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J Appl Physiol 102: 794-802, 2007. First published September 21, 2006; doi:10.1152/japplphysiol.00742.2006
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HIGHLIGHTED TOPIC
Physiological Imaging of the Lung

Evaluation of two-way protein fluxes across the alveolo-capillary membrane by scintigraphy in rats: effect of lung inflation

Nicolas de Prost,1 Didier Dreyfuss,2 and Georges Saumon1

1Institut National de la Santé et de la Recherche Médicale, U773, Centre de Recherche Bichat Beaujon CRB3, BP 416, and Université Paris 7 Denis Diderot, site Bichat, Paris; and 2Service de Réanimation Médicale, Hôpital Louis Mourier (Assistance Publique-Hôpitaux de Paris) and Université Paris 7 Denis Diderot, Paris, France

Submitted 4 July 2006 ; accepted in final form 18 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary microvascular and alveolar epithelial permeability were evaluated in vivo by scintigraphic imaging during lung distension. A zone of alveolar flooding was made by instilling a solution containing 99mTc-albumin in a bronchus. Alveolar epithelial permeability was estimated from the rate at which this tracer left the lungs. Microvascular permeability was simultaneously estimated measuring the accumulation of 111In-transferrin in lungs. Four levels of lung distension (corresponding to 15, 20, 25, and 30 cmH2O end-inspiratory airway pressure) were studied during mechanical ventilation. Computed tomography scans showed that the zone of alveolar flooding underwent the same distension as the contralateral lung during inflation with gas. Increasing lung tissue stretch by ventilation at high airway pressure immediately increased microvascular, but also alveolar epithelial, permeability to proteins. The same end-inspiratory pressure threshold (between 20 and 25 cmH2O) was observed for epithelial and endothelial permeability changes, which corresponded to a tidal volume between 13.7 ± 4.69 and 22.2 ± 2.12 ml/kg body wt. Whereas protein flux from plasma to alveolar space (111In-transferrin lung-to-heart ratio slope) was constant over 120 min, the rate at which 99mTc-albumin left air spaces decreased with time. This pattern can be explained by changes in alveolar permeability with time or by a compartment model including an intermediate interstitial space.

intermittent positive pressure ventilation; microvascular permeability; blood-air barrier

GAS IN ALVEOLAR SPACES is separated from blood by a thin membrane composed of epithelial and endothelial cell layers that are either closely juxtaposed or separated by a small interstitial space. Permeability to proteins of the epithelial and endothelial layers widely differs, alveolar epithelium being much less permeable than the endothelium (21). The alveolo-capillary membrane may face physical, chemical, or biological insults, and the response of the epithelial or endothelial layers may differ depending on insult type (1, 17, 25, 27, 32, 54). For example, Pseudomonas aeruginosa elastase on the alveolar side produces alveolar epithelium leakiness that manifests as an increase in the clearance of aerosolized albumin, without evidence of edema (1), whereas chronic heart failure due to large cardiac infarcts increases protein fluxes in both directions (11).

In vivo noninvasive measurement of protein fluxes across the pulmonary microvascular barrier has been proposed to detect lung injury in experimental models (22, 48) and in patients as well (2). The first attempt to estimate protein influx in lungs used 113mIn-transferrin as the tracer and a scintillation probe positioned over the thorax (22). This measurement was subsequently improved by the use of other tracers and gamma camera or positron emission tomography imaging (45). This latter method allows direct measurement of protein exchange rates, whereas simple scintigraphy imaging, which is of more widespread use, only provides (through lung-to-heart ratios) indexes that are proportional to protein fluxes (41).

