Early lung malformations in congenital diaphragmatic hernia

Lung hypoplasia, a leading contributor to the lethality of congenital diaphragmatic hernia (CDH), has been attributed to compression of the fetal lung by herniated abdominal viscera. Contested findings in experimental CDH suggest that lung malformation may precede diaphragmatic hernia. To address this unresolved question, we studied the pattern and progress of embryonic lung development in vivo and in vitro before diaphragmatic herniation in the nitrofen CDH model.

Sprague-Dawley rats were fed nitrofen on day 9.5 of pregnancy to induce pulmonary hypoplasia and CDH in newborns (term, day 22). Control rats received olive oil. Embryonic lungs were microdissected on day 13.5 gestation, 24 hours after lung primordia bud from the foregut (normal diaphragmatic closure, day 16.5). In vivo airway branching was measured by counting terminal lung buds at this stage. Lungs were cultured for up to 78 hours and longitudinal in vitro development studied by serial measurements of terminal bud count, area, and perimeter.

At 13.5 days of gestation in vivo, nearly 99% of normal lungs (n = 130) had > or = 6 terminal lung buds. In contrast, 36% of the nitrofen-exposed lungs (n = 170) fell short of this developmental milestone with less than 6 terminal buds (P < .001). In vitro, the nitrofen lungs had reduced area compared with controls after 6, 30, and 54 hours (P = .001, P < .001, and P = .001, respectively). Bud count and epithelial perimeter were reduced in the nitrofen lungs after 6 and 30 hours in vitro (P < .001 and P = .01 v normal terminal bud count; P < .001 and P= .002 v normal perimeter).

Before diaphragmatic herniation, nitrofen-exposed embryos in vivo have a 36% frequency of reduced airway branching that strikingly parallels the 30% to 40% term incidence of CDH in this model. The authors propose that this early lung anomaly is restricted to a subset of nitrofen-exposed embryos in which it is integral to an emerging CDH phenotype. In vitro data confirm that nitrofen-induced pulmonary hypoplasia precedes visceral herniation and persists to allow experimental manipulation in culture. The developmental biology underlying lung hypoplasia in CDH may now be addressed using this organ culture system.