Real-time visualization of partial liquid ventilation in a model of acute lung injury

Real-time visualization of partial liquid ventilation in a model of acute lung injury Shunsuke Endo, MD, Yasunori Sohara, MD, Fumio Murayama, MD, Tsutomu Yamaguchi, MD, Tsuyoshi Hasegawa, MD, and Yoshihiko Kanai, MD, Tochigi, Japan

Background. To clarify the effects of partial liquid ventilation, we visualized and morphologically analyzed real–time alveolar recruitment in a model of acute lung injury. Methods. Male Wistar rats were divided into 3 groups: a group that underwent hydrochloric acid aspiration and mechanical gas ventilation (ALI group, n = 15), a group that underwent acid aspiration and partial liquid ventilation beginning 90 minutes after acid aspiration (PLV group, n = 15), and a group that underwent mechanical ventilation without acid aspiration (control group, n = 5). The number of ventilated alveoli and the diameter of the largest ventilated alveolus in each of 10 high-power fields observed on fluorescence micrographs with a tracer of labeled albumin were determined and averaged from 90 to 210 minutes after acid aspiration. Results. The number of alveoli in the PLV group significantly increased in comparison to that in the ALI group. The diameter of the largest alveolus in the PLV group decreased from 103.7 ± 16.3 µm to 76.3 ± 6.5 µm until the end of the experiment. This diameter was equivalent to that in the control group. Conclusions. The excellent alveolar recruitment suggests that liquid ventilation ameliorates ventilatorassociated lung injury. (Surgery 2003;133:207-15.) From the Department of Thoracic Surgery, Jichi Medical School, Tochigi, Japan

ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is characterized by stiff lungs, hypoxemia, and diffuse pulmonary infiltrates.1 Both gas exchange and protection from lung injury are problematic in ARDS patients under critical care.2 Mechanical ventilation can exacerbate the underlying lung injury, producing what is known as ventilator-associated lung injury,3 but clinical investigators have recently reported improved outcomes in patients with ARDS who were ventilated with reduced tidal volumes.4,5 Lung protective strategies are working in patients at risk. Partial liquid ventilation (PLV) is another strategy that has been considered in ARDS patients.6-8 For this method of mechanical ventilation, the conventional ventilator is partially filled with liquid perfluorocarbon equivalent to the residual functional capacity of the lung.9 A number of studies in various models of acute respiratory failure have shown the efficacy of liquid ventilation for improvAccepted for publication August 26, 2002. Reprint requests: Shunsuke Endo, MD, Department of Thoracic Surgery, Jichi Medical School, Minamikawachi-machi, Kawachigun, Tochigi 329-0498, Japan. © 2003, Mosby, Inc. All rights reserved. 0039-6060/2003/$30.00 + 0 doi:10.1067/msy.2003.16

ing gas exchange,9-17 improvement of pulmonary compliance,10-12 reducing the volume of intrapulmonary shunting,13,14 avoiding hemodynamic compromise,15 reducing morphometric and histologic evidence of lung injury,16,17 and attenuating increases in pulmonary capillary permeability18-20 and inflammatory processes.21-25 However, we know of no clinical trial that has shown PLV to be advantageous over conventional mechanical ventilation. PLV is in fact a significant undertaking, and there is yet no in vivo morphometric data, either experimental or clinical, to substantiate the impression that a protective effect exists. In the present study, we visualized and morphometrically analyzed real-time alveolar recruitment under PLV in a rat ARDS model to clarify the morphologic effects of liquid ventilation. MATERIAL AND METHODS Animal preparation. The study was approved by the Jichi Medical School Review Board for the care and use of laboratory animals. Care and handling were in accord with the US National Institutes of Health guidelines. Rats, aged approximately 10 weeks and weighing approximately 300 g, were anesthetized by intraperitoneal administration of sodium pentobarbital (12.5 mg/kg) and diazepam (2.5 mg/kg). A tracheostomy was performed. SURGERY 207

