Fgf8^(P2A-3×GFP/+):A New Genetic Mouse Model for Specifically Labeling and Sorting Cochlear Inner Hair Cells

The cochlear auditory epithelium contains two types of sound receptors,inner hair cells(IHCs)and outer hair cells(OHCs).Mouse models for labelling juvenile and adult IHCs or OHCs exist;however,labelling for embryonic and perinatal IHCs or OHCs are lacking.Here,we generated a new knock-in Fgf8^(P2A-3×GFP/+)(Fgf8^(GFP)/+)strain,in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements.After confirming that GFP expression accurately reflects the expression of Fgf8,we successfully obtained both embryonic and neonatal IHCs with high purity,highlighting the power of Fgf8^(GFP)/+.Furthermore,our fate-mapping analysis revealed,unexpectedly,that IHCs are also derived from inner ear progenitors expressing Insm1,which is currently regarded as an OHC marker.Thus,besides serving as a highly favorable tool for sorting early IHCs,Fgf8^(GFP)/+will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells:Implications for Tinnitus Generation

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Forward masking in distinguishing inner and outer hair cell damage

Forward Masking Temporal audiotory resolution is the ability of the auditory system to resolve auditory signals in the time domain. Forward masking is a means of studying temporal resolution where one tone, the probe, is masked by a preceding tone, the masker. Forward masking is

PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

Studies have shown that phosphatase and tensin homolog deleted on chromosome ten(PTEN)participates in the regulation of cochlear hair cell survival.Bisperoxovanadium protects against neurodegeneration by inhibiting PTEN expression.However,whether bisperoxovanadium can protect against noise-induced hearing loss and the underlying mechanism remains unclear.In this study,we established a mouse model of noise-induced hearing loss by exposure to 105 dB sound for 2 hours.We found that PTEN expression was increased in the organ of Corti,including outer hair cells,inner hair cells,and lateral wall tissues.Intraperitoneal administration of bisperoxovanadium decreased the auditory threshold and the loss of cochlear hair cells and inner hair cell ribbons.In addition,noise exposure decreased p-PI3K and p-Akt levels.Bisperoxovanadium preconditioning or PTEN knockdown upregulated the activity of PI3K-Akt.Bisperoxovanadium also prevented H_(2)O_(2)-induced hair cell death by reducing mitochondrial reactive oxygen species generation in cochlear explants.These findings suggest that bisperoxovanadium reduces noise-induced hearing injury and reduces cochlear hair cell loss.

LOWER DOSE OF AMINOGLYCOSIDE OTOTOXIC EXPOSURE CAUSES PRESYNAPTIC ALTERATIONS ASSOICATED WITH HEARING LOSS

Objective To study presynaptic alternations of cochlear ribbons arising from aminoglycoside ototoxic stimuli in C57BL/6J mice. Methods Animals were injected with low dose gentamicin (100 mg/kg/day) for 14 days, From the 14th to 28th days, the mice were maintained free of gentamicin treatment. Immunohisto-chemistry labeling was employed to trace RIBEYE, a major presynaptic componment of ribbon synapses. RIBEYE/CtBP2 expression levels were assessed and compared with hearing threshold shifts. Auditory func-tion was assessed by auditory brainstem responses. The stereocilia of outer hair cells (OHCs) and IHCs was examined by scanning electron microscopy (SEM). Results Hearing thresholds were elevated with peak hearing loss observed on the 7th day after gentamicin exposure, followed by improvement after the 7th day. RIBEYE/CtBP2 expression directly correlated with observed hearing threshold shifts. Strikingly, we did not see any obvious changes in stereocilia in both OHCs and IHCs until the 28th day. Mild changes in stereocil-ia were only observed in OHCs on the 28th day. Conclusions These findings indicate that presynapse co-chlear ribbons, rather than stereocilia, may be sensitive to aminoglycoside ototoxic exposure in mice cochle-ae. A pattern of RIBEYE/CtBP2 expression changes seems to parallel hearing threshold shifts and suggests presynaptic response properties to lower dosage of aminoglycoside ototoxic stimuli.

