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Systemic Perfluorocarbons Suppress the Acute Lung Inflammation After Gastric Acid Aspiration in Rats

Nader, Nader D. MD, PhD*; Knight, Paul R. MD, PhD; Davidson, Bruce A. BS; Safaee, Saeid S. MD; Steinhorn, David M. MD§

doi: 10.1213/00000539-200002000-00022
CRITICAL CARE AND TRAUMA
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Perflurocarbons (PFCs) are used during liquid ventilation and as hemoglobin substitutes. PFCs reduce free radical generation and damage to the lung during liquid ventilation. Thus, we examined the effects of parenteral administration of PFCs on lung injury after acid aspiration. Rats were treated with intraperitoneal injection of either FC-77 or IV injection of Fluosol. Controls received intraperitoneal or IV normal saline (NS) before or at the time of injury and then were injured by instillation of NS + HCl (pH = 1.25) into their lungs via a tracheotomy. The animals were exposed to air or 98% oxygen, breathing spontaneously. The rats were injected with 0.05 μCi of 125I-albumin (bovine serum albumin) before injury. The extent of lung injury was assessed 5 h postinjury by compliance and lung albumin permeability index measurement. Myeloperoxidase (MPO) activity and histologic examination were used to assess neutrophilic infiltration. Both FC-77 and Fluosol decreased the permeability index compared with controls (1.05 ± 0.08; 1.08 ± 0.12, respectively, versus 1.34 ± 0.21) and improved lung compliance after intratracheal instillation of 1.2 mL/kg of HCl/NS, pH = 1.25 + hyperoxia injury (P < 0.05). Lung MPO activity decreased in the FC-77 group and was associated with a concomitant decrease in neutrophil infiltration. MPO activity of the spleen increased after FC-77 treatment. The administration of FC-77 decreased the severity of lung permeability changes associated with acid in the presence or absence of hyperoxia exposure. These data suggest that attenuation of neutrophilic infiltration by PFCs decreases lung injury.

Implications Intraperitoneally administered perfluorocarbons in rats attenuate the neutrophilic infiltration in the lung after acid aspiration, thereby decreasing the alveolar protein leakage and improving pulmonary compliance.

*Department of Anesthesiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas; †Department of Anesthesiology, SUNY at Buffalo, Buffalo, New York; ‡Polyclinic Hospital, Harrisburg, Pennsylvania; and §Northwestern University, Chicago, Illinois

October 27, 1999.

Supported by National Institutes of Health Grant H48889.

Address correspondence and reprint requests to Nader D. Nader, MD, PhD, Chief, Anesthesiology Service (110/LR), Central Arkansas VA Healthcare System, 4300 7th St., Little Rock, AR 72205. Address e-mail to: nnader@med.va.gov

Results of this study were presented in part at the annual meeting of the American Society of Anesthesiologists held in New Orleans 1996.

Adult respiratory distress syndrome (ARDS) carries a high risk of mortality (30%–70%), and requires aggressive therapy to maintain adequate oxygenation and ventilation (1–3). Low pulmonary compliance and progressive atelectasis after establishment of lung injury results in increased work of breathing and an inadequate pulmonary gas exchange. Treatment of ARDS with perfluorocarbons (PFCs) in the form of tidal or partial liquid ventilation has been suggested as a possible new therapeutic modality.

PFCs are biochemically inert liquids with a low surface tension and a high oxygen and carbon dioxide-carrying capacity. Liquid ventilation with PFCs has shown some success in the treatment of ARDS in the laboratory and in early clinical trials (4,5). The addition of PFCs to the airway improves oxygenation and pulmonary compliance and seems to enhance pulmonary surfactant function (6). Additionally, because of their low surface tension, PFCs have been used in the hope of enhancing lung volume recruitment. However, earlier studies suggest that regional distribution of PFCs may depend on the ventilatory strategy (e.g., tidal volume, inspiratory pressure), as well as the dose of PFCs (7).

