Animal preparation
The study was approved by the local Ethics Committee for Animal Research (Dnr 8401/2017). All animals received care according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, as well as to the USA Principles of Laboratory Animal Care of the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, published by the National Academies Press (1996).
Six Swedish landrace pigs with a mean weight of 63 ± 1.4 kg were fasted overnight with free access to water. Premedication was performed with an intramuscular injection of Xylazine (Rompun® vet. 20 mg/ml; Bayer AG, Leverkusen, Germany; 2 mg/kg) mixed with ketamine (Ketaminol® vet. 100 mg/ml; Farmaceutici Gellini S.p.A., Aprilia, Italy; 20 mg/kg) in their stables, and a peripheral IV access was established in the earlobe. The pig was then transferred to the laboratory and placed in a supine position on the operating table. Oral intubation was performed using a 7.5-size endotracheal tube after anaesthesia induction with sodium thiopental (Pentothal; Abbott Laboratories, North Chicago, Illinois, USA) and pancuronium bromide (Pavulon; N.V. Organon, Oss, the Netherlands). Anaesthesia was maintained with a ketamine (Ketaminol® vet), midazolam (Midazolam Panpharma®, Oslo, Norway) and fentanyl (Leptanal®, Lilly, France) infusion. Fluid loss was compensated for by continuous infusion of Ringer’s acetate. Mechanical ventilation was established with a Siemens-Elema ventilator (Servo Ventilator 300, Siemens, Solna, Sweden).
The animals were carefully turned on the sides about 30° in each direction according to a schedule to reduce the risk of atelectasis.
Abdominal surgery
A laparotomy was performed to mimic a clinical situation in an intensive care unit with mechanical ventilation on the subject with no previous lung injury, and all animals had a secondary temporary closure with negative pressure wound therapy (NPWT) in a standard way. A 30-cm-long midline incision was performed on each pig. The V.A.C.® Abdominal Dressing (KCI®, Inc., San Antonio, TX, USA) was used. The visceral protective layer was cut to an approximate size of 35 cm wide and 35 cm long, extending into the paracolic gutters on both sides. A layer of polyurethane Granu Foam was placed on top of the visceral protective layer between the edges of the wound. The wound was covered with a self-adhesive polyethylene drape, and a track pad was inserted through the drape (all from V.A.C., KCI, San Antonio, TX), and then connected to a continuous vacuum source with a negative pressure of − 120 mmHg. No manipulation of the abdominal organs was made.
Mechanical ventilation and pulmonary recruitment manoeuvre
All animals had endotracheal tubes size 7.5, ventilator settings with tidal volume of 6 ml/kg, positive end-expiratory pressure of 5 cmH2O and end-inspiratory pressures < 25 cmH2O with an inspiratory to expiratory ratio (I:E) of 1:2. These settings remained unchanged during the study period. Particle outflow was measured using a modified PExA 2.0 instrument (PExA, Gothenburg, Sweden) each day during two different ventilation modes: VCV and PCV.
The animals were randomised into two different groups: one group received VCV before PCV (n = 3) and the other group received PCV before VCV (n = 3). Each animal was monitored during VCV and PCV for 1 h each per day. Before the collection period began for the second ventilation mode, there was an equilibration period of 30 min with the second ventilation mode. Twice daily, a RM was performed for 60 s at PEEP 10, 4 breath/min and I:E 2:1. Measurements were done 3 min before the RM, 1 min during the RM and 3 min after the RM.
PExA measurements
The PExA 2.0 instrument (PExA, Gothenburg, Sweden) conducts measurements by optical particle counter and has been previously described in conjunction with mechanical ventilation. The instrument was connected to the outflow air of the mechanical respiratory circuit. The total accumulated number of particles from the airways was continuously measured by the PExA instrument during the two different ventilation modes each day. PExA measurements were made starting on day 1 until termination of mechanical ventilation on day 3. Measurements were made during each ventilation mode (VCV or PCV), each lasting 1 h in duration per day. Additionally, we monitored particle flow before, during and after RMs. The total particle count was measured for 3 min before the RM, for 1 min during the RM and for 3 min after the RM. Particles in the diameter range of 0.41–4.55 μm were measured by the PExA instrument. The particles are computationally divided into eight different size distributions according to their mean diameter. The PExA instrument does not take into count if the particles are proteins or lipids, endogenous or exogenous, the instrument measures only the particles diameter, no matter what the particles origin is. In this study, we only present the particles according to their mean diameter and not their physiological abilities. The mean diameter of the particles are as follows: particle 1, 0.48 μm; particle size 2, 0.59 μm; particle size 3, 0.75 μm; particle size 4, 0.98 μm; particle size 5, 1.22 μm; particle size 6, 1.67 μm; particle size 7, 2.52 μm; and particle size 8, 3.37 μm.
Blood gases and haemodynamic parameters
All animals had a central venous catheter (CVC). The blood gases were drawn from the CVC and analysed in a standard way. Haemodynamic parameters were continuously recorded in a standard way.
Experimental timeline
Mechanical ventilator settings were named as follows: VCV day 1 (VCV1), VCV day 2 (VCV2), VCV day 3 (VCV3), PCV day 1 (PCV1), PCV day 2 (PCV2) and PCV day 3 (PCV3).
Calculations and statistics
Descriptive statistics, in the form of the number of patients, mean and the standard error of the mean (SEM) for the different parameters were analysed. The results are presented for the different parameters divided into the different groups. Statistically significant differences between the groups were tested using a paired Student t test. All statistical analyses were performed, using GraphPad Prism Software, CA, USA. Significance was defined as p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), and p > 0.05 (not significant, n.s.).