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 (C_t) as the time elapsed from the end of alveolar filling to the appearance of 90% of the alveolar diameter. C_t 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.

	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.

View larger version (97K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (12K): [in this window] [in a new window]   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.

View larger version (17K): [in this window] [in a new window]   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.

View larger version (57K): [in this window] [in a new window]   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 C_t, 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 C_t under baseline conditions (P < 0.05), and it abrogated the posthyperinflation reduction of C_t (Fig. 6). When we pretreated alveoli with Ca²⁺-free HEPES solution alone, C_t was not different from baseline. However, similar to the BAPTA-AM experiments, Ca²⁺-free HEPES abrogated the hyperinflation-induced decrease of C_t, indicating that the inflation effect was also dependent on external Ca²⁺. These findings indicate that Ca²⁺-regulated processes determined C_t, although the nonspecific effects of BAPTA-AM may also have contributed to these results.

View larger version (13K): [in this window] [in a new window]   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.

C_t and alveolar diameter. Although all determinations were made at a constant PA of 5 cmH₂O, C_t differed in different alveoli. Under baseline conditions, C_t 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 C_t-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 C_t-diameter relationship.

View larger version (18K): [in this window] [in a new window]   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. , 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 C_t 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 C_t (data not shown), indicating that protein osmotic pressures were not a factor in the removal.

View larger version (14K): [in this window] [in a new window]   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 C_t 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 C_t, thereby ruling out active transport as the major underlying mechanism. Furthermore, in one experiment, C_t 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 C_t 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 C_t. 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 C_t at baseline and also blocked the posthyperinflation decrease of C_t. 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, C_t was almost two times higher than the control value and that hyperinflation failed to decrease C_t, indicating that the enhancement of liquid removal was blocked.

Third, depletion of extracellular Ca²⁺ also inhibited the posthyperinflation reduction of C_t. 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 (P_air) are constants, then the alveolar liquid pressure (P_liq) must increase as alveolar radius (r) increases. If P_liq is the force that convectively clears the liquid from the alveolus (see APPENDIX A), then we may expect a higher P_liq, hence faster removal in larger alveoli. The inverse C_t-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.