C-Phycocyanin Attenuates Noise-Induced Cochlear Synaptopathy via the Inhibition of Oxidative Stress and Intercellular Adhesion Molecule-1 in the Cochlea.

The synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are the most vulnerable structures in the noise-exposed cochlea. Cochlear synaptopathy results from the disruption of these synapses following noise exposure and is considered the main cause of poor speech understanding in noisy environments, even when audiogram results are normal. Cochlear synaptopathy leads to the degeneration of SGNs if damaged IHC-SGN synapses are not promptly recovered. Oxidative stress plays a central role in the pathogenesis of cochlear synaptopathy. C-Phycocyanin (C-PC) has antioxidant and anti-inflammatory activities and is widely utilized in the food and drug industry. However, the effect of the C-PC on noise-induced cochlear damage is unknown. We first investigated the therapeutic effect of C-PC on noise-induced cochlear synaptopathy. In vitro experiments revealed that C-PC reduced the H2O2-induced generation of reactive oxygen species in HEI-OC1 auditory cells. H2O2-induced cytotoxicity in HEI-OC1 cells was reduced with C-PC treatment. After white noise exposure for 3 h at a sound pressure of 118 dB, the guinea pigs intratympanically administered 5 µg/mL C-PC exhibited greater wave I amplitudes in the auditory brainstem response, more IHC synaptic ribbons and more IHC-SGN synapses according to microscopic analysis than the saline-treated guinea pigs. Furthermore, the group treated with C-PC had less intense 4-hydroxynonenal and intercellular adhesion molecule-1 staining in the cochlea compared with the saline group. Our results suggest that C-PC improves cochlear synaptopathy by inhibiting noise-induced oxidative stress and the inflammatory response in the cochlea.

Dynamic Heterogeneity Shapes Patterns of Spiral Ganglion Activity.

Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.

Salicylate Induced GABAAR Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons.

BACKGROUND Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABAARs). Our previous studies have shown that SS can increase the internalization of GABAARs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABAARs. MATERIAL AND METHODS The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABAARs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABAARs. Meanwhile, the phosphorylation level of GABAARγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABAARs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS D1Rs mediate the GABAAR internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.

Gap Junction-Mediated Intercellular Communication of cAMP Prevents CDDP-Induced Ototoxicity via cAMP/PKA/CREB Pathway.

In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.

Anti-PD-1 Therapy Does Not Influence Hearing Ability in the Most Sensitive Frequency Range, but Mitigates Outer Hair Cell Loss in the Basal Cochlear Region.

The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 µg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4-32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4-32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea.

Paeoniflorin protects spiral ganglion neurons from cisplatin-induced ototoxicity: Possible relation to PINK1/BAD pathway.

The objective of this study was to elucidate whether paeoniflorin (PF) exerted an effect on cisplatin-induced spiral ganglion neuron (SGN) damage, with special attention given to the role of PINK1/BAD pathway in this process. Middle cochlear turn culture and C57BL/6 mice were utilized to identify the character of PF in vitro and in vivo. We found that cisplatin treatment led to SGN damage, in which reactive oxygen species (ROS) generation increased, PINK1 expression decreased, BAD accumulation on mitochondria raised and mitochondrial apoptotic pathway activated. Conversely, we demonstrated that PF pre-treatment obviously mitigated cisplatin-induced SGN damage. Mechanistic studies showed that PF could reduce ROS levels, increase PINK1 expression, decrease the BAD accumulation on mitochondria and, thus, alleviate the activated mitochondrial apoptosis in SGNs caused by cisplatin. Overall, the findings from this work reveal the important role of PF and provide another strategy against cisplatin-induced ototoxicity.

Metformin mediates neuroprotection and attenuates hearing loss in experimental pneumococcal meningitis.

