Vacuolization and alterations of lysosomal membrane proteins in cochlear marginal cells contribute to hearing loss in neuraminidase 1-deficient mice

The neuraminidase-1 (Neu1) knockout mouse model is a phenocopy of the lysosomal storage disease (LSD) sialidosis, characterized by multisystemic and neuropathic symptoms, including hearing loss. We have characterized the auditory defects in Neu1^?/? mice and found that hearing loss involves both conductive and sensorineural components. Auditory brainstem response (ABR) thresholds were significantly elevated in Neu1^?/? mice at P21 (48  –55 dB), and hearing loss appeared progressive (53  –66 dB at P60). At these ages Neu1^?/? mice accumulated cerumen in the external ear canal and had a thickened mucosa and inflammation in the middle ear. In cochleae of adult wild-type mice, Neu1 was expressed in several cell types in the stria vascularis, the organ of Corti, and spiral ganglion. Progressive morphological abnormalities such as extensive vacuolization were detected in the Neu1^?/? cochleae as early as P9. These early morphologic changes in Neu1^?/? cochleae were associated with oversialylation of several lysosomal associated membrane proteins (Lamps) in the stria vascularis. A marked increase in the expression and apical localization of Lamp-1 in marginal cells of the stria vascularis predicts exacerbation of lysosomal exocytosis into the endolymph. Consequently, the endolymphatic potential in Neu1^?/? mice was reduced by approximately 20 mV at ages P31–P44, which would cause dysfunction of transduction in sensory hair cells. This study suggests a molecular mechanism that contributes to hearing loss in sialidosis and identifies potential therapeutic targets.     

Compound Heterozygosity of the Functionally Null Cdh23v-ngt and Hypomorphic Cdh23ahl Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23^ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23^v-ngt) null allele with mice carrying the hypomorphic Cdh23^ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23^v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23^ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23^v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23^v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.     

Developmental regulation of TRPC3 ion channel expression in the mouse cochlea

Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca²⁺ entry supporting Ca²⁺ homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16–adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner’s membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca²⁺ homeostasis and regulation of auditory neurotransmission via GPCR signaling.     

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.     

Pou3f4 deficiency causes defects in otic fibrocytes and stria vascularis by different mechanisms

DFN3, the most prevalent X-linked hearing loss, is caused by mutations in the POU3F4 gene. Previous studies in Pou3f4 knockout mice suggest that defective otic fibrocytes in the spiral ligament of the cochlear lateral wall may underlie the hearing loss in DFN3. To better understand the pathological mechanisms of the DFN3 hearing loss, we analyzed inner ears of Pou3f4-deficient mice during development. Our results indicate that compartmentalization of the spiral ligament mesenchyme setting up boundaries for specific otic fibrocytes occurs normally in Pou3f4-deficient cochlea. However, differentiation of the compartmentalized mesenchyme into specific otic fibrocytes was blocked in the absence of Pou3f4 function. In addition, we found that stria vascularis in the cochlear lateral wall was also affected in Pou3f4-deficient cochlea. Unlike the otic fibrocytes, differentiation of stria vascularis was completed in the absence of Pou3f4 function, yet expression of Kir4.1 channels in the strial intermediate cells, essential for the sound transduction, was lost afterwards. These results suggest that Pou3f4 deficiency causes defects in both otic fibrocytes and stria vascularis at different developmental stages and by different pathological mechanisms, which may account for the progressive nature of DFN3 hearing loss.     

Acoustic overstimulation activates 5'-AMP-activated protein kinase through a temporary decrease in ATP level in the cochlear spiral ligament prior to permanent hearing loss in mice

