Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp.

To determine whether trophozoites and lysates of pathogenic Acanthamoeba sp p, induce apoptosis in primary-culture microglial cells, transmission elect ron microscopic (TEM) examinations, assessment of DNA fragmentation by agar ose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni c ame in contact with a microglial cell, the digipodium was observed by TEM, Nuclear chromatin condensation was observed in 10% of microglial cells, whi le it was not revealed when they were cocultured with weakly pathogenic Aca nthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocul tured with the A. culbertsoni lysate was detected by electrophoresis, showi ng DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apop totic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate, In contrast, with microglial cells cocultured w ith the A. royreba lysate, only a background level of fluorescence of TdT-s tained apoptotic bodies was detected, These results suggest that some rat m icroglial cells cocultured with pathogenic A. culbertsoni undergo cytopathi c changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.