Live Imaging of Cochlear Organotypic Explants – a Tool for Studying Development, Pathology, and Regeneration

The organ of Corti is a strikingly complex organ that undergoes dramatic morphological and regulatory changes during development, as well as in later stages, in response to ototoxic drugs and aging. Understanding these dynamic processes in mouse model systems can improve our ability to develop strategies for hearing preservation and restoration. Current techniques are predominantly based on analysis of fixed samples, only providing snapshots of the state of the cells at each time point. Here, we report the development of a method for high resolution live imaging of cochlear organotypic explants using transgenic mice expressing the ZO1-GFP reporter that allows both short term (seconds to minutes) and long term (hours and days) tracking of cellular morphological changes and organization processes at different developmental stages. Time-lapse movies generated are analyzed using a custom-made image analysis platform, allowing for quantitative analysis of cellular morphologies. This platform allows for probing the role of different signaling pathways in the development of the organ of Corti by adding pharmacological inhibitors to the explants during imaging. Finally, the response of the tissue to local ablation of cells can be studied using a laser ablation system integrated into the microscopy system. To demonstrate the systems’ capabilities, we show the morphological effect of ablating a hair cell on surrounding supporting cells, and the effect of Blebbistatin, a non-muscle myosin II inhibitor, on tissue morphology. Overall this approach can be used to better understand developmental and pathological processes, including proliferation and trans-differentiation, in order to devise therapeutic strategies for hearing loss.