Alveolar permeability to proteins is affected by lung volume changes. Aerosolized albumin clearance was slightly (by 50%) increased by applying 10 cmH2O positive end-expiratory pressure (PEEP) in sheep (37, 38). It is, however, unclear whether this increase was due to increased exchange surface area, maybe by the unfolding of alveolar cells (20). Similar changes may also occur in humans since increasing functional residual capacity (29, 33) increased alveolar permeability to a small aerosolized solute like 99mTc-diethylenetriamine-pentaacetate (99mTc-DTPA). Static, supraphysiological, but not milder lung inflation (16, 28) increased alveolar epithelial permeability to proteins in liquid-filled lungs. Short periods of overinflation resulted in a reversible increase in pulmonary microvascular permeability to albumin and perhaps some increase in alveolar/airway epithelial permeability as radiolabeled albumin injected in the systemic circulation was recovered in bronchoalveolar lavage fluid (14). More sustained high tidal volume (VT) ventilation produced endothelial and epithelial cell alterations and a pulmonary edema of the permeability type (13).

The acute response to high-volume ventilation probably results more from biochemical events, such as the response to increased cellular calcium concentration because of the opening of stretch-activated cation channels (35, 36), than simple physical events. Higher lung stretch may result in cell membrane damage (stress failure) at the endothelial (19) or epithelial (51) level. Stretching alveolar epithelial cells in vitro resulted in membrane rearrangements (52) and increased the permeability to small molecules across alveolar cell monolayers (8).

We hypothesized that epithelial and endothelial cell layers might differently react to lung stretch as this was the case in other types of insults (1, 17, 25, 27, 32, 54). We locally instilled liquid in one lung and used two tracers, 99mTc-albumin (in the alveolar instillate) and 111In-transferrin (in the blood) to simultaneously evaluate alveolar epithelial and pulmonary microvascular permeability to proteins by a noninvasive method, scintigraphic imaging, in vivo. Instillation in one lung only allowed us to compare changes in microvascular permeability between liquid-filled and air-filled lungs. We examined the response of the alveolar epithelial and endothelial cell layers with this method with respect to the level of lung distension and duration of mechanical ventilation.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   All experiments were conducted in compliance with the recommendations for laboratory animal research of the European Union and the French Ministry of Agriculture. G. Saumon is recipient of an authorization (decree no. 2001–464; authorization no. 75–409; delivered June 28, 2001) issued by the French Veterinary Services to perform experiments on anesthetized rodents. Male Wistar rats (n = 22, 326.3 ± 13.31g, Elevage Janvier, Le Genest Saint Isle, France) were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital (Sigma, St. Quentin Fallavier, France). They were tracheostomized, and a cannula (Braun, Melsungen, Allemagne) was tightly secured to the trachea to avoid gas leaks.

Computed tomography scan.   Computed tomography (CT) scans were obtained in six rats with a dedicated scanner (Biospace, Paris, France) to verify that the liquid instilled in a bronchus homogenously filled a well-limited zone in one lung. Briefly, after airway instillation as described below, rats were ventilated with conventional ventilation [CV, 8 ml/kg VT; PEEP 2 cmH2O; respiratory rate (RR) 70 breaths/min; and fractional inspired O2 (FIO2) of 1] for 30 min and killed with an overdose of pentobarbital. Several CT scan acquisitions were immediately performed in the vertical position increasing lung volume by 0, 6, 14, 23, and 27 ml/kg that corresponded to airway pressures (Paw) of 0, 12.2 ± 0.86, 18.2 ± 0.87, 25.0 ± 0.48, and 31.5 ± 0.55 cmH2O. Airway pressure was measured with a piezoelectric transducer connected to the tracheal cannula (AST, Vanves, France). Tissue density was evaluated on slices performed at same levels of the lungs using a 256-level gray scale (Scion Image; http://www.scioncorp.com/pages/scion_image_windows.htm). Bone density was set to zero, and air density was set to 255.

