Temporary Neurotrophin Treatment Prevents Deafness-Induced Auditory Nerve Degeneration and Preserves Function.

After substantial loss of cochlear hair cells, exogenous neurotrophins prevent degeneration of the auditory nerve. Because cochlear implantation, the current therapy for profound sensorineural hearing loss, depends on a functional nerve, application of neurotrophins is being investigated. We addressed two questions important for fundamental insight into the effects of exogenous neurotrophins on a degenerating neural system, and for translation to the clinic. First, does temporary treatment with brain-derived neurotrophic factor (BDNF) prevent nerve degeneration on the long term? Second, how does a BDNF-treated nerve respond to electrical stimulation? Deafened guinea pigs received a cochlear implant, and their cochleas were infused with BDNF for 4 weeks. Up to 8 weeks after treatment, their cochleas were analyzed histologically. Electrically evoked compound action potentials (eCAPs) were recorded using stimulation paradigms that are informative of neural survival. Spiral ganglion cell (SGC) degeneration was prevented during BDNF treatment, resulting in 1.9 times more SGCs than in deafened untreated cochleas. Importantly, SGC survival was almost complete 8 weeks after treatment cessation, when 2.6 times more SGCs were observed. In four eCAP characteristics (three involving alteration of the interphase gap of the biphasic current pulse and one involving pulse trains), we found large and statistically significant differences between normal-hearing and deaf controls. Importantly, for BDNF-treated animals, these eCAP characteristics were near normal, suggesting healthy responsiveness of BDNF-treated SGCs. In conclusion, clinically practicable short-term neurotrophin treatment is sufficient for long-term survival of SGCs, and it can restore or preserve SGC function well beyond the treatment period. Significance statement: Successful restoration of hearing in deaf subjects by means of a cochlear implant requires a healthy spiral ganglion cell population. Deafness-induced degeneration of these cells can be averted with neurotrophic factors. In the present study in deafened guinea pigs, we investigated the long-term effects of temporary (i.e., clinically practicable) treatment with brain-derived neurotrophic factor (BDNF). We show that, after treatment cessation, the neuroprotective effect remains for at least 8 weeks. Moreover, for the first time, it is shown that the electrical responsiveness of BDNF-treated spiral ganglion cells is preserved during this period as well. These findings demonstrate that treatment of the auditory nerve with neurotrophic factors may be relevant for cochlear implant users.

Assessing the Firing Properties of the Electrically Stimulated Auditory Nerve Using a Convolution Model.

The electrically evoked compound action potential (eCAP) is a routinely performed measure of the auditory nerve in cochlear implant users. Using a convolution model of the eCAP, additional information about the neural firing properties can be obtained, which may provide relevant information about the health of the auditory nerve. In this study, guinea pigs with various degrees of nerve degeneration were used to directly relate firing properties to nerve histology. The same convolution model was applied on human eCAPs to examine similarities and ultimately to examine its clinical applicability. For most eCAPs, the estimated nerve firing probability was bimodal and could be parameterised by two Gaussian distributions with an average latency difference of 0.4 ms. The ratio of the scaling factors of the late and early component increased with neural degeneration in the guinea pig. This ratio decreased with stimulation intensity in humans. The latency of the early component decreased with neural degeneration in the guinea pig. Indirectly, this was observed in humans as well, assuming that the cochlear base exhibits more neural degeneration than the apex. Differences between guinea pigs and humans were observed, among other parameters, in the width of the early component: very robust in guinea pig, and dependent on stimulation intensity and cochlear region in humans. We conclude that the deconvolution of the eCAP is a valuable addition to existing analyses, in particular as it reveals two separate firing components in the auditory nerve.

Towards Clinical Application of Neurotrophic Factors to the Auditory Nerve; Assessment of Safety and Efficacy by a Systematic Review of Neurotrophic Treatments in Humans.

Animal studies have evidenced protection of the auditory nerve by exogenous neurotrophic factors. In order to assess clinical applicability of neurotrophic treatment of the auditory nerve, the safety and efficacy of neurotrophic therapies in various human disorders were systematically reviewed. Outcomes of our literature search included disorder, neurotrophic factor, administration route, therapeutic outcome, and adverse event. From 2103 articles retrieved, 20 randomized controlled trials including 3974 patients were selected. Amyotrophic lateral sclerosis (53%) was the most frequently reported indication for neurotrophic therapy followed by diabetic polyneuropathy (28%). Ciliary neurotrophic factor (50%), nerve growth factor (24%) and insulin-like growth factor (21%) were most often used. Injection site reaction was a frequently occurring adverse event (61%) followed by asthenia (24%) and gastrointestinal disturbances (20%). Eighteen out of 20 trials deemed neurotrophic therapy to be safe, and six out of 17 studies concluded the neurotrophic therapy to be effective. Positive outcomes were generally small or contradicted by other studies. Most non-neurodegenerative diseases treated by targeted deliveries of neurotrophic factors were considered safe and effective. Hence, since local delivery to the cochlea is feasible, translation from animal studies to human trials in treating auditory nerve degeneration seems promising.

