Liquid ventilation attenuates pulmonary oxidative damage

Liquid Ventilation Attenuates Pulmonary Oxidative Damage David

M. Steinhorn,

Michele

C. Papo, Alexandre Paresh

T. Rotta, Ahmed Dandona

Aljada,

Bradley

P. Fuhrman,

and

Purpose:Liquid per-fluorochemicals reduce the productioneaction oxygen species by alveolar macrophages. We sought to determine whether the use of liquid perfluorochemicals in vivo during liquid ventilation would attenuate oxidative damage to the lung. Materials and Methods: Healthy infant piglets fn = 16) were instrumented for mechanical ventilation and received intravenous oleic acid to create an acute lung injury. The animals were assigned to a nontreatment group receiving conventional mechanical ventilation or a treatment group receiving partial liquid ventilation with a liquid perfluorochemical. Following sacrifice, the bronchoalveolar lavage and lung parenchyma were analyzed for evidence of oxidative damage to lipids and proteins by determination of TBARS and carbonylated protein residues, respectively.

Results: Mortality in the control group was 50% at the &k&&on of the study compared with no deaths in the partial liquid ventilation group (P = .025). The alveolar-arterial oxygen difference was more favorable following injury in the partial liquid ventilation group. The liquid ventilation group demonstrated a 32% reduction in TBARS (P = .043) and a 14% reduction in carbonylated protein residues (P = .0611. Conc/usion:These data suggest that partial liquid ventilation supports gas exchange and reduces mortality in association with a reduction in the production of reactive oxygen species and the concomitant attenuation of tissue damage during the early phase of acute lung injury. Copyright 0 1999 by W.B. Saunders Company

R

ment in lung histology, gas exchange, and shortterm mortality seen following treatment of experimentally injured animals,’ we hypothesized that the diminished cellularity and permeability changes would be associated with a decrease in damage to the lung parenchyma as measured by biochemical parameters. The importance of this hypothesis lies in the central role of free-radical damage in the early phases of acute lung injuries, which may occur from either reperfusion injury or respiratory burst activity.r4 To study this question, we used a previously reported model of acute lung injury produced with intravenous oleic acid infusion in piglets.‘*i5 The outcome variables chosen for this study were measurement of biochemical markers of oxidative damage to the lung parenchyma.

ECENT INVESTIGATIONS of partial liquid ventilation (PLV) have demonstrated its effectiveness in a variety of experimental animal models of the adult respiratory distress syndrome (ARDS)“’ as well as in preliminary clinical studies in humans.6-8PLV represents a novel technique for supporting gas exchange in critically ill patients suffering from profound respiratory failure of many different origins. First reported in 1991, it is performed by filling the pulmonary airspace with an inert liquid perfluorochemical possessing high gascarrying capacity and then providing conventional mechanical ventilation through the liquid-filled airway.g Studies conducted in our laboratory over the last several years have suggested a wide range of effects of perflubron on cellular function in vitro.1o-‘3These studies have lacked the complexity seen during in vivo studies, thus limiting their applicability to clinical situations; however, it is evident from the in vitro data that alveolar macrophage response is attenuated following exposure to perflubron without a loss of viability.‘O In view of the apparent improve-

MATERIALS

AND METHODS

Experimental Preparation

From the Department of Pediatrics, Children’s Hospital of Buffalo; and the Department of Internal Medicine, MillardFilmore Hospital, State lJniversity of New York ut Buffalo, Buffalo, NY Received June 19, 1998. Accepted November 23, 1998. Address reprint requests to David M. Steinhorn, MD, Children S Memorial Hospital, 2300 Children’s Plaza, Box 73, Chicago, IL 60614. Copyright 0 1999 by WB. Saunders Company 0883-9441/99/1401-0004$10.00/0

