During lung expansion, the pattern of alveolar perimeter distension is likely to be an important determinant of lung functions as, for example, surfactant secretion. However, the segmental characteristics of alveolar perimeter distension remain unknown. Here, we applied real-time confocal microscopy in the isolated, perfused rat lung to determine the micromechanics of alveolar perimeter distension. To image the alveolar perimeter, we loaded alveolar epithelial cells with a fluorescent dye that we microinjected into the alveolus. Then we viewed single alveoli in a 2-microm-thick optical section at a focal plane 20 mum deep to the pleural surface at baseline. In each alveolus, we identified five to eight segments of the perimeter. For each segment, we determined length (L(seg)) by means of image analysis. At baseline alveolar pressure (P(alv)) of 5 cmH(2)O, L(seg) averaged 46 microm. We hyperinflated the lung to P(alv) of 20 cmH(2)O and identified the same optical section as referenced against morphological landmarks. Hyperinflation increased mean L(seg) by 14%. However, segment distension was heterogeneous, even within the single alveolus. Furthermore, distension was greater in alveolar type 1 than type 2 epithelial cells. These findings indicate that alveoli expand nonuniformly, suggesting that segments that distend the most might be preferred alveolar locations for injury in conditions associated with lung overdistension.

The decrease of lung compliance in pulmonary edema underlies ventilator-induced lung injury. However, the cause of the decrease in compliance is unknown. We tested the hypothesis that in pulmonary edema, the mechanical effects of liquid-filled alveoli increase tissue stress in adjacent air-filled alveoli. By micropuncture of isolated, perfused rat lungs, we established a single-alveolus model of pulmonary edema that we imaged using confocal microscopy. In this model, we viewed a liquid-filled alveolus together with its airfilled neighbor at different transpulmonary pressures, both before and after liquid-filling. Instilling liquid in an alveolus caused alveolar shrinkage. As a result, the interalveolar septum was stretched, causing the neighboring air-filled alveolus to bulge. Thus, the airfilled alveolus was overexpanded by virtue of its adjacency to a liquid-filled alveolus. Confocal microscopy at different depths of the liquid-filled alveolus revealed a meniscus. Lung inflation to neartotal lung capacity (TLC) demonstrated decreased compliance of the air-filled but not liquid-filled alveolus. However, at near TLC, the airfilled alveolus was larger than it was in the pre-edematous control tissue. In pulmonary edema, liquid-filled alveoli induce mechanical stress on air-filled alveoli, reducing the compliance of air-filled alveoli, and hence overall lung compliance. Because of increased mechanical stress, air-filled alveoli may be susceptible to overdistension injury during mechanical ventilation of the edematous lung.Keywords: alveolar edema; compliance; micromechanics; optical sectioning microscopy; fluorescence Pulmonary edema occurs in a stepwise manner such that in its initial stages, liquid-filled and air-filled alveoli lie juxtaposed to one another. This liquid-filling pattern is thought to result from the all-or-none mechanism (1), according to which the curved air-liquid interface in the alveolus establishes a transinterfacial force balance, governed according to the equation of Laplace. In this equation (P alv 2 P liq ) 5 2T/R, where P alv and P liq are pressures in the alveolar air and liquid, respectively, T is the airliquid interfacial surface tension, and R is the interfacial radius. The alveolar entry of edema liquid at constant P alv is thought to increase T, thereby reducing P liq and further promoting liquid entry from the interstitium. This process continues until the interfacial curvature, and thus the pressure difference across the interface, is abolished. Importantly, these force considerations may be sufficiently localized that a single alveolus fills with fluid, while its immediate neighbor remains air-filled.Although the all-or-none mechanism provides an explanation of how single alveoli become liquid-filled during the initiating phase of pulmonary edema, there is little understanding of how the juxtaposition of air-filled and liquid-filled alveoli affects lung mechanics. The critical questions involve whether meniscus formation occurs in the liquid-filled alveolus (1, 2), and whether a...

