Structural changes in the inner ear over time studied in the experimentally deafened guinea pig.

Today a cochlear implant (CI) may significantly restore auditory function, even for people with a profound hearing loss. Because the efficacy of a CI is believed to depend mainly on the remaining population of spiral ganglion neurons (SGNs), it is important to understand the timeline of the degenerative process of the auditory neurons following deafness. Guinea pigs were transtympanically deafened with neomycin, verified by recording auditory brainstem responses (ABRs), and then sacrificed at different time points. Loss of SGNs as well as changes in cell body and nuclear volume were estimated. To study the effect of delayed treatment, a group of animals that had been deaf for 12 weeks was implanted with a stimulus electrode mimicking a CI, after which they received a 4-week treatment with glial cell-derived neurotrophic factor (GDNF). The electrical responsiveness of the SGNs was measured by recording electrically evoked ABRs. There was a rapid degeneration during the first 7 weeks, shown as a significant reduction of the SGN population. The degenerative process then slowed, and there was no difference in the amount of remaining neurons between weeks 7 and 18. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Inactivation of Pmel alters melanosome shape but has only a subtle effect on visible pigmentation.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel⁻/⁻). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel⁻/⁻ melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

Membrane cholesterol modulates cochlear electromechanics.

Changing the concentration of cholesterol in the plasma membrane of isolated outer hair cells modulates electromotility and prestin-associated charge movement, suggesting that a similar manipulation would alter cochlear mechanics. We examined cochlear function before and after depletion of membrane cholesterol with methyl-ß-cyclodextrin (MßCD) in an excised guinea pig temporal bone preparation. The mechanical response of the cochlear partition to acoustic and/or electrical stimulation was monitored using laser interferometry and time-resolved confocal microscopy. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents. Exposure to MßCD increased the magnitude and asymmetry of the response, without changing the frequency tuning of sound-evoked mechanical responses or cochlear microphonic potentials. Sodium salicylate reversibly blocked the enhanced electromechanical response in cholesterol depleted preparations. The increase of sound-evoked vibrations during positive current injection was enhanced following MßCD in some preparations. Imaging was used to assess cellular integrity which remained unchanged after several hours of exposure to MßCD in several preparations. The enhanced electromechanical response reflects an increase in outer hair cell electromotility and may reveal features of cholesterol distribution and trafficking in outer hair cells.

Future risk for disability pension among people with sickness absence due to otoaudiological diagnoses: a population-based cohort study with a 12-year follow-up.

UNLABELLED: Hearing difficulties is a growing public health problem and more knowledge of consequences of those difficulties in working life is warranted. AIMS: To study the future risk of being granted a disability pension (DP) among people with sickness absence with an otoaudiological diagnoses (OAD) compared to other sickness absentees. METHODS: A population-based prospective cohort study of all 40,786 people in a Swedish county who in 1985 were aged 16-64 and had a new sick-leave spell >7 days. Those were followed for 12 years with regard to DP. Hazard ratios (HR) + 95% confidence intervals (CI) of being granted DP was calculated among those with sick leave due to OAD compared to people with sickness absence with other diagnoses. RESULTS: In 1985, 515 people had a new sick-leave spell with an OAD. Twelve years later, 36% of those had been granted DP, compared to 24% of all other sickness absentees. Their HR for DP was 1.42 (95% CI 1.23-1.64) adjusting for gender and age. Compared to men, women with an OAD had a HR of DP of 1.24 (95% CI 0.90-1.71), when adjusted for age. The HR for DP regarding those aged>45 years and sickness absent with OAD was 2.63 (95% CI 1.95-3.55) compared to the sickness absentees with OAD below 45 years of age, adjusted for gender. CONCLUSIONS: The risk for future DP was more than 40% higher among those initially on sickness absence due to OAD than among other sickness absentees.

Differential effects of salicylate, quinine, and furosemide on Guinea pig inner and outer hair cell function revealed by the input-output relation of the auditory brainstem response.

