compliance were performed during ventilatory and hemodynamic

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D. Pappert N. Gilliard G. Heldt T.A. Merritt P.D. Wagner R.G. Spragg

Effect of N.nitroso.N.methylurethane on gas exchange, lung compliance, and surfactant function of rabbits

Received: 12 October 1994 Accepted: 9 May 1995

This study was supported by grant -Pa 377/1-I 1 from the Deutsche

Forschungsgemeinschaft, Bonn, Germany, and grant HL23584 from the National In- stitutes of Health (SCOR Project 1) D. Pappert (~)

Klinik far Anaesthesiologie und operative Intensivmedizin, WE01 Universitatsklinikum

Rudolf Virchow, Freie Universit~it Berlin, D-13344 Berlin, Germany

N. Gilliard

Service d'Anesthesiologique,

Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland G. Heldt

Division of Neonatology, Department of Pediatrics, University of California, San Diego, California, USA T.A. Merritt

Division of Neonatology, Department of Pediatrics, University of California, Davis, California, USA R D. Wagner 9 R.G. Spragg Division of Pulmonary and Critical Care Medicine, Department of Medicine,

VA Medical Center and University of California,

Sand Diego, California, USA

Abstract Objectives: To define the effect of N-nitroso-N-methylurethane (NNNMU) on pulmonary gas exchange, compliance and the biochemical and functional properties of the lung surfactant system.

Design: Four days after inducing lung injury, gas exchange and pul- m o n a r y compliance were studied and a bronchoalveolar lavage was taken.

Setting." Experimental laboratory o f a university department of medicine, division of pulmonary and critical care medicine.

Animals: Ten rabbits after they had received an injection of N N N M U and five control animals.

Interventions: Controlled mechanical ventilation and bronchoalveolar lavage.

Measurements and results: Measure- ments of gas exchange (using the multiple inert gas elimination technique), hemodynamics and pulmonary compliance were performed during ventilatory and hemodynamic steady state. A bronchoalveolar lavage (BAL) was taken after sacrificing the animal. BAL samples were processed for cell count and biochemical and functional surfactant analysis. Animals injected with N N N M U developed mild, but significant reduction in PaO z, while maintaining eucapnia

during spontaneous air breathing.

fz/Q distributions and arterial blood gases were similar in all animals when ventilated mechanically with a fixed tidal volume. Compli- ance of the lung and phospholipid levels in lavage of N N N M U animals was significantly lower than in control animals (CON). Function o f surfactant recovered from animals receiving N N N M U was decreased significantly where compared to CON. Thus, N N N M U resulted in a lowered lavage surfactant phospholipid content, impaired surfactant function, decreased compliance and hypoxemia during spontaneous ventilation. However, gas exchange was similar to that of control animals during mechanical ventilation.

Conclusion: We conclude that NNNMU-induced gas exchange abnormalities present after 4 days are mild and are reversed by fixed volume mechanical ventilation despite marked alteration in surfactant function and lung compliance.

These observations further define properties of a lung injury model that is of value in the study of surfactant replacement.

Key words Pulmonary surfactants 9 Respiratory distress syndrome, adult 9 N-nitroso-N-methylurethane

9 Pulmonary c o m p l i a n c e . Rabbit

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Introduction

T h e a d u l t r e s p i r a t o r y distress s y n d r o m ( A R D S ) is a char- acteristic r e s p o n s e o f t h e l u n g to several t y p e s o f injuries.

A b n o r m a l i t i e s seen in t h e f u l l y d e v e l o p e d s y n d r o m e in- c l u d e severe i m p a i r m e n t o f gas e x c h a n g e p r e d o m i n a t e l y b e c a u s e o f s h u n t , n o n c a r d i o g e n i c a l v e o l a r a n d i n t e r s t i t i a l e d e m a , r e d u c e d c o m p l i a n c e o f the lung, a n d i n f l u x o f b l o o d a n d n e u t r o p h i l s [1]. T h e p a t h o g e n e t i c m e c h a n i s m s l e a d i n g to severe A R D S are n o t f u l l y u n d e r s t o o d . I m - p a i r e d f u n c t i o n o f t h e s u r f a c t a n t s y s t e m o f t h e l u n g m a y o c c u r e a r l y in t h e d e v e l o p m e n t o f A R D S , as a result o f b o t h b i o c h e m i c a l a l t e r a t i o n o f l u n g s u r f a c t a n t [ 2 - 4 ] a n d i n h i b i t i o n o f s u r f a c t a n t f u n c t i o n b y p l a s m a p r o t e i n s [5]

t h a t have g a i n e d access t o t h e a l v e o l a r space. Loss o f sur- f a c t a n t f u n c t i o n m a y l e a d to a d e c r e a s e in l u n g c o m p l i - a n c e a n d i n c r e a s e d a l v e o l a r collapse, s h u n t a n d h y p o x - e m i a .