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Volume-controlled continuous positive-pressure ventilation was instituted after intravenous injection of panchronium bromide (0.2 mL/kg). A pulsatile pump (Model 683 small luminal ventilator; Harvard Apparatus, South Natick, Mass) was set at 65 breaths/min, 5 cm H2O positive end-expiratory pressure, 10 mL/kg tidal volume. The animals were ventilated with oxygen (fraction of inspired oxygen of 1.0). Catheters made of polyethylene tube (inner diameter, 0.5 mm; outer diameter, 1.0 mm) were inserted into the right jugular vein for intravenous injection and right carotid artery for arterial blood pressure measurement and blood gas analysis. Continuous intravenous injection of saline (5 mL/kg/h) was performed with an injector (Model STC-521; Terumo, Tokyo, Japan). The catheter inserted into the right carotid artery was attached to a pressure transducer (Becton Dickinson, Sandy, Utah) and connected to a monitor (Polygraph System; Nihon Kohden, Tokyo, Japan) for monitoring systemic arterial pressure and heart rate. Acute lung injury. Acute lung injury was induced by instillation of 0.1 N/mL hydrochloric acid at 2 mL/kg via tracheostomy. The rats were placed in the semi–Fowler’s position so that the hydrochloric acid would be distributed after aspiration to an inferior portion of the lung under mechanical ventilation for 30 minutes. Experimental protocol. Thirty-five rats were divided into 3 groups. One group had no instillation of hydrochloric acid but underwent mechanical ventilation (control group, n = 5). An acute injury group underwent continuous mechanical ventilation after acid aspiration (ALI group, n = 15). A second acute lung injury group underwent mechanical ventilation after the acid aspiration as well as liquid ventilation starting at 90 minutes after the aspiration (PLV group, n = 15). Liquid ventilation was performed according to the conditions of mechanical ventilation described above except with instillation of perfluorocarbon (Cheminox, C8F17Br; Japan Mektoron, Tokyo, Japan) at 20 mL/kg. Visualization of ventilation on fluorescence micrographs. The left anterolateral chest wall of each animal was resected, and the lower pulmonary ligament was divided. The diaphragmatic portion of the left lung was stabilized in a glass suction chamber adjusted to the appropriate size at the mid-inspiratory phase and viewed under a fluorescence microscope. The pulmonary microcirculation including the capillary network between venules and arterioles was visualized with fluorescein isothiocyanate–labeled albumin tracer (0.25% FITC–albumin, molecular weight of 67,000 units;

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Sigma Chemical Co, St Louis, Mo). Ventilated alveoli were displayed as negative (dark) elliptical areas under the fluorescence microscope because labeled albumin encircled the alveoli. The pictures were amplified by a silicon–intensified television camera (Model C2400; Hamamatsu, Shizuoka, Japan) and viewed on a monitor via Super Video Home System video recorder. The observation system was described in a previous report.26, 27 Morphologic measurements. Arterial blood pressure and gasses as well as heart rate were monitored beginning at 90 minutes and continuing until 210 minutes after the acid aspiration. Measurements were obtained every 30 minutes. Ten high-power fields 1080  790 µm were selected in the center of the diaphragmatic portion of the left lung, and the number of ventilated alveoli encircled by capillary network was counted in each field. The largest alveolus in each field was measured at its greatest diameter to estimate overdistention of the alveoli. The data for the 10 fields were averaged. Statistical analysis. All data are presented as mean ± standard deviation. The physiologic data and the data obtained by morphologic analysis of fluorescent micrographs were evaluated by repeated measures analysis (ANOVA) with Bonferroni/Dunn correction for multiple comparisons. Differences found between the 2 treatment groups for each data point were analyzed. A P value of less than .0167 was considered significant. RESULTS Hemodynamic stability and oxygenation. The changes in blood pressure, heart rate, and arterial oxygen pressure (PaO2) in each group are shown in Fig 1. Blood pressure and heart rate at both 180 and 210 minutes decreased in the ALI and PLV groups, in comparison to those in the control group. However, differences between the ALI group and the PLV group were not significant at any time point. A significant decrease in PaO2 occurred in the ALI group at each time point in comparison to PaO2 in the control group. PaO2 in the PLV group was increased significantly beginning at 120 minutes after aspiration in comparison to PaO2 in the ALI group. Morphologic measurements. Fluorescent photographs in the control, ALI, and PLV groups are shown in Fig 2. In the control group, micrographs at every time point were same as those of the ALI group at 0 minutes (Fig 2, A). The albumin-labeled flooding in the alveoli and interstitial spaces of the ALI group was significant after 90 minutes (Fig 2,

