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Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the â¼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.
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Inner ear gene therapy using adeno-associated viruses (AAVs) has been successfully applied to several mouse models of hereditary hearing loss to improve their auditory function. While most inner ear gene therapy studies have focused on the mechanosensory hair cells and supporting cells in the organ of Corti, the cochlear lateral wall and the endolymphatic sac have not garnered much attention. The cochlear lateral wall and the endolymphatic sac play critical roles in inner ear ionic and fluid homeostasis. Mutations in genes expressed in the cochlear lateral wall and the endolymphatic sac are present in a large percentage of patients with hereditary hearing loss. In this study, we examine the transduction patterns and efficiencies of conventional (AAV2 and AAV8) and synthetic (AAV2.7m8, AAV8BP2, and Anc80L65) AAVs in the mouse inner ear. We found that AAV8BP2 and AAV8 are capable of transducing the marginal cells and intermediate cells in the stria vascularis. These two AAVs can also transduce the epithelial cells of the endolymphatic sac. Our data suggest that AAV8BP2 and AAV8 are highly useful viral vectors for gene therapy studies targeting the cochlear lateral wall and the endolymphatic sac.
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HYPOTHESIS: Three-dimensional (3D) printed temporal bones are comparable to cadaveric temporal bones as a training tool for otologic surgery. BACKGROUND: Cadaveric temporal bone dissection is an integral part of otology surgical training. Unfortunately, availability of cadaveric temporal bones is becoming much more limited and concern regarding chemical and biological risks persist. In this study, we examine the validity of 3D-printed temporal bone model as an alternative training tool for otologic surgery. METHODS: Seventeen otolaryngology trainees participated in the study. They were asked to complete a series of otologic procedures using 3D-printed temporal bones. A semi-structured questionnaire was used to evaluate their dissection experience on the 3D-printed temporal bones. RESULTS: Participants found that the 3D-printed temporal bones were anatomically realistic compared to cadaveric temporal bones. They found that the 3D-printed temporal bones were useful as a surgical training tool in general and also for specific otologic procedures. Overall, participants were enthusiastic about incorporation of 3D-printed temporal bones in temporal bone dissection training courses and would recommend them to other trainees. CONCLUSION: 3D-printed temporal bone model is a viable alternative to human cadaveric temporal bones as a teaching tool for otologic surgery.
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Approximately 3 in 1000 children in the US under 4 years of age are affected by hearing loss. Currently, cochlear implants represent the only line of treatment for patients with severe to profound hearing loss, and there are no targeted drug or biological based therapies available. Gene replacement is a promising therapeutic approach for hereditary hearing loss, where viral vectors are used to deliver functional cDNA to "replace" defective genes in dysfunctional cells in the inner ear. Proof-of-concept studies have successfully used this approach to improve auditory function in mouse models of hereditary hearing loss, and human clinical trials are on the immediate horizon. The success of this method is ultimately determined by the underlying biology of the defective gene and design of the treatment strategy, relying on intervention before degeneration of the sensory structures occurs. A challenge will be the delivery of a corrective gene to the proper target within the therapeutic window of opportunity, which may be unique for each specific defective gene. Although rescue of pre-lingual forms of recessive deafness have been explored in animal models thus far, future identification of genes with post-lingual onset that are amenable to gene replacement holds even greater promise for treatment, since the therapeutic window is likely open for a much longer period of time. This review summarizes the current state of adeno-associated virus (AAV) gene replacement therapy for recessive hereditary hearing loss and discusses potential challenges and opportunities for translating inner ear gene replacement therapy for patients with hereditary hearing loss.