BACKGROUND: Sensory hearing loss is predominantly caused by the destruction of cochlear outer hair cells (OHCs), inner hair cells (IHCs), or spiral ganglion cells (SGCs). There have been a number of attempts to differentiate between these etiologies of hearing loss, using various psychoacoustic and physiologic paradigms. PURPOSE: Here we investigate the potential of the auditory brainstem response (ABR) input/output function for such differential diagnosis. On the basis of the saturation of the OHC-based cochlear amplifier, it was hypothesized that selective impairment of OHCs would reduce ABR amplitudes at low to moderate but not at high sound levels. Selective impairment of IHCs or SGCs would reduce ABR amplitudes more or less uniformly across sound level. Finally, a mix of OHC and IHC or SGC impairment would reduce ABR amplitudes at all sound levels but less so at high levels depending on the relative contribution of OHC impairment to the hearing loss. RESEARCH DESIGN: To test these hypotheses, normal-hearing adult guinea pigs were intravenously injected with either salicylate, furosemide, or quinine, under ketamine anesthesia. ABRs, as well as distortion-product otoacoustic emissions (DPOAEs), were measured as a function of the sound stimulus level before and after drug injection. RESULTS: Following salicylate injection, ABR amplitudes were reduced only at low-moderate stimulus levels. Following furosemide or quinine injection, ABR amplitudes were reduced at all levels but less so at high ones. This is in accord with the expectation that acute salicylate administration selectively affects the OHCs, while furosemide and quinine affect both OHCs and IHCs/SGCs. Such differential diagnosis was not possible solely on the basis of DPOAE amplitudes, which were unchanged at high stimulus levels after the injection of each of the three drugs. Comparison of ABR and DPOAE threshold shifts could also differentiate the effects of salicylate from those of furosemide and quinine but could not, for example, unequivocally point to salicylate's selective impairment of OHCs. CONCLUSIONS: ABR amplitudes appear suitable for differentiating between damage to OHCs and IHCs/SGCs, at least in a controlled experimental setting where pre- and postmanipulation data are available. This could be useful for noninvasively testing the effects of drugs or acoustic overstimulation on the cochlea, at least in the laboratory. Clinical applicability would seem to be limited by the high variability in ABR amplitudes among normal-hearing humans but might be feasible in the future if regular ABR testing entered into routine clinical practice.

Signaling through erbB receptors is a critical functional regulator in the mature cochlea.

Noise, ototoxic substances and various genetic factors are common causes of profound hearing loss. Cochlear implants can often restore hearing in these cases, but only if a sufficient number of responsive auditory nerve fibers remain. Over time, these nerve fibers degenerate in the damaged ear, and it is therefore important to establish factors that control neuronal survival and maintain neural excitability. Recent studies show that neuregulins and their receptors are important for survival and proper targeting of neurons in the developing inner ear. A role for neuregulins as maintainers of the neuronal population in the mature inner ear was therefore hypothesized. Here, this hypothesis was directly tested by chronic local application of substances that block neuregulin receptors. Using auditory brainstem response measurements, we demonstrate that such receptor block leads to a progressive hearing impairment that develops over the course of weeks. This impairment occurs despite a normal number of auditory neurons and preserved outer hair cell function. Real-time quantitative reverse transcriptase-polymerase chain reaction shows alterations in neurotrophin-3 expression, suggesting that this growth factor participates in regulating cochlear sensitivity. The present work demonstrates the critical importance of neuregulin/erbB signaling in long-term functional regulation in the mature guinea pig hearing organ.

In vivo infusion of UTP and uridine to the deafened guinea pig inner ear: effects on response thresholds and neural survival.

