Closing the Gap between the Auditory Nerve and Cochlear Implant Electrodes: Which Neurotrophin Cocktail Performs Best for Axonal Outgrowth and Is Electrical Stimulation Beneficial?

Neurotrophins promote neurite outgrowth of auditory neurons and may help closing the gap to cochlear implant (CI) electrodes to enhance electrical hearing. The best concentrations and mix of neurotrophins for this nerve regrowth are unknown. Whether electrical stimulation (ES) during outgrowth is beneficial or may direct axons is another open question. Auditory neuron explant cultures of distinct cochlear turns of 6-7 days old mice were cultured for four days. We tested different concentrations and combinations of BDNF and NT-3 and quantified the numbers and lengths of neurites with an advanced automated analysis. A custom-made 24-well electrical stimulator based on two bulk CIs served to test different ES strategies. Quantification of receptors trkB, trkC, p75NTR, and histological analysis helped to analyze effects. We found 25 ng/mL BDNF to perform best, especially in basal neurons, a negative influence of NT-3 in combined BDNF/NT-3 scenarios, and tonotopic changes in trk and p75NTR receptor stainings. ES largely impeded neurite outgrowth and glia ensheathment in an amplitude-dependent way. Apical neurons showed slight benefits in neurite numbers and length with ES at 10 and 500 µA. We recommend BDNF as a potent drug to enhance the man-machine interface, but CIs should be better activated after nerve regrowth.

Phoenix auditory neurons as 3R cell model for high throughput screening of neurogenic compounds.

Auditory neurons connect the sensory hair cells from the inner ear to the brainstem. These bipolar neurons are relevant targets for pharmacological intervention aiming at protecting or improving the hearing function in various forms of sensorineural hearing loss. In the research laboratory, neurotrophic compounds are commonly used to improve survival and to promote regeneration of auditory neurons. One important roadblock delaying eventual clinical applications of these strategies in humans is the lack of powerful in vitro models allowing high throughput screening of otoprotective and regenerative compounds. The recently discovered auditory neuroprogenitors (ANPGs) derived from the A/J mouse with an unprecedented capacity to self-renew and to provide mature auditory neurons offer the possibility to overcome this bottleneck. In the present study, we further characterized the new phoenix ANPGs model and compared it to the current gold-standard spiral ganglion organotypic explant (SGE) model to assay neurite outgrowth, neurite length and glutamate-induced Ca2+ response in response to neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) treatment. Whereas both, SGEs and phoenix ANPGs exhibited a robust and sensitive response to neurotrophins, the phoenix ANPGs offer a considerable range of advantages including high throughput suitability, lower experimental variability, single cell resolution and an important reduction of animal numbers. The phoenix ANPGs in vitro model therefore provides a robust high-throughput platform to screen for otoprotective and regenerative neurotrophic compounds in line with 3R principles and is of interest for the field of auditory neuroscience.

ExplantAnalyzer: An advanced automated neurite outgrowth analysis evaluated by means of organotypic auditory neuron explant cultures.

BACKGROUND: Neuronal outgrowth assays using organotypic explant cultures are commonly utilized to study neuroregenerative and -protective effects of drugs such as neurotrophins. While this approach offers higher organized tissue compared to single cell cultures and less experimental effort than in-vivo studies, quantitative evaluation of the neuronal network is often time consuming. Thus, we developed ExplantAnlayzer, a time-saving high-throughput evaluation method, yielding numerous metrics to objectively describe neuronal outgrowth. NEW METHOD: Spiral ganglion explants were cultured in 24-well plates, mechanically fixed in a collagen matrix and immunolabeled against beta-III-tubulin. The explants were imaged using a fluorescent tile-scan microscope and resulting images were stitched. The evaluation was developed as an open-source MATLAB routine and involves several image processing steps, including adaptive thresholding. The neurite network was eventually converted to a graph to track neurites from their terminals back to the explant body. COMPARISON WITH EXISTING METHOD(S): We compared ExplantAnlayzer quantitatively and qualitatively to common existing methods, such as Sholl analyses and manual fiber tracing, using representative explant images. ExplantAnlayzer is able to achieve similar and as detailed results as manual tracing while decreasing manual interaction and required time dramatically. RESULTS: After an initial setup phase, the explant images could be batch-processed altogether. Bright bundles as well as faint fibers were reliably detected. Several metrics describing the outgrowth morphology, including total outgrowth, neurite numbers and length estimations, as well as their growth directions, were computed. CONCLUSIONS: ExplantAnalyzer is a time-saving and objective method for an in-depth evaluation of organotypic explant outgrowth.

Intrinsically Self-renewing Neuroprogenitors From the A/J Mouse Spiral Ganglion as Virtually Unlimited Source of Mature Auditory Neurons.

