EDITORIAL FOCUS
Rapid alveolar liquid removal by a novel convective mechanism

P. M. Wang, Y. Ashino, H. Ichimura, and J. Bhattacharya

Departments of Medicine and Physiology and Cellular Biophysics, College of Physicians and Surgeons and St. Luke's-Roosevelt Hospital Center, Columbia University, New York, New York 10019


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

Although alveoli clear liquid by active transport, the presence of surface-active material on the alveolar surface suggests that convective mechanisms for rapid liquid removal may exist. To determine such mechanisms, we held the isolated blood-perfused rat lung at a constant alveolar pressure (PA). Under videomicroscopy, we micropunctured a single alveolus to infuse saline or Ringer solution in ~10 adjacent alveoli. Infused alveoli were lost from view. However, as the infused liquid cleared, the alveoli reappeared and their diameters could be quantified. Hence the time-dependent determination of alveolar diameter provided a means for quantifying the time to complete liquid removal (Ct) in single alveoli. All determinations were obtained at an PA of 5 cmH2O. Ct, which related inversely to alveolar diameter, averaged 4.5 s in alveoli with the fastest liquid removal. Injections of dye-stained liquid revealed that the liquid flowed from the injected alveoli to adjacent air-filled alveoli. Lung hyperinflations instituted by cycling PA between 5 and 15 cmH2O decreased Ct by 50%. Chelation of intracellular Ca2+ prolonged Ct and abolished the inflation-induced enhancement of liquid removal. We conclude that when liquid is injected in a few alveoli, it rapidly flows to adjacent air-filled alveoli. The removal mechanisms are dependent on alveolar size, inflation, and intracellular Ca2+. We speculate that removal of liquid from the alveolar surface is determined by the curvature and surface-active properties of the air-liquid interface.

surfactant; pulmonary edema; cell calcium; Laplace equation; inflation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

IN PULMONARY EDEMA, the failure of gas exchange results from alveolar liquid accumulation. Alveoli are capable of removing the liquid, although the only known removal mechanism is by active salt transport across the alveolar membrane (4). However, active transport mechanisms are slow, effecting liquid transport with a time course of hours. Faster mechanisms of alveolar liquid removal that are protective in the early stages of pulmonary edema may coexist but remain unidentified.

We considered the possibility that surface-active forces in the alveolus effect rapid convective removal of alveolar liquid. Although airway continuity provides a potential route for convective liquid removal from the alveoli to the distal airways, potential flow-supportive forces remain unidentified. Nevertheless, classic evidence for the formation of edema foam in the airway (18) and nonuniform alveolar liquid accumulation (20) suggests that alveolar-airway liquid flow exists in early pulmonary edema. However, such flows have not been directly confirmed.

Classically, the distribution of alveolar liquid forces is understood on the basis of the Laplace equation for curved surfaces (3, 12, 19). However, it is not clear that air-liquid interface curvatures undergo sufficient alterations in alveolar liquid filling as to generate convective forces for alveolar liquid removal. We considered that if convective mechanisms exist, then liquid should drain rapidly from a filled alveolus. On the other hand, lack of convection should be evident in the retention of alveolar liquid to the extent that it is slowly removed by active transport. We tested this hypothesis by microinjecting liquid into small groups of alveoli on the surface of the constantly inflated lung. Our findings indicate that alveoli thus filled eject the liquid in seconds, thereby indicating the existence of a novel convective mechanism for alveolar liquid removal.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

Lung microscopy. The isolated blood-perfused rat lung was prepared as previously described (2). Briefly, lungs excised from anesthetized Sprague-Dawley rats were pump perfused with autologous rat blood at 14 ml/min at 37°C. Pulmonary arterial and left atrial pressures were held at 10 and 5 cmH2O, respectively. At baseline, the lungs were constantly inflated at an alveolar pressure (PA) of 5 cmH2O. The lungs were positioned on a vibration-free table and superfused with saline at 37°C to prevent drying. The lung surface was viewed through a stereomicroscope (SZH, Olympus) as previously described (2), and images taken with a microscope-mounted video camera (T123A CDD camera, NEC) connected to a video timer (VTG-33, FOR.A Company) were displayed on a video monitor (PM-127 REK.B, Ikegami). All images were recorded with a videocassette recorder (BR-S378U, JVC) at 30 frames/min.

