Methods

Pregnant rodent model. All studies were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (20342). Pregnant Sprague Dawley rats (10 wk old, n= 12) were exposed to mainstream cigarette smoke for 10 days before fertilization and throughout their 21-to 23-day gestation using a whole-body exposure chamber and smoking apparatus (InExpose, SCIREQ). Smoke exposure was applied 4 hours daily, 5 days a week, with an average of 800 mg/L particulate matter per day. Air-exposed pregnant control rats (n= 10) were placed in identical chambers for the same duration. Tracheas were excised from neonates at Day 0 (within 12 h of delivery) and Day 3 of life. There was no ongoing cigarette smoke exposure to neonates postpartum.

CFTR expression and function. CFTR activity was measured ex vivo in excised tracheas from Day 0 and Day 3 neonates using short-circuit current (Isc) analysis under voltage-clamp conduits as previously described (11). CFTR mRNA was extracted with the RNeasy Mini Kit (Qiagen). qRT-PCR was performed using the QuantStudio 3 system (Life Technologies) with the TaqMan RNA-to-Ct 1-Step Kit and TaqMan gene expression assays (Thermo Scientific)(b-actin and CFTR). PCR amplifications were performed in triplicate and gene expression was determined by the comparative cycle threshold (DDCt) method using b-actin as an internal control as described previously (4).