Scintigraphic imaging.   Continuous planar thoracic acquisitions were performed using a dedicated small animal gamma camera ({gamma}-Imager, Biospace, Paris) equipped with a parallel collimator. Acquisition lasted 120 min without interruption. Acquisition windows were 140 keV ± 15% for 99mTc and 245 keV ± 20% for 111In (99mTc does not emit in this energy window). Spillover of 111In in the 99mTc energy window (~60% 111In counts in the 245-keV window) was subtracted, and 99mTc activity decay was taken into account. 99mTc/111In lung count ratio in the 99mTc window was always ~10 so that the correction always remained well below 10% of 99mTc counts.

Dynamic series of images of 150 s each were computed from the registered scintigraphy data.

Measurement of pulmonary microvascular permeability.   About 400 µCi of 111In chloride were injected in the dorsal penile vein. Transferrin (76 kDa) strongly binds 111In, and the 111In-transferrin complex is a good tracer of passive protein movement because it does not bind to transferrin receptors (34). A region of interest (ROI) was delineated on the cardiac cavities using the early images before any significant 111In-transferrin diffusion in the lungs. Radioactivity counts in the ROIs corresponding to the instilled and contralateral lung (ROIE and ROICL) and to cardiac cavities were averaged per pixel. 111In-transferrin accumulation in lungs relative to blood (or plasma) was estimated using the normalized lung-to-heart activity ratio (41). This index (lung-to-heart ratio divided by its initial value) accurately reflects 111In-transferrin plasma to lung flux assuming that lung and heart blood volumes do not vary during the observation period. The slope of this 111In-transferrin accumulation index was calculated by linear regression.

Measurement of alveolar epithelial permeability.   99mTc-labeled albumin (69 kDa) was prepared using a commercial kit (Vasculocis; Cis Bio International, Gif sur Yvette, France). Paper chromatography using methanol as a solvent was performed to verify the amount of free 99mTc (12); 99.7 ± 0.08% 99mTc was bound to albumin. The solution was instilled in one lung so that pulmonary microvascular permeability could be compared between the instilled and the noninstilled lung. We were unable to reproducibly produce a localized zone of alveolar "flooding" during preliminary experiments using 500 µl of an isotonic solution. A smaller volume might have not uniformly filled distal airspaces. We obtained better results with 250 µl of a solution in which osmolarity was made about twice that of plasma by adding mannitol (120 mg/ml). This solution was supplemented with 600 µCi 99mTc-albumin, bovine serum albumin (80 mg/ml), 1 mM amiloride (an epithelial sodium channel inhibitor), and 1 mM phloridzin (a sodium-glucose cotransport inhibitor) to inhibit alveolar liquid absorption (3). It was slowly instilled in a distal airway after a short period of ventilation with FIO2 = 1. A ROI was drawn over the thorax (ROIT). Two other ROIs were drawn over the flooded zone (ROIE) and the contralateral lung (ROICL). The surface enclosed in these two ROIs was slightly smaller than ROIT. Activity in each ROI was integrated over 150-s steps and divided by initial total, i.e., ROIT, activity.

The decrease in 99mTc-albumin activity in ROIT during ventilation with 25 or 30 cmH2O end-inspiratory plateau pressure (Pplat) followed a two-phase exponential decay. Pooled data were fitted with a two-exponential decay equation

Formula 1(1)
where M0 is initial lung 99mTc-albumin content. The same time course was also observed during severe oleic-acid pulmonary edema for aerosolized 99mTc-albumin (37), the presence of a fast compartment being explained by the presence of more damaged lung regions. However, this time course can also be due to an intermediate, interstitial compartment as shown in Fig. 1. Indeed, interstitial space may play a significant role in the clearance of alveolar solutes during lung injury (24). Proteins are assumed to pass through a unique airway/alveolar membrane and enter the microcirculation directly or via the pulmonary interstitium. We will simply give the solution for this classic compartment system because it has been published many times (see, for example, Ref. 40). Let FA be the fraction of the tracer in the alveolar compartment and FI the fraction in the interstitial compartment, then, with the notation kij = k from compartment j to compartment i:

Formula 2(2)

Formula 3(3)
where

Formula 3

Formula 3

Formula 3

Formula 3

Formula 3


Figure 1
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  		Fig. 1. Compartment model of 99mTc alveolar albumin clearance. A, alveoli; I, interstitium; O, plasma.

