Departments of Anesthesia and Medicine and Cardiovascular Research Institute, University of California, San Francisco, California 94143; and Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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Endogenous release of catecholamines is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. The objective of this study was to determine whether this catecholamine-dependent mechanism upregulates alveolar liquid clearance after prolonged hemorrhagic shock. Rats were hemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 min and then resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later as the concentration of protein in the distal air spaces over 1 h after instillation of a 5% albumin solution into one lung. There was no upregulation of alveolar liquid clearance after prolonged hemorrhagic shock and fluid resuscitation despite a significant increase in plasma epinephrine levels. The intravenous or intra-alveolar administration of exogenous catecholamines did not upregulate alveolar liquid clearance. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored either by depletion of neutrophils with vinblastine, by the normalization of the concentration of reduced glutathione in the alveolar epithelial lining fluid by N-acetylcysteine, or by the inhibition of the conversion from xanthine dehydrogenase to xanthine oxidase. These experiments provide the first in vivo evidence that a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the upregulation of alveolar fluid clearance by catecholamines in the absence of a major alteration in paracellular permeability to protein after prolonged hemorrhagic shock.
alveolar epithelium; -adrenergic receptor; alveolar liquid
clearance; neutrophil; oxidative injury
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INTRODUCTION |
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TRAUMA AND HEMORRHAGIC SHOCK are two of the most common causes of acute lung injury in patients (26). Even though the mechanisms of lung endothelial injury after hemorrhagic shock have been studied by several investigators (19, 25, 36, 46, 58), the critical importance of the alveolar epithelial barrier in the pathogenesis and recovery from acute lung injury secondary to hemorrhagic shock has not been well examined. In addition, in patients with acute lung injury who develop pulmonary edema, the preservation of the capacity of the alveolar epithelium to actively remove fluid from the air spaces is crucial for their survival (34).
The results from earlier experimental studies from our laboratory (37, 45) indicate that the endogenous release of catecholamines is a major mechanism that prevents alveolar flooding after the onset of septic or short-term hemorrhagic shock by upregulating vectorial, sodium-dependent fluid transport across the alveolar epithelium. However, the impact of this protective mechanism on lung fluid balance and alveolar fluid transport several hours after fluid resuscitation from prolonged hemorrhagic shock has not been studied.
Therefore, the first objective of these studies was to determine
whether the endogenous release of catecholamines can upregulate alveolar fluid clearance after prolonged hemorrhagic shock and fluid
resuscitation. Because there was no upregulation of alveolar fluid
clearance 6 h after prolonged hemorrhagic shock despite a 20-fold
increase in plasma levels of epinephrine, the second objective was to
maximize the response of -adrenergic receptors by the administration
of exogenous
-adrenergic agonists in hemorrhaged and
fluid-resuscitated rats. Because exogenous
-adrenergic agonists did
not stimulate alveolar fluid clearance, the third objective was to test
the hypothesis that a neutrophil-dependent injury to the alveolar
epithelium might account for the absence of upregulation of alveolar
fluid clearance after prolonged hemorrhagic shock. Because neutrophil
depletion with vinblastine before hemorrhagic shock was associated with
restoration of the response of the alveolar epithelium to
-adrenergic agonists, the fourth objective was to test the
hypothesis that oxidant-mediated injury may account for the inability
of the alveolar epithelium to respond to
-adrenergic-agonist stimulation after prolonged hemorrhagic shock and fluid resuscitation by two different approaches. First,
N-acetylcysteine (NAC), a glutathione
precursor and an oxygen radical scavenger, restored the capacity of the
alveolar epithelium to respond to
-adrenergic agonists by
upregulating alveolar fluid clearance and normalized the concentration
of glutathione in the alveolar epithelial lining fluid. Second, the
inhibition of the conversion from xanthine dehydrogenase to xanthine
oxidase (XO) by a tungsten-enriched, molybdenum-deficient diet or an
allopurinol-supplemented diet restored the
-adrenergic-mediated
upregulation of alveolar fluid clearance in hemorrhaged rats.
Therefore, taken together, these data provide the first in vivo
evidence of an oxidant-mediated neutrophil-dependent decrease in the
fluid transport capacity of the alveolar epithelium in the absence of a
major alteration in paracellular permeability to protein after
prolonged hemorrhagic shock.
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METHODS |
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The protocol for these studies was approved by the University of California, San Francisco Animal Research Committee.
Lung Barrier Function Studies
Surgical preparation and ventilation. Male Sprague-Dawley rats (n = 102) weighing 300-350 g were anesthetized with pentobarbital sodium (60 mg/kg ip), and anesthesia was maintained with 30 mg/kg of pentobarbital sodium every 2 h. An endotracheal tube (PE-220) was inserted through a tracheotomy. Pancuronium bromide (0.3 mg · kgPreparation of instillate. A 5% bovine albumin solution was prepared with Ringer lactate and adjusted with NaCl to be isosmolar with the rat's circulating plasma as previously published (37). Anhydrous Evan's blue dye (0.5 mg) was added to the albumin solution to confirm the location of the instillate at the end of the study, and 1 µCi of 125I-labeled human serum albumin (Frosst Laboratories) was also added to the albumin solution. 125I-albumin served as the alveolar protein tracer in all experiments. A sample of the instilled solution was saved for total protein measurement, radioactivity counts, and water-to-dry weight ratio measurements so that the dry weight of the protein solution could be subtracted from the final lung water calculation. In addition, in some studies, epinephrine, salmeterol, or propranolol was added to the instillate.
