Animals, handling, anaesthesia, surgery and euthanasia
Eight outbred pigs (25% Duroc, 25% Yorkshire, 50% Norwegian Landrace, 29–37 kg) were included in the study after approval from the Norwegian State Commission for Animal Experimentation. All the animals received human care in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.
The animals were premedicated with intramuscular diazepam 10 mg and azaperone 400 mg. Anaesthesia was induced through an intravenous access on the external ear with atropine 1.0 mg, fentanyl 8.0 μg/kg, thiopenthal sodium 4.0 mg/kg and ketamine hydrochloride 8.0 mg/kg. Before intubation, 5 ml of 40 mg/ml lidocaine was applied to the larynx. The animals were ventilated in PRVC mode on a Servo-i respirator (Maquet, Getinge Group, Gothenburg, Sweden) with initial values of FiO2 at 0.30, a tidal volume of 10 ml/kg, PEEP at 6 cmH2O and minute ventilation adjusted to maintain PaCO2 at 4.5–5.5 kPa. Anaesthesia was maintained by an infusion of fentanyl 20 μg/(kg h) and midazolam 0.40 mg/(kg h). Based on clinical response this was supplemented with boluses of fentanyl 50 μg/ml as needed. Intravascular volume was maintained by a bolus of acetated Ringer’s solution 10 ml/kg, followed by a continuous infusion of 10 ml/(kg h) throughout the experiment. 5000 IU heparin was administered i.v. to prevent clot formation.
Before cannulation we surgically prepared the left carotid artery and the right and left internal jugular vein. Two mono lumen catheters were inserted into the left carotid artery and the left internal jugular vein for invasive blood pressure monitoring, arterial blood gases and intravenous injections. A flow-directed pulmonary artery catheter (PAC; Swan Ganz CCOmbo 7.5 Fr, Edwards Lifescience, USA) was inserted in the right internal jugular vein and advanced into the pulmonary artery using classical pressure observations and fluoroscopic guidance and validation. The PAC provided central venous pressure (CVP), pulmonary artery pressure (PAP) and was used to sample mixed venous blood gases. The PAC was connected to a Vigilance II monitor for continuous cardiac output (CO) measurements (Edwards Lifescience, USA). A bladder catheter was inserted by a mini-laparotomy.
At the end of the experiment, the animals were euthanized with pentobarbital 100 mg/kg.
Study protocol and respirator manipulations
To induce HPV, the FiO2 had to be reduced to levels below the physiological range (the oxygen content in air at sea level is 20.9%, and equals an FiO2 of 0.21). In order to reduce the FiO2 to subnormal values, nitrogen was plugged into the air inlet of the respirator, and the animals were then ventilated with an oxygen and nitrogen mixture. The actual FiO2 was monitored via a side stream multi-gas analyser.
After end of surgery the animals stabilized for 30 min before baseline measurements were recorded. PH was then induced by slowly reducing FiO2 on the respirator while monitoring the response in mPAP. When mPAP levels above 25 mmHg were reached, the animal rested for 30 new minutes before new measurements were recorded.
Data material, measurements and statistics
Vital variables [mean pulmonary artery pressure (mPAP), pulmonary wedged pressure (PWP), central venous pressure (CVP), heart rate (HR), cardiac output (CO)] and arterial and mixed venous blood gases were measured at baseline and after establishing PH. Blood gases were processed by a Radiometer ABL 720 blood gas analyser (Radiometer, Brønshøj, Denmark).
Equations for calculating PVR, DO2 and VO2:
$$Pulmonary\,\,vascular\,\,resistance \,\,(PVR) = \frac{{80\left( {mPAP - PWP} \right)}}{CO}{\text{ Unit: dyn}} \,{\text{s}}/ {\text{cm}}^{5}$$
$$\begin{aligned} Arterial\,\,oxygen\,\,content\,\, ( {CaO_{2} }) & = \left( {Hgb \times 1.34} \right)SaO_{2}\\&\quad + ( {PaO_{2} \times 0.0031}) \\ &\quad\quad{\text{ Unit: ml O}}_{ 2} / {\text{dl blood}} \end{aligned}$$
$$Mixed \,\, venous\,\, oxygen \,\,content\,\, ( {CvO_{2} }) = \left( {Hgb \times 1.34} \right)SvO_{2} + \left( {PvO_{2} \times 0.0031} \right)$$
$$Oxygen\,\,delivery \,\, ( {DO_{2} } ) = CaO_{2} \times CO \times 10{\text{ Unit: ml O}}_{ 2} / {\text{min}}$$
$$Oxygen\,\, consumption \,\, ( {VO_{2} }) = C\left( {a - v} \right)O_{2} \times CO \times 10$$
Only simple descriptive medical statistics and paired sample t tests were used in this study. All datas were analysed in Excel (MS Excel for Mac 2011, Microsoft Corporation, USA) and R (version 3.1.1, The R Foundation for Statistical Computing, Vienna, Austria).