Historical Aspects of Gene Therapy and Stem Cell Therapy in the Treatment of Hearing and Balance Disorder.

This review presents many but not all the major historical events that have led to our current understanding of gene and stem cell therapies for the treatment of hearing and balance disorders in animal models of these disorders. In order to better understand the application of these emerging therapies to the treatment of inner ear disorders in a clinical setting, it has been necessary to provide some genetic and pathobiology backgrounds from both animal models and clinical disorders. The current focus and goal of gene and stem cell therapies are directed toward understanding the effective treatment of animal models that mimic human disorders of hearing and balance. This approach not only addresses the most effective ways to deliver the gene or stem cell therapies to affected inner ears, it also provides an assessment of the efficacy of the applied therapy(s) in achieving either partial or full restoration of either hearing and/or balance within the animal models receiving these therapeutic interventions. This review also attempts to present a realistic assessment of how close the research fields of gene and stem cell therapies are to application for the treatment of human disorders in a clinical setting. Progress made in developing these novel therapies toward clinical applications would not have been possible without the many pioneering studies and discoveries achieved by the investigators cited in this review. There were also many other excellent studies performed by gifted investigators that were not able to be included within this review. Anat Rec, 303:390-407, 2020. © 2019 American Association for Anatomy.

A Regenerative Medicine Approach to the Treatment of Hearing, Balance, and Olfactory Disorders: What Is in the Future for Otolaryngology?

Regenerative medicine is being applied to many fields of medicine and is now starting to be considered and developed for application to treat hearing, balance, olfaction, and voice disorders. This special issue of the Anatomical Record with a series of over 20 papers covers many aspects of gene and stem cell therapies as they are developed for clinical applications in both in vitro and in vivo laboratory studies. These studies cover a wide range of approaches from gene editing in zebrafish with the latest technology (i.e., CRISPR/Cas9) to the isolation of human inner ear progenitor cells, to tracking transplanted human umbilical cord stem cells in mini pigs, to the in vitro building of graft tissues to repair tracheal defects with adipose tissue-derived stem cells. Anat Rec, 303:385-389, 2020. © 2019 American Association for Anatomy.

Isolation and Characterization of Mammalian Otic Progenitor Cells that Can Differentiate into Both Sensory Epithelial and Neuronal Cell Lineages.

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy.

TGF ß-1 and WNT Signaling Pathways Collaboration Associated with Cochlear Implantation Trauma-Induced Fibrosis.

The crosstalk between TGF-ß1 and WNT pathways has been proven to regulate aspects of the development and tissue homeostasis processes. Recently, it has been demonstrated this collaboration also takes place during fibrotic diseases, where TGF-ß1 activates the WNT/ß-catenin pathway that results in dedifferentiation of fibroblasts into myofibroblasts, increased production of extracellular matrix components and fibrosis. Independent studies show the functions of these molecules during the development of the inner ears in several different species. However, little is known about the collaboration between TGF-ß1 and WNT in the injured inner ear and particularly how this collaboration affects the fibrotic process that often occurs following cochlear implantation. First, we used a cochlear explant model to study the effect of electrode insertion trauma and TGF-ß1 signaling in activation of the WNT pathway. Finally, adult TopGal mutant mice were used in vivo to track the activation of the WNT/ß-catenin in response to EIT. A chronic inflammatory response, increased cell proliferation and tissue remodeling are hallmarks of fibrotic disease. This study explores and highlights the collaboration between the TGF-ß1 and WNT pathways in the trauma-initiated fibrotic process within the implanted cochlea. WNT signaling is involved in the development of the inner ear and therefore a potential target in hair cell regeneration therapies. However, in light of our observations from the current study, manipulation of the WNT pathway by gene therapy techniques in the pathological ear seems a very complex process with an increased risk of inducing excessive fibrosis thereby compromising the efficacy of implant function. Anat Rec, 303:608-618, 2020. © 2019 American Association for Anatomy.

Ultrastructural Characterization of Stem Cell-Derived Replacement Vestibular Hair Cells Within Ototoxin-Damaged Rat Utricle Explants.

