Combinatorial Atoh1, Gfi1, Pou4f3, and Six1 gene transfer induces hair cell regeneration in the flat epithelium of mature guinea pigs.

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.

USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Purpose: Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). Methods: CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. Results: The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Conclusions: Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. Translational Relevance: To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea.

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Small Arms Fire-like noise: Effects on Hearing Loss, Gap Detection and the Influence of Preventive Treatment.

A noise-induced loss of inner hair cell (IHC) - auditory nerve synaptic connections has been suggested as a factor that can trigger the progression of maladaptive plastic changes leading to noise-induced tinnitus. The present study used a military relevant small arms fire (SAF)-like noise (50 biphasic impulses over 2.5 min at 152 dB SPL given unilaterally to the right ear) to induce loss (∼1/3) of IHC synaptic ribbons (associated with synapse loss) in rat cochleae with only minor (less than 10%) loss of outer hair cells. Approximately half of the noise-exposed rats showed poorer Gap Detection post-noise, a behavioral indication suggesting the presence of tinnitus. There was significantly greater loss of IHC ribbons in noise-exposed rats with reduced Gap Detection compared to noise-exposed rats retaining normal Gap Detection. We have previously shown systemic administration of piribedil, memantine, and/or ACEMg significantly reduced loss of IHC ribbons induced by a 3 h 4 kHz octave band 117 dB (SPL) noise. The present study examined if this treatment would also reduce ribbon loss from the SAF-like noise exposure and if this would prevent the reduced Gap Detection. As in the previous study, piribedil, memantine, and ACEMg treatment significantly reduced the noise-induced loss of ribbons, such that it was no longer significantly different from normal. However, it did not prevent development of the reduced Gap Detection indication of tinnitus in all treated noise-exposed rats, reducing the incidence but not reaching significance.

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.

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.

Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae.

Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing.

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.

Connexin 26 null mice exhibit spiral ganglion degeneration that can be blocked by BDNF gene therapy.

Mutations in the connexin 26 gene (GJB2) are the most common genetic cause of deafness, leading to congenital bilateral non-syndromic sensorineural hearing loss. Here we report the generation of a mouse model for a connexin 26 (Cx26) mutation, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the auditory epithelium. We determined that these conditional knockout mice, designated Gjb2-CKO, have a severe hearing loss. Immunocytochemistry of the auditory epithelium confirmed absence of Cx26 in the non-sensory cells. Histology of the organ of Corti and the spiral ganglion neurons (SGNs) performed at ages 1, 3, or 6 months revealed that in Gjb2-CKO mice, the organ of Corti began to degenerate in the basal cochlear turn at an early stage, and the degeneration rapidly spread to the apex. In addition, the density of SGNs in Rosenthal's canal decreased rapidly along a gradient from the base of the cochlea to the apex, where some SGNs survived until at least 6 months of age. Surviving neurons often clustered together and formed clumps of cells in the canal. We then assessed the influence of brain derived neurotrophic factor (BDNF) gene therapy on the SGNs of Gjb2-CKO mice by inoculating Adenovirus with the BDNF gene insert (Ad.BDNF) into the base of the cochlea via the scala tympani or scala media. We determined that over-expression of BDNF beginning around 1 month of age resulted in a significant rescue of neurons in Rosenthal's canal of the cochlear basal turn but not in the middle or apical portions. This data may be used to design therapies for enhancing the SGN physiological status in all GJB2 patients and especially in a sub-group of GJB2 patients where the hearing loss progresses due to ongoing degeneration of the auditory nerve, thereby improving the outcome of cochlear implant therapy in these ears.

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.

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.

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.

Delayed neurotrophin treatment following deafness rescues spiral ganglion cells from death and promotes regrowth of auditory nerve peripheral processes: effects of brain-derived neurotrophic factor and fibroblast growth factor.

