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CRITICAL CARE AND TRAUMA

Perfluorohexane Vapor Has Only Minor Effects on Spatial Pulmonary Blood Flow Distribution in Isolated Rabbit Lungs

From the Department of Anesthesiology and Intensive Care Medicine, Technical University Dresden, Germany

Address correspondence and reprint requests to Matthias Hübler, MD, DEAA, Department of Anesthesiology and Intensive Care Medicine, Carl Gustav Carus University Hospital, Fetscherstr. 74, 01307 Dresden, Germany. Address e-mail to matthias.huebler{at}mailbox.tu-dresden.de.

	Abstract

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We tested the hypothesis that administration of perfluorohexane (PFH) vapor does not significantly affect the relative pulmonary blood flow (_rel) distribution in isolated rabbit lungs. Fourteen isolated rabbit lungs were perfused with a Krebs-Henseleit buffer solution (flow 150 mL/min). Pulmonary afterload was set to 3 mm Hg. The lungs were ventilated with 4% CO₂ in room air using a small animal ventilator (respiratory rate, 30 breaths/min; tidal volume, 12 mL/kg body weight; positive end-expiratory pressure, 2 cm H₂O). After a steady-state period, 18 vol. % of PFH vapor was administered to 9 lungs for 30 min. In a second set of experiments five lungs served as controls. Change in _rel distribution was assessed using fluorescent-labeled microspheres. The unpaired Student's t-test was used to compare variables between groups. The paired Student's t-test, the one-sample Student's t-test, the Anderson-Hauck test of equivalence, and Pearson correlation were used to analyze changes within groups. The mean correlation coefficients of _rel were 0.564 ± 0.182 for the PFH group and 0.502 ± 0.295 for the control group, respectively. No significant changes in _rel distribution over time and between groups were found. However, in the PFH group a tendency towards redistribution of _rel to more ventral lung areas was noted. Our results suggest that PFH vapor has no significant effects on redistribution of _rel in isolated rabbit lungs.

	Introduction

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Experimental data increasingly suggest that the beneficial effects of perfluorocarbons (PFCs) are not limited to gas exchange and that PFCs also attenuate the inflammatory response after oleic acid (1) or endotoxin injury (2), acid aspiration, or viral infection (3). As a consequence of the disappointing results of the first large clinical trial with partial liquid ventilation (PLV) (4), research is now focused on early start of treatment to modulate the initial inflammatory response.

Initiation of PLV in healthy lungs typically leads to deterioration in pulmonary gas exchange. This is probably due to a redistribution of regional pulmonary blood flow to nondependent lung areas (5) and to diffusion limitation, which leads to incomplete equilibration of capillary and alveolar gas partial pressures (6). It has been shown that perfluorohexane (PFH) vapor lacks similar negative effects on gas exchange in noninjured lungs (7). This less invasive application mode of PFC might therefore be suitable for an early start of treatment.

Our aim was to further investigate the effects of PFH vapor in noninjured lungs. After having shown that gas exchange is not negatively affected (7), we looked at the pulmonary blood flow distribution. Our hypothesis was that application of PFH vapor does not influence the spatial distribution of relative pulmonary blood flow (_rel).

	Methods

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The study was approved by the University Animal Care Committee and the responsible governmental institution (Regierungspräsidium Dresden, AZ 74–9168.11–1–2001–18). National Institutes of Health guidelines for animal use and care were followed throughout.

The experiments were performed on 14 chinchilla rabbits (orticolagus caniculus) of both genders, ranging in weight from 2.0 to 2.9 kg. In a first set of experiments, nine isolated lungs were exposed to PFH. The five control experiments were performed thereafter. The rabbits were anesthetized with ketamine (50 mg/kg) and xylazine (4 mg/kg) and anticoagulated with heparin-sodium (1000 U/kg), injected in an ear vein. A tracheotomy tube was placed under local anesthesia (lidocaine 1%). The animals were mechanically ventilated with room air using a small animal ventilator (Hugo Sachs Elektronik, March, Germany). The ventilator settings were as follows: respiratory frequency 30 breaths/min; tidal volume (Vt) 12 mL/kg; positive end-expiratory pressure 2 cm H₂O. The ventilation was never disrupted thereafter.

