The investigation conformed to the Danish law for animal research (Act no. 1306 of 23/11/2007, Danish Ministry of Justice) and the guidelines from Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).
The study was an experimental animal study performed at a university research laboratory. Twenty-four pigs were allocated to four groups: IAH with CO2 pneumoperitoneum [CO2] (n = 8), CO2 pneumoperitoneum sham (n = 4), IAH with intra-abdominal volume addition [VOL] (n = 8), and intra-abdominal volume addition sham (n = 4). After all experiments were performed, we evaluated the data from the CO2 sham group and the VOL sham group at T = 0 h and T = 10 h for the following parameters: weight, diuresis, pH, partial pressure of CO2 [pCO2], base excess, K+, lactate, heart rate, mean arterial pressure [MAP], mean pulmonary artery pressure [MPAP], central venous pressure [CVP], and creatinine. The groups were similar for all measured values except lactate T = 0 h (mean value for the three groups: control = 0.963, CO2 = 0.863, VOL = 1.125) (P = 0.03). Data from the CO2 sham group and the VOL sham group were therefore pooled into one control group (n = 8). After baseline measurements (T = 0 h), the pigs were randomized to one of the four study groups, and the IAH induction procedure or the sham operation was performed. Sampling started at baseline (T = 0 h) and continued with one sample per hour for 12 h (T = 1 h to T = 12 h). The following data were collected: (1) physiological parameters: intra-bladder pressure [IBP], heart rate, MAP, MPAP, CVP, abdominal perfusion pressure (APP = MAP - IAP), and tidal volume; (2) arterial blood samples: pH, pCO2, base excess, K+, and lactate; and (3) venous blood samples: creatinine.
The animals were female Danish Landrace pigs that had fasted for 24 h prior to the experiment and had free access to water. Prior to transportation to the institute, they received 0.5 mg/kg midazolam (Janssen Pharmaceutica, Beerse, Belgium) and 4 mg/kg azeperone (Janssen-Cilag GmbH, Neuss, Germany) intramuscularly. General anesthesia was induced with an intramuscular injection of 4.35 mg/kg (S)-ketamine (Pfizer ApS, Ballerup, Denmark) and 0.375 mg/kg midazolam (Hameln Pharmaceuticals GmbH, Hameln, Germany) intravenously, followed by intubation. After intubation, the animals were ventilated via a respirator (Datex-Ohmeda S/5 Avance, GE Healthcare, Brøndby, Denmark). Sedation was maintained with sevoflurane (Abbott Scandinavia AB, Solna, Sweden) inhalation to obtain a minimal alveolar concentration of approximately 1.5. Fentanyl was infused intravenously at a constant rate of 12.5 μg/kg/h. No muscular relaxant was used. Pressure-controlled respirator settings were used. Initial settings were a fraction of inspired oxygen [FiO2] of 0.3; positive end-expiratory pressure [PEEP] of 4 cm H20; inspiratory pressure [P
insp] of 12 cm H2O, restricting peak airway pressure to a maximum of 16 cm H2O; respiratory frequency of 12/min, and inhalation/expiration ration [I/E] of 1:2. Saline was infused at a constant rate of 1.5 ml/kg/h.
A 6Fr catheter was placed in the carotid artery using a cutdown technique for the measurement of arterial blood pressure and arterial blood sampling. An 8Fr catheter was placed in the right external jugular vein for infusions and to introduce a Swan-Ganz catheter (CCOmbo®, Edwards Lifesciences LLC, Irvine, CA, USA) connected to a Baxter Vigilance monitor (Edwards Life Sciences LLC, Irvine, CA, USA) in order to measure MPAP and CVP. A 7Fr catheter was placed in the left external jugular vein for venous blood sampling. The urinary bladder was catheterized using a 12G Foley catheter connected to an IBP measuring catheter (UnoMeter Abdo-Pressure, Unomedical, Birkerød, Denmark) and a sample tube to monitor diuresis. A rectal thermometer was inserted to measure core temperature. A pulse oximetry device was attached to the pig's tail to observe arterial blood oxygen saturation.