Nearly 460 million individuals are affected by sensorineural hearing loss (SNHL), one of the most common human sensory disorders. In mammals, hearing loss is permanent due to the lack of efficient regenerative capacity of the sensory epithelia and spiral ganglion neurons (SGN). Sphere-forming progenitor cells can be isolated from the mammalian inner ear and give rise to inner ear specific cell types in vitro. However, the self-renewing capacities of auditory progenitor cells from the sensory and neuronal compartment are limited to few passages, even after adding powerful growth factor cocktails. Here, we provide phenotypical and functional characterization of a new pool of auditory progenitors as sustainable source for sphere-derived auditory neurons. The so-called phoenix auditory neuroprogenitors, isolated from the A/J mouse spiral ganglion, exhibit robust intrinsic self-renewal properties beyond 40 passages. At any passage or freezing-thawing cycle, phoenix spheres can be efficiently differentiated into mature spiral ganglion cells by withdrawing growth factors. The differentiated cells express both neuronal and glial cell phenotypic markers and exhibit similar functional properties as mouse spiral ganglion primary explants and human sphere-derived spiral ganglion cells. In contrast to other rodent models aiming at sustained production of auditory neurons, no genetic transformation of the progenitors is needed. Phoenix spheres therefore represent an interesting starting point to further investigate self-renewal in the mammalian inner ear, which is still far from any clinical application. In the meantime, phoenix spheres already offer an unlimited source of mammalian auditory neurons for high-throughput screens while substantially reducing the numbers of animals needed.

Age-Dependency of Neurite Outgrowth in Postnatal Mouse Cochlear Spiral Ganglion Explants.

BACKGROUND: The spatial gap between cochlear implants (CIs) and the auditory nerve limits frequency selectivity as large populations of spiral ganglion neurons (SGNs) are electrically stimulated synchronously. To improve CI performance, a possible strategy is to promote neurite outgrowth toward the CI, thereby allowing a discrete stimulation of small SGN subpopulations. Brain-derived neurotrophic factor (BDNF) is effective to stimulate neurite outgrowth from SGNs. METHOD: TrkB (tropomyosin receptor kinase B) agonists, BDNF, and five known small-molecule BDNF mimetics were tested for their efficacy in stimulating neurite outgrowth in postnatal SGN explants. To modulate Trk receptor-mediated effects, TrkB and TrkC ligands were scavenged by an excess of recombinant receptor proteins. The pan-Trk inhibitor K252a was used to block Trk receptor actions. RESULTS: THF (7,8,3'-trihydroxyflavone) partly reproduced the BDNF effect in postnatal day 7 (P7) mouse cochlear spiral ganglion explants (SGEs), but failed to show effectiveness in P4 SGEs. During the same postnatal period, spontaneous and BDNF-stimulated neurite outgrowth increased. The increased neurite outgrowth in P7 SGEs was not caused by the TrkB/TrkC ligands, BDNF and neurotrophin-3 (NT-3). CONCLUSIONS: The age-dependency of induction of neurite outgrowth in SGEs was very likely dependent on presently unidentified factors and/or molecular mechanisms which may also be decisive for the age-dependent efficacy of the small-molecule TrkB receptor agonist THF.

Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging.

Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol's iodine potassium iodine (I2KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 µm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 µm voxel size in human and 0.5 µm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I2KI and imaged in toto at 25 µm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition.

Analysis of Vestibular Labyrinthine Geometry and Variation in the Human Temporal Bone.

Stable posture and body movement in humans is dictated by the precise functioning of the ampulla organs in the semi-circular canals. Statistical analysis of the interrelationship between bony and membranous compartments within the semi-circular canals is dependent on the visualization of soft tissue structures. Thirty-one human inner ears were prepared, post-fixed with osmium tetroxide and decalcified for soft tissue contrast enhancement. High resolution X-ray microtomography images at 15 µm voxel-size were manually segmented. This data served as templates for centerline generation and cross-sectional area extraction. Our estimates demonstrate the variability of individual specimens from averaged centerlines of both bony and membranous labyrinth. Centerline lengths and cross-sectional areas along these lines were identified from segmented data. Using centerlines weighted by the inverse squares of the cross-sectional areas, plane angles could be quantified. The fit planes indicate that the bony labyrinth resembles a Cartesian coordinate system more closely than the membranous labyrinth. A widening in the membranous labyrinth of the lateral semi-circular canal was observed in some of the specimens. Likewise, the cross-sectional areas in the perilymphatic spaces of the lateral canal differed from the other canals. For the first time we could precisely describe the geometry of the human membranous labyrinth based on a large sample size. Awareness of the variations in the canal geometry of the membranous and bony labyrinth would be a helpful reference in designing electrodes for future vestibular prosthesis and simulating fluid dynamics more precisely.