The auditory apparatus of the inner ear does not show turnover of sensory hair cells (HCs) in adult mammals; in contrast, there are many observations supporting low-level turnover of vestibular HCs within the balance organs of mammalian inner ears. This low-level renewal of vestibular HCs exists during normal conditions and it is further enhanced after trauma-induced loss of these HCs. The main process for renewal of HCs within mammalian vestibular epithelia is a conversion/transdifferentiation of existing supporting cells (SCs) into replacement HCs.In earlier studies using long-term organ cultures of postnatal rat macula utriculi, HC loss induced by gentamicin resulted in an initial substantial decline in HC density followed by a significant increase in the proportion of HCs to SCs indicating the production of replacement HCs. In the present study, using the same model of ototoxic damage to study renewal of vestibular HCs, we focus on the ultrastructural characteristics of SCs undergoing transdifferentiation into new HCs. Our objective was to search for morphological signs of SC plasticity during this process. In the utricular epithelia, we observed immature HCs, which appear to be SCs transdifferentiating into HCs. These bridge SCs have unique morphological features characterized by formation of foot processes, basal accumulation of mitochondria, and an increased amount of connections with nearby SCs. No gap junctions were observed on these transitional cells. The tight junction seals were morphologically intact in both control and gentamicin-exposed explants. Anat Rec, 303:506-515, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Laminin-coated electrodes improve cochlear implant function and post-insertion neuronal survival.

The benefits of Cochlear implant (CI) technology depend among other factors on the proximity of the electrode array to the spiral ganglion neurons. Laminin, a component of the extracellular matrix, regulates Schwann cell proliferation and survival as well as reorganization of actin fibers within their cytoskeleton, which is necessary for myelination of peripheral axons. In this study we explore the effectiveness of laminin-coated electrodes in promoting neuritic outgrowth from auditory neurons towards the electrode array and the ability to reduce acoustic and electric auditory brainstem response (i.e. aABR and eABR) thresholds. In vitro: Schwann cells and neurites are attracted towards laminin-coated surfaces with longer neuritic processes in laminin-coated dishes compared to uncoated dishes. In vivo: Animals implanted with laminin-coated electrodes experience significant decreases in eABR and aABR thresholds at selected frequencies compared to the results from the uncoated electrodes group. At 1 month post implantation there were a greater number of spiral ganglion neurons and neuritic processes projecting into the scala tympani of animals implanted with laminin-coated electrodes compared to animals with uncoated electrodes. These data suggest that Schwann cells are attracted towards laminin-coated electrodes and promote neuritic outgrowth/ guidance and promote the survival of spiral ganglion neurons following electrode insertion trauma.

l-N-acetylcysteine protects outer hair cells against TNFα initiated ototoxicity in vitro.

OBJECTIVE: The present study is aimed at determining the efficacy and exploring the mechanisms by which l-N-acetylcysteine (l-NAC) provides protection against tumor necrosis factor-alpha (TNFα)-induced oxidative stress damage and hair cell loss in 3-day-old rat organ of Corti (OC) explants. Previous work has demonstrated a high level of oxidative stress in TNFα-challenged OC explants. TNFα can potentially play a significant role in hair cell loss following an insult to the inner ear. l-NAC has shown to provide effective protection against noise-induced hearing loss in laboratory animals but mechanisms of this otoprotective effect are not well-defined. DESIGN: Rat OC explants were exposed to either: (1) saline control (N = 12); (2) TNFα (2 µg/ml, N = 12); (3) TNFα+l-NAC (5 mM, N = 12); (4) TNFα+l-NAC (10 mM, N = 12); or (5) l-NAC (10 mM, N = 12). Outer hair cell (OHC) density, levels of reactive oxygen species (ROS), lipid peroxidation of cell membranes, gluthathione activity, and mitochondrial viability were assayed. RESULTS: l-NAC (5 and 10 mM) provided protection for OHCs from ototoxic level of TNFα in OC explants. Groups treated with TNFα+l-NAC (5 mM) showed a highly significant reduction of both ROS (p < 0.01) and 4-hydroxy-2-nonenal immunostaining (p < 0.001) compared to TNFα-challenged explants. Total glutathione levels were low in TNFα-challenged explants compared to control and TNFα+l-NAC (5 mM) treated explants (p < 0.001). CONCLUSIONS: l-NAC is a promising treatment for protecting auditory HCs from TNFα-induced oxidative stress and subsequent loss via programmed cell death.

Recent Advancements in the Regeneration of Auditory Hair Cells and Hearing Restoration.