Induction of intra-abdominal hypertension
Induction of IAH with the CO2 group
Verres Cannula was inserted below the umbilicus and attached to a laparoscopic CO2 insufflator (Thermoflator 26432020, Karl Storz, Holte, Denmark or Vision F103, Lemke, Berlin, Germany). The insufflator was set to IAP = 25 mmHg.
Induction of IAH with the VOL group
Via a 15-cm incision above the umbilicus, seven 1-l ordinary saline infusion bags were placed in the abdominal cavity. Bags were positioned into the small pelvis until IBP was above 20 mmHg. The abdominal wall was closed in two layers, including the fascia, using running sutures.
CO2 pneumoperitoneum sham group
These animals underwent a procedure similar to the CO2 group, but Verres Cannula remained unattached to the insufflator.
Intra-abdominal volume addition sham group
The animals underwent a procedure similar to the VOL group, but empty saline bags were inserted into the abdominal cavity.
Anesthesia maintenance and ventilator settings during experiment
IAH decreases lung capacity and requires the adjustment of respirator settings to ensure sufficient ventilation. Our priorities were to maintain arterial oxygen saturation > 90%, pCO2 < 5 kPa, MAP > 60 mmHg, and a tidal volume of 400 ml. Initial respirator settings could be increased up to FiO2 = 0.7; PEEP = 8 cm H2O; P
insp = 24 cm H2O, restricting peak airway pressure to a maximum of 32 cm H2O; respiratory frequency = 24; and I/E = 1:1.5. Immediately following IAH generation, all respirator settings, except FiO2, were adjusted to maximally allowed settings in order to avoid respiratory complications such as atelectasis. A 500-ml bag of Rheomacrodex® 100 mg/ml with saline was infused when arterial pressure was < 60 mmHg. Two bags of Rheomacrodex® (Meda A/S, Alleroed, Denmark) were allowed for each pig.
A fixed point at bladder level was marked on the animals. Prior to IBP measurements, the bladder pressure-measuring catheter was elevated. This installed the fluid inside the catheter into the bladder, ensuring that the bladder was not empty when IBP was measured. The zero point of the IBP-measuring catheter was aligned with the marked point on the pig, and the catheter was held vertically. End-expiratory bladder pressure was noted. The animals were in supine position.
Arterial blood samples were analyzed on a blood gas analyzer (ABL 700 series, Radiometer, Copenhagen, Denmark). Heart rates and blood pressures were sampled and recorded through a computer connected to the respirator using software (Datex-Ohmeda S5 Collect Program, version 4.0, Datex-Ohmeda Division, Instrumentarium Corp, Helsinki, Finland). Venous blood samples were stored at -20°C, and the creatinine content was measured by trained personnel at Aarhus University Hospital. APP was calculated as MAP - IBP. Dynamic lung compliance [Cdyn] was calculated as tidal volume/(P
insp - PEEP).
Repeated serial measurements were tested for group differences over time by univariate repeated measures using analysis of variance [ANOVA]. MedCalc 184.108.40.206 (MecCalc Software, Mariakerke, Belgium) and Intercooled STATA 9.2 (College Station, TX, USA) were used in the analyses. Furthermore, a Kruskal-Wallis one-way ANOVA on ranks using Dunn's method for multiple comparisons was performed between the three groups at corresponding moments. Sigmastat™ version 3.5 (Systat Software, San Jose, California, USA) was used in the analyses. The similarity between the two sham groups (CO2 pneumoperitoneum sham group (n = 4) and the intra-abdominal volume addition sham group (n = 4)) was tested with the Mann-Whitney rank sum test using SigmaStat™ version 3.11 (Systat Software, San Jose, California, USA) at two points: T = 0 h and T = 10 h. For all analyses, a P value of less than 0.05 was considered significant.