Thereafter the lungs were isolated. The techniques of preparing and perfusing isolated rabbit lungs have been described previously (8). Briefly, a median sternotomy was performed and a catheter was inserted into the pulmonary artery. The left atrium was cannulated via the left ventricle to collect the pulmonary outflow. The lung organ-preparation was removed and suspended vertically from a weight transducer in a temperature-controlled (37°C) and humidified chamber. The average time required to isolate the lungs was approximately 15–20 min. After the cannulation procedure, the lungs were perfused with a cell-free and plasma-free perfusion medium. A 40-µm filter and a bubble trap prevented pulmonary embolism by circulating particles or air. The perfusate consisted of a Krebs-Henseleit hydroxyethyl starch buffer solution with a colloid oncotic pressure between 23 and 25 mm Hg, yielding final concentrations of: Na⁺ 138 mmol/L; K⁺ 4.5 mmol/L; Mg⁺⁺ 1.33 mmol/L; Cl^– 135 mmol/L; Ca²⁺ 2.38 mmol/L; glucose 12 mmol/L; and HCO₃^– 12 mmol/L. The osmolality was approximately 330 mosm/kg (Mikro-Osmometer, Roebling Meßtechnik, Berlin, Germany). The pH of the buffer solution was adjusted to 7.4 with 1 M NaHCO₃.

First, slow flow rates were used in an open circulatory system and the perfusion fluid was exchanged 2 times via 2 separate perfusion circuits, 2 and 15 min after the beginning of extracorporeal circulation. Flow was then increased to 150 mL/min. Because of a constant perfusion flow, alterations of perfusion pressure directly reflect alterations of pulmonary vascular resistance. Pulmonary afterload was set to 3 mm Hg using a height-adjustable fluid bridge open to ambient pressure. The lungs were ventilated with 4% CO₂ in air with unchanged ventilator settings. As it has been shown that oxygenation of the lung by ventilation alone is satisfactory (9), no additional oxygenator unit in the perfusion circuit was required. The mean pulmonary artery pressures (mPAP) and left atrial pressures were continuously recorded using pressure transducer sets (Becton Dickinson, Sandy, UT) zero referenced at hilum level. The lungs showed a homogenous white appearance with no signs of hemostasis or edema formation and had no increase in weight during the steady-state period of 30 min. The lungs had a constant mPAP of 12 ± 3 mm Hg (mean ± sd).

Thereafter, 18 vol. % of PFH was administered to 9 lungs for 30 min (PFH group), whereas 5 lungs served as controls (CTL group). _rel distributions were assessed immediately before (time point t₀) and after the vaporization period (time point t₃₀) using fluorescent microspheres. The microspheres were injected over 1 min into the pulmonary artery catheter.

All experiments were performed using PFH (ABCR, Karlsruhe, Germany) with a purity of 95%. Its chemical and physical properties and similarity to volatile anesthetics are described elsewhere (12). Vaporization was achieved using two modified vaporizers (type 19n; Dräger Werke, Lübeck, Germany). The PFH-adapted vaporizers were connected in series in the inspiratory limb of the animal ventilator. In pilot experiments, we confirmed that an inspiratory concentration of 18 vol.% of PFH was delivered to the lungs as described previously (12). As PFH vapor may affect the delivered Vt, we measured Vt before and after opening the vaporizers to guarantee a stable experimental condition.

Fluorescent polystyrene microspheres (red, orange, yellow-green, and crimson) of 15 µm diameter (Molecular Probes, Eugene, OR) were used to measure regional flow to the lungs. Details of the method are described elsewhere (10). When the number of injected microspheres is sufficiently large, and when appropriately sized microspheres are used, regional blood flow is proportional to the number of trapped microspheres. Immediately before injection, the microspheres were vortexed and then sonicated for 90 s. The number of microspheres per injection was 600,000. The order that the different microspheres were injected was randomized in every experiment.

After completion of the experiments, lungs were inflated, and dried by constant tracheal airflow for 2 days (pressure limit, 20 cmH₂O). The lungs were then coated with a one-component polyurethane foam (BTI Befestigungstechnik GmbH & Co.KG, Ingelfingen, Germany), suspended vertically in a square box, and embedded in rapidly setting urethane foam (Polyol and Isocyanate, kind gift of Elastogran GmbH, Lemförde, Germany). The foam block was cut into cubes of 1 cm³ in volume. Each cube was weighed and assigned a three-dimensional coordinate. Samples with airways occupying >25% of the cube's volume were discarded (3 to 5 cubes per lung). The amount of large airways within one cube was checked visually. The samples were then soaked for 2 days in 2 mL of 2-ethoxyethyl acetate (Aldrich Chemical CO, Milwaukee, WI) to retrieve the fluorescent dye. The fluorescence was read in a luminescence spectrophotometer (LS-50B; Perkin-Elmer, Beaconsfield, Buckinghamshire, UK) fitted with a flow cell and a standard photomultiplier tube.

