neutral position. After preparation, the abdominal incision was closed and the animals were allowed to recover from instrumentation and stabilize for 60 minutes.
Throughout the entire study, all animals received a basal infusion of 3 mL/kg per hour of Ringer lactate (RL) to avoid excessive fluid administration. This fixed fluid administration resulted in a low central venous and pulmonary capillary wedge pressure (PCWP) of between 2 and 4 mm Hg at baseline. Body temperature of the animals was maintained at 38.0 ± 0.5°C with a forced-air patient air warming system (Warm Touch 5700; Mallinckrodt, Hennef, Germany). Baseline measurements were performed after stabilization at t = 0 minutes. Subsequently, all hemodynamic measurements were repeated every 30 minutes for 4 hours. Blood samples were drawn hourly after the measurements of the hemodynamic parameters.
Immediately after baseline measurements, the pigs were randomly assigned to one of three fluid treatment groups using a reproducible set of computer-generated random numbers. The assignments were kept in sealed, opaque, and sequentially numbered envelopes until used. Once the fluid therapy was assigned, the investigators were not blinded anymore. The assigned fluid therapy was started 15 minutes after the first measurement. The fluid treatment groups were as follows.
Groups
The 'restricted Ringer lactate' (R-RL) group (n = 9) received a fixed administration of 3 mL/kg per hour of lactated Ringer solution throughout the experiment without additional fluids.
The 'goal-directed Ringer lactate' (GD-RL) group (n = 9) received a fixed administration of 3 mL/kg per hour of lactated Ringer solution throughout the experiment. Additionally, this group received an administration of 250 mL of lactated Ringer solution as a bolus (within 3 to 4 minutes) if the mixed venous oxygen saturation (SvO2) was less than 60% ('lockout' time between two boluses = 30 minutes).
The 'goal-directed colloid' (GD-C) group (n = 9) received a fixed administration of 3 mL/kg per hour of lactated Ringer solution throughout the experiment. Additionally, this group received an administration of 250 mL of hydroxyethyl starch (HES) (130/0.4) as a bolus (within 3 to 4 minutes) if the SvO2 was less than 60% (lockout time between two boluses = 30 minutes).
Measurements
Respiratory monitoring
Expired minute volume, tidal volume, respiratory rate, peak and other respiratory pressures, positive end-expiratory pressure, inspired and end-tidal carbon dioxide fraction, and inspired/expired oxygen fraction were monitored (S/5 Critical Care Monitor; Datex-Ohmeda, GE Healthcare) throughout the study.
Hemodynamic monitoring
Mean arterial blood pressure (MAP) (mm Hg), central venous pressure (CVP) (mm Hg), mean pulmonary artery pressure (PAP) (mm Hg), hepatic vein pressure (HVP) (mm Hg), and PCWP (mm Hg) were recorded with quartz pressure transducers. Pulse pressure variation (PPV) and stroke volume (SV) were measured with a PiCCO (pulse contour cardiac output) plus hemodynamic monitor (Pulsion Medical Systems GmbH, Munich, Germany) connected to the arterial pressure transducer. Heart rate was measured from the electrocardiogram. Heart rate, MAP, PAP, and CVP were displayed continuously on a multi-modular monitor (S/5 Critical Care Monitor). A thermodilution method was used to measure cardiac output at each measurement point (mean value of three consecutive manually performed measurements with 5 mL of cold saline). Core temperature was measured from the thermistor in the pulmonary artery catheter. Regional blood flow in the SMA, the celiac trunk, and the hepatic artery was continuously measured throughout the experiments with ultrasonic transit time flowmetry (mL per minute) using two double-channel HT 206 flowmeters (Transonic Systems).