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Dependovirus , Oído Interno , Animales , Sordera/terapia , Dependovirus/genética , Terapia Genética , Vectores Genéticos , Pérdida Auditiva/genética , Pérdida Auditiva/terapia , Pérdida Auditiva Sensorineural , Humanos , Enfermedades del Laberinto/genética , Enfermedades del Laberinto/terapiaRESUMEN
BACKGROUND: Recessive mutations of coding regions and splice sites of the SLC26A4 gene cause hearing loss with enlargement of the vestibular aqueduct (EVA). Some patients also have a thyroid iodination defect that can lead to multinodular goiter as part of Pendred syndrome. A haplotype of variants upstream of SLC26A4, called CEVA, acts as a pathogenic recessive allele in trans to mutations affecting the coding regions or splice sites of SLC26A4. Our first hypothesis is that CEVA, acting as a pathogenic recessive allele, is correlated with a less severe phenotype than mutations affecting the coding regions and splice sites of SLC26A4. Our second hypothesis is that CEVA acts as a modifier of the phenotype in patients with EVA caused by mutations affecting the coding regions or splice sites of both alleles of SLC26A4 or EVA caused by other factors. METHODS: This was a prospective cohort study of 114 individuals and 202 ears with EVA. To test our first hypothesis, we compared the thyroid and auditory phenotypes of subjects with mutations affecting coding regions of both alleles of SLC26A4 with those of subjects carrying CEVA in trans to mutations affecting the coding regions. To test our second hypothesis, we compared the phenotypes associated with the presence versus absence of CEVA among subjects with no coding region mutations, as well as among subjects with mutations affecting coding regions of both alleles. RESULTS: Subjects carrying CEVA in trans to a mutation of SLC26A4 have a normal thyroid phenotype and less severe hearing loss in comparison to individuals with mutations affecting coding regions of both alleles of SLC26A4. In subjects with no mutant alleles of SLC26A4, hearing loss was more severe in subjects who carry the CEVA haplotype in comparison to non-carriers. There was no correlation of CEVA with the phenotype of subjects with mutations affecting coding regions of both alleles. CONCLUSIONS: CEVA, acting as a likely pathogenic recessive allele, is associated with a less severe phenotype than alleles with a mutation affecting the coding regions or splice sites of SLC26A4. CEVA may act as a genetic modifier in patients with EVA caused by other factors.
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Bocio Nodular/genética , Haplotipos , Pérdida Auditiva Sensorineural/genética , Mutación , Fenotipo , Transportadores de Sulfato/genética , Acueducto Vestibular/anomalías , Acueducto Vestibular/patología , Adolescente , Adulto , Alelos , Audiometría , Niño , Preescolar , Cromosomas Humanos Par 7/genética , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Genotipo , Audición/genética , Pérdida Auditiva/genética , Heterocigoto , Homocigoto , Humanos , Masculino , Estudios Prospectivos , Sitios de Empalme de ARN , Glándula Tiroides , Adulto JovenRESUMEN
Inner ear gene therapy offers great potential as a treatment for hearing loss and dizziness. The surgical method used to deliver gene therapy into the inner ear is a critical step in determining the success of inner ear gene therapy. Here we describe two commonly used surgical methods for gene delivery in neonatal mouse inner ear: the round window approach and the posterior semicircular canal approach. Both of these approaches are effective at delivering gene therapy to the neonatal mouse inner ear.
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Vectores Genéticos/administración & dosificación , Ventana Redonda/cirugía , Canales Semicirculares/cirugía , Animales , Animales Recién Nacidos , Oído Interno/cirugía , Técnicas de Transferencia de Gen , Terapia Genética , Ratones , Procedimientos Quirúrgicos OperativosRESUMEN
Adeno-associated virus (AAV) has been successfully used to deliver gene therapy to improve auditory function in mouse models of hereditary hearing loss. Many forms of hereditary hearing loss have mutations which affect the cochlear hair cells, the mechanosensory cells which allow for sound detection and processing. While most conventional AAVs infect inner hair cells (IHCs) with various efficiencies, they infect outer hair cells (OHCs) and supporting cells at lower levels in the cochlea. Here we examine the infection patterns of two synthetic AAVs (AAV2.7m8 and AAV8BP2) in the mouse inner ear. AAV2.7m8 infects both IHCs and OHCs with high efficiency. In addition, AAV2.7m8 infects inner pillar cells and inner phalangeal cells with high efficiency. Our results suggest that AAV2.7m8 is an excellent viral vector for inner ear gene therapy targeting cochlear hair cells and supporting cells, and it will likely greatly expand the potential applications for inner ear gene therapy.
