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1 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130; and 2 Department of Animal Physiology, Lund University, S-223 63 Lund, Sweden
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our recent report (Garat C, Carter EP, and Matthay MA. J Appl Physiol 84: 1763-1767, 1998) described a new method to measure alveolar fluid clearance (AFC) in an in situ mouse preparation. However, in vivo preparations may be more suitable for studying alveolar fluid transport under some pathological conditions. Therefore, we developed a ventilated mouse model and compared AFC in the ventilated and the in situ mouse models. After 15 min, AFC was similar in both groups, but, after 30 min, AFC was 38% slower in the in situ mice (P < 0.05). Bilateral adrenalectomy and propranolol did not inhibit AFC after 15 min. Amiloride inhibited 90% of AFC in both groups. To evaluate the mechanism for the slower AFC in the in situ mouse preparation, we measured the extravascular lung water and calculated interstitial fluid volume. Extravascular lung water and interstitial fluid volume were greater in the in situ mice than in the ventilated mice at 30 min (P < 0.05). These results indicate that mouse AFC is fast, highly amiloride sensitive, and independent of endogenous catecholamines during the first 15 min. Accumulation of interstitial fluid probably plays an important role in slowing AFC in the in situ mouse lung model at later time intervals. These mouse models will be useful to quantify alveolar epithelial fluid transport under pathological conditions.
pulmonary edema; acute lung injury; epithelial transport; lung fluid balance
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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STUDIES OF ALVEOLAR FLUID clearance in the mouse make it possible to test the contribution of specific transport genes to the regulation of alveolar fluid clearance. Although we developed murine models to study alveolar fluid clearance in the in situ mouse lung (16, 20) and the isolated perfused lung (2), prior studies in ex vivo rat and human lung models indicate that these ex vivo preparations (4, 32) underestimate in vivo rates (38) of alveolar fluid clearance. Furthermore, in vivo preparations may be more suitable for studying alveolar fluid transport under pathological conditions (5, 6, 15, 17, 23, 27, 30, 31, 38).
Therefore, the first objective was to develop a new, ventilated mouse model to compare alveolar fluid clearance with that in our recent in situ mouse model (16). Once the ventilated model was successfully established, the second objective was to test the hypothesis that the rate of alveolar fluid clearance would be slower in the in situ mouse model than in the ventilated mice. Because we discovered that alveolar fluid clearance slowed in the in situ mouse model at 30 min compared with that in the ventilated model, the third objective was to determine the mechanisms of the slower clearance. We hypothesized that the slower alveolar fluid clearance rate in the in situ mouse lung was directly related to the accumulation of interstitial fluid in the nonperfused lung.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Male CD-1 mice (Benton-Kingman, Fremont, CA) that weighed 25-35 g were used for all experiments. The mice were housed in air-filtered, temperature-controlled units with food and water. All procedures conformed to the guidelines of the University of California at San Francisco Committee on Animal Research.