Does vestibular end-organ function recover after gentamicin-induced trauma in Guinea pigs?

Until 1993 it was commonly accepted that regeneration of vestibular hair cells was not possible in mammals. Two histological studies then showed structural evidence for spontaneous regeneration of vestibular hair cells after gentamicin treatment. There is less evidence for functional recovery going along with this regenerative process; in other words, do regenerated hair cells function adequately? This study aims to address this question, and in general evaluates whether spontaneous functional recovery may occur, in the short or long term, in mammals after ototoxic insult. Guinea pigs were treated with gentamicin for 10 consecutive days at a daily dose of 125 mg/kg body weight. Survival times varied from 1 day to 16 weeks. Vestibular short-latency evoked potentials (VsEPs) to linear acceleration pulses were recorded longitudinally to assess otolith function. After the final functional measurements we performed immunofluorescence histology for hair cell counts. Auditory brainstem responses (ABRs) to click stimuli were recorded to assess cochlear function. As intended, gentamicin treatment resulted in significant loss of utricular hair cells and accompanying declines in VsEPs. Hair cell counts 8 or 16 weeks after treatment did not significantly differ from counts after shorter survival periods. Maximal functional loss was achieved 1-4 weeks after treatment. After this period, only 2 animals showed recovery of VsEP amplitude - all other animals did not reveal signs of regeneration or recovery. In contrast, after initial ABR threshold shifts there was a small but significant recovery. We conclude that spontaneous recovery of otolith function, in contrast to cochlear function, is very limited in guinea pigs. These results support the concept of intratympanic gentamicin treatment where gentamicin is used for chemoablation of the vestibular sensory epithelia.

Effect of KTP laser cochleostomy on morphology in the guinea pig inner ear.

BACKGROUND: The main advantage of using the KTP (potassium-titanyl-phosphate) laser for stapedotomy instead of the conventional micropick instrument is the smaller risk for mechanical damage. However, the KTP laser could theoretically inflict damage to inner ear structures. We hypothesize that KTP laser light [wavelength (λ) = 532 nm] is hardly absorbed in perilymph but well absorbed in solid structures. The aim of this pilot study was to assess if damage occurred after KTP laser cochleostomy in an animal model and, if so, to what extent and at which settings. MATERIALS AND METHODS: In six guinea pigs, a KTP laser cochleostomy at the basal turn was created. Laser settings of 1, 3 and 5 W and 100 ms pulse time (n = 2 each) were used. Histological preparations were studied for damage to neuroendothelial cells and intrascalar blood. RESULTS: No damage to inner ear neuroendothelial cells was observed, even at the highest power. Blood clots in the scala tympani from vessels in the cochlear wall were seen. The effects were minimal in the lowest, currently clinically used settings. CONCLUSION: KTP laser cochleostomy gives no damage to inner ear neuroendothelial cells but may cause intrascalar hemorrhages.

Ocular vestibular evoked myogenic potentials: frequency tuning to air-conducted acoustic stimuli in healthy subjects and Ménière's disease.

Ocular vestibular evoked myogenic potentials (oVEMP) in response to 250-, 500- and 1000-Hz air-conducted short tone bursts were studied in 22 healthy subjects and 37 Ménière's disease patients. The goal of this study was to investigate normal tuning characteristics of the oVEMP and the possible oVEMP changes with respect to frequency dependence in Ménière's disease. In unilateral Ménière's disease patients, a distinction was made between affected ears and unaffected ears. It was found that in normal subjects, the oVEMP tunes to a stimulus frequency of 500 Hz, with the highest amplitude and lowest threshold at this particular frequency. Generally, Ménière's disease patients showed lower amplitudes and higher thresholds than normal subjects at all 3 stimulus frequencies in both the affected and the unaffected ear. Additionally, for ears affected by Ménière's disease, the best stimulus frequency was 1000 Hz. With the use of this altered tuning for these ears, we tried to find a criterion for distinguishing normal from Ménière's disease ears.