T h e t i m e c o u r s e o f A R D S is n o t p r e d i c t i b l e a n d varies f r o m a c u t e to p r o t r a c t e d [6, 7]. M o s t e x p e r i m e n t a l m o d e l s o f A R D S result in t h e a c u t e o n s e t o f p u l m o n a r y d y s f u n c - t i o n w i t h i n a few h o u r s , r a t h e r t h a n in t h e slow d e v e l o p - m e n t o f d y s f u n c t i o n over t h e c o u r s e o f several days. P r e - v i o u s w o r k b y B a r r e t t et al. [8] has d e m o n s t r a t e d , however, t h a t t h e s u b c u t a n e o u s a d m i n i s t r a t i o n o f N - n i t r o s o - N - m e t h y l u r e t h a n e ( N N N M U ) to d o g s results in a p u l m o - n a r y i n j u r y t h a t r e s e m b l e s h u m a n A R D S b o t h h i s t o l o g i - c a l l y a n d in t e m p o o f o n s e t . E x p e r i m e n t s b y Lewis et al.

p r o v i d e d evidence t h a t N N N M U causes a s i m i l a r i n j u r y to r a b b i t s [9]. T h e p r e s e n t s t u d y was u n d e r t a k e n to d e f i n e f u r t h e r t h e a b n o r m a l i t i e s o f gas e x c h a n g e a n d a l t e r a t i o n s in l u n g c o m p l i a n c e a n d s u r f a c t a n t c o m p o s i t i o n a n d func- t i o n in r a b b i t s receiving N N N M U . T h i s i n f o r m a t i o n is critical for t h e d e s i g n o f f u r t h e r studies e v a l u a t i n g a p - p r o a c h e s to the t h e r a p e u t i c use o f e x o g e n o u s s u r f a c t a n t .

Materials and methods

NNNMU preparation

Just prior to use, N-nitroso-N-methylurethane (Pfaltz & Bauer Inc., Canterbury, Conn.) was suspended in 0.15 M NaC1 at 12 mg/ml by vigorously mixing the components in a glass tube for 5 rain.

Animal preparation

New Zealand white rabbits (2.0-2.5 kg; n = 15), were randomly as- signed either to the control (CON; n = 5) or to the study group (NNNMU; n = 10). Weight and rectal temperature were recorded for 4 days prior to the study, and an arterial blood sample for a daily blood gas analysis was drawn from the central ear artery after re- straining the animals in a squeeze cage. On day 0 the rabbits received either 12 mg NNNMU/kg (NNNMU group), or the same volume of saline (CON group), subcutaneously in two divided dos- es to the left and right dorsal pelvic area. Thereafter, the weight, temperature, ABG and hematocrit were monitored daily. If the rab-

bits showed evidence of hypovolemia (weight loss _>5070 and hematocrit _> 40~ then 25 ml/kg of NaC1 0.15 M containing 5~

glucose, was given subcutaneously for fluid replacement.

On day 4, all rabbits were weighed, and blood gas analysis was performed on an arterial blood sample obtained while the animal was spontaneously breathing room air. Each rabbit then received ketamine (35 mg/kg) and xylazine (5 mg/kg) for the induction of anesthesia, each was placed in a supine position at an angle of approximately 22 ~ head up, and a tracheostomy was performed. An- esthesia was achieved with droperidol-fentanyl (Innovar-Vet 0.2 ml/kg i.m.) and diazepam (1 mg/kg i.m.) and maintained with repeated injections of half this dose every 30 rain. Ventilation was maintained with a volume-cycled ventilator (Model 6665, Harvard Apparatus, South Natick, Mass.) at a rate of 25/rain, a V T of 25 ml/kg, FIO 20.4 and an I : E ratio of i : 2 using a tight fitting uncuffed endotracheal tube (3.0 mm i.d.). The expiratory loop was connected to a specially designed heated sampling box for mixed expired air. During mechanical ventilation the animal received a positive end-expiratory pressure of I cm H20 measured with a U-manometer attached to the endotracheal tube.

