Fgf8P2A-3×GFP/+: A New Genetic Mouse Model for Specifically Labeling and Sorting Cochlear Inner Hair Cells / 神经科学通报·英文版

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 Fgf8P2A-3×GFP/+ (Fgf8GFP/+) 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 Fgf8GFP/+. 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, Fgf8GFP/+ 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 / 神经科学通报·英文版

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 Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ 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.

Aligned Organization of Synapses and Mitochondria in Auditory Hair Cells / 神经科学通报·英文版

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.

Differential Expression of Ca²⁺-buffering Protein Calretinin in Cochlear Afferent Fibers: A Possible Link to Vulnerability to Traumatic Noise

The synaptic contacts of cochlear afferent fibers (CAFs) with inner hair cells (IHCs) are spatially segregated according to their firing properties. CAFs also exhibit spatially segregated vulnerabilities to noise. The CAF fibers contacting the modiolar side of IHCs tend to be more vulnerable. Noise vulnerability is thought to be due to the absence of neuroprotective mechanisms in the modiolar side contacting CAFs. In this study, we investigated whether the expression of neuroprotective Ca²⁺-buffering proteins is spatially segregated in CAFs. The expression patterns of calretinin, parvalbumin, and calbindin were examined in rat CAFs using immunolabeling. Calretinin-rich fibers, which made up ~50% of the neurofilament (NF)-positive fibers, took the pillar side course and contacted all IHC sides. NF-positive and calretinin-poor fibers took the modiolar side pathway and contacted the modiolar side of IHCs. Both fiber categories juxtaposed the C-terminal binding protein 2 (CtBP2) puncta and were contacted by synaptophysin puncta. These results indicated that the calretinin-poor fibers, like the calretinin-rich ones, were afferent fibers and probably formed functional efferent synapses. However, the other Ca²⁺-buffering proteins did not exhibit CAF subgroup specificity. Most CAFs near IHCs were parvalbumin-positive. Only the pillar-side half of parvalbumin-positive fibers coexpressed calretinin. Calbindin was not detected in any nerve fibers near IHCs. Taken together, of the Ca²⁺-buffering proteins examined, only calretinin exhibited spatial segregation at IHC-CAF synapses. The absence of calretinin in modiolar-side CAFs might be related to the noise vulnerability of the fibers.

Locacorten Vioform Ototoxicity Upon Guinea Pig Middle Ear Application

BACKGROUND AND OBJECTIVES: Locacorten Vioform (Novartis UK) is frequently prescribed for otomycosis. Its component, Clioquinol, also has anti-bacterial properties. Up to this point, its ototoxic potential has not been evaluated. Our objective aims to evaluate Locacorten Vioform’s potential ototoxicity when applied directly to the middle ear cavity. MATERIALS AND METHODS: We performed an experimental prospective animal study in our animal research center with 20 Hartley guinea pigs divided into 2 groups. The first group (experimental) was treated with Locacorten Vioform in one ear and with a physiologic saline solution in the other. The second group (positive control) was treated with concentrated gentamycin in one ear and physiologic saline in the other. Auditory brainstem response measurements were obtained before and after three sets of injections. Statistics were analyzed using a variance analysis with repeated measures. The histological state of cochlear outer hair cells was compared between the two groups using scanning electron microscopy. RESULTS: Average hearing loss in ears treated with Locacorten Vioform was 32.1 dB, compared with a 2.5 dB average loss in the saline-treated ears. Ears treated with gentamycin lost an average of 33.0 dB. There were clinically and statistically significant differences between the two ears of the guinea pigs in both groups (p < 0.001). Scanning electron microscopy revealed severe pericochlear and cochlear inflammation and ossification in the Locacorten Vioform-treated ears. Gentamycin caused significant destruction of outer hair cell architecture. CONCLUSIONS: Locacorten Vioform induces a hearing loss similar to that caused by gentamycin when applied directly to the middle ear of a guinea pig model. Electron microscopy indicates a pericochlear and cochlear inflammatory reaction with ossification.

