David M. Steinhorn

A liquid perfluorochemical decreases the in vitro production of reactive oxygen species by alveolar macrophages

OBJECTIVE To determine whether reactive oxygen metabolite production by alveolar macrophages is affected by liquid perfluorochemical exposure. DESIGN Controlled, animal laboratory investigation of alveolar macrophage function in vitro. SETTING Animal research facility of a health sciences university. SUBJECTS Six adult male New Zealand white rabbits and six young piglets. INTERVENTIONS Alveolar macrophages were obtained after sacrifice from both species by total lung lavage. Macrophages were divided into control and experimental groups. Macrophages in the experimental groups were exposed to perfluorooctylbromide. To determine production of reactive oxygen metabolites, hydrogen peroxide production and chemiluminescence were measured in both experimental and control groups after chemical stimulation. MEASUREMENTS AND MAIN RESULTS Perfluorooctylbromide-exposed alveolar macrophages produced significantly less hydrogen peroxide (1.4 +/- 1.5 vs. 2.4 +/- 1.6 nmol/10(6) cells; p = .002). Perfluorooctylbromide-exposed alveolar macrophages demonstrated significantly less chemiluminescence activity compared with nonexposed cells (0.70 +/- 0.2 vs. 1.5 +/- 0.2 mV of relative activity per 3.5 x 10(5) cells; p = .005). CONCLUSIONS Exposure of alveolar macrophages to perfluorooctylbromide in vitro decreases the responsiveness of macrophages to potent stimuli. This finding may partially explain the decrease in pulmonary inflammation seen in animals treated with partial liquid ventilation during experimentally induced lung injury.

Perfluorocarbon-associated gas exchange in gastric aspiration.

ObjectivesTo test whether perfluorocarbon-associated gas exchange (gas ventilation of the perfluorocarbon-liquid filled lung) could support oxygenation better than conventional positive pressure breathing in a piglet model of gastric aspiration-induced adult respiratory distress syndrome (ARDS). DesignProspective, randomized, blinded, controlled study. SettingA critical care research laboratory in a university medical school. SubjectsFourteen healthy piglets. InterventionsUnder α-chloralose anesthesia and metocurine iodide neuromuscular blockade, 14 piglets underwent tracheostomy; central venous, systemic and pulmonary arterial catheterizations; and volume-regulated continuous positive-pressure breathing. Homogenized gastric aspirate (1 mL/kg) titrated to pH of 1.0 was instilled into the tracheostomy tube of each subject at 0 min to induce ARDS. Hemodynamics, lung mechanics, and gas exchange were evaluated every 30 mins for 6 hrs. Seven piglets were treated at 60 mins by tracheal instillation of perflubron, a volume selected to approximate normal functional residual capacity, and were supported by perfluorocarbon-associated gas exchange without modifying ventilatory settings. Perflubron was added to the trachea every hour to replace evaporative losses. Measurements and Main ResultsThere was a significant difference in oxygenation over time when tested by repeated-measures analysis of variance (F test = 8.78, p < .01). On further analysis, the differences were not significant from baseline to 2.5 hrs but became increasingly significant from 2.5 to 6 hrs after injury (p < .05) in the inflammatory phase of gastric aspiration-induced ARDS. Histologic evidence for ARDS in the treated group 6 hrs after injury was lacking. ConclusionsIn the piglet model, perfluorocarbon-associated gas exchange with perflubron facilitates oxygenation in the acute phase of gastric aspiration-induced inflammatory ARDS when compared with conventional positive-pressure breathing. Histologic and physiologic data suggest that perfluorocarbon-associated gas exchange with perflubron might prevent ARDS if instituted after aspiration in the time window before the acute inflammatory process is manifest. (Crit Care Med 1994; 22:1445–1452)

Comparison of lung protective ventilation strategies in a rabbit model of acute lung injury.

