Etiology of the pulmonary pathophysiology associated with inhalation injury

43

Resuscitation, 14 (1986) 43-59 Elsevier Scientific Publishers Ireland Ltd.

ETIOLOGY OF THE PULMONARY WITH INHALATION INJURY

D.N. HERNDON, L.D. TRABER, and D.L. TRABER

PATHOPHYSIOLOGY

ASSOCIATED

H. LINARES, J.D. FLYNN, G. NIEHAUS, G. KRAMER

The University of Texas Medical Branch and Shriners Burns Institute, Galveston, TX, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA and Northeastern Ohio University College of Medicine, Rootstown, OH (U.S.A.)

SUMMARY

This study describes an experimental model of smoke inhalation injury in sheep in which the same pathophysiologic alterations occur as with clinical inhalation injury in man. Diffuse pulmonary mucosal sloughing with atelectasis and emphysema with concommitant development of pulmonary edema results in a decrease in arterial oxygen and progressive pulmonary deterioration which results in a substantial mortality. Increased pulmonary edema fluid is shown to be caused by an increased microvascular permeability to protein with pulmonary lymph and tracheobronchial fluid, a filtrate of plasma. Concommitant with this increase in microvascular permeability is an influx of neutrophils, release of proteolytic enzymes and an identified presence of the metabolite of the prostanoid thromboxane A2 which are postulated as contributors to the progressive pulmonary dysfunction post inhalation injury. Key words: Bum - Inhalation Sheep - Smoke

injury - Lung - Lung lymph - Pulmonary

-

INTRODUCTION

There has been a marked decrease in mortality from bum injury in all age groups over the last 5 years provided the individuals did not simultaneously inhale significant amounts of smoke. Mortality from severe parenchymal pulmonary injury despite the advent of advanced support techniques has not improved. The incidence of inhalation injury as diagnosed by bronchoscopy

Abbreviations: 6-keto PGF,,, 6-keto prostaglandin F,,; RIA, radioimmunoassay; total body surface area burned; TxB,, thromboxane B,. 0 1986 Elsevier Scientific Publishers Ireland Ltd. 0300-9572/86/$03.50 Printed and Published in Ireland

TBSAB,

44 TABLE I INCIDENCE

OF INHALATION

The incidence

of inhalation

INJURY

AS DIAGNOSED

WITH BRONCHOSCOPY

injury is about 20%.

Institution

Author

Year

No. of patients

Cornell

Herndon

80-81

277

20.9%

U.T.M.B. Galveston

Thompson

81-84

1015

19.1%

Incidence of inhalation injury

has been found to be as high as 19-21s (Hemdon et al., 1983; Thompson et a&1985) (Table I). The mortality from inhalation injury has been reported to be between 50 and 77% (Stone and Martion, 1969; Venus et al., 1981; Vincenti et al., 1971) as demonstrated in Table II. The incidence and mortality from inhalation injury increases as related to total body surface area burned (TBSAB) as demonstrated in Table III (Thompson et al., 1985) and and increases with increasing age as demonstrated in Table IV (Thompson, 1985). The presence of inhalation injury is as significant a contributor to mortality as either age or % TBSAB. The purpose of this study was to develop an animal model that would simulate the salient clinical features of inhalation injury in patients and to investigate the pathogenesis of inhalation injury to allow future modulation of the host response to this trauma which might reduce mortality. MATERIALS

AND METHODS

Fifty range ewes of Suffolk or Merino breed, average body weight 50 f 1.3 kg, were prepared in two separate operations: first, a Silastic (R) catheter TABLE II MORTALITY

FROM INHALATION

INJURY

The mortality

among patients with inhalation

injury is high.

Institution

Author

Year

No. of inhalation injury patients

Mortality %

Emory

Stone Venus Vincenti Hemdon Thompson

61-68 76-78 56-68 80-81 81-84

197 84 66 58 88

47.7 54.7 57.6 77 56

COOk

Brooke Cornell U.T.M.B. Galve&.on

.-

45 TABLE III INCIDENCE AND MORTALITY BURN Inhalation

OF INHALATION

injury was a better predictor of mortality

% TBSA burn

No. of

patients

O-20 21-40 41-60 61-80 81-100 Total

% with inhalation injury (N)

INJURY AS RELATED

TO TBSA

than was burn size.