BACKGROUND: Pneumococcal meningitis is associated with high risk of neurological sequelae such as cognitive impairment and hearing loss. These sequelae are due to parenchymal brain and inner ear damage primarily induced by the excessive inflammatory reaction in response to bacterial brain invasion. Metformin-a biguanide drug to treat diabetes mellitus type 2-was recently found to suppress neuroinflammation and induce neuroregeneration. This study evaluated the effect of metformin adjunctive to antibiotics on neuroinflammation, brain and inner ear damage, and neurofunctional outcome in experimental pediatric pneumococcal meningitis. METHODS: Eleven-day-old Wistar rats were infected intracisternally with 5.22 ± 1.27 × 103 CFU Streptococcus pneumoniae and randomized for treatment with metformin (50 mg/kg, i.p., once daily for 3 weeks) plus ceftriaxone (100 mg/kg, i.p., bid, n = 61) or ceftriaxone monotherapy (n = 79). Cortical damage and hippocampal apoptosis were evaluated histomorphometrically 42 h post infection. Cerebrospinal fluid cytokine levels were analyzed during acute infection. Five weeks post infection, auditory brainstem responses were measured to determine hearing thresholds. Spiral ganglion neuron density and abundance of recently proliferated and integrated hippocampal granule neurons were assessed histologically. Additionally, the anti-inflammatory effect of metformin was studied in primary rat astroglial cells in vitro. RESULTS: Upon pneumococcal infection, metformin treatment significantly reduced levels of inflammatory cytokines and nitric oxide production in cerebrospinal fluid and in astroglial cell cultures in vitro (p < 0.05). Compared to animals receiving ceftriaxone monotherapy, adjunctive metformin significantly reduced cortical necrosis (p < 0.02) during acute infection and improved median click-induced hearing thresholds (60 dB vs. 100 dB, p < 0.002) 5 weeks after infection. Adjuvant metformin significantly improved pure tone hearing thresholds at all assessed frequencies compared to ceftriaxone monotherapy (p < 0.05) and protected from PM-induced spiral ganglion neuron loss in the inner ear (p < 0.05). CONCLUSION: Adjuvant metformin reduces brain injury during pneumococcal meningitis by decreasing the excessive neuroinflammatory response. Furthermore, it protects spiral ganglion neurons in the inner ear and improves hearing impairments after experimental pneumococcal meningitis. These results identify adjuvant metformin as a promising therapeutic option to improve the outcome after pediatric pneumococcal meningitis.

Neurotoxicity of sodium salicylate to the spiral ganglion neurons: GABAA receptor regulates NMDA receptor by Fyn-dependent phosphorylation.

The purpose of this study was to observe the regulatory effects of GABAA (γ-aminobutyric acid A) receptor on the N-methyl-D-aspartate (NMDA) receptor during excitotoxicity in spiral ganglion neurons in the rat cochlea induced by sodium salicylate (SS). Western blot illustrated SS decreased the expression of NMDA receptor 2B subunit (NR2B) surface protein through affecting GABAA receptor, but the total protein content did not significantly change. Y1472 and S1480 are important phosphorylation sites in NR2B, SS downregulated the Fyn-dependent phosphorylation of Y1472 in a manner not related to the CK2 (Casein Kinase 2) dependent phosphorylation of S1480, thus regulating the surface distribution and internalization of NMDA receptor through GABAA receptor. These results suggest that the modified pattern of dynamic balance between excitation and inhibition by coactivation of the GABAA receptor can attenuate the excitatory NMDA receptor under the action of SS, via inhibiting the Fyn-dependent phosphorylation of Y1472.

Sustained-Release Triamcinolone Acetonide Hydrogels Reduce Hearing Threshold Shifts in a Model for Cochlear Implantation with Hearing Preservation.