Combined administration of kanamycin and furosemide does not result in loss of vestibular function in Guinea pigs.

Aminoglycoside antibiotics are known to damage the vestibular and auditory sensory epithelia. Although loop diuretics enhance the cochleotoxic effect of aminoglycosides, it is not known whether concomitant administration of an aminoglycoside and a loop diuretic affects the vestibular system. The aim of our study was to investigate the effect of co-administration of kanamycin and furosemide upon the otolith organs and to compare it to the known vestibulotoxic effect of gentamicin. Five guinea pigs were injected with a single dose of both kanamycin (400 mg/kg, s.c.) and furosemide (100 mg/kg, i.v.), 5 animals received gentamicin (100 mg/kg, i.p.) for 10 days, and 5 untreated animals served as controls. After 7 days, vestibular function was assessed by measuring vestibular short-latency evoked potentials (VsEPs) to linear acceleration stimuli and cochlear function by auditory brainstem responses (ABRs) to clicks. Hair cell densities were determined in phalloidin-stained whole mounts of the utricles and saccules, and in midmodiolar sections of resin-embedded cochleae. Co-administration of kanamycin and furosemide had no significant effect on VsEPs and hair cell densities in the utricles and saccules were not reduced. ABR thresholds were increased to a great extent (by ∼60 dB), and histologically a severe loss of cochlear hair cells was observed. The effect of gentamicin, both on vestibular and cochlear function, was just the opposite. VsEP thresholds to horizontal stimulation were elevated and suprathreshold amplitudes showed a decrease, whereas cochlear function was not reduced. With this protocol, we have a tool to selectively induce cochlear or vestibular damage, which may be of interest to researchers and clinicians alike.

Changes in the Electrically Evoked Compound Action Potential over time After Implantation and Subsequent Deafening in Guinea Pigs.

The electrically evoked compound action potential (eCAP) is a direct measure of the responsiveness of the auditory nerve to electrical stimulation from a cochlear implant (CI). CIs offer a unique opportunity to study the auditory nerve's electrophysiological behavior in individual human subjects over time. In order to understand exactly how the eCAP relates to the condition of the auditory nerve, it is crucial to compare changes in the eCAP over time in a controlled model of deafness-induced auditory nerve degeneration. In the present study, 10 normal-hearing young adult guinea pigs were implanted and deafened 4 weeks later, so that the effect of deafening could be monitored within-subject over time. Following implantation, but before deafening, most examined eCAP characteristics significantly changed, suggesting increasing excitation efficacy (e.g., higher maximum amplitude, lower threshold, shorter latency). Conversely, inter-phase gap (IPG) effects on these measures - within-subject difference measures that have been shown to correlate well with auditory nerve survival - did not vary for most eCAP characteristics. After deafening, we observed an initial increase in excitability (steeper slope of the eCAP amplitude growth function (AGF), lower threshold, shorter latency and peak width) which typically returned to normal-hearing levels within a week, after which a slower process, probably reflecting spiral ganglion cell loss, took place over the remaining 6 weeks (e.g., decrease in maximum amplitude, AGF slope, peak area, and IPG effect for AGF slope; increase in IPG effect for latency). Our results suggest that gradual changes in peak width and latency reflect the rate of neural degeneration, while peak area, maximum amplitude, and AGF slope reflect neural population size, which may be valuable for clinical diagnostics.

BDNF-mediated preservation of spiral ganglion cell peripheral processes and axons in comparison to that of their cell bodies.

Treatment with neurotrophins prevents degeneration of spiral ganglion cells (SGCs) after severe hair cell loss. In a previous study we demonstrated a long-lasting effect with brain-derived neurotrophic factor (BDNF) after cessation of treatment. In that study the survival of the SGC cell bodies was examined. Here we address the question whether their peripheral processes and central processes (axons) were protected by this treatment as well in the cochleas of the aforementioned study. Guinea pigs were deafened by co-administration of kanamycin and furosemide. Two weeks after deafening the right cochleas were implanted with an intracochlear electrode array combined with a cannula connected to an osmotic pump filled with BDNF solution. Four weeks later the treatment was stopped by surgically removing the osmotic pump. At that point, or another four or eight weeks later, the animals were sacrificed for histological analysis. Control groups consisted of normal-hearing animals, and three groups of deafened animals: two-weeks-deaf untreated animals, and six- and fourteen-weeks-deaf sham-treated animals. Cochleas were processed for analysis of: (1) the myelinated portion of peripheral processes in the osseous spiral lamina, (2) the cell bodies in Rosenthal's canal, and (3) axons in the internal acoustic meatus. Packing densities and cross-sectional areas were determined using light microscopy. Up to eight weeks after treatment cessation the numbers of peripheral processes and axons were significantly higher than in untreated cochleas of control animals. Whereas the numbers of cell bodies and axons were similar to those at the start of treatment, the peripheral processes were significantly less well preserved. This smaller protective effect was found mainly in the apical turns. Strategies to prevent SGC degeneration after hair cell loss should consider the differential effects on the various neural elements.