Mortality % without inhalation injury 0’)

% with inhalation (N)

injury

627 200 102 56 33

2 11 20 32 55

(11) (21) (20) (18) (18)

1 2 18 24 47

(6) (4) (15) (9) (7)

36* 3a* 50* 67* 83*

(4) (8) (10) (12) (15)

1018

9

(88)

4

(38)

56*

(49)

*P-value < 0.01 for increase in mortality

with inhalation

injury.

was placed in the left atrium through a thoracotomy in the fifth intercostal space. During this operation, the borders of the diaphragm and posterior aspect of the left thoracic cavity were cauterized to sever systemic afferent lymphatics that might enter the caudal mediastinal lymph node. In 36 of the 50 sheep, 1 week after the left thoracotomy, the afferent lymphatic vessel TABLE IV INCIDENCE InhaIation patient.

Age

AND MORTALITY

injury was a better predictor of mortality

No. of patients

% with inhalation injury (N)

<4 5L14

317 195

75

15-44 45-59 L59

394 67 45 1018

Total

OF INHALATION

9 18 27

(16) (13) (35) (12) (12)

9

(88)

*P-value < 0.01 for increase in mortaIity

INJURY AS RELATED

TO AGE

than was age of the thermally injured

Mortality % without inhalation injury 0’) 12

% with inhalation (N) 44* 38*

5 15 24

(5) (2) (17) (8) (8)

54* 58* 92*

(7) (5) (19) (7) (11)

4

(38)

56*

(49)

with inhalation

injury.

injury

46

from the caudal mediastinal lymph node was cannulated by a modification of the technique of Staub et al. (1975). A right thoracotomy was performed through the sixth intercostal space. The caudal mediastinal node was isolated and a single afferent vessel of the node cannulated. All other afferents were identified and ligated. A second incision was made at the ninth intercostal space. The distal end of the caudal mediastinal lymph node was identified and ligated. The borders of the diaphragm and the posterior aspects of the right hemithorax were cauterized to prevent systemic contamination of the afferent lymph flow. In all 50 sheep, a flow-directed thermal-dilution Swan-Ganz catheter (Model 93-A-131-7F, Edwards Laboratories) was positioned in the pulmonary artery through an incision in the femoral triangle. An arterial catheter was placed through the same incision into the femoral artery and advanced into the thoracic aorta. These catheters were tunneled under the skin from the femoral triangle to the surface of the flank. Tracheostomies were performed 5 cm below the thyroid cartilage and a cuffed tracheostomy tube (10 mm diameter, Shiley Corporation, California) was inserted. All surgical procedures were performed under halothane anesthesia. One week after the last surgical procedure, baseline data were collected for 2 h, and the sheep were then anesthetized. Twelve of the 50 sheep were insufflated with room air and 38 were insufflated with smoke by a technique previously described by Walker et al. (1981). For the smoke insufflations, the combustion chamber of a bee smoker was filled with four 10-g segments (total 40 g) of ignited cotton toweling and the container sealed. The bee smoker was attached to the tracheostomy tube by an &inch segment of lo-mm tubing containing an indwelling thermistor (Yellowspring Instruments Corporation), and the smoke was delivered by depressing the bellows of the bee smoker. (The bellows had been converted to a closed system by attaching them to the combustion chamber with tubing) (Fig. 1). A series of 14 breaths of smoke were delivered. The sheep were then given a 2-min break, during which time they received 2% halothane and 98% oxygen via the anesthetic apparatus. Four replicates of this sequence were used for each smoke inhalation challenge so that the sheep received a total of 56 breaths of smoke. With each 14 breaths the combustion chamber was emptied of its contents, cooled, and recharged with an additional 10 g of toweling material. The temperature of the smoke was monitored at the level of the endotracheal connector and was not allowed to exceed 39°C (the body temperature of the sheep). The residues of combustion were weighed, 15 + 2 g were found to have been burned during the 56th breath sequence. Arterial carboxyhemoglobin saturations of 20 + 5% were achieved. Arterial and pulmonary artery pressure were monitored during control periods and at various times thereafter, with pressure transducers (P231D, Statham Instrument, Oxnard, CA), connected to a physiologic recorder (OM9, Electronics for Medicine, Pleasantville, NY). Blood and lymph cells

f

Fig. 1, Modified bee smoker - the bee smoker is modified by the placement of a l-way air intake and sealed top. The smoker is connected to the trachea with a plastic hose. A thermister is connected to the plastic hose to measure the heat of the smoke as it enters the animal.

