The studies described in this section reflect not only work performed in the Miller Laboratory but also substantial efforts by Drs. Richard Altschuler and Yehoash Raphael and their students. These colleagues have provided invaluable help in all of these studies, in particular those involving the regrowth of peripheral processes of the auditory nerve (Altschuler) and studies of the effectiveness of adenoviral delivered genes to upregulate the production of neurotrophic factors in the inner ear to enhance nerve survival (Raphael). Important contributions to this work have also been made by Timo Stiver, a post-doctoral research fellow.
The purpose of this program of research has been to enhance the preservation of the auditory nerve following destruction of the sensory cells of the inner ear and to initiate a regeneration and regrowth of peripheral processes of the auditory nerve. Following loss of sensory cells of the inner ear, the peripheral processes of the auditory nerves die back to the cell bodies (spiral ganglion cells, SGC), and subsequently these cell bodies and proximal projections of the auditory nerve retreat into the brain stem, degenerate, and die. We know that the benefits of the cochlear prosthesis depend significantly upon the preservation of the auditory nerve and fibers, and there is strong evidence that there is an enhanced benefit from close contact between the stimulating electrodes and the auditory nerves and peripheral processes. Our work in this area began some years ago with the demonstration that chronic electrical stimulation (ES) following deafness could prevent the degeneration of the SGC. On the basis of this, we hypothesized that activity in the auditory nerve was a necessary survival factor for these cells and ES via prosthesis could provide an appropriate substitute for normal activity generated in these cells by the sensory cells of the inner ear, leading us to hypothesize we could obtain similar beneficial protective effects on the auditory nerve and peripheral processes with the application of neurotrophins.
Neurotrophins have been demonstrated as survival factors for neurons in vitro, particularly in the de-affected visual and auditory systems. In vivo, neurotrophins have been demonstrated as survival factors to enhance the survival of primary visual and auditory neurons following destruction of the receptor cells. In the auditory system, these findings may have direct clinical application for enhancement of the benefits of the cochlear implant for the profoundly deaf. This neural prosthesis functions by directly stimulating the auditory nerve, bypassing the damaged inner ear receptor. It is important from both a basic and a clinical perspective to demonstrate, in vivo, that enhanced survival of neurons is associated with enhanced responsiveness. We have now shown enhanced survival and/or regrowth of auditory neurons with multiple neurotrophins delivered alone or in combination. Importantly, we have shown improved function in neurotrophin-treated ears when we have used the auditory brainstem response to measure threshold sensitivity. The protective effects of neurotrophins have been evident with chronic infusion into the scala tymapni, as well as delivery using adenoviral gene therapy.
Previously, we performed studies with ES and observed that with the right intensity and frequency we were able to preserve SGCs. Based on this observation and results from other laboratories, we hypothesized 1) that the ES and activity of the auditory nerve up-regulates the expression of proteins that can provide long-term survival support for these cells, 2) that growth factors in particular may be up regulated by ES induced activity in these cells, and, 3) that this upregulation by ES may be dependent upon calcium channel pathways. We tested this hypothesis by delivering calcium channel blockers into the inner ear of chronically electrically stimulated animals to assess the change in auditory nerve survival. Collaborative work with other laboratories has demonstrated that depolarization of the auditory nerve in vitro results in increased cell survival. However, loss of Ca2+ to these cells is fatal, regardless of depolarization activity. With verapamil (a L-type Ca2+ channel blocker) chronically infused via an implantable mini-osmotic pump and microcannulation into the inner ear of chronically stimulated and unstimulated animals, we were able to reduce the protective effects of chronic stimulation on the auditory nerve cells without altering the evoked functional response of the ear. This supports our hypothesis that the protective effects of ES are dependent upon L-type calcium channels. This knowledge provides an important understanding of stimulation-induced nerve survival and makes an important advance in basic research, which will one day lead to new treatments with clinical application.
We have also investigated potential survival-enhancing effects of neurotrophin application on immature dorsal root ganglion (DRG) neurons and stem cells transplanted into the cochlea. Survival of DRG neurons and stem cells transplanted into the cochlea is enhanced by neurotrophic factors. In addition, we have found that stem cells differentiate into neurons, migrate to the auditory nerve, and project into the organ of Corti as well as the CNS. We have now identified these cells as immunoreactive to anti-glutamatergic antibodies.
Regeneration or replacement of the auditory nerve would provide a major new clinical intervention to reduce hearing loss. These results will thus have significant impact on the use of electrical stimulation and neurotrophins with and without viral vectors in future human clinical trials.