Preparation of Instillate
The instillate consisted of 5 g/100 ml bovine serum albumin (Sigma Chemical, St. Louis, MO) with Ringers lactate that was adjusted to 340 mosmol/kgH2O to be isosmolar with mouse plasma. We adjusted the osmolality of the instillate because a recent study from our laboratory showed that normal plasma osmolality is higher (336 ± 12 mosmol/kgH2O) in mice than in other species (1). To evaluate an effect of plasma osmolality of the instillate in murine alveolar fluid clearance, we compared the 275 mosmol/kgH2O instillate that we had used in our prior mouse study (16) with the 340 mosmol/kgH2O instillate in the in situ mice at 60 min. The 340 mosmol/kgH2O instillate showed a 20% slower clearance than the 275 mosmol/kgH2O instillate. Our 275 mosmol/kgH2O clearance data were similar to our previously reported values (16). A recent report suggested that an osmolality difference may have an interesting role in alveolar fluid clearance (20). This difference in clearance was caused by the osmolality differences; thus we have used the 340 mosmol/kgH2O instillate in all of our subsequent mouse studies. Amiloride and propranolol (Sigma Chemical) were added to the instillate in selected studies, as described in the specific protocols. We also added 0.1 µCi of 131I-labeled albumin (Merck-Frosst, Montreal, PQ, Canada) to the instillate as a labeled alveolar protein tracer.Surgical Preparation and Ventilation
In situ mouse model. Mice were killed by an overdose of pentobarbital sodium (200 mg/kg ip). A tracheostomy was done with a 20-gauge, trimmed Angiocath plastic needle (Becton Dickinson, Sandy, UT). The lungs were inflated with 7 cmH2O continuous positive airway pressure with 100% oxygen throughout the experiment. Body temperature was maintained at 37-38°C by an infrared lamp (Fisher, Santa Clara, CA) placed 30 cm above the body. The lamp was cycled on and off to maintain the core temperature at 37-38°C. A temperature probe (Yellow Springs Instrument, Yellow Springs, OH) was inserted via a 0.5-cm incision into the abdominal cavity to monitor the core temperature throughout the experiment. In preliminary experiments, we compared the core temperature between the thoracic cavity and abdominal cavity. The difference between abdominal and thoracic temperatures showed less than a 0.3°C difference throughout the experiments.
Ventilated mouse model. Mice were anesthetized with pentobarbital sodium (50 mg/kg ip). A tracheostomy was done, and the lungs were mechanically ventilated by a small-animal ventilator (Harvard, Millis, MA) with an inspired oxygen fraction of 1.0, tidal volume of 9-10 ml/kg, a respiratory rate of 90 breaths/min, peak airway pressure of 8-12 cmH2O, and a positive end-expiratory pressure of 2-3 cmH2O. Body temperature was kept constant at 37-38°C by the infrared lamp. It was cycled on and off to maintain the core temperature of mice.
Adrenalectomy. To test the possible contribution of endogenous epinephrine to alveolar fluid clearance in mice, one group of mice was anesthetized with inhaled methoxyflurane (Mallinckrodt Veterinary, Mundelein, IL). Both adrenal glands were resected via a dorsal skin incision. After surgery, the wounds in both the muscle layer and the skin were closed. After 24 h, the mice were used for experiments. In three studies, the plasma epinephrine level was not detectable in plasma samples obtained at 24 h (data not shown).
General Protocol
In situ mouse model. For all studies in both the in situ and the ventilated models, 10-13 ml/kg of the 5% albumin Ringer lactate solution with 131I-albumin was used. The instillate was delivered over 30 s into both lungs through the tracheal cannula. All mice were given 0.1 ml of 125I-albumin intraperitoneally as a vascular tracer 2 h before the experiment. At the end of experiment, the lungs were removed through a median sternotomy and a blood sample was obtained from the heart. An alveolar fluid sample (0.05-0.10 ml) was aspirated with a 1-ml syringe that was connected directly to a 20-gauge Angiocath. The aspirate was centrifuged at 3,000 g for 10 min, and the supernatant of the fluid was used to measure the total protein concentration and radioactivity.
Ventilated mouse model. All mice were injected with 125I-albumin (0.1 µCi) intraperitoneally 2 h before the experiments. This tracer was used to calculate the flux of plasma protein into the air spaces as in our prior studies (17, 31, 36). In all experiments, a 20-min baseline was required before alveolar fluid instillation. Half of the instillate volume was delivered to both lungs every 5 min over 30 s via the trachea with a small catheter (PE-10) and a syringe to prevent backward flow into the tracheostomy tube. We used the end of first instillation as time 0 in this model. At the end of experimental period, the aspirate was collected from the distal air spaces of the lung (see above) and centrifuged to obtain the supernatant. Simultaneously, the blood sample was obtained by cardiac puncture. The lungs were removed through a sternotomy. The total protein concentration and radioactivity were measured on the alveolar samples.