Neurosensory responses of hearing and balance are mediated by receptors in specialized neuroepithelial sensory cells. Any disruption of the biochemical and molecular pathways that facilitate these responses can result in severe deficits, including hearing loss and vestibular dysfunction. Hearing is affected by both environmental and genetic factors, with impairment of auditory function being the most common neurosensory disorder affecting 1 in 500 newborns, as well as having an impact on the majority of elderly population. Damage to auditory sensory cells is not reversible, and if sufficient damage and cell death have taken place, the resultant deficit may lead to permanent deafness. Cochlear implants are considered to be one of the most successful and consistent treatments for deaf patients, but only offer limited recovery at the expense of loss of residual hearing. Recently there has been an increased interest in the auditory research community to explore the regeneration of mammalian auditory hair cells and restoration of their function. In this review article, we examine a variety of recent therapies, including genetic, stem cell and molecular therapies as well as discussing progress being made in genome editing strategies as applied to the restoration of hearing function.

Comparison of packing material in an animal model of middle ear trauma.

PURPOSE: To compare the performance of absorbable gelatin sponge (AGS) with polyurethane foam (PUF) as middle ear packing material after mucosal trauma. MATERIALS AND METHODS: Using a randomized, controlled and blinded study design fifteen guinea pigs underwent middle ear surgery with mucosal trauma performed on both ears. One ear was packed with either PUF or AGS while the contralateral ear remained untreated and used as non-packed paired controls. Auditory brainstem response (ABR) thresholds were measured pre-operatively and repeated at 1, 2, and 6weeks postoperatively. Histological analysis of middle ear mucosa was done in each group to evaluate the inflammatory reaction and wound healing. Another eighteen animals underwent middle ear wounding and packing in one ear while the contralateral ear was left undisturbed as control. Twelve guinea pigs were euthanized at 2weeks postoperatively, and six were euthanized at 3days post-operatively. Mucosal samples were collected for analysis of TGF-ß1 levels by enzyme-linked immunosorbent assay. RESULTS: ABR recordings demonstrate that threshold level changes from baseline were minor in PUF packed and control ears. Threshold levels were higher in the AGS packed ears compared with both control and PUF packed ears for low frequency stimuli. Histological analysis showed persistence of packing material at 6weeks postoperatively, inflammation, granulation tissue formation, foreign body reaction and neo-osteogenesis in both AGS and PUF groups. TGF-ß1 protein levels did not differ between groups. CONCLUSION: PUF and AGS packing cause inflammation and neo-osteogenesis in the middle ear following wounding of the mucosa and packing.

Electrode array-eluted dexamethasone protects against electrode insertion trauma induced hearing and hair cell losses, damage to neural elements, increases in impedance and fibrosis: A dose response study.

We evaluated the effects of dexamethasone base (DXMb) containing electrode arrays in a guinea pig model of cochlear implantation to determine if eluted DXMb could protect the cochlea against electrode insertion trauma (EIT)-induced: 1) loss of hair cells; 2) disruption of neural elements; 3) increases in hearing thresholds; 4) increased electrical impedance and 5) fibrosis. A guinea pig model of EIT-induced hearing and hair cell losses was used to test silicone electrode arrays that contained either 10%, 1%, 0.1%, or 0% levels of micronized DXMb. These four types of electrode arrays were implanted into the scala tympani via basal turn cochleostomies and left in place for 3 months. Hearing thresholds were determined by ABR and CAP recordings in response to a series of defined pure tone stimuli (i.e. 16-0.5 kHz). Changes in impedance were measured between the implant electrode and a reference electrode. Hair cell counts and neural element integrity were determined by confocal microscopy analyses of stained organ of Corti whole mounts obtained from 90 day post-implantation animals. Fibrosis was measured in Masson trichrome stained cross-sections through the organ of Corti. The results showed that either 10% or 1.0% DXMb eluting electrode arrays protected; hearing thresholds, hair cells, and neural elements against EIT-induced losses and damage. Electrode arrays with 0.1% DXMb only partial protected against EIT-induced hearing loss and damage to the cochlea. Protection of hearing thresholds and organ of Corti sensory elements by electrode-eluted DXMb was still apparent at 3 months post-EIT. All three concentrations of DXMb in the electrode arrays prevented EIT-induced increases in impedance. EIT-initiated fibrosis was significantly reduced within the implanted cochlea of the two DXMb concentrations tested. In conclusion, DXMb eluting electrodes protected the cochlea against long term increases in hearing thresholds, loss of hair cells, damage to neural elements and increases in impedance and fibrosis that result from EIT-initiated damage. The protection achieved by DXMb-eluting electrodes was dose dependent. Establishing a significant level of trauma induced elevation in hearing thresholds was important for the determination of the otoprotective effects of array-eluted DXMb.