Objective To determine the impact of different protective and nonprotective mechanical ventilation strategies on the degree of pulmonary inflammation, oxidative damage, and hemodynamic stability in a saline lavage model of acute lung injury. Design A prospective, randomized, controlled, in vivo animal laboratory study. Setting Animal research facility of a health sciences university. Subjects Forty-six New Zealand White rabbits. Interventions Mature rabbits were instrumented with a tracheostomy and vascular catheters. Lavage-injured rabbits were randomized to receive conventional ventilation with either a) low peak end-expiratory pressure (PEEP; tidal volume of 10 mL/kg, PEEP of 2 cm H2O); b) high PEEP (tidal volume of 10 mL/kg, PEEP of 10 cm H2O); c) low tidal volume with PEEP above Pflex (open lung strategy, tidal volume of 6 mL/kg, PEEP set 2 cm H2O > Pflex); or d) high-frequency oscillatory ventilation. Animals were ventilated for 4 hrs. Lung lavage fluid and tissue samples were obtained immediately after animals were killed. Lung lavage fluid was assayed for measurements of total protein, elastase activity, tumor necrosis factor-&agr;, and malondialdehyde. Lung tissue homogenates were assayed for measurements of myeloperoxidase activity and malondialdehyde. The need for inotropic support was recorded. Measurements and Main Results Animals that received a lung protective strategy (open lung or high-frequency oscillatory ventilation) exhibited more favorable oxygenation and lung mechanics compared with the low PEEP and high PEEP groups. Animals ventilated by a lung protective strategy also showed attenuation of inflammation (reduced tracheal fluid protein, tracheal fluid elastase, tracheal fluid tumor necrosis factor-&agr;, and pulmonary leukostasis). Animals treated with high-frequency oscillatory ventilation had attenuated oxidative injury to the lung and greater hemodynamic stability compared with the other experimental groups. Conclusions Both lung protective strategies were associated with improved oxygenation, attenuated inflammation, and decreased lung damage. However, in this small-animal model of acute lung injury, an open lung strategy with deliberate hypercapnia was associated with significant hemodynamic instability.

Partial liquid ventilation in premature lambs with respiratory distress syndrome: Efficacy and compatibility with exogenous surfactant

OBJECTIVE To determine the efficacy of partial liquid ventilation (PLV) by means of a medical-grade perfluorochemical liquid, perflubron (LiquiVent), in premature lambs with respiratory distress syndrome (RDS). Further, to determine the compatibility of perflubron with exogenous surfactant both in vitro and in vivo during PLV. DESIGN Prospective, randomized, controlled study, with in vitro open comparison. SUBJECTS Twenty-two premature lambs with RDS. INTERVENTIONS In vitro assays were conducted on three exogenous surfactants before and after combination with perflubron. We studied four groups of lambs, which received one of the following treatment strategies: conventional mechanical ventilation (CMV); surfactant (Exosurf) plus CMV; PLV; or surfactant plus PLV. MEASUREMENTS AND MAIN RESULTS In vitro surface tension, measured for three exogenous surfactants, was unchanged in each animal after exposure to perflubron. Lung mechanics and arterial blood gases were serially measured. All animals treated with PLV survived the 5 hours of experiment without complication; several animals treated with CMV died. During CMV, all animals had marked hypoxemia and hypercapnia. During PLV, arterial oxygen tension increased sixfold to sevenfold within minutes of initiation, and this increase was sustained; arterial carbon dioxide tension decreased to within the normal range. Compliance increased fourfold to fivefold during PLV compared with CMV. Tidal volumes were increased during PLV, with lower mean airway pressure. Resistance was similar for both CMV and PLV; there was no difference with surfactant treatment. CONCLUSIONS We conclude that PLV with perflubron improves lung mechanics and gas exchange in premature lambs with RDS, that PLV is compatible with exogenous surfactant therapy, and that, as a treatment for RDS in this model, PLV is superior to the surfactant studied.

Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury

OBJECTIVE To determine whether pulmonary neutrophil sequestration and lung injury are affected by partial liquid ventilation with perfluorocarbon in a model of acute lung injury (ALI). DESIGN A prospective, controlled, in vivo animal laboratory study. SETTING An animal research facility of a health sciences university. SUBJECTS Forty-one New Zealand White rabbits. INTERVENTIONS Mature New Zealand White rabbits were anesthetized and instrumented with a tracheostomy and vascular catheters. Animals were assigned to receive partial liquid ventilation (PLV, n = 15) with perflubron (18 mL/kg via endotracheal tube), conventional mechanical ventilation (CMV, n = 15) or high-frequency oscillatory ventilation (HFOV, n = 5). Animals were ventilated, using an FIO2 of 1.0, and ventilatory settings were required to achieve a normal PaCO2. Animals were then given 0.9 mg/kg of Escherichia coli endotoxin intravenously over 30 mins. Partial liquid ventilation, conventional mechanical ventilation, or high-frequency oscillatory ventilation was continued for an additional 4 hrs before the animals were killed. A group of animals not challenged with endotoxin underwent conventional ventilation for 4.5 hrs, serving as the control group (control, n = 6). Lungs were removed and samples were frozen at -70 degrees C. Representative samples were stained for histology. A visual count of neutrophils per high-power field (hpf) was performed in five randomly selected fields per sample in a blinded fashion by light microscopy. Lung samples were homogenized in triplicate in phosphate buffer, ultrasonified, freeze-thawed, and clarified by centrifugation. Supernatants were analyzed for myeloperoxidase (MPO) activity by spectrophotometry with o-dianisidine dihydrochloride and hydrogen peroxide at 460 nm. MEASUREMENTS AND MAIN RESULTS Histologic analysis of lung tissue obtained from control animals showed normal lung architecture. Specimens from the PLV and HFOV groups showed a marked decrease in alveolar proteinaceous fluid, pulmonary vascular congestion, edema, necrotic cell debris, and gross inflammatory infiltration when compared with the CMV group. Light microscopy of lung samples of animals supported with PLV and HFOV had significantly lower neutrophil counts when compared with CMV (PLV, 4 +/- 0.3 neutrophils/hpf; HFOV, 4 +/- 0.5 neutrophils/hpf; CMV, 10 +/- 0.9 neutrophils/hpf; p < .01). In addition, MPO activity from lung extracts of PLV and HFOV animals was significantly lower than that of CMV animals (PLV, 61 +/- 13.3 units of MPO activity/lung/kg; HFOV, 43.3 +/- 6.8 units of MPO activity/lung/kg; CMV, 140 +/- 28.5 units of MPO activity/lung/kg; p < .01). MPO activity from lungs of uninjured control animals was significantly lower than that of animals in the PLV, HFOV, and CMV groups (control, 2.2 +/- 2 units of MPO activity/lung/kg; p < .001). CONCLUSIONS Partial liquid ventilation decreases pulmonary neutrophil accumulation, as shown by decreased neutrophil counts and MPO activity, in an experimental animal model of ALI induced by systemic endotoxemia. The attenuation in pulmonary leukostasis in animals treated with PLV is equivalent to that obtained by a ventilation strategy that targets lung recruitment, such as HFOV.

Perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome

OBJECTIVE To compare the effectiveness of perfluorocarbon-associated gas exchange to volume controlled positive pressure breathing in supporting gas exchange, lung mechanics, and survival in an acute lung injury model. DESIGN A prospective, randomized study. SETTING A university medical school laboratory approved for animal research. SUBJECTS Neonatal piglets. INTERVENTIONS Eighteen piglets were randomized to receive perfluorcarbon-associated gas exchange with perflubron (n=10) or volume controlled continuous positive pressure breathing (n=8) after acute lung injury was induced by oleic acid infusion (0.15 mL/kg iv). MEASUREMENTS AND MAIN RESULTS Arterial and venous blood gases, hemodynamics, and lung mechanics were measured every 15 mins during a 3-hr study period. All animals developed a metabolic and a respiratory acidosis during the infusion of oleic acid. Following randomization, the volume controlled positive pressure breathing group developed a profound acidosis (p<.05), while pH did not change in the perfluorocarbon-associated gas exchange group. Within 15 mins of initiating perfluorocarbon-associated gas exchange, oxygenation increased from a PaO2 of 52 +/- 12 torr (6.92 +/- 1.60 kPa) to 151 +/- 93 torr (20.0 +/- 12.4 kPa) and continued to improve throughout the study (p<.05). Animals that received volume controlled positive pressure breathing remained hypoxic with no appreciable change in PaO2. Although both groups developed hypercarbia during oleic acid infusion, PaCO2, steadily increased over time in the control group (p<.01). Static lung compliance significantly increased postrandomization (60 mins) in the animals supported by perflurocarbon-associated gas exchange (p<.05), whereas it remained unchanged over time in the volume controlled positive pressure breathing group. However, survival was significantly higher in the perfluorocarbon-associated gas exchange group with eight (80%) of ten animals surviving the entire study period. Only two (25%) of the eight animals in the volume controlled positive pressure breathing group were alive at the end of the study period (log-rank statistic, p=.013). CONCLUSIONS Perflurocarbon-associated gas exchange enhanced gas exchange, pulmonary mechanics, and survival in this model of acute lung injury.