Alveolar microinjection. For each imaged alveolus, the microscope focus was set at the maximum alveolar diameter. For alveolar micropuncture and microinfusion, we used previously described methods (2). Briefly, we prepared beveled glass micropipettes with a tip diameter of 5 µm. We backfilled each micropipette with the injection solution, and then, using a micropipette manipulator (Leitz), we micropunctured single alveoli that we viewed with a stereomicroscope and microinfused it with saline (0.9% sodium chloride injection USP) or Ringer solution (pH 7.4; lactated Ringer injection USP, B.BRAUN Medical) for 1-2 s to fill ~10 adjacent alveoli. After microinfusion, the micropipette was withdrawn, and the alveoli were imaged. No alveolus was punctured more than once.

Quantification of liquid removal. Before alveolar microinjection, we quantified diameters of several alveoli in the imaged field as the means of the longest and the shortest diameters in each alveolus. After microinfusion, loss of visible alveolar margins denoted alveolar liquid filling. As the microinfused liquid drained away from each alveolus, the alveolar margins became evident. Hence we quantified the liquid removal time (Ct) as the time elapsed from the end of alveolar filling to the appearance of 90% of the alveolar diameter. Ct was quantified first under baseline conditions at an PA of 5 cmH2O and then after a 1-min hyperinflation period in which the lungs were given 10 inflations by cycling PA between 5 and 15 cmH2O.

In separate groups, we quantified Ct in control alveoli that received liquid injections in the absence of pretreatment and in alveoli given a 15-min pretreatment with an infusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 40 µM; Calbiochem, San Diego, CA) in Ca2+-free HEPES solution or one of the inhibitors of active transport, ouabain (1 mM, pH 7.4) or amiloride (10 µM, pH 7.4; Sigma, St. Louis, MO). Ca2+-free HEPES solution (pH 7.4, osmolarity of 295 mosM) contained (in mM) 20 HEPES, 150 NaCl, 5 KCl, and 10 glucose. The vehicle was lactated Ringer solution.

Data are means ± SE shown in plots or means ± SD shown in text; n is the number of lungs. Significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

Rate of liquid removal. Figure 1 exemplifies our alveolar microinfusion procedure. It shows that the light-reflecting air-liquid interface, which defined the alveolar margin under air-filled conditions (baseline), was lost after liquid instillation (0 s) in the marked region. Hence alveoli were not visible while liquid filled. However, as depicted over time in Fig. 1, as the liquid drained away, the alveolar margins became progressively evident.


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Fig. 1.   Removal of liquid in microinjected alveoli. Shown is the sequence of events after microinfusion of Ringer solution in a group of 9 alveoli (nos. in circles). Objects evident in each video image (left) are highlighted in the corresponding sketch (right). Baseline, a single alveolus is micropunctured; 0 s, immediately after infusion, alveoli are no longer visible in the microinfused region (dashed line); 3-34 s, alveoli appear in the microinfused region as the injected liquid drains away.

Because the light reflections at the air-liquid interface clearly denoted the perimeter of each alveolus, we quantified alveolar diameter as the equatorial distance across an alveolus using either microcalipers placed on the alveolar image or the line scan tool of our image analysis program (LSM Image Examiner, Zeiss). Light reflections at the alveolar wall provided sufficient edge contrast as to mark the alveolar perimeter (Figs. 2, A and B). The distance between edge contrasts at opposite poles of the alveolar equator denoted alveolar diameter (Fig. 2B). Loss of edge contrast signified alveolar liquid filling. Progressive reappearance of the edge contrasts indicated progressive removal of the alveolar liquid. Alveolar diameter quantifications obtained by these image analysis methods did not differ from those with microcalipers (APPENDIX B); hence we have not distinguished between the determinations.


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Fig. 2.   Quantification of alveolar diameter. A: single alveolus before (baseline) and at different time points after (postinjection) alveolar liquid instillation. Dotted line, alveolar perimeter; solid line, equator. B: tracings are computer-generated line scan profiles obtained by placing an image analysis line tool on the alveolar equator. Arrowheads, edge artifacts caused by light reflections at the perimeter of the air-filled alveolus. Note that the distance (double-headed arrow) between the edge artifacts gives the alveolar diameter. Edge artifacts are not detectable 0-3 s after alveolar liquid filling. Subsequent tracings denote return of edge artifacts as the liquid is removed. C: time course of diameter (d) changes in a single liquid-filled alveolus. Dashed line, alveolar diameter before microinjection (baseline). After microinjection (arrow), the alveolar diameter could not be recorded for a short period because the alveolus was not visible. However, as liquid drained and the alveolus became air filled, the diameter rapidly increased. As shown for this alveolus, the baseline diameter was reestablished within 7-8 s of the end of microinfusion. We quantified the liquid removal time (Ct) as the time elapsed from the end of microinfusion to the reestablishment of the baseline alveolar diameter.

