Increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine cells in an experimental model of congenital diaphragmatic hernia

Abstract

A congenital diaphragmatic hernia (CDH) model was induced in pregnant rats following administration of 100 mg nitrofen. The fetuses were stored and fixed in Bouin's solution for 24 h after caesarean section at term. After fixation, the lungs were dissected out. Immunostaining of the CDH lungs and controls with rabbit anti-rat calcitonin gene-related peptide (CGRP) antibody at "optimal" and "supraoptimal" dilution levels was obtained by examining the intensity of staining with a series of dilutions of the antisera from 1: 1,000 to 1: 20,000. Supraoptimal dilution detects variations in antigen concentration that may be masked if the routine optimal dilution is used. Immunostaining of the lung by antisera to platelet-derived growth factor (PDGF) and alpha-smooth-muscle actin (ASMA) was performed to examine vascular remodelling. The number of CGRP-immunoreactive cells was significantly (P <0.001) greater in the lungs of CDH rats (n = 26) (0.74 +-0.19 NEB [neuroepithelial bodies]/mm(2); mean +- SEM) compared with controls (n = 21) (0.30+-0.16 NEB/mm(2)) seen at supraoptimal dilution (1:20,000). Since CGRP is a vasodilator, this could have important implications in the development of pulmonary hypertension. The pattern of ASMA and PDGF immunostaining was similar in CDH lungs and controls, and therefore, vascular remodelling is not a feature of CDH lungs in fetuses delivered by caesarean section and not exposed to hypoxia.