This study was approved by the Institutional Animal Care and Utilization Committee of the State University of New York at Buffalo in conformity with NIH and USDA guidelines for the use of animals in medical research. Sixteen piglets (3.55 + .73 kg) were sequentially assigned to a nontreatment group consisting of oleic acid injury with conventional ventilation (n = 8) or a treatment group consisting of oleic acid injury supported subsequently with partial liquid ventilation (n = 8). Four healthy piglets were killed in a similar fashion without instrumentation to serve as pure controls for the subsequent biochemical analyses. The animals were anesthetized with o-chloralose (50 mg/kg, intravenously [iv]), paralyzed with metacurine (0.3 mgikg, iv), and the airway controlled with a tracheostomy placed under local anesthesia (1% lidocaine). They were supported with volume-controlled ventilation (VT = 1215 ml/kg), Fio, 1.0, positive end-expiratory pressure (PEEP) 4 cm H,O, and 20 breaths per minute (Servo 9OOC, Siemens

20

Journal

of Critical

Care, Vol 14, No 1 (March),

1999: pp 20-28

PLV ATTENUATES

OXIDATIVE

DAMAGE

21

Elena, Solno, Sweden). No attempt was made subsequently in either experimental group by adjustment of ventilator setting to correct the metabolic or gas exchange abnormalities that developed during the protocol. A 4-Fr thermodilution pulmonary artery catheter was placed via the right jugular vein to monitor intravascular pressures and to measure cardiac output. A femoral venous line was inserted for administration of medications, and a femoral arterial line was placed for obtaining arterial blood samples and pressure monitoring. Blood temperature was monitored continuously and maintained in a normothermic range with electric heating pads. All animals received Hespan (6% hetastarch in 0.9% NaCl) 10 to 20 mL/kg as needed to achieve a right atria1 pressure of 5 mm Hg before randomization. Hespan (6% hetastarch in 0.9% NaCl) was infused at a constant rate of 1.5 mL/kg/h throughout the study to maintain a central venous pressure >4 mm Hg during oleic acid injury. Maintenance fluid was provided with D5 0.2 NS at 4 ml/kg/h. Previous experience with this injury model indicated that supplemental volume expanders (Hespan) were required to compensate for the systemic capillary leak induced by this injury.2 Baseline measurements of intravascular pressures were recorded, and cardiac output was determined by triplicate 5-mL injections of iced saline. Baseline venous and arterial blood samples were analyzed for Pao,, PacoZ, and pH (ABL-3 Radiometer, Westlake, OH) as well as hemoglobin and oxygen saturation (OSM-3 Radiometer, Westlake, OH). Following instrumentation and before injury, 2 mL of blood with ethylenediaminetetraacetic acid (EDTA) was obtained and immediately spun down at 4°C. The plasma was removed, aliquoted, and immediately frozen at -70°C for subsequent biochemical analysis. The experimental injury consisted of an IV infusion of 0.15 mL/kg of oleic acid (Sigma Chemical, St. Louis, MO) over 30 minutes as we have previously reported.* The nontreatment group received conventional mechanical ventilation throughout the study period. The treatment group received room temperature perflubron (30 mL/kg, Liquivent; Alliance Pharmaceutical Corp., San Diego, CA) via the endotracheal tube. An additional 2 mL/kg/h of pefflubron was instilled to replace evaporative losses. Dopamine (10 pg/kg/min) was started in both groups at the same time oleic acid was infused and maintained at this dose for the duration of the experiment. Vital signs and measurements as performed at baseline were recorded every 15 minutes for 1 hour, then every 30 minutes for a total study duration of 3 hours. As noted earlier, the ventilator settings were not manipulated during the protocol in response to changes in measured variables during the protocol. Barometric pressure (Pbar) was noted on the day of the experiment. The alveolar-arterial oxygen difference (A-a do,) was calculated by conventional formula as an indicator of the intrapulmonary shunt [A-a do2 = (Fio? X (Pbar-PH,O) - (PacoJ0.8) - paoJ]. During partial liquid ventilation, the partial pressure of perflubron (PWRFLUBRON) was included in the calculation of A-a do2 [A-a doZ = (Fio, X (Pbar-PH,O - PPERFLUBR& - (PacoJ 0.8) - Pao,)]. In the event of death before completion of the study, the survival time was recorded in minutes. The animals were killed by potassium chloride injection while under general anesthesia.

