The research concerns the morphological and biochemical events that lead o and accompany the regeneration/repair of the organ of Corti in cultures explanted from the neonatal mouse and injured by laser beam. Specifically, it is proposed to study: 1) Cellular interactions between injured hair cells and their supporting cells. Ultrastructural evidence indicate that supporting cells participate in hair cell survival and recovery. The study will attempt to elucidate if sprouting of supporting cells and wrapping on injured hair cells depends on a degree of hair cell damage (by varying intensity of laser beam) or the site of injury, targeted either at hair cell or selectivity at supporting cells; 2) Hair cell degeneration following laser ablation of their afferent neurons. Both hair cell death and damage have been observed upon the elimination of spiral neurons. The study aims to understand if these changes are caused by the process of laser irradiation that led tot he increase in voltage of membrane potential of the radial fibers or by the sudden denervation. The attenuation of the laser beam during the ablation and mechanical elimination of spiral neurons should help in distinguishing the factor that led to the secondary destruction of hair cells; 3) Selective destruction of hair cells by laser ablation of spiral neurons. The study raises provocative possibility of the trans-synaptic transfer of damage from the nerve cell to its target cell. By varying the site of ablated neurons we will try to induce the selective degeneration of inner versus outer hair cells; 4) Reinnervation of hair cells by collateral neuronal sprouting. It is expected that elimination of some spiral neurons will induce the collateral sprouting from the surviving ganglion cells. These experiments are designed to study the acquisition of new synaptic contacts; 5) Reformation of hair cell apical specializations after injury. The extent and constancy of hair cell repair will be examined: a) the regrowth of the stereociliary bundle will be studied by high resolution scanning electron microscope; b) the biochemical reconstruction of the hair cell cuticular plates will be examined by ultrastructural immunocytochemistry of the cytoskeletal proteins (in collaboration with N.B. Slepecky). A long-term goal of the project is to develop and in vitro model of defined reproducible injury to the hair cell in order to understand mechanism(s) and factors that lead to survival and recovery of the receptor cells. The significance of the research lies in bringing to light the plasticity of the mammalian hearing organs and identifying cellular processes that facilitate the preservation of its integrity.
