457 - Timing of Permanent Remodeling of the Ductus Arteriosus (DA) in Mice and Development of Mouse Models to Study Drug-induced Reopening of the Closed DA

Publication Number: 3442.457

Edith M. Charron, Monroe Carell Jr. Children's Hospital at Vanderbilt, Brentwood, TN, United States; Deanna Sekulich, Vanderbilt University School of Medicine, Charlottesville, VA, United States; Alexus J.. Brown, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States; Charmaine R. Rock, Vanderbilt University Medical Center, Nashville, TN, United States; Cameron Nichols, Lipscomb University, Nashville, TN, United States; Naoko Boatwright, Vanderbilt University Medical Center, Franklin, TN, United States; Elaine L.. Shelton, Vanderbilt University School of Medicine, Nashville, TN, United States; Jeff Reese, Vanderbilt University Medical Center, Nashville, TN, United States

Background: The ductus arteriosus (DA) closes after birth in two steps and failure to close results in patent DA (PDA). “Functional” closure (first few hours of life) is followed by “anatomic” closure (days-weeks), where the DA remodels into the ligamentum arteriosum. Although furosemide, gentamicin and other drugs in the neonatal formulary can *prevent* DA closure, we hypothesized that drug exposures in the NICU may also lead to DA *re-opening*.

Objective: 1) Identify the timeframe for functional closure in the mouse DA before irreversible anatomical remodeling. 2) Evaluate the ability of putative DA dilators to reverse functional DA closure.

Design/Methods: DAs of newborn CD-1 mice were examined on postnatal day 0 (P0) to P2. In ex vivo pressure myography studies, DAs at 4h, 10h, 24-26h, 30-33h, or 48-51h of age were mounted and sequentially exposed to 10-5 M SNP, 10-6 M PGE2, and 10-4 M papaverine (PAP). Responses to furosemide and/or gentamicin were examined at the 24h time point. DAs were video-monitored for lumen re-opening and re-establishment of flow, defined as buffer passage through the distal stopcock. In vivo data were collected following single or multiple intraperitoneal injections of PGE2 (100µg/kg) at 4h, 6h, 8h, 10-12h, and 24h of life. Offspring were euthanized 30 mins after injection, and DA patency was visually scored (categorically, from 0,10, 50, 75, 90, or 100% patency compared to pulmonary artery diameter). Data were analyzed by t-test or ANOVA.

Results: At 4-10h, the closed ex vivo DA re-opened with rewarming and exposure to pressure but by 24h, vessels only reopened with vasodilators (SNP< PGE< PAP) reaching 40% of fetal DA caliber. After 30h, the DA could not be reopened. At 24-26h, gentamicin (10-3 to 10-2 M) reliably re-opened the DA, whereas furosemide (10-3M) was ineffective. Combined treatment was no better than gentamicin alone. In vivo studies showed that DA closure was irreversible after 8h of age in response to single< multiple PGE2 injections. In vivo gentamicin- and furosemide-induced DA re-opening rates were equivalent.

Conclusion(s): We showed that postnatal DA closure in mice has time-dependent reversibility and developed both ex vivo and in vivo systems to monitor lumen re-opening. 24-26h of age ex vivo and 6h in vivo were established as optimal time points to test other drugs. Proof-of-concept studies showed furosemide (via Cl- mechanisms) was unable to re-open the ex vivo DA, while gentamicin (via PKC and Ca++ mechanisms) effectively re-opened the closed DA, suggesting that some drugs used in the NICU may contribute to PDA through unexpected mechanisms.