was removed, aliquoted, and immediately frozen at -70°C for subsequent biochemical analysis. The lungs were excised en bloc, weighed, and separated into right lung for lavage of the airspace and left lung for histological and biochemical analysis of the parenchyma. There was no gross asymmetry in the appearance of the lungs, thus we believed that error was unlikely to be introduced in this process. The right lung was lavaged with a measured volume of normal saline (approximately 100 mL/ kg body weight in 25 mL/kg aliquots). The pooled lavage was spun at 600 g for 8 minutes, and the cell-free supernatant was frozen at -70°C for subsequent biochemical analysis. Following lavage, samples of the lung were immediately removed, snap frozen in liquid nitrogen, and stored at -70°C for subsequent analysis. Tissue samples were stored as multiple replicates from the same area of each lung to permit subsequent handling without repeated freeze-thawing of each sample before biochemical analysis.

Sample Collection

Statistical Analysis

Immediately following with EDTA was obtained

death of the animals, 2 mL of blood and spun down at 4°C. The plasma

Biochemical Analysis of Lung Tissue A portion of the previously frozen lung tissue representing approximately 20% to 30% of the entire lung was homogenized in double distilled H,O at 4°C using a Polytron tissue homogenizer, All samples were analyzed in batch fashion and the thiobarbituric acid reactive substances (TBARS) were determined in an aliquot of the crude homogenate. Oxidative damage to lipids was performed following the method of Yagi16 to measure the presence of TBARS using a malondialdehyde (MDA) standard. In brief, this assay is based on the reaction of thiobarbituric acid (TBA) with MDA or other lipid peroxides present in the sample during heating of the sample with thiobarbituric acid under acidic conditions. The adduct was extracted with butanol/ pyridine and the amount of TBARS was determined fluorometrically. The total protein content of the homogenate was determined by the method by Lowry et al” to permit normalization of the biochemical results. Oxidative damage to proteins was assessed by quantitative determination of carbonylated protein residues by the method of Levine et alI8 In this assay, tissue samples were homogenized in a lysate buffer and the protein precipitated by the addition of trichloroacetic acid. Derivitization of the proteins with 2,4dinitrophenylhydrazine was performed under acidic conditions. After agitation and incubation for 1 hour, the mixture was centrifuged and the resulting pellet washed repeatedly with ethanol-ethyl acetate to remove the free agent. The pellet was subsequently redissolved in 6 mol/L guanidine and the carbonyl content determined spectrophotometrically. The total protein content of the homogenate was determined by the method by Lowry et al” to permit normalization of the results.

Biochemical Analysis of Plasma and Lavage The samples of plasma and bronchoalveolar lavage were analyzed without homogenization for the presence of TBARS as noted earlier. The protein content of each sample was determined by Lowry et al’s methodi to permit normalization of the results. The presence of TBARS in the perflubron recovered from the airspace during the bronchoalveolar lavage was analyzed as noted earlier for the aqueous lavage fraction.

The data are expressed as mean ? SD. Comparisons between the PLV group and control group for TBARS and carbonylated

STEINHORN

22

protein residues in lung tissue, plasma lavage, plasma were analyzed by Mann-Whitney U analysis (Statview 4.1, SAS Institute Inc., San Francisco, CA) with significance taken at P < .05. Because the baseline value of plasma TBARS varied between animals, the postinjury values were indexed to the preinjury value; thus, the results for plasma samples are expressed as the ratio of post-to-pre TBARS levels. Two-way repeated measures analysis of variance was used to assess differences between and within groups over time. The Bonferroni correction was used to correct for multiple comparisons over time. The Huynh-Feldt epsilon was used to adjust the degree of freedom for the univariate F statistic P values. The data were analyzed only to 150 minutes because fewer than five animals were surviving at 165 and 180 minutes in the control group. Kaplan-Meier survival analysis was used to assess differences in survival between groups. Statistical significance was assigned at P < .05.