were counted by an electronic cell counter (Coulter Counter, Model ZF, Coulter Electronics, Hialeah, FL). Differential cell counts were performed by standard histologic staining techniques. Blood gas and pH were measured with a Model 1303 Blood Gas Analyzer (Instrumentation Laboratory). Total protein was measured with a Protometer (National Protometer, Baltimore, MD). Albumin, IGG and IGM were determined by rocket immunoelectrophoresis technique (Niehaus et al., 1980). Activated neutrophils are capable of releasing proteolytic enzymes. Antiproteases bind rapidly to the released enzymes. Changes in enzyme levels are difficult to measure directly. The consumptive depletion of the antiproteinases is an index of enzyme release. The ability of the plasma or lymph to inhibit proteases in vitro was therefore determined as an index of antiprotease activity. Ten milliliters of plasma were diluted 990 to 1 with Tris (pH 7.6) buffer and incubated for 1 h at 37°C with 1 ml of 0.001% trypsin solution in Tris buffer. Three milliliters of 0.1 M solution of N-a:-benzoyl-Dtargininepara-nitroanitride hydrochloride was added and the mixture incubated for 30 min. The reaction was stopped by adding 1 ml of 0.2 N hydrochloric acid and the extinction was read with a spectrophotometer at A400nm.The specific activity of the trypsin was determined by active site titration. The quantity of trypsin inhibited per ml ww determined by using a standard curve of various concentrations of trypsin incubated with buffer instead of lymph and exudate. Specimens for light microscopic study were fixed with 10% neutral phosphate-buffered formalin, embedded in paraplast and sectioned at 5

48

micron. They were stained with hematoxylineosin, periodic acid Schiff and Mason’s trichrome’and Giemsa for morphologic studies. Eicosanoids levels were determined by the content of thromboxane Bz (TxB,) and 6-keto prostaglandin F1, (6-keto PGF& in the plasma as determined by specific radioimmunoassay (RIA), Antibodies to these eicosanoids were produced in rabbits by the subcutaneous foot pad injection of the carbodiimide, conjugate of either TxB? or 6-keto PGFI, emulsified with complete Freunds adjuvant. The resultant TxBz antibody was used at a final dilution of 1:20 000 while the 6-keto PGFI, antibody was used at a final dilution of 1: 5000. Both of these antibodies exhibit a linear working range of between 20 and 2500 pg of the respective lipid. As has previously been described, the cross-reactivity for these antibodies with other major prostanoids is negligible (Head, 1980). Both radioimmunoassays were carried out at pH 7.3 in 0.01 M Tris buffer containing 0.1% gelatin. A l-h incubation at 20°C was terminated by adding dextran-coated charcoal and centrifuging the samples. Aliquots of the clear supematant were dissolved in a suitable counting medium and counted to a 2.0% confidence limit in a Beckman Model laboratory microcomputer was used to calculate a linear regression equation for an &point standard curve, the ratio of binding constant counts to sample counts and dpm. All samples were performed in duplicate. Data for unknown samples were calculated by the RIA program, fit to the regression line, corrected for aliquot volume, and expressed as nanograms of eicosanoid per ml of fluid. The lowest limits of detection of both eicosanoids was calculated as the lowest limit of detection in the RIA times the plasma aliquot assayed (0.02 ng/O.Ol ml = <0.20 ng/ml). ANALYSIS

The determination of arterial and pulmonary artery pressure, blood and lymph cell counts, blood gases and total protein were compared to sheep’s baseline before insufflation as well as controls. Thirty-two of the sheep that were insufflated with smoke were not put on respirators. Sixteen of these sheep died within 12-72 h after injury an LDsO was achieved. Survivors were compared to non-survivors and controls by a 3-way analysis of variance. Five of the smoked animals were treated separately. Ventilatory assistance with a servo M740 ventilator was instituted for all animals in this subgroup when arterial oxygen saturation fell to 80% on an inspired oxygen tracheostomy collar of 0.4. In this group of 5 animals, tracheobronchial fluid was collected in a gravity drip chamber attached to the tracheotomy tube for analysis of components of tracheobronchial exudate. RESULTS

