From morphological heterogeneity at alveolar level to the overall mechanical lung behavior: an in vivo microscopic imaging study

Abstract

In six male anesthetized, tracheotomized, and mechanically ventilated rabbits, we imaged subpleural alveoli under microscopic view (60×) through a "pleural window" obtained by stripping the endothoracic fascia and leaving the parietal pleura intact. Three different imaging scale levels were identified for the analysis on increasing stepwise local distending pressure (P ld) up to 16.5 cmH2O: alveoli, alveolar cluster, and whole image field. Alveolar profiles were manually traced, clusters of alveoli of similar size were identified through a contiguity-constrained hierarchical agglomerative clustering analysis and alveolar surface density (ASD) was estimated as the percentage of air on the whole image field. Alveolar area distributions were remarkably right-skewed and showed an increase in median value with a large topology-independent heterogeneity on increasing P ld. Modeling of alveolar area distributions on increasing P ld led to hypothesize that absolute alveolar compliance (change in surface area over change in P ld) increases fairly linearly with increasing initial alveolar size, the corollary of this assumption being a constant specific compliance. Clusters were reciprocally interweaved due to their highly variable complex shapes. ASD was found to increase with a small coefficient of variation (CV <25%) with increasing P ld. The CV of lung volume at each transpulmonary pressure was further decreased (about 6%). The results of the study suggest that the considerable heterogeneity of alveolar size and of the corresponding alveolar mechanical behavior are homogenously distributed, resulting in a substantially homogenous mechanical behavior of lung units and whole organ.

Keywords: Alveolar mechanics; in vivo microscopy; multiscale.

Figure 1.

Experimental preparation. (A) Macrophotography on creating a “pleural window”: (a) intact chest wall, (b) intercostal muscle layer, (c) denuded portion of parietal pleura (“pleural window”). (B) Part of a pleural window under the microscopic view with a magnification of 60×: alveoli are clearly visible where the endothoracic fascia was stripped off the denuded parietal pleura (c1), as compared to the unstripped portion (c2). (C) An example of manually segmented alveolar units (digital zoom of a 60× magnified microscopic image) at P_ld = 4.5 cmH₂O. Four nonoverlapped ROI are shown together with the corresponding percentage values of alveolar surface (alveolar surface density, ASD). (D) The same alveolar population of (C) with manually traced alveolar clusters (black lines) obtained by the contiguity‐constrained hierarchical clustering analysis. Alveoli not belonging to any clusters are not encircled.

Figure 2.

(A and B) Distributions of alveolar areas at P_ld = 3 and 16.5 cmH₂O (P₀ and P₁, respectively), obtained by pooling data from all rabbits. (C) Box plot describing the alveolar surface areas from all rabbits at different levels of alveolar pressure (only the inflation limb is shown). For each pressure, the gray box shows a 25–75% percentile range; the median value is represented by the horizontal bar. The whiskers above and below the gray boxes encompass 80% of the variability in area values. (D) Semivariogram of alveolar areas at P₀.

Figure 3.

Histogram of circularity values for alveolar clusters at P₀.

Figure 4.

(A) Alveolar surface density (ASD) as a function of the local distending pressure. Mean values of data pooled from all rabbits are presented with ±SD. (B) Transpulmonary pressure versus lung volume curve (normalized to TLC), obtained as the average of individual rabbit pressure–volume curves.

Figure 5.

(A) Absolute compliance estimated by comparing subpopulations drawn from alveolar area distributions at P₀ and P₁ (continuous line). (B) Specific compliance derived from the absolute compliance, as detailed in the text (continuous line). Dashed line in (A) and (B) correspond to the assumption of a constant specific compliance. (C) Modeling of the experimental alveolar area distribution at P₁ (white dotted points), assuming either a variable (black dashed line) or fixed (gray dashed line) specific compliance.