Pattern of hair cell loss and delayed peripheral neuron degeneration in inner ear by a high-dose intratympanic gentamicin

To gain insights into the ototoxic effects of aminoglycoside antibiotics (AmAn) and delayed peripheral ganglion neuron death in the inner ear, experimental animal models were widely used with several different approaches including AmAn systemic injections, combination treatment of AmAn and diuretics, or local application of AmAn. In these approaches, systemic AmAn treatment alone usually causes incomplete damage to hair cells in the inner ear. Co-administration of diuretic and AmAn can completely destroy the cochlear hair cells, but it is impossible to damage the vestibular system. Only the approach of AmAn local application can selectively eliminate most sensory hair cells in the inner ear. Therefore, AmAn local application is more suitable for studies for complete hair cell destructions in cochlear and vestibular system and the following delayed peripheral ganglion neuron death. In current studies, guinea pigs were unilaterally treated with a high concentration of gentamicin (GM, 40 mg/ml) through the tympanic membrane into the middle ear cavity. Auditory functions and vestibular functions were measured before and after GM treatment. The loss of hair cells and delayed degeneration of ganglion neurons in both cochlear and vestibular system were quantified 30 days or 60 days after treatment. The results showed that both auditory and vestibular functions were completely abolished after GM treatment. The sensory hair cells were totally missing in the cochlea, and severely destroyed in vestibular end-organs. The delayed spiral ganglion neuron death 60 days after the deafening procedure was over 50%. However, no obvious pathological changes were observed in vestibular ganglion neurons 60 days post-treatment. These results indicated that a high concentration of gentamycin delivered to the middle ear cavity can destroy most sensory hair cells in the inner ear that subsequently causes the delayed spiral ganglion neuron degeneration. This model might be useful for studies of hair cell regenerations, delayed degeneration of peripheral auditory neurons, and/or vestibular compensation. In addition, a potential problem of ABR recording for unilateral deafness and issues about vestibular compensation are also discussed.     

Time course of inner ear degeneration and deafness in mice lacking the Kir4.1 potassium channel subunit

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

CONCLUSION: Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. OBJECTIVES: At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. MATERIALS AND METHODS: Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. RESULTS: Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Distribution of beta-tubulin in guinea pig inner ear

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

Conclusion. Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. Objectives. At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. Materials and methods. Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. Results. Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Intermediate filaments in the newborn inner ear of the mouse

The presence of intermediate filaments in the inner ear of the newborn mouse was analyzed with immunofluorescence techniques using antibodies against the five classes of intermediate filaments: cytokeratins, vimentin, desmin, neurofilaments and glial fibrillary acid protein (GFA). Neurofilaments were found in all nerve fibers from the ganglion cell to the hair cell. In the vestibular ganglion two subpopulations of ganglion cells were identified: a minor part staining intensively with neurofilament and the major part of cells lacking this immunofluorescence. Vimentin occurred in a number of supporting structures in the membranous labyrinth, but not in vestibular or cochlear ganglion cells. Cytokeratins, desmin or GFA were not identified in the inner ear.     

Localization of histamine (H1, H2, H3 and H4) receptors in mouse inner ear

Conclusion The present findings show that all four types of histamine receptors (H1R, H2R, H3R, and H4R) are present in the inner ear, thus supporting the hypothesis that histamine plays a physiological role in the inner ear. Objective To analyse the presence of histamine receptors in the normal mouse inner ear. Methods CBA/J mice were used in this study. The localization of H1R, H2R, H3R, and H4R in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion, and endolymphatic sac, was studied by real-time PCR and immunohistochemistry. Results The mRNA for each receptor sub-type was detected in the inner ear. In the immunohistochemical study, the organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory epithelium, and endolymphatic sac cells showed an immunofluorescent reaction to all histamine receptors.     

Distribution of frequenin in the mouse inner ear during development, comparison with other calcium-binding proteins and synaptophysin

Frequenin is a calcium-binding protein previously implicated in the regulation of neurotransmission. We report its immunocytochemical detection in the mouse inner ear, in the adult, and during embryonic (E) and postnatal (P) development. The distribution of frequenin was compared with those of other calcium-binding proteins (calbindin, calretinin, parvalbumin) and synaptophysin. In the adult mouse inner ear, frequenin immunostaining was observed in the afferent neuronal systems (vestibular and cochlear neurons, their processes and endings) and in the vestibular and cochlear efferent nerve terminals. Frequenin colocalized with synaptophysin in well characterized presynaptic compartments, such as the vestibular and cochlear efferent endings, and in putative presynaptic compartments, such as the apical part of the vestibular calyces. Frequenin was not found in vestibular hair cells and in cochlear inner and outer hair cells. During development, frequenin immunoreactivity was first detected on E11 in the neurons of the statoacoustic ganglion. On E14, frequenin was detected in the afferent neurites innervating the vestibular sensory epithelium, along with synaptophysin. On E16, frequenin was detected in the afferent neurites below the inner hair cells in the organ of Corti. The timing of frequenin detection in vestibular and cochlear afferent neurites was consistent with their sequences of maturation, and was earlier than synaptogenesis. Thus in the inner ear, frequenin is a very early marker of differentiated and growing neurons and is present in presynaptic and postsynaptic compartments.     