All 38 of the animals insufflated with smoke demonstrated clinical signs of pulmonary damage between 6 and 15 h after injury. A productive cough

49

expectorating large pseudomembrane casts was found. The casts were fir‘m, gray-white replicas of the mainstem and sometimes lobar bronchi as dc!mcInstrated in Fig. 2. Autopsies were performed on the 16 animals that ; diied within 72 h of smoke inhalation. Pathological lesions similar to thexe

Fig. 2. The trachea-bronchial

cast - cast removed from asheep 36 h post inhalation injury.

50

described by Walker et al. (1981) and Hemdon et al, (1984) were found, Histopathologic changes were present around the trachea and mainstem bronchi with lesions present in the lower airways as well, characterized by focal areas of congestion and edema, alternating with areas of collapse, pneumonia and hyperaeration involving primarily areas adjacent to the bronchial-bronchiolar tree. This produced a typical gross anatomical picture of areas of ateledasis alternating with areas of hyper-inflation (Fig. 3). Microscopic areas were found in which the greater part of the lining epithehum of the upper airways had been shed via progressive separation of the epithelium and formation of a pseudomembranous casts, which could be found free in the bronchial lumen. These casts cause complete or partial obstruction of the airway. Pseudomembranes were not found in bronchioles. At lower levels acute inflammatory changes were present with marked infiltration of leukocytes into the airways and into the peribronchiole interstitial spaces. The pulmonary parenchyma demonstrated marked degrees of interstitial as well as alveolar edema. Occasional hyalin membranes and patchy areas of dense atelectasis were characteristic. In the 32 sheep that were not placed on respirators and insufflated with smoke arterial oxygen fell from 108 f 4 mmHg before injury to 78 f 6 mmHg 36 h after injury. Heart rate, core temperature and total peripheral neutrophil count were increased in all sheep after inhalation injury. Peripheral arterial pressure, left atrial pressure, cardiac output were unchanged in these sheep from preinjury control values all sheep receiving 70 ml/m, per h or of % Dextrose in Ringer’s Lactate solution. Smoke inhalation increased the pulmonary lymph flow from 7.4 f 1.4 ml/ h at the beginning of the experiment to 29 f 2 ml/h, 12 h post inhalation injury. Lymph to plasma total protein concentration ratios increased from 0.59 f 0.03 before to 0.70 + 0.02 8 h after the injury. Sixteen of the 32 sheep that were not put on ventilators died between 12 and 72 h after the injury and 16 survived. All of the sham-treated controls survived. The survivors, non-survivors and controls are compared in terms of PaO, total lymph’ flow and lung transvascular flux of protein in Figs. 3,4 and 5. Cardiac output and left atrial pressure did not differ significantly between the smoke insufflated and the control sheep. The air insufflated group demonstrated no change in Pa& lymph flow or transvascular protein flux. Pa& decreased significantly (Fig. 4) and lung lymph flow increased (Fig. 5) and transvascular flux of protein increased (Fig. 6) in the non-survivors as compared to survivors and controls and in the survivors compared to controls. Trypsin inhibitory capacity decreased significantly after injury in the lung lymph of the sheep that died in contrast with the surviving sheep (734 f 40505 + 92 pg/ml, vs. 778 + 38-746 + 44 gg/ml;P < 0.05). Tracheobronchial fluid was collected from five animals post-smoke inhalation injury. It was produced at a rate of 20 + 2 ml/h. The total protein content of the tracheobronchial fluid was 4.5 f 0.6 g/d1 relative to 6.2 f 0.2 g/d1 in the plasma, a ratio of 0.66 f 0.08. Albumin, IGG and IGM ratios in the

Fig. 3, Lungs after inhalation ation of smoke (48 h).