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Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Pérdida Auditiva Sensorineural/terapia , Miosinas/genética , Animales , Animales Recién Nacidos , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas Internas/patología , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patología , Audición/genética , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Ratones , Miosina VIIa , Miosinas/metabolismoRESUMEN
Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.
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Oído Interno/metabolismo , Terapia Genética/métodos , Canales Semicirculares/irrigación sanguínea , Animales , Oído Interno/citología , Femenino , Humanos , Ratones , EmbarazoRESUMEN
Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.
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Terapia Genética , Audición/genética , Equilibrio Postural/genética , Síndromes de Usher/genética , Síndromes de Usher/fisiopatología , Animales , Conducta Animal , Modelos Animales de Enfermedad , Expresión Génica , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestructura , Pruebas Auditivas , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fenotipo , Estereocilios/metabolismo , Estereocilios/ultraestructura , Síndromes de Usher/terapiaRESUMEN
OBJECTIVES/HYPOTHESIS: To characterize the severity and natural history of hearing loss, and the prevalence of having a cochlear implant in a maturing cohort of individuals with enlarged vestibular aqueduct (EVA) and zero or one mutant allele of SLC26A4. STUDY DESIGN: Prospective cohort study of subjects ascertained between 1998 and 2015 at the National Institutes of Health Clinical Center. METHODS: Study subjects were 127 individuals (median age, 8 years; range, 0-59 years) with EVA in at least one ear. RESULTS: Ears with EVA and zero or one mutant allele of SLC26A4 had mean 0.5/1/2/4-kHz pure-tone averages of 62.6 and 52.9 dB HL, respectively, in contrast to EVA ears with two mutant alleles of SLC26A4 (88.1 dB HL; P < .01). This association was independent of age, sex, or side of EVA (P < .001). Natural history of hearing loss was not associated with number of mutant alleles (P = .94). The prevalence of having a cochlear implant was nine (12%) of 76, two (13%) of 15, and 12 (38%) of 32 subjects with zero, one, and two mutant alleles, respectively (P = .00833). This association was not independent (P = .534) but reflected underlying correlations with age at time of first audiogram (P = .003) or severity of hearing loss (P = .000). CONCLUSIONS: Ears with EVA and zero or one mutant allele of SLC26A4 have less severe hearing loss, no difference in prevalence of fluctuation, and a lower prevalence of cochlear implantation in comparison to ears with two mutant alleles of SLC26A4. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E238-E243, 2017.
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Alelos , Análisis Mutacional de ADN , Sordera/genética , Proteínas de Transporte de Membrana/genética , Acueducto Vestibular/anomalías , Adolescente , Adulto , Umbral Auditivo , Niño , Preescolar , Estudios de Cohortes , Sordera/rehabilitación , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Transportadores de Sulfato , Adulto JovenRESUMEN
Designer nucleases have gained widespread attention for their ability to precisely modify genomic DNA in a programmable manner. These genome-editing nucleases make double-stranded breaks at specified loci, and desired changes can be made to modify, ablate, or excise target genes. This technology has been used widely to develop human disease models in laboratory animals and to study gene functions by silencing, activating, or modifying them. Furthermore, the recent discovery of a bacterially derived programmable nuclease termed clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) has revolutionized the field because of its versatility and wide applicability. In this article, we discuss various modalities used to achieve genome editing with an emphasis on CRISPR-Cas9. We discuss genome-editing strategies to either repair or ablate target genes, with emphasis on their applications for investigating dermatological diseases. Additionally, we highlight preclinical studies showing the potential of genome editing as a therapy for congenital blistering diseases and as an antimicrobial agent, and we discuss limitations and future directions of this technology.
