Vestibular sensory epithelium regeneration after ototoxic damage

Aims. The auditory and vestibular sensory epithelia are characterized by the presence of hair cells, playing a crucial role in transduction of mechanical stimuli (sound waves or head movements) into neural impulses. The hair bundle deflection gates transduction channels generating a membrane potential that results in neurotransmitter release at the basal pole of the cell and afferent fibers stimulation. In mammals, hair cells can be produced only during embryonic and neonatal development and, if lost, they can be restored only at very low rates, with permanent damage of the vestibular and acoustic function. Lower vertebrates, not only produce hair cells at low rates throughout all adult life, but they retain the ability to regenerate the sensory epithelium after hair cell loss induced by chemical or physical agents. We induced hair cell degeneration in frog crista ampullaris via intraotic gentamicin administration, and we used this animal model to study hair cell recovery in vivo. Materials and methods. Adult frogs (rana esculenta) were injected intraotically with 15 µl of 5 mM gentamicin and sacrificed at different times after treatment to dissect the ampulla of the posterior semicircular canal. For the electrophysiological studies the ampullae were thermically stimulated and the afferent activity was derived from the ampullar nerve. For the immunohistochemistry the ampullae were fixed and paraffin embedded; 5 µm sections were then blocked and treated with primary antibodies against specific hair cell or supporting cell markers (calretinin, calmodulin, cytokeratins, etc.). Degenerating and regenerating samples were also treated and examined by electron microscopy. Results and Conclusions. Hair cell degeneration is evident at 24-48 hrs after gentamicin injection and is maximal 4-5 days after treatment. The damage consists initially in the loss of the stereociliary apparatus and then of the whole hair cell, while supporting and basal cells seems to be unaffected. Regeneration partially overlaps degeneration and is almost complete 15-20 days post-treatment both at the morphological and functional level. Three main mechanisms have been proposed for hair cell regeneration: repair of sublethally damaged hair cell, regenerative proliferation and phenotypic conversion. In order to discriminate between supporting cells trans-differentiation and activation of quiescent precursors, we combined the analysis of mitotic markers and the immunolabeling for hair cell and supporting cell specific markers, with a fine morphological analysis of the degenerating and regenerating epithelia. In our model system we observed, nearby hair cell degeneration sites, supporting cells assuming hair cell-like characteristics (appearance of a stereociliary apparatus, calretinin expression etc.) and also undifferentiated cells spanning the whole epithelium. Our data, indicating a scarce mitotic activity during the early phases of the regenerative process, suggest that regeneration might involve two distinct mechanisms: hair cell loss is initially recovered by supporting cell trans-differentiation in new hair cells; then the reduced number of supporting cells might induces regenerative proliferation of a pseudo-staminal precursor, that we identify with basal cells, to restore the original number of cells and the receptor function.