As liquid was removed from an instilled alveolus, alveolar diameter increased abruptly, with an S-shaped time course (Fig. 2C). In some instillations, the loss of alveolar margin was incomplete, leaving a residual margin that probably reflected the alveolar opening into the respiratory duct. The punctured alveolus was neither the first nor the last liquid-clearing alveolus. To determine the rate of liquid removal, we quantified time-dependent changes of alveolar diameter in the postinfusion period. As shown in Fig. 2C, we quantified Ct as the duration from the end of the microinjection to the appearance of the baseline alveolar diameter.

On average, the fastest liquid removal occurred within 4.5 s (Fig. 3). All determinations were obtained at a PA of 5 cmH2O, before (baseline) and after (hyperinflation) lung expansions established by cyclically varying the PA from 5 to 15 cmH2O 10 times in 1 min. Hyperinflation markedly enhanced Ct (P < 0.03; Fig. 3). For alveoli with the fastest liquid removal, mean Ct decreased from 4.5 s at baseline to 1.9 s in the postinflation period, indicating that the intervening hyperinflation period enhanced liquid removal by >50%. These rapid removal rates indicate that the alveolar liquid was removed convectively.


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Fig. 3.   Ct from the 3 fastest liquid-clearing alveoli. Baseline determinations were made at alveolar pressure (PA) of 5 cmH2O. Postinflation determinations were made at PA of 5 cmH2O after the lungs were subjected to 10 hyperinflations. N, no. of lungs. * P < 0.01 compared with bar on left.

Route of removal. To determine the fate of the instilled liquid, we included fluorescent latex beads (diameter 1 µm) in the alveolar microinfusion. Figure 4 shows that despite a constant PA of 5 cmH2O, single beads showed translational displacement out of the injected alveolus within 3-5 s and then continued along a trajectory of spontaneous movement across alveoli for up to 80 s. To the extent that we could determine, beads that exited a given alveolus moved in approximately the same direction, indicating that their movements were not random but were probably actuated by bulk flow out of the instilled alveolus.


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Fig. 4.   Spontaneous bead movement from liquid-infused alveolus. A: images taken at times indicated after injection of fluorescent microbead (diameter 1 µm). Alveolar margins are sketched. Arrows: left, fluorescent bead in an alveolus; middle, 25 s later, the bead has moved to the adjoining alveolus; right, 90 s later, the bead has traveled further along to the next alveolus. B: detailed trajectory of bead movement. Trajectory was drawn from bead positions determined in multiple images obtained during bead movement. Experiment was replicated 3 times.

In a single experiment, we instilled an Evans blue-saline solution (0.7 mg/ml). As shown in Fig. 5, the perimeter of the dye-instilled region increased progressively, suggesting that convective flow occurred out of the injected alveoli toward the surrounding alveoli. Consistent with liquid removal from the alveoli, dye intensity progressively decreased. The dye could be removed by intra-alveolar microinjections of dye-free saline but not by superfusing the area with saline, indicating that alveolar microinjections did not leak across the alveolar membrane.


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Fig. 5.   Disappearance of injected Evans blue dye from alveoli. Images were recorded at indicated times after Evans blue (0.67 mg/ml) injection in a single alveolus. Dotted lines, perimeter of dye-stained zone.

Role of Ca2+. To determine the role of intracellular Ca2+ as a determinant of Ct, we treated alveoli with 20-min microinfusions of BAPTA-AM in Ca2+-free HEPES. BAPTA-AM chelates intracellular Ca2+, and as Ashino et al. (2) have previously shown, it blocks the Ca2+ increases and surfactant secretion in type II cells of intact alveoli. BAPTA-AM markedly prolonged Ct under baseline conditions (P < 0.05), and it abrogated the posthyperinflation reduction of Ct (Fig. 6). When we pretreated alveoli with Ca2+-free HEPES solution alone, Ct was not different from baseline. However, similar to the BAPTA-AM experiments, Ca2+-free HEPES abrogated the hyperinflation-induced decrease of Ct, indicating that the inflation effect was also dependent on external Ca2+. These findings indicate that Ca2+-regulated processes determined Ct, although the nonspecific effects of BAPTA-AM may also have contributed to these results.