RESULTS

Of the 16 piglets entered into the study, 12 completed the protocol with 4 piglets dying in the control group apparently due to hypotension, hypoxia, or acidosis following the oleic acid injury (Fig 1). The log-rank statistic demonstrated improved survival in the PLV treated animals (P = .025). As demonstrated in Table 1, there was no difference in hemodynamics at baseline. Throughout the study, there was no pattern of difference in hemodynamics in the surviving animals despite the increased mortality seen in the control group. In general, the pulmonary gas exchange and hemodynamic profiles of the PLV-treated animals was better than the control group aspreviously reported.* Although the pulmonary mechanics were similar in the surviving animals at each time point throughout the study as shown in Table 2, the alveolar-

1.0 PLV

--

2a 0.82 2 w 0.6$

I

‘7

CONTROL

0.2I 0

I 30

60

150 TIME &N”TE$

180

Fig 1. Cumulative survival during PLV and during volumecontrolled mechanical ventilation. Kaplan-Meier survival analysis found the PLV group of animals to have improved survival over the control group Hog-rank statistic, P = ,025).

arterial oxygen difference and shunt fraction demonstrated less derangement in the partial liquid ventilation group compared with control animals (Fig 2). The biochemical analysis of the lung tissue revealed a 32% reduction of TBARS in the PLV group compared with the conventional ventilation group (P = .043; respectively, 0.18 t 0.05 v 0.27 -+ 0.07 nanomoles/mg protein). Furthermore, there was a strong statistical trend towards a 14% decrease in oxidative damage to proteins in the partial liquid ventilated group compared with controls (P = ,061; respectively, 2.0 2 0.9 v 2.3 5 0.9 nanomoles/mg protein). These data are depicted in Figure 3. The quantification of TBARS and carbonylated proteins in healthy, uninjured animals was 0.086 2 0.04 nanomoleslmg protein and 0.4 2 0.09 nanomoles/mg protein, respectively. Analysis of the Perflubron layer recovered with the bronchoalveolar lavage revealed no detectable TBARS (data not presented). The histological appearance of the lungs was markedly better in the partial liquid ventilation group (Fig 4). Examination of random fields under light microscopy revealed a generalized reduction in alveolar hemorrhage and hyaline material, reduced interstitial edema and inflammatory response, and an improved appearance of aerated, open alveoli compared with the gas-ventilated control group. The amount of TBARS in the plasma was not statistically different between groups. When analyzed as the ratio of postinjury to preinjury plasma, the TBARS levels demonstrated a mild trend towards a 21% decrease in TBARS (P = .24). Similarly, the amount of TBARS in the bronchoalveolar lavage fluid demonstrated a trend towards a 19% decrease in the treated animals (P = .ll). These findings are shown in Table 3. DISCUSSION

L-I--

35 0.4-

ET AL

Although the pathogenic mechanisms of acute lung injury are incompletely understood, the role of reactive oxygen species in setting the stage for acute lung injury, producing ongoing tissue injury and amplifying the immune system’s response to injury, is a recurring theme in organ failure research.‘4J9-21 Investigations of experimental, antioxidant treatments in attenuating tissue damage lend further credence to the important role of reactive oxygen species in contributing to acute injury.22-24 Many sources exist in cells for the generation of

PLV ATTENUATES

Table

OXIDATIVE

1. Hemodynamics

DAMAGE

During

Partial

23

Liquid

HR (beatsimin)

Time

Baseline PLV (n = 8)

272 t

16

Ventilation

MAP (mm Hg)

and Volume-Controlled CVP (mm Hg)

Ventilation SW32 (%)

(Control)

(mean

SW, (%)

2 SD) Hgb WdLl

6+2

19 + 2

98 i

1

Control (n = 8) 15 minutes

241 F 40

93?

13

4?1

25 ir 9

98 i

1

PLV (n = 8) Control (n = 8)

216 t 31t

76 ir IO* 75 + 12$

622 522

34 + 11 39 t Jt

98 2 1

206 2 31 237 + 35

73 -t 8$ 78~

Ilt

622 5?1

38 + 5 40 t 6t

97 t

218 + 32

PLV (n = 8)

239 i

46

80 i- J+

722

35 + 4

97 t

1

31 i 9t

6.0 t 0.8

Control

212 t 34

83 + 19

522

36?

