Defective parasympathetic innervation is associated with airway branching abnormalities in experimental CDH.
Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models of CDH. We hypothesize that defective parasympathetic innervation may contribute to airway branching abnormalities and, therefore, lung hypoplasia, during lung development in CDH. The murine nitrofen model of CDH was utilized to study the effect of the cholinergic agonist carbachol on embryonic day 11.5 (E11.5) lung explant cultures. Airway branching and contractions were quantified. In a subset of experiments, verapamil was added to inhibit airway contractions. Sox9 immunostaining and 5-bromo-2-deoxyuridine incorporation were used to identify and quantify the number and proliferation of distal airway epithelial progenitor cells. Intra-amniotic injections were used to determine the in vivo effect of carbachol. Airway branching and airway contractions were significantly decreased in nitrofen-treated lungs compared with controls. Carbachol resulted in increased airway contractions and branching in nitrofen-treated lungs. Nitrofen-treated lungs exhibited an increased number of proliferating Sox9-positive distal epithelial progenitor cells, which were decreased and normalized by treatment with carbachol. Verapamil inhibited the carbachol-induced airway contractions in nitrofen-treated lungs but had no effect on the carbachol-induced increase in airway branching, suggesting a direct carbachol effect independent of airway contractions. In vivo treatment of nitrofen-treated embryos via amniotic injection of carbachol at E10.5 resulted in modest increases in lung size and branching at E17.5. These results suggest that defective parasympathetic innervation may contribute to airway branching abnormalities in CDH.
Department of Surgery, University of Pittsburgh and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
Julie
J
Rhodes
Department of Surgery, University of Pittsburgh and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
Deeksha
D
Saxena
Department of Surgery, University of Pittsburgh and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
GuangFeng
G
Zhang
Department of Surgery, University of Pittsburgh and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
George K
GK
Gittes
Department of Surgery, University of Pittsburgh and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania douglas.potoka@chp.edu.
Douglas A
DA
Potoka
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology; D004195:Disease Models, Animal; D004622:Embryo, Mammalian / Q000187:drug effects / Q000473:pathology*; D005260:Female; D065630:Hernias, Diaphragmatic, Congenital / Q000139:chemically induced / Q000196:embryology / Q000473:pathology*; D006801:Humans; D007124:Immunoenzyme Techniques; D008168:Lung / Q000002:abnormalities* / Q000187:drug effects / Q000473:pathology*; D051379:Mice; D010275:Parasympathetic Nervous System / Q000196:embryology / Q000378:metabolism / Q000473:pathology*; D010575:Pesticides / Q000633:toxicity; D010647:Phenyl Ethers / Q000633:toxicity; D012137:Respiratory System / Q000187:drug effects / Q000196:embryology / Q000473:pathology*; D013234:Stem Cells / Q000187:drug effects; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002316:Cardiotonic Agents; D010575:Pesticides; D010647:Phenyl Ethers; D002217:Carbachol; D014700:Verapamil; C007350:nitrofen
congenital diaphragmatic hernia; lung development; nitrofen model; pulmonary innervation