Bronchial ligation enhances murine fetal lung development in whole-organ culture

Abstract

Evidence exists from both congenital anomalies and animal models that normal fetal lung development is dependent on maintenance of fluid pressure within the developing “airways.” Fetal tracheostomy, allowing free egress of airway fluids, results in lung hypoplasia, indicating that some airway distending pressure is required for normal lung development 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 culture. Fetuses from time-dated pregnant CD-1 mice at day 14 of gestation 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°C (95% air, 5% CO 2) in BGJb media supplemented with vitamin C and antibiotics. Some fetal lungs were 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 paraffin embedding for light microscopic evaluation and morphometric data collection, or were freshly minced and aliquots taken for total protein and DNA content. Several more ligated and unligated lungs were processed for ultrastructural analysis. Morphometric analysis on transverse sections of lungs showed significant differences in the lung tissue size, thickness, epithelial cell height, luminal areas, perimeters, and total number of airspaces (airway + primordial alveolar airspaces). It was evident that bronchial ligation promoted lung development. The ligated lungs displayed thinning of the primordial alveolar walls with cuboidal epithelial cells. The total number of airspaces per field was lower for better developed ligated lungs because of the increased area of airspaces compared with that of the unligated lungs. The dorsoventral tissue thickness (in micrometers) of the ligated lungs was significantly greater than that of the unligated lungs (124.1 ± 7.0 v 89.6 ± 8.0); the average outer perimeter of the primordial alveolar airspaces was greater for ligated lungs (404.56 ± 19.0 μm v 256.85 ± 17.0 μm). Similarly, the luminal diameter of the spaces of ligated lungs was almost double that of the unligated lungs (38.0 ± 2.0 μm v 20.3 ± 2.0 μm), as was the luminal surface area. The morphometric data, which suggest enhanced maturation of the ligated lungs, are supported by results of ultrastructural studies. Ligated lungs had significantly more lamellar bodies. Although total protein and DNA content were greater among the ligated lungs, the protein/DNA ratios did not differ among the groups. The intraluminal pressure (airway pressure) of ligated lungs was 2.9 mm Hg and 3.1 mm Hg at 2 and 4 days in organ culture; the respective pressures for unligated lungs were 1.0 mm Hg and 0.8 mm Hg. These data support the hypothesis that mechanical distending pressure 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 proposed in vitro organ culture model. This model may prove to be a powerful tool for the study of molecular mechanisms of mammalian lung development with respect to mechanical and chemical (cytokines, hormones) stimuli.