injury - these are lungs from an animal following

the inhal-

52

4

5

%02

Lymph Flow

120

1

+* + L +*

A’

d

4

o-

i2

8

24 Time in Hours Post Inhalation Injury *

SURVIVORS DIFFERENT NON-SURVIVORS

FROM SHAM

DIFFERENT

FROM SHAM

0

4

$------. 8

12

24

Time in Hours Post Inhalation Injury + *

NON-SURVIVORS DIFFERENT FROM SURVIVORS SURVIVORS DIFFERENT FROM SHAM NON-SURVIVORS DIFFERENT FROM SHAM

Fig. 4. These are data obtained from sheep which were insufflated with air (controls) or smoke. In the animals insufflated with smoke, the non-survivors show a much lower PRO, than the survivors. 0, control; A, survivors; ?? , non-survivors. Fig. 5. These are data obtained from sheep which were insufflated with air (controls) or smoke. In the animals insufflated with smoke, the non-survivors show a much higher lymph flow than the survivors. ?? , control; A, survivors; ?? , non-survivors.

tracheobronchial fluid relative to plasma were determined and were 0.8 f. 0.03, 0.59 f 0.07 and 0.17 ? 0.02, respectively (Fig. 7). When apherogram of the rocket immunoelectrophoreses of the plasma and tracheobronchial exudate were compared, they were found to be similar (Fig. 8). Prostacycline and TxBz reflected by their stable metabolites in the tracheobronchial fluid were 0.498 ?r 0.305 ng/ml and 2.50 + 0.886 ng/ml, respectively .

In contrast levels of TxBz were lower in lymph and 6-keto F1, was elevated in lymph (Fig. 9) 5.1 + 2.9 X 106/ml white blood cells with 61 * 11% polymorphonuclear leukocytes were found in the tracheobronchial fluid 24-48 h post injury. At this same time plasma B glucuronidase levels increased 3-fold from control levels (Table V). The ratio of B glucuronidase/ total protein in the tracheobronchial exudate was higher than in plasma (0.59-0.40%) (Table V). A fall in elastase inhibitory capacity and trypsin inhibitors capacity in lymph from the animals after inhalation injury is seen to parallel increases in neutrophil numbers present in the lymph as depicted in Fig. 10.

53

6

, Cdmparisun of Puknonary

Lung Transvascular Flux of Protein

Lymph and Exudate LO-

.I?

O&

‘0 u

Eo.s% a 2 ;

0.4-

0.2Time in Hours Post Inhalation

*

SURVIVORS

DIFFERENT

NON-SURVIVORS

FROM

DIFFERENT

SHAM FROM

SHAM

*

40

80

Molecular Radius (81 Fig. 6. These are data obtained from sheep which were insufflated with air (controls) or smoke. In the animals insufflated with smoke, the non-survivors show a much higher lung transvascular flux of protein than the survivors. 0, control; A, survivors; 9, non-survivors. Fig. 7. Comparison of pulmonary lymph and exudate - comparison of pulmonary lymph and exudate shows higher molecular weight proteins are more abundant in the lymph than the exudate. This size selectivity suggests movement through pores. The pores through which the exudate was formed are smaller than those of the lymph. ?? , exudate; A, lymph. Pulmonary lymph and exudate have a similar composition. The fluid-to-plasma ratios of specific proteins decreases as molecular size increases.

DISCUSSION

The inhalation of smoke produces severe pulmonary injury (Feller and Hendrix, 1964; Shook et al., 1968) which greatly increases morbidity and mortality (Moylan, 1981; Moylan et al., 1978) in combination with cutaneous boms. After smoke inhalation bronchoscopy discloses an edematous inflamed tracheobronchial tree (Moylan et al., 1972; Petroff et al., 1979). As this syndrome progresses tracheobronchial edema becomes more severe, mucosa separates in the upper airways and forms casts (Moylan et al., 1972; Petroff et al., 1979) which cause patchy areas of atelectasis alternating with emphyzematous segments and resultant ventilation to perfusion mismatch. Arterial oxygen (Pa&) decreases (Moylan, 1981; Pietznan et al., 1981) from a combination of upper airway obstruction and parenchymal edema. In this study, an animal model with smoke inhalation exhibited pathophysiological changes in a time course that was quite similar to that observed