Zinc-finger protein A20 protects hair cells from damage made by high-power microwave

Inner ear hair cells are important for maintaining hearing.Irreversible damage to hair cells is an important cause of sensorineural deafness.Electromagnetic radiation,especially high-power microwave,is an important threat to human health in modern society and war.However,it is not clear whether high-power microwave has an effect on cochlea hair cells.This study aimed to assess the effects of high-power microwave on cochlear hair cells in guinea pigs,and investigate the potential protection of these cells against high-power microwave-induced damage by recombinant adenovirus A20.Based on experimental results,a 65 W/cm^(2) irradiation density applied to guinea pigs in this study to establish a high-power microwave inner ear injury model.In addition,pAdEeay-1/A20 was injected via a round window into experimental guinea pig cochlea,whereas artificial perilymph was injected into the control group.Auditory function was assessed by testing the auditory brainstem response threshold,and damage to cochlear hair cells was investigated by cell counting and scanning electron microscopy observations of the basilar membrane.Inner ear injury was observed 6 hours after 65 W/cm^( 2 ) of irradiation and the auditory brainstem response threshold was significantly higher in the irradiation group(P<0.05)compared with other groups.Propidium iodide staining and scanning electron microscopy results indicated that significant morphological changes occurred after radiation,especially to inner hair cells,which exhibited remarkable damage and the presence of several unknown spherical substances.Auditory brainstem response threshold was decreased in the pAdEeay-1/A20 group compared with the artificial perilymph group;moreover,damage to hair cells was milder in the pAdEeay-1/A20 group compared with the control group(P<0.01).Thus,high-power microwave can cause damage to cochlear hair cells,as well as hearing loss with prolonged exposure and/or high dosage.In this regard,65 W/cm^( 2 ) of irradiation for 6 hours is a reliable target dose for observation of damage.The zinc finger protein A20 can protect cochlear hair cells from high-power microwave-induced damage and prevent further hearing loss.This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University,China on April 18,2017.

Aligned Organization of Synapses and Mitochondria in Auditory Hair Cells

Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell(IHC).This feature is believed to be critical for audition over a wide dynamic range,but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear.By means of three-dimensional electron microscopy and artificial intelligence-based algorithms,we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice.We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization.Moreover,our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole.

Ototoxic effects of mefloquine in cochlear organotypic cultures

Mefloquine is a widely used anti-malarial drug. Some clinical reports suggest that mefloquine may be ototoxic and neurotoxic, but there is little scientific evidence from which to draw any firm conclusion. To evaluate the ototoxic and neurotoxic potential of mefloquine, we treated cochlear organotypic cultures and spiral ganglion cultures with various concentrations of mefloquine. Mefloquine caused a dose-dependent loss of cochlear hair cells at doses exceeding 0.01 mM. Hair cell loss progressed from base to apex and from outer to inner hair cells with increasing dose. Spiral ganglion neurons and auditory nerve fibers were also rapidly destroyed by mefloquine in a dose-dependent manner. To investigate the mechanisms underlying mefloquine-induced cell death, cochlear cultures were stained with TO-Pro-3 to identify morphological changes in the nucleus, and with carboxyfluorescein FAM-labeled caspase inhibitor 8, 9 or 3 to determine caspase-mediated cell death. TO-Pro-3-labeled nuclei in hair cells, spiral ganglion neurons and supporting cells were shrunken or fragmented, morphological features characteristic of cells undergoing apoptosis. Both initiator caspase 8 (membrane damage) and caspase 9 (mitochondrial damage), along with executioner caspase 3, were heavily expressed in cochlear hair cells and spiral ganglions after mefloquine treatment. These three caspases were also expressed in support cells, although labeling was less widespread and less intense. These results indicate that mefloquine damages both the sensory and neural elements in the postnatal rat cochlea by initially activating cell death signaling pathways on the cell membrane and in mitochondria.