78i

16

22 t 9t

6.0 I

1.3

243 t

6t2 5kl

35 + 5 39 + 4

94 2 2 75 i 20

34 2 13t

5.9 i

0.9

25 + 14t

5.8 k 1.0

30 minutes PLV (n = 8) Control (n = 8) 45 minutes (n = 8)

60 minutes PLV (n = 8) Control

(n = 8)

75 minutes PLV (n = 8)

103 t IO

Mechanical

MPAP (mm HG)

43

81 + 15

215 t 31

78 k 23

4

97 _f 1 1

80 i 23

60 2 10 60 t 7

5.6 t

34 t 9t 38 i IOt

5.9 t

5.9 i

0.7 1.3

6.0 + 0.8 0.9

30 i 9t

6.2 k 0.8

28 t

6.4 t

IOt

1.0

252 + 35

81 2 13

Control (n = 7) 90 minutes

216 i 36

81 k IO

7i2 5+2

36 i 9 41 ? 4

94 +- 2 742 18

42 f 242

16 Ilt

5.7 + 0.7 5.6 2 1.0

PLV (n = 8) Control (n = 7) 105 minutes

256 t 36 208 i 20

83 i- 15 79 F 17

7i-2 512

39 + 8

95 i 2 68 t 21

41 t 23 t

15

5.7 + 0.7

40 k 6

IOt

5.6 t 0.8

PLV (n = 8) Control (n = 6)

251 2 38 199 f 18

86 k 23 79 2 27

7i2 522

39 + 7 40 t 4

95 2 1

37 t

11"

5.3 i 0.8

63 2 22

25 i 21t

5.3 2 0.8

254 t 36 200 i 20

88 t 26 82 i 27

723 5i2

42 t 2 42 ? 3

95 i

37 _f II" 18 2 IOt

5.1 + 0.7t

254 i 35 203 f 20

88 _f 24 92 t 15

712 622

44 i- 3 41 i: 4

95 I

59 2 20

37 2 9* 18 i IOt

5.0 f 0.7* 4.8 -t 0.5

260 i- 27‘

87 t 26

44 i: 3 43 i 5

38 +- 9

4.7 + 0.5t

87 i 29

8 t 3$ 622

94 2 1

185 i 32

56 + 24

16 k 6

4.3 2 0.3

249 i 32

86?

26 11

44 t 4 45 i 3

37 i- 13

982

823 622

95 k 1

195 IL 23

61 2 6

19 i

5

4.5 2 0.7 4.0 t 1.0

255 t 27

90 t 15

824

41 + 5

92 + 4

34215

4.4 t 0.4

191 + 21

86 k 25

622

45 t 3

58 k 7

17 2 4

4.0 ir 0.4

120 minutes PLV (n = 8) Control (n = 5)

1

68 +- 20

4.8 t 0.7

135 minutes PLV (n = 8) Control (n = 5) 150 minutes PLV (n = 9) Control (n = 4) 165 minutes PLV (n = 8) Control (n = 4) 180 minutes PLV (n = 8) Control There

(n = 4) were

no differences

between

groups

at baseline.

Abbreviations: HR, heart rate; MAP, mean arterial Sao,, arterial oxygen saturation; Svo,, mixed venous *P <.05 versus control. ‘P 1.05

versus

baseline.

*P c.01

versus

baseline.