54 *

P herograms Plasma Albumin

Thromboxane

8,

6 Keto F;a

0 Exudate

Exudate

?? Lymph

I. r.,,,,,. 1.0 0.8 0.6 0.4 0.2

1 I a0

MOBILITY Fig. 8. Pherograms of plasma and exudate .- the similarities between these two pherograms with the absence of higher molecular weight proteins suggests that the tracheobronchial exudate formed after inhalation injury is a filtrate of plasma, Exudate is a filtrate of plasma. Plasma and exudate samples were subjected to polyacrylamide gel electrophoresis and stained with Poncreau S. All bonds present in plasma were also found in exudate. Fig. 9. Eicosanoid levels in lung lymph and tracheobronchial exudate - TxB, is more abundant in the exudate than lymph, whereas the prostacycline is more dominant in the plasma following inhalation injury,

TABLE V PLASMA LEVELS OF fl-GLUCURONIDASE Plasma glucuronidase was elevated after smoke inhalation with peak values occurring at 24-48 h. Glucuronidase concentrations in exudate were similar. Normalized for total protein concentration the exudate concentrations were higher. p-Glucuronidase-plasma

(N = 5)

Control (units/ml)

24-48 h (units/ml)

10.5 + 3.8

34.5 + 1.9

Ratio ~glucuronidase/Total

protein

Plasma

Exudate

0.40 f 0.07

0.69 f 0.13

55

,~,Elastase

lnbibitory

Capocity

750- Trypsin Inhibitory Capacity

~~ Neutrophils

in the Lymph

I1

0

48 Hours

Post Inhalation

Fig. 10. Antiproteases following inhalation injury - following inhalation injury, the lack of antiproteases fall in the lung lymph and the level of neutrophils rise. * indicates statistically significant changes as determined by analysis of variance and Waller-Duncan Kratio t-test.

patients with inhalation injury. All of the sheep demonstrated diffuse upper airway mucosal injury within 8 h of the time of smoke inhalation with concurrent development of pulmonary edema. Lung lymph flow increased markedly within 12 h of injury. The lymph flow was positively correlated with an increase in transvascular flux of protein, indicating this to be a microvascular permeability-caused edema. All of the observed changes were more prominent in sheep that died, than in the surviving animals. Inhalation injury in this model increased lung lymph flow, lymph to plasma total protein ratios and transvascular protein flux. All of the lymph changes were consistent with an increase in pulmonary microvascular permeability. The lung lymph flow changes may be influenced by changes in systemic bronchial microvasculature. The pulmonary microvasculature might remain intact, while there is an elevation in fluid formation from the systemic microvasculature of the lung. A further preliminary study (Kramer et al., 1984) in

56

shows that there is a lo-fold elevation in blood flow in the tracheobronchial areas following inhalation injury. Such a hyperemia would certainly result in edema formation. We have also accumulated, however, information in another study to support the hypothesis that the increase in pulmonary microvascular permeability is the primary source of edema (Traber et al., 1984). In this study, pulmonary interestitial edema was evaluated by the thermal dye technique edema was seen in the parenchyma. In addition, blood levels of angiotensin converting enzyme, a pulmonary vascular endothelial marker was elevated and abundant numbers of neutrophils were sequestered in the pulmonary microvascular areas. Thus, both systemic and pulmonary microvascular areas are injured. The present study further demonstrates that the tracheobronchial exudate following smoke inhalation is a filtrate of plasma with a composition similar to pulmonary lymph and presumably interstitial edema fluid. This again supports the hypothesis of pulmonary microvascular damage. Relatively more of the smaller molecules such as albumin are present in the tracheobronchial fluid than IGG or even more so than IGM which is an even larger molecule. The pheograms of plasma and exudate are relatively similar in absolute terms. As molecular size increases from albumin to IGG and finally IGM, less of the substance crosses the endothelium as a filtrate. We have demonstrated the presence of a markedly increased level of leukocytes and their proteolytic enzymes as well as the potent vasoconstrictor thromboxane in the lymph draining the lung after inhalation injury, as well as in the tracheobronchial fluid produced post-inhalation injury. It is our hypothesis that these substances cause local tissue destruction in both the lower and upper airways, which result in increased microvascular permeability. This contributes to progressive pulmonary failure which can be potentially modulated by pharmacologic manipulation. Peitzman et al. (1981) have reported clinical data to substantiate the fact that there is an elevation in extravascular lnng water following thermal injury. Transbaugh et al. (1980) have clinical data to the contrary. This difference is certainly of importance and requires further evaluation. This apparent conflict in data could be related to the degree of fluid resuscitation that the patients receive, their cardiac outputs and perhaps the time post injury at which measurements were made. Zawaki et al. (1977), using a rat model of inhalation injury, found that the ensuing pulmonary edema was augmented by fluid restriction. Those animals that received fluid resuscitation had less lung injury, We have preliminary data to support this in the sheep model (Hendron et al., 1984). In this present study the animals were allowed 70 ml/ m2 per h and their cardiac output, left atria1 and pulmonary artery pressures were within 10% of the control values. The mechanisms responsible for the increased permeability demonstrated by our study remains to be completely elucidated. Other investigators have similarly implicated the neutrophil in the development of lung injury after