An arterial catheter (G 21) was inserted into the left carotid artery, and venous catheters (G 21) were inserted into a marginal ear vein and a femoral vein. A 4 F single-lumen pulmonary artery catheter (no. 93-116F, Edwards Laboratories, Irvine, Calif.) was intro- duced via the right external jugular vein under fluoroscopic control and continuously flushed with NaC1 0.15 M (3 ml/h) and heparin (2 IU/ml).

Experimental protocol

Arterial blood pressure and pulmonary artery pressure were recorded continuously (model 77543, Hewlett-Packard, Palo Alto, Calif.).

Temperature was maintained within+0.5 ~ of the prestudy range using a heating pad; a continuous infusion of NaC10A5M (10 ml kg -1 h -1) was given. Blood losses due to sampling were re- placed with the same volume of unmatched banked rabbit blood, preserved in 14~ CPD (citrate phosphate dextrose) and stored no longer than 14 days.

Ventilation was monitored intermittently by attaching a mass- spectrometer (Perkin-Elmer 1100, Perkin Elmer Corp., Norwalk Conn.) to the endotracheal tube. Respiratory rate was adjusted to achieve an end-tidal expiratory CO 2 of 3.8070; inspiratory oxygen concentration was 40070. Blood gases were checked repeatedly using a Coming Blood Gas Analyzer i78 Ph/ABG (Corning, Corning N.Y.). A baseline was established over a 10 rain period during which heart rate, arterial pressure and pulmonary artery pressure changed by an average of less than 6.3070. After this baseline, hemodynamics, gas exchange and inert gas elimination were recorded in duplicate and repeated again after 40 min to study the effect of ventilation on gas exchange prior to surfactant replacement in future studies. Ven- tilator settings were not changed during this period to ensure consistent conditions for the repeated measurements of ventilation-perfusion distribution.

VA/ Q Distribution

Continuous ventilation-perfusion distributions (12A/Q) were determined using the multiple inert gas elimination technique (MIGET) described by Wagner et al. [10, 11]. On the first day of the study, an infusion bag with NaC10.15 M was equilibrated with trace amounts of six inert gases (sulfur hexafluoride (SF6), ethane, cyclopropane, enflurane, ether and acetone). This solution was in- fused at a constant rate of 0.2 ml/min via a catheter in the femoral vein. After at least 15 min of infusion, arterial, mixed venous and

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mixed expired samples were obtained and immediately assayed.

During the infusion period, end tidal CO2, heart rate, and mean arterial blood pressure varied by less than+ 5%. Inert gas concen- trations in blood and gas samples were measured using a gas chromatograph (Hewlett-Packard 5890 Series II), equipped with a flame ionization detector and an electron capture detector.

Compliance

Compliance was measured in live rabbits after the blood sampling for the second MIGET by connecting the tracheal tube to a U-manometer. First the total compliance of chest and lung (CRs) was determined with the chest closed, then after a median sternotomy and exposure of the lungs, pulmonary compliance (CL) was measured in situ. Compliance was measured in both cases by inflating the lungs with air to a pressure of 30 cm H20 for 10 s. After passive deflation, they were reinflated in increments of 5 cm H20 up to 25 cm H20 and then slowly deflated in the same manner. The compliance data were corrected for the intrinsic compliance of the manometer used.

Bronchoalveolar lavage (BAL)

After sacrificing the rabbit with 100 mg/kg Nembutal i.v., the lungs were lavaged via the endotracheal tube with nine 50-ml aliquots of 0.15 M CaC1 at 4~ Each aliquot was instilled tracheatly three times. BAL was kept at 4 ~ until further processing.

Differential centrifugation of BAL was performed in three steps using a modification of the protocol described by Ikegami et al.

[121. After the BAL was filtered through gauze, the filtrate was centrifuged (250 g, 4 ~ 15 min) to generate a pellet (P0 of cells. The supermatant (S~) was centrifuged (10000 g, 15 rain, 4 ~ and the resulting surfactant-rich pellet (Pz) was saved. The supernatant ($2) was then spun (40000 g, 15 min, 4 ~ to produce a final pellet (P3) and supernatant ($3). A differential cell count was performed on the filtered BAL. The DNA content of the native BAL was measured [13] to provide a reflection of the absolute number of nucleat- ed cells in the BAL.