General protocol. In all experiments,
after surgery, heart rate and systemic blood pressure were allowed to
stabilize for 60 min (Fig.
1A).
The rat was placed in the left lateral decubitus position to facilitate
liquid deposition into the left lung 300 min after the onset of
hemorrhagic shock. Hemorrhagic shock was induced by withdrawing blood
from the carotid artery to maintain a mean systemic arterial pressure
of 30-35 mmHg for 60 min. This corresponded to the removal of
9-12 ml of blood. After 1 h of hemorrhagic shock, the rats were
resuscitated with an intravascular 4% albumin solution in 0.9% NaCl
over 30 min to maintain a central venous pressure < 8 mmHg as done
before by Modelska et al. (37). The volume of 4% albumin
solution administered was twice the amount of blood withdrawn.
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A vascular tracer, 1 µCi of 131I-labeled human albumin, was injected into the blood 270 min after the onset of hemorrhagic shock to calculate the flux of plasma protein into the lung interstitium as previously published (37). An alveolar tracer, 1 µCi of 125I-albumin in 3 ml/kg of the 5% bovine albumin solution, was instilled into the left lower lobe 30 min later to calculate the flux of protein from the air spaces into the circulating plasma. This tracer was instilled with a 1-ml syringe and a polypropylene tube (0.5-mm ID) over 20 min.
At the end of the experiment (1 h after the beginning of the alveolar instillation), the abdomen was opened, and the rats were exsanguinated by transecting the abdominal aorta. Urine was obtained for radioactivity counts. The lungs were removed through a median sternotomy. An alveolar fluid sample (0.1-0.2 ml) from the distal air spaces was obtained by gently passing the sampling catheter (PE-50 catheter, 0.5-mm ID) into a wedged position in the instilled area of the left lower lobe. After centrifugation, the total protein concentration and radioactivity of the liquid samples were measured. The right and left lungs were homogenized separately for water-to-dry weight ratio measurements and radioactivity counts.
In experiments designed to achieve XO depletion (Fig. 1B), inhibition of conversion from xanthine dehydrogenase to XO was achieved by either a tungsten-enriched, molybdenum-deficient diet (0.7 g sodium tungstate/kg chow; ICN Biochemicals) given for 4 wk or an allopurinol-supplemented diet (50 mg/kg chow; ICN Biochemicals) given for 1 wk before hemorrhagic shock (11). Control rats that were fed a regular protein diet were not XO depleted.
Plasma levels of epinephrine were measured at three time points: just before the onset of hemorrhagic shock, at the end of the ischemic phase of hemorrhagic shock, and at the end of each experiment.
Specific Protocols
Group 1: Effect of prolonged hemorrhagic shock and fluid resuscitation on alveolar liquid clearance. The rats were hemorrhaged and resuscitated to determine the effect of prolonged hemorrhagic shock and fluid resuscitation on fluid transport across the lung epithelium. Five hours after the onset of hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution with 1 µCi of 125I-albumin were instilled into the left lung (n = 6 rats). Control studies included rats (n = 10) that underwent the same surgical preparation and were studied for the same period of time but were neither hemorrhaged nor fluid resuscitated. Additional experiments that included control and hemorrhaged rats (n = 6) were done to measure myeloperoxidase activity in the lung as a quantitative assessment of neutrophil infiltration into the lung 6 h after the onset of hemorrhagic shock.Group 2: Stimulation with exogenous
-adrenergic agonists after prolonged hemorrhagic
shock. GROUP 2A:
ADMINISTRATION OF EPINEPHRINE. Because there was no upregulation
of alveolar fluid clearance after prolonged hemorrhagic shock,
epinephrine was either given intravenously at a concentration of 0.08 mg · kg
1 · min
1
(n = 5 rats) or added to the protein
solution instilled into the distal air spaces at a concentration of
10
5 M
(n = 4 rats). This concentration of
intravenous epinephrine has previously been shown in pilot experiments
to be sufficient to upregulate alveolar liquid clearance. Control
studies included rats that were not hemorrhaged nor fluid resuscitated
but either received an intravenous infusion of epinephrine for 6 h at a
concentration of 0.08 mg · kg
1 · min
1
(n = 4 rats) or had their distal air
spaces instilled with an albumin solution containing
10
5 M epinephrine for the
last hour of the experiment (n = 3 rats).
Bronchoalveolar Lavage Studies
To determine whether oxidative injury to the alveolar epithelium secondary to prolonged hemorrhagic shock was associated with a decrease in the concentration of reduced glutathione (GSH) in the alveolar epithelial lining fluid, a first series of experiments included rats (n = 4) that were hemorrhaged and fluid resuscitated as described in General protocol. Then the rats were exsanguinated, and their lungs were lavaged through a tracheotomy with HEPES (10.3 mM)-phosphate buffer (2.59 mM), pH 7.4, containing NaCl (133 mM), KCl (5.2 mM), CaCl2 (1.89 mM), and MgCl2 (1.29 mM). The lungs were then inflated to total lung capacity (10 ml) and lavaged once. The lavage samples were then centrifuged at 800 g for 10 min at 4°C to remove cells and quickly frozen atA second series of experiments included rats (n = 4) that were pretreated with NAC before they underwent prolonged hemorrhagic shock and fluid resuscitation as described in General protocol. At the end of the experiments, the lungs were then inflated to total lung capacity (10 ml) and lavaged once.