 
The amount of 99mTc-albumin in the lungs at time t would then be:

Formula 4(4)

Equation 4 is similar to Eq. 1, with M0 = 1 and A = ({alpha} – kIA/Z).

When t -> 0, FI -> 0, and then the initial slope of the curve (albumin clearance rate from alveoli) would be:

Formula 5(5)

This initial slope was calculated by linear regression using the first six data points (first 15 min) after the beginning of high-pressure ventilation. The alveolar/airway epithelium permeability-surface area product (PSA) for 99mTc-albumin is thus Ka x VA, where VA is the volume of the flooded zone, ~0.5 ml because we instilled 250 µl of a x2 hypertonic solution.

Ventilation modalities.   Rats were paralyzed by injection of 15 mg/kg succinyl-choline (Sigma) and ventilated using a Harvard rodent volume ventilator (Ealing, Courtaboeuf, France). Conventional ventilation was applied for 30 min, followed by different ventilation modalities for 120 min. Four end-inspiratory Pplat pressures were tested, 15, 20, 25, and 30 cmH2O (n = 16), corresponding to VT of 7.8 ± 0.31, 13.7 ± 4.69, 22.2 ± 2.12, and 25.9 ± 1.76 ml/kg, respectively, in the absence of PEEP. RR was adjusted so that minute ventilation was about the same for all ventilation modalities.

Wet-to-dry lung weight ratio.   Lungs were removed from the thorax, weighed, and placed at –20°C for 1 mo to allow radioactivity to decay. They were then weighed one more time to verify the absence of loss, and dry weight was determined after 7-day desiccation at 80°C.

Statistical methods.   All results are expressed as means ± SE. Comparisons were made by ANOVA using Bonferroni's post hoc test or the Student's t-test. Nonlinear regressions were made using GraphPad Prism (San Diego, CA). Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CT scan imaging.   CT scans (Fig. 2) showed that the instillation protocol resulted in localized lung condensation containing an air bronchogram. Instillation was performed four times in the left lung and two times in the right lung. Average surface areas were similar at functional residual capacity for lung slices taken at the same slice level. They were (cm2) for instilled left lungs 0.321, 0.446, 0.385, and 0.352 (average 0.376), and for uninstilled left lungs 0.422 and 0.281 (average 0.352). For instilled right lungs, they were 0.356 and 0.305 (average 0.331), and for uninstilled right lungs 0.301, 0.414, 0.378, and 0.319 (average 0.353).


Figure 2
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  		Fig. 2. Left to right: examples of computerized tomography (CT) scan slices performed at functional residual capacity (FRC) airway pressure (Paw) = 0, 12, and 17 cmH2O, showing the recruitment of the zone of alveolar flooding. Inflation volumes were 0, 6.3, and 15.6 ml/kg, respectively. Arrow shows air bronchogram. Analyzed slices were on the same plane positioned on the ribs. The heart moved with lung inflation and disappeared from the slice as acquisitions were performed in the vertical position because of the scanner design.

 
There was an exponential decrease in lung density (inverse of grayscale) in the zone of alveolar flooding as lung volume increased (Fig. 3). Both lungs received about the same relative volume of gas, as differences in grayscale at low and high volume did not differ significantly.


Figure 3
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  		Fig. 3. Density (gray scale) changes of the CT scan slices obtained at different inflation pressures in 6 rats. {circ}, Instilled lung; bullet, contralateral lung. Density of the flooded lung became closer to that of the contralateral lung at high Paw, reflecting recruitment.