Inner ear disorders are known to be elicited by mitochondrial dysfunction, which decreases the ATP level in the inner ear. 5′-AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by metabolic stress and by an increase in the AMP/ATP ratio. To elucidate the involvement of AMPK-derived signals in noise-induced hearing loss, we investigated whether in vivo acoustic overstimulation would activate AMPK in the cochlea of mice. Std-ddY mice were exposed to 8 kHz octave band noise at a 90-, 110- or 120-dB sound pressure level (SPL) for 2 h. Exposure to the noise at 110 or 120 dB SPL produced outer hair cell death in the organ of Corti and permanent hearing loss. Exposure to the noise at 120-dB SPL elevated the level of the phospho-AMPK α-subunit (p-AMPKα), without affecting the protein level of this subunit, immediately and at 12-h post-exposure in the lateral wall structures including the spiral ligament and stria vascularis. In the hair cells and spiral ganglion cells, no marked change in the level of p-AMPKα was observed at any time post-exposure. The level of phospho-c-Jun N-terminal kinase (p-JNK) was increased in the lateral wall structures at 2- to 4-h post-exposure at 120 dB SPL. Noise exposure significantly, but temporarily, decreased the ATP level in the spiral ligament, in an SPL-dependent manner at 110 dB and above. Likewise, elevation of p-AMPKα and p-JNK levels was also observed in the lateral wall structures post-exposure to noise at an SPL of 110 dB and above. Taken together, our data suggest that AMPK and JNK were activated by ATP depletion in the cochlear spiral ligament prior to permanent hearing loss induced by in vivo acoustic overstimulation.     

Integrity and Regeneration of Mechanotransduction Machinery Regulate Aminoglycoside Entry and Sensory Cell Death

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23^+/+ and Cdh23^v2J/+ mice, but failed to detect GTTR uptake in Cdh23^v2J/v2J hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N'',N''-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23^v2J/v2J and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.     

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.     

Attenuation of Progressive Hearing Loss in DBA/2J Mice by Reagents that Affect Epigenetic Modifications Is Associated with Up-Regulation of the Zinc Importer Zip4

Various factors that are important for proper hearing have been identified, including serum levels of zinc. Here we investigated whether epigenetic regulatory pathways, which can be modified by environmental factors, could modulate hearing. RT-PCR detected expression of genes encoding DNA methyltransferase and histone deacetylase (Hdac) in the postnatal as well as adult mouse auditory epithelium. DBA/2J mice, which are a model for progressive hearing loss, were injected subcutaneously with one or a combination of the following reagents: <smallcaps>L</smallcaps>-methionine as a methyl donor, valproic acid as a pan-Hdac inhibitor, and folic acid and vitamin B12 as putative factors involved in age-related hearing loss. The mice were treated from ages 4 to 12 weeks (N ≥ 5), and auditory brainstem response (ABR) thresholds were measured at 8, 16, and 32 kHz. Treatment of the mice with a combination of <smallcaps>L</smallcaps>-methionine and valproic acid (M+V) significantly reduced the increase in the ABR threshold at 32 kHz. Treatment with any of these reagents individually produced no such effect. Microarray analyses detected 299 gene probes that were significantly up- or down-regulated in the cochleae of mice treated with M+V compared with the control vehicle-treated mice. Quantitative RT-PCR confirmed significant up-regulation of a zinc importer gene, Zip4, in the cochleae of mice treated with M+V. Immunohistochemistry demonstrated an intense Zip4 signal in cochlear tissues such as the lateral wall, organ of Corti, and spiral ganglion. Finally, mice treated with the Zip4 inducer (–)-epigallocatechin-3-O-gallate showed a significant reduction in the increase of the ABR threshold at 32 kHz and up-regulation of Zip4 expression in the cochlea. This study suggests that epigenetic regulatory pathways can modify auditory function and that zinc intake in the cochlea via Zip4 mediates maintenance of mammalian hearing.     

Involvement of calpain-I and microRNA34 in kanamycin-induced apoptosis of inner ear cells

Inner ear cells, including hair cells, spiral ganglion cells, stria vascularis cells and supporting cells on the basilar membrane, play a major role in transducing hearing signals and regulating inner ear homoeostasis. However, their functions are often damaged by antibiotic-induced ototoxicity. Apoptosis is probably involved in inner ear cell injury following aminoglycoside treatment. Calpain, a calcium-dependent protease, is essential for mediating and promoting cell death. We have therefore investigated the involvement of calpain in the molecular mechanism underlying ototoxicity induced by the antibiotic kanamycin in mice. Kanamycin (750 mg/kg) mainly induced cell death of cochlear cells, including stria vascularis cells, supporting cells and spiral ganglion cells, but not hair cells within the organ of Corti. Cell death due to apoptosis occurred in a time-dependent manner with concomitant up-regulation of calpain expression. Furthermore, the expression levels of two microRNAs, mir34a and mir34c, were altered in a dose-dependent manner in cochlear cells. These novel findings demonstrated the involvement of both calpain and microRNAs in antibiotic-induced ototoxicity.     