57

sepsis (Meyrick and Brigham, 1983) embolization (Klick et, al., 1981) hemodialysis (Craddock et al., 1977) disseminated intravascular coagulation (Tahamount and Malik, 1983). Our observation of an increase in pulmonary extravascular neutrophil concentration along with their proteolytic enzyme products suggests that the neutrophils and/or the mediators which they release may contribute to the decreased lung microvascular integrity observed after smoke inhalation. It is well known that activated neutrophils release proteolytic enzymes (Curreri et al., 1980) and oxygen free radicals (Weiss et al., 1978) which are capable of damaging lung tissue. Antiproteases in the lung combine with neutrophil proteases rapidly and reversibly to prevent tissue destruction. Smoke-induced pulmonary sequestration of neutrophils may result in the release of enzymes in quantitites that exceed the inhibitory capacity of local antiproteases. We feel that the uninhibited proteases and free radicals can then be available to interact with lung tissue which can result in disruption of pulmonary integrity (Janoff et al., 1970). The significant decrease in trypsin and elastase inhibitory capacity that occurred in our sheep is consistent with release of large amounts of proteases by neutrophils that have been sequestered in the lung. In addition, oxygen-free radicals can likewise inactivate antiproteases or directly disrupt cells (Carp and Janoff, 1979). The evidence of thromboxane A2 release into the trachea-bronchial areas is the opposite of what was noted in the lymph where prostacycline is the most dominant eicosanoid following inhalation injury. This supports the concept that edema in the alveolar area is the result of damage to the pulmonary microvasculature rather than flooding from the bronchial areas. Were the latter true, one would have expected to see thromboxane as the dominant prostanoid. Thromboxane is a potent vasoconstrictor of smooth muscle, and its presence in the trachobronchial area may account for some of the bronchial constriction an ventilatory perfusion mismatching. The evidence of increased amounts of leukocytes, proteases and prostanoids lead us to the postulate that smoke inhalation stimulates sequestration of neutrophils in the lungs which release proteolytic enzymes and perhaps free radicals, which cause microvascular injury. The resulting structural damage causes an increase in transvascular flux of protein and fluid. The resulting edema progressively depresses pulmonary function and results in decreasing arterial oxygenation. This animal model subjected to smoke inhalation closely mimicks the clinical situation that markedly increases mortality from thermal injury. Smoke inhalation causes tracheobronchial mucosal destruction and atelectasis and emphysema and an increase in pulmonary microvascular permeability, which may be mediated by proteolytic enzymes released,by neutrophils which are sequestered in the lungs. This animal model will be extremely useful in allowing us to modulate these potential mediators of progressive pulmonary dysfunction post smoke inhalation.