 
End-inspiratory pressures (Pplat) and wet-to-dry lung weight ratio during ventilation.   Figure 4A shows Pplat values at 30 min (t30) and 120 min (t120). Pplat did not vary significantly during the test ventilation period when initial value was 15 or 20 cmH2O, whereas it increased significantly from 25 to 32.5 ± 1.44 cmH2O (P < 0.05) and from 30 to 44.0 ± 1.68 cmH2O (P < 0.01) in the two other groups.


Figure 4
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  		Fig. 4. A: plateau pressures (Pplat) at 30 min (t30) and 120 min (t120) during the different ventilation modalities. B: wet-to-dry lung weight ratios in instilled and contralateral lungs. Pressures (for the 25 and 30 cmH2O Pplat ventilation modalities) and wet-to-dry ratios (for the 30 cmH2O Pplat ventilation modality) increased, reflecting the occurrence of a pulmonary edema. **P < 0.01, *P < 0.05.

 
Wet-to-dry lung weight ratios (Fig. 4B) were ~61% (flooded lungs) and 89% (air-filled lungs) higher in rats ventilated with 30 cmH2O Pplat compared with those ventilated with 15 cmH2O Pplat (P < 0.01 and P < 0.05 for instilled and contralateral lungs, respectively). There was no significant difference between other wet-to-dry lung weight ratios.

Scintigraphic imaging.   Examples of images integrating the first and last 15 min of an experiment are shown on Fig. 5. Alveolar flooding remained localized and stable during 120 min (99mTc window) with 15 cmH2O Pplat (Fig. 5A), and there was a slow 111In-transferrin lung accumulation. By contrast, 30-cmH2O Pplat ventilation induced a contralateral dispersion of the tracer and an increase in alveolo-capillary barrier permeability as attested by the obvious decrease in overall 99mTc-albumin activity over the thorax (Fig. 5B) and the significant lung 111In-transferrin uptake (Fig. 5C).


Figure 5
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  		Fig. 5. Examples of scintigraphy images integrating the 15 min following instillation ( t0–t15; left) and in the 15 last min of the experiment (t105–t120; right). A: 99mTc energy window; rat ventilated with 15 cmH2O Pplat: the tracer remained confined in the right lung. B and C: ventilation with 30 cmH2O Pplat. B: 99mTc energy window. C: 111In energy window. There was a strong contralateral dispersion and an obvious leakage of 99mTc-albumin from the lungs (decrease in overall activity) and an increase in lung 111In-transferrin content. Note the absence of significant 99mTc activity over the liver region that suggests that circulating 99mTc-albumin concentration was low and could not account for the activity in the contralateral lung (B, right). ROIE, ROICL, ROIT are instilled lung, contralateral lung, and total region of interest, respectively.

 
Lung 111In-transferrin accumulation.   111In-transferrin lung-to-heart activity ratio increased linearly during the test ventilation period (between t30 and t120) in all groups (Fig. 6). Slopes of 111In-transferrin accumulation in lungs were thus calculated by linear regression. 111In-transferrin accumulated in lungs slowly in rats ventilated with 15 and 20 cmH2O Pplat but much faster in rats ventilated with 25 and 30 cmH2O Pplat (see Figs. 6 and 9A). Accumulation slope increased, although nonsignificantly, with 25-cmH2O Pplat (0.05 < P < 0.1 compared with 15 cmH2O Pplat) and was significantly higher in rats ventilated with 30 cmH2O Pplat (P < 0.001). 111In-transferrin accumulated at the same rate in the instilled and contralateral lung, whatever the Pplat (Fig. 6). 111In-transferrin slopes correlated with lung wet-to-dry ratios in the instilled lung (r2 = 0.68, P < 0.001) and contralateral lung as well (r2 = 0.82, P < 0.001). These slopes were averaged to provide a unique index of lung 111In-transferrin accumulation because they did not significantly differ.