Somatic mtDNA mutations cause progressive hearing loss in the mouse

Mitochondrial dysfunction has been implicated in the commonly occurring age-associated hearing loss (presbyacusis). We have previously generated mtDNA mutator mice with increased levels of somatic mtDNA point mutations causing phenotypes consistent with premature ageing. We have now utilized these mice to investigate whether elevated levels of somatic mtDNA mutations affect the auditory system. The mtDNA mutator mice develop a progressive impairment of hearing (ABR thresholds). Quantitative assessment of hair cell loss in the cochlea did not show any significant difference between the mutator and wild-type mice. The mtDNA mutator mice showed progressive apoptotic cell loss in the spiral ganglion and increased pathology with increasing age in the stria vascularis. The neurons in the cochlear nucleus showed an accelerated progressive degeneration with increasing age in the mutator mice compared to the wild-type mice. Both physiological and histological characterization thus reveals a striking resemblance between the auditory system pathology of mtDNA mutator mice and humans with presbyacusis. Somatic mtDNA mutations accumulate during normal ageing and further studies in humans are now warranted to investigate whether presbyacusis can be linked to mitochondrial dysfunction.     

A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart

Background

Hearing impairment is the most common sensory impairment in humans, affecting 1∶1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade.

Methodology/Principal Findings

The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2 ^N538K mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2 ^N538K/N538K) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent.

Conclusions/Significance

Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.     

PMCA2 mutation causes structural changes in the auditory system in deafwaddler mice

Homozygous deafwaddler mice (dfw/dfw) have a mutation in the gene encoding plasma membrane Ca²⁺ATPase isoform 2 (Pmca2). They walk with a hesitant and wobbly gait, display head bobbing and are deaf. Light microscopy and transmission electron microscopy were used to evaluate the nature and relationship of morphological changes in the cochlea, spiral ganglion cells and spherical cells of the cochlear nucleus in homozygous and heterozygous mice of different ages and controls. Ultrastructural findings showed that in 7 week old homozygous (dfw) mice, inner hair cells and their afferent terminals were present although outer hair cells appeared apoptotic. Stereocilia were absent from the second and third rows of outer hair cells. Ganglion cells were also present although abnormal in appearance. In older homozygous mutants there was a loss of hair cells and spiral ganglion cells. Remaining ganglion cells in this group contained very few cytoplasmic organelles apart from a few hypertrophied mitochondria. In the anteroventral cochlear nucleus, spherical cell soma size was smaller in all homozygous (dfw) mutants than in heterozygous mice and controls. The ultrastructural appearance of the end bulbs of Held in homozygous mutants was abnormal compared with controls, and in the younger group were seen to be swollen, with less distinct synaptic densities and containing large numbers of small synaptic vesicles arranged in clumps. In the older group these synapses were distorted and contained hypertrophied mitochondria and no synaptic densities could be seen, suggesting that these synapses may be non-functional. This study has shown that in homozygous (dfw) mice structural abnormalities occurred not only in cochlear hair cells but also in the spiral ganglion neurones and spherical cells in the cochlear nucleus. It seems likely that these changes are the result of the Pmca2 mutation and the subsequent accumulation of toxic levels of calcium that may lead to alterations in their functional integrity.     

Expression of EFR3A in the Mouse Cochlea during Degeneration of Spiral Ganglion following Hair Cell Loss

Retrograde degeneration of spiral ganglion cells in the cochlea following hair cell loss is similar to dying back in pathology. The EFR3A gene has recently been discovered to be involved in the pathogenesis of dying back. The relationship of EFR3A and spiral ganglion degeneration, however, was rarely investigated. In this study, we destroyed the hair cells of the mouse cochlea by co-administration of kanamycin and furosemide and then investigated the EFR3A expression during the induced spiral ganglion cell degeneration. Our results revealed that co-administration of kanamycin and furosemide quickly induced hair cell loss in the C57BL/6J mice and then resulted in progressive degeneration of the spiral ganglion beginning at day 5 following drug administration. The number of the spiral ganglion cells began to decrease at day 15. The expression of EFR3A increased remarkably in the spiral ganglion at day 5 and then decreased to near normal level within the next 10 days. Our study suggested that the change of EFR3A expression in the spiral ganglion was coincident with the time of the spiral ganglion degeneration, which implied that high expression of EFR3A may be important to prompt initiation of spiral ganglion degeneration following hair cell loss.     

Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue

Large-scale deletions of mitochondrial DNA (mtDNA) have been associated with aging and disease in post-mitotic tissues. These post-mitotic tissues, including skeletal muscle, heart and brain, are heavily dependent on intact functional mitochondria. The cochlear tissues are known to contain an abundance of mitochondria. This observation stimulated a search for mtDNA deletions in the cochlea and its elements using a sensitive nested PCR methodology and long range PCR to explain the functional deficits observed in age-related hearing loss. The presence of the so-called “common” deletion (CD) was detected in cochlear tissue from two individuals with age-related hearing loss, 73 and 78 years of age. Three additional deletions, that to our knowledge have not been previously reported, were also identified in these two individuals, including a 5354 bp deletion flanked with a 3 bp repeat, a 9682 bp deletion flanked by a 10 bp repeat and a 5142 bp deletion without a flanking repeat. The 9682 and 5142 bp deletions were also detected in an individual 39 years of age with normal hearing, however, these two deletions were not detected in a normal hearing individual 9 years of age. In contrast, the 5354 bp deletion was detected in all four of the individuals studied. To localize the deletions within the cochlea, the cochlear elements were removed by laser capture microdissection (LCM) and the mtDNA from these tissues was studied. The 5142 and 5354 bp deletions were detected in the organ of corti, spiral ligament, and ganglion cells, but not in the stria vascularis. These findings correlate with the reduction in the number of spiral ganglion cells and outer hair cells, and the normal stria vascularis volume observed in this individual. All four of these deletions involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. These observations suggest that multiple mtDNA deletions may contribute to a deficit in mitochondrial function in the cochlea and result in hearing loss if a level of physiological significance is reached.     

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23^ahl) and ahl8 (Fscn2^ahl8) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2^ahl8 allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3–54.5, 64.6–119.9, and 119.9–137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80–3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F₁ × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2^ahl8 to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23^ahl allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.     

顺铂耳毒性大鼠耳聋模型的研究

摘要 目的：探讨顺铂对大鼠造成的听力损伤及耳蜗细胞形态学变化。方法：体内实验,运用顺铂腹腔注射的方法,连续七天注射,通过听性脑干反应检测,观察顺铂对不同日龄的大鼠听力损伤情况;测听后取耳蜗,通过基底膜铺片和冰冻切片的免疫荧光染色,观察听力损伤后对耳蜗毛细胞和螺旋神经元的影响。体外实验,耳蜗器官培养免疫荧光染色,观察顺铂对耳蜗毛细胞和螺旋神经元的影响。结果：顺铂具有耳毒性,会对大鼠听力造成损伤,高频听力损伤更加严重,而且对不同日龄的大鼠造成的听力损失不同,小日龄的大鼠对顺铂耳毒性更加敏感。体内实验,顺铂耳毒性造成听力损失,会引起大鼠耳蜗毛细胞的缺失,但未观察到明显的螺旋神经元缺失,也没有观察到明显的Cleaved caspase-3阳性螺旋神经元细胞。体外实验,可以观察到顺铂同时引起毛细胞和螺旋神经元产生明显的损伤。结论：体、内外实验,都可以建立稳定的顺铂耳毒性大鼠耳聋模型,对研究顺铂损伤耳蜗毛细胞的发生机制和保护奠定了实验基础。     

Expression and function of pannexins in the inner ear and hearing

Pannexin (Panx) is a gene family encoding gap junction proteins in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required.     

Role of Somatostatin Receptor-2 in Gentamicin-Induced Auditory Hair Cell Loss in the Mammalian Inner Ear

Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin—a peptide with hormone/neurotransmitter properties—can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin) on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.