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Supplementary Information on Materials and Methods
JCMM-06-2012-002 R1
Manuscript title: Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine
Authors: Preuß S. et al.
Animal preparation and clinical care
The experimental protocol was approved by the review board of the Schleswig-Holstein government for the care of animal subjects (letter of acceptance V312-72241.121-24) in accordance with the German law for animal protection (BGBI 1, page 1319), and the European Community guidelines (2007/526/EC). 30 piglets (mixed country breed from 13 litters) of either sex were studied between days 2 and 6 of life. Piglets were left with their sows until the morning of the first study day to avoid any periods of food abstinence.
Initially 0.025 mg/kg atropine, 15 mg/kg ketamine and 1.5 mg/kg midazolam hydrochloride were administered intramuscularly to allow intubation with an uncuffed 3.5 mm endotracheal tube. For initial surgical preparation, each piglet received 10 µg/kg fentanyl intravenously. Anesthesia was maintained by continuous infusion of 5 mg/kg/h ketamine, 0.5 mg/kg/h midazolam, and 0.4 mg/kg/h vecuronium bromide throughout the study. To prevent leakage the endotracheal tube (ETT) was tightly secured in place by a peritracheal ligature. A thermodilution catheter was inserted into a femoral artery by cut-down for monitoring of hemodynamic parameters and arterial blood gases. An internal jugular vein was catheterized for cold bolus injection to perform thermodilution measurements.
Heating pads were used to maintain a constant core temperature between 38 and 39°C. The piglets were provided with feedings via an orogastric tube using special piglet milk (Panto™, Hamburger Leistungsfutter, Hamburg, Germany). The milk was given every 4 h at 25 mL/kg summing up to 150 mL/kg/d of water and 129 kcal/kg/d of energy. Approximately four hours after the first intervention all piglets received a trans-abdominal urinary bladder catheter for urine output monitoring. Two hours after the first intervention and then every 12 hours apart, 50 mg/kg of ampicillin/sulbactam was administered intravenously to prevent bacterial infections.
To prevent nosocomial infections only sterile material (ETT, ventilator tubings, suction catheters, indwelling catheters, syringes, lines) was used. In addition, after completion of a single study, the whole intensive care facility was carefully cleansed, and all surfaces desinfected with Bacillol AF (Bode, Hamburg, Germany), containing propane-1-ol 45%, propane-2-ol 25%, and ethanol 5%. All manipulations were carried out with sterile gloves and sterile gowns, and face masks added when doing the initial surgical preparations.
Mechanical ventilation and airway lavage
Mechanical ventilation was provided by time-cycled pressure-limited infant ventilators (Babylog 1, Dräger, Germany). The following ventilator settings were initially used: FiO2 = 0.5, positive end-expiratory pressure (PEEP) = 6 mbar, flow = 8 L/min, inspiratory time = 0.5 sec, ventilator rate = 25/min before lavage and 35/min during the sequence of repeated airway lavage until the first intervention. Peak inspiratory pressure (Peak) was adjusted to keep tidal volume (VT) at 7 mL/kg.
Repeated airway lavage (rBAL, Figure 1) was carried out by the instillation and removal of 30 mL/kg of warmed normal saline via the ETT over a 30 sec period. After the first lavage the ventilator rate was switched to 35/min and VT was adjusted. Airway lavage was then repeated every 3-5 min until both the PaO2/FiO2 decreased to ~100 mmHg, and a Peak ≥19 mbar was required to maintain VT at 7 mL/kg. We then waited for an additional 20 min for clearance of lavage fluid from the small airways, and two more lavages were carried out in case of improved oxygenation (Δ PaO2/FiO2 ³ 20 mmHg).
During the sequence of lavage and for the whole observation period of 72 h, ventilator rate, Peak, and FiO2 were adjusted every hour according to changes in PaCO2, VT, and PaO2, respectively. To keep PaCO2 within a range of 35-50 mm Hg, ventilator rate was changed according to the following protocol: reduction of 5 breaths/min for PaCO2 <35 mm Hg; reduction of 10 breaths/min for PaCO2 <30 mm Hg; increase of 5 breaths/min for PaCO2 >50 mm Hg; increase of 10 breaths/min for PaCO2 >60 mm Hg. Peak was adjusted to keep VT at 7 mL/kg. FiO2 was reduced of 0.1 for PaO2 >150 mm Hg, and was increased of 0.2 for PaO2 <50 mm Hg.
Experimental protocol and randomization
24 h after repeated airway lavage, a second protocol for induction of lung injury was carried out by injurious ventilation (inj. vent. in Figure 1) consisting of a 1 h period of zero-PEEP ventilation followed by another 1 h period of doubled VT (i.e. 14 mL/kg at PEEP 6 mbar). To complete triple-hit lung injury, another 24 h apart, all piglets received a tracheal instillation of 2.5 mg lipopolysaccharide dissolved in 0.5 mL normal saline (E. coli serotype O127:B8; Sigma-Aldrich, München, Germany) via the second lumen of the ETT (LPS in Figure 1).