Figure 6
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  		Fig. 6. 111In-transferrin lung-to-heart activity ratios. A–D: rats ventilated with 15, 20, 25, and 30 cmH2O Pplat, respectively. bullet, Instillated lung; {circ}, contralateral lung. Slopes after t30 were significantly higher during 30 cmH2O Pplat ventilation than during 15 or 20 cmH2O Pplat ventilation (P < 0.001) and 25 cmH2O Pplat ventilation (P < 0.01).

 

Figure 7
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  		Fig. 7. 99mTc-albumin changes in the different regions of interest (ROIs) expressed as percentages of initial total activity. A–D: rats ventilated with 15, 20, 25, and 30 cmH2O Pplat, respectively. Ventilation with 15 and 20 cmH2O Pplat were accompanied by a low systemic leakage and no pulmonary dispersion of the tracer. In contrast, ventilation with 25 and 30 cmH2O Pplat induced an increase in ROICL and a decrease in ROIE and ROIT activities.

 

Figure 8
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  		Fig. 8. Averaged 99mTc-albumin activity in ROIT (continuous lines) during ventilation with Pplat = 25 cmH2O (A) and ventilation with Pplat = 30 cmH2O (B). 99mTc-albumin activity displayed a two-phase exponential decay (discontinuous lines). Exponent values were K1 = 5.449 x 10–3 min–1, K2 = 9.936 x 10–5 min–1 (A); and K1 = 1.053 x 10–1 min–1, K2 = 1.232 x 10–3 min–1 (B).

 

Figure 9
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  		Fig. 9. A: relationship between Pplat and 111In-transferrin lung-to-heart ratio slopes (left axis, {circ}) and alveolar 99mTc-albumin permeability-surface area product (PS; right axis, bullet). B: linear regression of 111In-transferrin lung-to-heart ratio slope and 99mTc-albumin PS (r2 = 0.87, P < 0.0001) for rats ventilated with 15, 20, 25, and 30 cmH2O Pplat. The strong correlation between alveolar albumin PS product and 111In-transferrin lung-to-heart ratio slopes was still significant (r2 = 0.90, P < 0.0001) when lung-to-heart ratio slopes below 0.0025 min–1 were discarded.

 
Alveolar 99mTc-albumin permeability.   Ventilation with 15 and 20 cmH2O Pplat did not induce 99mTc-albumin dispersion in the lungs (variation in ROIE and ROICL activities) or leakage from airspaces (change in ROIT activity). These results are summarized in Fig. 7. In contrast, ventilation with 25 and 30 cmH2O Pplat was associated with a dispersion of the tracer in the lungs that manifested as an increase in ROICL activity. There was also a decrease in ROIT activity that reflected the leakage of 99mTc-albumin from air spaces (Fig. 7, C and D). This leakage did not appreciably increase blood 99mTc-albumin content, as no significant activity was found over the liver (data not shown, but this is obvious from Fig. 5B). There was, however, no correlation between the amount of 99mTc-albumin that redistributed in the contralateral lung and the leakage of this tracer from the lungs during the period of ventilation under test (r2 < 0. 01, NS).

The two-exponential decay curves adjusted over mean (for better legibility) ROIT values obtained during 25 and 30 cmH2O Pplat ventilations are shown in Fig. 8. Mean Ka values and albumin clearance rates calculated with individual data are given in Table 1.


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  		Table 1. Mean Ka values and albumin clearance rates

 
Relationships between Pplat and 111In-transferrin lung-to-heart ratio slopes or 99mTc-albumin clearance were similar (Fig. 9A). There was a linear relationship between albumin PS product and 111In-transferrin lung-to-heart ratio slope (PSA = 67.20 x slope – 0.06, r2 = 0.87, P < 0.0001). There thus was a strong correlation between these indexes of endothelial and epithelial permeability (Fig. 9B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study shows that the simultaneous monitoring by scintigraphy of pulmonary capillary and alveolar/airway permeability to proteins may provide further insight into the physiological alterations due to high lung stretch. Ventilation with airway pressures above 25 cmH2O (that corresponded to an increase in lung volume of 22.2 ± 2.12 ml/kg above FRC) immediately increased pulmonary microvascular permeability, an observation we already made with much higher distending pressures (14). This increase was accompanied by a simultaneous, immediate increase in alveolar/airway epithelial permeability to proteins, which is a new observation.