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Fig. 6.   Ct during Ca2+ depletion. N, number of lungs; Ca2+(+) and Ca2+(-), Ca2+-containing and Ca2+-depleted solutions, respectively; BAP, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 40 µM). * P < 0.05 compared with baseline.

Ct and alveolar diameter. Although all determinations were made at a constant PA of 5 cmH2O, Ct differed in different alveoli. Under baseline conditions, Ct correlated inversely with alveolar diameter (Fig. 7A), indicating a strong dependence of the liquid removal rate on alveolar size. However, after a period of cyclic hyperinflations in which PA was increased to 15 cmH2O 10 times in 1 min, the correlation was lost (Fig. 7B), suggesting the involvement of surfactant-dependent surface forces in the liquid removal (see DISCUSSION). Similar to control, the inverse Ct-alveolar diameter relationship was also evident in BAPTA-AM-treated alveoli (Fig. 7C). However, different from control, hyperinflation failed to abolish the relationship (Fig. 7D), indicating that the possible inhibition of surfactant secretion by BAPTA-AM rescued the Ct-diameter relationship.


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Fig. 7.   Alveolar diameter determined Ct at baseline (A and C) and hyperinflation (B and D) before (A and B) and after (C and D) 15-min alveolar infusion of BAPTA-AM. AD, alveolar diameter. open circle , Determinations at PA of 5 cmH2O; , determinations at PA of 5 cmH2O after lungs were subjected to 10 hyperinflations. Lines were drawn by exponential regression.

Active transport. Figure 8 summarizes the experiments in which we determined the extent to which active transport plays a role in the observed liquid removal. As shown, the active transport blockers ouabain and amiloride failed to affect Ct at either baseline or postinflation conditions, indicating that alveolar liquid removal was not determined by active transport mechanisms across the alveolar wall. In two experiments, we confirmed that the presence of albumin (5% human albumin solution) in the injected liquid did not affect Ct (data not shown), indicating that protein osmotic pressures were not a factor in the removal.


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Fig. 8.   Effect of active transport inhibitors on Ct. N, no. of lungs; Baseline, determinations at PA of 5 cmH2O; postinflation, determinations at PA of 5 cmH2O after lungs were subjected to 10 hyperinflations. Inhibitors were given as 15-min alveolar infusions before Ct determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

We show here for the first time that in an air-filled lung, when a few alveoli become liquid filled, as might occur during the initiation of pulmonary edema (20), the alveoli rapidly eject the liquid. This was evident in our experiments in which we modeled the pattern of alveolar filling in early edema by microinjecting a single alveolus with Ringer lactate to fill 6-10 adjoining alveoli. In the immediate postinstillation period, the alveoli were lost from view because each liquid-filled alveolus lost its light-reflecting air-liquid interface that defines the alveolar margin and allows the alveolus to be visualized by light microscopy. However, although in the liquid-filled condition the alveoli were not discernible, each liquid-filled alveolus ejected its liquid load and became visible as it refilled with air.

The delay to reappearance of a liquid-filled alveolus provided a measure of the Ct that, although varying over a 10-fold range, occurred as rapidly as 5 s. In every case, alveolar reappearance was a rapid event such that each alveolus seemed to "pop" open as air replaced the instilled liquid. This was evident in the rapid rate at which the alveolar diameter regained its preinfusion value (Fig. 2C). In an overwhelming majority of experiments, the micropunctured alveolus was neither the first nor the last to clear liquid. In alveoli microinjected with Evans blue solution, no liquid leakage was evident (Fig. 5). These findings rule out the possibility that liquid leaked into the interstitium at the micropuncture site or elsewhere. Ouabain and amiloride, which are blockers of NaCl transport, failed to affect Ct, thereby ruling out active transport as the major underlying mechanism. Furthermore, in one experiment, Ct was not modified by the inclusion of albumin in the instilled Ringer lactate (5% human albumin solution), indicating that colloid osmotic forces did not determine removal. The dependence of Ct on alveolar size, shown in Fig. 7, rules out a gravitational effect on the removal. Hence, based on these considerations as well as on the rapidity of the removal, we interpret that the instilled liquid was removed convectively.

