Abstract
Because cochlear implants function by stimulating the auditory nerve, it is assumed that the condition of the nerve plays an important role in the efficacy of the prosthesis. Thus, considerable research has been devoted to methods of preserving the nerve following deafness. Neurotrophins have been identified as a potential contributor to neural health, but most of the research to date has been done in young animals and for short periods (less than 3 to 6 months) after the onset of treatment. The first objective of the current experiment was to examine the effects of a neurotrophin gene therapy delivery method on spiral ganglion neuron (SGN) preservation and function in the long term (5 to 14 months) in mature guinea pigs with cochlear implants. The second objective was to examine several potential non-invasive monitors of auditory nerve health following the neurotrophin gene therapy procedure. Eighteen mature adult male guinea pigs were deafened by cochlear perfusion of neomycin and then one ear was inoculated with an adeno-associated viral vector with an Nft3-gene insert (AAV.Ntf3) and implanted with a cochlear implant electrode array. Five control animals were deafened and inoculated with an empty AAV and implanted. Data from 43 other guinea pig ears from this and previous experiments were used for comparison: 24 animals implanted in a hearing ear, nine animals deafened and implanted with no inoculation, and ten normal-hearing non-implanted ears. After 4 to 21 months of psychophysical and electrophysiological testing, the animals were prepared for histological examination of SGN densities and inner hair cell (IHC) survival. Seventy-eight percent of the ears deafened and inoculated with AAV.Ntf3 showed better SGN survival than the 14 deafened-control ears. The degree of SGN preservation following the gene therapy procedure was variable across animals and across cochlear turns. Slopes of psychophysical multipulse integration (MPI) functions were predictive of SGN density, but only in animals with preserved IHCs. MPI was not affected by the AAV.Ntf3 treatment, but there was a minor improvement in temporal integration (TI). AAV.Ntf3 treatment had significant effects on ECAP and EABR amplitude growth func-tion (AGF) slopes; the reduction in slope in deafened ears was ameliorated by the AAV.Ntf3 treatment. Slopes of the ECAP and EABR AGFs were predictive of SGN density in a broad area near and just apical to the implant. The highest ensemble spontaneous activity (ESA) values were seen in animals with surviving IHCs, but AAV.Ntf3 treatment in deafened ears resulted in slightly higher ESA values compared to deafened untreated ears. Overall, a combination of the psychophysical and electrophysiological measures can be useful for monitoring the health of the implanted cochlea in guinea pigs. These measures should be applicable for assessing cochlear health in human subjects.
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Acknowledgements
This work was supported by NIH grants R01 DC007634, R01 DC010412, R56 DC010786, R01 DC015809, and P30 DC 005188, the University of Michigan Center for Organogenesis, and a contract from MED-EL. We express appreciation to Cameron Budenz and James Wiler for assistance with AAV inoculations, Lisa Kabara and Melissa Watts for assistance with the electrophysiology, and Lisa Beyer for histological preparation.
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One of the authors, Stefan B. Strahl, is an employee of MED-EL GmbH, which was one of the sponsors of this research. None of the other authors have any financial relationship with any of the sponsors.
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Fig. S1
In a set of animals not formally included in this study, we tested the pattern of transduction of an AAV2 vector with a reporter gene (AAV.GFP) and determined that 20 days after the vector was injected into the perilymph of mature deafened guinea pigs, GFP-labeled cells (arrows) were sparsely distributed in the mesothelium lining the scala tympani in both the apical and basal areas of the cochlear partition. GFP was seen in the basal turn (a and b) and as far apically as the third turn (c). In every whole-mount inspected, a small number of cells was transduced, indirectly suggesting that transduction with an AAV2 carrying the Ntf3 gene leads to low levels of NT-3 secreted into cochlear fluids. In a control ear that was not injected with the virus, GFP positive cells were absent (d). These whole-mount preparations include the bone surrounding the osseous spiral lamina which introduces a low level of auto-fluorescence seen in all images. All panels are the same scale (bar = 20 μm). (GIF 101 kb).
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Pfingst, B.E., Colesa, D.J., Swiderski, D.L. et al. Neurotrophin Gene Therapy in Deafened Ears with Cochlear Implants: Long-term Effects on Nerve Survival and Functional Measures. JARO 18, 731–750 (2017). https://doi.org/10.1007/s10162-017-0633-9
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DOI: https://doi.org/10.1007/s10162-017-0633-9