Partial liquid ventilation enhances surfactant phospholipid production

OBJECTIVE To study the effect of partial liquid ventilation on phospholipid metabolism. DESIGN Prospective, controlled laboratory study. SETTING University-affiliated animal research facility. SUBJECTS Mature New Zealand white rabbits (n = 17). INTERVENTIONS The rabbits were sedated, anesthetized, and instrumented with tracheostomy and the insertion of an arterial catheter. The rabbits were sequentially assigned to receive conventional mechanical ventilation or partial liquid ventilation with Perflubron (18 mL/kg by bolus fill). Ventilator strategies were identical in both groups and consisted of an FiO2 of 0.5, positive end-expiratory pressure of 4 cm H2O, effective tidal volume of 8 to 13 mL/kg, and rate to maintain Pco2 of 30 to 40 torr (4.0 to 5.3 kPa). Phosphatidylcholine was labeled in vivo by injection of 3H-methylcholine (25 microCi/kg iv). Ventilation was continued for 5.5 hrs. MEASUREMENTS AND MAIN RESULTS When animals were killed, phosphatidylcholine was extracted from the total lung lavage and from the pulmonary parenchyma. After the separation of phospholipids by thin-layer chromatography, the 3H activity was determined by liquid scintillation counting. Inorganic phosphorus was also determined to assess the enrichment of the phosphatidylcholine. The 3H-phosphatidylcholine activity in the partial liquid ventilation treated- vs. control rabbits demonstrated a 53% increase (p = .051) in the lavage and a 48% increase (p = .013) in the parenchyma for a net 50% (p = .012) total pulmonary increase. The phospholipid content of the partial liquid ventilation treated- vs. the control rabbits demonstrated a 78% increase (p = .046). CONCLUSIONS We conclude that partial liquid ventilation with Perflubron appears to have no negative impact on phospholipid metabolism but rather enhances surfactant phospholipid synthesis and secretion.

PARTIAL LIQUID VENTILATION INFLUENCES PULMONARY HISTOPATHOLOGY IN AN ANIMAL MODEL OF ACUTE LUNG INJURY

PURPOSE The aim of this study was to assess the effect of partial liquid ventilation (PLV) and conventional mechanical ventilation (CMV) in the pattern of distribution of lung injury in a rabbit model of acute lung injury. MATERIALS AND METHODS Animals (1.5 to 3.5 kg) were assigned to receive CMV (tidal volume of 10 mL/kg and a PEEP of 5 cm H2O) or PLV with 18 mL/kg of intratracheal perflubron (tidal volume of 10 mL/kg and a PEEP of 5 cm H2O). Lung injury was elicited by intravenous administration of Escherichia coliendotoxin. Uninjured animals ventilated as the CMV group served as controls. After 4 hours of mechanical ventilation, the lungs were removed and tissue injury was assessed by light microscopy using a scoring system. RESULTS Animals in the CMV group had higher lung injury scores in comparison to the PLV group (10+/-4.5 vs. 5+/-3.3, respectively, P < .05). The injury scores were similar for nondependent lung regions (CMV: 8+/-4.3, PLV: 6+/-2.9) but significantly different for the dependent regions (CMV: 12+/-4.6, PLV: 5+/-3.8, P< .05). CONCLUSIONS PLV is associated with significant attenuation of lung injury, in comparison to CMV. This effect is predominantly due to attenuation of injury in the dependent region of the lung.