Although the mechanisms underlying this rapid removal of alveolar liquid remain unclear, alveolar surfactant is implicated by three findings. First, hyperinflation, which stimulates alveolar surfactant secretion (9, 14) decreased Ct. Ashino et al. (2) recently showed that even a single brief period of lung expansion induced alveolar surfactant secretion for a prolonged period. Hence it is possible that in the posthyperinflation period, alveoli were surfactant rich and that the faster liquid removal was attributable to enhancement of surface forces by hyperinflation-induced surfactant secretion.

Second, the intracellular Ca2+ chelator BAPTA-AM markedly prolonged Ct at baseline and also blocked the posthyperinflation decrease of Ct. Surfactant secretion is a Ca2+-dependent process (6, 8). Alveolar stretch due to lung expansion increases alveolar cell Ca2+ and thereby causes surfactant secretion (2, 8, 21). BAPTA-AM chelates intracellular free Ca2+ and prevents both the stretch-induced Ca2+ increase as well as the subsequent enhancement of surfactant secretion (2). If surfactant were responsible for the rapid liquid removal at baseline, then inhibition of surfactant secretion should delay the removal. Furthermore, the inhibition of surfactant secretion should block the hyperinflation effect of increasing surfactant in the alveolus and thereby further delaying liquid removal. Both predictions were supported by our findings that in BAPTA-AM-treated alveoli, Ct was almost two times higher than the control value and that hyperinflation failed to decrease Ct, indicating that the enhancement of liquid removal was blocked.

Third, depletion of extracellular Ca2+ also inhibited the posthyperinflation reduction of Ct. Because Ca2+ determines the organization and maintenance of tubular myelin (11), extracellular Ca2+ is an essential requirement for surfactant function. It is likely that the depletion of extracellular Ca2+ caused disruption of the lining and therefore loss of surface-active forces that may have driven the liquid flow.

In brief, we implicate surfactant in these findings because hyperinflation, a surfactant stimulant, speeded removal, whereas BAPTA-AM, an inhibitor of surfactant secretion, slowed removal. In addition, another important determining factor for removal was alveolar diameter. As predicted by the Laplace equation (see APPENDIX A), if the interfacial tension (T) and the air pressure (Pair) are constants, then the alveolar liquid pressure (Pliq) must increase as alveolar radius (r) increases. If Pliq is the force that convectively clears the liquid from the alveolus (see APPENDIX A), then we may expect a higher Pliq, hence faster removal in larger alveoli. The inverse Ct-alveolar diameter relationship in Fig. 7 supports this expectation.

Despite these considerations, other interpretations may apply to our data. For example, a different form of the Laplace equation may be valid (10). Also, the pores of Kohn that form potential interconnecting pathways between adjoining alveoli (5) could provide a route of liquid flow. Furthermore, a potential explanation may lie in a liquid equivalent of the phenomenon of air "pendulluft" in which out-of-phase airflow occurs between adjacent lung regions because of imbalances in airway impedance (13, 15, 17). Differences in impedance to liquid flow attributable to differences in alveolar diameter may account for the present patterns of liquid removal from instilled alveoli. Although we interpret the BAPTA-AM effects as being due to inhibition of surfactant secretion, other processes inhibited by intracellular Ca2+ chelation may have played a role. These issues require further investigation.

To determine the direction in which the alveolar liquid was removed, we instilled fluorescent latex beads. After instillation, single beads were evident as fluorescent spots and could be followed by time-based fluorescence microscopy. The fluorescent beads were expelled within 3-5 s from the instilled alveolus and, subsequently, continued to move spontaneously across four alveolar diameters for up to 90 s. The evidence for such spontaneous translation is consistent with the notion that the beads were convectively transported in the postinstillation period. A similar interpretation may be drawn from our experiments in which we instilled dye-labeled albumin solution. Here as well, convective flow was evident in that spread of the dye was evident as a spot of increasing perimeter on the lung surface. To the extent that the beads and the albumin solution marked the pattern of alveolar liquid removal, we interpret that the instilled alveolar liquid flowed out of the filled alveolus and then traversed several adjacent alveoli, possibly en route to the respiratory duct.

