Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the XXXX Brain.

@entity19 @entity3158 @entity51 @entity3612 @entity544 @entity460 @entity413
Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various @entity51 . The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: @entity19 euthanasia with @entity544 according to protocols of The University of Toledo's Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain's slices in a customized glass-bottom plate containing Dulbecco's Modified Eagle's Medium (DMEM)/High- @entity413 at 37   C in the presence of 95%/5% @entity3612 / @entity3158 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular @entity460 oscillation properties of ependymal cells as well as the effect of pharmacological agents on the @entity460 oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.