PFC emulsions have also been systemically used as hemoglobin substitutes (8–10). Systemic PFCs are primarily eliminated by evaporation through the lungs and, to a lesser extent, uptake by the reticulo-endothelial system. Transpulmonary elimination of PFCs depends on their physicochemical properties (water and lipid solubility), and emulsion particle diameter (11). An uneven distribution of PFCs in the lung during partial liquid ventilation is a theoretic concern. Because of poor ventilation/perfusion match, the access of these agents to the site of the injury may be limited because of a regional alveolar collapse. However, despite a segmental hypoperfusion in the atelectatic area, some level of blood flow is maintained to the collapsed alveoli. Therefore, the administration of systemic PFCs may be advantageous in delivering PFCs to injured areas of the lung in hope of improving pulmonary compliance and reexpansion.

Studies conducted in our laboratory over the last several years have suggested a wide range of effects of PFCs on cellular function in vitro (12). These studies are highly controlled and lack the complex multifactorial condition present in vivo, however, their applicability to clinical situations is limited. The role of acute inflammation in the pathogenesis of aspiration pneumonitis has been previously demonstrated (13–15). In view of the apparent improvement in pulmonary function with partial liquid ventilation (16), we hypothesized that systemically administered PFCs would inhibit the protein leakage and improve lung compliance after acid aspiration and decrease pulmonary leukostasis. To test this hypothesis, we used a standard model of acid aspiration pneumonitis and hyperoxia in rats, and studied the response to the systemic administration of FC-77, an industrial grade of PFCs (14,15,17,18). FC-77 has been used during liquid ventilation in animal experimental settings (19,20).

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Methods

The experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of the State University of New York at Buffalo and based on National Institutes of Health guidelines. Pathogen-free male Long-Evans rats (Harlan, Indianapolis, IN) were used. Animals received either 15 mL/kg FC-77 (3M Corp., St. Paul, MN) or a similar amount of normal saline (NS) (treatment controls), intraperitoneally (IP) 48 h before injury to produce steady state systemic levels of FC-77 (Figure 1). Another group of rats was treated with 15 mL/kg Fluosol® (Alliance Pharmaceutical, San Diego, CA), IV grade of PFC, IV administered at the time of lung injury. Controls for this group received an equal volume of IV NS. The lungs of rats were injured under halothane anesthesia by intratracheal instillation of 1.2 mL/kg of HCl/NS, pH = 1.25 (acid), or left uninjured after a sham operation of anesthesia plus surgery with no intratracheal instillation of fluids (SHAM), as described before (15). The rats were injected with 0.05 μCi of 125I-albumin (bovine serum albumin) via the penile vein. In an additional set of studies, the acid-injured animals were permitted to spontaneously breathe 98% oxygen after instillation of the injury vehicle. All animals were killed by exsanguination under halothane anesthesia and then, lung injury and function were assessed.

Figure 1

Figure 1

The extent of lung injury was determined by assessing pulmonary alveolar leakage of radiolabeled protein from the intravascular space (wet to dry weight) and by determining lung compliance (17). After the animals were killed, their abdomens were opened and 1 mL of blood was collected from the inferior vena cava for determination of 125I activity. The pulmonary vasculature was rinsed with 10 mL of NS via the right ventricle to remove residual 125I-labeled albumin and the lungs were then excised. Radioactivity in the lungs and blood was determined by using a 1282 CompuGamma CS gamma counter (Walace, Gaithersburg, MD). The protein permeability index (PI) of the alveolar-capillary membrane was defined as the ratio of radioactivity (cpm) in the harvested lungs to the radioactivity of 1 mL of blood from the same animal. Previous data indicated a very close correlation between PI and the extent of lung injury as assessed by histologic criteria (21–23). Lung injury was further assessed by measuring the ratio of wet weight of the lung to the dry weight (obtained after 24 h drying at 56°C autoclave) as an indicator of interstitial water.

Pulmonary compliance was dynamically assessed by continuous electronic acquisition of the volume changes per centimeter of water pressure while the lungs were inflated up to 30 cmH2O and deflated by a programmed constant flow injector. The maximum dynamic lung compliance (dV/dP) of the lungs was measured during the inspiratory phase, as described previously (17).