Spiral ganglion cells and macrophages initiate neuro-inflammation and scarring following cochlear implantation.

Conservation of a patient's residual hearing and prevention of fibrous tissue/new bone formation around an electrode array are some of the major challenges in cochlear implant (CI) surgery. Although it is well-known that fibrotic tissue formation around the electrode array can interfere with hearing performance in implanted patients, and that associated intracochlear inflammation can initiate loss of residual hearing, little is known about the molecular and cellular mechanisms that promote this response in the cochlea. In vitro studies in neonatal rats and in vivo studies in adult mice were performed to gain insight into the pro-inflammatory, proliferative, and remodeling phases of pathological wound healing that occur in the cochlea following an electrode analog insertion. Resident Schwann cells (SC), macrophages, and fibroblasts had a prominent role in the inflammatory process in the cochlea. Leukocytes were recruited to the cochlea following insertion of a nylon filament in adult mice, where contributed to the inflammatory response. The reparative stages in wound healing are characterized by persistent neuro-inflammation of spiral ganglion neurons (SGN) and expression of regenerative monocytes/macrophages in the cochlea. Accordingly, genes involved in extracellular matrix (ECM) deposition and remodeling were up-regulated in implanted cochleae. Maturation of scar tissue occurs in the remodeling phase of wound healing in the cochlea. Similar to other damaged peripheral nerves, M2 macrophages and de-differentiated SC were observed in damaged cochleae and may play a role in cell survival and axonal regeneration. In conclusion, the insertion of an electrode analog into the cochlea is associated with robust early and chronic inflammatory responses characterized by recruitment of leukocytes and expression of pro-inflammatory cytokines that promote intracochlear fibrosis and loss of the auditory hair cells (HC) and SGN important for hearing after CI surgery.

Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma.

Loss of auditory sensory hair cells (HCs) is the most common cause of hearing loss. This review addresses the signaling pathways that are involved in the programmed and necrotic cell death of auditory HCs that occur in response to ototoxic and traumatic stressor events. The roles of inflammatory processes, oxidative stress, mitochondrial damage, cell death receptors, members of the mitogen-activated protein kinase (MAPK) signal pathway and pro- and anti-cell death members of the Bcl-2 family are explored. The molecular interaction of these signal pathways that initiates the loss of auditory HCs following acoustic trauma is covered and possible therapeutic interventions that may protect these sensory HCs from loss via apoptotic or non-apoptotic cell death are explored.

Mechanisms of programmed cell death signaling in hair cells and support cells post-electrode insertion trauma.

CONCLUSION: Programmed cell death (PCD) initially starts in the support cells (SCs) after electrode insertion trauma (EIT), followed by PCD in hair cells (HCs). Activation of caspase-3 was observed only in SCs. Protecting both SCs and HCs with selective otoprotective drugs at an early stage post implantation may help to preserve residual hearing. OBJECTIVES: Cochlear implant EIT can initiate sensory cell losses via necrosis and PCD within the organ of Corti, which can lead to a loss of residual hearing. PCD appears to be a major factor in HC loss post-EIT. The current study aimed to: (1) determine the onset of PCD in both SCs and HCs within the traumatized organ of Corti; and (2) identify the molecular mechanisms active within the HCs and SCs that are undergoing PCD. METHODS: Adult guinea pigs were assigned to one of two groups: (1) EIT and (2) unoperated contralateral ears as controls. Immunostaining of dissected organ of Corti surface preparations for phosphorylated-Jun, cleaved caspase-3, and 4-hydroxy-2,3-nonenal (HNE) were performed at 6, 12, and 24 h post-EIT and for contralateral control ears. RESULTS: At 6 h post-EIT the SCs immunolabeled for the presence of phosphorylated-Jun and activated caspase-3. Phosphorylated p-Jun labeling was observed at 12 h in both the HCs and SCs of middle and basal cochlear turns. Cleaved caspase-3 was not observed in HCs of any cochlear turn at up to 24 h post-EIT. Lipid peroxidation (HNE immunostaining) was first observed at 12 h post-EIT in both the HCs and SCs of the basal turn, and reached the apical turn by 24 h post-EIT.

Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants.

We have studied aminoglycoside-induced vestibular hair-cell renewal using long-term culture of utricular macula explants from 4-day-old rats. Explanted utricles were exposed to 1 mM of gentamicin for 48 h, during 2nd and 3rd days in vitro (DIV), and then recovering in unsupplemented medium. Utricles were harvested at specified time points from the 2nd through the 28th DIV. The cellular events that occurred within hair cell epithelia during the culture period were documented from serial sectioned specimens. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) with the assistance of morphometric software. Ultrastructural observations were made from selected specimens with transmission electron microscopy (TEM). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs was 11% of the number of HCs observed in non-gentamicin-exposed control explants. At 28 DIV the HC density was 61% of the number of HCs observed in the control group explant specimens. Simultaneously with this increase in HCs there was a corresponding decline in the number of SCs within the epithelium. The proportion of HCs in relation to SCs increased significantly in the gentamicin-exposed explant group during the 5th to the 28th DIV period of culture. There were no significant differences in the volume estimations of the gentamicin-exposed and the control group explants during the observed period of culture. Morphological observations showed that gentamicin exposure induced extensive loss of HCs within the epithelial layer, which retained their intact apical and basal linings. At 7 to 14 DIV (i.e. 3-11 days after gentamicin exposure) a pseudostratified epithelium with multiple layers of disorganized cells was observed. At 21 DIV new HCs were observed that also possessed features resembling SCs. After 28 DIV a new luminal layer of HCs with several layers of SCs located more basally characterized the gentamicin-exposed epithelium. No mitoses were observed within the epithelial layer of any explants. Our conclusion is that direct transdifferentiation of SCs into HCs was the only process contributing to the renewal of HCs after gentamicin exposure in these explants of vestibular inner ear epithelia obtained from the labyrinths of 4-day-old rats.

Otoprotective properties of mannitol against gentamicin induced hair cell loss.

BACKGROUND: Gentamicin is a widely used antibiotic, which causes hearing loss because of destruction of auditory hair cells. Mannitol has been shown to have cytoprotective properties in the cochlea both in vitro and in vivo. Mannitol has been shown to be safe in concentrations up to 100 mM in organ of Corti explants. It is proposed as an otoprotective agent against gentamicin ototoxicity. METHODS: Organ of Corti were dissected from P-3 rat pups and cultured under the following conditions for 96 hours: 1) control, 2) gentamicin (10 µM for all hair cell count experiments), 3) gentamicin + mannitol 10 mM, 4) gentamicin + mannitol 50 mM, and 5) gentamicin + mannitol 100 mM. The tissues were then fixed and stained, and hair cells were counted for segments of the apex, middle, and basal turns. Quantitative RT-PCR (qRT-PCR) was performed on organ of Corti explant extracted RNA after 24 hours in vitro: 1) control; 2) gentamicin (100 µM for all gene expression and CellRox experiments); 3) gentamicin +mannitol 100 mM; and 4) mannitol 100 mM for tumor necrosis factor-alpha (TNF-α), TNF-α receptor (TNFR1A), interleukin-1 beta (IL-1ß) and cyclooxygenase-2 (COX-2). In vitro examination of oxidative stress was performed for the same test groups at 24 hours using CellRox Deep Red assay. RESULTS: Gentamicin induced loss of both inner hair cells and outer hair cells with increasing severity from apex to middle to basal segments (Pearson r = -0.999 for inner hair cells and -0.972 for outer hair cells). Mannitol demonstrated dose-dependent otoprotection of IHCs and outer hair cells (p < 0.001 for mannitol at 100 mM). CellRox demonstrated increased oxidative stress induced by gentamicin exposure, and this effect was attenuated by treatment of gentamicin-exposed explants with mannitol (p < 0.05). TNF-α, IL-1ß TNFR1A, and COX-2 mRNA levels were upregulated by gentamicin (p < 0.05). Mannitol treatment of gentamicin explants downregulated the gene expression of the proinflammatory cytokines, but this difference did not achieve significance. Interestingly, in gentamicin-challenged organ of Corti explants, Mannitol upregulated the expression of TNFR1A, but this increase did not achieve significance (p > 0.05). CONCLUSION: Gentamicin ototoxicity is increasingly severe from the apex to basal turn of the cochlea. Treatment with mannitol prevents gentamicin-induced hair cell loss in a dose-dependent manner, protecting both IHCs and outer hair cells. Mannitol appears to act as a free radical scavenger to reduce the cytotoxic effects of gentamicin by reducing the level of oxidative stress.

