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J Appl Physiol 103: 1037-1044, 2007. First published June 21, 2007; doi:10.1152/japplphysiol.00160.2007
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Alveolar expansion imaged by optical sectioning microscopy

Carrie E. Perlman and Jahar 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

Submitted 7 February 2007 ; accepted in final form 20 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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-µm-thick optical section at a focal plane 20 µm 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 (Lseg) by means of image analysis. At baseline alveolar pressure (Palv) of 5 cmH2O, Lseg averaged 46 µm. We hyperinflated the lung to Palv of 20 cmH2O and identified the same optical section as referenced against morphological landmarks. Hyperinflation increased mean Lseg 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.

alveolar distension; alveolar fluorescence; heterogeneity; lung hyperinflation; type 2 cell

THE EFFECT OF LUNG VOLUME (V1) on alveolar geometry impacts several aspects of lung function. For example, high V1 decreases width and red cell content of septal capillaries (17), affecting the lung's vascular resistance and blood flow. Alveolar expansion induces Ca2+ conduction between alveolar type 1 and type 2 epithelial cells, resulting in surfactant secretion (2). High tidal volume ventilation induces inflammatory signaling in lung endothelial cells (6) that might underlie ventilator-induced lung injury (VILI). However, the effect of lung expansion on alveolar segmental geometry has not been determined.

Since the polygonal alveolar perimeter comprises five to eight segments, segmental heterogeneities might determine the pattern of alveolar distension. The transpulmonary pressure, which is the difference between the alveolar and the pleural pressures, provides the force for alveolar expansion. For a single alveolus, pressure is spatially uniform. Hence, if transpulmonary pressure were the sole determinant, an alveolus of roughly uniform geometry might expand uniformly as classically proposed (11, 18, 38). However, alveolar geometry is not uniform, and several factors other than the transpulmonary pressure may potentially delimit uniform alveolar expansion.

Studies based on computational modeling indicate that alveolar geometry and nonuniform septal stiffness determine the alveolar expansion pattern (10, 23). Spatially nonuniform distribution of the alveolar wall liquid (3) might induce differences in the air-liquid surface tension on different perimeter segments, causing differences in distension induced by transpulmonary pressure increase. Variations in thickness or composition of the alveolar epithelial basement membrane (8, 25, 31, 36) could vary segmental stiffness, thereby affecting the extent of segmental distension. However, direct quantification of alveolar segmental distension is lacking.

The difficulty is that most studies of alveolar expansion employed lung histology and electron microscopy (e.g., Refs. 14, 16), in which tissue fixation precluded determinations in the same alveolus at different lung inflation levels. Real-time fluorescence imaging (RFI), combined with optical sectioning microscopy (OSM), offers a potential solution, since lung imaging at defined locations (30) can be repeated at varying inflation levels. Here, applying RFI-OSM to the alveolus at a morphologically landmarked focal plane, we tested the hypothesis that alveoli expand nonuniformly. We report nonuniformity of expansion, regarding both segments of the alveolar perimeter and individual alveolar epithelial cell types. Our data indicate that heterogeneities of alveolar perimeter segments pattern alveolar expansion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents

Fluorophores used were the cytosolic dyes calcein AM and calcein red-orange AM (10–13 µM; Molecular Probes, Eugene, OR) in Ringer solution (HEPES buffered Ringer solution with 4% dextran and 1% fetal bovine serum). Antibodies used were a blocking Ab [F(ab')2 goat anti-mouse IgG from Santa Cruz Biotechnology, Santa Cruz, CA; 4 µg/ml in 5% albumin], a primary anti-rat type 2 cell-recognizing MAb (80 µg/ml in Ringer; gift of Dr. Leland Dobbs, University of California at San Francisco), and a fluorescent secondary Ab [AlexaFluor 488 conjugated F(ab')2 goat anti-mouse IgG from Molecular Probes; 10 µg/ml in Ringer].

