Fig 4. Nischedsn/edsn mice display embryonic and adult lung abnormalities.
(A-D) At E16.5, E18.5 and P0, some Nischedsn/edsn embryos display lung abnormalities. H&E stained lung sections from (A) Nisch+/+ and (B) severely affected Nischedsn/edsn mice at P0, show thickened interstitial mesenchyme and smaller airspaces in Nischedsn/edsn lungs. (C) Number of airspaces was counted in three different 6.5 x 105 μm2 regions for all embryos and new born mice, with no significant difference between genotypes. (D) Airspace diameters were measured for 30 airspaces in three different regions for all embryos and new born mice. The mean airspace width in severely affected Nischedsn/edsn animals was significantly smaller. Nisch+/+ n = 6–9; Nischedsn/+ n = 11–15; Nischedsn/edsn n = 6–10; mildly affected Nischedsn/edsn n = 4–7; severely affected Nischedsn/edsn n = 2–3. (E-H) Adult Nischedsn/edsn mice exhibit an emphysema-like phenotype. H&E stained lung sections from (E) Nisch+/+ and (F) Nischedsn/edsn animals at 20 wk, show enlargement of air spaces in Nischedsn/edsn lungs accompanied by disruption of normal alveolar architecture. (G) In Nischedsn/edsn lungs quantification of the number of airspaces indicated a significant decrease compared to wild-type and (H) the mean airspace width in Nischedsn/edsn lungs was significantly larger than in wild-type tissue. Nisch+/+ n = 10; Nischedsn/+ n = 10; Nischedsn/edsn n = 18. (I-M) The severity of the emphysema-like lung phenotype observed in Nischedsn/edsn replicates the severity of the OM phenotype in the middle ear. H&E stained lung sections from Nischedsn/edsn animals at 20 wk show the increasing severity of the emphysema-like phenotype from mice with (I) no OM phenotype, to (J) unilateral OM and to (K) bilateral OM. (L) The mean number of airspaces in Nischedsn/edsn animals with no OM phenotype was significantly increased compared to Nischedsn/edsn animals with bilateral OM. (M) The mean airspace width in Nischedsn/edsn mice with no OM phenotype was significantly smaller than Nischedsn/edsn animals with bilateral OM. Bilateral OM n = 10; Unilateral OM n = 6; Clear n = 2. (N, O) Immunohistochemical staining of lung sections using an F4/80 antibody. Lung sections from (N) Nisch+/+ and (O) Nischedsn/edsn animals at 20 wk stained with F4/80 show collections of enlarged alveolar macrophages (brown) in Nischedsn/edsn lungs compared to wild-type littermates. Representative images from four mice per genotype. N, O scale bar = 200 μm; A, B, E, F, I, J, K scale bar = 100 μm. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. Error bars indicate standard error of mean. Embryonic lung data in panels C and D were analysed by one-way ANOVAs and Holm-Sidak’s multiple comparison procedures for post-hoc testing. Adult lung data (G, H, L, M) was not normally distributed and a Kruskall-Wallis test was performed followed by Dunn’s multiple comparison tests for post-hoc analysis.