Signaling molecules in the fetal rabbit model for congenital diaphragmatic hernia

Abstract

Summary. Rationale and objectives: Little is known about molecular changes in lungs of fetal rabbits with surgically induced diaphragmatic hernia (DH). Therefore, we examined in this model gene expressions of pivotal molecules for the developing lung. Methods: At day 23 of gestation, DH was created in 12 fetuses from 4 does. Both lungs from six live DH fetuses and from six unoperated controls were harvested and weighed at term. Transcription of 15 genes involved in alveolarization, angiogenesis, regulation of vascular tone, or epithelial maturation was investigated by real-time quantitative polymerase chain reaction. Main results: DH decreased lung-to-body weight ratio (P < 0.001). A bilateral downregulation was seen for genes encoding for tropoelastin (P < 0.01), lysyl oxidase (P < 0.05), fibulin 5 (P < 0.05), and cGMP specific phosphodiesterase 5 (P < 0.05). Lower mRNA levels for endothelial nitric oxide synthase occurred in the ipsilateral lung (P < 0.05). Conclusions: Experimental DH in fetal rabbits disrupted transcription of genes implicated in lung growth and function. Similarities with the human disease make this model appropriate for investigation of new prenatal therapies. Pediatr Pulmonol. ß

Excessive Reversal of Epidermal Growth Factor Receptor and Ephrin Signaling Following Tracheal Occlusion in Rabbit Model of congenital Diaphragmatic Hernia

“…Vukovich, et al reported both elastogenesis and transforming growth factor-β signaling were increased in DHTO to levels beyond those of DH or control (6,7). Again, our findings regarding these processes did not reach statistical significance; however their findings are consistent with the observed pattern that processes impacted by DH are excessively reversed in DHTO.…”

“…While TO for CDH may gain more widespread acceptance in the coming years, there remains an urgent need to understand the cellular responses to CDH and TO so that targeted therapies may be developed to improve fetal lung development and to limit any undesirable effects of TO. There have been reports of how CDH and TO impact lung matrix synthesis (6)(7)(8)(9), lung epithelial function and surfactant (10)(11)(12)(13) and microvascular development (14)(15)(16), and that tracheal occlusion reverses some of resulting in severe pulmonary hypoplasia (2). Despite optimal medical and surgical care, mortality remains high with survivors experiencing severe restrictive lung disease and significant pulmonary hypertension (1).…”

Increased TGF-β: a drawback of tracheal occlusion in human and experimental congenital diaphragmatic hernia?

“…Time-dated pregnant New Zealand White rabbits were obtained at 15 days of gestational age from Charles River Laboratories (Germany) with approval by the Ethics Committee for Animal Experimentation of the District Government of Upper Bavaria (Munich, Germany; number 55.2-1-54-2531-149-08). Animals were housed as reported (63). All experiments were conducted in agreement with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health.…”

“…Due to high fetal losses and the small size of neonatal rabbit lungs, left and right lungs from each pup were differently allocated to postmortem analyses. We previously showed that biological and histological features of lung hypoplasia were also present in the right lung, i.e., contralateral to the hernia (62,63). The right upper lobe was gently expanded with 4% paraformaldehyde under vacuum and stored for 24 h at 4°C in fixative solution.…”

“…Total RNA from rabbit and human lungs was extracted and quantified by absorbance (63). In rabbit samples, integrity of extracted RNA was tested by gel electrophoresis (63). RNA degradation was expected in human specimens because of the unavoidable postmortem interval.…”