Surfactant analysis

Lipids in the pellets P2 and P3 and in the supernatant S 3 were extracted following the procedure described by Bligh and Dyer [14], and phospholipid concentration of each extract was determined by measuring the total phosphorus content [15]. From this the phospholipid content was calculated using the formula: gg phosphorus• = mg phospholipid [16]. The pellets P2 and P3 were analyzed for protein content (Pierce Chemical, Rackford, Ill.).

Two-dimensional thin-layer chromatography was used for as- sessment of the phospholipid composition of the surfactant in the fractions P2, P3 and S 3, Precoated silica gel 60 TLC plates (Merck, Darmstadt, Germany), were sprayed with 0.4 M boric acid and run in a solvent system described by Poorthuis et al. [17]. For identifica- tion of the different phospholipids, reference phospholipids were run in the same system. The phospholipids were visualized by ex- posing the TLC-plates to iodine vapor. The silica gel of each spot and the incorporated lipids were scraped, and the phosphorus content of each spot was determined as described.

In vitro surface activity was measured in the surfactant from combined pellets Pz and P3 that were resuspended (4 mg phospholipid/ml 0.15 M NaC1) by vigorous mixing. Homogeneity was ac- complished by sonicating the suspension for 10 rain at 4~ and

50 W (model MS-50, Ultrasonics Heat Systems, Farmingdale, N.Y.).

The samples were tested at 37 ~ and 20 cycles/rain, using the oscil- lating bubble technique [18] (Pulsating Bubble Surfactometer, Elec- tronetics, Amherst, N.Y.).

Statistics

All data are presented discretely or as means_+SD. Testing for the significance of differences between the CON group and the NNNMU group was performed using either a paired or unpaired t- test. For data not normally distributed, the Mann-Whitney rank sum test was used. P values are given as the calculated value and differences with a P_0.05 considered to be significant.

Results

T e m p e r a t u r e a n d weight o f a n i m a l s i n b o t h the N N N M U g r o u p a n d the C O N g r o u p did n o t c h a n g e s i g n i f i c a n t l y over the course o f 4 days. T h e r e were n o s i g n i f i c a n t differences i n b l o o d gases b e t w e e n the two groups o n day 0.

Over the same t i m e interval, P a O z o f the N N N M U g r o u p d u r i n g s p o n t a n e o u s v e n t i l a t i o n decreased s i g n i f i c a n t l y ( P = 0.016) f r o m 9 9 . 9 + 2 . 8 m m H g to 8 7 . 9 + 2 . 3 m m H g , resulting i n a s i g n i f i c a n t ( P = 0.016) c h a n g e o f the alveolar-arterial P a O 2 g r a d i e n t (AaDOz) f r o m 4 . 0 + 4 . 0 to 1 6 . 8 + 9 . 1 m m H g . T h e P a C O 2 a n d p H r e m a i n e d u n - c h a n g e d (Table 1). Values o f PaO2, PaCO2 a n d p H i n the c o n t r o l g r o u p r e m a i n e d stable, a n d A a D O 2 did n o t c h a n g e s i g n i f i c a n t l y b e t w e e n day 0 a n d day 4 (11.3 +6.3 vs 11.5 + 12.0 m m H g ) . A n i m a l s i n b o t h g r o u p s were ventilated with a F I O z o f 0.4. There was n o s i g n i f i c a n t difference i n PaO2 b e t w e e n the two groups after establishing m e c h a n i c a l v e n t i l a t i o n . T h e N N N M U g r o u p ' s P a O 2 was 163.9_+ 61.0 a n d the C O N g r o u p ' s P a O 2 was 150.9 + 35.2.

B l o o d gas values i n b o t h groups were n o t s i g n i f i c a n t l y c h a n g e d after 40 r a i n o f m e c h a n i c a l v e n t i l a t i o n .

A f t e r establishing m e c h a n i c a l v e n t i l a t i o n , gas ex- c h a n g e a n d c a r d i o v a s c u l a r f u n c t i o n were assessed u s i n g the m u l t i p l e i n e r t gas e l i m i n a t i o n technique. T h e cardiac

Table 1 Arterial blood gas values

NNNMU CON

Day 0 pH 7.38 +_ 0.05 7.42 _+ 0.03

PaO 2 99.9 +8.5 89.3 _+2.8

P a C Q 39.8 _+ 3.6 41.5 • 6.5

Day 4 pH 7.36 _+ 0.12 7.40-+ 0.01

PaO 2 87.9 -+2.3* 90.0 _+2.8

PaCO 2 37.6 _+7.7 40.5 _+2.9

Mechanical pH 7.57 _+ 0.15 7.49 _+ 0.12

ventilation PaO 2 163.9 _+61.0 150.9 _+35.2

PaCO 2 31.9 +5.2 33.6 -+3.4

NNNMU (n = 10) and control subjects (n = 5) during spontaneous and mechanical ventilation (FIO z 0.4)