Prolonged studies of perfluorocarbon associated gas exchange and of the resumption of conventional mechanical ventilation

OBJECTIVE To determine whether oxygenation and lung mechanics are preserved during perfluorocarbon associated gas exchange of 24 hrs duration and after evaporation of perfluorocarbon. DESIGN Prospective, experimental animal trials. SETTING Animal laboratory in a university setting. SUBJECTS Ten normal, neonatal piglets weighing 2.5 to 4.5 kg. INTERVENTIONS Ten piglets were anesthetized with fentanyl (25 micrograms/kg/hr), paralyzed with metocurine iodide (0.3 mg/kg) and placed on volume regulated continuous positive pressure breathing instituted at an FIO2 setting of 1.0, tidal volume of 15 mL/kg, respiratory rate of 25 breaths/min and positive end-expiratory pressure of 4 cm H2O. Perfluorocarbon associated gas exchange was initiated by intratracheal instillation of perflouorooctylbromide (30 mL/kg) followed by gas ventilation at the same settings. Evaporative losses were replaced by intratracheal instillation of 2.5 mL/kg/hr of perfluorocarbon. In one group of five piglets, evaporative losses were replaced for 24 hrs until the end of the study. In the other group of five piglets, replacement of perfluorocarbon was discontinued after 2 hrs, although gas ventilation was continued for 24 hrs. Blood gases and lung mechanics were measured in both groups. Histologic evaluation of lungs from both groups of animals was performed. MEASUREMENTS AND MAIN RESULTS Airway pressures and blood gases were stable throughout the 24-hr study period in both groups. Airway pressures in the evaporative group increased as evaporation of perfluorocarbon neared completion. There was no hemodynamic deterioration during the 24-hr study period. Histology showed good preservation of lung architecture in both groups. CONCLUSIONS Perfluorocarbon associated gas exchange was safe and effective in normal piglets for a period of 24 hrs. Evaporation of perfluorocarbon and resumption of continuous positive pressure breathing was well tolerated.

Partial liquid ventilation with perflubron attenuates in vivo oxidative damage to proteins and lipids.

Objective: To determine the impact of partial liquid ventilation on the degree of pulmonary damage by reactive oxygen species in a model of acute lung injury caused by systemic endotoxemia. Design: A prospective, controlled, in vivo, animal laboratory study. Setting: Animal research facility of a health sciences university. Subjects: Forty New Zealand White rabbits. Interventions: Mature rabbits were anesthetized and instrumented with a tracheostomy and vascular catheters. Animals were assigned to receive either partial liquid ventilation (n = 16) with perflubron (18 mL/kg via endotracheal tube) or conventional mechanical ventilation (n = 16). Both groups were ventilated using similar strategies, with an FIO2 of 1.0 and tidal volume as required to obtain a normal PaCO2. Animals were then given 0.9 mg/kg Escherichia coli endotoxin intravenously over 30 mins. Eight uninjured instrumented and mechanically ventilated animals served as controls. Partial liquid ventilation or conventional ventilation was continued for 4 hrs before the animals were killed. Lung homogenates were analyzed for malondialdehyde (MDA) and 4‐hydroxy‐2(E)‐nonenal (4‐HNE) concentrations using a colorimetric assay. To assess protein oxidative damage, carbonyl groups in protein side chains were derivatized with 2,4‐dinitrophenylhydrazine followed by Western blotting with a dinitrophenylated‐specific primary antibody. Measurements and Main Results: MDA (713.42 ± 662 vs. 1601.4 ± 1156 nmol/g protein; p = .023) and MDA plus 4‐HNE (1480.24 ± 788 vs. 2675.2 ± 1628 nmol/g protein; p = .038) concentrations were lower in animals treated with partial liquid ventilation compared with conventionally ventilated animals, respectively. Animals treated with partial liquid ventilation exhibited attenuation of dinitrophenylated‐derivatized protein bands by Western blotting, indicating a reduction in protein oxidative damage. The presence of perfluorocarbon did not interfere with the MDA assay when assessed by independent analysis in vitro. Perflubron did not serve as a sink for peroxyl radicals produced in the aqueous phase during separate in vitro oxidation experiments. Conclusions: Partial liquid ventilation attenuates oxidative damage to lipids and proteins during experimental acute lung injury. This finding is not caused by binding of lipid peroxidation products to perflubron or by the peroxyl radical scavenging properties of perflubron.