Isolated, Perfused Lung Preparation

All animals were treated in accord with a protocol approved by the Institutional Animal Care and Use Committee of the St. Luke's-Roosevelt Institute for Health Science. We established the isolated, perfused lung preparation as per our established protocol (2). Briefly, we anesthetized adult, male Sprague-Dawley rats (n = 14, 300–550 g) with 5% halothane and 40 mg/kg pentobarbital sodium. We cannulated the trachea through a tracheotomy. After withdrawing blood (10 ml) by cardiac puncture, we accessed the lungs through a midline sternotomy. We cannulated the pulmonary artery through the right ventricle and the left atrium through the left ventricle. Then we excised the heart and lungs en bloc and inflated the lungs with 30% O2, 6% CO2, and 64% N2 at constant positive airway pressure (Palv, as measured at the trachea. Using autologous rat blood diluted with 5% albumin (Hct 15%), we pump-perfused the lungs (10 ml/min, 37°C) at constant pulmonary artery and left atrial pressures of 10 and 3 cmH2O, respectively, and at constant Palv as stated. All Palv were established after lung deflation from Palv of 20 cmH2O.

Alveolar Microinfusion

To deliver dyes and antibodies to the alveolus, we micropunctured single alveoli using our reported methods (20). Briefly, we used micropipettes of 6- to 8-µm tip diameter. We gave each microinfusion in four boluses at ~3-min intervals. For each bolus, we microinfused the alveolus for ~3 s, to instill ~0.5 nl. Each bolus filled six to eight alveoli, but the liquid drained completely from the alveoli in seconds (37). No data were obtained in micropunctured alveoli.

Microscopy

We imaged lungs by laser scanning confocal microscopy (LSM 510, Carl Zeiss Microscopy, Heidelberg, Germany) using a x40 water immersion objective (numerical aperture 0.80, Achroplan, Carl Zeiss Microscopy), except where stated. To immerse the objective in water, we placed a water drop on a coverslip that we held in a metal O-ring, as described previously (5). We positioned the O-ring such that the coverslip made light contact with the pleural surface without indenting the surface. We removed the coverslip before inducing changes of Vl.

We excited and collected fluorescence at, respectively, 543 and 560 nm for calcein red-orange, and at 488 and 505–530 nm for both calcein and Alexa 488. For alveoli, we imaged 2.1-µm-thick optical sections (1,024 x 1,024 pixels) at vertical intervals of 2 µm from the pleural surface to a depth of 30 µm. For a single type 2 cell, we used a similar imaging protocol, except we imaged optical sections at 1-µm intervals through the depth of the cell.

Experimental Protocol

To view the epithelial cells lining the alveolar margin, we micropunctured an alveolus on the diaphragmatic surface of the left lower lobe and microinfused calcein or calcein red-orange. After 5 min, we adjusted Palv from 20 to 5 to 20 cmH2O. Then we imaged the alveolus at sequential Palv values of 20 and 5 cmH2O, unless otherwise stated. In any protocol, approximately 5 min elapsed between imaging at sequential pressures. We imaged two to four alveolar fields per lung. Total imaging time was 4–6 h per experiment. Our determinations did not vary with experiment duration. Experimental groups were as follows.

Alveolar expansion.   We imaged alveoli (x0.7 zoom magnification) at Palv of 20 cmH2O (hyperinflation) and then 5 cmH2O (baseline), as well as at intermediate pressures as stated (n = 6 lungs). To determine autofluorescence, we imaged lungs under experimental settings but before dye loading, which resulted in black images (data not shown). To determine the effect of using a coverslip, we imaged alveoli with a x10 air objective in the presence and absence of a coverslip.

Alveolar deflation.   We imaged alveoli (x0.7 zoom magnification) at Palv of 5 cmH2O, 0 cmH2O, and specified re-inflation pressures up to 20 cmH2O (n = 3 lungs).

Type 2 cells.   To view single type 2 cells, we microinfused type 2 cell-identifying antibodies in addition to calcein red-orange. We imaged type 2 cells (x5 zoom magnification) at Palv of 20 and 5 cmH2O (n = 4 lungs).

