Marta Roccio

Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

Response profiles of murine spiral ganglion neurons on multi-electrode arrays.

OBJECTIVE Cochlear implants (CIs) have become the gold standard treatment for deafness. These neuroprosthetic devices feature a linear electrode array, surgically inserted into the cochlea, and function by directly stimulating the auditory neurons located within the spiral ganglion, bypassing lost or not-functioning hair cells. Despite their success, some limitations still remain, including poor frequency resolution and high-energy consumption. In both cases, the anatomical gap between the electrode array and the spiral ganglion neurons (SGNs) is believed to be an important limiting factor. The final goal of the study is to characterize response profiles of SGNs growing in intimate contact with an electrode array, in view of designing novel CI devices and stimulation protocols, featuring a gapless interface with auditory neurons. APPROACH We have characterized SGN responses to extracellular stimulation using multi-electrode arrays (MEAs). This setup allows, in our view, to optimize in vitro many of the limiting interface aspects between CIs and SGNs. MAIN RESULTS Early postnatal mouse SGN explants were analyzed after 6-18 days in culture. Different stimulation protocols were compared with the aim to lower the stimulation threshold and the energy needed to elicit a response. In the best case, a four-fold reduction of the energy was obtained by lengthening the biphasic stimulus from 40 μs to 160 μs. Similarly, quasi monophasic pulses were more effective than biphasic pulses and the insertion of an interphase gap moderately improved efficiency. Finally, the stimulation with an external electrode mounted on a micromanipulator showed that the energy needed to elicit a response could be reduced by a factor of five with decreasing its distance from 40 μm to 0 μm from the auditory neurons. SIGNIFICANCE This study is the first to show electrical activity of SGNs on MEAs. Our findings may help to improve stimulation by and to reduce energy consumption of CIs and thereby contribute to the development of fully implantable devices with better auditory resolution in the future.

NANOCI—Nanotechnology Based Cochlear Implant With Gapless Interface to Auditory Neurons

Cochlear implants (CI) restore functional hearing in the majority of deaf patients. Despite the tremendous success of these devices, some limitations remain. The bottleneck for optimal electrical stimulation with CI is caused by the anatomical gap between the electrode array and the auditory neurons in the inner ear. As a consequence, current devices are limited through 1) low frequency resolution, hence sub-optimal sound quality and 2), large stimulation currents, hence high energy consumption (responsible for significant battery costs and for impeding the development of fully implantable systems). A recently completed, multinational and interdisciplinary project called NANOCI aimed at overcoming current limitations by creating a gapless interface between auditory nerve fibers and the cochlear implant electrode array. This ambitious goal was achieved in vivo by neurotrophin-induced attraction of neurites through an intracochlear gel-nanomatrix onto a modified nanoCI electrode array located in the scala tympani of deafened guinea pigs. Functionally, the gapless interface led to lower stimulation thresholds and a larger dynamic range in vivo, and to reduced stimulation energy requirement (up to fivefold) in an in vitro model using auditory neurons cultured on multi-electrode arrays. In conclusion, the NANOCI project yielded proof of concept that a gapless interface between auditory neurons and cochlear implant electrode arrays is feasible. These findings may be of relevance for the development of future CI systems with better sound quality and performance and lower energy consumption. The present overview/review paper summarizes the NANOCI project history and highlights achievements of the individual work packages.