Microcirculatory blood flow was monitored continuously in the mucosa and the muscularis of the jejunum using a multi-channel laser Doppler flowmeter system (Oxford Optronix). A detailed description of the theory of LDF operation and practical details of LDF measurements have been published previously [17, 18]. The regional blood flow and the LDF data were acquired online with a sampling rate of 10 Hz via a multi-channel interface (MP 150; Biopac Systems Inc., Goleta, CA, USA) with acquisition software (Acqknowledge 3.9; Biopac Systems Inc.) and saved on a portable computer. Laser Doppler flowmeters are not calibrated to measure absolute blood flow but indicate microcirculatory blood flow in arbitrary perfusion units. Due to a relatively large variability of baseline values, the results usually are expressed as changes relative to baseline [19–22] and that was also the case in the present study.
The jejunal intramucosal carbon dioxide pressure was measured with air tonometry (Tonocap® Monitor; Datex-Ohmeda, GE Healthcare). The jejunal mucosal-to-arterial carbon dioxide pressure gap (CO2 gap) was calculated at each measurement point.
Arterial, mixed venous, mesenteric, and hepatic venous blood samples were withdrawn hourly from the indwelling catheters and immediately analyzed in a blood gas analyzer (ABL 620; Radiometer, Copenhagen, Denmark) for oxygen partial pressure (pO2) (kPa), carbon dioxide partial pressure (pCO2) (kPa), pH, lactate (mmol/L), and base excess (BE). Arterial oxygen saturation (SO2) (percentage) and total hemoglobin concentration (Hb) (g/dL) were measured with an analyzer specially adjusted to porcine blood (OSM 3; Radiometer). All values were adjusted to body temperature. Mixed and hepatic venous saturations were displayed continuously on two continuous cardiac output monitors (Vigilance; Edwards Lifesciences LLC, Baxter, Irvine, CA, USA).
CI (mL/kg per minute), SMA flow index (SMAI) (mL/kg per minute), and systemic vascular resistance index (SVRI) (mm Hg/kg per minute) were indexed to body weight. SVRI was calculated as: SVRI = (MAP - CVP)/CI [20, 23].
Systemic oxygen delivery index (sDO2I) (mL/kg per minute), systemic oxygen consumption index (sVO2I) (mL/kg per minute), and the corresponding mesenteric (splanchnic) variables (mDO2I and mVO2I) (mL/kg per minute) were calculated using the following formulas: Systemic (total body) oxygen delivery index (sDO2) = (CI × CaO2), where CaO2 is the arterial oxygen content. Systemic (total body) oxygen consumption index (sVO2) = (CI × [CaO2 - CvO2]), where CvO2 is the mixed venous oxygen content. Mesenteric (splanchnic) oxygen delivery index (mDO2) = SMAI × CaO2. Mesenteric (splanchnic) oxygen consumption index (mVO2) = SMAI × (CaO2 - CmO2), where CmO2 is the mesenteric vein oxygen content. Oxygen content (mL of O2/mL of blood) = ([pO2 × 0.0031] + [Hb × SO2 × 1.36])/100.
In the same animals an additional hypothesis was tested regarding the changes of microcirculatory blood flow in healthy colon and in a critically perfused colon anastomosis. This data is published elsewhere [24].
Statistical analysis
Data were tested for normality by QQ-plot and Kolmogorov-Smirnov test. All baseline data (that is, before the start of the respective treatment at t = 0 minutes) were compared with analysis of variance (ANOVA) or Kruskal-Wallis test to exclude initial group discrepancies. Differences between the three fluid treatment groups were assessed by ANOVA for repeated measurements using group as between-subject factor and time as within-subject factor. If a significant difference between the groups was detected, a Tukey post hoc test was performed to assess differences at individual time points. Additionally, the area under the variable-time curve for each variable of interest was calculated and compared with ANOVA for group differences. A Tukey post hoc test was performed to compare individual treatments if the ANOVA had detected significant differences between the groups. Measurements of microcirculatory blood flow (LDF) were transformed with baseline set to 100% (t = 0 minutes) prior to statistical analysis. Absolute values were used for all other calculations. Data are presented as means ± standard deviations unless otherwise specified. A P value of less than 0.05 was considered significant. For statistical calculations, SAS version 8 (SAS Institute Inc., Cary, NC, USA) was used.