Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

The Role of Glia in the Peripheral and Central Auditory System Following Noise Overexposure: Contribution of TNF-α and IL-1ß to the Pathogenesis of Hearing Loss.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1ß are implicated in regulating the initiation and progression of noise-induced hearing loss.

Treatment with Piribedil and Memantine Reduces Noise-Induced Loss of Inner Hair Cell Synaptic Ribbons.

Noise overstimulation can induce loss of synaptic ribbons associated with loss of Inner Hair Cell - Auditory Nerve synaptic connections. This study examined if systemic administration of Piribedil, a dopamine agonist that reduces the sound evoked auditory nerve compound action potential and/or Memantine, an NMDA receptor open channel blocker, would reduce noise-induced loss of Inner Hair Cell ribbons. Rats received systemic Memantine and/or Piribedil for 3 days before and 3 days after a 3 hour 4 kHz octave band noise at 117 dB (SPL). At 21 days following the noise there was a 26% and 38% loss of synaptic ribbons in regions 5.5 and 6.5 mm from apex, respectively, elevations in 4-, 8- and 20 kHz tonal ABR thresholds and reduced dynamic output at higher intensities of stimulation. Combined treatment with Piribedil and Memantine produced a significant reduction in the noise-induced loss of ribbons in both regions and changes in ABR sensitivity and dynamic responsiveness. Piribedil alone gave significant reduction in only the 5.5 mm region and Memantine alone did not reach significance in either region. Results identify treatments that could prevent the hearing loss and hearing disorders that result from noise-induced loss of Inner Hair Cell - Auditory Nerve synaptic connections.

Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this "toughening" effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with "toughening" and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

ACEMg Diet Supplement Modifies Progression of Hereditary Deafness.

Dietary supplements consisting of beta-carotene (precursor to vitamin A), vitamins C and E and the mineral magnesium (ACEMg) can be beneficial for reducing hearing loss due to aminoglycosides and overstimulation. This regimen also slowed progression of deafness for a boy with GJB2 (CONNEXIN 26) mutations. To assess the potential for treating GJB2 and other forms of hereditary hearing loss with ACEMg, we tested the influence of ACEMg on the cochlea and hearing of mouse models for two human mutations: GJB2, the leading cause of childhood deafness, and DIAPH3, a cause of auditory neuropathy. One group of mice modeling GJB2 (Gjb2-CKO) received ACEMg diet starting shortly after they were weaned (4 weeks) until 16 weeks of age. Another group of Gjb2-CKO mice received ACEMg in utero and after weaning. The ACEMg diet was given to mice modeling DIAPH3 (Diap3-Tg) after weaning (4 weeks) until 12 weeks of age. Control groups received food pellets without the ACEMg supplement. Hearing thresholds measured by auditory brainstem response were significantly better for Gjb2-CKO mice fed ACEMg than for the control diet group. In contrast, Diap3-Tg mice displayed worse thresholds than controls. These results indicate that ACEMg supplementation can influence the progression of genetic hearing loss.

Persistence, distribution, and impact of distinctly segmented microparticles on cochlear health following in vivo infusion.

Delivery of pharmaceuticals to the cochleae of patients with auditory dysfunction could potentially have many benefits from enhancing auditory nerve survival to protecting remaining sensory cells and their neuronal connections. Treatment would require platforms to enable drug delivery directly to the cochlea and increase the potential efficacy of intervention. Cochlear implant recipients are a specific patient subset that could benefit from local drug delivery as more candidates have residual hearing; and since residual hearing directly contributes to post-implantation hearing outcomes, it requires protection from implant insertion-induced trauma. This study assessed the feasibility of utilizing microparticles for drug delivery into cochlear fluids, testing persistence, distribution, biocompatibility, and drug release characteristics. To allow for delivery of multiple therapeutics, particles were composed of two distinct compartments; one containing polylactide-co-glycolide (PLGA), and one composed of acetal-modified dextran and PLGA. Following in vivo infusion, image analysis revealed microparticle persistence in the cochlea for at least 7 days post-infusion, primarily in the first and second turns. The majority of subjects maintained or had only slight elevation in auditory brainstem response thresholds at 7 days post-infusion compared to pre-infusion baselines. There was only minor to limited loss of cochlear hair cells and negligible immune response based on CD45+ immunolabling. When Piribedil-loaded microparticles were infused, Piribedil was detectable within the cochlear fluids at 7 days post-infusion. These results indicate that segmented microparticles are relatively inert, can persist, release their contents, and be functionally and biologically compatible with cochlear function and therefore are promising vehicles for cochlear drug delivery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1510-1522, 2016.

