Experiments in two groups of adult white-New Zealand rabbits of 3(0.5)kgr were performed in accordance with the European Community and NIH guidelines for using experimental animals. All procedures were approved by our institution's Animal Studies Committee [EL 42 BIO 04]. The animals were maintained in room air and temperature and after induction of anesthesia with intramuscular administration of 1 ml ketamine 100 mg/ml and 1 ml midazolam 5 mg/ml, a peripheral vein cannula was inserted and administration of N/S 0.9% 20 ml/hour was commenced. Tracheostomy was performed based on previous reports, animals were subsequently endotracheally intubated; animals were subjected to neuro-muscular blocade and were mechanically ventilated to reach stable respiratory conditions with a volume controlled mode (FiO2 = 0.3, frequency 35–40/minute, tidal volume (Vt) = 6 ml/kgr, positive end-expiratory pressure (PEEP) = 2 cm H2O - Drager Respirator). Animals were subjected to neuro-muscular blocade by administering 1 ml cis-atracurium 2 mg/ml iv and thereafter anesthesia and muscle relaxation were maintained with periodic intravenous infusions of midazolam and cis-atracurium. Subsequently, the femoral artery was exposed and cannulated and the right jugular vein was prepared and the pulmonary artery was cannulated based on a previously described technique.[10, 11] Briefly, an angled polypropylene introducer made from standard tubing 15 cm long, external diameter 3 mm and lumen diameter 2 mm was used. The distal 1.5 cm was heat angled at 90 degrees to the shaft and a marker made to indicate the direction of the angle. The introducer was filled with heparinized saline and a No. 4.5 French gauge right side coronary angiography catheter - which has its tip angled - was inserted so that the tip lied just inside the distal end of the introducer. The catheter was then filled with heparinized saline and connected to a pressure transducer. Following the exposure of the right jugular vein, the introducer-catheter assembly was inserted and passed into the right ventricle through the superior vena cava and the right atrium using the pressure signals for guidance. Correct placement in the right ventricle was confirmed by the pressure signal and then the angled tip was rotated to point anteriorly and slightly to the left and withdrawn until the angle impinges on the tricuspid valve. One ml of cold saline was flushed through the catheter and the catheter was advanced to pass directly into the pulmonary artery. Correct placement was confirmed by the change in the pressure signal.
Samples of arterial blood were obtained from the femoral artery. End expiratory carbon dioxide (EtPCO2) was continuously monitored using a capnograph (RESPIRONICS CO2SMO MONITOR, USA) adapted to the endotracheal tube.
Animals were ventilated with the aforementioned baseline settings to obtain stable physiological conditions and were then exposed to different experimental conditions in terms of ventilation settings and inhaled gas mixtures. Initially, animals were randomly allocated in two groups of different tidal volumes either 6 ml/Kgr (LowVt group) or 9 ml/Kgr (HighVt group) and were ventilated with FiO2 0.3 (Normocapnic Phase (NP1)). Subsequently, animals in each Vt group inhaled an enriched in CO2 gas mixture (FiO2 0.3, FiCO2 0.10.) in order to develop hypercapnia (PCO2 was targeted between 70–90 mmHg - Hypercapnic Phase-1(HP1)). Animals were then re-ventilated with FiO2 0.3 (Normocapnic Phase-2 (NP2)) and then re-exposed to enriched in CO2 gas mixture (FiO2 0.3, FiCO2 0.10.) (Hypercapnic Phase-2 (HP2) to assess the impact of hypercapnic preconditioning in pulmonary pressures. All animals were exposed to each setting for 30 minutes to obtain stable condition before measurements and between different conditions were ventilated with the baseline settings for 30 min (Fig. 1). At the end of the experiments, animals were sacrificed by administering potassium chloride 5% iv while under anesthesia.
In order to assess the impact of hypoxic conditions to pulmonary pressures, six animals under mechanical ventilation inhaled a gas mixture with low O2 concentration (FiO2 0.15, FiCO2 0.0) whereas in three of them, heart and lungs were exposed for macroscopic inspection of the placement of the catheter and for measurements of the mechanical properties of the exposed lungs.
Pulmonary arterial pressures were recorded at expiration; pressure signal was recorded with a commercially available polygraphic system (NIHON KOHDEN POLYGRAPH SYSTEM RM-6000, Japan) and simultaneously displayed on the screen of the recording system. Pressures were measured with piezo pressure transducers integrated in the polygraphic system. Pressure transducer was calibrated immediately before, after, and when necessary, during each procedure.
Mean pulmonary arterial pressure changes (PAPmean – mmHg) was the primary outcome of this investigation.
Results are expressed as mean (SD). Data were analyzed for normality with the Shapiro-Wilk test and by the paired T-test or the Wilcoxon matched pair test as appropriate. The statistical tests were 2-sided. A result was considered statistically significant when p < 0.05. Analysis was performed using statistical software, SPSS v.15 for Windows.