The presence of a threshold volume/pressure for microvascular permeability increase with lung tissue stretch has already been evoked (13), but information on the in vivo acute response of the alveolar/airway epithelial barrier to stretch is lacking, even if in vitro experiments (8, 51) suggest that stretch may similarly affect epithelial and endothelial layer permeability. The acute changes in alveolar/airway permeability appear to be biphasic, which may correspond to a decrease in permeability with time, or more probably, as will be discussed below, to the participation of an interstitial compartment.

Roselli and Riddle (41) have shown that the many indexes that have been used to describe plasma to lung protein fluxes using radioactive tracers and an external detection method were all related to a single one that they called the normalized slope index. This index is independent of the amount of tracer injected and of interstitial lung volume, tends to be independent of microvascular pressure, and increases with microvascular permeability. However, this normalized slope index requires an intravascular marker (usually 99mTc-erythrocytes) together with the diffusible protein (often 111In-transferrin). This technique could not be used here as we used 99mTc-albumin for our double-isotope imaging of protein fluxes across the alveolo-capillary barrier. We then used the normalized lung-to-heart ratio slope of 111In-transferrin that is related to the normalized slope index in the absence of lung and blood volume change (41, 47, 49). We already showed that the 111In-transferrin lung-to-heart ratio slope correlates well with protein accumulation rate in lungs during ventilator-induced lung injury (6). The correlation found between this slope and the wet-to-dry ratio further confirmed that it reflected permeability edema severity.

We instilled a hypertonic solution (about twice that of rat plasma) so that a large part of the alveolar flooding came from the circulation, as this occurs during actual pulmonary edema. Lung epithelium was not altered by this solution as the permeability of the alveolar barrier to 99mTc-labeled albumin remained low during the 30 min of conventional ventilation, in agreement with previous reports that hypertonic solutions were not injurious, either for epithelial or endothelial cells (7, 9). Hyperosmolarity may protect lung endothelial barrier properties during various insults (42) after a transient increase in permeability because of cell shrinkage (39). However, we found no difference between instilled and noninstilled lungs (Fig. 6), suggesting that the protective effect of hyperosmolarity was weak. Mannitol was used as the osmotic agent as it is not actively absorbed by epithelia, and sodium transport inhibitors were added to the solution to decrease the rate of liquid absorption (43), thus stabilizing the volume of the flooded lung.

Some studies have used the pulmonary clearance of aerosolized 99mTc-albumin to study alveolar permeability to proteins (see, for example, Refs. 26, 50). We choose to deliver the tracer as a bulk solution as this has been done for a long time to study alveolar epithelium protein transport (16, 31). Although it is possible that filling lungs with liquid may modify active and passive transport across the alveolar epithelial barrier (44), many studies have used liquid filled lungs to study lung protein transport (23). Further, bulk instillation may better reflect what occurs during pulmonary edema. CT scans showed that lung density varied in about the same proportion (Fig. 3) in both lungs as they were inflated with air, which suggested that they underwent comparable distension during ventilation as their FRC were comparable. Liquid-filled lungs are theoretically more distensible than air-filled lungs because of the absence of an air-liquid interface. However, in lungs ventilated with air, the air-surfactant interface is replaced by places by an air-water interface with higher surface tension. It is therefore difficult to predict the stress to which these instilled lungs are subjected at the tissue level. On the whole, the observation that 111In-transferrin accumulation, and thus microvascular permeability alterations, were similar in the flooded and contralateral lung suggests that tissue stress was similar in both lungs. The immediate increase in lung-to-heart ratio slope once high-volume ventilation was applied suggested that acute lung distension was responsible for this increase in permeability rather than cellular injury resulting from opening/reopening of distal lung units or the displacement of liquid in airways (5). An increase in exchange surface area was also unlikely to explain this observation as there was no correlation between 99mTc-albumin leakage from lungs and the amount that redistributed in contralateral lung.

