Fig. 4: Aging-associated fibrosis limits alveolar expansion and surfactant secretion.

a, b Two-photon images (i and ii) show collagen fluorescence by second harmonic generation in the subpleural interstitium of an 18 (left) and an 86 (right) year-old lung. Adjacent panels show collagen fluorescence in the depth plane (y-z) along the indicated lines (dashed lines). Tracings in (b) represent fluorescence intensity along the lengths of the dashed lines (“distance”). c–e Imaging was carried out across a tissue volume calculated as the product of the area and the depth of the imaged field (see sketch). Tracings in (c) are from two representative fields and quantify collagen fluorescence along the depth axis from the pleural margin. In (d) “collagen density” was calculated as the summed fluorescence per unit volume for multiple fields in each lung. (Each color indicates a separate lung.) Group data are shown in (e). f, g Images and group data show peri-alveolar collagen. h Confocal images show an alveolus (alv) stained with the intracellular dye, calcein-AM. Alveoli were imaged at alveolar pressures 5 (green) and 20 cmH20 (red). The alveolar diameter at 5 cmH2O is marked by the line “a”. The septal displacement at 20 cmH2O is marked by the line “b” (see a magnified image of the region selected by the rectangle). i, j Alveolar expansion was calculated from the distances of the lines exemplified in h, as (b/a)%, and plotted for individual imaged fields against donor age (i) and subpleural collagen densities (j). k The images show an alveolus stained with calcein-AM (green) and the extracellular lipid dye FM1-43 (red). A selected region (rectangle in left image) was magnified in the middle and right images. Alveolar stretch caused surfactant secretion as indicated by a time-dependent increase of red fluorescence (arrow). l, m Plots show determinants of surfactant secretion. For a: determinations replicated 9 times in an 18- and an 86-year-old lung gave similar results. For e and g: data were mean ± SEM; n = 7 lungs each group; P value is for two-tailed Student’s t-test. For i, j, l, and m: each dot shows the response for a single alveolus. For f, h, and k: determinations were repeated in seven lungs in each group. For i, j, and m: P values were computed by linear regression. For boxplots in l: the middle line shows the median, the lower and upper hinges are the first and third quartiles, the lower and upper whiskers extend to the value at most 1.5 IQR below the first quartile or 1.5 IQR above the third quartile, respectively, the data beyond the ends of the whiskers are outlying points, and the P value shown is for two-tailed Student’s t-test.