58 REFERENCES Carp, J. and Janoff, A. (1979) In vitro suppression of serum elastase inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes. J. Clin. Invest., 63,793-797. Craddock, P.R., Fehr, J. and Brigham, K.L. (1977) Complement and leukocyte mediated pulmonary dysfunction in hemodialysis. N. Engl. J. Med., 296,744-769. Curreri, P.W., Luterman, A. and Braun, D.W., Jr. (1980) Burn injury: Analysis of survival and hospitalization time for 937 patients. Ann. Surg., 192,472-478. Feller, I. and Hendrix, R.C. (1964) Clinical pathology study of sixty fatally burned patients. Surg. Gynecol. Obstet., 119, l-5. Flick, M.R., Perel, A. and Staub, N.C. (1981) Leukocytes are required for increased lung microvascular permeability after microembolization in sheep. Circ. Res., 48, 344-351. Head, J.M. (1980) Inhalation injury in burn. Am. J. Surg., 139,508-512. Herndon, D.N., Kraft, E.R. and Braun, A. (1983) The effect of inhalation injury on mortality from burn injury. Proc. Am. Burn Assoc., 15,77. Herndon, D.N., Traber, D.L. and Niehaus, G.D. (1984) The pathophysiology of smoke inhalation injury in a sheep model. J. Trauma, 24, 1044-1052. Herndon, D.N., Traber, D.L. and Traber, L.D. (1984) The effect of resuscitation on postsmoke inhalation pulmonary fluid formation. Circ. Shock, 13,79. Janoff, A., Sloan, B. and Weinbaum, G. (1970) Experimental emphysema induced with purified human neutrophil elastase: Tissue localization of the instilled protease. Am. Rev. Resp. Dis., 115,461-470. Kramer, G.C., Cross, C.E. and Herndon, D.N. (1984) Unanesthetixed sheep measured with a double microsphere technique. Fed. Proc., 43, 921. Meyrick, B. and Brigham, K.L. (1983) Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep structure : Function relationships. Lab. Invest., 48,458-470. Moylan, J.A. (1981) Inhalation injury: A primary determinant of survival following major burns. J. Burn Care Rehab., 3,78-84. Moylan, J.A. and Alexander, L.G., Jr. (1978) Diagnosis and treatment of inhalation injury. World J. Surg., 2, 185-191. Moylan J.A., Wilmore, D.W. and Mouton, D.E. (1972) Early diagnosis of inhalation injury using Xenon lung scan. Ann. Surg., 176,477-484. Niehaus, G.D., Schumacker, P. and Saba, T.M. (1980) Influence of opsonic fibronectin deficiency on lung fluid balance during bacterial sepsis. J. Appl. Physiol., 39,693-699. Petroff, P.A., Pruitt, B.A., Jr. and Pruitt, B.A. (1979) Pulmonary disease in the burn patient. In: Burns: A team approach. pp. 95-105. Editors: C.P. Artz, J.A. Moncrief and B.A. Pruitt. Sauderna, Philadelphia. Pietzman, A.B., Shires, G.T., III and Corbett, W.A. (1981) Measurement of lung water in inhalation injury. Surgery, 90, 305-319. Shook, C.D., MacMillan, B.G., Altemeier, W.A. (1968) Pulmonary complications of the burn patient. Arch. Surg., 97,215-224. Staub, N.C., Bland, R.D. and Brigham, K.L. (1975) Preparation of chronic lung lymph fiitulas in sheep, J. Surg. Res., 19, 315-320. Stone, H.H. and Martion, J.D., Jr. (1969) Pulmonary injury associated with thermal burns. Surg. Gynecol. Obstet., 129,1242-1246. Tahamont, M.V. and Malik, A.B. (1983) Granulocytes mediate the increase in PUlmOmY after thrombin embolism. J. Appl. Physiol., 54, 1489-1495. vascular permeability Thompson, P.B., Herndon, D.N. and Abston, S. (1985) Effect on mortality of inhalation injury. J. Trauma, in press. Traber, D.L., Schlag, G. and Redl, H. (1984) The mechanism of the pulmonary edema of smoke inhalation. Circ. Shock, 13, 77.

59 Tranbaugh, R.E., Lewis, F.R. and Christensin, L.M. (1980) Lung water changes after thermal injury. Ann. Surg., 192,479-490. Venus, A., Matsuda, T. and Copioxo, J.B. (1981) Prophylactic intubation and continuous positive airway pressure in the management of inhalation injury in burn victims. Crit. Care Med., 9,519-523. Vincenti, F.C., Pruitt, B.A., Reckler, J.M. (1971) Inhalation injury. J. Trauma, 22, 109. Waiker, H.S., McLean, C.C., Jr. and McManus, W.F. (1981) Experimental inhalation injury in the goat. J. Trauma, 21,962-964. Weiss, S.J., Rustagi, P.K. and LoBuglio, A.F. (1978) Human granulocyte generation of hydroxyl radical. J. Exp. Med., 147, 316-323. Zawacki, B.E., Jung, R.C. and Joyce, J. (1977) Smoke, burns, and the natural history of inhalation injury in fiie victims: A correlation of experimental and clinical data, Ann. Surg., 185,100-110.