A third series of experiments included control rats (n = 4) that were neither hemorrhaged nor fluid resuscitated. At the end of experiments, the lungs were then inflated to total lung capacity (10 ml) and lavaged once.
In each group, GSH and oxidized glutathione (GSSG) levels were measured in the alveolar epithelial lining fluid.
Measurements
Hemodynamics, pulmonary gas exchange, and protein concentration. Systemic arterial, central venous, and airway pressures were continuously measured. Arterial blood gases were measured at 1-h intervals. Samples from the instilled protein solution, the final distal air space fluid, and the initial and final blood were collected to measure total protein concentration with an automated analyzer (AA2 Technicon, Tarrytown, NY).Albumin flux across endothelial and epithelial barriers. Two different methods were used to measure the flux of albumin across the lung endothelial and epithelial barriers as done before by our laboratory (37, 45). The first method measures residual 125I-albumin (the air space protein tracer) in the lungs as well as the accumulation of 125I-albumin in plasma. The second method measures 131I-albumin (the vascular protein tracer) in the extravascular space of the lungs.
The total quantity of 125I-albumin
(the air space protein tracer) instilled into the lung was determined
by measuring duplicate samples of the instilled solution for total
radioactivity counts (in
counts · min1 · g
1)
and multiplying this value by the total volume instilled into the lung.
To calculate the residual
125I-albumin in the lungs at the
end of the study, the average radioactivity counts of two 0.5-g samples
obtained from the lung homogenate was multiplied by the total weight of
the lung homogenate. The 125I-albumin count in the lung
homogenate was added to the recovered count in the final aspirated
distal air space fluid to calculate the quantity of instilled
125I-albumin that remained in the
lungs at the end of the study. 125I-albumin in the circulating
plasma was measured from a sample of plasma obtained at the end of the
experiment. The plasma fraction was accounted for by multiplying the
counts per minute per gram times the plasma volume [body weight × 0.07(1
hematocrit)] (37, 45).
The second method requires measurement of the vascular protein tracer 131I-albumin in the extravascular space of the lungs. We estimated the quantity of plasma that entered the instilled lungs by measuring the accumulation of the vascular protein tracer 131I-albumin into the extravascular space of the instilled lung using the equation of plasma equivalents previously described (37, 45). The extravascular lung plasma equivalents were determined by the total count of 131I-albumin in the lung, subtracting the fraction in the blood in the lung as measured with the gravimetric method, and then dividing by the mean count in the circulating plasma over the duration of the experiment as previously done (37, 45).
Alveolar liquid clearance. Changes in the concentration of the nonlabeled bovine albumin and the instilled 125I-albumin over the study period (1 h) were used to measure the liquid clearance from the distal air spaces as done before by our laboratory (37, 45). There is a good correlation between the changes in the concentration of instilled nonlabeled bovine albumin and those of 125I-albumin. Because some reabsorption may have occurred across distal bronchial epithelium, the term alveolar does not imply that all fluid reabsorption occurred at the alveolar level.
Tracer binding measurement. To determine 125I binding to albumin, trichloroacetic acid (20%) was added to all tubes, which were then centrifuged to obtain the supernatant for the measurement of free 125I radioactivity. The results are expressed as a percentage of the unbound 125I radioactivity to the total amount of 125I-albumin radioactivity instilled. These fluid samples always had <1% of unbound iodine present.
Determination of plasma concentration of
epinephrine. In all experiments, the plasma
concentration of epinephrine was measured. One milliliter of blood was
collected in a heparinized tube just before the onset of hemorrhagic
shock, at the end of the ischemic phase of hemorrhagic shock, and at
the end of each experiment. Blood samples were immediately centrifuged
at 14,000 rpm for 10 min at 4°C; 0.5 ml of plasma was transferred
to an Eppendorf tube and quickly frozen to 70°C in acetone
and dry ice. Samples were stored at
70°C until analyzed.
Plasma and distal air space fluid samples were spiked with an internal
standard and absorbed on activated alumina at pH 8.6. Epinephrine was
eluted with 0.1 M perchloric acid, analyzed by reverse-phase HPLC with
a C8 column, and measured by the
amperometric method with an electrochemical detector. The recovery
efficiency of plasma epinephrine was 60-70%. The correlation
coefficient and detection limit of this method were 0.96 and 10 pg/ml, respectively.
Measurement of blood neutrophil count. On the day before the experiment, depletion of circulating neutrophils in vinblastine-pretreated rats was confirmed by evidence of <200 polymorphonuclear leukocytes/ml blood on a blood smear stained with modified May-Grünwald Giemsa.