Synergistic effects of free radical scavengers and cochlear vasodilators: a new otoprotective strategy for age-related hearing loss.

The growing increase in age-related hearing loss (ARHL), with its dramatic reduction in quality of life and significant increase in health care costs, is a catalyst to develop new therapeutic strategies to prevent or reduce this aging-associated condition. In this regard, there is extensive evidence that excessive free radical formation along with diminished cochlear blood flow are essential factors involved in mechanisms of other stress-related hearing loss, such as that associated with noise or ototoxic drug exposure. The emerging view is that both play key roles in ARHL pathogenesis. Therapeutic targeting of excessive free radical formation and cochlear blood flow regulation may be a useful strategy to prevent onset of ARHL. Supporting this idea, micronutrient-based therapies, in particular those combining antioxidants and vasodilators like magnesium (Mg(2+)), have proven effective in reducing the impact of noise and ototoxic drugs in the inner ear, therefore improving auditory function. In this review, the synergistic effects of combinations of antioxidant free radicals scavengers and cochlear vasodilators will be discussed as a feasible therapeutic approach for the treatment of ARHL.

Antioxidant vitamins and magnesium and the risk of hearing loss in the US general population.

BACKGROUND: The protective effects of antioxidant vitamins on hearing loss are well established in animal studies but in few human studies. Recent animal studies suggest that magnesium intake along with antioxidants may act in synergy to prevent hearing loss. OBJECTIVE: We examined associations between intake of antioxidant vitamins (daily ß-carotene and vitamins C and E) and magnesium and hearing thresholds and explored their joint effects in US adults. DESIGN: We analyzed cross-sectional data from 2592 participants aged 20-69 y from NHANES 2001-2004. Hearing thresholds as pure tone averages (PTAs) at speech (0.5, 1, 2, and 4 kHz) and high frequencies (3, 4, and 6 kHz) were computed. RESULTS: When examined individually, modeled as quartiles, and after adjustment for potential confounders, higher intakes of ß-carotene, vitamin C, and magnesium were associated with lower (better) PTAs at both speech and high frequencies. High intakes of ß-carotene or vitamin C combined with high magnesium compared with low intakes of both nutrients were significantly associated with lower (better) PTAs at high frequencies (-14.82%; 95% CI: -20.50% to -8.74% for ß-carotene + magnesium and -10.72%; 95% CI: -16.57% to -4.45% for vitamin C + magnesium). The estimated joint effects were borderline significantly larger than the sums of the individual effects [high ß-carotene/low magnesium (-4.98%) and low ß-carotene/high magnesium (-0.80%), P-interaction = 0.08; high vitamin C/low magnesium (-1.33%) and low vitamin C/high magnesium (2.13%), P-interaction = 0.09]. CONCLUSION: Dietary intakes of antioxidants and magnesium are associated with lower risks of hearing loss.

Nestin-expressing cells in the developing, mature and noise-exposed cochlear epithelium.

The auditory sensory epithelium in non-mammalian vertebrates can replace lost hair cells by transdifferentiation of supporting cells, but this regenerative ability is lost in the mammalian cochlea. Future cell-based treatment of hearing loss may depend on stem cell transplantation or on transdifferentiation of endogenous cells in the cochlea. For both approaches, identification of cells with stem cell features within the mature cochlea may be useful. Here we use a Nestin-ß-gal mouse to examine the presence of Nestin positive cells in the mature auditory epithelium, and determine how overstimulation of the ear impacts these cells. Nestin positive cells were found in the apical turn of the cochlea lateral to the outer hair cell area. This pattern of expression persisted into mature age. The area of Nestin positive cells was increased after the noise lesion. This increase in area coincided with an increase in expression of the Nestin mRNA. The data suggest that cells with potential stem cell features remain in the mature mammalian cochlea, restricted to the apical turn, and that an additional set of signals is necessary to trigger their contribution to cell replacement therapy in the ear. As such, this population of cells could serve to generate cochlear stem cells for research and potential therapy, and may be a target for treatments based on induced transdifferentiation of endogenous cochlear cells.

Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3.

Wnt/ß-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, Neurogenin1 (Ngn1), NeuroD, and Brn3a, in the nervous system. As neurons in cranial sensory ganglia and dorsal root ganglia transiently express Ngn1, NeuroD, and Brn3a during embryonic development, we hypothesized that Wnt proteins could instructively promote a sensory neuronal fate from mesenchymal stem cells (MSCs) directed to differentiate into neurons. Consistent with our hypothesis, Wnt1 induced expression of sensory neuron markers including Ngn1, NeuroD, and Brn3a, as well as glutamatergic markers in neurally induced MSCs in vitro and promoted engraftment of transplanted MSCs in the inner ear bearing selective loss of sensory neurons in vivo. Given the consensus function of T-cell leukemia 3 (Tlx3), as a glutamatergic selector gene, we postulated that the effects of canonical Wnt signaling on sensory neuron and glutamatergic marker gene expression in MSCs may be mediated by Tlx3. We first confirmed that Wnt1 indeed upregulates Tlx3 expression, which can be suppressed by canonical Wnt inhibitors. Next, our chromatin immunoprecipitation assays revealed that T-cell factor 3/4, Wnt-activated DNA binding proteins, interact with a regulatory region of Tlx3 in MSCs after neural induction. Furthermore, we demonstrated that forced expression of Tlx3 in MSCs induced sensory and glutamatergic neuron markers after neural induction. Together, these results identify Tlx3 as a novel target for canonical Wnt signaling that confers somatic stem cells with a sensory neuron phenotype upon neural induction.

Post-treatment effects of local GDNF administration to the inner ears of deafened guinea pigs.

For patients with profound hearing loss, a cochlear implant is the only treatment available today. The function of a cochlear implant depends in part on the function and survival of spiral ganglion neurons. Following deafferentation, glial cell-derived neurotrophic factor (GDNF) is known to affect spiral ganglion neuron survival. The purpose of this study was to assess delayed GDNF treatment after deafening, the effects of cessation of GDNF treatment, and the effects of subsequent antioxidants on responsiveness and survival of the spiral ganglion neurons. Three-week deafened (by local neomycin administration) guinea pigs were implanted in the scala tympani with a combined cochlear implant electrode and cannula. GDNF (1 µg/mL) or artificial perilymph was then delivered for 4 weeks, following which the animals received systemic ascorbic acid + Trolox or saline for an additional 4 weeks. Thresholds for electrically-evoked auditory brain stem responses (eABRs) were significantly elevated at 3 weeks with deafness, stabilized with GDNF, and showed no change with GDNF cessation and treatment with antioxidants or saline. The populations of spiral ganglion neurons were reduced with deafness (by 40% at 3 weeks and 70% at 11 weeks), and rescued from cell death by GDNF with no further reduction at 8 weeks following 4 weeks of cessation of GDNF treatment equally in both the antioxidant- and saline-treated groups. Local growth factor treatment of the deaf ear may prevent deterioration in electrical responsiveness and rescue auditory nerve cells from death; these effects outlast the period of treatment, and may enhance the benefits of cochlear implant therapy for the deaf.

Safety of ciprofloxacin and dexamethasone in the guinea pig middle ear.