Specific Protocols
Group 1 consisted of time-course studies of alveolar fluid clearance in the in situ mouse model (n = 60). Alveolar fluid clearance in the in situ mice was determined at 15 min (n = 10), 30 min (n = 9), 60 min (n = 9), 90 min (n = 10), 120 min (n = 9), 180 min (n = 10), and 240 min (n = 3).
Group 2 consisted of time-course studies of alveolar fluid clearance in ventilated mice (n = 20). Alveolar fluid clearance was measured in ventilated mice at 15 min (n = 9), 30 min (n = 7), and 60 min (n = 4).
Group 3 tested the effect of adrenalectomy on alveolar fluid clearance in both ventilated and in situ mouse models (n = 25). To determine the effect of endogenous epinephrine on alveolar fluid clearance, we used adrenalectomized mice. Alveolar fluid clearance was measured at 15 min in both ventilated (n = 8) and in situ (n = 9) mice and at 30 min in ventilated mice (n = 8).
Group 4 tested the effect of mechanical ventilation on alveolar fluid clearance in the in situ mice (n = 10). To determine the effect of mechanical ventilation on alveolar fluid clearance, in situ mice were ventilated according to the ventilation protocol.
Group 5
tested the effect of amiloride (10
3 M) on alveolar fluid
clearance in both ventilated mice (n = 5) and in situ
mice (n = 8). The concentration of amiloride was used
according to our prior studies (16, 21). To
study the contribution of amiloride-sensitive sodium uptake during the
initial clearance period, we studied both the ventilated and the in
situ mice at 15 min.
Group 6
tested the effect of propranolol on alveolar fluid clearance
(n = 6). Propranolol (104 M) was added to
the instillate to determine whether the high rate of basal alveolar
fluid clearance over 15 min in the in situ mice was mediated by
stimulation of â-adrenergic receptors by endogenous epinephrine.
Measurement of Alveolar Fluid Clearance
According to our prior studies (6, 16, 21, 28), alveolar fluid clearance (% of instilled fluid) was calculated by measuring the increase in tracer-labeled albumin (131I-albumin) concentration of the instilled solution.Alveolar fluid clearance (AFC) was calculated as follows
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(1) |
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(2) |
1 · g1 (cpm/g) of the instilled protein tracer] and the
final (TPf) alveolar fluid protein concentration (expressed as cpm/g of
the concentration of the protein tracer in the final alveolar sample) and g is grams of fluid. Fr is the fraction of alveolar tracer (131I-albumin) protein that remains in the lung at the end
of experiment. To be sure that no major osmotic-induced change in
131I-albumin concentration occurred, we sampled the
alveolar compartment 1-2 min after instillation in the in situ mice.
Protein concentration was measured by gamma counting of the tracer protein. The concentrations of 131I-albumin and 125I-albumin were determined by gamma counting. 131I-albumin has a comparatively broader spectrum and overlapped the spectrum from 125I-albumin. Therefore, we subtracted the overlap from the 125I-albumin count.
Plasma Equivalents
To estimate clearance of plasma into the air spaces in selected studies, the plasma equivalents in the alveolar fluid were calculated with Eq. 3 (below) according to our prior studies (21, 36). This method was modified for the murine model. 125I-albumin was injected intraperitoneally 2 h before the experiment. To evaluate the circulating level of 125I-albumin in plasma, blood samples were obtained at the beginning of the experiment and the end of the experiment in both ventilated and in situ mice.