Simultaneous rather than retrograde spiral ganglion cell degeneration following ototoxically induced hair cell loss in the guinea pig cochlea.

Severe damage to the organ of Corti leads to degeneration of the spiral ganglion cells (SGCs) which form the auditory nerve. This degeneration starts at the level of synaptic connection of the peripheral processes (PPs) of SGCs with the cochlear hair cells. It is generally thought that from this point SGC degeneration progresses in a retrograde fashion: PPs degenerate first, followed by the SGC soma with a delay of several weeks to many months. Evidence for this course of events, both in animals and in humans, is not unambiguous, while this knowledge is important since the presence or absence of the different neural elements may greatly influence the response to electrical stimulation with a cochlear implant (CI). We therefore aimed to provide a comprehensive account of the course of SGC degeneration in the guinea pig cochlea after ototoxic treatment. Histological analysis of eighteen healthy and thirty-three deafened cochleas showed that the degeneration of SGCs and their peripheral processes was simultaneous rather than sequential. As the site of excitation for electrical stimulation with a CI may depend on the course of degeneration of the various neural elements, this finding is relevant both for understanding the electrophysiological mechanisms behind cochlear implantation and for recent efforts to induce PP resprouting for improved electrode-neural interface. Since excitation of the PPs is often thought to result in (secondary) longer-latency activity, we tested the hypothesis that having relatively many PPs produces a larger N2 peak in the electrically evoked compound action potential (eCAP); the present findings however do not support this theory. The course of the degeneration process may vary among species, and may depend on the cause of deafness, but the present findings at least indicate that gradual retrograde degeneration of the auditory nerve is not an elemental process following severe damage to the organ of Corti.

Morphological changes in spiral ganglion cells after intracochlear application of brain-derived neurotrophic factor in deafened guinea pigs.

When guinea pigs are deafened with ototoxic drugs spiral ganglion cells (SGCs) degenerate progressively. Application of neurotrophins can prevent this process. Morphological changes of rescued SGCs have not been quantitatively determined yet. It might be that SGCs treated with neurotrophins are more vulnerable than SGCs in cochleae of normal-hearing guinea pigs. Therefore, the mitochondria and myelinisation of type-I SGCs were studied and the perikaryal area, cell circularity and electron density were determined. Guinea pigs were deafened with a subcutaneous injection of kanamycin followed by intravenous infusion of furosemide. Brain-derived neurotrophic factor (BDNF) delivery was started two weeks after the deafening procedure and continued for four weeks. Four cohorts of cochleae were studied: (1) cochleae of normal-hearing guinea pigs; (2) of guinea pigs two weeks after deafening; (3) six weeks after deafening; (4) cochleae treated with BDNF after deafening. The deafening procedure resulted in a progressive loss of SGCs. Six weeks after deafening the size of mitochondria, perikaryal area and cell circularity of the remaining untreated SGCs were decreased and the number of layers of the myelin sheath was reduced. In the basal part of the cochlea BDNF treatment rescued SGCs from degeneration. SGCs treated with BDNF were larger than SGCs in normal-hearing guinea pigs, whereas circularity had normal values and electron density was unchanged. The number of layers in the myelin sheath of BDNF-treated SGCs was reduced as compared to the number of layers in the myelin sheath of SGCs in normal-hearing guinea pigs. The morphological changes of SGCs might be related to the rapid loss of SGCs that has been reported to occur after cessation of BDNF treatment.

Quality of reporting of otorhinolaryngology articles using animal models with the ARRIVE statement.