Strategies to preserve or regenerate spiral ganglion neurons

PURPOSE OF REVIEW: Degeneration of spiral ganglion neurons following hair cell loss carries critical implications for efforts to rehabilitate severe cases of hearing loss with cochlear implants or hair cell regeneration. This review considers recently identified neurotrophic factors and therapeutic strategies which promote spiral ganglion neuron survival and neurite growth. Replacement of these factors may help preserve or regenerate the auditory nerve in patients with extensive hair cell loss. RECENT FINDINGS: Spiral ganglion neurons depend on neurotrophic factors supplied by hair cells and other targets for their development and continued survival. Loss of this trophic support leads to spiral ganglion neuron death via apoptosis. Hair cells support spiral ganglion neuron survival by producing several peptide neurotrophic factors such as neurotrophin-3 and glial derived neurotrophic factor. In addition, neurotransmitter release from the hair cells drives membrane electrical activity in spiral ganglion neurons which also supports their survival. In animal models, replacement of peptide neurotrophic factors or electrical stimulation with an implanted electrode attenuates spiral ganglion neuron degeneration following deafferentation. Cell death inhibitors can also preserve spiral ganglion neuron populations. Preliminary studies show that transfer of stem cells or neurons from other ganglia are two potential strategies to replace lost spiral ganglion neurons. Inducing the regrowth of spiral ganglion neuron peripheral processes to approximate or contact cochlear implant electrodes may help optimize signaling from a diminished population of neurons. SUMMARY: Recent studies of spiral ganglion neuron development and survival have identified several trophic and neuritogenic factors which protect these specialized cells from degeneration following hair cell loss. While still preliminary, such strategies show promise for future clinical applications.     

Expression of intermediate filament proteins in the mature inner ear of the rat and guinea pig.

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes.     

Stem cells for the treatment of hearing loss

One of the greatest challenges in the treatment of inner ear disorders is to find a cure for the hearing loss caused by the loss of cochlear hair cells or spiral ganglion neurons. The recent discovery of stem cells in the adult inner ear that are capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatments.     

Stammzellbasierte Ansätze zur Therapie von Innenohrerkrankungen

The capacity of stem cells to regenerate lost tissue cells has gained recognition among physicians. Despite the successful use of blood stem cells for treating blood cancers, other stem cell types have not yet been widely introduced into clinical practice. Therapy options involving stem cells for inner ear diseases consequently have not been implemented. Nonetheless, several reports have recently been published describing the generation of morphologically and immunologically distinctive inner ear cell types-such as hair cells, supporting cells, and spiral ganglion neurons-from stem cells. Although promising, all of these studies still lack functional results regarding hearing restoration or vestibular function.     

Gentamicin uptake in the chinchilla inner ear

Studies of transtympanic gentamicin have focused on clinical use and outcomes. This study presents evidence of bilateral uptake and retention of gentamicin in certain inner ear cells and structures following transtympanic gentamicin application. Middle ear application of gentamicin was performed by either minipump (Alza model, 2002) or transtympanic injection in a chinchilla model. Histological sections of decalcified temporal bones were stained to identify the distribution of gentamicin. Using both anti-gentamicin immunohistochemistry and autoradiography of tracer amounts of tritiated gentamicin, Scarpa’s and spiral ganglion cells, stria vascularis, and vestibular dark cells of the injected ear were found to have higher levels of gentamicin and retain it within cell bodies while staining levels fell to background levels in the rest of the injected ear over the course of 14 days. There was no evidence of an apical to basal gradient of anti-gentamicin staining within the spiral ganglion. Contralateral inner ear cells showed light anti-gentamicin staining. Cell bodies in the ipsilateral dorsal cochlear nucleus bordering the cochlear aqueduct (CA) showed a lateral to medial gradient of gentamicin staining, suggesting the CA as a potential site of transfer of gentamicin to the contralateral ear. Direct effects of aminoglycosides on ganglion cells may have implications on both the success of cochlear implantation in patients deafened following systemic aminoglycoside therapy and on the advisability of clinical practices of transtympanic gentamicin therapy and ototopic aminoglycoside treatment.     

Advances in inner ear gene therapy: exploring cochlear protection and regeneration

PURPOSE OF REVIEW: To review the application of gene therapy in the inner ear. Gene delivery to the inner ear was first reported in 1996. Since then the field has progressed on multiple fronts. RECENT DEVELOPMENTS: More diverse and sophisticated vectors are improving the efficiency of delivery to the inner ear. Research is transitioning from the delivery of marker genes to the delivery of therapeutic genes in animal models of inner ear disease. Three distinct areas of research are developing: (1) delivery of genes for protection of spiral ganglion neurons with potential application in cochlear implantation, (2) delivery of genes for protection of hair cells and hearing preservation in degenerative diseases and cochlear insults and (3) the use of gene therapy to transform cells from one phenotype to another and replace lost cells, potentially restoring lost function. SUMMARY: Currently, no specific drugs are targeted at inner ear disease. The use of gene therapy in the inner ear is being applied in animal models of ototoxicity and ischemia reperfusion injury. Gene therapy can protect the inner ear from damage and even restore function through the regeneration of hair cells.     

