Activating Influences on Supporting Cell Proliferation in the Regenerating Avian Cochlea

Abstract

Aminoglycoside treatment is associated with hair cell death and hearing loss. In the avian inner ear, it has been well-documented that this hair cell death stimulates the proliferation of adjacent supporting cells, leads to a structural regeneration of hair cells, and results in a subsequent recovery of auditory function. We have been studying the mechanisms that lead to hair cell death in the avian cochlea following aminoglycoside treatment and how this process reactivates the cell cycle in a normally quiescent population of supporting cells. Hair cell death appears to be caused by the induction of apoptosis, or programmed cell death. This apoptotic pathway is initiated within the first 12 hours after a single systemic injection of gentamicin (300 mg/kg), as identified by the nuclear-to-cytoplasmic translocation of the T cell-restricted intracellular antigen-related protein (TIAR). TIAR is an RNA binding protein that is normally found in the cell nucleus but that has been shown to exhibit increased cytoplasmic localization in a variety of tissues undergoing apoptosis. Confocal microscopy demonstrated that within 12 hours of gentamicin injection, punctate cytoplasmic TIAR labeling was seen only in those hair cells in the gentamicin-damaged region of the cochlea that would later proceed to die. Even though these early signs of apoptosis could be detected by 12 hours, signs of structural damage to the hair cells and expression of activated forms of execution stage apoptotic enzymes such as caspase-3 were not seen until 24 to 36 hours after the gentamicin injection. These terminal-stage changes in hair cells were accompanied by an intensification and alteration in the immunocytochemical labeling for myosin VIIa, a hair cell-specific unconventional myosin. The final expulsion of hair cells in the gentamicin-damaged region of the cochlea occurred between 48 and 72 hours after the gentamicin injection and was accompanied by pyknotic and TUNEL-labeled hair cell nuclei. Concurrently, the supporting cells in the damaged region of the cochlea began to enter the S (DNA synthesis) phase of the cell cycle by 65 hours and reached a peak in S phase between 72 and 96 hours. The period of S phase was associated with a loss of connexin43-containing gap junctions between supporting cells in the damaged region of the cochlea. New hair cells were identified in the damaged region by their expression of myosin VIIa at 96 hours. The number of new hair cells increased dramatically by 120 hours and was accompanied by a restoration of connexin43-containing gap junctions between supporting cells in the damaged region of the cochlea. These data reinforce the role of apoptosis in the death of cochlear hair cells after gentamicin treatment and suggest that TIAR, myosin VIIa, and caspase-3 are key components in this apoptotic pathway. Moreover, our data suggest that steps in the hair cell apoptotic pathway directly stimulate supporting cell gap junction disassembly, and this leads to proliferation of a subset of these supporting cells.