Nucleotides and nucleosides are known to function as neurotransmitters and neuromodulators but have recently been shown to have a trophic effect on neurons. It has previously been shown, in an animal model for cochlear implants, that local infusion of neurotrophic factors intervenes with the degenerative processes occurring after deafening and protects the auditory spiral ganglion neurons so that electrical responsiveness is maintained. Here we test the hypothesis that nucleosides and nucleotides have a similar effect on the acutely damaged inner ear. Pigmented guinea pigs received a cochlear implant electrode for measuring electrically evoked auditory brainstem responses and a miniosmotic pump for delivering drugs directly to the cochlea. The animals were deafened by a 48-hr infusion with 10% neomycin, followed by 23 days of treatment with primarily UTP, uridine nucleotides, or as control artificial perilymph. Electrically evoked responses were measured weekly, and at the end of the experiment the cochleae were collected and processed for morphological analysis and spiral ganglion neuron counting. Both UTP- and uridine-treated groups showed significantly better response after 23 days of treatment compared with the control group. The densities of spiral ganglion neuron were significantly higher for both treated groups compared with the control group treated with artificial perilymph. The results demonstrate that UTP and uridine rescue auditory neurons and suggest that drugs acting on purinoceptors could be of clinical importance.

Middle ear application of a sodium hyaluronate gel loaded with neomycin in a Guinea pig model.

OBJECTIVE: Establishing methods for topical administration of drugs to the inner ear have great clinical relevance and potential even in a relatively short perspective. To evaluate the efficacy of sodium hyaluronate (HYA) as a vehicle for drugs that could be used for treatment of inner ear disorders. METHODS: The cochlear hair cell loss and round window membrane (RWM) morphology were investigated after topical application of neomycin and HYA into the middle ear. Sixty-five albino guinea pigs were used and divided into groups depending on the type of the treatment. Neomycin was chosen as tracer for drug release and pharmacodynamic effect. HYA loaded with 3 different concentrations of neomycin was injected to the middle ear cavity of guinea pigs. Phalloidin stained surface preparations of the organ of Corti were used to estimate hair cell loss induced by neomycin. The thickness of the midportion of the RWM was measured and compared with that of controls using light and electron microscopy. All animal procedures were pe rformed in accordance with the ethical standards of Karolinska Institutet. RESULT: Neomycin induced a considerable hair cell loss in guinea pigs receiving a middle ear injection of HYA loaded with the drug, demonstrating that neomycin was released from the gel and delivered to the inner ear. The resulting hair cell loss showed a clear dose-dependence. Only small differences in hair cell loss were noted between animals receiving neomycin solution and animals exposed to neomycin in HYA suggesting that the vehicle neither facilitated nor hindered drug transport between the middle ear cavity and the inner ear. One week after topical application, the thickness of the RWM had increased and was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal. CONCLUSION: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss.

Spatiotemporal loss of K+ transport proteins in the developing cochlear lateral wall of guinea pigs with hereditary deafness.

Genetic deafness is one of the most common human genetic birth defects. To understand the molecular mechanisms underlying human hereditary deafness, deaf animal strains have proved to be invaluable models. The German waltzing guinea pig is a new strain of animals with unidentified gene mutation(s), displaying recessively inherited cochleovestibular impairment. Histological investigations of the homozygous animals (gw/gw) revealed a collapse of the endolymphatic compartment and malformation of stria vascularis. RT-PCR showed a significant reduction in expression of the strial intermediate cell-specific gene Dct and the tight-junction gene Cldn11 in the embryonic day (E)40 and adult gw/gw cochlear lateral wall. Immunohistochemical analysis of the gw/gw cochlea showed loss of the tight junction protein CLDN11 in strial basal cells from E40, loss of the potassium channel subunit KCNJ10 in strial intermediate cells from E50, and loss of the Na-K-Cl cotransporter SLC12A2 in strial marginal cells from E50. In addition, a temporary loss of the gap junction protein GJB2 (connexin 26) between fibrocytes in the spiral ligament of the E50 gw/gw cochlea was observed. The barrier composed of tight junctions between strial basal cells was disrupted in the gw/gw cochlea as indicated by a biotin tracer permeability assay. In conclusion, spatiotemporal loss of K+ transport proteins in the cochlear lateral wall is caused by malformation of the stria vascularis in the developing German waltzing guinea pig inner ear. This new animal strain may serve as a good model for studying human genetic deafness due to disruption of inner ear ion homeostasis.

Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cells.

The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology.

Activation of JNK in the inner ear following impulse noise exposure.