Expression of Adaptin-2 in Different Stages of Mouse Cochlea / 听力学及言语疾病杂志

Objective To investigate the expression of adaptin-2(AP-2) in different stages of mouse coch‐lea and its probable role in the auditory generation and formation .Methods Mice were divided into 4 experimental groups by age (7 days old ,15 days old ,35 days old ,16 months old) ,which respectively represented the newborn mice ,developmental mice ,mature mice and old mice .Auditory brainstem response (ABR) ,laser scanning confocal microscopy (LSCM) ,immune-fluroscence histochemistry and qRT - PCR were used in this study .Results For the 15 days old ,35 days old ,16 months old groups ,ABR average threshold was 18 .67 ± 1 .21 dB nHL ,13 .83 ± 1 .47 dB nHL ,37 .83 ± 7 .68 dB nHL ,respectively ,for the 7 days old groups no responses were observed .The AP-2 im‐munoreactivity was found in all the stages of mice cochlea inner hair cell (IHC) cytoplasm ,especially in IHC basal part ,nearby the ribbon synapse .For the 7 days old ,15 days old ,35 days old ,16 months old groups ,immune-flu‐roscence histochemistry IMV(intensity mean value)were 190 .91 ± 17 .27 ,494 .06 ± 27 .63 ,838 .41 ± 38 .23 ,682 .65 ± 72 .22 ,respectively .For the 7 days old ,15 days old ,35 days old ,16 months old groups ,mRNA RQ(relative quantity)were 0 .53 ± 0 .09 ,1 .03 ± 0 .02 ,1 .00 ± 0 .09 ,1 .03 ± 0 .06 ,respectively .Developmental mice expressed significantly higher than those of the newborn in the AP -2 protein expression level which was measured by immuno -fluores‐cence histochemistry and qRT PCR(P 0 .05) ,but mature mice had significant advantages by immuno-fluorescence histochemistry .Conclusion AP-2 protein expression level may closely related to the au‐ditory formation and maintenance because its expression gradually increases with age in mice .

The Changes of the Number of Ribbon Synapse in Inner Hair Cells of C57BL/6J Mice with Age / 听力学及言语疾病杂志

Objective To investigate the number changes of the ribbon synapses(RS) in the inner hair cell of C57BL/6J mice with age .Methods The cochlear basilar membrane was obtained from C57BL/6J mice in 2 ,6 ,10 , 12 months old .RIBEYE on the presynaptic membrane and AMPA receptor on the postsynaptic membrane were double labeled by immunofluorescence histochemical technique .The stained sections were observed under a confocal laser-scanning microscope .Then the number of RS of the basilar membrane was calculated by three dimensional reconstruction images using 3DS max software .Results The numbers of RS reduced gradually from the cochlear a‐pex to base in the same developing stages .The numbers of RS in apex and middle turn of cochlea were gradually re‐duced with the age increasing .The number of RS in basilar turn reduced first and increased slowly then .Conclusion The decreases of the number of RS maybe the main pathological change at the early stage of presbycusis .In addi‐tion ,there may be a kind of compensatory mechanism by increaseing the number of RS to delay the hearing deficits .

Protective Effect of Hexane and Ethanol Extract of Piper Longum L. on Gentamicin-Induced Hair Cell Loss in Neonatal Cultures

OBJECTIVES: Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures. METHODS: The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. RESULTS: SPL extract at a concentration of 1 microg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 microg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay. CONCLUSION: An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.

Temperature Enhances Activation and Inactivation Kinetics of Potassium Currents in Inner Hair Cells Isolated from Guinea-Pig Cochlea

OBJECTIVES: Until recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs. METHODS: IHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25degrees C) and physiological temperatures (35degrees C-37degrees C). RESULTS: IHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36degrees C than at room temperature. Half-time for activation was shorter at 36degrees C than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q10 for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36degrees C than at room temperature between the membrane potentials of -20 and +60 mV. Q10 for the inactivation time constant was 3.19. CONCLUSION: The results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.

N-Acetylcysteine and N-Nitroarginine Methyl Ester Attenuate Carboplatin-Induced Ototoxicity in Dissociated Spiral Ganglion Neuron Cultures

OBJECTIVES: Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs). METHODS: Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored. RESULTS: Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME. CONCLUSION: The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.

N-Acetylcysteine and N-Nitroarginine Methyl Ester Attenuate Carboplatin-Induced Ototoxicity in Dissociated Spiral Ganglion Neuron Cultures

OBJECTIVES: Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs). METHODS: Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored. RESULTS: Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME. CONCLUSION: The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.