Measurement of GSH and GSSG levels in lung epithelial
lining fluid. For determining the levels of GSH in lung
epithelial lining fluid, we used a variation of the HPLC method used by
Martin and White (32). Briefly, bronchoalveolar lavage fluid was
collected, and the cells were spun down at 14,000 rpm for 10 min at
4°C. Each sample was then extracted in 5% perchloric acid with 0.2 M boric acid and -glutamyl-glutamate (10 µM) as an
internal standard. Iodoacetic acid (final concentration 7 mM) was
added, and the pH was adjusted to 9.0 ± 0.2. After incubation for
20 min to obtain S-carboxymethyl
derivatives of thiols, dansyl chloride was added, and the samples were
incubated for 24 h in the dark. The samples were then separated on an
amine column with the solvents described by Reed et al. (49).
Fluorescence detection was used for separation and quantification of
the dansyl derivatives. The dilution of the lung epithelial lining
fluid from the saline lavage was estimated by concomitant measurements
of urea in the plasma and lavage fluid (50). Additionally, levels of
GSSG were measured in the lung lavage fluid samples in parallel with
the levels of GSH. From the lung lavage fluid, glutathione levels (GSH
and GSSG) are expressed as a corrected concentration (in µM) in the
epithelial lining fluid.
Myeloperoxidase activity assay. Myeloperoxidase (MPO) activity in the lung was measured with a modification of the method described by Mullane et al. (39). Briefly, lungs isolated from the rats were washed, blotted dry, and frozen in liquid nitrogen. After being weighed, the frozen lungs were homogenized in 2 ml of 50 mM phosphate buffer, pH 6.0, containing hexadecyltrimethylammonium bromide (0.5 g/100 ml). The samples were then freeze-thawed four times with liquid nitrogen and a 37°C water bath, sonicated, and centrifuged at 16,000 g for 20 min at 4°C. The supernatant was assayed in triplicate for activity in a hydrogen peroxide-o-dianisine buffer at 460 nm. The intra-assay coefficient of variation was 8 ± 2%. Results are expressed as units of MPO activity per gram of lung tissue.
Statistics
All the data are summarized as means ± SE. One-way analysis of variance and the Fisher's exact t-test were used to compare experimental with control groups. Nonparametric tests were used to compare plasma epinephrine values measured between experimental and control groups and concentrations of GSH and GSSG in alveolar epithelial lining fluid. A P value of <0.05 was considered significant. ![]() |
RESULTS |
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Effects of Prolonged Hemorrhagic Shock and Fluid Resuscitation
To induce severe hemorrhagic shock and maintain a systemic mean arterial pressure of 30-35 mmHg for 60 min, 35 ± 3% of the blood volume was withdrawn. This corresponded to the removal of 9.1 ± 1.4 ml of blood. The development of prolonged hemorrhagic shock resulted in severe systemic arterial hypotension (Fig. 2A) and metabolic acidosis (Fig. 2B). The final calculated base deficit of the arterial blood at the end of the ischemic phase of prolonged hemorrhagic shock was
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Prolonged hemorrhagic shock was associated with a significant increase
in the endogenous release of catecholamines. There was a 20-fold
increase in the epinephrine plasma levels at the end of the ischemic
phase of hemorrhagic shock (2,414 ± 381 vs. 83 ± 15 pg/ml;
P < 0.05; Table
1). Mean plasma levels of epinephrine remained significantly elevated until the end of the study in the
hemorrhaged and resuscitated rats compared with those in the control
rats (Table 1). There was also a significant threefold increase in lung
MPO activity in rats that were hemorrhaged and fluid resuscitated
compared with that in control rats (9.46 ± 1.02 vs. 3.22 ± 0.51 U/g lung tissue; P < 0.05).
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However, in contrast to our results in rats that underwent short-term
hemorrhagic shock and fluid resuscitation (37), there was no
upregulation of alveolar liquid clearance after prolonged hemorrhagic
shock. The final-to-initial distal air space unlabeled protein
concentration was 1.27 ± 0.07 g/100 ml in control rats and 1.26 ± 0.07 g/100 ml in hemorrhaged rats (Table
2). Alveolar liquid clearance was
comparable in control and hemorrhaged rats measured either with the
nonlabeled bovine albumin (34 ± 4 vs. 36 ± 3%; Table 2) or
with 125I-albumin (26 ± 0.07 vs. 27 ± 0.07%).
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Hemorrhagic shock and fluid resuscitation were associated with a modest
increase in the endothelial permeability to protein as indicated by the
significant increase in extravascular plasma equivalents of the
noninstilled lung in hemorrhaged rats compared with that in control
rats (Table 3). Although the water-to-dry weight ratio of the noninstilled lung was not significantly different between hemorrhaged and control rats, the values measured after hemorrhagic shock and fluid resuscitation were significantly increased compared with those measured immediately after the ischemic phase of
hemorrhagic shock (Table 4).
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Finally, there was no increase in the protein flux
(125I-albumin) from the air spaces
to the plasma. The percentage of instilled 125I-albumin recovered from the
lungs and blood at the end of the experiments was comparable in
hemorrhaged and control rats (Table 5).
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Stimulation with Exogenous -Adrenergic Agonists
After Prolonged Hemorrhagic Shock
In rats that underwent prolonged hemorrhagic shock and fluid
resuscitation, when epinephrine was administered intravenously (Fig.
3A) or
in the distal air spaces (Fig. 3B),
the expected increase in alveolar liquid clearance did not occur.
Comparable results were obtained when salmeterol
105 M
(n = 4 rats) was added to the protein
solution instilled in the distal air spaces of the lung. In contrast,
there was a significant increase in alveolar liquid clearance in
control rats that were treated with epinephrine either intravenously
(34 ± 4 vs. 44 ± 3%; Fig.