The extent to which neurotrophic factors are able to not only rescue the auditory nerve from deafferentation-induced degeneration but also promote process regrowth is of basic and clinical interest, as regrowth may enhance the therapeutic efficacy of cochlear prostheses. The use of neurotrophic factors is also relevant to interventions to promote regrowth and repair at other sites of nerve trauma. Therefore, auditory nerve survival and peripheral process regrowth were assessed in the guinea pig cochlea following chronic infusion of BDNF + FGF(1) into scala tympani, with treatment initiated 4 days, 3 weeks, or 6 weeks after deafferentation from deafening. Survival of auditory nerve somata (spiral ganglion neurons) was assessed from midmodiolar sections. Peripheral process regrowth was assessed using pan-Trk immunostaining to selectively label afferent fibers. Significantly enhanced survival was seen in each of the treatment groups compared to controls receiving artificial perilymph. A large increase in peripheral processes was found with BDNF + FGF(1) treatment after a 3-week delay compared to the artificial perilymph controls and a smaller enhancement after a 6-week delay. Neurotrophic factor treatment therefore has the potential to improve the benefits of cochlear implants by maintaining a larger excitable population of neurons and inducing neural regrowth.

Tolerability of N-chlorotaurine in the guinea pig middle ear: a pilot study using an improved application system.

The tissue tolerance of N-chlorotaurine (NCT), a mild endogenous antimicrobial oxidant, has been investigated by application to the guinea pig middle ear. The animals were implanted with a novel cannula system that allows chronic external drug delivery to the round window niche. In the first part of the study, 3 animals each received 100 microL of 0.1% NCT (5.5 mmol/L) and 1% NCT, respectively, in aqueous solution twice daily for 8 days. In the second part, NCT was dissolved in phosphate-buffered saline solution to 300 milliosmolar (isotonic), and 27 microL was injected in 3 additional animals twice daily for 7 days. The guinea pigs injected with 100 microL of NCT developed immediate dizziness and nystagmus and did not thrive. Other reactions included mucosal thickening in the middle ear, rupture of the tympanic membrane, and blood and gelatinous material in the cochlea accompanied by hair cell loss and a 10- to 90-dB elevation of the hearing threshold as determined by auditory brain stem responses. The effects seemed to be dose-dependent, but the rate of variability was high across animals. In contrast, the guinea pigs treated with 27 microL of isotonic NCT showed no signs of discomfort, no or only moderate thickening of the middle ear mucosa, no shift of the hearing threshold, and no hair cell loss. Positive control animals injected with 10% neomycin sulfate developed extensive hair cell loss. Provided that the membranes of the inner ear are intact and that low single-dose volumes are used to avoid increased middle ear pressure, isotonic NCT seems to be well tolerated in the tympanic cavity. The new drug delivery system proved to be advantageous for ototoxicity studies.

Systemic co-treatment with alpha-melanocyte stimulating hormone delays hearing loss caused by local cisplatin administration in guinea pigs.

It has previously been demonstrated that ototoxicity induced by systemic administration of cisplatin is reduced by concomitant administration of melanocortins, like alpha-melanocyte stimulating hormone (alpha-MSH). However, these experiments were hampered by large interanimal variability. Therefore, we re-investigated the effects of systemically administered alpha-MSH during local (intracochlear) administration of cisplatin. Guinea pigs, implanted with a round-window electrode, allowing daily monitoring of the compound action potentials (CAPs), and a mini-osmotic pump, pumping either 0.5 microl/h physiological saline or cisplatin solution (15 microg/ml), were co-treated daily with a subcutaneous bolus injection of either alpha-MSH (75 microg/kg) or physiological saline for 1 week or until the electrocochleogram showed a persistent decrease in CAP amplitude (40 dB threshold shift at 8 kHz). Next, the animals were sacrificed and the cochleas were processed for histology. After 2-3 days, cisplatin alone caused a threshold shift at all frequencies (2-16 kHz). Co-administration with alpha-MSH consistently delayed the criterion threshold shift by 1 day. When the 40 dB criterion had been reached, similar outer hair cell losses in both the cisplatin/alpha-MSH- and cisplatin/saline-treated groups were observed. This experiment confirms that direct administration of cisplatin into the cochlea results in considerably less interanimal variability than systemic administration and that co-treatment with alpha-MSH delays cisplatin ototoxicity. Since cisplatin was delivered directly to the cochlea, the ameliorating effect of alpha-MSH probably involves a cochlear target.