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(3) |
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Gravimetric Lung Water and Calculation of Interstitial Fluid Volume
To determine the water-to-dry weight ratio in the lungs of the mice in the ventilated and in situ models, standard methods were used as in prior studies (21, 36). Before the end of the mouse experiment, a blood sample was obtained for measurement of hemoglobin concentration and water-to-dry weight ratio of blood for the lung water calculation. The lungs were homogenized, and the extravascular lung water (E) was determined by calculating the water-to-dry weight ratio. The dry weight of the experimental lung was corrected for the dry weight of the instilled protein remaining in the lungs. This value was subtracted from the total dry weight of the experimental lung. The equation for extravascular lung water is
![E (g)<IT>=</IT>[We/(De<IT>−</IT>P)<IT>−</IT>Wc/Dc]<IT>×</IT>(De<IT>−</IT>P)](/content/vol89/issue2/fulltext/672/img006.gif)
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(4) |
A calculation of interstitial fluid volume was used to estimate the
fluid in the interstitial space of the lung. Interstitial fluid volume
should be equal to the total extravascular lung water minus the
alveolar fluid volume. Therefore, we measured extravascular lung water
and then calculated the residual alveolar fluid volume (instilled
volume minus the volume of fluid cleared from the air space)
![Interstitial fluid volume (g)<IT>=</IT>[We/(De<IT>−</IT>P)<IT>−</IT>Wc/Dc]](/content/vol89/issue2/fulltext/672/img007.gif)
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(5) |
Statistical Analysis
The data are summarized as means ± SE. Analysis of variance was used to compare the different animal groups. Where appropriate, an unpaired t-test was used. We regarded a P value of <0.05 as statistically significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Ventilated Vs. In Situ Mouse Alveolar Fluid Clearance and Effect of Amiloride and Endogenous Epinephrine Over 15 Min
Over 15 min, alveolar fluid clearance was very fast in both the in situ mice as well as in the ventilated mice (Fig. 1). Propranolol did not affect basal clearance in the in situ mice over 15 min (Fig. 1). Furthermore, amiloride inhibited over 90% of the fast clearance in both the ventilated and in situ mice at 15 min (Fig. 2).
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Ventilated Mice Vs. In Situ Mice Over Longer Time Periods
In the ventilated mice after 30 min, alveolar fluid clearance continued at an even faster rate so that nearly 30% of the alveolar fluid was removed by 30 min (Figs. 3 and 4). In contrast, the rate of alveolar fluid clearance slowed significantly in the in situ mice by 30 min (Fig. 4). Mechanical ventilation of the in situ mice for 30 min did not increase alveolar fluid clearance (Fig. 3). Also, adrenalectomized mice had slower clearance in the ventilated 30-min studies (Fig. 3).
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Mechanisms Responsible for Slower Alveolar Fluid Clearance in the In Situ Mouse
The rapid clearance continued over 60 min in the ventilated mice (Fig. 4). However, interestingly, alveolar fluid clearance slowed significantly in the in situ mice after 30 min. What mechanism accounted for the slower alveolar and lung fluid clearance in the in situ mouse model after 15 min? One potential explanation was that interstitial fluid was cleared more rapidly in the presence of perfusion. This hypothesis was suggested by the observation that extravascular lung water was lower in the ventilated mice at 15 min (P = 0.066) and at 30 min (P < 0.05) compared with that in the in situ mice (Fig. 5). In support of this hypothesis, longer time-course studies indicated that extravascular lung water declined to 6.0 ± 0.9 g H2O/g dry lung in the living ventilated mouse at 60 min, indicating that over 50% of the instilled fluid had been removed (Fig. 5). In contrast, in the in situ mouse studies, the extravascular lung water (Fig. 5) and alveolar fluid clearance (Fig. 4) declined more slowly. Finally, the in situ mice required 240 min to match the level of extravascular lung water measurement at 60 min in the ventilated mice (Fig. 5). Thus the half-time for lung liquid clearance was 15 min in the ventilated mice vs. 120 min in the in situ mice.
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The presence of positive-pressure ventilation in the ventilated mice
could have stimulated alveolar fluid clearance. Therefore, we
ventilated the in situ mice using the same protocol as in the ventilated mice. Alveolar fluid clearance in the in situ mice was
19.5% with mechanical ventilation for 30 min, a value that was not
higher than that in the nonventilated in situ mice (21.3%). Theoretically, it is also possible that there could have been increased
leakage of intravascular plasma into the lungs of the in situ mice, but
there was no evidence for this (Table 1).