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Sistemas CRISPR-Cas/genética , Dermatología/métodos , Edición Génica/métodos , Ingeniería Genética/tendencias , Animales , Predicción , Ingeniería Genética/métodos , Humanos , Proyectos de InvestigaciónRESUMEN
OBJECTIVES: Melanin pigmentation is present in the human inner ear. In this study, we quantify the melanin pigmentation in the vestibular system and examine racial differences of vestibular melanin pigmentation using human cadaveric temporal bone specimens. STUDY DESIGN: Basic research. SETTING: Laboratory. SUBJECTS AND METHODS: Light microscopy was used to examine specimens from 40 left temporal bones from the Johns Hopkins Human Temporal Bone Collection. Color images of (1) ampulla of the horizontal canal, (2) utricular wall, (3) endolymphatic duct, and (4) posterior ampullary nerve as it enters the posterior canal were acquired with a digital camera attached to the microscope and image acquisition software. Acquired images of each anatomic area of interest were processed offline through ImageJ. Melanin content was then compared according to ethnicity, age, sex, and location. RESULTS: Fifteen African American and 25 Caucasian specimens were analyzed. Mean age was 68.8 years. African American specimens had a significantly greater amount of pigment at all 4 sampled locations as compared with Caucasian specimens (P < .01). Between sexes, there was a statistically significant difference (P < .05) at the posterior ampullary nerve, with males having more than females. Melanin content was not associated with age. CONCLUSIONS: There is greater melanin pigmentation within the vestibular system of African Americans than in Caucasians, similar to what has been described in the cochlea. Racial differences in vestibular physiologic function have been observed in the literature and may be explained by differences in melanin pigmentation.
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Negro o Afroamericano , Melaninas/análisis , Pigmentación , Hueso Temporal/metabolismo , Vestíbulo del Laberinto/metabolismo , Población Blanca , Anciano , Cadáver , Femenino , Humanos , Masculino , Programas InformáticosRESUMEN
Cochlear implant (CI) surgery in the setting of an open mastoid cavity is evolving. Two strategies are commonly pursued: a staged approach, clearing the disease, closing the meatus or the external auditory canal (EAC), and reevaluating in 3 to 6 months prior to implantation, or a single-stage procedure with mastoid obliteration without EAC closure. Meatal closure is often employed in the setting of an open mastoid cavity to reduce the risk of electrode extrusion and infection. An open cavity offers the advantages of being a single-stage procedure, permitting direct surveillance for recurrent cholesteatoma, and reducing the need for repeat computed tomography scans. We describe an approach to the coverage of a CI within a dry, open mastoid cavity using an anteriorly-based postauricular pericranial flap.
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Implantación Coclear/métodos , Conducto Auditivo Externo/cirugía , Apófisis Mastoides/cirugía , Cráneo/trasplante , Colgajos Quirúrgicos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del TratamientoAsunto(s)
Alérgenos/inmunología , Factor de Transcripción GATA3/genética , Haploinsuficiencia , Hipersensibilidad Inmediata/genética , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad Inmediata/diagnóstico , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Pruebas Cutáneas , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.
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Sordera/terapia , Oído Interno/metabolismo , Terapia Genética/métodos , Proteínas de la Membrana/genética , Estereocilios/ultraestructura , Animales , Supervivencia Celular , Sordera/metabolismo , Sordera/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Oído Interno/citología , Vectores Genéticos/administración & dosificación , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestructura , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Estereocilios/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVES/HYPOTHESIS: Hearing loss and enlarged vestibular aqueduct (EVA) can be inherited as an autosomal recessive trait caused by mutant alleles of the SLC26A4 gene. In some other families, EVA does not segregate in a typical autosomal recessive pattern. The goal of this study was to characterize the SLC26A4 genotypes and phenotypes of extended families with atypical segregation of EVA. STUDY DESIGN: Prospective study of cohort of families ascertained between 1998 and 2014 at the National Institutes of Health Clinical Center. METHODS: Study subjects were members of eight families segregating EVA in at least two members who were not related as siblings. Evaluations included pure-tone audiometry, temporal bone imaging, SLC26A4 nucleotide sequence analysis, SLC26A4-linked marker genotype and haplotype analysis, and pedigree analysis. RESULTS: One family had members with EVA caused by different etiologies, and two families had pseudodominant inheritance of recessive mutations of SLC26A4. In five families, the etiology remained unknown and could include inheritance of mutant alleles at another genetic locus, nongenetic influences, or a combination of these factors. CONCLUSIONS: Familial EVA can demonstrate a variety of atypical segregation patterns. Pseudodominant inheritance of SLC26A4 mutations or recessive alleles of other hearing loss genes may be more likely to occur in families in which deaf individuals have intermarried. The etiologic basis of atypical segregation of EVA without detectable SLC26A4 mutations remains unknown. Future studies of these families may reveal novel genes for EVA. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E240-E247, 2016.
