Inhaled aerosolised recombinant human activated protein C ameliorates endotoxin-induced lung injury in anaesthetised sheep.

INTRODUCTION: We recently demonstrated that intravenously infused recombinant human activated protein C (APC) attenuates ovine lipopolysaccharide (LPS)-induced lung injury. In this study, our aim was to find out whether treatment with inhaled aerosolised APC (inhAPC) prevents formation of increased lung densities and oedema and derangement of oxygenation during exposure to LPS. METHODS: Sheep were anaesthetised during placement of intravascular introducers. After one to four days of recovery from instrumentation, the animals were re-anaesthetised, endotracheally intubated and mechanically ventilated throughout a six-hour experiment where the sheep underwent quantitative lung computed tomography. Sheep were randomly assigned to one of three groups: a sham-operated group (n = 8) receiving inhaled aerosolised saline from two hours after the start of the experiment; a LPS group (n = 8) receiving an intravenous infusion of LPS 20 ng/kg per hour and, after two hours, inhaled aerosolised saline over the next four hours; a LPS+inhAPC group (n = 8) receiving an intravenous infusion of LPS 20 ng/kg per hour and, after two hours, aerosolised APC 48 microg/kg per hour inhaled throughout the experiment. Data were analysed with analysis of variance; P less than 0.05 was regarded as significant. RESULTS: An infusion of LPS was associated with a reduction of well-aerated lung volume and a rapid fall in arterial oxygenation that were both significantly antagonised by inhaled APC. Pulmonary vascular pressures and extravascular lung water index increased significantly during exposure to LPS, but inhaled APC had no effect on these changes. CONCLUSIONS: Inhalation of aerosolised APC attenuates LPS-induced lung injury in sheep by preventing a decline in the volume of aerated lung tissue and improving oxygenation.

Recombinant human activated protein C ameliorates oleic acid-induced lung injury in awake sheep.

INTRODUCTION: Acute lung injury (ALI) may arise both after sepsis and non-septic inflammatory conditions and is often associated with the release of fatty acids, including oleic acid (OA). Infusion of OA has been used extensively to mimic ALI. Recent research has revealed that intravenously administered recombinant human activated protein C (rhAPC) is able to counteract ALI. Our aim was to find out whether rhAPC dampens OA-induced ALI in sheep. METHODS: Twenty-two yearling sheep underwent instrumentation. After 2 days of recovery, animals were randomly assigned to one of three groups: (a) an OA+rhAPC group (n = 8) receiving OA 0.06 mL/kg infused over the course of 30 minutes in parallel with an intravenous infusion of rhAPC 24 mg/kg per hour over the course of 2 hours, (b) an OA group (n = 8) receiving OA as above, or (c) a sham-operated group (n = 6). After 2 hours, sheep were sacrificed. Hemodynamics was assessed by catheters in the pulmonary artery and the aorta, and extravascular lung water index (EVLWI) was determined with the single transpulmonary thermodilution technique. Gas exchange was evaluated at baseline and at cessation of the experiment. Data were analyzed by analysis of variance; a P value of less than 0.05 was regarded as statistically significant. RESULTS: OA induced profound hypoxemia, increased right atrial and pulmonary artery pressures and EVLWI markedly, and decreased cardiac index. rhAPC counteracted the OA-induced changes in EVLWI and arterial oxygenation and reduced the OA-induced increments in right atrial and pulmonary artery pressures. CONCLUSIONS: In ovine OA-induced lung injury, rhAPC dampens the increase in pulmonary artery pressure and counteracts the development of lung edema and the derangement of arterial oxygenation.

Recombinant human activated protein C attenuates endotoxin-induced lung injury in awake sheep.