Adult human nasal mesenchymal-like stem cells restore cochlear spiral ganglion neurons after experimental lesion.

A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes deafness, affecting millions of people. Currently, there is no effective therapy to repair the inner ear sensory structures in humans. Cochlear implantation can restore input, but only if auditory neurons remain intact. Efforts to develop stem cell-based treatments for deafness have demonstrated progress, most notably utilizing embryonic-derived cells. In an effort to bypass limitations of embryonic or induced pluripotent stem cells that may impede the translation to clinical applications, we sought to utilize an alternative cell source. Here, we show that adult human mesenchymal-like stem cells (MSCs) obtained from nasal tissue can repair spiral ganglion loss in experimentally lesioned cochlear cultures from neonatal rats. Stem cells engraft into gentamicin-lesioned organotypic cultures and orchestrate the restoration of the spiral ganglion neuronal population, involving both direct neuronal differentiation and secondary effects on endogenous cells. As a physiologic assay, nasal MSC-derived cells engrafted into lesioned spiral ganglia demonstrate responses to infrared laser stimulus that are consistent with those typical of excitable cells. The addition of a pharmacologic activator of the canonical Wnt/ß-catenin pathway concurrent with stem cell treatment promoted robust neuronal differentiation. The availability of an effective adult autologous cell source for inner ear tissue repair should contribute to efforts to translate cell-based strategies to the clinic.

Short interfering RNA against Bax attenuates TNFα-induced ototoxicity in rat organ of Corti explants.

OBJECTIVE: Evaluate the effectiveness of short interfering RNA against Bax (Bax siRNA) as a treatment for tumor necrosis factor α (TNFα)-induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants. STUDY DESIGN: Basic science. SETTING: Basic science laboratory, University of Miami Ear Institute. METHODS: Organ of Corti explants dissected from 3-day-old rats were cultured in control media, TNFα, and TNFα + Bax siRNA at 0, 48, and 72 hours in vitro. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, HC viability studies, immunofluorescence, and confocal microscopy were performed. Analysis of variance with post hoc testing was used with P < .05 considered significant. RESULTS: The TNFα-damaged explants demonstrated significant decreases in viable HCs in the basal turn with corresponding increased levels of Bax gene and protein expression, compared with control explant levels. The levels of viable HCs and Bax gene and protein expression in TNFα + Bax siRNA-treated explants approached levels measured in control explants. Immunolocalization studies showed increased Bax protein expression in basal turn HCs in TNFα-treated explants, whereas control explants and TNFα + Bax siRNA-treated explants had low levels of Bax expression. CONCLUSION: TNFα initiates the programmed cell death of auditory HCs in OC explants through upregulation of proapoptotic Bax gene and protein expression. Bax siRNA blocks TNFα-induced apoptosis of HCs by decreasing the TNFα-induced levels of Bax mRNA and protein expression in treated explants. Bax siRNA is an effective treatment for TNFα-induced ototoxicity in OC explants in vitro and has great potential to be a therapeutic agent against trauma/inflammation-induced hearing loss.

Molecular mechanisms involved in cochlear implantation trauma and the protection of hearing and auditory sensory cells by inhibition of c-Jun-N-terminal kinase signaling.