PaO 2 and PaCO 2 are expressed in mmHg; values are mean_+ SD;

* P = 0.02 compared to day 0 value

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output (0.10+ 0.05 1/kg/min) measured by the Fick prin- ciple, using data derived from MIGET, showed no significant differences within the groups after 40 rain. Thus, the data obtained with multiple inert gas elimination technique reflect steady-state conditions.

lkA/Q distribution analysis showed a shunt fraction

(I?A/Q

= 0) o f 0.03_+0.01 and areas with a low

lkA/Q

ratio (0 __ Q ___ o. 1) o f 0.01 + 0.01 for both groups. There were no significant differences between groups, consistent with the results o f arterial blood gas analysis. Areas o f increased

12A/Q

ratios ( 1 0 - - V A / Q ) were also not significantly different between groups (Table 2).

The static compliance of the lungs o f animals receiving N N N M U was significantly less than that of the lungs o f control animals (Fig. 1). Compliance o f the respiratory system was 1.65_+0.62 vs 2.99_+0.49 m l . c m H z O - l . k g -1 (P = 0.02); compliance o f the lung, measured after thora- ctomy was 2.49 + 1.15 vs 4.73 _+ 0.66 ml. cm H 2 0 - 1. k g - 1 (P = 0.02). Calculated compliance o f the chest wall was 5.37_+ 1.37 vs 8.52_+ 2.76 ml. cm HzO - 1. k g - 1 for N N N M U and C O N animals, respectively, and was not significantly different between groups. The a m o u n t of saline recovered from the lung after BAL was also not significantly different between groups ( N N N M U 404_+35.0ml vs C O N 396+_24 ml).

D N A content in total BAL from NNNMU-treated rabbits was significantly increased compared to healthy rabbits (148.9+107.7 gg vs 25.0+7.8 gg, respectively, P = 0.04). Consistent with increased cellularity o f the lavage fluid, the percent cells present as neutrophil granulocytes was also increased in the N N N M U group relative to the C O N group (41.1_+28.1~ vs 7.4_+ 12.8070;

P = 0.02) (Table 3).

The BAL of the N N N M U group had a significantly higher protein content in both fractions P2 and P3 (58.9+ 17.4 mg vs 36.7+_ 12.3 mg, P = 0.02) than the BAL o f the C O N group; the phospholipid/protein ratio changed from 0.31_+0.10 in control animals to 0.08 + 0.012 in rabbits reeeiving N N N M U (P = 0.003) (Ta- ble 3).

Phospholipid contents o f pooled pellets P2 and P3 from N N N M U animals were significantly reduced relative to C O N animals. Because P z + P 3 contains the larger, functionally more active surfactant aggregates [9], further analyses were done on the pooled pellets. Pellets from P z + P 3 from the N N N M U group contained 4.4+1.2 mg phospholipids vs 10.7_+2.6mg from the C O N group (P = 0.003). We estimated the ratio o f surfactant phospholipid in small and large aggregates by calculating the ratio of phospholipid in $3 to that in P2 +P3- This ratio was 5.95+3.77 and 2,17+0.76 in the N N N M U and C O N groups, respectively (P = 0.007). As there was no difference between the S 3 phospholipid content (25.6+12.2 and 22.0_+0.76 in the N N N M U and C O N groups, respectively, this difference is due entirely to differences in the phospholipid content of P2 +P3.

Table 2 Ventilation/perfusion distribution and gas exchange during mechanical ventilation

NNNMU CON

P.aO2/FIO 2 373.2 + 127.8 449.7 _+63.0

V/Q

= 0 0.03 _+ 0.04 0.03 + 0.01

V/Q<_O.1

0.06_+0.10 0.03_+0.14

~/Q >_

10 0.26 -+ 0.06 O. 33 _+ 0;08

V/Q

= oo 0.26+0.06 0.33-+0.08

BF mean 1.50 -+ 0.41 1.48 _+ 0.21

SD Q 1.00-+0.56 0.93_+0.17

VENT mean 3.39 _+ 1.04 3.40 _+ 0.65

SD 12 0.82_+0.16 0.86_+0.16

RSS 4.18 _+ 2.23 2.76 _+ 1.51

Data obtained by multiple inert gas technique in NNNMU animals (n = 10) and CON animals (n = 5). Values are mean_+ SD; no significant differences exist between any of the four sets of observations