Whole lung.   To determine the lung pressure-volume relation, we cannulated the trachea (n = 4 lungs). By means of air injection/withdrawal through a syringe, we inflated the lung to total lung capacity (TLC) and deflated it to Palv of 5 cmH2O. Subsequently, we inflated the lung to Palv of 20 cmH2O and then deflated it in 1-ml increments to 0 cmH2O. From the volume withdrawn, we determined Vl at Palv of 20 and 5 cmH2O. To determine TLC, we reinflated the lung to Palv of 30 cmH2O and then withdrew air in 1-ml increments to Palv of 0 cmH2O.

Length Measurements and Calibrations

For image analysis, we used commercial software (Metamorph, Universal Imaging, Downingtown, PA). Imaging a micrometer under experimental settings demonstrated that pixel lengths were 0.32 and 0.05 µm at x0.7 and 5 magnification, respectively. To detect a 5% change in length, we analyzed dimensions of only alveolar perimeter segments or type 2 cells that were at least 40 or 9.5 µm in length, respectively, at baseline.

To calibrate length data, we placed green fluorescent latex beads (Molecular Probes) on silicone tape (Radio Shack, Fort Worth, TX). We fixed the silicone tape to calipers and increased total tape length from a baseline of 30 mm by 5%. At baseline and after stretching the silicone tape, we imaged the beads stuck to the tape using the same microscope settings as in our alveolar experiments. At x0.7 magnification, we determined change in distance between pairs of beads (0.78-µm diameter) separated by 43 ± 3 µm at baseline. Tape stretch increased interbead distance by 2.2 ± 0.2 µm (n = 12, P < 0.01). At x5 magnification, we determined change in distance between pairs of beads (0.36-µm diameter) separated by 11 ± 1 µm at baseline. Tape stretch increased interbead distance by 0.53 ± 0.04 µm (n = 9, P < 0.01). Thus the accuracy of our methods enabled us to determine a 5% change in length.

Statistics

Data from the same alveolus or type 2 cell were compared by paired t-test. Deflation/reinflation data from multiple inflation pressures were compared by ANOVA with Bonferroni correction. Other data were compared by unpaired t-test. All data are means ± SE. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Within the plane of an optical section, the perimeters of calcein-loaded alveoli were evident as continuous, fluorescent lines that circumscribed air-filled, nonfluorescent alveolar lumens (Fig. 1A). At high magnification, alveolar septa appeared as pairs of fluorescent lines that separated adjacent air-filled alveoli (Fig. 1B, brackets). The fluorescence marked the epithelial cytosol and indicated the contour of the alveolar perimeter. The total septum comprised the two fluorescent epithelial layers and the nonfluorescent capillaries and interstitium located between them. At each alveolar corner, we used a morphological landmark (Fig. 1B, inset, arrow) to define a point on the alveolar perimeter. Between landmarked points at successive corners, we used the cursor of the analysis software to trace the alveolar perimeter. From the pixel length of the traced line, we determined the length of the perimeter segment (Lseg). For each alveolus, the sum of Lseg for all perimeter segments gave the total alveolar perimeter length (L). We determined the alveolar luminal area (A) from the number of pixels enclosed by the perimeter tracing. We calculated the alveolar diameter (D), which we used as a length scale for the alveolus, from the relation D = (4A/{pi})1/2.


Figure 1
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  		Fig. 1. Alveolar morphology. A: low-magnification image of an alveolar field. Calcein AM (green pseudocolor) was loaded into the cytosol of epithelial cells. B, left: 2.1-µm-thick optical section of three alveoli (a1, a2, and a3) at a subpleural depth of 20 µm. Airway inflation pressure Palv was 5 cmH2O. White dotted line traces the perimeter of alveolus a1. The length of the line is the alveolar perimeter length (L); the area enclosed by the line is the cross-sectional luminal area (A). Brackets indicate the septum between alveoli a2 and a3. Arrows indicate two corners of alveolus a1. Red dotted line marks one perimeter segment of alveolus a1. Perimeter segment has length Lseg. Note, septal width 14 µm (*) is possibly due to capillary congestion. Right: detail of boxed area from left image shows alveolar corner and example characteristic fluorescent pattern used as a morphological landmark (arrow) to define the perimeter segment endpoint. C: optical sections of a septum between two alveoli (a) at baseline (Palv, 5 cmH2O; green pseudocolor) and hyperinflation (Palv, 20 cmH2O; red pseudocolor). Baseline optical section was located 20 µm below the pleural surface. Epithelial morphology, for example the distinct fluorescence pattern in the region circled by the solid line and the decreased septal thickness in the region circled by dotted line, landmarked the baseline section. Note, the same landmarks were evident at hyperinflation, although at a subpleural depth of 22 µm, thus identifying the same optical section.