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Fig 1. Heart rate (HR), blood pressure (BP), and arterial oxygen pressure (PaO2,) in the 3 study groups: the control group (), the ALI group (), and the PLV group (). Data are shown as the mean ± standard deviation. (*P < .0167 compared with values in the ALI group by ANOVA with Bonferroni-Dunn correction for multiple correction).

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Fig 2. Representative fluorescence micrographs after injection of labeled albumin as a tracer in the ALI group at 0 minutes (A), 30 minutes (B), 90 minutes (C). (continued)

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Fig 2—cont’d. 180 minutes (D) and in the PLV group at 180 minutes (E).

B-D). Subsequently, the number of the ventilated alveoli decreased markedly. Marked alveolar recruitment was observed after PLV in the PLV group (Fig 2, E). The albumin-labeled exudates did not increase in the alveoli and interstitial spaces of the PLV. The capillary microcirculation decreased, whereas alveoli were recruited after application of liquid ventilation in the PLV group. The numbers of ventilated alveoli at 90 minutes after acid aspiration decreased significantly in the ALI group (44.3 ± 15.8) and the PLV group (43.0 ± 16.6), in comparison to the number in the control group (159.9 ± 10.4), as shown in Fig 3. The numbers of ventilated alveoli in the PLV group increased to 102.1 ± 17.6 at 120 minutes after aspiration (ie, at 30 minutes after liquid ventilation) and gradually increased to 122.4 ± 20.3 at 150 minutes, to 133.4 ± 18.2 at 180 minutes, and to 130.3 ±

19.1 at 210 minutes. The largest ventilated alveolus in the ALI group increased gradually from 106.8 ± 14.5 µm in diameter at 90 minutes to 119.6 ± 14.4 µm in diameter at 210 minutes. The diameter in the PLV group decreased to 86.5 ± 7.4 µm at 120 minutes after aspiration (ie, at 30 minutes after liquid ventilation), 79.5 ± 9.7 µm at 150 minutes, 76.4 ± 7.8 at 180 minutes, and 76.3 ± 6.5 at 210 minutes (P < .0167 vs diameter in the ALI group at all time points after 120 minutes). The diameter remained constant until the end of the experiment and equivalent to the diameter in the control group after 150 minutes. DISCUSSION The objective of this study was to evaluate, via real-time visualization of ventilation on fluorescence micrographs, the efficacy of fluorochemicals

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Fig 3. The number of ventilated alveoli encircled by the capillary network and the diameters of the largest alveolus in the 3 groups: the control group (), the ALI group (), and the PLV group (). Data are shown as the mean ± standard deviation based on 10 high-power fields (hpf) (*P < .0167 compared with values in the ALI group by ANOVA with Bonferroni-Dunn correction for multiple comparisons). Note that the number of the ventilated alveoli in the PLV group increased significantly after liquid ventilation. The diameter of the largest alveolus in the PLV group decreased to the diameter in the control group at 180 minutes.