Research involving animal models is crucial for the advancement of science, provided that experiments are designed, performed, interpreted, and reported well. In order to investigate the quality of reporting of articles in otorhinolaryngology research using animal models, a PubMed database search was conducted to retrieve eligible articles. The checklist of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines was used to assess the quality of reporting of articles published in ear, nose and throat (ENT) and multidisciplinary journals. Two authors screened titles, abstracts, and full texts to select articles reporting otorhinolaryngology research using in vivo animal models. ENT journals ( n = 35) reported a mean of 57.1% adequately scored ARRIVE items (median: 58.3%; 95% confidence interval [CI; 53.4-60.9%]), while articles published in multidisciplinary journals ( n = 36) reported a mean of 49.1% adequately scored items (median: 50.0; 95% CI [46.2-52.0%]). Articles published in ENT journals showed better quality of reporting of animal studies based on the ARRIVE guidelines ( P < 0.05). However, adherence to the ARRIVE guidelines is generally poor in otorhinolaryngology research using in vivo animal models. The endorsement of the ARRIVE guidelines by authors, research and academic institutes, editorial offices and funding agencies is recommended for improved reporting of scientific research using animal models.

Ouabain Does Not Induce Selective Spiral Ganglion Cell Degeneration in Guinea Pigs.

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea.

Time course of cochlear electrophysiology and morphology after combined administration of kanamycin and furosemide.

In animal models of deafness, administration of an aminoglycoside in combination with a loop diuretic is often applied to produce a rapid loss of cochlear hair cells. However, the extent to which surviving hair cells remain functional after such a deafening procedure varies. In a longitudinal electrocochleographical study, we investigated the variability of cochlear function between and within guinea pigs after combined administration of kanamycin and furosemide. Concurrently, histological data were obtained at 1, 2, 4 and 8 weeks after deafening treatment. The main measures in our study were compound action potential (CAP) thresholds, percentage of surviving hair cells and packing density of spiral ganglion cells (SGCs). One day after deafening treatment, we found threshold shifts widely varying among animals from 0 to 100dB. The variability decreased after 2 days, and in 18 out of 20 animals threshold shifts greater than 55dB were found 4-7 days after deafening. Remarkably, in the majority of animals, thresholds decreased by up to 25dB after 7 days indicating functional recovery. As expected, final thresholds were negatively correlated to the percentage of surviving hair cells. Notably, the percentage of surviving hair cells might be predicted on the basis of thresholds observed one day after deafening. SGC packing density, which rapidly decreased with the period after deafening treatment and correlated to the percentage of surviving inner hair cells, was not a determining factor for the CAP thresholds.

Systematic review of compound action potentials as predictors for cochlear implant performance.

OBJECTIVES/HYPOTHESIS: The variability in speech perception between cochlear implant users is thought to result from the degeneration of the auditory nerve. Degeneration of the auditory nerve, histologically assessed, correlates with electrophysiologically acquired measures, such as electrically evoked compound action potentials (eCAPs) in experimental animals. To predict degeneration of the auditory nerve in humans, where histology is impossible, this paper reviews the correlation between speech perception and eCAP recordings in cochlear implant patients. DATA SOURCES: PubMed and Embase. REVIEW METHODS: We performed a systematic search for articles containing the following major themes: cochlear implants, evoked potentials, and speech perception. Two investigators independently conducted title-abstract screening, full-text screening, and critical appraisal. Data were extracted from the remaining articles. RESULTS: Twenty-five of 1,429 identified articles described a correlation between speech perception and eCAP attributes. Due to study heterogeneity, a meta-analysis was not feasible, and studies were descriptively analyzed. Several studies investigating presence of the eCAP, recovery time constant, slope of the amplitude growth function, and spatial selectivity showed significant correlations with speech perception. In contrast, neural adaptation, eCAP threshold, and change with varying interphase gap did not significantly correlate with speech perception in any of the identified studies. CONCLUSIONS: Significant correlations between speech perception and parameters obtained through eCAP recordings have been documented in literature; however, reporting was ambiguous. There is insufficient evidence for eCAPs as a predictive factor for speech perception. More research is needed to further investigate this relation. Laryngoscope, 2016 127:476-487, 2017.

Degeneration of auditory nerve fibers in guinea pigs with severe sensorineural hearing loss.