3A) or into the distal air spaces
(34 ± 4 vs. 44 ± 3%; Fig.
3B). Comparable results were
obtained when salmeterol
(10
5 M;
n = 3 rats) was instilled in the
distal air spaces of control rats (34 ± 4 vs. 42 ± 2%).
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The severity of systemic arterial hypotension, metabolic acidosis, and
calculated base deficit was comparable in rats that underwent prolonged
hemorrhagic shock and were receiving exogenous -adrenergic agonists
and in hemorrhaged rats that did not have any treatment.
Aministration of an Exogenous -Adrenergic Antagonist
After Prolonged Hemorrhagic Shock
The severity of systemic arterial hypotension, metabolic acidosis, and calculated base deficit was comparable in rats that underwent prolonged hemorrhagic shock and were instilled with propranolol and in hemorrhaged rats that did not have any treatment.
Neutrophil Depletion Experiments
Because the administration of exogenousNeutrophil depletion with vinblastine 4 days before the onset of
hemorrhagic shock completely restored the ability of the alveolar
epithelium to upregulate alveolar liquid clearance in response to the
instillation of epinephrine
(105 M) into the distal air
spaces of the lung (Fig.
4B).
Comparable results were obtained from the first control group of rats
that were neutrophil depleted but did not undergo prolonged hemorrhagic shock (Fig. 4A). To rule out an
effect of vinblastine separate from its neutrophil depletion effect,
rats that were given vinblastine 1 day before hemorrhagic shock and had
a normal blood neutrophil count were studied. These rats did not show
an upregulation of alveolar liquid clearance compared with rats that
underwent a prolonged hemorrhagic shock (36 ± 1 vs. 36 ± 3%).
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Interestingly, neutrophil depletion with vinblastine 4 days before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were depleted in neutrophils before the onset of shock compared with the values measured in hemorrhaged rats with a normal neutrophil count (Table 3).
There was no difference in the severity of hemorrhagic shock (mean arterial pressure, arterial pH, and base deficit; data not shown) in the plasma epinephrine levels (Table 1) and in the protein flux (125I-albumin) from the air spaces to the plasma (Table 5) between rats depleted of neutrophils 4 days before hemorrhagic shock and in rats with normal neutrophil counts. Comparable results were obtained for the rats that were given vinblastine 1 day before hemorrhagic shock (second control group).
Pretreatment With an Oxygen Radical Scavenger, NAC
Because neutrophil depletion with vinblastine before hemorrhagic shock was associated with a restoration of the response of the alveolar epithelium toProlonged hemorrhagic shock significantly decreased GSH levels and the
GSH-to-GSSG ratio in alveolar epithelial lining fluid (Table
6). However, there was a complete
normalization of these glutathione levels after pretreatment with NAC.
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Pretreatment with NAC 30 min before the onset of hemorrhagic shock
completely restored the ability of the alveolar epithelium to
upregulate alveolar fluid clearance in response to the instillation of
epinephrine (105 M) in the
distal air spaces (49 ± 3 vs. 36 ± 3%; Fig.
5B).
Control studies of the effect of NAC in nonhemorrhaged rats treated
with epinephrine showed no effect of NAC under these conditions (Fig. 5A).
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Interestingly, pretreatment with NAC 30 min before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were pretreated with NAC 30 min before the onset of hemorrhagic shock compared with the values measured in hemorrhaged rats that were not pretreated with NAC (Table 3).
Systemic arterial hypotension, metabolic acidosis, calculated base deficit (data not shown), plasma epinephrine levels (Table 1), and protein flux (125I-albumin) from the air spaces to the plasma (Table 5) were comparable in rats that underwent prolonged hemorrhagic shock and were pretreated with NAC and in hemorrhaged rats that did not have any pretreatment.
XO Depletion Experiments
To exclude an effect of NAC that was not primarily related to its oxidant-scavenging properties, an additional series of experiments was done to inhibit the conversion from xanthine dehydrogenase to XO with a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet in hemorrhaged and fluid-resuscitated rats.There was a restoration of the normal upregulation of alveolar liquid
clearance in hemorrhaged rats that were fed either a tungsten-enriched
or an allopurinol-supplemented diet before the onset of hemorrhagic
shock (Fig.
6B).
Control studies of the effect of XO depletion in nonhemorrhaged rats
treated with epinephrine showed no effect of XO depletion under these
conditions (Fig. 6A).
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Depletion of XO before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were fed either a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet before the onset of hemorrhagic shock compared with the values measured in hemorrhaged nonpretreated rats (Table 3).
There was no difference in the severity of hemorrhagic shock (mean arterial pressure, arterial pH, and base deficit) and in the protein flux (125I-albumin) from the air spaces to the plasma (Table 5) between rats fed either a tungsten-enriched or an allopurinol-supplemented diet before the onset of hemorrhagic shock and in hemorrhaged rats that did not have any pretreatment.