for alveolar recruitment in acute lung injury under gas ventilation. The investigation was based on the assumption that the hydrochloric acid injury occurred uniformly in the dependent diaphragmatic regions, which were the area of focus in this study. Perfluorocarbon, with its relatively high specific gravity of approximately 2 g/cm3, preferentially fills the dependent regions. On the fluorescence micrographs, the tracer allowed us to observe vessels enhanced with labeled albumin, surrounding the ventilated alveoli, which are displayed as negative elliptical regions (Fig 2, A). When alveoli are damaged, their outline disappears in the flood of labeled albumin in the alveo-

lar and interstitial spaces (Fig 2, B-D). The capillary networks, where unlabeled erythrocytes circulated and were visualized as negative dots, stood out as dark and mesh-like conduits. The perfluorochemical eliminated labeled exudates in the alveoli, and the alveolar outlines reappeared (Fig 2, E). The surface tension in the injured ARDS lung is reported to be increased from 40 dynes/cm to approximately 70 dynes/cm.28 Perfluorocarbon liquid (surface tension, 18 dynes/cm) eliminates air-fluid interfaces in the lung and so reduces alveolar surface tension, contributing to alveolar recruitment. Thus, the ventilated alveoli both with and without PLV could be visualized.

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We elected to use the acid aspiration model because the aspiration of gastric acid is a factor predisposing to ARDS.2 Aspiration of hydrochloric acid causes significant injury to the alveolocapillary membrane and subsequent pulmonary edema.29 The pattern of lung injury appeared to be typically biphasic.30 The impairment within the first hour after injury is associated with the direct effects of the chemical on the alveolar and capillary membranes, whereas changes in the second phase (after 4 to 6 hours) including microcirculatory derangement can be attributed mainly to the inflammatory response. It was mainly the first phase of lung injury that was shown on the fluorescence micrographs. The first phase of injury was characterized by significant amounts of labeled albumin exudate within the alveoli as well as in the interstitial spaces. Decrease of ventilated alveoli and their overdistention reached their peak at 90 minutes after acid aspiration on the fluorescence micrographs. Many studies have suggested the usefulness of liquid ventilation with perfluorocarbon. The mechanism of action has been investigated theoretically, experimentally, and histologically. To date, no dynamic visualization of liquid ventilation has been performed. We demonstrated not only improved oxygenation but also excellent recruitment of alveoli in the damaged lung under mechanical gas ventilation. Blood gas oxygenation did not significantly improve, but the number of recruited alveoli increased markedly. PLV in the nondependent region in which the impairment is mild is not always a significant undertaking. Decreased oxygenation is reported in healthy lungs under PLV.9 Interstitial edema may contribute to a decline in the diffusion capacity of oxygen between the alveoli and capillaries. The results of our study indicate an advantage in PLV not only with respect to impaired gas exchange, ie, ventilation-perfusion mismatching, but also with respect to lung injury. A decrease was shown in the diameter of the largest alveoli under PLV. A previously reported morphometric analysis showed that the average diameter of damaged alveoli under gas ventilation decreased to 67.7 µm and that under PLV it increased to 82.4 µm.16 In contrast, the mean thickness of the alveolar wall decreased under PLV, compared to that under gas ventilation.31 Those data suggested tamponade effects of PLV.20,25 Our results were based on realtime measurements and showed the size of the ventilated alveoli in vivo, not the size of the regional flooded alveoli. The average diameter of all alveoli including the ventilated and flooded alveoli under the ALI treatment might be smaller than that under PLV.

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Recent lung protective strategies have focused on ventilator-associated lung injury. This type of injury is characterized by regional overdistention that occurs under a high tidal volume and is seen on fluorescent micrographs. From this standpoint, PLV should be an excellent lung protection strategy. However, clinical study associated with PLV has not shown an advantage. The appropriate doses of perfluorocarbons are still unclear. Dose-dependent improvement of gas exchange was demonstrated in experimental ARDS.32 Clinicians may instill too much liquid to improve oxygenation, leading instead to PLV-associated lung barotraumas.33 Recent study reported that PLV with small volumes of liquid increased survival time of lung injury models.34 Further investigation is needed to determine the appropriate amount of perfluorochemical or whether adequate gas exchange can be achieved with alternative ventilation strategies in combination with PLV.35 The appropriate timing is also unclear. As described above, the second phase of acute lung injury induced by acid aspiration is microcirculatory derangement due to the inflammatory response. PLV may not be significantly effective in the late phase,8,36,37 although perfluorochemical can provide alveolar recruitment and attenuate the inflammatory response of cytokines and neutrophils.21-25 Furthermore, PLV was shown to reduce pulmonary neutrophil activity without any effect on the lung water concentration in an oleic acid–induced injury model.38 This model simulated late injury effected by the inflammatory reaction. In the present study, which focused on the early phase, excellent recruitment was readily achieved after liquid ventilation, and labeled albumin exudates did not increase. These results support the notion that the effects on filtration are limited to the early phase of capillary injury.18 The physical properties of perfluorocarbon make tamponade of damaged alveoli possible.20,25 Beneficial effects of PLV on pulmonary fluid filtration have been observed in several injury models.18-20 PLV attenuates pulmonary-capillary permeability20 and induces resorption of exudates.19 Reduction of capillary blood flow velocity around the recruited alveoli has also been shown to be advantageous for oxygenation in terms of blood contact time.39 Pulmonary blood flow and lung water distribution are suggested to shift from dependent regions to nondependent regions, and this attenuation of increased lung water may contribute not only to improved ventilation-perfusion matching but also to amelioration of transalveolar exudation.13,14