1

pressure; CVP, central venous pressure; oxygen saturation; Hgb, hemoglobin.

reactive oxygen species; however, the most significant sources during acute injury and disease are believed to be superoxide anion generated via xanthine-oxidase during reperfusion” and via activation of phagocytes. 26-28 Reactive oxygen species appear to overwhelm the local antioxidant defenses leading to tissue injury through their ability to directly alter protein, carbohydrate, lipid, and nucleic acid molecules as well as their ability to affect

MPAP,

pulmonary

artery

pressure;

enzyme activity and induce gene transcription. Thus, acute lung injury is frequently associated with an increase in the production of reactive oxygen species with the resultant damage to end organs by both oxygen-centered14 and nitrogen-centered freeradical species.29-30 Most studies investigating PLV in intact animal preparations represent short-term, acute investigations and have evaluated physiological parameters

STEINHORN

24

Table

2. Pulmonary

Time

Mechanics

During

Partial

Liquid (Control)

Ventilation and Volume-Controlled (mean k SD)

Peak Airway Pressure (cm H,O)

Peak End-lnspiratory Pressure (cm H,O)

24.7 -t 1.7 24.7 t 2.7

16.8 lr 3.7

Mean Airway

Mechanical

Pressure

ET AL

Ventilation

Minute Ventilation

km H,O)

(Urnin)

17.9 t 3.9

6.9 i 0.8 6.0 i 0.8

1.4 i 0.3 1.2 i 0.2

36.1 i- 4.lt

28.6 t 2.9t

10.4 + 4.0

32.0 + 4.4"

34.7 + 5.6*

43.6 '- 6.3t

27.1 + 3.2t

39.5 k 7.8t

27.0 t 7.7t

8.2 + 1.4t

Baseline PLV (n = 8) Control In = 8) 15 minutes PLV (n = 8) Control (n = 8) 30 minutes PLV (n = 8) Control In = 8) 45 minutes PLV (n = 8)

7.1 t

1.0"

11.5 ? 3.6

1.5 IO.3 1.2 2 0.2 1.4 k 0.3 1.2 ?r 0.2

42.5 2 5.9t

26.4 k 2.5t

41.1 ? 4.5t

25.6 t 6.3t

11.6 ir 3.4 8.6 i- 1.6t

1.4 2 0.3

In = 8)

60 minutes PLV (n = 8) Control (n = 8)

41.2 C 6.5t

24.4 i 4.lt

11.3 It 3.4

41.4 k 3.8t

27.0 i 6.0t

8.6 2 1.6t

1.4 i 0.3 1.2 i 0.2

40.1 i 5.7t 41.0 2 3.5t

25.6 + 3.2t

10.0 2 0.6t

1.4 i 0.3

39.8 + 5.8t 41.6 t 2.7t

Control

75 minutes PLV (n = 8) Control In = 7) 90 minutes PLV (n = 8) Control (n = 7) 105 minutes PLV (n = 8)

28.2 2 6.8t

8.6 i

1.2 i 0.3

1.6t

1.3 ? 0.3

25.3 t 3.0t

10.1 + 0.8t

1.4 i 0.3

28.2 t 6.8t

8.8 + 1.6t

1.3 i 0.3

39.4 t 3.7t

26.1 t 3.0t

10.1 2 0.8t

1.4 + 0.3

Control (n = 7) 120 minutes

42.8 t 2.4t

29.5 i 7.1 t

9.0 2 1.7t

1.2 i 0.3

PLV In = 8) Control (n = 6)

39.4 + 3.5t 42.9 i- 3.7t

26.3 i 2.6t 29.7 i 6.9*

9.9 + 0.9t 9.2 i 1.9*

1.4 t 0.3 1.3 i 0.3

135 minutes PLV (n = 8) Control (n = 5)

38.9 -t 3.7t 43.2 ? 2.9t

27.0 + 2.3t 29.6 k 6.8

9.8 2 l.Ot 9.2 i 2.1*

1.4 + 0.3 1.3 k 0.3

150 minutes PLV (n = 8) Control (n = 5)

39.2 + 3.6t 44.6 t 3.lt

28.1 2 2.5t 28.9 i 9.4

9.8 + l.Ot 9.4 f 1.8t

1.4 ir 0.3 1.2 i 0.3

39.9 i 4.0 47.8 2 4.5

23.6 + 3.6 23.9 + 6.5

9.8 2 1.1

1.4 k 0.3 1.4 IO.1

39.1 k 4.1

28.3 i 3.8

9.8 ? 1.1

45.5 ir 4.5

36.2 2 5.7

9.9 t 2.1

165 minutes PLV (n = 8) Control (n = 4) 180 minutes PLV (n = 8) Control

(n = 4)