Table 3 Differential cell count, total DNA content, total phospholipid content and total protein of the pellets PE and P3

NNNMU CON

Granulocytes (%) 41.1 +28.1' 7.4 _+ 12.8

Macrophages (~ 58.3 _+28.2* 92.6 _+ 12.8

Lymphocytes (~ 0.6 _+ 1.7 0.0 _+0.0

DNA (~tg/BAL) 148.9 _+107.7" 25.0 _+7.8 Phospholipid

(mg/BAL pellets Pz and P3) 4.4 + 1.2"* 10.7 _+ 1.8 Protein

(mg/BAL pellets P2 and P~) 58.9 _+ 17.4" 36.7 _+ 12.3 Phospholipid/protein 0.08 _+0.01 ** 0.31 _+ 0.1

BAL recovery (ml) 408 _+ 36 396 -+ 27

NNNMU animals (n = 10) and CON animals (n = 5); values are mean+SD; * P_<0.05; ** P_<0.01

45 0) 40 E

= 35 g 30

~ 25 2o 15 10 5

I t I I I

0 5 10 15 2o 25

Pressure (cm H20)

Fig. 1 Representative semistatic pressure-volume curve from an animal receiving NNNMU

(dashed line)

and a control animal

(solid

line)

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The surfactant phospholipid fractional composition o f the pellet P2 and pellet P3 from N N N M U animals was similar; likewise, the phospholipid fractional composition from P2 and P3 of CON animals was similar. Therefore, we calculated the phospholipid composition of the pooled pellets P2+P3 (Table 4). Surfactant o f the N N N M U group revealed a significantly reduced fractional content of phosphatidylglycerol (PG) and lysophosphophatidyl- choline (LPC) when compared to the CON group (4.26-+2.36~ vs 7.72-+1.60070 for P G and 2.80-+0.790/0 vs 4.70+ 1.83 for LPC). Although a lower content of phosphatidylcholine (PC) was demonstrable in the N N N M U group (71.28-+ 9.3907o compared to 74.47 + 3.9207o for the CON group), this difference was not significant. Phos- phatidylserine ( P S ) a n d phosphatidylethanolamine (PE) increased significantly (4.51+1.69070-2.21+0.70070 for PS and 2.91 _+ 1.83-7.38+4.08070) in the N N N M U group.

Surfactant function of the resuspended surfactant from pellet P2 was measured at 4 m g / m l phospholipid content in a pulsating bubble surfactometer. There was a significant increase in minimal surface tension in samples

Table4 Surfactant phospholipid composition in the bronchoalveolar lavage fluid

N N N M U CON

Phosphatidylcholine 71.28 + 9.39 74.47 _+ 3.92 Phosphatidylglycerol 4.26 + 2.36" 7.72 + 1.60 Phosphatidylserine 4.51 + 1.69 * 2.21 + 0.70 Phosphatidylethanolamine 7.38 _+ 4.08 * 2.91 + 1.83 Phosphatidylinositol 4.45 +_ 2.54 5.31 _+ 1.57 Sphingomyelin 5.32 _+ 4.98 2.69 _+ 1.17 Lysophosphatidylcholine 2.80 _+ 0.79 * 4.70 +_ 1.83

~ Total phospholipids in the p o o l e d pellets P2 and P3 (n = 5 in each group). Values are mean__+SD; * P<_O.05

f - , E

~" 25

20

9 o 15 't:

o~ lO

1

N N N M U

i i i i i ~ ' - ' - q l . . . . i

2 3 4 5 6 7 8 9 10 11

Time [min]

Fig. 2 Minimal surface tension of chloroform extracted pellet P2 measured with a pulsating bubble surfactometer in NNNMU-inj ect- ed animals (filled diamond) and control animals (empty circles)

from the N N N M U group expressed in m N / m after 10 min of pulsation as compared to the CON group, (12.70 + 5.41 vs 0.34+0.37, respectively (P = 0.003), indicating a significant reduction of surface tension lowering ability in the BAL of rabbits receiving N N N M U (Fig. 2).

The histology o f the lung in the injured rabbits taken before bronchoalveolar lavage showed the development of patchy atelectatic areas with an infiltration of neutrophils. Although we did not undertake a quantita- tive study, examination of representative sections dis- closed edema present in the perialveolar space and the development of hyaline membranes in the alveolar space (Fig. 3). To exclude failure of other organs as a cause of the distress syndrome, we examined sections of heart, liv- er and kidney as well, but found no evidence of abnormality.