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Finally, a direct calculation of interstitial fluid volume showed that
interstitial fluid volume was markedly elevated at 30 min in the in
situ mice compared with the ventilated mice (Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Recently, functional genomics have become useful for determining the role of specific phenotypes in lung fluid balance (2, 24). Because most of the studies resulting from functional genomic interventions are carried out in mouse models, it is important to develop functional lung models to study lung fluid balance in mice. We recently developed an in situ model (16) as well as an isolated mouse lung model (2) to evaluate mouse alveolar fluid clearance, but it was also important to develop a new ventilated mouse model to evaluate in vivo alveolar and lung fluid clearance to use in physiological genomics research. We achieved our first objective, which was to establish an in vivo preparation for studying alveolar fluid clearance in ventilated mice. Our working hypothesis was that alveolar fluid clearance in the ventilated mice would be faster than the in situ mice. The hypothesis was true but only after 15 min. Therefore, we developed a new working hypothesis that an increase of interstitial fluid volume might slow alveolar fluid clearance in the in situ mice.
The major findings of this study can be summarized as follows. 1) It is feasible to use ventilated or in situ mice for investigations of alveolar epithelial fluid transport. 2) Over 15 min, mouse alveolar fluid clearance was equal and fast in both the in situ mice and the live ventilated mice. 3) Basal fluid clearance was inhibited by 90% with amiloride in both the ventilated and the in situ mice. Also, during the first 15 min, basal alveolar fluid clearance was not dependent on endogenous epinephrine release. 4) Over 30 min, alveolar fluid clearance slowed in the in situ mice compared with the ventilated mice. The in situ mice had a higher extravascular lung water and interstitial fluid volume compared with measurements in the ventilated mice at the later time points.
Relation Between Composition of Instillate and Alveolar Fluid Clearance in Mice
In our previous mouse study, we used a 275 mosmol/kgH2O instillate for the in situ mouse preparation (16). However, subsequently, we discovered that the plasma osmolality in mice was 336 ± 12 mosmol/kgH2O, higher than in other mammalian species (1). Therefore, we used an instillate of 340 mosmol/kgH2O for these studies. We used 10-13 ml/kg of instillate volume in both the ventilated and the in situ mouse models. Previous investigators have used different relative instillate volumes, ranging from 1.5 to 20 ml/kg body wt (4, 16, 28). A potential contribution of the volume of the instillate was considered in a recent mouse study (16). The reason we selected this volume was to prevent death from hypoxia in the ventilated mice. To exclude a possible contribution of different instillate volumes (16), we used the same volume in the in situ model.Rapid Alveolar Fluid Clearance in Mice
In this study, alveolar fluid clearance was measured over 1 h in the ventilated mice and over 4 h in the in situ mice. Alveolar fluid clearance in the ventilated mice was 11.8 ± 1.6% in first 15 min. This is a remarkably high rate of alveolar fluid clearance compared with other mammalian species (5, 28, 29, 36). After 60 min, alveolar fluid clearance in ventilated mice was 50% of the instilled volume. We were not able to measure alveolar fluid clearance in the ventilated mouse model beyond 60 min because fluid could not be collected from the distal air spaces at later time points, probably due to the rapid rate of alveolar fluid clearance.Interestingly, alveolar fluid clearance in the in situ mice was similar to that in the ventilated mice during the first 15 min. The rapid alveolar fluid clearance in both the ventilated and in situ models was 140% faster than the alveolar fluid clearance in the isolated mouse lung model (2). Similar differences between the in vivo models and isolated models have been reported in rats (4, 21). Possible explanations for slower clearance in the isolated lung include altered lymphatic function (34), declining levels of ATP, and increased lung vascular permeability (14).