The inflammatory response was assessed by peripheral blood neutrophil count (PMN) and myeloperoxidase (MPO) activity in the lung and spleen. Neutrophil and differential white blood cell counts were determined on EDTA anticoagulated blood obtained at the time of death by an automated cell counter in the clinical hematology lab.

Myeloperoxidase activity was measured as a marker of lung neutrophil infiltration in the lung and spleen. Harvested lungs were homogenized on ice three times for 3-s pulses, by using a Polytron TP-2000 tissue homogenizer (Brinkman Instruments, Westbury, NY) in 3 mL buffer containing 50 mM KH2PO4, 13.7 mM hexadecyltrimethylammonium bromide, and 5 mM EDTA, pH = 6.0 (Sigma Chemical, St. Louis, MO). The homogenate was then sonicated on ice four times for 10-s pulses by using a Sonifier Cell Disruptor 350 with microtip probe (Branson Ultrasonics, Danbury, CT) and centrifuged at 2300 ×g for 30 min. at 4°C. The resultant supernatant was assayed for MPO activity by combining 50 μL of sample with 1.5 mL assay buffer containing 50 mM KH2PO4, 176 μM H2O2, and 525 μM o-dianisidine dihydrochloride, pH = 6.0 (Sigma Chemical), in a cuvette and continually recording the absorbency at 460 nm for 2 min by using a DU-650 spectrophotometer (Beckman Instruments, Palo Alto, CA). The spectrophotometer was blanked with 50 mM of phosphate buffer before measurement. MPO activity was normalized to the mg protein concentrations of lung homogenates and expressed as the absorbency change (at 460 nm) per min (ABS · min−1 · mg−1 protein) over the linear portion of the curve (24).

To confirm our findings from the MPO assay, tissue slices from the lung and spleen were evaluated by an experienced pathologist (SSS) in a blinded fashion. Approximately 200 mg of lung tissue samples was fixed in 4% formaldehyde and embedded in Tissue-TeK® O.C.T. compound (Sakura Fintek, Torrance, CA) for sectioning. Tissue sections (5 μ) were mounted on a slide and stained with hematoxylin and eosin. Histopathologic changes were quantified from 0–3. Decimal points were used to express the intermediate changes. Histopathologic changes were defined as alveolar damage, mixed inflammatory cells infiltration (predominantly neutrophils), and vascular congestion. We also examined the spleen for the presence of congestion and expansion of white pulp.

Accepting 20% β error, power analysis was performed to calculate the number of rats in each group. Significant change in protein leakage and compliance was set to 30% of the controls. Our previous results indicate that the standard deviation for PI and dV/dP ranges from 10% to 15% of the mean value, therefore 8–10 rats were used in each group. Data were expressed as means ± SD. Simple regression analysis was performed to compare the microscopic findings with MPO activity in the lungs and spleen. Analysis of variance was used to identify the difference between the groups. Bonferroni/Dunn modification was used for post hoc analysis of the injury and the treatment. Null hypotheses were rejected at P values <5%.

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Results

Intrapulmonary instillation of acid produced significant inflammatory lung injury. Acid injury predictably increased PI and wet/dry weight of the lungs from 0.29 ± 0.02 and 4.52 ± 0.05, respectively, in the SHAM-injury group to 0.90 ± 0.11 and 5.69 ± 0.06, respectively, in the acid injury group (P < 0.05 for both measurements). Intraperitoneal administration of FC-77 resulted in a decrease in PI when compared with NS-treated animals to 0.72 ± 0.09 (P < 0.05). IV Fluosol administration at the time of injury, however, did not result in a decrease in PI in the lungs from rats injured with acid. There was also no change in wet/dry weight. FC-77 treatment produced a similar decrease in PI in rats exposed to hyperoxia > 90% for 5 h after acid injury (from 1.34 ± 0.21 to 1.05 ± 0.08, P < 0.02). IV Fluosol treatment at the time of injury similarly decreased PI to 1.08 ± 0.16 compared with hyperoxia-exposed controls (P < 0.05) (Figure 2).