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In conclusion, our study indicates that PLV with perfluorocarbon can provide for excellent alveolar recruitment immediately after application in hydrochloric acid–injured lungs. PLV can minimize overdistention of residual ventilated alveoli and may also ameliorate ventilator-associated lung injury. REFERENCES 1. Anderson WR, Thielen K. Correlative study of adult respiratory distress syndrome by light, scanning, and transmission electron microscopy. Ultrastruct Pathol 1992; 16:615-28. 2. Suchyta MR, Clemmer TP, Elliot CG, Orme JF, Weaver LK. The adult respiratory distress syndrome: a report of survival and modifying factors. Chest 1992;101:1074–9. 3. Dreyfuss D, Saumon G. Ventilator-induced lung injury: lessons from experimental studies. Am J Respir Crit Care Med 1998;157:294-323. 4. Amato MB, Barbas CS, Medeiros DM, Magaldi RB, Scettino GP, Lorenzi-Filho G, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory syndrome. N Engl J Med 1998;338:347-54. 5. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000;342: 1301-8. 6. Leach CL, Greenspan JS, Rubenstein D, Shaffer TH, Wolfson MR, Jackson C, et al. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome. N Engl J Med 1996;335:761-7. 7. Hirschl RB, Pranikoff T, Wise C, Overbeck MC, Gauger P, Schreiner RJ, et al. Initial experience with partial liquid ventilation in adult patients with the acute respiratory syndrome. JAMA 1996;275:383-9. 8. Croce MA, Fabian TC, Patton JH, Melton SM, Moore M, Trenthem LL. Partial liquid ventilation decreases the inflammatory responses in the alveolar environment of trauma patients. J Trauma 1998;45:273-85. 9. Fuhrman BP, Paczan PR, DeFrancisis M. Perfluorocarbonassociated gas exchange. Crit Care Med 1991;19:712-22. 10. Hirschl RB, Tooley R, Parent AC, Johnson K, Bartlet RH. Improvement of gas exchange, pulmonary function, and lung injury with partial liquid ventilation. Chest 1995;108:500-8. 11. Papo MC, Paczan PR, Fuhrman BP, Steinhorn DM, Hernan LJ, Leach CL, et al. Perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome. Crit Care Med 1996;24:466-74. 12. Hirschl RB, Tooley R, Parent A, Johnson K, Bartlett RH. Evaluation of gas exchange, pulmonary compliance, and lung injury during total and partial liquid ventilation in the acute respiratory distress syndrome. Crit Care Med 1996;24:1001-8. 13. Gauger PG, Overbeck MC, Koeppe RA, Shulkin BL, Hrycko JN, Weber ED, et al. Distribution of pulmonary blood flow and total lung water during partial liquid ventilation in acute lung injury. Surgery 1997;122:313-23. 14. Doctor A, Ibla JC, Grenier BM, Zurakowski D, Ferretti ML, Thompson JE, et al. Pulmonary blood flow distribution during partial liquid ventilation. J Appl Physiol 1998;84: 1540-50.

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