Mechanisms of Rapid Clearance in Mice
What mechanism accounts for the rapid clearance in both the ventilated and the in situ mice? Active transport of sodium is the major mechanism for removal of alveolar fluid (11, 25, 26). To evaluate the contribution of amiloride-sensitive sodium channels, we added 103 M
amiloride in the instillate. There was a 90% inhibition of alveolar
fluid clearance in both the ventilated and the in situ mouse models
during the first 15 min. A number of in vitro and in vivo studies have
documented an inhibitory effect of amiloride on alveolar fluid
clearance in other species (16, 21,
29, 36). However, the fraction of
amiloride-sensitive transport in the mouse was significantly higher
than in other species (4, 21,
29, 33, 36). Knock out of the
-subunit of ENaC (epithelial sodium channel) in the mouse (3)
indicated that this subunit is critical for removal of alveolar fluid
at birth in mice. Because this subunit is amiloride sensitive,
we speculate that the -subunit of ENaC may be more important in the
mouse than in other species for regulation of alveolar fluid clearance.
In rat lung (21) and human lung (36), for
instance, amiloride-sensitive alveolar fluid clearance accounts for
~40-50% of alveolar epithelial fluid transport. The high
dependence on amiloride-sensitive sodium uptake may be a unique
characteristic of alveolar epithelial fluid transport in mice.
Effect of Endogenous Epinephrine on Alveolar Fluid Clearance in Mice
To evaluate the contribution of endogenous epinephrine in this model, we tested adrenalectomized mice in both the ventilated and the in situ models. Bilateral adrenalectomy did not affect alveolar fluid clearance during the first 15 min in either model. Also, the concentration of plasma epinephrine was undetectable in the in situ mice. The addition of propranolol, a standard
-antagonist, to the
instillate in normal mice also did not inhibit basal alveolar fluid
clearance in the in situ mice. However, bilateral adrenalectomy decreased alveolar fluid clearance by 15% (P < 0.05)
in the ventilated mice at 30 min. Several reports have shown that
endogenous epinephrine can accelerate alveolar fluid clearance during
canine neurogenic pulmonary edema (23), in newborn guinea
pigs (15), and during septic shock in rats
(30). Newborn guinea pigs also showed high clearance rates
that depended on a -adrenergic mechanism (15). Mice
have comparatively faster clearances than other mammalian species, but
there was no apparent contribution of endogenous epinephrine to basal
alveolar fluid clearance in the first 15 min. In the next 15 min,
endogenous epinephrine in the ventilated mice upregulated alveolar
fluid clearance. Epinephrine may have increased in these mice after 15 min because mechanical ventilation was perhaps insufficient to maintain
a normal pH, an effect that would not have occurred during the first 15 min; because we did not measure arterial blood gases, we cannot
verify this possibility. This is one mechanism to explain part of the
difference between the ventilated mice and the in situ mice (Fig. 3).
Mechanisms for Slower Clearance in the In Situ Mouse Model
In this study, the ventilated model maintained a rapid fluid clearance over 1 h, but alveolar fluid clearance began to slow in the in situ model by 30 min. Is the in situ model useful to evaluate alveolar fluid clearance over several hours? Several studies have reported intact alveolar fluid clearance in several different preparations. For example, alveolar fluid clearance is maintained in sheep for 4 h with perfusion (34), for 1 h in an isolated perfused rat lung preparation (35), and also over 4 h in an ex vivo nonperfused human lung (33). In this study, mouse alveolar fluid clearance continued over 4 h in the in situ lung. Why did alveolar fluid clearance slow in the in situ mouse model? First, we tested for a change on lung vascular permeability using a protein vascular tracer at 30, 60, and 180 min in the in situ model. Several reports suggested that an increase in vascular permeability by oleic acid (10), septic shock (31), and endotoxin (17, 30, 37) could induce pulmonary edema and alveolar flooding (10, 31). Thus a change of vascular permeability could have influenced our extravascular lung water, interstitial fluid volume, and alveolar fluid clearance. However, vascular permeability in the in situ mouse lung did not change over the 3 h. Second, we tested for an effect of the lack of mechanical ventilation in the in situ mice compared with the ventilated mice. A group of the in situ mice were ventilated with the same protocol as the ventilated mouse for 30 min. However, the