OBJECTIVES/HYPOTHESIS: To investigate the molecular mechanisms involved in electrode insertion trauma (EIT) and to test the otoprotective effect of locally delivered AM-111. STUDY DESIGN: An animal model of cochlear implantation. METHODS: Guinea pigs' hearing thresholds were measured by auditory brainstem response (ABR) before and after cochlear implantation in four groups: EIT; pretreated with hyaluronate gel 30 minutes before EIT (EIT+Gel); pretreated with hyaluronate gel/AM-111 30 minutes before EIT (EIT+AM-111); and unoperated contralateral ears as controls. Neurofilament, synapsin, and fluorescein isothiocyanate (FITC)-phalloidin staining for hair cell counts were performed at 90 days post-EIT. Immunostaining for 4-hydroxy-2-nonenal (HNE), activated caspase-3, CellROX, and phospho-c-Jun were performed at 24 hours post-EIT. RESULTS: ABR thresholds increased post-EIT in the cochleae of EIT only and EIT+Gel treated animals. There was no significant increase in hearing thresholds in cochleae from either EIT+AM-111 treated or unoperated control ears. AM-111 protection of organ of Corti sensory elements (i.e., hair cells [HCs], supporting cells [SCs], nerve fibers, and synapses) was documented at 3 months post-EIT. Immunostaining of 24-hour post-EIT specimens demonstrated increased levels of HNE in HCs and SCs; increased levels of CellROX and activation of caspase-3 was observed only in SCs, and phosphorylation of c-Jun occurred only in HCs of the EIT-only and EIT+Gel specimens. There was no immunostaining for either HNE, CellROX, caspase-3, or phospho-c-Jun in the organ of Corti specimens from AM-111 treated cochleae. CONCLUSIONS: Molecular mechanisms involved in programmed cell death of HCs are different than the ones involved in programmed cell death of SCs. Local delivery of AM-111 provided a significant level of protection against EIT-induced hearing losses, HC losses, and damage to neural elements.

A novel organ of corti explant model for the study of cochlear implantation trauma.

This study presents a novel in vitro model of electrode insertion trauma-induced hair cell (HC) damage and loss and its application for testing the efficacy of otoprotective drugs. In the cochlear implant (CI) procedure as a treatment for profound deafness, an electrode array is surgically inserted to provide electrical stimulation to the auditory nerve. Mechanical trauma from insertion of a CI electrode into the scala tympani can lead to inflammation and a high level of oxidative stress, which can initiate the apoptosis of auditory HCs and intracochlear fibrosis. HC apoptosis and intracochlear fibrosis are thought to be causes of poor CI functional outcomes. In order to gain insight into the molecular mechanisms that initiate HC apoptosis and scala tympani fibrosis following electrode insertion trauma (EIT), and the otoprotective effects of dexamethasone (DXM) observed in previous studies, an in vitro model of EIT was designed. Here we present and characterize a novel, reproducible in vitro model for the study of cellular and molecular events that occur following an EIT procedure. Cochleae from 3-day-old rats were subjected to a cochleostomy and were then divided into three groups: (1) control, (2) EIT, and (3) EIT + DXM (20 µg/mL). In Groups 2 and 3, a 0.28-mm diameter monofilament fishing line was introduced through the small cochleostomy located next to the round window area, allowing for an insertion of between 110° and 150°. HC counts, gene expression for pro-inflammatory cytokines (i.e., TNFα and IL-1ß), pro-inflammatory inducible enzymes (i.e., iNOS and COX-2) and growth factors (i.e., TGFß1, TGFß3 and CTGF), oxidative stress (i.e., CellROX), and analyses of apoptosis pathways (i.e., caspase-3, apoptosis induced factor and Endonuclease G) were carried out on all explants at different time points. The results of this EIT in vitro model show the initiation of wound healing in which an inflammatory response is followed by a proliferative-fibrosis phase. Moreover, DXM treatment of EIT explants inhibited the inflammatory response and promoted a nonscarring wound healing process. The novel in vitro model described here will improve our understanding of mechanisms underlying CI insertion trauma and protective strategies such as DXM treatment.

Conservation of hearing and protection of hair cells in cochlear implant patients' with residual hearing.

This review covers the molecular mechanisms involved in hair cell and hearing losses which can result from trauma generated during the process of cochlear implantation and the contributions of both the intrinsic and extrinsic cell death signaling pathways in producing these trauma/inflammation induced losses. Application of soft surgical techniques to conserve hearing and protect auditory sensory cells during the process of cochlear implantation surgery and insertion of the electrode array during the process of cochlear implantation are reviewed and discussed. The role of drug therapy and mode of drug delivery for the conservation of a cochlear implant patient's residual hearing is presented and discussed.