No differences *P 5.05 versus +P 5.01 versus

existed between baseline. baseline.)

groups

9.8 -t 2.0

1.4 t 0.3 1.3 2 0.2

at baseline.

as the major outcome variables.‘-5x9x31 Investigations of tissue injury have been predominately limited to These reports demonhistological observation. 1~2s,31 strate diminished alveolar hemorrhage, less alveolar hyaline material, improved alveolar recruitment, and diminished interstitial cellularity.‘~31Although some reports have suggested a decrease in actual lung injury as implied by a decreased permeability to proteins, ‘,14they provide no further insight into possible mechanisms leading to the attenuated lung

injury during PLV. Papo et al2 have speculated that the simple presence of the dense (1.8 g/mL) perfluorochemical provides a mechanical stenting of the injured alveoli. This study clearly demonstrates the effect of PLV in the oleic acid injured animal with improved survival and better oxygenation. The data we present in the current study are the first documentation of a decrease in actual tissue damage during the early process of injury. Although the hemodynamic and

PLV ATTENUATES

OXIDATIVE

DAMAGE

25

700

600

I

I

I

I

I

I

I

I

0

30

60

90

120

150

180

OA injury

p g 6

80

-

70

-

60

-

Minutes

5040

-

20

10 I

OA Injury

that we might be able to alter the trajectory of acute lung injury through a timely intervention with PLV In a recent report on the effects of PLV on neutrophi1 accumulation in rabbits following the IV administration of endotoxin, we have demonstrated a significant decrease in neutrophil accumulation and myeloperoxidase activity during treatment with PLV.‘l This finding strengthens our speculation that the suppression of respiratory burst activity in phagocytes may be contributing to the improved histological appearance and function of the injured lung when perflubron is present within the airspace. In this study, we have analyzed biological samples obtained from an experimental model that is known to be associated with oxidative tissue damage15 in order to investigate a possible mechanism by which end organ injury might be decreased by treatment with partial liquid ventilation. The rationale for this approach is based on our current understanding of the role of neutrophils in the early phase of organ system injury28,32,33 and our recent demonstration of the attenuation of alveolar macrophage responsiveness following in vitro exposure to perflubron.‘0-13As hypothesized, filling of the airway with perflubron was associated with a decrease in oxidative damage to lipids and proteins. One possible explanation for this finding would be that the perflubron had absorbed the lipid peroxides and effectively leached them from the lung parenchyma. Perflubron is a biochemically inert material with relatively high lipophilicity. To investigate the possibility that lipid peroxides might be absorbed by the perflubron leading to the decreased TBARS

Minutes

Fig 2. Alveolar-arterial oxygen difference (A-a do,, A) and WQt (B) during PLV (solid circles) and in the control group (open circles). Values expressed are mean k SD. Data were analyzed with two-way analysis of variance with post-hoc Bonferroni correction for multiple comparisons. [*Significant difference from controls; mdifference from baseline.)

gas exchange data indicate that the PLV-treated animals also were clearly affected by the oleic acid injury, it is exciting to speculate that the improved survival and reduced oxidative damage alone with the decreased leukostasis demonstrated in other models3’ might translate into a genuine change in outcome. Although the model we examined is not an ideal model of human disease nor are the results of a relatively short-term study easily interpreted in terms of human disease, it is intriguing to think

350 300

P d 250 mh % E 200 FZ $ 150 5 100 50 0

control TBARS

PLV

Control

PLV

Carbonylated Proteins

Fig 3. Composite graph representing the thiobarbituric acid reactive substances (TBARS) and carbonylated protein residues recovered from lung homogenates of control (shaded bars) animals versus PLV-treated animals (open bars). There was a 32% reduction in TBARS f*P = ,043) and a 14% reduction in carbonylated protein residues If = .061).

