FLUORESCENCE ANISOTROPY AS AN INDEX OF FETAL LUNG MATURATION INVITRO

Fluorescence techniques have been used to assess the viscosity of surf actant-containing fluids in vivo and have been successfully employed c linically as indices of lung maturity. However, fluorescence measureme nts have not been previously used as indicators of fetal lung maturati on in an in vitro system. Lung explants derived from 19-, 20-, and 21- day fetal rats were cultured in F-12 medium for 24-72 h. Tissue homoge nates and culture medium were eluted on Sephacryl S-300 columns, a dip henylhexatriene (DPH) probe was added to each fraction, and fluorescen ce anisotropy and intensity were measured after excitation at 357 nm a nd emission at 435 nm. Elution fractions containing the major fluoresc ence peak were demonstrated to correspond to the phosphatidylcholine-c ontaining fraction and were shown to contain lamellar bodies. Fluoresc ence anisotropy of tissue homogenates obtained from 19-day lung explan ts decreased after 72 h in culture, suggesting lower microviscosity of the surfactant-containing fractions. Assessment of culture media coll ected at 24-h intervals revealed significant decreases in anisotropy h y 48 h for the 19-day explants, and hy 24 h for the 20- and 21-day exp lants. Anisotropy of the final (48-72 h) culture media aliquots was si gnificantly lower for 2 1-day explants (0.144 +/- 0.004, SE), than for 20-day (0.172 +/- 0.013) or 19-day explants (0.197 +/- 0.008), p < .0 05. Anistropy of culture medium tended to be lower than anisotropy of corresponding tissue homogenates, suggesting that viscosity of recentl y secreted surfactant may be different from viscosity of surfactant wi thin lamellar bodies in type II cells. Relative fluorescence intensity of tissue homogenates also increased with time in culture. These resu lts indicate that fluorescence anisotropy can be used to assess the vi scosity of surfactant in vitro and serve as another index of fetal lun g maturation in in vitro systems. Estimation of the microviscosity of the surfactant phospholipid bilayer using anisotropy measurements may provide additional insight into such roles of surfactant function as a dsorption and spreading.