Abnormal development of tracheal innervation in rats with experimental diaphragmatic hernia

We previously demonstrated that tracheobronchial innervation, originated from the vagus nerve and hence of neural crest origin, is deficient in rats with experimental congenital diaphragmatic hernia (CDH). The present study examines the development of this innervation during fetal life in an attempt to understand the nature of these deficiencies.

Pregnant rats were given either 100 mg nitrofen or vehicle on E 9.5. Embryos were recovered on E15 and E18. Control and nitrofen/CDH pups (n = 10 each) were studied on each of these days and compared with our previous results on E21. Whole mount preparations of tracheas stained for anti-protein gene product 9.5 (PGP 9.5) and smooth muscle contractile alpha-actin were examined under confocal microscopy for the morphology of intrinsic neural network. Sections of tracheas were immunostained with anti-low-affinity neurotrophin receptor (p75(NTR)), neural cell marker PGP 9.5, and anti-glial cell marker S100 antibodies. The proportions of sectional areas occupied by neural and glial structures were measured in the proximal and distal trachea. PGP 9.5 protein, and mRNA expressions were determined. Mann-Whitney tests with a threshold of significance of P < 0.05 were used for comparison.

Positive staining for p75(NTR) confirmed the neural crest origin of tracheal neural cells. The neural network appeared less organized on E15, and it was less dense on E18 in nitrofen-exposed embryos than in controls. The proportions of section surface occupied by neural elements were similar in both groups on E15, but that of glial tissue was significantly increased in nitrofen-exposed embryos. On E18, the relative neural surface was significantly reduced in CDH embryos in contrast with increased glial tissue surface. On E21 the proportion of neural tissue was reduced only in the distal trachea. The expression of PGP 9.5 protein was decreased in CDH fetuses on E18 and E21. In contrast, PGP 9.5 mRNA levels were increased in CDH fetuses on E18 and E21.

The development of intrinsic innervation of the trachea in rats with CDH is abnormal with reduction of neural tissue accompanied by increase of glial tissue that could represent a response to neural damage. The significance of increased PGP 9.5 mRNA levels is unclear.