Discussion

The pathophysiologic and histopathologic changes in laboratory animals secondary to subcutaneous injection of N N N M U closely resemble those seen in the early stages of ARDS, except for the unaltered gas exchange capabili- ties of the lung. We found that rabbits receiving N N N M U developed only a very mild impairment of respiratory function by the 4th day after injection, as demonstrated by a small, but significant decrease in PaO2 and AaDO>

Pathologic findings paralleling those in ARDS were evi- dent. Lung compliance in the N N N M U group was significantly decreased (Fig. 1). Analysis of BAL revealed a decrease in total phospholipid content, a change in phospholipid composition and reduced biophysical properties of the surfactant. The histology findings were similar to those in the lungs of patients with ARDS, revealing profound alveolar damage [1, 19-22].

The gas exchange abnormality that we observed during spontaneous ventilation was limited to a mild, but significant decrease in PaO2 in animals receiving NNNMU. As the N N N M U group had a mean PaO 2 at baseline that was inexplicably higher than the mean PaO2 at baseline of the CON group, the true significance o f this observation is unknown. The deterioration in gas exchange in these animals was apparent during spontaneous ventilation and normalized, as determined by blood gas measurement and

12A/Q

distribution when the animals were mechanically ventilated in a volume control mode. In contrast, Lewis et al. [9] found a significant decrease in PaO2/FIO2 of ventilated rabbits receiving 12 m g / k g NNNMU. The mild reduction of PaO2/FIO 2 from 343_+ 13.2 in the control group versus 288_+4.6 two days after receiving N N N M U also contrasts the severity o f gas exchange deterioration, as seen in h u m a n ARDS.

Measurements in that study were made during pressure- limited ventilation where peak inspiratory pressure was

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adjusted to control P a C O 2, Given the decrease in lung compliance caused by NNNMU treatment, it is likely that the difference in ventilation strategies was responsible for the contrasting observations. For this reason we attempt- ed to demonstrate abnormal

12A/Q

by MIGET in spontaneously breathing rabbits after receiving NNNMU, but found that the study of spontaneously breathing animals was very difficult and that we were unable to achieve reliable results.

Compliance of the respiratory system was significantly reduced in animals receiving NNNMU (Fig. 1), and in agreement with other studies, we found NNNMU to have an insignificant effect on chest wall compliance [8, 23].

This change is likely due to loss of pulmonary com- plicance. Values for both control and treated animals were approximately threefold those reported by Lewis et al. [9], consistent with the use of volume-controlled ventilation and effective recruitment of gas-exchange units. Al- though direct measurement of pulmonary compliance did not include absolute measurement of lung volume, results were consistent with a significant fall in pulmonary compliance. Compliance of the lung may be decreased as a consequence of interstitial edema, but may also be reduced as a result of loss of surface tension lowering the properties of surfactant. The influence of quantified pulmonary edema, fibrosis and surfactant dysfunction on pulmonary compliance cannot be separated from each other in this type of injury. All three were seen in this study. The consequent atelectasis that develops results in a further reduction of gas exchange. Increased shunting of intrapulmonary blood flow, as described by Lewis et al. [9], is likely to be largely responsible for the decrease of PaO 2 during spontaneous breathing; interstitial pulmonary edema by itself is unlikely to affect substantially the diffusion of 02 and CO2 [24, 25]. The unaffected alveolar-arterial PO2 difference and the similarity between the PaO 2 predicted by MIGET and that which we observed are consistent with this observation. The relatively mild decrease in oxygenation may in part be explainable by the histologic demonstration of patchy atelectatic zones. The resulting impairment of gas exchange in these areas may be compensated by pulmonary hypoxic vasoconstriction, thus leading to a redistribution of blood flow to ventilated areas and thereby normalizing

(ZA/Q

ratios. As ARDS is a clinical diagnosis based on gas exchange criteria, it is not known whether these findings parallel early stages of human ARDS prior to clinical diagnosis. Reopening of atelectatic alveoli by positive pressure ventilation may explain the disparity between the alterations in histology and compliance, on the one hand, and normal gas exchange during mechanical ventilation on the other.