The weight-normalized relative blood flows at the two time points were calculated for each lung piece as follows:

where _rel,i is the weight-normalized relative blood flow of the piece i, x_i is the fluorescence divided by the weight of the piece, and n is the number of pieces of the lung. The mean normalized relative flow was therefore 1.0.

Our primary hypothesis was that PFH vapor does not influence spatial distribution of _rel. A priori, an Anderson-Hauck test of equivalence was used to estimate the necessary group size (11). It assesses the necessary difference in expected values, which would lead to rejection of equivalence between compared values. We used 100% of the sd of the differences in expected values as maximum level of equivalence. The sd values for this analysis were taken from former blood flow studies performed in rabbits (12). A sample size of eight would then allow an 80% chance of showing equivalence in measured values at a fixed time point with a two-tailed level of statistical significance ( < 0.05). However, analysis of the lungs of the PFH group yielded only a mean correlation of _rel distribution between t₀ and t₃₀ of 0.564. As a consequence, we performed a set of control experiments to exclude minor effects of PFH vapor on _rel distribution.

Analysis of variance for repeated measures was used to compare changes in lung weights, mPAP, and left atrial pressures. The unpaired, two-sided Student's t-test was used to compare variables between groups at t₀ and t₃₀. The paired, two-sided Student's t-test was used to compare changes in blood flow distribution within each group over time. Bonferroni's correction for multiple tests was applied when appropriate. A one-sample, two-tailed Student's t-test was used to compare changes in _rel distribution to a hypothesized mean of zero. Post hoc, an Anderson-Hauck test of equivalence was used to further analyze the blood flow distribution (11). Failure to reject the null hypothesis does not lead to the conclusion of equivalence because the strength of the evidence against and not for the null hypothesis is measured. Equivalence tests are usually used to evaluate whether an experimental treatment is sufficiently similar to an established treatment to justify its use (13). In our study, we used the Anderson-Hauck equivalence test to evaluate the strength of the absent effects of PFH vapor application on _rel distribution. This test, as described by the authors (14), uses group size, measured mean values, and sd as input parameters. It provides a way of quantifying (with P values) what was actually determined from the study instead of saying what the study may or may not have accomplished with some degree of certainty (power). A possible outcome of the equivalence testing approach is the conclusion at the 5% level that two means do not differ by more than some specified amount.

All data are presented as means ± sd. Throughout this study, P < 0.05 is considered to represent statistical significance. All analyses were performed using SPSS Version 10.0.7. (SPSS, Chicago, IL) and SAS Version 8 (SAS, Cary, NC).

	Results

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During the study period, lung weight and mPAP values were comparable between the two groups. Analysis of variance yielded no significant changes in these variables within groups and between groups over time (Fig. 1). Sixty-seven ± 10 samples were obtained from each rabbit lung. The average weight of the samples was 0.019 ± 0.012 g. The mean Pearson correlations of _rel at t₀ and t₃₀ were 0.564 ± 0.182 for the PFH group and 0.502 ± 0.295 for the CTL group, respectively. Table 1 shows how spatial _rel distributions were affected by time and by vaporized PFH, respectively. Statistical analysis did not yield significant differences when comparing the two groups at the time points t₀ and t₃₀ or when comparing each group over time. However, there was a tendency towards redistribution of _rel to more ventral lung areas in the PFH group (P = 0.054). Table 2 shows the results of the one-sample, two-tailed Student's t-tests comparing differences in slopes at t₃₀ minus t₀ to a hypothesized mean of zero and the results of the Anderson-Hauck tests of equivalence. Again, the tendency towards redistribution of _rel to more ventral lung areas in the PFH group was confirmed. Figure 2 and Figure 3 show _rel distribution versus spatial axes at the two time points in one representative animal of the PFH group and in one representative animal of the CTL group, respectively.

View larger version (36K): [in this window] [in a new window]   Figure 1. Lung weights (upper lines, right scale) and mPAP values (lower lines, left scale) during the study period. Data are means ± sd. mPAP, mean pulmonary artery pressure; CTL, control group; PFH, perfluorohexane group.