Figure 2

Figure 2

The reduction in protein leakage was associated with an improvement in lung compliance. Compliance changes were only tested in rats treated with FC-77 because of its maximal protective effects. Dynamic lung compliance was 2.4 ± 0.5 mL · kg−1 · cm H2O−1 in SHAM-injured rats, whereas acid-injured rats yielded a maximum dV/dP of 1.3 ± 0.3 mL · kg−1 · cm H2O−1 in NS-treated animals (P < 0.01). Systemic administration of FC-77 increased lung compliance to 1.9 ± 0.6 mL · kg−1 · cm H2O−1 after acidic injury (P < 0.05), representing a 46% increase. In hyperoxia-exposed rats, the dynamic lung compliance improved approximately 110% in the FC-77-treated group to 1.5 ± 0.5 mL · kg−1 · cm H2O−1 from 0.71 ± 0.12 mL · kg−1 · cm H2O−1 in NS-treated controls P < 0.05 (Figure 3).

Figure 3

Figure 3

FC-77 treatment decreased neutrophils to 800 ± 190 PMN/μL in the peripheral blood from 3800 ± 200 PMN/μL in the SHAM-injury group (P < 0.001). Acid-injured controls receiving NS treatment had a decreased PMN to 1600 ± 220 PMN/μL compared with SHAM-injury NS treatment group, P < 0.05. Contrary to its effect in the SHAM-injury group, FC-77 treatment in the acid-injured group, increased the PMNs to 3000 ± 140 PMN/μL (P < 0.05) (Figure 4).

Figure 4

Figure 4

The MPO activity of the lungs increased predictably after acid injury. However, FC-77 treatment was associated with a lower MPO activity in the lungs of acid-injured rats (0.80 ± 0.12 ABS · min−1 · mg−1 protein) compared with those from NS-treated animals (1.52 ± 0.26 ABS · min−1 · mg−1 protein, P < 0.05). MPO activity increased in the spleen of animals after intratracheal instillation of acid (1.66 ± 0.22 ABS · min−1 · mg−1 protein) when compared with SHAM-injured controls 0.84 ± 0.06 ABS · min−1 · mg−1 protein, (P < 0.05). FC-77 treatment increased MPO activity in the spleen to 1.82 ± 0.30 in the SHAM-injury group and to 3.48 ± 0.72 ABS · min−1 · mg−1 protein after acid injury (P < 0.05) (Figure 5).

Figure 5

Figure 5

Microscopic histopathologic scoring was strongly correlated with MPO activity in the lungs (r2 = 0.79, P < 0.001) and in the spleen samples (r2 = 0.80, P < 0.0001). FC-77 did not cause any alteration of the lung structure in SHAM-injured rats, however, there was a sporadic foamy appearance in the alveoli compared with uninjured, untreated lungs. Acid injury predictably resulted in moderate to severe neutrophilic infiltration and proteinaceous exudation and obliteration of alveolar space (mean pathologic score = 2.5 ± 0.5) when compared with the normal structure in the SHAM-injury group. FC-77 treatment decreased the neutrophilic infiltration, as well as alveolar exudate from moderate-severe in control rats to a moderate level (mean pathologic score = 1.7 ± 0.3, P < 0.05). A mild-to-moderate hypercellularity and expansion of the white pulps was noted in the spleen of SHAM-injured, FC-77-treated animals (mean pathologic score = 2.0 ± 0.3) when compared with NS-treated controls. Intrapulmonary deposition of acid resulted in an additional increase in splenic congestion and expansion of the white pulp in NS-, as well as FC-77-treated animals.

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Discussion

We have demonstrated that systemic treatment with PFCs diminishes the protein leakage and improves dynamic lung compliance in injured animals. We have histologic evidence that FC-77 is present in the alveoli of rats within 48 hour after IP administration. This finding is not novel, because the lung is an important elimination route for PFCs. The presence of PFCs in the alveoli affects dynamic lung compliance similar to partial liquid ventilation, possibly, by reducing the surface tension. Furthermore, improvement of alveolar-capillary wall integrity and lung compliance after treatment with PFCs is associated with decrease in inflammatory response after the acid injury.