Using a micropuncture technique, Schurch et al. (16) deposited liquid droplets of different surface tensions on the alveolar surface. Droplets of low surface tension rapidly spread on the surface, whereas those of high surface tension tended to remain rounded, indicating that the balance between the alveolar surface pressure and the surface tension of the deposited liquid droplet was the critical determinant of surface wetting, hence droplet stability (7). In our experiments, the situation was somewhat different because we deposited not a droplet but a volume of liquid that filled the entire lumen of the injected alveolus. If the considerations of the droplet experiment of Schurch et al. (16) were applicable, then given the high surface tension of water, we would expect the injected liquid to remain as a stabilized drop. Because the liquid flowed away, we interpret that subinterfacial pressures generated by the Laplace considerations were the likely factor in this liquid removal.

In conclusion, our findings are the first evidence that alveoli are capable of rapidly ejecting instilled liquid, which indicates the presence of a novel liquid removal mechanism in the lung and suggests that in addition to active transport, alveoli possess the potential for convective removal. Staub microinjected a relatively large volume (1-2 µl) of dyed saline into subpleural alveoli and concluded that the liquid was removed by interstitial drainage (19). Our findings are different because, as we discuss above, the present liquid removal occurred by an intra-alveolar and not an interstitial route. Although the exact mechanisms need to be clarified, our findings indirectly implicate surface-active alveolar forces in the liquid removal. The physiological significance bears on the mechanisms by which alveoli may be protected from liquid filling in early stages of pulmonary edema. As alveoli begin to fill with liquid, rapid convection of the liquid away from the alveoli may be beneficial not only for maintaining liquid-free alveoli to protect gas exchange but also as a provision for washing out cells or particles that may have been deposited on the alveolar surface. The extent to which such a removal mechanism applies in later stages of pulmonary edema when a large number of alveoli become liquid filled remains uncertain. However, we believe that in early pulmonary edema when alveolar liquid entry is incipient and the alveoli are largely still air-filled, the present convective mechanisms may provide a crucial liquid elimination process from the gas exchange area.


    APPENDIX A
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

Laplacian considerations. Because convective flow occurs down a pressure gradient, we considered the possibility that a flow-inducing pressure gradient develops between two adjacent alveoli when one of them becomes liquid filled. This situation is illustrated in Fig. 9 in which a fully liquid-filled alveolus (alveolus A) lies adjacent to an air-filled alveolus (alveolus B). According to the Laplace equation as classically applied to pressure distribution across the alveolar air-liquid interface (3, 12)
(Pair<IT>−</IT>Pliq)<IT>=</IT>2T<IT>/r</IT>
where Pair = PA.


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Fig. 9.   Pressure distribution between 2 adjacent alveoli when 1 becomes liquid filled. Pair, air pressure; r, alveolar radius; Pliq, alveolar liquid pressure.

In alveolus A, the air-liquid interface that occurs at the alveolar mouth has a curvature that is almost flat, resulting in a large increase in r. According to the Laplace equation, the increase in r decreases the transinterfacial pressure drop. Therefore, because Pair is constant, Pliq increases. In fact, in the absence of interfacial curvature, there is no transinterfacial pressure drop and Pair = Pliq as shown. In contrast, the adjacent air-filled alveolus B maintains the normal curvature of the air-liquid interface such that Pliq is less than Pair as classically predicted by the Laplace equation. Thus Pliq in alveolus A is greater that that in alveolus B and thereby establishes the pressure gradient for driving liquid flow, alveolus A right-arrow alveolus B.


    APPENDIX B
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

Because the manual determination of alveolar diameter by microcalipers involved the subjective assessment of alveolar edge detection, we constructed a kappa statistic according to standard methods (1). Two observers determined whether alveoli were air or liquid filled before and after, respectively, alveolar microinjection. Table 1 shows the data that generated a kappa of 0.934, indicating excellent agreement between observers. Hence microcaliper measurements provided dependable quantification of the state of alveolar liquid filling.

                              
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Table 1.   Data used to generate kappa statistic


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grants HL-36024, HL-64896, and HL-57556 (to J. Bhattacharya), and HL-10142 (to P. M. Wang).


    FOOTNOTES

Address for reprint requests and other correspondence: J. Bhattacharya, St. Luke's-Roosevelt Hospital Center, 1000 10th Ave., New York, NY 10019 (E-mail: jb39{at}columbia.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. Section 1734 solely to indicate this fact.

Received 4 May 2001; accepted in final form 30 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX A
APPENDIX B
REFERENCES

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