Retinoic acid rescues lung hypoplasia in nitrofen-induced hypoplastic foetal rat lung explants

Abstract

There is increasing evidence to suggest that the retinoid pathway is involved in the pathogenesis of congenital diaphragmatic hernia (CDH). We hypothesised that retinoids are involved in the pathogenesis of associated pulmonary hypoplasia in CDH and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 of gestation. Foetal lungs were harvested on embryonic day 13.5 and were cultured for 96 h with or without exogenous retinoic acid (RA) (1 muM) added daily to the culture medium. Lungs were divided into four study groups: control (n=31); control + RA (n=19); nitrofen (n=19); and nitrofen + RA (n=12). Lung growth was assessed in each group by measuring branching morphogenesis, total DNA content and the proportion of proliferating cells stained by immunohistochemistry. One-way ANOVA test was used for statistical analysis. Retinoic acid significantly increased the growth of nitrofen-induced hypoplastic lungs, whilst growth of control lungs did not change. The number of lung buds and lung area of nitrofen-exposed hypoplastic lungs after 96 h of culture significantly increased after the addition of RA compared to the non-treated hypoplastic lungs (25.75+/-6.47 vs 15.11+/-3.29 and 0.98+/-0.18 mm(2) vs 0.65+/-0.13 mm(2), respectively; P<0.0001). Lung perimeter was also higher when RA was added to hypoplastic lungs compared to the non-treated ones, although it did not reach significance (12.51+/-2.53 mm vs 11.19+/-2.56 mm; P=0.17). Conversely, the addition of RA to control lungs did not affect the number of lung buds, lung area or lung perimeter after 96 h in culture compared to the non-treated ones (31.28+/-4.66 vs 31.81+/-6.67; 1.29+/-0.18(2) vs 1.29+/-0.23 mm(2) and 18.47+/-3.47 mm vs 17.89+/-2.94 mm, respectively; P=NS). Retinoic acid also increased the total DNA content and the proportion of proliferating cells in hypoplastic lungs compared to the non-treated ones (2.59+/-0.58 mug vs 1.96+/-0.31 mug and 57.89+/-9.46% vs 36.76+/-8.15%, respectively; P<0.001). The addition of RA did not affect either total DNA content or the proportion of proliferating cells in control lungs compared to the non-treated ones (4.04+/-0.64 mug vs 3.79+/-0.85 mug and 58.67+/-11.23% vs 56.03+/-10.36%, respectively; P=NS). This study demonstrates for the first time that RA rescues lung hypoplasia in nitrofen-induced hypoplastic lungs. These results suggest that retinoid pathway may be involved in the pathogenesis of associated pulmonary hypoplasia in CDH.