BRONCHIAL LIGATION ENHANCES MURINE FETAL LUNG DEVELOPMENT IG WHOLE-ORGAN CULTURE

Evidence exists from both congenital anomalies and animal models that normal fetal lung development is dependent on maintenance of fluid pre ssure within the developing ''airways.'' Fetal tracheostomy, allowing free egress of airway fluids, results in lung hypoplasia, indicating t hat some airway distending pressure is required for normal lung develo pment to occur. In contrast, fetal tracheal ligation, which increases fetal airway pressure, reverses lung hypoplasia in animal models. The authors' experiments test the hypothesis that large airway obstruction accelerates the development of murine lungs in vitro in whole-organ c ulture. Fetuses from time-dated pregnant CD-1 mice at day 14 of gestat ion were removed (term, 20 days), and the lungs were excised. The left bronchus of each lung was ligated (n = 26), after which the left lung was isolated and cultured at 37 degrees C (95% air, 5% CO2) in BGJb m edia supplemented with vitamin C and antibiotics. Some fetal lungs wer e cultured under similar conditions without bronchial ligation (n = 11 ). After 7 days in culture, the lungs were taken for various analyses. The lungs were fixed in either formaldehyde and processed for paraffi n embedding for light microscopic evaluation and morphometric data col lection, or were freshly minced and aliquots taken for total protein a nd DNA content. Several more ligated and unligated lungs were processe d for ultrastructural analysis. Morphometric analysis on transverse se ctions of lungs showed significant differences in the lung tissue size , thickness, epithelial cell height, luminal areas, perimeters, and to tal number of airspaces (airway + primordial alveolar airspaces). It w as evident that bronchial ligation promoted lung development. The liga ted lungs displayed thinning of the primordial alveolar walls with cub oidal epithelial cells. The total number of airspaces per field was lo wer for better developed ligated lungs because of the increased area o f airspaces compared with that of the unligated lungs. The dorsoventra l tissue thickness (in micrometers) of the ligated lungs was significa ntly greater than that of the unligated lungs (124.1 +/- 7.0 v 89.6 +/ - 8.0); the average outer perimeter of the primordial alveolar airspac es was greater for ligated lungs (404.56 +/- 19.0 mu m v 256.85 +/- 17 .0 mu m). Similarly, the luminal diameter of the spaces of ligated lun gs was almost double that of the unligated lungs (38.0 +/- 2.0 mu m v 20.3 +/- 2.0 pm), as was the luminal surface area. The morphometric da ta, which suggest enhanced maturation of the ligated lungs, are suppor ted by results of ultrastructural studies. Ligated lungs had significa ntly more lamellar bodies. Although total protein and DNA content were greater among the ligated lungs, the protein/DNA ratios did not diffe r among the groups. The intraluminal pressure (airway pressure) of lig ated lungs was 2.9 mm Hg and 3.1 mm Hg at 2 and 4 days in organ cultur e; the respective pressures for unligated lungs were 1.0 mm Hg and 0.8 mm Hg. These data support the hypothesis that mechanical distending p ressure resulting from airway obstruction not only improves pulmonary architecture but also accelerates lung development in vitro. Although these effects have been seen in in vivo models, this is the first prop osed in vitro organ culture model. This model may prove to be a powerf ul tool for the study of molecular mechanisms of mammalian lung develo pment with respect to mechanical and chemical (cytokines, hormones) st imuli. Copyright (C) 1996 by W.B. Saunders Company