Fine control of drug delivery for cochlear implant applications

Cochlear implants are neuroprostheses that are inserted into the inner ear to directly electrically stimulate the auditory nerve, thus replacing lost cochlear receptors, the hair cells. The reduction of the gap between electrodes and nerve cells will contribute to technological solutions simultaneously increasing the frequency resolution, the sound quality and the amplification of the signal. Recent findings indicate that neurotrophins (NTs) such as brain derived neurotrophic factor (BDNF) stimulate the neurite outgrowth of auditory nerve cells by activating Trk receptors on the cellular surface (1–3). Furthermore, small-size TrkB receptor agonists such as di-hydroxyflavone (DHF) are now available, which activate the TrkB receptor with similar efficiency as BDNF, but are much more stable (4). Experimentally, such molecules are currently used to attract nerve cells towards, for example, the electrodes of cochlear implants. This paper analyses the scenarios of low dose aspects of controlled release of small-size Trk receptor agonists from the coated CI electrode array into the inner ear. The control must first ensure a sufficient dose for the onset of neurite growth. Secondly, a gradient in concentration needs to be maintained to allow directive growth of neurites through the perilymph-filled gap towards the electrodes of the implant. We used fluorescein as a test molecule for its molecular size similarity to DHF and investigated two different transport mechanisms of drug dispensing, which both have the potential to fulfil controlled low-throughput drug-deliverable requirements. The first is based on the release of aqueous fluorescein into water through well-defined 60-μm size holes arrays in a membrane by pure osmosis. The release was both simulated using the software COMSOL and observed experimentally. In the second approach, solid fluorescein crystals were encapsulated in a thin layer of parylene (PPX), hence creating random nanometer-sized pinholes. In this approach, the release occurred due to subsequent water diffusion through the pinholes, dissolution of the fluorescein and then release by out-diffusion. Surprisingly, the release rate of solid fluorescein through the nanoscopic scale holes was found to be in the same order of magnitude as for liquid fluorescein release through microscopic holes.

The Severity of Infection Determines the Localization of Damage and Extent of Sensorineural Hearing Loss in Experimental Pneumococcal Meningitis

Hearing loss is an important sequela of pneumococcal meningitis (PM), occurring in up to 30% of survivors. The role of the severity of infection on hearing function and pathomorphological consequences in the cochlea secondary to PM have not been investigated to date. Using a well-established model of PM, we systematically investigated the functional hearing outcome and the long-term fate of neurosensory cells in the cochlea, i.e., hair cells and spiral ganglion neurons (SGNs), with a focus on their tonotopic distribution. Intracisternal infection of infant rats with increasing inocula of Streptococcus pneumoniae resulted in a dose-dependent increase in CSF levels of interleukin-1β, interleukin-6, tumor necrosis factor α, interleukin-10, and interferon-γ in acute disease. The severity of long-term hearing loss at 3 weeks after infection, measured by auditory brainstem response recordings, correlated to the initial inoculum dose and to the levels of proinflammatory cytokines determined in the acute phase of PM. Quantitative cochlear histomorphology revealed a significant loss of SGNs and outer hair cells that strongly correlated to the level of infection, with the most severe damage occurring in the basal part of the cochlea. Inner hair cells (IHCs) were not significantly affected throughout the entire cochlea. However, surviving IHCs lost synaptic connectivity to remaining SGNs in all cochlear regions. These findings provide evidence that the inoculum concentration, i.e., severity of infection, is the major determinant of long-term morphological cell pathologies in the cochlea and functional hearing loss. SIGNIFICANCE STATEMENT Hearing loss is a neurofunctional deficit occurring in up to 30% of patients surviving pneumococcal meningitis (PM). Here, we analyze the correlation between the severity of infection and the inflammatory response in the CSF, the tonotopic distribution of neurosensory pathologies in the cochlea, and the long-term hearing function in a rat model of pneumococcal meningitis. Our study identifies the severity of infection as the key determinant of long-term hearing loss, underlining the importance of the prompt institution of antibiotic therapy in patients suffering from PM. Furthermore, our findings reveal in detail the spatial loss of cochlear neurosensory cells, providing new insights into the pathogenesis of meningitis-associated hearing loss that reveal new starting points for the development of otoprotective therapies.