 
To determine alveolar dimension in a single optical section, we used the section 20 µm below the pleural surface at baseline. From the z-stack imaged at baseline, we identified up to five morphological landmarks around the alveolus (Fig. 1C shows two example landmarks) that defined this one section. From the z-stack imaged at hyperinflation, we used the landmarks to identify the same section. Thus the landmarks enabled us to identify one section and track it at different inflation pressures.

Increasing Palv increased L and D. However, at any Palv, L and D were each greater during deflation than inflation (Fig. 2A). Furthermore, decreasing Palv to 0 cmH2O markedly decreased L. Subsequently, increasing Palv reestablished baseline L only at Palv in excess of 15 cmH2O (Fig. 2, B and C). These findings indicated the presence of hysteresis in the Palv-L and Palv-D relations.


Figure 2
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  		Fig. 2. Alveolar hysteresis. A: inflation pressure (Palv)-perimeter length (L) and inflation pressure-alveolar diameter (D) curves for a typical alveolus. B: in separate experiments, the lung was deflated from Palv of 5 to 0 cmH2O and reinflated, sequentially, to Palv of 5, 10, 15, and 20 cmH2O. Images are examples from Palv of 5 cmH2O before deflation, Palv of 0 cmH2O, and reinflation Palv of 15 and 20 cmH2O. C: response of L to decrease and increase of Palv (*P < 0.01 compared with baseline Palv of 5 cmH2O and reinflation Palv values of 15 and 20 cmH2O; #P < 0.01 compared with all other data points). D: hyperinflation from Palv of 5 to 20 cmH2O increased both L and D (*P < 0.01; n = 30). The increase in D was greater than that in L (#P < 0.01; n = 30).

 
Uniform alveolar expansion should increase L and D equally. However, hyperinflation to Palv of 20 cmH2O induced greater increase in D than in L (Fig. 2D). These data indicate that alveolar perimeter distension was nonuniform.

Inflation also increased Vl from 8 ± 1 ml (19 ml/kg body wt) at baseline to 13 ± 2 ml (31 ml/kg) at hyperinflation (n = 4, P < 0.01). Since TLC was 16 ± 2 ml (38 ml/kg), we expanded Vl by 12 ml/kg to 82% of TLC. Hyperinflation increased Vl1/3 by 18 ± 3% (n = 4, P < 0.01). Therefore, the percent increase in Vl1/3 did not differ from the percent increases in L or D. This similarity indicates that single alveolar expansion was of the same order of magnitude as whole lung expansion.

The alveolar perimeter comprised five to eight segments (Fig. 3A). At baseline, in 70% of segments, Lseg ranged between 30 and 80 µm (Fig. 3B). In each alveolus, hyperinflation to Palv of 20 cmH2O caused nonuniform perimeter segment distension, as measured in a morphologically landmarked optical section. In the alveolus shown in Fig. 3A, hyperinflation increased L and D by 13 and 15%, respectively. However, individual perimeter segment distension varied. Hyperinflation distended perimeter segments 1 and 2 by 7 and 19%, respectively. Group data indicate that the hyperinflation-induced increase in Lseg was heterogeneous (Fig. 3C). Additionally, segment distension failed to correlate with baseline Lseg (Fig. 3D).