Noise exposure is known to induce cell death signaling in the cochlea. Since c-Jun N-terminal kinase (JNK) signaling is known to induce both cell survival and apoptosis, the present study focused on early changes (within 24 h) after impulse noise exposure, inquiring whether cell death is always related to phosphorylation of JNK in the inner ear. Anesthetized adult albino rats were exposed to a single impulse noise exposure (194 kPa) and sacrificed 3 or 24 h later. Paraffin-embedded sections were examined for positive staining of phosphorylated JNK and the presence of cells with fragmented DNA (TUNEL staining). Positive TUNEL staining was observed at the spiral limbus and in the stria vascularis at 24 h following impulse noise exposure, but no correlation with JNK activation was found at these locations. In the hearing organ (organ of Corti) and in the lateral wall, TUNEL-reactive cells were observed at 24 h following trauma. This was preceded by p-JNK staining at 3 h, indicating JNK-activated cell death in these regions. Finally, p-JNK reactivity was observed in the spiral ganglion with no correlation to TUNEL staining within the time frame of this study. These results suggest that JNK activation following impulse noise exposure may not always be related to cell death, and conversely, some cells may die through JNK-independent signaling.

Type I hair cell degeneration in the utricular macula of the waltzing guinea pig.

Waltzing guinea pigs are an inbred guinea pig strain with a congenital and progressive balance and hearing disorder. A unique rod-shaped structure is found in the type I vestibular hair cells, that traverses the cell in an axial direction, extending towards the basement membrane. The present study estimates the total number of utricular hair cells and supporting cells in waltzing guinea pigs and age-matched control animals using the optical fractionator method. Animals were divided into four age groups (1, 7, 49 and 343 day-old). The number of type I hair cells decreased by 20% in the 343 day-old waltzing guinea pigs compared to age-matched controls and younger animals. Two-photon confocal laser scanning microscopy using antibodies against fimbrin and betaIII-tubulin showed that the rods were exclusive to type I hair cells. There was no significant change in the length of the filament rods with age. Taken together, our data show that despite rod formation in the type I hair cells and deformation of hair bundles being present at birth, the type I hair cell population is not affected quantitatively until a year after birth.

Augmented ototoxic effect of cisplatin in heterozygotes of the German waltzing guinea pig.

It has previously been demonstrated that the carriers of the German waltzing guinea pig are less susceptible to noise trauma. To explore whether this represents a general resistance to inner ear trauma, carriers of the German waltzing guinea pig were exposed to the ototoxic agent cisplatin. Two doses of cisplatin were injected intravenously into anesthetized carriers and weight-matched control animals. Prior to and 96 h after the injections hearing thresholds were established by recording the auditory brainstem responses at 3.5, 7, 14, and 28 kHz. The cochleae were harvested to estimate hair cell loss and to analyze total platinum content. The carriers of the German waltzing guinea pig strain suffered from a more pronounced cisplatin-induced hearing loss compared to the control animals. The results suggest that mechanisms responsible for the protection against acoustic stress do not provide any protection against cisplatin in carriers of the German waltzing guinea pig.

The feasibility of an encapsulated cell approach in an animal deafness model.

For patients with profound hearing loss a cochlear implant (CI) is the only treatment today. The function of a CI depends in part of the function and survival of the remaining spiral ganglion neurons (SGN). It is well known from animal models that inner ear infusion of neurotrophic factors prevents SGN degeneration and maintains electrical responsiveness in deafened animals. The purpose with this study was to investigate the effects of a novel encapsulated cell (EC) device releasing neurotrophic factors in the deafened guinea pig. The results showed that an EC device releasing glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) implanted for four weeks in deafened guinea pigs significantly preserved the SGNs and maintained their electrical responsiveness. There was a significant difference between BDNF and GDNF in favour of GDNF. This study, demonstrating positive structural and functional effects in the deafened inner ear, suggests that an implanted EC device releasing biologically protective substances offers a feasible approach for treating progressive hearing impairment.

Image reversal for direct electron beam patterning of protein coated surfaces.