STEINHORN

26

ET AL

Fig 4. Photomicrograph of oleic acid injured piglet lungs. (A) Representative histology from the control group reveals marked alveolar and septal hemorrhage, hyaline material within alveoli, edema, and lack of aerated alveoli. IB) Histological appearance of oleic acid injured piglet lung following treatment with PLV revealing well-aerated alveoli, minimal cellularity, and tissue damage compared with control.

seen in our study, we determined the TBARS content of the perflubron layer recovered during the bronchoalveolar lavage. This analysis revealed undetectable amounts of TBARS suggesting that absorption of lipid peroxides into the perfluorocarbon does not account for the decreased recovery of TBARS from the lung. In view of perflubron’s volatility, it is unlikely that residual perflubron within the reaction tube during the TBARS analysis played any role in these findings. A further explanation of our findings might be that the perflubron had served as a sink or scavenger for reactive oxygen

Table

3. TBARS

Lavage”

Uninjured CMV PLV

‘Micromoles

0.058

? 0.031

0.14 2 0.053 0.11 t 0.037 P=.ll per milligram

protein.

in Bronchoalveolar Parenchyma*

0.074

species. It is unlikely that significant amounts of highly polar molecules, such as oxygen-centered free radicals, could dissolve in the perflubron in view of its high hydrophobicity. Thus, the most likely explanation for our findings is that the production of superoxide anion was decreased in the treated animals. We have previously reported the decrease in superoxide anion and hydrogen peroxide production by alveolar macrophages exposed directly to perflubron.lO The mechanism responsible for this observation has not yet been elucidated. One possi-

Lavage,

Lung Plasmat

Parenchyma,

and Plasma

Pre

Plasmat

? 0.036

48 i- 63

0.270 + 0.07 0.18 i 0.047 P = .07

140 I! 70 140 I200 NS

Post

Post I Pre Ratio

280 2 100 240 ?I 120 NS

2.14 t .47 1.69 -t 52 P=

.24

PLV ATTENUATES

OXIDATIVE

27

DAMAGE

ble explanation proposed was the paralysis of macrophage function through the endocytosis of the perflubron, which could not be degraded leading to a state of so-called frustrated phagocytosis. A further mechanism proposed for this finding was alteration in membrane fluidity mediated through the intercalation of perfluorochemical molecules into the cell membranes. This proposed mechanism has been demonstrated in the case of volatile anesthetic agents. 34.35 We speculate that through an alteration in membrane fluidity, the assembly of the NADPH-oxidase complex in the cell membrane following activation of the cell might be adversely affected leading to diminished respiratory burst activity. Further investigations will be required to determine the specific cause of the decreased freeradical production as well as to determine whether recovery of respiratory burst function occurs following removal of the cells from the perfluorochemical compounds. The injury model used in this study is well established in pulmonary research and is known to elicit significant free-radical damage.15 A limitation of this model is that it creates significant systemic derangement of hemodynamics and endothelial function through its nonselective effects. We had hoped to demonstrate that PLV might attenuate the overall systemic effects by measurement of plasma TBARS before and after receiving oleic acid. Per-

flubron has a vapor pressure of - 11 mm Hg at 37°C and has good lipid solubility; thus, perflubron is known to distribute throughout the body through equilibration of the alveolar perflubron with the pulmonary circulation. Although the findings of the postinjury to preinjury TBARS ratio did not achieve significance, the data hint at the possibility of a decrease in systemic oxidative damage although it is more likely that they represent a decrease in pulmonary oxidative damage. Many further questions are raised by this preliminary observation including the optimal timing of treatment as well as what the best experimental model is to study the effects of PLV on systemic free-radical production and associated tissue injury. In conclusion, PLV with perflubron appears to be a novel and previously unreported mechanism for reducing free-radical damage to lipids and proteins in tissues with which it is in direct contact. Insufficient data exist at the present time to indicate a systemic effect; however, models of injury other than IV oleic acid may be more appropriate to answer the further questions raised by these findings. ACKNOWLEDGMENTS The authors thank Beverly Bun&-Kahn and Mark Dowhy for their skilled assistance with the animal protocol and Dr. Kuldip Thusu for his assistance with the biochemical analyses.

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