Prior studies found that increasing lung volume by 10 cmH2O PEEP slightly accelerated the clearance of aerosolized 99mTc-albumin in normal lungs (37). We also observed that distension produced an increase in 99mTc-albumin clearance in normal, flooded lungs. Clearance became biphasic, as this has been previously observed for aerosolized 99mTc-DTPA, a small solute (37) with 10 cmH2O PEEP or during oleic-acid edema (25), and for aerosolized 99mTc-albumin, during severe oleic-acid edema (37) or surfactant inactivation (26, 50). The explanation put forward for the biphasic shape of 99mTc DTPA clearance in the presence of 10 cmH2O PEEP was that the aerosol deposited in lung areas with different permeability-surface area products (38), whereas, during pulmonary edema, 99mTc-albumin clearance biphasic shape was thought to be due to the presence of lung regions with different levels of injury (37). It may also be due during high volume ventilation to a decrease in alveolar permeability with time, but this seems unlikely as increasing duration of ventilation usually worsens lung injury (13). It is worth noting that the biphasic shape was only seen when solute clearance was high, whatever solute size. Another simple explanation for these observations is that DTPA or albumin removal from the lungs by the circulation occurs both directly, probably through the thin part of the alveolo-capillary barrier, and via lung interstitium, with a possible significant backflux from interstitium to alveolus when the tracer is not rapidly enough cleared from the interstitium. This backflux may increase with interstitium hydration during pulmonary edema development (46). Simulation of the model depicted in Fig. 1 (VBA for Excel, available on request, data not shown) shows that when alveolar permeability (k0A + kIA) is low, as was the case for 15 and 20 cmH2O Pplat ventilation, it is impossible to distinguish between a fast and a slow compartment. The albumin clearance rate we observed during low and moderate Pplat ventilation (1.44%/h, averaging 15 and 20 Pplat values) was comparable to that previously reported using a similar model [1.6%/h (4)].

One of the main findings of our study is that there seems to be a threshold for Pplat or VT value above which alveolo-capillary membrane becomes permeable to proteins during mechanical ventilation, resulting in acute lung distension. This pressure is lower than that found during static lobe distension in the rabbit (15). Interestingly, the threshold pressure value was about the same (above 20 cmH2O) for the epithelial and endothelial barrier. It is worth noting that this threshold approximately corresponds to the pressure at which a decrease in the respiratory system pressure-volume curve slope (the so called "upper inflection point") is observed in rats (30). This decrease in compliance has been ascribed to the beginning of "hyperinflation" (10). Thus ventilation above the upper inflection point may rapidly increase epithelial permeability to proteins and decrease alveolar epithelium reflection coefficient for all solutes, which may have a negative impact on alveolar liquid clearance in addition to producing a permeability type edema. Alveolar liquid clearance is important for the improvement of patients suffering from the acute respiratory distress syndrome (53). These abnormalities may rapidly reverse if the challenge is not sustained long enough (14, 18).

In conclusion, a simple double-isotope imaging technique can be used to noninvasively explore the simultaneous changes of lung microvascular and alveolar permeability to proteins in vivo. Lung distension during mechanical ventilation is associated with an increase in alveolo-capillary barrier permeability to proteins that is characterized by epithelial and endothelial permeability alterations that occur at about the same threshold pressure value.


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This work has been supported by a grant from the Comité de l’assistance respiratoire à domicile d’Ile de France (CARDIF).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Saumon, INSERM U773, Equipe 11, CRB3, BP 416, F-75018, Paris, France (e-mail: saumon{at}bichat.inserm.fr)

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. Section 1734 solely to indicate this fact.


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Protein permeability in lung injury: now in real time again?
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