Supplementary Material for: Retinoic Acid Rescues Deficient Airway Innervation and Peristalsis of Hypoplastic Rat Lung Explants

<i>Background:</i> Bronchial peristalsis modulates lung growth and is deficient in hypoplastic nitrofen-exposed rat lung explants. Retinoic acid (RA) rescues lung hypoplasia. This study examines whether decreased bronchial innervation contributes to this developmental deficiency and if RA is able to recover bronchial innervation and motility. <i>Material and Methods:</i> After IRB approval, pregnant rats received either 100 mg nitrofen or vehicle on gestational day 9.5 (E9.5). Embryonic lung primordia harvested on E13 were cultured for 72 h and RA was added daily to the medium when appropriate. Lung growth was assessed by counting the number of terminal buds and measuring explant surface, total DNA and protein in control, control + RA, nitrofen and nitrofen + RA groups. Peristaltic contractions were recorded for 10 min under an inverted microscope. Lung explants stained for anti-protein gene product 9.5 (PGP 9.5) and smooth muscle α-actin were examined under a confocal microscope for depicting the specific relationship between neural and smooth muscle cells. PGP 9.5 and smooth muscle α-actin levels were quantified by Western blot analysis for assessing the neural and muscle cell expressions. Comparisons between groups were made with non-parametric tests. <i>Results:</i> The number of terminal buds, the explants’ surface and the DNA and protein contents were significantly decreased in nitrofen-exposed lungs in comparison with controls. In contrast, these measurements were normal in explants exposed to both nitrofen and RA. Bronchial peristalsis (contractions/min) was significantly decreased in nitrofen-exposed lungs in comparison with controls; in contrast, in nitrofen + RA lungs it was similar to controls. In all study groups, the airways were surrounded by smooth muscle and ensheathed in a plexus of nerve fibers containing ganglia. PGP 9.5 protein levels were decreased in nitrofen-exposed lungs, but they normalized when RA was added. No differences were found in α-actin protein levels. Explants exposed only to RA were similar to control. <i>Conclusions:</i> Lung growth, bronchial innervation and peristalsis are decreased in nitrofen-exposed lung explants and are rescued by RA. If deficient airway innervation contributing to dysmotility and pulmonary hypoplasia can be pharmacologically rescued, new relatively simple prenatal interventions could be envisioned.背景：支气管蠕动可调节肺生长，而暴露于硝基酚的发育不全大鼠肺外植体中，支气管蠕动功能存在缺陷。维甲酸（retinoic acid，RA）可挽救肺发育不全。本研究旨在探讨支气管神经支配减少是否为该发育缺陷的诱因，以及维甲酸能否恢复支气管神经支配与蠕动功能。 材料与方法：本研究经伦理审查委员会（Institutional Review Board，IRB）批准后，于妊娠第9.5天（E9.5）对孕鼠给予100 mg硝基酚或溶剂对照。于妊娠第13天（E13）收取胚胎肺原基并体外培养72小时，按需每日向培养基中添加维甲酸。通过计数终末芽（terminal bud）数量、测量外植体表面积、检测总DNA与蛋白含量，对对照组、对照组+维甲酸组、硝基酚组、硝基酚+维甲酸组的肺生长情况进行评估。于倒置显微镜下记录10分钟的蠕动收缩情况。采用抗蛋白基因产物9.5（PGP 9.5）与平滑肌α-肌动蛋白（smooth muscle α-actin）抗体对肺外植体进行染色，通过共聚焦显微镜观察神经细胞与平滑肌细胞的特异性关联。采用蛋白质印迹（Western blot）分析定量检测PGP 9.5与平滑肌α-肌动蛋白的表达水平，以评估神经细胞与肌细胞的表达情况。组间比较采用非参数检验。 结果：与对照组相比，硝基酚暴露组肺组织的终末芽数量、外植体表面积以及DNA与蛋白含量均显著降低。与之相反，硝基酚联合维甲酸处理组的上述指标均恢复至正常水平。硝基酚暴露组的支气管蠕动频率（收缩次数/分钟）较对照组显著降低；而硝基酚+维甲酸组的蠕动频率与对照组无显著差异。所有研究组的气道均被平滑肌包裹，并被含有神经节的神经纤维丛所包绕。硝基酚暴露组的PGP 9.5蛋白水平降低，但添加维甲酸后可恢复至正常水平。平滑肌α-肌动蛋白的蛋白水平在各组间无显著差异。仅接受维甲酸处理的外植体与对照组无显著差异。 结论：暴露于硝基酚的肺外植体中，肺生长、支气管神经支配与蠕动功能均出现降低，且可被维甲酸挽救。若气道神经支配缺陷所致的运动障碍与肺发育不全可通过药理学手段挽救，则有望开发出相对简便的产前干预新策略。