Figure 3
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  		Fig. 3. Heterogeneous perimeter segment distension. A: an alveolus was imaged at baseline (Palv, 5 cmH2O; green pseudocolor) and hyperinflation (Palv, 20 cmH2O; red pseudocolor). Numbers in baseline image label two perimeter segments. An overlay of the images demonstrates inflation-induced alveolar expansion, which increased L and D by 13 and 15%, respectively. However, perimeter segment distension was heterogeneous. The two labeled perimeter segments are shown at low and high magnification; magnification levels are the same for both perimeter segments. In the low-magnification images, baseline Lseg is given, and arrows indicate the perimeter segment endpoint at which the baseline and hyperinflation images are aligned. High-magnification images show details of the other endpoints. Green and red marks indicate the separation between the baseline and hyperinflation images. Distance between marks, labeled in micrometers and as a percentage of baseline length, indicates perimeter segment distension. B: histogram of baseline Lseg. Y-axis shows frequency per alveolus (n = 30). C: histogram of perimeter segment distension. Perimeter segments are grouped, on the x-axis, by the extent to which hyperinflation increased their length, Lseg. Y-axis shows frequency per alveolus (n = 30). Less than 5% distension was not detectable by our methods. D: inflation-induced distension did not correlate with baseline Lseg (R2 = 0.02, n = 115).

 
We determined Lseg in the 61% of all perimeter segments that exceeded 40 µm in length at baseline (Fig. 3B). These segments comprised 74% of the alveolar perimeter. In the alveolus, hyperinflation increased average Lseg by 14 ± 1% (n = 30, P < 0.01). For the remaining 26% of the perimeter, we determined the global length Lrem. Hyperinflation increased Lrem 16 ± 2% (n = 26, P < 0.01). There was no difference between the increases in Lseg and Lrem. Therefore, the segments we analyzed were representative of all perimeter segments.

We identified type 2 epithelial cells by methods previously reported by our group (2), namely by means of in situ immunofluorescence using a cell-specific, surface-epitope recognizing antibody (Fig. 4A). We loaded the cells with calcein red-orange and determined cell length by means of optical sectioning (Fig. 4B). Increasing Palv from 5 to 20 cmH2O distended approximately one-half the imaged type 2 cells (Fig. 4C) and distended type 2 cells, on average, significantly less (P < 0.01) than perimeter segments lined primarily by type 1 cells (Fig. 3C). Furthermore, hyperinflation distended even the subset of perimeter segments adjacent to nondistended type 2 cells (Fig. 4, D and E). While most type 2 cells were located at the alveolar corner, 24% were located on an alveolar septum (Fig. 4F). However, no correlation existed between cellular location and distension (Fig. 4G).


Figure 4
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  		Fig. 4. Alveolar type 2 epithelial cell detection and distension. A, left: surface immunofluorescence indicated type 2 cell. Center: calcein red-orange loading of type 1 and 2 epithelial cells. Right: overlay of left and center images confirmed the identity of the type 2 cell. B: baseline (Palv, 5 cmH2O; green pseudocolor), hyperinflation (Palv, 20 cmH2O; red pseudocolor), and overlay images of the type 2 cell from A. Line in baseline image shows an example of type 2 cell length measurement parallel to the alveolar margin. C: histogram of hyperinflation-induced type 2 cell distension (n = 25). Less than 5% distension was not detectable by our methods. D: baseline, hyperinflation, and overlay images of the area containing the type 2 cell from A and B. Green and red lines mark the endpoints of the adjacent perimeter segment at baseline and hyperinflation, respectively. Inflation distended the adjacent perimeter segment by 12%. E: inflation distended type 1 cell-lined perimeter segments adjacent to nondistended type 2 cells (*P < 0.01, n = 9). This type 1 cell distension was greater than that of the adjacent, nondistended type 2 cells (#P < 0.01, n = 9). F: images of two representative type 2 cells. Images show calcein red-orange loaded epithelium (type 1 and 2 cells) and antibody identification of type 2 cells, which are outlined with dotted white lines. Type 2 cells were located at the corner of an alveolus (a) as in left image or in the middle of a septum as in right image. G: type 2 cell distension did not correlate with location.