Electron beam lithography (EBL) is used to create surfaces with protein patterns, which are characterized by immunofluorescence and atomic force microscopies. Both negative and positive image processes are realized by electron beam irradiation of proteins absorbed on a silicon surface, where image reversal is achieved by selectively binding a second species of protein to the electron beam exposed areas on the first protein layer. Biofunctionality at the cellular level was established by culturing cortical cells on patterned lines of fibronectin adsorbed on a bovine serum albumin background for 7 days in culture.

Rapid confocal imaging for measuring sound-induced motion of the hearing organ in the apical region.

We describe a novel confocal image acquisition system capable of measuring the sound-evoked motion of the organ of Corti. The hearing organ is imaged with a standard laser scanning confocal microscope during sound stimulation. The exact temporal relation between each image pixel and the sound stimulus is quantified. The motion of the structures under study is obtained by fitting a Fourier series to the time dimension of a continuous sequence of acquired images. Previous versions of this acquisition system used a simple search to find pixels with similar phase values. The Fourier series approach permits substantially faster image acquisition with reduced noise levels and improved motion estimation. The system is validated by imaging various vibrating samples attached to a feedback-controlled piezoelectric translator. When using a rigid sample attached to the translator, the system is capable of measuring motion with peak-to-peak amplitudes smaller than 50 nm with an error below 20% at frequencies between 50 and 600 Hz. Examples of image sequences from the inner ear are given, along with detailed performance characteristics of the method.

A cell therapy approach to substitute neural elements in the inner ear.

Three different donor tissues were tested for their capacity to survive, integrate and differentiate in the adult inner ear. Surviving embryonic dorsal root ganglion cells were found within the spiral ganglion neuron region and along the auditory nerve fibers. In the presence of exogenous nerve growth factor (NGF), the dorsal root ganglion cells formed extensive growth of neurites that seemed to contact the host neurons. Adult neural stem cells survived relative poorly in the inner ear whereas embryonic stem cells showed a somewhat greater capacity for survival and integration. Overall, the survival rate of implanted tissue was quite low in the cochlea. It is concluded that an inner ear cell therapy approach based on the implantation of exogenous cells will require that important survival factors are identified and supplied. In addition, it is possible that the physical properties of the cochlea, e.g., fluid-filled compartments and very limited space for cell proliferation, are unfavorable, at least in the normal cochlea.

Exploring efferent-mediated DPOAE adaptation in three different guinea pig strains.

The aims of this study were to explore the correlation between DPOAE adaptation magnitude in three different guinea pig strains to examine if the genetic component affects the DPOAE adaptation magnitude. It was also to investigate the correlation between strains with certain characteristics i.e. reduced susceptibility to noise, and early onset of age-dependent hearing loss and the DPOAE adaptation magnitude. The animals were anaesthetized and the 2f1-f2 DPOAE (f1=8k Hz, and f2/f1=1.2) adaptation was established with a minimum of 144 combinations of f1; f2 where f1 was held fixed and f2 was varied in 1 dB or 0.4 dB steps. The DPOAE adaptation magnitude was defined as the difference between maximum positive level and the maximum negative level. ABRs were conducted at different age-groups (at 4, 6.3, and 12.5k Hz) to evaluate the progress of hearing thresholds by age. There was a significant difference between strains regarding the hearing loss at one year of age. There was no significant difference in DPOAE adaptation magnitude between strains included in this study and from this we conclude that the DPOAE adaptation magnitude is not a predictor for the susceptibility to noise trauma, or early onset of age-dependent hearing loss, using the methods described in this paper.

Cell replacement therapy in the inner ear.

The mammalian inner ear is vulnerable to genetic disorders and aging, as well as to injuries caused by overstimulation, ototoxic drugs, and viral infections. Due to the poor regeneration of the sensory epithelium and the spiral ganglion neurons in the adult mammalian inner ear, cell replacement therapy strategies have been proposed to compensate for degeneration and loss of sensory and neuronal cells. Transplantation of stem cells and embryonic neurons into the inner ear has revealed that exogenous cells can survive, migrate, differentiate, and extend neuritic projections in the auditory system of adult mammals. These results suggest that cell replacement therapy could provide an effective future treatment alternative for hearing loss and other inner ear disorders.