OBJECTIVE: To investigate the ototoxic potential of ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%, after administration to the guinea pig middle ear. DESIGN: Fifty guinea pigs were randomly assigned to 4 test groups of 10 animals each and 2 control groups of 5 animals each. The 4 test groups were treated twice daily for 4 weeks with 10 muL of (1) ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%; (2) ciprofloxacin hydrochloride, 1.0%, plus dexamethasone, 0.3%; (3) ciprofloxacin hydrochloride, 0.3%, or (4) vehicle. The positive and negative control groups were treated with neomycin sulfate, 10%, or isotonic sodium chloride solution, respectively. SETTING: Academic research laboratory. INTERVENTIONS: Study animals were implanted with a drug delivery cannula to the middle ear, terminating in the round window niche for direct delivery to the round window membrane. MAIN OUTCOME MEASURES: Auditory brainstem responses were collected at baseline and following 2 and 4 weeks of dosing. At the termination of the study, inner ear tissues were evaluated microscopically. RESULTS: No biologically relevant hearing losses were observed after either 2 or 4 weeks of treatment with vehicle, ciprofloxacin alone, or combinations of ciprofloxacin plus dexamethasone. Examination of the organ of Corti revealed normal hair cell counts in all animals that received isotonic sodium chloride solution, vehicle, ciprofloxacin, or combinations of ciprofloxacin and dexamethasone. Conversely, the neomycin sulfate positive control group demonstrated a significant elevation in hearing threshold and profound hair cell loss (P <.001, P = .02, and P <.001 at 2, 8, and 16 kHz, respectively). CONCLUSION: The results of this preclinical study support the safety of ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%, for clinical use in the open middle ear cavity.

Functional evaluation of a cell replacement therapy in the inner ear.

HYPOTHESIS: Cell replacement therapy in the inner ear will contribute to the functional recovery of hearing loss. BACKGROUND: Cell replacement therapy is a potentially powerful approach to replace degenerated or severely damaged spiral ganglion neurons. This study aimed at stimulating the neurite outgrowth of the implanted neurons and enhancing the potential therapeutic of inner ear cell implants. METHODS: Chronic electrical stimulation (CES) and exogenous neurotrophic growth factor (NGF) were applied to 46 guinea pigs transplanted with embryonic dorsal root ganglion (DRG) neurons 4 days postdeafening. The animals were evaluated with the electrically evoked auditory brainstem responses (EABRs) at experimental Days 7, 11, 17, 24, and 31. The animals were euthanized at Day 31, and the inner ears were dissected for immunohistochemistry investigation. RESULTS: Implanted DRG cells, identified by enhanced green fluorescent protein fluorescence and a neuronal marker, were found close to Rosenthal canal in the adult inner ear for up to 4 weeks after transplantation. Extensive neurite projections clearly, greater than in nontreated animals, were observed to penetrate the bony modiolus and reach the spiral ganglion region in animals supplied with CES and/or NGF. There was, however, no significant difference in the thresholds of EABRs between DRG-transplanted animals supplied with CES and/or NGF and DRG-transplanted animals without CES or NGF supplement. CONCLUSION: The results suggest that CES and/or NGF can stimulate neurite outgrowth from implanted neurons, although based on EABR measurement, these interventions did not induce functional connections to the central auditory pathway. Additional time or novel approaches may enhance functional responsiveness of implanted cells in the adult cochlea.

Effects of delayed treatment with combined GDNF and continuous electrical stimulation on spiral ganglion cell survival in deafened guinea pigs.

Electrical stimulation (ES) of spiral ganglion cells (SGC) via a cochlear implant is the standard treatment for profound sensor neural hearing loss. However, loss of hair cells as the morphological correlate of sensor neural hearing loss leads to deafferentation and death of SGC. Although immediate treatment with ES or glial cell line-derived neurotrophic factor (GDNF) can prevent degeneration of SGC, only few studies address the effectiveness of delayed treatment. We hypothesize that both interventions have a synergistic effect and that even delayed treatment would protect SGC. Therefore, an electrode connected to a pump was implanted into the left cochlea of guinea pigs 3 weeks after deafening. The contralateral untreated cochleae served as deafened intraindividual controls. Four groups were set up. Control animals received intracochlear infusion of artificial perilymph (AP/-). The experimental groups consisted of animals treated with AP in addition to continuous ES (AP/ES) or treated with GDNF alone (GDNF/-) or GDNF combined with continuous ES (GDNF/ES). Acoustically and electrically evoked auditory brain stem responses were recorded. All animals were killed 48 days after deafening; their cochleae were histologically evaluated. Survival of SGC increased significantly in the GDNF/- and AP/ES group compared with the AP/- group. A highly significant increase in SGC density was observed in the GDNF/ES group compared with the control group. Additionally, animals in the GDNF/ES group showed reduced EABR thresholds. Thus, delayed treatment with GDNF and ES can protect SGC from degeneration and may improve the benefits of cochlear implants.