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Segregación Cromosómica/genética , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva/genética , Proteínas de Transporte de Membrana/genética , Linaje , Acueducto Vestibular/anomalías , Adolescente , Adulto , Anciano , Alelos , Audiometría de Tonos Puros , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Estudios Prospectivos , Transportadores de Sulfato , Hueso Temporal/diagnóstico por imagen , Adulto JovenRESUMEN
BACKGROUND: Delivery of therapeutic agents directly through the round window (RW) offers promise for treating sensorineural hearing loss. However, hearing loss can result from the surgical approach itself, and the reasons for this are poorly understood. We examined the hearing loss following the 3 major steps involved with the RW approach to access the mouse cochlea: bullostomy, RW puncture, and RW injection. METHODS: Twenty-one adult CBA/J mice underwent bullostomy alone, 10 underwent RW puncture, and 8 underwent RW injection with PBS with 5% glycerol. Auditory brainstem responses (ABR) and otoscopy were performed preoperatively and up to 6 weeks postoperatively. Hair cells were stained, and survival was assessed using immunofluorescence. RESULTS: One week postoperatively, mice in all groups showed significant threshold shifts. Otoscopy revealed approximately half of all mice had middle ear effusion (MEE), with a higher incidence of effusion in the RW puncture and RW injection groups. Those with MEE had significant ABR threshold shifts, whereas those without MEE had minimal hearing loss. MEE persisted through 6 weeks in a majority of cases, but in those mice with MEE resolution, there was at least partial improvement in hearing. Immunohistochemistry showed minimal loss of hair cells in all animals. CONCLUSION: MEE is highly correlated with hearing loss in mice undergoing RW surgery. Otoscopy is an important adjunct to consider after ear surgery in mice, as MEE may contribute to postsurgical hearing loss.
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Potenciales Evocados Auditivos del Tronco Encefálico , Pérdida Auditiva/etiología , Otitis Media con Derrame/etiología , Procedimientos Quirúrgicos Otológicos/efectos adversos , Complicaciones Posoperatorias/etiología , Ventana Redonda/cirugía , Animales , Supervivencia Celular , Células Ciliadas Auditivas , Pérdida Auditiva/fisiopatología , Inyecciones , Ratones , Ratones Endogámicos CBA , Otitis Media con Derrame/fisiopatología , Complicaciones Posoperatorias/fisiopatología , Punciones , Recuperación de la FunciónRESUMEN
The effect of small amounts of air on sound-induced umbo velocity in an otherwise saline-filled middle ear (ME) was investigated to examine the efficacy of a novel balloon-like air-filled ME implant suitable for patients with chronically non-aerated MEs. In this study, air bubbles or air-filled implants were introduced into saline-filled human cadaveric MEs. Umbo velocity, a convenient measure of ME response, served as an indicator of hearing sensitivity. Filling the ME with saline reduced umbo velocity by 25-30 dB at low frequencies and more at high frequencies, consistent with earlier work (Ravicz et al., Hear. Res. 195: 103-130 (2004)). Small amounts of air (â¼30 µl) in the otherwise saline-filled ME increased umbo velocity substantially, to levels only 10-15 dB lower than in the dry ME, in a frequency- and location-dependent manner: air in contact with the tympanic membrane (TM) increased umbo velocity at all frequencies, while air located away from the TM increased umbo velocity only below about 500 Hz. The air-filled implant also affected umbo velocity in a manner similar to an air bubble of equivalent compliance. Inserting additional implants into the ME had the same effect as increasing air volume. These results suggest these middle-ear implants would significantly reduce conductive hearing loss in patients with chronically fluid-filled MEs.