Inner ear anomalies in Waardenburg's syndrome associated with Hirschsprung's disease

Congenital inner ear anomalies are reported in temporal bones of a 22-month-old boy with Waardenburg's syndrome and Hirschsprung's disease. Although no changes in the central auditory pathway were identified, peripheral lesions of the cochlear and vestibular membranous labyrinth were observed. Bilateral atrophy of the organ of Corti and stria, and a sparsity of spiral ganglion cells were observed in the cochlea. Degeneration of the vestibular end organs, including a loss of Scarpa's ganglion cells, was also seen. This is the first report of temporal bone histopathology associated with Waardenburg's syndrome and Hirschsprung's disease. The pathoembryology of these inner ear anomalies associated with aganglionosis of the colon supports the hypothesis that Waardenburg's syndrome and Hirschsprung's disease are hereditary defects of neural crest cells.     

Diameter of the cochlear nerve in deaf humans: implications for cochlear implantation.

Although the parameters that are most important for postoperative speech perception in cochlear implantation have not been identified, it is assumed that the numbers of remaining cochlear neurons and spiral ganglion cells in the implanted deaf ears are critical. In this study, we evaluated the correlation of the maximum diameter of the cochlear and vestibular nerve trunks with the number of spiral ganglion cells in horizontal sections of the temporal bone of 42 patients who were profoundly deaf during life, and in 5 patients with normal hearing. The maximum diameters of the cochlear, vestibular, and eighth cranial nerves were significantly smaller in the deaf population as compared to normal-hearing controls. In addition, the counts of the remaining spiral ganglion cells were significantly correlated with the maximum diameter of the cochlear (p = .0006), vestibular (p = .001), and eighth cranial nerves (p = .0003). The regression equation estimated that 25% of the variance of the spiral ganglion cell count was predicted by the maximum diameter of the eighth nerve. Although the results of this study suggest that preoperative radiographic imaging of the diameter of the eighth nerve may be helpful in predicting the residual spiral ganglion cell count, the wide variability of diameters of the eighth nerve in hearing and deaf subjects militates against this theoretic usefulness.     

Die Wirkung von Fibroblastenwachstumsfaktor-1 (FGF-1) auf Spiralganglienzellen der Säugetiercochlea

Transient expression by hair cells, increasing levels of FGF-1 mRNA in neonatal rat spiral ganglion neurons and strong expression in adulthood, make FGF-1 a candidate to be associated with development and maintenance of the mammalian spiral ganglion. To test this hypothesis, dissociated spiral ganglion cells from 5 day old rats were cultured in the presence of FGF-1 at 100 ng/ml plus heparan sulfate proteoglycans (HSPG) at 500 ng/ml for 72 hours. Spiral ganglion cells incubated with FGF-1/HSPG achieved an average neurite length of 323 microns while control cells gained an average neurite length of 203 microns. The results of this study are consistent with our previous findings in whole spiral ganglion explants (3) where FGF-1 incubation significantly stimulated neurite outgrowth at about the same range. However, stimulation of neurite outgrowth in dissociated spiral ganglion cells suggests that FGF-1 directly binds to FGF receptors on the surface of spiral ganglion neurons and/or neurites instead of acting via intermediate cells such as glia. Since FGF receptor mRNA was found to be expressed only at very low levels in neonatal spiral ganglion neurons (7) it is possible that the receptors are highly localized, perhaps to neurite growth cones. Alternatively, an unknown FGF receptor or splice variant may be expressed in these cells. Adequate FGF-1 application to the human inner ear may stimulate spiral ganglion cell survival and neurite extension after hair cell loss in patients suitable for cochlear implant treatment. By creating a closer contact between spiral ganglion cells and the electrode, FGF-1 might also improve the efficacy of cochlear implants.     

Histopathology of the ears, eyes, and brain in Norrie's disease (oculoacousticocerebral degeneration)

Norrie's disease is an x-linked recessive disorder characterized by progressive oculoacousticocerebral degeneration. The light and electron microscopic changes in the temporal bones, eyes, and brain of an affected 77-year-old man who suffered from bilateral profound sensorineural hearing loss, blindness, and mental retardation are described. The inner ears showed marked atrophy of the stria vascularis, severe degeneration of hair cells and cochlear neurons, and connective tissue proliferation in the spiral ganglion, osseous spiral lamina, and walls of the membranous vestibular labyrinth. The eyes showed detached retinae, dense proliferation of fibrillary glial cells in the retina and vitreous, severe atrophy of the optic nerves, and degenerative hyalinization of blood vessels. This case is the first published report of the histopathology of the inner ear in Norrie's disease.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.