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DISCUSSION |
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The overall objective of these studies was to determine the mechanisms that regulate fluid transport across the alveolar epithelium after prolonged hemorrhagic shock, one of the common causes of acute lung injury in humans (26). Because preservation of the capacity of the alveolar epithelium to actively remove fluid from the air spaces is critical for the survival of patients with acute lung injury (34), we developed an experimental animal model to investigate the effect of hemorrhagic shock on the function of the alveolar epithelium. The results of a previous study by Pittet et al. (44) indicate that the capacity of alveolar epithelium to remove fluid from the air spaces was maintained during the ischemic phase of hemorrhagic shock. In fact, there was an upregulation of the vectorial, sodium-dependent fluid transport across the alveolar epithelium secondary to the shock-mediated release of endogenous catecholamines (44). This protective mechanism against alveolar flooding was also sustained during the first 6 h after fluid resuscitation in the clinical setting of brief, severe hemorrhagic shock (37). However, the effect of prolonged hemorrhagic shock and fluid resuscitation on the function of the alveolar epithelium has not been studied.
Therefore, the first objective of these studies was to determine
whether the endogenous release of catecholamines can upregulate alveolar fluid clearance after prolonged hemorrhagic shock and fluid
resuscitation. The results indicate that although basal alveolar fluid
clearance was maintained, there was no upregulation of alveolar fluid
clearance across the alveolar epithelium in rats that underwent
prolonged hemorrhagic shock (1 h) followed by fluid resuscitation
despite a 20-fold increase in the epinephrine plasma levels at the end
of the ischemic phase of hemorrhagic shock. Mean plasma levels of
epinephrine remained significantly elevated until the end of the study
in the hemorrhaged and resuscitated rats compared with those in control
rats. In addition, there was no upregulation of alveolar liquid
clearance when the response of -adrenergic receptors was maximized
by the administration of exogenous
-adrenergic agonists in
hemorrhaged and fluid-resuscitated rats. Thus the results of these
studies indicate a failure of the alveolar epithelium to respond to
-adrenergic agonists, with the upregulation of vectorial fluid
transport after prolonged hemorrhagic shock and fluid resuscitation.
The inability of the alveolar epithelium to respond to -adrenergic
agonists could be explained by functional desensitization and
downregulation of alveolar epithelial
-adrenergic receptors after
prolonged stimulation by those agonists. In vivo (59) and in vitro (28)
exposure of lung epithelial cells to
-adrenergic agonists can result
in desensitization and downregulation of
2-adrenergic receptors by
mechanisms involving an increase in the rate of receptor protein
degradation. In contrast, the cAMP response to forskolin was unaffected
by prior in vivo stimulation with
-adrenergic agonists (59), and
exposure to forskolin did not affect the
2-adrenergic-receptor density
of cultured human airway epithelial cell monolayers (28). These results
suggest that the activation of
-adrenergic-receptor kinases is
involved in this mechanism because these kinases require receptor
occupancy to exert their effects, are highly expressed in the lung, and
play an important role in mediating rapid catecholamine-induced
-adrenergic-receptor desensitization in human airway epithelial
cells (29). However, there are several reasons why it is unlikely that
the exposure to
-adrenergic agonists in these studies is responsible
for the failure of the alveolar epithelium to upregulate alveolar fluid clearance in response to those agonists after prolonged hemorrhagic shock. First, catecholamine-induced downregulation of
2-adrenergic receptors was
observed after a longer time exposure to
-adrenergic agonists (24 h
or more) than in our present experiments (6 h), although some
2-adrenergic-receptor
desensitization may already occur in humans after a shorter exposure to
-adrenergic agonists (42). Second, it has recently been reported
(16) that the catecholamine-dependent upregulation of alveolar fluid
transport is reversible when plasma epinephrine levels return to normal values after the administration of a constant intravenous epinephrine infusion in rats is stopped. Moreover, in that study (16), the administration of a second infusion of epinephrine 120 min after the
first infusion was stopped and the plasma levels of epinephrine were
normalized caused an upregulation of the vectorial fluid transport
across the alveolar epithelium comparable with that observed after the
first epinephrine infusion. Third, in the present experiments, control
nonhemorrhaged rats that were stimulated with endogenous and/or
exogenous epinephrine for 6 h had a significant upregulation of
alveolar fluid clearance that was associated with a >20-fold increase
in plasma levels of epinephrine (Table 1, Fig. 3). Fourth, Modelska et
al. (37) previously reported that shock-mediated endogenous release of
epinephrine caused a significant upregulation of alveolar fluid
clearance in rats that underwent a brief period of severe hemorrhagic
shock followed by fluid resuscitation. Therefore, these results and the
data reported here indicate that desensitization of the
-adrenergic
receptors is not likely to be responsible for the failure of the
alveolar epithelium to respond to
-adrenergic agonists by
upregulating alveolar fluid clearance. Interestingly, the results of a
recent study (40) indicate that catecholamines and agents that increase
cAMP cause a transient increase in chloride secretion by the alveolar
epithelium of rabbits in vivo. The importance of this
catecholamine-mediated chloride secretion by the alveolar epithelium in
the present experimental model of hemorrhagic shock is unknown and
needs to be further studied. Therefore, the mechanisms responsible for
this lack of effect of
-adrenergic agonists on alveolar fluid
clearance after prolonged hemorrhagic shock are not completely
understood and need to be elucidated in future studies.