PaO2 and PaCO2 were measured every 1-6 h, as were lung function parameters [tidal volume (VT), specific (dynamic) compliance of the respiratory system (sCrs), resistance of the respiratory system (Rrs)], and hemodynamic parameters [cardiac index (CI), pulmonary blood volume (PBV), intrathoracic blood volume (ITBV), global end-diastolic volume (GEDV) and extravascular lung water index (EVLWI)].
2 h after repeated airway lavage, the animals were randomized to one of the four intervention groups by drawing lots. The clinical investigators (SP, FDO, JS, SaS, PvB) were blinded to the types of intervention:
- a control group (C) received 2.5 mL/kg of air into a second lumen of the ETT;
- an intervention group (S) received 50 mg/kg of porcine surfactant at a volume of 2.5 mL/kg;
- an intervention group (S+Imi) received 5 mg imipramine admixed with 50 mg/kg surfactant;
- an intervention group (S+PIP2) received 2 mg phosphatidyl-inositol-3,5-bisphosphate (Cayman, Tallinn, Estonia) admixed with 50 mg/kg surfactant.
All interventions were carried out three times, each following a specific lung injury protocol 2 h apart. A time table of all lung injuries, interventions and diagnostics is provided in Figure 1.
Surfactant administration
The surfactant preparation used in this study was poractant alfa (Chiesi Farmaceutici, Parma, Italy), that is isolated from minced porcine lungs by lipid extraction. It consists of approximately 98% phospholipids and 1% surfactant proteins B and C. The original preparation was diluted with warmed normal saline to a final concentration of 20 mg/mL. 50 mg/kg surfactant together with the additives (i.e. Imi and PIP2) were given three times (at 2h, 26h, and 50h). Surfactant was applied via a second lumen of the ETT within 2 min and without interrupting mechanical ventilation.
In the intervention group S+Imi, 5 mg (~ 2 mg/kg) crystalline imipramine was added to the surfactant preparation immediately before its application by gentle stirring. The same technique was used for admixture of 2 mg PIP2 (~0.8 mg/kg) to the diluted surfactant preparation. Surfactant was therefore used as a therapeutic and as a carrier substance fpr Imi and PIP2.
Measurements of gas exchange, hemodynamics and lung function
PaO2 was used for calculation of an oxygenation index (OI = mean airway pressure x %O2 / PaO2). PaCO2 was used for calculation of a ventilation efficiency index (VEI = 3800/(Peak-PEEP)x f x PaCO2) according to Notter et al [1].
Cardiac index (CI) monitoring (PC 8000 PiCCO monitor, Pulsion, München, Germany) was performed using the transpulmonary indicator dilution technique. In short, a 4-Fr thermodilution catheter was introduced into a femoral artery with the thermistor tip placed into the lower abdominal aorta. After injection of a cold bolus of 2 mL normal saline <8°C, cardiac output (CO) was determined by calculating the area under the curve using the Stuart-Hamilton method:
CO = ((Tb-Ti) * Vi * C) / (òDTb*dt) (1)
where Tb is the blood temperature before cold injection, Ti is the temperature of the cold bolus, Vi is the cold bolus volume, C is a constant composed of specific weights and temperatures of blood and cold bolus, and òDTb*dt is the area under the thermodilution curve.
Extravascular lung water (EVLW) is equivalent to the extravascular thermo-accessible volume in the lung as assessed by the mean transit time method (needle to needle volume):
EVLW = ITTV – ITBV (2)
where ITTV is the intrathoracic thermo-accessible volume, and ITBV is the intrathoracic blood volume. Pulmonary blood volume (PBV) is calculated by subtracting ITBV from global end-diastolic volume (GEDV):
PBV = ITBV – GEDV (3)
with GEDV being approximately 80% of ITBV [2].
To calculate VT, sCrs and Rrs we used the single breath least squares method for lung mechanics by fitting airflow and VT signals to proximal airway pressure using the standard equation of motion. Airflow and pressure signals were sampled at a rate of 200 Hz, digitized, and stored in a PC for subsequent analysis [3].
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REFERENCES
1. Notter RH, Egan EA, Kwong MS, et al. Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age. Pediatr Res. 1985; 19: 569-77.
2. Effros RM, Pornsuriyasak P, Porszasz J, et al. Indicator dilution measurements of extravascular lung water: basic assumptions and observations. Am J Physiol Lung Cell Mol Physiol. 2008; 294: L1023-L31.
3. Krause MF, Jäkel C, Haberstroh J, et al. Alveolar recruitment promotes homogeneous surfactant distribution in a piglet model of lung injury. Pediatr Res. 2001; 50: 34-43.