Electrically evoked auditory nerve responses in the cochlea with normal outer hair cells.

As hybrid cochlear implant devices are increasingly used for restoring hearing in patients with residual hearing it is important to understand electrically evoked responses in cochleae having functional hair cells. To test the hypothesis that extracochlear electrical stimulation (EES) from sinusoidal current can provoke an auditory nerve response with normal frequency selectivity, the EES-evoked compound action potential (ECAP) was investigated in this study. Brief sinusoidal electrical currents, delivered via a round window electrode, were used to evoke ECAP. The ECAP waveform was observed to be the same as the acoustically evoked CAP (ACAP), except for a shorter latency. The input/output and intensity/latency functions of ACAPs and ECAPs were also similar. The maximum acoustic masking for both ACAP and ECAP occurred near probe frequencies. Since the masked tuning curve of a CAP reflects the frequency selectivity of neural excitation, these data demonstrate a highly specific activation of the auditory nerve, which would result in high degree of frequency selectivity. This frequency selectivity likely results from the cochlear traveling wave caused by electrically stimulated outer hair cells.

Glutamatergic neuronal differentiation of mouse embryonic stem cells after transient expression of neurogenin 1 and treatment with BDNF and GDNF: in vitro and in vivo studies.

Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50-75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.

Stem cell transplantation for auditory nerve replacement.

The successful function of cochlear prostheses depends on activation of auditory nerve. The survival of auditory nerve neurons, however, can vary widely in candidates for cochlear implants and influence implant efficacy. Stem cells offer the potential for improving the function of cochlear prostheses and increasing the candidate pool by replacing lost auditory nerve. The first phase of studies for stem cell replacement of auditory nerve has examined the in vitro survival and differentiation as well as in vivo differentiation and survival of exogenous embryonic and tissue stem cells placed into scala tympani and/or modiolus. These studies are reviewed and new results on in vivo placement of B-5 mouse embryonic stem cells into scala tympani of the guinea pig cochleae with differentiation into a glutamatergic neuronal phenotype are presented. Research on the integration and connections of stem cell derived neurons in the cochlea is described. Finally, an alternative approach is considered, based on the use of endogenous progenitors rather than exogenous stem cells, with a review of promising findings that have identified stem cell-like progenitors in cochlear and vestibular tissues to provide the potential for auditory nerve replacement.

Deafferentation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor.

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-week-delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate- and delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers.

Bcl-2 genes regulate noise-induced hearing loss.

Proteins of the Bcl-2 family have been implicated in control of apoptotic pathways modulating neuronal cell death, including noise-induced hearing loss. In this study, we assessed the expressions of anti- and proapoptotic Bcl-2 genes, represented by Bcl-xL and Bak following noise exposures, which yielded temporary threshold shift (TTS) or permanent threshold shift (PTS). Auditory brainstem responses (ABRs) were assessed at 4, 8, and 16 kHz before exposure and on days 1, 3, 7, and 10 following exposure to 100 dB SPL, 4 kHz OBN, 1 hr (TTS) or 120 dB SPL, 4 kHz OBN, 5 hr (PTS). On day 10, subjects were euthanized. ABR thresholds increased following both exposures, fully recovered following the TTS exposure, and showed a 22.6 dB (4 kHz), 42.5 dB (8 kHz), and 44.9 dB (16 kHz) mean shift on day 10 following the PTS exposure. PTS was accompanied by outer hair cell loss progressing epically and basally from the 4-kHz region. Additional animals were euthanized for immunohistochemical assessment. BcL-xL was robustly expressed in outer hair cells following TTS exposure, whereas Bak was expressed following PTS exposure. These results indicate an important role of the Bcl-2 family proteins in regulating sensory cell survival or death following intense noise. Bcl-xL plays an essential role in prevention of sensory cell death following TTS levels of noise, and PTS exposure provokes the expression of Bak and, with that, cell death.