Damage to and loss of the organ of Corti leads to secondary degeneration of the spiral ganglion cell (SGC) somata of the auditory nerve. Extensively examined in animal models, this degeneration process of SGC somata following deafening is well known. However, degeneration of auditory nerve axons, which conduct auditory information towards the brainstem, and its relation to SGC soma degeneration are largely unknown. The consequences of degeneration of the axons are relevant for cochlear implantation, which is applied to a deafened system but depends on the condition of the auditory nerve. We investigated the time sequence of degeneration of myelinated type I axons in deafened guinea pigs. Auditory nerves in six normal-hearing and twelve deafened animals, two, six and fourteen weeks (for each group four) after deafening were histologically analyzed. We developed a semi-automated method for axon counting, which allowed for a relatively large sample size (20% of the total cross-sectional area of the auditory nerve). We observed a substantial loss of auditory nerve area (29%), reduction in axon number (59%) and decrease in axoplasm area (41%) fourteen weeks after deafening compared to normal-hearing controls. The correlation between axonal degeneration and that of the SGC somata in the same cochleas was high, although axonal structures appeared to persist longer than the somata, suggesting a slower degeneration process. In the first two weeks after induction of deafness, the axonal cross-sectional area decreased but the axon number did not. In conclusion, the data strongly suggest that each surviving SGC possesses an axon.

Scar formation in mice deafened with kanamycin and furosemide.

In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium-thiocarbohydrazide-osmium (OTO), tannic acid-arginine-osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766-772, 2016. © 2016 Wiley Periodicals, Inc.

Acceleration of cisplatin ototoxicity by perilymphatic application of 4-methylthiobenzoic acid.

The antitumor agent cisplatin has dose-limiting side effects such as ototoxicity. Systemical co-treatment with anti-oxidants like 4-methylthiobenzoic acid (MTBA) and sodium thiosulfate (STS) provides protection against cisplatin ototoxicity. However, systemically administered protective agents may reduce the chemotherapeutic effect of cisplatin. Local application of the protective agents could avoid this undesirable effect. In the present study, we aimed at suppressing cisplatin-induced ototoxicity in guinea pigs by administering MTBA or STS perilymphatically through cochlear perfusion. Guinea pig cochleas were perfused for 10 min with artificial perilymph (ArtP) containing cisplatin at 0.3 mg/ml, either alone, or in combination with MTBA (0.1 or 1.0 mg/ml) or STS (0.75 or 3.0 mg/ml). The compound action potential (CAP) and the summating potential (SP), evoked by 8 kHz tone bursts, and the endocochlear potential (EP; MTBA only) were measured just before and 1, 2, 3 and 4 h after perfusion. Cisplatin gradually reduced the CAP amplitude in time. Adding MTBA only accelerated this ototoxic effect. After cisplatin treatment a decline was found in the EP, irrespective of co-treatment, i.e., addition of MTBA did not accelerate the EP decrease. In contrast to MTBA, STS ameliorated the ototoxic effect of cisplatin. In conclusion, local application of anti-oxidants can ameliorate cisplatin ototoxicity but this is not a feature of all anti-oxidants.

The cochlear targets of cisplatin: an electrophysiological and morphological time-sequence study.

Cisplatin ototoxicity has at least three major targets in the cochlea: the stria vascularis, the organ of Corti, and the spiral ganglion. This study aims to differentiate between these three targets. In particular, we address the question of whether the effects at the level of the organ of Corti and spiral ganglion are mutually dependent or whether they develop in parallel. This question was approached by studying the ototoxic effects while they develop electrophysiologically and comparing these to earlier presented histological data [Van Ruijven et al., 2004. Hear. Res. 197, 44-54]. Guinea pigs were treated with intraperitoneal injections of cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. This time sequence has not revealed any evidence of one ototoxic process triggering another. Therefore, we have to stay with the conclusion of Van Ruijven et al. (2004) that both processes run in parallel.

The Wnt and Notch signalling pathways in the developing cochlea: Formation of hair cells and induction of regenerative potential.

The Wnt and Notch signalling pathways control proliferation, specification, and cell fate choices during embryonic development and in adult life. Hence, there is much interest in both signalling pathways in the context of stem cell biology and tissue regeneration. In the developing ear, the Wnt and Notch signalling pathways specify otic cells and refine the ventral boundary of the otic placode. Since both signalling pathways control events essential for the formation of sensory cells, such as proliferation and hair cell differentiation, these pathways could hold promise for the regeneration of hair cells in adult mammalian cochlea. Indeed, modulating either the Wnt or Notch pathways can trigger the regenerative potential of supporting cells. In the neonatal mouse cochlea, Notch-mediated regeneration of hair cells partially depends on Wnt signalling, which implies an interaction between the pathways. This review presents how the Wnt and Notch signalling pathways regulate the formation of sensory hair cells and how modulating their activity induces regenerative potential in the mammalian cochlea.