INTRODUCTION: Acute lung injury often complicates severe sepsis. In gram-negative sepsis, bacterial endotoxin activates both coagulation and inflammation. Enhanced lung vascular pressures and permeability, increased extravascular lung water content and deteriorated gas exchange characterize ovine endotoxin-induced lung injury, a frequently used model of acute lung injury. Recombinant human activated protein C (rhAPC), with its anticoagulant, anti-inflammatory, fibrinolytic and antiapoptotic effects, reportedly reduces the respirator-dependent days and the mortality of patients with severe sepsis. We speculate whether rhAPC antagonizes endotoxin-induced lung injury in sheep. METHODS: Two groups of sheep were exposed to Escherichia coli endotoxin (lipopolysaccharide) 15 ng/kg/minute intravenously from 0 to 24 hours; one group received only lipopolysaccharide throughout (n = 8), and the other group received lipopolysaccharide in combination with rhAPC 24 microg/kg/hour from 4 to 24 hours (n = 9). In addition, one group received rhAPC as above as the only intervention (n = 4), and four sham-operated sheep were used for determination of the alpha and epsilon isoforms of protein kinase C in pulmonary tissue. Data were assessed by one-way analysis of variance for repeated measurements. Biochemical data were analyzed using Student's t test, or using the Mann-Whitney U test when appropriate. RESULTS: Infusion of endotoxin caused lung injury, manifested by increments in pulmonary artery pressure, in pulmonary micro-occlusion pressure, in pulmonary vascular downstream resistance, in pulmonary vascular permeability index, in extravascular lung water index and in deterioration of oxygenation that were all attenuated by infusion of rhAPC. Endotoxemia led to changes in inflammation and coagulation, including pulmonary neutrophil accumulation paralleled by increased TNFalpha and decreased protein C and fibrinogen in animal plasma, which all improved following infusion of rhAPC. Moreover, rhAPC prevented the translocation of protein kinase C alpha and epsilon isoforms from the cytosolic fraction of lung tissue extracts. CONCLUSION: In awake sheep, rhAPC alleviates endotoxin-induced lung injury--as characterized by improvements of oxygenation, coagulation and inflammation, as well as by reversal of pulmonary hemodynamic and volumetric changes.

Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep.

INTRODUCTION: Acute lung injury is associated with accumulation of extravascular lung water (EVLW). The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLWST) and postmortem gravimetric (EVLWG). METHODS: Eighteen instrumented and awake sheep were randomly assigned to one of three groups. All groups received Ringer's lactate (5 ml/kg per hour intravenously). To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours (n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min (n = 7). A third group (n = 4) was subjected to sham operation. Haemodynamic variables, including EVLWST, were measured using a PiCCOplus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLWST was compared with EVLWG. RESULTS: At the end of experiment, values for EVLWST (mean +/- standard error) were 8.9 +/- 0.6, 11.8 +/- 1.0 and 18.2 +/- 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively (P < 0.05). The corresponding values for EVLWIG were 6.2 +/- 0.3, 7.1 +/- 0.6 and 11.8 +/- 0.7 ml/kg (P < 0.05). Ranges of EVLWIST and EVLWIG values were 7.5-21.0 and 4.9-14.5 ml/kg. Regression analysis between in vivo EVLWST and postmortem EVLWG yielded the following relation: EVLWST = 1.30 x EVLWG + 2.32 (n = 18, r = 0.85, P < 0.0001). The mean bias +/- 2 standard deviations between EVLWST and EVLWG was 4.9 +/- 5.1 ml/kg (P < 0.001). CONCLUSION: In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry.

Extravascular lung water after pneumonectomy and one-lung ventilation in sheep.