Conductive hybrid carbon nanotube (CNT)–polythiophene coatings for innovative auditory neuron-multi-electrode array interfacing

Cochlear implants (CIs) are neuroprosthetic devices that restore hearing in deaf patients. They consist of a linear electrode array surgically inserted into the scala tympani of the cochlea. In this position the array directly stimulates the spiral ganglion neurons (SGNs), bypassing lost or nonfunctioning sensory hair cells. In order to reduce the energy needed to stimulate the SGNs, we attempted to improve the conductivity of the CI electrodes by creating conductive nanocomposites. We assessed the functional modification of CI platinum (Pt) pads by using multi-walled carbon nanotubes (MWCNTs) either alone or in combination with a conductive polythiophene phase, to obtain conductive nanocomposites (NCs). A novel functional poly(EDOT3:EDOT-COOH1) copolymer was synthesized by liquid-phase oxidative polymerization (LPP) of both 3,4-ethylenedioxythiophene (EDOT) and carboxylated EDOT (EDOT–COOH) monomers. Molar ratios of monomers EDOT3:EDOT-COOH1 were obtained and a poly(EDOT3:EDOT-COOH1) polymeric phase was further incorporated with oxidized MWCNTs (ox-MWCNTs) to obtain a homogeneous conductive NC phase. Immobilization of single components or NC onto Pt electrodes using cysteamine-based covalent modification has been achieved and characterized. Impedance spectroscopy of Pt-modified electrodes showed a reduced resistance at different frequencies for the specific NC consisting of poly(EDOT3:EDOT-COOH1)/SH/ox-MWCNTs/SH (1 : 1 wt%). Finally, we revealed the functional outcome of these nanostructured chemical modifications concerning the stimulation of SGN in vitro using multi-electrode arrays (MEAs). We aimed at reducing the energy needed to stimulate the SGNs and obtain an effective neuronal response. This has been observed for Pt electrodes modified with NC.

Streptococcus pneumoniae-induced ototoxicity in organ of Corti explant cultures.

Abstract Hearing loss remains the most common long‐term complication of pneumococcal meningitis (PM) reported in up to 30% of survivors. Streptococcus pneumoniae have been shown to possess different ototoxic properties. Here we present a novel ex vivo experimental setup to examine in detail the pattern of hair cell loss upon exposure to different S. pneumoniae strains, therefore recapitulating pathogen derived aspects of PM‐induced hearing loss. Our results show a higher susceptibility towards S. pneumoniae‐induced cochlear damage for outer hair cells (OHC) compared to inner hair cells (IHC), which is consistent with in vivo data. S. pneumoniae‐induced hair cell loss was both time and dose‐dependent. Moreover, we have found significant differences in the level of cell damage between tissue from the basal and the apical turns. This shows that the higher vulnerability of hair cells located at high frequency regions observed in vivo cannot be explained solely by the spatial organisation and bacterial infiltration from the basal portion of the cochlea. Using a wild type D39 strain and a mutant defective for the pneumolysin (PLY) gene, we also have shown that the toxin PLY is an important factor involved in ototoxic damages. The obtained results indicate that PLY can cause both IHC and OHC loss. Finally, we are reporting here for the first time a higher vulnerability of HC located at the basal and middle cochlear region to pneumolysin‐induced damage. The detailed description of the susceptibility of hair cells to Streptococcus pneumoniae provided in this report can in the future determine the choice and the development of novel otoprotective therapies during pneumococcal meningitis. HighlightsEx‐vivo co‐culture system of the organ of corti together with S. pneumoniae is presented.Outer hair cells show higher susceptibility to S. pneumoniae secreted toxins.Secreted pneumolysin is a crucial factor responsible for the observed hair cell death.Hair cells located at the base show a higher vulnerability to pneumolysin‐induced damage.

Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture

The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGFβ) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Molecular characterization and prospective isolation of human fetal cochlear hair cell progenitors

Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.Hearing requires mechanosensitive hair cells in the organ of Corti, which derive from progenitors of the cochlear duct. Here the authors examine human inner ear development by studying key developmental markers and describe organoid cultures from human cochlear duct progenitors for in vitro hair cell differentiation.

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.