 
To rule out artifacts, we imaged an alveolus with a x10 air objective at hyperinflation and baseline in both the presence and absence of a coverslip. The coverslip caused no change in alveolar geometry (Fig. 5).


Figure 5
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  		Fig. 5. Coverslip effects. A calcein-loaded alveolus imaged at Palv of 5 cmH2O with a x10 air objective in the absence (green pseudocolor) and presence (red pseudocolor) of a coverslip is shown. Overlay image shows the coverslip did not alter alveolar geometry.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Using RFI-OSM, we report here the first detailed micromechanics of alveolar shape change. This new approach provides the advantage of repeat imaging of the same sample at different points in the Vl history. Lung hyperinflation to 82% of TLC distended the alveolar perimeter by 14%. However, different segments within the same alveolus distended as little as 5% or as much as 25% of initial. We also observed significantly greater inflation-induced distension of type 1 than type 2 epithelial cells. Hence, when the lung is inflated to near maximum, we conclude that alveolar expansion is not uniform as previously proposed (11, 18, 38). Rather, we find that alveolar expansion is markedly nonuniform.

The advantage of RFI-OSM is that this approach uniquely affords the detection of alveolar dimensional changes in a defined subpleural plane. We applied RFI-OSM in a cross-sectional alveolar plane that we could consistently identify at different lung inflation levels by morphological landmarking, a critical feature of our approach. Our findings in two dimensions need to be interpreted with caution with respect to three-dimensional alveolar expansion. However, despite alveolar expansion, we were able to identify specific landmarks and track them at different levels of lung inflation (Fig. 1C). If the alveolus as a whole expanded uniformly, then we would expect it to expand uniformly within the plane of our analysis. In contrast, in a plane 20 µm below the pleural surface at baseline, alveolar expansion was nonuniform. We believe that RFI-OSM is a novel and effective approach for quantification of alveolar micromechanics.

Two sets of results pointed to nonuniform alveolar expansion. First, Lseg increased heterogeneously within the alveolus. To ensure that we could detect at least 5% distension, we determined distension for only perimeter segments with Lseg ≥ 40 µm at baseline. These segments comprised 74% of the alveolar perimeter, and distension of these segments was heterogeneous. However, global distension did not differ between this 74% of the perimeter and the Lrem. This result and the lack of correlation between septal baseline length and distension (Fig. 3D) indicate that the Lseg distensions that we analyzed likely represented distensions for all perimeter segments in the alveolus.

Second, lung inflation increased D relatively more than L. The parameter D was intended to be a length scale for the alveolus that was proportional to A1/2, and the equivalent circle model we used to determine D has been used previously (13). However, this choice of geometry was arbitrary, since, regardless of the geometric shape chosen, uniform alveolar expansion in the plane of our analysis would be expected to cause equal percent increases in L and D. Thus our finding that inflation increased D more than L is independent of our application of the equivalent circle model.

Possible reasons for nonuniform alveolar expansion include heterogeneous septal stiffness, heterogeneous levels of force supported by septa, and inflation-induced unfolding of basement membrane pleats (28, 35). Further experiments will be required to elucidate the cause of the heterogeneity. However, our data from measurements of Lseg and of total L together indicate that alveolar expansion is nonuniform.

The relevance of our findings requires consideration in the context of whole lung expansion. The present hysteresis in alveolar expansion affirms previous findings that acinar expansion occurs with hysteresis (27). Although alveolar expansion pattern might differ during dynamic ventilation, we suggest that the expansion behavior of single alveoli underlies the well-known hysteresis of whole lung expansion. Furthermore, lung hyperinflation from baseline to near TLC increased D by 20% and Vl1/3 by 18%. Given this similarity, the present alveolar expansion may underlie lung expansion. The present expansion by 12 ml/kg body wt is equivalent to a tidal volume that might exacerbate lung injury and cause VILI (7). Thus, in the presence of lung injury, the most distended perimeter segments might be the most susceptible to VILI.