Kharge AB, Wu Y, Perlman CE. Surface tension in situ in flooded alveolus unaltered by albumin. J Appl Physiol 117: 440 -451, 2014. First published June 26, 2014 doi:10.1152/japplphysiol.00084.2014.-In the acute respiratory distress syndrome, plasma proteins in alveolar edema liquid are thought to inactivate lung surfactant and raise surface tension, T. However, plasma protein-surfactant interaction has been assessed only in vitro, during unphysiologically large surface area compression (%⌬A). Here, we investigate whether plasma proteins raise T in situ in the isolated rat lung under physiologic conditions. We flood alveoli with liquid that omits/includes plasma proteins. We ventilate the lung between transpulmonary pressures of 5 and 15 cmH2O to apply a near-maximal physiologic %⌬A, comparable to that of severe mechanical ventilation, or between 1 and 30 cmH2O, to apply a supraphysiologic %⌬A. We pause ventilation for 20 min and determine T at the meniscus that is present at the flooded alveolar mouth. We determine alveolar air pressure at the trachea, alveolar liquid phase pressure by servo-nulling pressure measurement, and meniscus radius by confocal microscopy, and we calculate T according to the Laplace relation. Over 60 ventilation cycles, application of maximal physiologic %⌬A to alveoli flooded with 4.6% albumin solution does not alter T; supraphysiologic %⌬A raise T, transiently, by 51 Ϯ 4%. In separate experiments, we find that addition of exogenous surfactant to the alveolar liquid can, with two cycles of maximal physiologic %⌬A, reduce T by 29 Ϯ 11% despite the presence of albumin. We interpret that supraphysiologic %⌬A likely collapses the interfacial surfactant monolayer, allowing albumin to raise T. With maximal physiologic %⌬A, the monolayer likely remains intact such that albumin, blocked from the interface, cannot interfere with native or exogenous surfactant activity. alveolar edema; albumin; plasma proteins; surfactant; surface tension IN THE ACUTE RESPIRATORY DISTRESS syndrome (ARDS), high alveolar-capillary barrier permeability results in pulmonary edema.1 The liquid that floods the alveolus contains plasma proteins that can inactivate lung surfactant by adsorbing faster than surfactant and, once present at the interface, blocking further surfactant adsorption (21,45,47,53). Plasma proteins have been shown in vitro to increase surface tension, T, in a dose-dependent fashion (18,41,42,45,53). Elevated surface tension may, in turn, underlie a decrease in lung compliance in ARDS (29) and thus contribute to the need for mechanical ventilation. Intratracheal delivery of exogenous surfactant has been tested in six randomized, controlled clinical trials as a therapy for ARDS, a means of reversing surfactant inactivation, but has failed to reduce mortality (5), which remains Ͼ35% (33).The evidence for increased surface tension in ARDS stems from the reduced surface activity of bronchoalveolar lavage fluid (BALF) from ARDS patients (14,18,19,32,34). This reduced surface activity is principally attri...

The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr ؊/؊ mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr ؊/؊ mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease.Keywords: lung; mouse; CFTR; alveolar secretion; optical-sectioning microscopyThe thin liquid layer lining the alveolar wall of the lung constitutes an important component of the systemic defense against inhaled pathogens. The epithelial liquid establishes a liquid phase adjacent to the alveolar epithelium, enabling secreted phospholipids and proteins to maintain alveolar patency (1) and to establish alveolar immune defense (2). However, mechanisms underlying formation of the alveolar wall liquid (AWL) remain unidentified. In fact, the bulk of the evidence favors the view that in the adult lung, active mechanisms in the alveolar wall remove, rather than secrete, alveolar liquid (3).The difficulty is that studies of AWL have employed conventional morphologic approaches that are likely to alter physiologic liquid conditions in the alveolus (4, 5). To some extent, the liquid morphology was preserved by the application of lowtemperature electron microscopy that revealed continuity of the AWL (6). However, understanding of liquid formation and flow in the alveolar wall continues to be hampered by the lack of direct, real-time methods.Here, we address these issues through the first direct visualization of AWL by means of optical-sectioning microscopy of the mouse lung. Liquid secreted in proximal airways might drain (Received in original form September 13, 2006 and in final form January 25, 2007 ) The studies reported here were supported by NIH grants HL36024, HL64896, and HL78645 (J.B.); HL-75503 (K.P.); and HL080878 (C.E.P).Correspondence and requests for reprints should be addressed to Jahar