Volume 279, June 2000

Volume 48, June 2000

Pages R1496-R1505: Yasushi Takahashi, Christine Roman, Sylvain Chemtob, Mary M. Tse, Emil Lin, Michael A. Heymann, and Ronald I. Clyman. "Cyclooxygenase-2 inhibitors constrict the fetal lamb ductus arteriosus both in vitro and in vivo."

The accepted version of the In Vitro Studies section of METHODS is shown below instead of the earlier version that was published.

In Vitro Studies

Nineteen fetal lambs (between 125 and 137 days) were delivered by cesarean section. The ewe was anesthetized with a constant intravenous infusion of ketamine HCl and diazepam throughout the procedure. The fetus was given ketamine HCl (30 mg/kg im) before rapid exsanguination. The ductus arteriosus was dissected free of loose adventitial tissue and divided into 1-mm-wide rings that were placed in separate 10-ml organ baths and kept in a dark room, as we described previously (7). Tissue oxygen concentration is significantly lower in isolated ductus rings incubated in bath solutions containing "fetal PO₂" when compared with the fetal ductus in vivo. In vivo, the fetal lamb ductus relies on vasa vasorum that perfuse 60-70% of the muscle media. The measured average tissue O₂ concentration in the late gestation fetal lamb ductus in vivo is 1.5-2% O₂ (PO₂ = 10-14 mmHg) (H. Kajino, C. Koch, and R. Clyman, unpublished results). In vitro, the ductus wall receives oxygen only from the lumen and adventitial surface (as well as the 2 cut faces). Average tissue O₂ concentrations in rings of fetal lamb ductus incubated with bath solutions containing fetal PO₂ (4% O₂, PO₂ = 28 mmHg) is only 0.6% (PO₂ = 4 mmHg). An average tissue O₂ concentration of 1.5-2% (as observed in the fetal ductus in vivo) is not achieved in vitro until the oxygen concentration of the organ bath solution is increased to 30% O₂ (Kajino et al., unpublished results). Therefore, we equilibrated our bath solution with 30% O₂ to mimic average fetal tissue O₂ concentration in vivo. Although there are different absolute tensions that develop in ductus rings that are incubated in bath solution equilibrated with 30% O₂ compared with those incubated at 4% O₂ (because of differences in oxygen-induced tension), the contractile response to nonselective (indomethacin) and selective COX-2 inhibitors is the same at these two bath oxygen concentrations. This is consistent with our unpublished observations that PGE₂ or 6-keto-PGF₁ (6ketoPGF₁) production rates are similar at these two bath solution oxygen concentrations (Kajino et al., unpublished results).