in situ mice with mechanical ventilation showed similar alveolar fluid clearance compared with the nonventilated in situ mice, 30% lower than in the ventilated mice. Therefore, an effect of mechanical ventilation did not contribute to the difference between the ventilated mice and the in situ mice, at least during the first 30 min.Extravascular lung water in the ventilated mice was significantly lower than the in situ mice at 30 min. Therefore, we calculated the lung interstitial fluid volume. The in situ mice had a statistically greater interstitial fluid volume than the ventilated mice after 30 min, suggesting that accumulation of interstitial fluid may have slowed alveolar fluid clearance in the in situ mice. Accumulation of fluid around perivascular cuffs correlated with extravascular lung water in prior histological studies (9, 13). Also, accumulation of interstitial fluid increased interstitial pressure in dogs (8, 12). However, no studies have assessed the potential effect of interstitial fluid accumulation on alveolar fluid clearance. It is known that several factors can influence lung interstitial pressure (7, 8, 18). The pulmonary circulation is probably critical for removing the lung interstitial fluid. An effect of pulmonary blood flow on alveolar fluid clearance was examined in dogs (5, 19) and sheep (22). Alveolar fluid clearance was equivalent over 4 h in the ventilated dogs compared with the in situ dog (5, 19). However, alveolar fluid clearance is much slower in dogs than in sheep (5, 27). In sheep, occlusion of pulmonary artery for 4 h did not affect basal alveolar fluid clearance (22). However, the rate of alveolar fluid clearance in sheep is intermediate compared with that in other species, and the volume of the instillate in the sheep studies was only 3-4 ml/kg, much lower than those measured in murine studies (16, 20, 22, 27, 28). Thus the volume of fluid transported to the interstitial space in mice is greater and therefore may result in higher lung interstitial pressures.
Thus our results support two possible explanations for the observed slowing of clearance at 30 min in the in situ mice. First, endogenous epinephrine did accelerate clearance in live ventilated mice at 30 min. However, adrenalectomized, ventilated mice had higher clearance rates than the in situ mice, suggesting that epinephrine plays only a limited role. Second, alveolar fluid clearance is limited by a higher interstitial fluid volume in the in situ mice. Because murine alveolar fluid clearance was much faster than in other species (5, 20, 21, 28, 29, 36), especially during the first 15 min, the rapid fluid removal from the distal air spaces to the interstitium may increase lung interstitial fluid volume to a level that limits alveolar fluid clearance or causes reflooding of interstitial fluid into the air spaces, thus slowing net alveolar fluid clearance. This observation may have relevance to the variable rates of alveolar fluid clearance that we have measured recently in patients during the resolution of hydrostatic pulmonary edema (38). Some patients with hydrostatic pulmonary edema have submaximal rates of alveolar fluid clearance, even when exposed to elevated plasma epinephrine levels (38). One mechanism to explain the slower alveolar fluid clearance rates could be the persistence of extensive lung interstitial edema.
In summary, both in situ and ventilated mouse models can be used for studying lung fluid balance and alveolar epithelial fluid transport. In the in situ model, alveolar fluid clearance slows after 15 min, probably because of the lack of endogenous epinephrine stimulation and the accumulation of a larger interstitial fluid volume in the lung. Because in situ alveolar fluid clearance was similar to that in the ventilated mouse at 15 min, the simpler in situ model should be useful for short-term studies of alveolar fluid clearance.
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ACKNOWLEDGEMENTS |
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We appreciate the assistance of Dr. Lorraine Ware in critically reading this manuscript. This WORF was supported by HL51854.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-51854.
Address for reprint requests and other correspondence: M. A. Matthay, Cardiovascular Research Institute, Univ. of California, 505 Parnassus Ave., HSW-825, San Francisco, CA 94143-0130 (E-mail: mmatt{at}itsa.ucsf.edu).
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.
Received 21 October 1999; accepted in final form 25 April 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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