Acidic pH is a major contributing factor to the pathology of aspiration pneumonitis (15). Aspiration of acid produces acute inflammation characterized by pulmonary infiltration of neutrophils (25). Neutrophil mediators (e.g., reactive species of oxygen and proteases) play a major role in the pathogenesis of low-pH injury to the lungs (17). We have demonstrated that systemic PFC treatment reduces the protein leakage and improves lung compliance. We speculate that the decrease in protein leakage (representing an approximately 60% decrease in the neutrophilic component of the injury) is caused by the effect of PFCs on neutrophil sequestration. This hypothesis is consistent with our MPO activity and pulmonary histopathology data.

Our results clearly demonstrate that intrapulmonary deposition of acid decreases the number of circulating neutrophils, which is consistent with a previous report (26). An important new finding of our study is that FC-77 treatment by IP is capable of reducing the number of circulating neutrophils in uninjured animals. We believe this is caused by filtration of FC-77-engorged neutrophils in the spleen. However, in acid-injured animals, FC-77 treatment fails to induce neutropenia indicating the presence of an overriding stimulus to leukocytosis. We believe that inhibition of leukosequestration into the lungs, as well as other nonreticuloendothelial tissues by FC-77, is the underlying mechanism for this relative leukocytic response.

Activation of neutrophils and their sequestration within the lung after pulmonary acid deposition is a primary mechanism in the pathogenesis of acid aspiration. We have tested this hypothesis by examining the pulmonary histopathology and MPO activity. An acidic insult significantly increases neutrophil sequestration within the lung, secondary to the release of chemokines (e.g., MIP-2) (27), increases expression of integrins, and may be responsible for the subsequent low peripheral neutrophil count (28). PFCs are phagocytized by neutrophils and other cells of the reticuloendothelial system. We have examined the spleen for evidence of sequestration of FC-77-containing neutrophils and hypercellularity as a marker of the neutrophil scavenging function of the spleen that could account for the neutropenia associated with FC-77 administration. Our data clearly indicate that FC-77 treatment results in increased MPO activity in the spleen and congestion and expansion of white pulps in this organ. This is seen both in injured and uninjured rats after treatment with FC-77, although it was more pronounced in acid-injured animals.

A reduction in pulmonary leukostasis has also been reported in endotoxin-induced lung injury in rabbits treated with partial liquid ventilation (29). In that study, the lung was exposed directly to PFC, a fact that may have led to the diminished local accumulation of neutrophils. In our study, the FC-77 may have affected the lung through its excretion via the lung during respiration. The quantity of PFCs in tissues and blood is directly related to its vapor pressure and lipid solubility, therefore, FC-77 with a vapor pressure of 75 torr at 37°C (versus perflubron [Liquivent®; Alliance Pharm., San Diego, CA]: 10.5 torr at 37°C) may achieve a sufficient concentration in both blood and tissues to exert an effect on neutrophil accumulation.

Although our study was not designed to answer specific cellular mechanistic questions, our findings raise novel and interesting issues regarding the potential of systemically administered PFCs to modify systemic inflammatory responses. We speculate that IP administration of PFCs may attenuate the systemic response to invading organisms or shock states through an, as yet, unclear mechanism of immunomodulation. Although factors which seem to suppress inflammation may be viewed with great skepticism in patients at risk of sepsis, during a controlled study of rabbits supported with partial liquid ventilation, it has been recently demonstrated that there is no increased risk of bacterial infection (30).

In conclusion, FC-77 reduces the circulating neutrophil count in healthy animals with apparent sequestration by the reticuloendothelial system. Decreased pulmonary neutrophil infiltration and reduced permeability changes in acid-injured animals pretreated with FC-77 suggest a possible novel therapeutic approach to acute lung injury and perhaps systemic inflammation in general.

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