To further address the mechanism of whole lung expansion, we decreased Palv to 0 cmH2O, which decreased L by 60%. Such a marked decrease in transpulmonary pressure might not occur in vivo. Here, increase of Palv to greater than 15 cmH2O was necessary to return L to baseline. We cannot rule out some possible surfactant loss with microinfusion. However, previous work from our laboratory (2) has shown that our present inflation protocol causes surfactant secretion; therefore, we believe surfactant was present at the interface. While we maintained Palv at or above 5 cmH2O, we detected no alveolar recruitment or derecruitment during Vl cycling. Hence we agree with reported data (28) that alveolar recruitment plays no role in physiological lung inflation.

The order of magnitude of our determinations agrees with those of past studies of alveoli deep in the lung's parenchyma (18, 26, 33). Gil et al. (16) showed there is no difference in the alveolar surface area-volume relation between subpleural and parenchymal alveoli. Furthermore, the model of Mead et al. (24) showed subpleural and internal alveoli are subjected to the same pressures. Together, these considerations support the notion that micromechanics of expansion might be similar between subpleural and deeper alveoli.

An important finding was that lung inflation distended type 1 cell-lined alveolar perimeter segments almost two times as much as single type 2 cells. Thus distension heterogeneity was evident not only between segments, but also within segments. These heterogeneities potentially bear on the manner in which alveolar expansion induces surfactant secretion, as reported by our (2) and other (39) groups. Although the reason for the differential distension of type 1 and 2 cells is unclear, we suggest differences in the composition and mechanical properties of the matrix underlying the two cell types (8, 21, 31, 36) might play a role. Since the stiffness modulus of the collagen-containing extracellular matrix (1, 8, 12, 22, 29) is four orders of magnitude greater than the epithelial cell modulus (4, 19), matrix mechanical properties might be expected to dominate the pattern of alveolar perimeter distension. Structural considerations include the fact that type 1 cells tend to cover "thin" sections of the extracellular matrix, and type 2 cells, with pseudopodia that penetrate the matrix, tend to locate on "thick" sections (9, 22, 36). These issues potentially underlying differential distensions of type 1 and type 2 cells require further clarification.

Several aspects of our methodology require consideration. No hemorrhage was evident. There was no loss of intracellular dye and no air leakage at the micropuncture site. These observations are consistent with rapid resealing of the plasma membrane following transient membrane injury (15, 32, 34). Previous work from our laboratory demonstrated the presence of surfactant at the air-liquid interface following microinfusion and lung inflation (2) and constant alveolar liquid lining layer thickness following microinfusion (20). The absence of diffuse interstitial fluorescence ruled out the presence of edema. Therefore, our micropuncture procedure did not damage the alveolus.

Finally, our length measurements were calibrated against images of a micrometer. At x0.7 zoom magnification, pixel length was 0.32 µm. Taking into account error at each end of a length measurement, we determined distension of only perimeter segments measuring at least 40 µm at baseline so that we could detect a 5%, or 2 µm, increase. At x5 zoom magnification, pixel length was 0.05 µm. We determined distension of only type 2 cells measuring at least 9.5 µm at baseline, so that we could detect a 5%, or 0.5 µm, increase. This consideration of measurement accuracy, along with our calibrations using beads on silicone tape distended by 5%, indicates that a 5% change in length was detectable by our methods.

In conclusion, our findings reveal a novel feature of alveolar micromechanics, in that the alveolar perimeter possesses markedly different mechanical properties at different locations. We show here that these mechanical differences lead to at least two unpredicted alveolar consequences, namely uneven expansion of the alveolus as a whole and differences in the extents to which major epithelial cell types in the alveolar perimeter undergo distension. The physiological significance underlying these micromechanical properties remains unclear, but is likely to be important. Thus, uneven alveolar expansion might impact septal vascular mechanics, while differences in cellular micromechanics might influence the regulation of surfactant secretion. The role played by site-specific differences in alveolar micromechanics in the regulation of alveolar function requires investigation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-80878 (to C. E. Perlman) and HL-64896 (to J. Bhattacharya).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Bhattacharya, 432 W. 58th St., AJA 509, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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