Fluorescence anisotropy as an index of fetal lung maturation in vitro

Fluorescence techniques have been used to assess the viscosity of surfactant-containing fluids in vivo and have been successfully employed clinically as indices of lung maturity. However, fluorescence measurements have not been previously used as indicators of fetal lung maturation 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 homogenates and culture medium were eluted on Sephacryl S-300 columns, a diphenylhexatriene (DPH) probe was added to each fraction, and fluorescence anisotropy and intensity were measured after excitation at 357 nm and emission at 435 nm. Elution fractions containing the major fluorescence peak were demonstrated to correspond to the phosphatidylcholine-containing fraction and were shown to contain lamellar bodies. Fluorescence anisotropy of tissue homogenates obtained from 19-day lung explants decreased after 72 h in culture, suggesting lower microviscosity of the surfactant-containing fractions. Assessment of culture media collected at 24-h intervals revealed significant decreases in anisotropy by 48 h for the 19-day explants, and by 24 h for the 20- and 21-day explants. Anisotropy of the final (48-72 h) culture media aliquots was significantly lower for 21-day explants (0.144 +/- 0.004, SE), than for 20-day (0.172 +/- 0.013) or 19-day explants (0.197 +/- 0.008), p < .005. Anisotropy of culture medium tended to be lower than anisotropy of corresponding tissue homogenates, suggesting that viscosity of recently secreted surfactant may be different from viscosity of surfactant within lamellar bodies in type II cells. Relative fluorescence intensity of tissue homogenates also increased with time in culture. These results indicate that fluorescence anisotropy can be used to assess the viscosity of surfactant in vitro and serve as another index of fetal lung 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 adsorption and spreading.