Fig. 3 Microscopic sections of a lung from a a control animal and b - d from animals receiving NNNMU (hematoxylin-esoin, X 200)

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The findings of increased protein, increased cellularity (as reflected by total DNA), and markedly increased neutrophil fraction of BAL cells suggest an acute inflam- matory alveolar injury. Histologic evidence of neutrophil infiltration, alveolar edema, and hyaline membranes con- firms this suggestion. Accompanying this pulmonary in- flammation were changes in surfactant composition and function. We found a decrease in the total amount of surfactant, a decrease in the phospholipid/protein ratio, an altered surfactant composition, and a reduction in surfactant function, confirming previous reports for the NNNMU animal model [9, 24, 26, 27] similar to ARDS [19, 20, 28]. Analysis of the surfactant system was performed from bronchoalveolar lavage fluid recovered from injured and non-injured areas may thus represent alterations averaged for the whole lung. Biochemical and functional alterations of the surfactant system may not devel- op synchronous to the reduction of arterial oxygenation because of the compensatory mechanisms already dis- cussed.

Several factors may contribute to the loss of surfactant function we observed. First, the composition of surfac- rant is important for its biophysical properties. Lung injury is accompanied by a change in composition of human lung surfactant [3, 4], and such changes are well described for lung injury induced by NNNMU in animals [9, 23, 26, 27] and for humans suffering from ARDS [4, 28, 29]. A reduction in phosphatidylcholine is a common finding in most studies. Although we did not observe a significant decrease in phosphatidylcholine content in animals receiving NNNMU, the relatively large variance in the measurements may have obscured detection of such a decrease. Liau et al. also demonstrated a decrease of phosphatidylglycerol content following NNNMU injury which, in contrast to other phospholipid alterations, is persistent even after physiologic recovery [26].

Changes in structure of surfactant aggregate also ac- company changes in surfactant function. Lewis et al. reported that physiologic alterations induced in rabbits by NNNMU were accompanied by an increase in the number of "small" surfactant aggregates and a decrease in the number of "large" aggregates (estimated by determining DSPC content of the sedimented and supernatant fractions of extracted pellets). In vitro and in vivo measurement of surface tension lowering function of both large and small aggregates, exhibited impaired function [9]. Us- ing total phospholipid content of the extracted pellet or supernatant, we also found a decrease in the large aggregate pool size and a significant increase in the ratio of small to large aggregates, accompanied by a loss of func-

tion, by in vitro measurements, of surfactant in pellet P2, which contains "large" aggregates described by these in- vestigators. In contrast to the prior report, we did not find a significant increase in small aggregate content. Consis- tent with the prior report, we found no difference in the fractional phosphatidylcholine (PC) content of small and large aggregates between samples from control rabbits and samples from rabbits receiving NNNMU.

An additional, and critically important cause of surfactant inactivation may be an increase in total protein and a resulting change in the ratio of phospholipid to protein [5, 30]. The inhibitory effects of plasma protein that gain access to the alveolar space are well described. Final- ly, we found a marked accumulation of neutrophils in the injured lungs. Release of neutrophilic proteases and reac- tive oxygen species may alter surfactant function [2, 26, 27, 31-33].

In summary, these results show that the early changes of lung injury induced in the rabbit by NNNMU closely resemble the spectrum of changes seen in the lungs of humans with ARDS. Despite marked alterations of surfac- rant function and compliance, gas exchange abnormalities were minimal and were not apparent during volume- controlled mechanical ventilation. This reversibility sug- gests that the altered gas exchange may be primarily the result of alveolar instability and atelectasis due to surfac- rant abnormalities. Thus, in this model, alterations in surfactant function, lung compliance and histopathological changes could be dissociated from severe irreversible impairment of gas exchange, as observed late in the course of ARDS. Although the NNNMU-induced lung injury does not parallel all features of ARDS, the observed abnormalities may precede the clinical Onset of ARDS and its diagnosis. This animal model is only one in a series of lung injury models useful to investigate ARDS of different etiology and at different stages. When compared to more acute injury models, the NNNMU lung injury model may provide information regarding ARDS with a slow evolution of the clinical syndrome in contrast to a direct chemical or physical type injury, e.g., blunt chest trauma or aspiration.

The sensitivity of this model to mechanical ventilatory pressure is an important consideration in interpreting studies of the acute lung injury induced by NNNMU. As factors that influence the mechanical properties of the lung have a substantial effect on gas exchange, additional studies will be required to determine whether administra- tion of exogenous lung surfactant results in an increase in compliance of the lungs of rabbits receiving NNNMU.

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