INTRODUCTION: In recent years, the preservation of residual hearing has become a major factor in patients undergoing cochlear implantation (CI). In studies attempting to pharmaceutically improve hearing preservation rates, glucocorticoids (GCs) applied perioperatively in many institutions have emerged as a promising treatment regimen. Although dexamethasone is most commonly used and has been applied successfully by various research groups, recently pharmacological properties have been reported to be relatively unsuitable for topical delivery to the inner ear. Consequently other glucocorticoids merit further evaluation. The aim of this study was therefore to evaluate the otoprotective effects of the topical application of a sustained-release triamcinolone acetonide (TAAC) hydrogel in CI with hearing preservation. METHODS: Normal-hearing pigmented guinea pigs were randomized into a group receiving a single dose of a 6% TAAC poloxamer 407 hydrogel, a group receiving a 30% TAAC hydrogel and a control group. All hydrogel applications were performed 1 day prior to CI. After a cochleostomy was drilled, a specifically designed silicone electrode was inserted into the scala tympani for 5 mm. Frequency-specific compound action potentials of the auditory nerve (0.5-32 kHz) were measured pre- and directly postoperatively as well as on days 3, 7, 14, 21, and 28. Finally, temporal bones were harvested for histological evaluation. RESULTS: Application of the TAAC hydrogels resulted in significantly reduced hearing threshold shifts in low, middle and high frequencies and improved spiral ganglion cell survival in the second turn of the cochlea. Outer hair cell numbers in the basal and second turn of the cochlea were slightly reduced after TAAC application. CONCLUSION: In summary, we were able to demonstrate functional benefits of a single preoperative application of a TAAC hydrogel in a guinea pig model for CI, which persisted until the end of the observational period, that is, 28 days after surgery.

Sphingosine 1-phosphate signaling axis mediates fibroblast growth factor 2-induced proliferation and survival of murine auditory neuroblasts.

Hearing loss affects millions of people in the world. In mammals the auditory system comprises diverse cell types which are terminally differentiated and with no regenerative potential. There is a tremendous research interest aimed at identifying cell therapy based solutions or pharmacological approaches that could be applied therapeutically alongside auditory devices to prevent hair cell and neuron loss. Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid that plays key role in the regulation of many physiological and pathological functions. S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P1-5. In this study, murine auditory neuroblasts named US/VOT-N33 have been used as progenitors of neurons of the spiral ganglion. We demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action was dependent on SK1, SK2 as well as S1P1 and S1P2. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. Additionally, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively. Taken together, these findings demonstrate a crucial role for S1P signaling axis in the proliferation and the survival of otic vesicle neuroprogenitors, highlighting the identification of possible novel therapeutical approaches to prevent neuronal degeneration during hearing loss.

Valproic acid promotes the neuronal differentiation of spiral ganglion neural stem cells with robust axonal growth.

Hearing loss occurs with the loss of hair cells of the cochlea and subsequent degeneration of spiral ganglion neurons (SGNs). Regeneration of SGNs is a potentially promising therapeutic approach to hearing loss in addition to the use of a cochlear implant (CI), because this device stimulates SGNs directly to restore hearing bypassing the missing hair cells. The presence of SGN-neural stem cells (NSCs) has been reported in adult human and mice. These cells have the potential to become SGNs and thus represent a cellular foundation for regeneration therapies for hearing loss. Valproic acid (VPA) has been shown to influence the neural differentiation of NSCs through multiple signaling pathways involving glycogen synthase kinase3ß (GSK3ß). Our present study therefore aimed to modulate the neural differentiation potential of SGN-NSCs by treatment with VPA. We here report that a clinically relevant concentration of 1 mM VPA induced the differentiation of basic fibroblast growth factor (bFGF)-treated P1- and P14-SGN-NSCs into neuronal and glial cells, confirmed by neuronal marker (Tuj1 and MAP2) and glial cell marker (GFAP and S100ß) detection. VPA-treated cells also promoted much longer neurite outgrowth compared to differentiated cells cultured without bFGF. The effects of VPA on the regulation of differentiation may be related to the activation of the Wnt/ß-catenin signaling pathway, but not the inhibition of histone deacetylases (HDACs). We propose that VPA has the potential to convert SGN-NSCs into SGNs and thereby restore hearing when combined with a CI.

Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss.

Hearing loss affects more than two-thirds of the elderly population, and more than 17% of all adults in the U.S. Sensorineural hearing loss related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs, stimulating intense interest in therapies to regenerate synaptic function. 7,8-Dihydroxyflavone (DHF) is a selective and potent agonist of tropomyosin receptor kinase B (TrkB) and protects the neuron from apoptosis. Despite evidence that TrkB agonists can promote survival of SGNs, local delivery of drugs such as DHF to the inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached SGNs. The results suggest that bisphosphonate-TrkB agonist conjugates have promise as a novel approach to targeted delivery of drugs to treat sensorineural hearing loss.

Mimic Cochlear Implant Surgery-Induced Cochlear Infection Fails to Further Damage Auditory Pathway in Deafened Guinea Pigs.

BACKGROUND Kanamycin and subsequent furosemide administration was applied to the healthy guinea pigs to induce deafness. MATERIAL AND METHODS Of the deafened guinea pigs, 10 were further infused with anti-infection procedures (Group B) and the other 10 animals did not undergo anti-infection procedures (Group C). In Group B, the deafened animals were able to restore cochlear and middle ear functions following the anti-infection procedure. In Group C, all animals developed cochlear and middle ear infections. RESULTS Compared to the healthy guinea pigs, hair cells and spiral ganglion neurons (SGN) of deafened animals (in Group B and Group C) were severely damaged. SGN density of deafened animals was significantly lower than that of healthy control animals in all ear turns except the basal turn. There was no significant difference between Group B and Group C in SGN density. The average optical density value of neurofilaments of deafened animals was also significantly decreased after the ototoxic drug administration. Notably, the density of the neurons in the cochlear nucleus region (CNR) of the brainstem were not significantly different between the healthy control guinea pigs and deafened animals. CONCLUSIONS Mimic cochlear implant surgery-induced cochlear infection caused no significant damage to the auditory pathway in ototoxic drug-induced deafened guinea pigs.

Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl- channel TMEM16A.

The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.

Contralateral Suppression of DPOAEs in Mice after Ouabain Treatment.

Medial olivocochlear (MOC) efferent feedback is suggested to protect the ear from acoustic injury and to increase its ability to discriminate sounds against a noisy background. We investigated whether type II spiral ganglion neurons participate in the contralateral suppression of the MOC reflex. The application of ouabain to the round window of the mouse cochlea selectively induced the apoptosis of the type I spiral ganglion neurons, left the peripherin-immunopositive type II spiral ganglion neurons intact, and did not affect outer hairs, as evidenced by the maintenance of the distorted product otoacoustic emissions (DPOAEs). With the ouabain treatment, the threshold of the auditory brainstem response increased significantly and the amplitude of wave I decreased significantly in the ouabain-treated ears, consistent with the loss of type I neurons. Contralateral suppression was measured as reduction in the amplitude of the 2f1-f2 DPOAEs when noise was presented to the opposite ear. Despite the loss of all the type I spiral ganglion neurons, virtually, the amplitude of the contralateral suppression was not significantly different from the control when the suppressor noise was delivered to the treated cochlea. These results are consistent with the type II spiral ganglion neurons providing the sensory input driving contralateral suppression of the MOC reflex.

Otoprotective effects of mouse nerve growth factor in DBA/2J mice with early-onset progressive hearing loss.

