Experiments In Pathogenesis And Treatment Of Lung Hypoplasia With Congenital Diaphragmatic Hernia

Purpose: To investigate the mechanism of glucocorticoids treatment, the role of alteration in nitric oxide synthesis and prenatal artificial gastroschisis treatment in congenital diaphragmatic hernia (CDH).Materials and methods:1 Intervention of glucocorticoid: Eighteen pregnant Sprague-Dawley(SD) rats were divided into five groups randomly: control group(C group n=3) , nitrofen group(N group n=5), nitrofen+0.25mg/kg dose of dexamethasone (DEX) group(N+DEX group n=4), nitrofen+1mg/kg dose of dexamethasone group(N+4DEX group n=4) and nitrofen+ Saline group(N+S group n=2). Timed pregnant rats received intragastrically either 200 mg 2,4-dichlorophenyl-p-nitrophenylether (nitrofen) or oil on day 9.5. Antenatal dexamethasone was given intraperitoneally on days 18.5 and 19.5 of gestation. Lung tissue weight (LW) and body weight (B W) of each fetus were recorded, lung histologic and morphometric evaluations were performed, and image analysis was combined after lung processing. Internal diameter (ID), external diameter (ED), and medial thickness (%MT) were also detected . Transmission electron microscopy was used for ultrastructural observation, especially type II pneumocytes. Transforming growth factor β₁ (TGF-β₁)and Thyroid transcription factor -1 (TTF-1) were detected before and after the dexamethasone administration with immunohistochemistry and real-time reverse transcription polymerase chain reaction (real-time RT-PCR) techniques.2. Intervention of L-nitro-arginine methyl easter (L-NAME): CDH was induced in the offspring by maternal exposure to nitrofen during pregnancy. Saline solution and L-NAME (250mg/kg) were infused subcutaneously to rats model from day 14 to day 20 of gestation. Internal diameter (ID), external diameter (ED), and medial thickness (%MT) were analysed with the results of immunohistochemistry test of nitric oxide synthetase (eNOS)..3. Experimentally induced gastroschisis: Twenty-one pregnant rabbits underwent fetal surgery on gestational day 23. A left diaphragmatic hernia was created in 1 end fetus (DH group) from each rabbit, and the other end fetus with sham thoracotomy as the control (CR group). Eleven pregnant rabbits were used in artificial gastroschisis experiments. Left diaphragmatic hernia operations on both end fetuses were also done on gestational day 23. Three days after the first operation, gastroschisis was created inone end fetus (GS group) and the other end as the control (CGS group). Weight of liver and lung were recorded. Lung histologic and morphometric evaluations were performed. Results:1. Intervention of glucocorticoid:1) Lw/Bw, RAC, S% were decreased and MTBD, MT% were increased significantly in N group when compared with C group (PO.05). N-S group showed no difference from N group.2) When the dexamethasone administration was given, N+DEX group showed higher Lw/Bw, RAC\ S% and lower MTBD. MT% than those of N group. But it was statistically significant just in N+4DEX group (P<0.05) .3) Positive stained area ratio of TTF-1 antibody was 39.23 + 1.14%in C group, but decreased to 26.84±3.39% in N group. After the dexamethasone administration was given ,the ratio increased to 28.65 + 0.65 % in N+DEX group and 33.81 +4.36% in N+4DEX group(P<0.05 vs N group). Real time RT-PCR showed the same results.2. Intervention of L-NAME: There were 2.49+1.62 lung vessels in each scope in L -NAME group, and average of ED was 45.87+ 13.51um,ID 21.28 + 13.26 um and MT% 52.9+2.41 %.These data showed no difference between L—NAME group and control group. Positive stained area ratio of eNOS antibody was decreased in L— NAME group with controls(21.35 + 3.02%/35.47 + 2.65%).3 The effect of experimentally induced gastroschisis on pulmonary hypoplasia in fetal rabbits with congenital diaphragmatic hernia (CDH): Lw/Bw was 0.0262 + 0.005, RAC 3.17 + 1.00, MTBD 3.42 + 1.20 and S% 58.12 + 11.80% in CR group. CGS group and DH group had decreased Lw/Bw, RAC, S%,and increased MTBD. MT%. In the GS group, the alveoli was diminished than that of CR group, and air space decreased significantly. The pulmonary arterial wall was markedly thickened in the DH group and kept this tendency in the GS group. Conclusion:1 Experiments are done on rats to induce congenital diaphragmatic hernia. It is found that there are hypoplasia in alveoli, bronchi, and arterioles in this model. Type II pneumocyte depletion can not be found through transmission electron microscopy, whereas surfactant protein granules excretion is decreased.2. The prenatal administration of dexamethasone has positive effects on the lung structural changes promoted by congenital diaphragmatic hernia. TTF-1 is a possible...