Anatomy and physiology of peripheral auditory system and commen causes of hearing loss

For Otolaryngologist,it is the most important to know the principle of anatomy,physiology and common ototoxicity.Short but more concise summary has been sum up in the following review in order to help young ENT doctor to understand the importance of this basic knowledge.

Planar cell polarity regulators in asymmetric organogenesis during development and disease

The phenomenon of planar cell polarity is critically required for a myriad of morphogenetic processes in metazoan and is accurately controlled by several conserved modules.Six“core”proteins,including Friz zled,Flamingo(Celsr),Van Gogh(Vangl),Dishevelled,Prickle,and Diego(Ankrd6),are major components of the Wnt/planar cell polarity pathway.The Fat/Dchs protocadherins and the Scrib polarity complex also function to instruct cellular polarization.In vertebrates,all these pathways are essential for tissue and organ morphogenesis,such as neural tube closure,left-right symmetry breaking,heart and gut morphogenesis,lung and kidney branching,stereociliary bundle orientation,and proximal-distal limb elongation.Mutations in planar polarity genes are closely linked to various congenital diseases.Striking advances have been made in deciphering their contribution to the establishment of spatially oriented pattern in developing or gans and the maintenance of tissue homeostasis.The challenge remains to clarify the complex interplay of different polarity pathways in organogenesis and the link of cell polarity to cell fate specification.Inter disciplinary approaches are also important to understand the roles of mechanical forces in coupling cellular polarization and differentiation.This review outlines current advances on planar polarity regulators in asymmetric organ formation,with the aim to identify questions that deserve further investigation.

D-AP5 blocks the increase of intracellular free Ca^(2+) induced by glutamate in isolated cochlear IHCs

Objective To investigate the effect of D-AP5 (D-2-amino-5-phosphonopentanoate, a specific NMDA-antagonist) on the increase of intracellular free Ca 2+ concentration ([Ca 2+ ] i) induced by glutamate in isolated cochlear inner hair cells (IHCs), and to detect the autoreceptors of the IHC membrane. Methods When a laser scanning confocal microscope (LSCM) was used, the exogenous glutamate (Glu)-induced changes in [Ca 2+ ] i of isolated IHCs and OHCs of guinea pig cochlea were observed with fluo-3, a fluorescent probe for [Ca 2+ ] i. After D-AP5 or CNQX (6--cyano--7--nitroguinoxaline--2, 3--dione, a specific AMPA- antagonist) was administrated, the exogenous glutamate (Glu)-induced changes in [Ca 2+ ] i of isolated IHCs were recorded. Results In the presence of a low concentration Glu (3.85?μmol/L), there was an increase of [Ca 2+ ] i in IHCs, whereas there was no change in OHCs. When 50?μmol/L of D-AP5 was administrated in advance, Glu did not induce a corresponding increase in [Ca 2+ ] i in IHCs, and 50?μmol/L of CNQX did not completely block the increase of [Ca 2+ ] i in IHCs. Conclusions These results suggest that the autoreceptors existing in the IHC membrane are mainly of NMDA type, while there are relatively few AMPA receptors. Exogenous Glu is capable of increasing [Ca 2+ ] i in IHCs by acting on the NMDA autoreceptor of IHCs in a positive feedback manner.

Changes of summating potentials in the guinea pig cochlea after impulse sound exposure

Compound action potentials (CAP) and action potential-summating potential complex (AP-SP) evoked by click and tone burst respectively were recorded from the round window in guinea pigs. Correlations between the positive and negative summating potentials (SP+ and SP-) were examined before and after impulse sound exposure. The results show that the SP- is related to the threshold shift of CAP. In comparison to normal condition, it appears with a high incidence when the auditory threshold shift reaches 30 dB or more. SP-increases in amplitude when SP+ decreases. The smaller the amplitude of SP+, the larger that of SP-. This suggests that in normal hearing condition SP- may be suppressed by SP+. Such suppression may be released if OHCs are injured. The dominant SP- as a sign of recruitment may be due to the change in the nonlinear character of IHC following OHC damage.