As it displays progressive hair-cell loss and degeneration of spiral ganglion neurons (SGNs) characterized by early-onset progressive hearing loss (ePHL), DBA/2J is an inbred mouse strain widely used in hearing research. Mouse nerve growth factor (mNGF), as a common exogenous nerve growth factor (NGF), has been studied extensively for its ability to promote neuronal survival and growth. To determine whether mNGF can ameliorate progressive hearing loss (PHL) in DBA/2J mice, saline or mNGF was given to DBA/2J mice of either sex by daily intramuscular injection from the 1st to the 9th week after birth. At 5, 7, and 9 weeks of age, in comparison with vehicle groups, mNGF groups experienced decreased auditory-evoked brainstem response (ABR) thresholds and increased distortion product otoacoustic emission (DPOAE) amplitudes, the prevention of hair cell loss, and the inhibition of apoptosis of SGNs. Downregulation of Bak/Bax and Caspase genes and proteins in cochleae of mice receiving the mNGF treatment was detected by real-time PCR, Western blot, and immunohistochemistry. This suggests that the Bak-dependent mitochondrial apoptosis pathway may be involved in the otoprotective mechanism of mNGF in progressive hearing loss of DBA/2J mice. Our results demonstrate that mNGF can act as an otoprotectant in the DBA/2J mice for the early intervention of PHL and, thus, could become of great value in clinical applications. © 2017 Wiley Periodicals, Inc.

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.

Carbaryl-induced ototoxicity in rat postnatal cochlear organotypic cultures.

Carbaryl, a widely used carbamate-based insecticide, is a potent anticholinesterase known to induce delayed neurotoxicity following chronic exposure. However, its potential toxic effects on the cochlea, the sensory organ for hearing that contains cholinergic efferent neurons and acetylcholine receptors on the hair cells (HC) and spiral ganglion neurons has heretofore not been evaluated. To assess ototoxic potential of carbaryl, cochlear organotypic cultures from postnatal day 3 rats were treated with doses of carbaryl ranging from 50 to 500 µM for 48 h up to 96 h. Carbaryl damaged both the sensory HC and spiral ganglion neurons in a dose- and duration-dependent manner. HC and neuronal damage was observed at carbaryl concentrations as low as 50 µM after 96-h treatment and 100 µM after 48-h treatment. Hair cell was greatest in the high frequency basal region of the cochlea and progressively decreased towards the apex consistent with the majority of ototoxic drugs. In contrast, damage to the spiral ganglion neurons was of similar magnitude in the basal and apical regions of the cochlea. Carbaryl damage was characterized by soma shrinkage, nuclear condensation and fragmentation, and blebbing, morphological features of programmed cell death. Carbaryl upregulated the expression of executioner caspase-3 in HC and spiral ganglion neurons indicating that cellular damage occurred primarily by caspase-mediated apoptosis. These results suggest that chronic exposure to carbaryl and other carbamate anticholinesterases may be ototoxic. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 956-969, 2017.

[A study on toxic effects of sodium salicylate on rat cochlear spiral ganglion neurons: dopamine receptors mediate expressions of NMDA and GABAA receptors].

The aim of the present study was to observe whether dopamine receptor (DR) was involved in the effects of sodium salicylate (SS) on the expressions of N-methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid (GABA) receptors in rat cochlear spiral ganglion neurons (SGNs). Forty-eight hours after primary culture of rat SGNs, immunofluorescence technique was applied to detect expressions of DR1 and DR2, the two subtypes of dopamine receptors. Western blot was performed to assess NMDA receptor NR1 subunit and GABAA receptor subunit α2 (GABRα2) protein expressions in the SGNs after the treatments of SS alone or in combination with DR antagonists. The results demonstrated that: (1) The DR1 and DR2 were expressed in the bodies and axons of the SGN; (2) After the treatment with SS, the surface protein expressions of GABRα2 and NR1 were decreased by 44.69% and 21.57%, respectively, while the total protein expressions showed no significant changes; (3) Neither SS + SCH23390 (DR1 antagonist) group nor SS + Eticlopride (DR2 antagonist) group showed significant differences in GABRα2 and NR1 surface protein expressions compared with the control group. These results suggest that SS regulates the surface GABAA and NMDA receptors trafficking on SGN, and the mechanism may involve DR mediation.

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.