View this table: [in this window] [in a new window]   Table 1. Slopes of rel Versus Spatial Axis

View this table: [in this window] [in a new window]   Table 2. Differences in Slopes of rel Versus Spatial Axis

View larger version (42K): [in this window] [in a new window]   Figure 2. Spatial relative pulmonary blood flow (rel) distribution along the y-axis (dorsal-to-ventral), z-axis (caudal-to-cranial), and h-axis (hilus-to-peripheral) in one representative lung of the perfluorohexane (PFH) group. t0 and t30, time points before and after the vaporization period.

View larger version (43K): [in this window] [in a new window]   Figure 3. Spatial relative pulmonary blood flow (rel) distribution along the y-axis (dorsal-to-ventral), z-axis (caudal-to-cranial), and h-axis (hilus-to-peripheral) in one representative lung of the control (CTL) group. rel distribution was assessed at the end of the stabilization period (t0) and 30 min thereafter (t30).

	Discussion

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The pulmonary effects of PFC are numerous and include large oxygen-carrying capacity of PFC liquids, effects on surface tension at the air-liquid and liquid-tissue interfaces, antiinflammatory effects, and effects on pulmonary ventilation and perfusion distribution (2,15–18). Many of these effects can also be achieved when PFCs are administered to the lungs as vapor or aerosol (12,19–21). These application modes have the advantage that they are less invasive than PLV and can easily be used in an intensive care unit. PLV is indicated in patients with fully established adult respiratory distress syndrome (ARDS). However, the prognosis of these patients is often very poor. In a prospective, randomized, controlled pilot study PLV did not prove to be superior to modern ventilatory strategies in ARDS patients (4). A possible therapeutic strategy might therefore be to attenuate the inflammatory process by an earlier start of treatment before the ARDS is fully established. In this context, the use of less invasive application modes of PFCs theoretically offers several advantages.

The main result of the present study is that a change of _rel distribution does not occur when PFH vapor is administered to isolated rabbit lungs. It therefore confirms the hypothesis that no significant amount of PFH vapor condenses within the lungs (22), which is also supported by the lack of weight increase over time. Thus, a diffusion limitation seems very unlikely. This could explain why no deterioration of gas exchange is observed when this mode of application is used in noninjured animals (7). Treatment with PFH vapor might therefore be started in a very early phase of lung injury without causing negative effects on gas exchange. This is especially interesting because an early start of treatment would also have effects on noninjured lung regions. PLV, when applied to healthy lungs, typically worsens gas exchange. This is likely attributable to ventilation/perfusion heterogeneity and diffusion limitation (6). Liquid PFC probably layers within the alveoli and causes diffusion limitation, leading to incomplete equilibration of capillary/alveolar oxygen and carbon dioxide partial pressures. Additionally, PLV causes a redistribution of pulmonary blood flow to nondependent lung areas (5). This effect of PLV on _rel distribution has also been confirmed in isolated lung studies (23).

The isolated lung model is widely used to study a variety of physiological phenomena, including pulmonary vasoactivity, microvascular permeability, enzyme activity, and blood flow distributions. It is especially useful in studying gravitational changes of _rel (24). However, results of perfusion studies from isolated lung models have to be interpreted carefully because the isolation and suspension of the lung does not represent a physiological situation. Factors that influence the pulmonary vascular tone and might influence the spatial distribution of pulmonary blood flow include neural activity, partial pressures of inspiratory gases, gravitational forces, pleural and alveolar pressures, and numerous humoral factors (e.g., nitric oxide). Nevertheless, we chose to use an ex vivo model to control some of the known factors that influence pulmonary blood flow distribution (e.g., flow rates, anesthesia, mechanical ventilation) (25,26).

In conclusion, PFH vapor has no statistically significant effect on _rel distribution in noninjured isolated rabbit lungs. Further studies should confirm these data in nonisolated lungs, ideally also looking at effects on spatial ventilation distributions.

The authors thank Rainer Koch, PhD (Professor, Institute of Medical Informatics and Biometrics, University Dresden, Dresden, Germany) for expert statistical support.

	Footnotes

 
Supported, in part, by Deutsche Forschungsgemeinschaft (Bonn, Germany) Grant HU 818/3–1.

Presented, in part, at the 3rd European Symposium on Perfluorocarbon Application in Berlin, Germany, November 30, 2002, and at the 11th Meeting of the European Society of Anaesthesiology in Glasgow, Great Britain, May 4, 2003.

Accepted for publication September 29, 2004.

This work was part of the medical thesis of Tobias Kroll.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hübler, M. Articles by Koch, T. Search for Related Content PubMed PubMed Citation Articles by Hübler, M. Articles by Koch, T. Related Collections Critical Care Airway