We hypothesized that a neutrophil-dependent injury to the alveolar
epithelium might account for the absence of upregulation of alveolar
fluid clearance after prolonged hemorrhagic shock because there was a
significant increase in the lung MPO activity of hemorrhaged and
fluid-resuscitated rats compared with the values measured in control
rats. Therefore, rats were pretreated with vinblastine, which caused a
99% depletion of circulating blood neutrophils. This pretreatment has
previously been shown to markedly decrease the marginated pool of lung
neutrophils as measured by MPO content in the lung and by histology
(2). The results indicate that depletion of neutrophils by
administration of vinblastine 4 days before the induction of prolonged
hemorrhagic shock restored the ability of the alveolar epithelium to
respond to -adrenergic agonists by upregulating alveolar fluid
clearance (Fig. 4). This protective effect was not due to a nonspecific
effect of vinblastine because it did not occur in hemorrhaged rats that
were pretreated only 1 day before hemorrhagic shock and had a normal
blood neutrophil count. Also, plasma epinephrine levels measured in
hemorrhaged rats depleted of their neutrophils were in the same range
as those measured in henorrhaged rats that had a normal neutrophil
count, thus excluding that these results could simply be explained by a
difference in plasma levels of epinephrine between both groups (Table
1). Thus these results indicate a neutrophil-dependent mechanism to
explain the decrease in alveolar epithelial fluid transport after
prolonged hemorrhagic shock.
In a model of hemorrhagic shock in rats that is comparable to the model used in the present study, neutrophil sequestration occurred in the lungs within 1 h after fluid resuscitation by an XO-related mechanism as indicated by a significant increase in lung MPO activity in hemorrhaged compared with control rats (3). Importantly, there was no increase in lung neutrophil sequestration in carefully matched sham control rats because anesthesia, catheter placement, and surgical stress could have caused a transient accumulation of neutrophils in the lungs (3). Moreover, neutrophil sequestration in the lung has been shown to contribute to diffuse lung endothelial injury induced by hemorrhagic shock (7, 17, 60). Also, the degree of activation of circulating neutrophils before the induction of shock is associated with the severity of shock-induced endothelial injury (6). However, the data in this study are the first results that demonstrate a link between neutrophil-mediated endothelial injury (as shown in this study by a modest increase in the endothelial permeability to protein) and a decrease in alveolar epithelial fluid transport that occurred without a major increase in alveolar epithelial permeability to protein. The data may be germane to human acute lung injury because both lung endothelial and epithelial injuries occur in patients with acute lung injury (5). Also, the severity of neutrophilic alveolar inflammation correlates with the mortality rate in a study of patients with acute lung injury (57). Finally, the functional status of the lung epithelium is likely to be a major factor that determines which patients develop alveolar flooding and prolonged respiratory failure after acute lung injury (33, 34).
The final objective of these studies was to test the hypothesis that neutrophil-induced injury to the fluid transport capacity of the alveolar epithelium was associated with an oxidative stress to the lung. Because glutathione has been shown to be one of the major oxidant scavengers in the alveolar epithelial lining fluid and a good marker for oxidative stress in the lung (51), GSH and GSSG levels were measured in the alveolar epithelial lining fluid of control and hemorrhaged rats. Prolonged hemorrhagic shock significantly decreased GSH levels and the GSH-to-GSSG ratio in alveolar epithelial lining fluid (Table 6), indicating the presence of an oxidative stress in the lung of hemorrhaged and fluid-resuscitated rats.
Evidence of oxidative stress has previously been reported in the lungs of animals and patients who died with clinical lung injury (21). Interestingly, the results of a morphological study (1) demonstrate that one of the predominant localizations of oxidizing species in the lung after ischemia-reperfusion was in alveolar epithelial type II cells. Reactive oxygen intermediates (e.g., H2O2) in vitro cause a significant depletion in intracellular GSH associated with an efflux of GSSG and a significant increase in the formation of protein-mixed disulfides (48). In humans, there is evidence of a significant reduction in the GSH-to-GSSG ratio in the epithelial lining fluid of patients with acute lung injury compared with normal subjects and patients with cardiogenic edema (15, 41). Similarly, plasma-oxidized glutathione is a marker of ischemia-reperfusion associated with single-lung transplantation (27). Moreover, there was a depletion in total GSH in the alveolar epithelial lining fluid of patients with a lung allograft (8). Thus the detection of a decrease in GSH and in the GSH-to-GSSG ratio in alveolar epithelial lining fluid of hemorrhaged rats indicates that the shock-mediated alveolar epithelial dysfunction was associated with the presence of oxidative injury to this barrier.
Therefore, to determine whether the inability of the
-adrenergic-receptor agonists to upregulate vectorial fluid
transport across the alveolar epithelium after prolonged hemorrhagic
shock and fluid resuscitation was mediated through oxidant-mediated mechanisms, hemorrhaged rats were pretreated or treated with NAC, a
glutathione precursor and an oxygen radical scavenger. The results indicate that pretreatment with NAC restored the ability of the alveolar epithelium to respond to
-adrenergic agonists by
upregulating alveolar fluid clearance in hemorrhaged and
fluid-resuscitated rats. The restoration of the response of the
alveolar epithelium to
-adrenergic agonists after the administration
of NAC was associated with a normalization of GSH levels and the
GSH-to-GSSG ratio in alveolar epithelial lining fluid of hemorrhaged
rats. Administration of NAC has previously been shown to decrease
interleukin-1-induced neutrophil influx and lung leak in rats (30), to
reduce endotoxin-induced neutrophil activation in sheep (9), to protect
against phosgene lung injury in rabbits (55), and to reduce pulmonary
endothelial cell damage induced by immune complexes deposition in rats
(52). However, the present results are the first to demonstrate that NAC is able to correct the shock-mediated decrease in alveolar epithelial fluid transport function.