OBJECTIVE: To compare the single thermodilution and the thermal-dye dilution techniques with postmortem gravimetry for assessment of changes in extravascular lung water after pneumonectomy and to explore the evolution of edema after injurious ventilation of the left lung. DESIGN: Experimental study. SETTING: University laboratory. SUBJECTS: A total of 30 sheep weighing 35.6 +/- 4.6 kg. The study included two parts: a pneumonectomy study (n = 18) and an injurious ventilation study (n = 12). METHODS: Sheep were anesthetized and mechanically ventilated with an FiO2 of 0.5, tidal volume of 6 mL/kg, and positive end-expiratory pressure of 2 cm H2O. In the pneumonectomy study, sheep were assigned to right-sided pneumonectomy (n = 7), left-sided pneumonectomy (n = 7), or lateral thoracotomy only (sham operation, n = 4). In the injurious ventilation study, right-sided pneumonectomy was followed by ventilation with a tidal volume of 12 mL/kg and positive end-expiratory pressure of 0 cm H2O (n = 6) or by ventilation with a tidal volume of 6 mL/kg and positive end-expiratory pressure of 2 cm H2O for 4 hrs (n = 6). Volumetric variables, including extravascular lung water index (EVLWI), were measured with single thermodilution (STD; EVLWI(STD)) and thermal-dye dilution (TDD; EVLWI(TDD)) techniques. We monitored pulmonary hemodynamics and respiratory variables. After the sheep were killed, EVLWI was determined for each lung by gravimetry (EVLWI(G)). RESULTS: In total, the study yielded strong correlations of EVLWI(STD) and EVLWI(TDD) with EVLWI(G) (n = 30; r = .83 and .94, respectively; p < .0001). After pneumonectomy, both the left- and the right-sided pneumonectomy groups displayed significant decreases in EVLWI(STD) and EVLWI(TDD). The injuriously ventilated sheep demonstrated significant increases in EVLWI that were detected by both techniques. The mean biases (+/-2 SD) compared with EVLWI(G) were 3.0 +/- 2.6 mL/kg for EVLWI(STD) and 0.4 +/- 1.6 mL/kg for EVLWI(TDD). CONCLUSIONS: After pneumonectomy and injurious ventilation of the left lung, TDD and STD displayed changes in extravascular lung water with acceptable accuracy when compared with postmortem gravimetry. Ventilator-induced lung injury seems to be a crucial mechanism of pulmonary edema after pneumonectomy.

Extravascular lung water determined with single transpulmonary thermodilution correlates with the severity of sepsis-induced acute lung injury.

OBJECTIVE: To find out if the extravascular lung water index (EVLWI) and the derived permeability indexes determined by the single transpulmonary thermodilution technique are associated with markers of acute lung injury in human septic shock. DESIGN: Prospective, observational study. SETTING: Mixed intensive care unit of a 900-bed university hospital. PATIENTS: Thirty-eight consecutive adult patients with septic shock and acute lung injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The variables were assessed over a 72-hr period and included hemodynamics, EVLWI, and pulmonary vascular permeability indexes determined with the single indicator transpulmonary thermodilution technique, lung compliance, oxygenation ratio (Pao2/Fio2), lung injury score, cell counts, and the plasma concentration of endothelin-1. At day 1, EVLWI was elevated (>or=7 mL/kg) in 28 (74%) patients and correlated with lung compliance (r=-.48, p=.002), Pao2/Fio2 (r=-.50, p=.001), lung injury score (r=.46, p=.004), roentgenogram quadrants (r=.39, p=.02), and platelet count (r=-.43, p=.007). At day 3, EVLWI correlated with compliance (r=-.51, p=.002), Pao2/Fio2 (r=-.49, p = .006), and lung injury score (r=.53, p=.003). At day 3, EVLWI and pulmonary vascular permeability indexes were higher in nonsurvivors (p<.05). The plasma concentration of endothelin-1 (mean+/-sd) was significantly higher in patients with elevated EVLWI (>or=7 mL/kg) (3.85+/-1.40 vs. 2.07+/-0.38 pg/mL, respectively). Twenty-two (59%) patients died before day 28. CONCLUSIONS: In human septic shock, EVLWI demonstrated moderate correlation with markers of acute lung injury, such as lung compliance, oxygenation ratio, roentgenogram quadrants, and lung injury score. In nonsurvivors, EVLWI and permeability indexes were significantly increased at day 3. Thus, EVLWI might be of value as an indicator of prognosis and severity of sepsis-induced acute lung injury.