Cyclosporine Effects on Chick Limb Bud Mesenchyme and DNA Content

Background: Cyclosporine A (CsA) is a powerful immunosuppressive agent, which is used for the prevention of allograft rejection and for the treatment of autoimmune diseases.  Many transplant recipients must take this medicine for the rest of their lives.  Females in reproductive age group on prolonged CsA therapy have legitimate concerns about drug effects on pregnancy. Objective: To explore CsA's teratogenicity in embryonic limb development. Methods: Mesenchymal cells obtained from stage 23-24 chick embryo limb buds were grown in 96-well plates using chemically defined tissue culture medium.  Cultures were treated with a range of CsA concentrations and incubated (at 37 ºC, 5% CO2) with daily medium changes for 4 days.  After incubation, each well received Hoechst 33342 and DNA content was assayed using a 96-well fluorometer. Results: It was found that high concentrations of CsA caused cell loss and intermediate concentrations decreased DNA content.  Low CsA concentrations however had no significant effect on DNA content in these cultures.  Thus, the decrease in DNA content was dose-dependent. Conclusion: CsA teratogenicity may involve reducing the number of dividing cells or reducing the proliferation rate in developing structures.