There are several mechanisms that may explain the protective effect of
NAC against the shock-mediated oxidative injury to the alveolar
epithelium. First, NAC may act by increasing intracellular and
epithelial lining fluid GSH levels because it is a thiol-containing compound that is easily deacetylated to form cysteine, which can contribute to the synthesis of GSH (38). In fact, restoration of normal
GSH levels in the alveolar epithelial lining fluid has been observed
after intravenous administration of NAC in patients with idiopathic
pulmonary fibrosis (35). Also, increases in intracellular and
epithelial lining fluid GSH levels have been reported to protect in
vitro rat alveolar type II cells against paraquat-induced cytoxicity
(13, 23, 24) and in vivo preterm rabbits from oxidative lung injury
(14). In addition to its direct extracellular antioxidant effect, GSH
may protect signal transduction in alveolar epithelial type II cells by
preventing oxidant-mediated inhibition of second messenger generation
(inositol triphosphate and cAMP), events that are important for
surfactant secretion and active ion transport by these cells (12).
Also, NAC may have reversed the oxidant-mediated inhibition in ATP
synthesis in alveolar epithelial cells (22). Second, NAC could act as a
direct scavenger of oxygen radicals. NAC effectively inactivates hypochlorous acid and hydroxyl radical while minimally scavenging superoxide anion and
H2O2
in vitro (4). Because all of these oxidants are produced by
neutrophils, direct scavenging of neutrophil-derived oxygen radicals
may contribute to protection of the alveolar epithelial transport
function after the administration of NAC in hemorrhaged rats. Third,
NAC may also inhibit oxidant-mediated nuclear factor (NF)-B
activation, cytokine and chemokine gene activation, and neutrophilic
lung inflammation. Exposure of cells to reactive oxygen species has
been shown to activate the transcription factor NF-
B (53). Moreover,
NAC may block activation of NF-
B in vitro (53, 56) and in vivo after
intraperitoneal administration of Escherichia
coli endotoxin in rats (10). This mechanism may play an
important role in our experimental animal model because it has recently
been reported (31) that prolonged hemorrhagic shock (1 h) causes
activation of NF-
B and a secondary increase in the expression of
proinflammatory cytokines such as tumor necrosis factor-
,
interleukin-1
, and transforming growth factor-
in lung
mononuclear cells in mice.
To confirm the contribution of oxidative injury to the alveolar
epithelium in these studies, we carried out additional experiments in
which the conversion of xanthine dehydrogenase to reactive oxygen
species-generating XO was inhibited by two different strategies, either
by a tungsten-enriched diet or by pretreatment with allopurinol. Production of XO in hemorrhagic shock causes neutrophil sequestration in the lung (3) and activation of proinflammatory cytokine expression
in lung mononuclear cells (54). Then, activated neutrophils induce
conversion of xanthine dehydrogenase to XO by causing secretion of
elastase in close proximity to lung endothelial cells (43). The results
of these studies showed that the inhibition of XO formation by either
of the inhibitors restored the ability of the alveolar epithelium to
respond to -adrenergic stimulation after prolonged hemorrhagic shock
(Fig. 6). Therefore, taken together, these data provide in vivo
evidence for the presence of oxidative injury of the alveolar
epithelium after prolonged hemorrhagic shock. Other investigators (20)
have reported that oxidant injury to isolated alveolar epithelial type
II cells decreases active transport of sodium and water across these
epithelial monolayers. The present results may also have important
clinical implications. It has recently been reported that plasma
hypoxanthine levels, a prooxidant substrate for XO, are significantly
higher in patients with acute lung injury than in mechanically
ventilated control patients. The higher plasma hypoxanthine levels were
associated with a loss of protein thiol groups and correlate with a
poor outcome in these patients (47).
In summary, the present data provide the first in vivo evidence for a neutrophil-dependent decrease in alveolar epithelial fluid transport without a major alteration in the paracellular permeability to protein after prolonged hemorrhagic shock and fluid resuscitation. Because the expected catecholamine-mediated upregulation of alveolar liquid clearance was restored by normalization of the concentration of GSH in the alveolar epithelial lining fluid by NAC or by inhibition of the conversion of xanthine dehydrogenase to XO, these experiments also indicate that the decrease in the fluid transport capacity of the alveolar epithelium is mediated by an oxidant stress.
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ACKNOWLEDGEMENTS |
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We thank Ricardo Ciriales for technical assistance in carrying out these studies.
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FOOTNOTES |
---|
This work was primarily supported by National Heart, Lung, and Blood Institute Grant HL-51854. This project was also supported in part by a research grant (to J. F. Pittet) and a fellowship grant (to K. Modelska) from the American Lung Association, California.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. F. Pittet, Dept. of Anesthesia, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110 (E-mail: jean_pittet{at}quickmail.ucsf.edu).
Received 20 August 1998; accepted in final form 27 January 1999.
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