Cochlea cell-specific marker expression upon in vitro Hes1 knockdown

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Solution Using Lagrange's Equation to the Model of Cochlear Micromechanics / Solución del modelo de la micromecánica de la cóclea usando la ecuación de Lagrange

Abstract In this paper a new solution to micromechanical model of the cochlea developed by Neely and Kim is presented using Lagrange's equation. This solution has the advantage over previous methodologies to provide a mathematical model for the displacement exercised on the outer hair cells in the organ of Corti that only depends of the mechanical characteristics in the system and the value of the excitation frequency in the inner ear. For the evaluation of this new model the parameters developed by Ku are used and is considers that the amplitude of the excitation frequency is normalized. The model developed is satisfactorily compared with the impedance method and its numerical solution proposed by Neely and Kim, the state space analysis developed by Elliot, Ku and Lineton and the physiological measurements taken from Békésy.

Resumen En este trabajo se presenta una nueva solución utilizando la ecuación de Lagrange al modelo micromecánico de la cóclea desarrollado por Neely y Kim. Esta solución tiene la ventaja respecto a las ya existentes de proporcionar un modelo matemático del desplazamiento ejercido a los cilios en el órgano de Corti que sólo depende de las características mecánicas del sistema y del valor de la frecuencia de excitación en el oído interno. Para su evaluación se utilizan los parámetros desarrollados por Ku y se considera que la amplitud de la frecuencia de excitación está normalizada. El modelo desarrollado se compara satisfactoriamente con el método de impedancias y su solución numérica propuesta por Neely y Kim, el método de análisis de espacio estado desarrollado por Elliot, Ku y Lineton y con las mediciones fisiológicas realizadas por Békésy.

The Study of Otoacoustic Emissions and the Suppression of Otoacoustic Emissions in Subjects with Tinnitus and Normal Hearing: An Insight to Tinnitus Etiology

Introduction Analysis of the suppression effect is a simple method to evaluate cochlear status and central auditory mechanisms and, more specifically, the medial olivocochlear system. This structure may be involved in the generation of mechanisms that cause tinnitus and in the pathophysiology of tinnitus in patients with tinnitus and normal hearing. Objective To review the literature of the etiology of tinnitus on the lights of otoacoustic emissions in patients with normal hearing. Data Synthesis Individuals with tinnitus and normal hearing have a higher prevalence of alterations in transient-evoked otoacoustic emissions and distortion-product otoacoustic emissions than normal subjects. This fact suggests that dysfunctions of the outer hair cells (OHCs) might be important in the generation of the tinnitus; however, this feature is not always present in those who have the symptoms of tinnitus. Final Comments These findings suggest that OHC dysfunction is not necessary for tinnitus development—that is, there might be mechanisms other than OHC damage in the tinnitus development. On the other hand, OHC dysfunction alone is not sufficient to cause the symptom, because a great many individuals with OHC dysfunction did not complain about tinnitus. .

Potassium Currents in Isolated Deiters' Cells of Guinea Pig

Deiters' cells are the supporting cells in organ of Corti and are suggested to play an important role in biochemical and mechanical modulation of outer hair cells. We successfully isolated functionally different K+ currents from Deiters' cells of guinea pig using whole cell patch clamp technique. With high K+ pipette solution, depolarizing step pulses activated strongly outward rectifying currents which were dose-dependently blocked by clofilium, a class III anti-arrhythmic K+ channel blocker. The remaining outward current was transient in time course whereas the clofilium-sensitive outward current showed slow inactivation and delayed rectification. Addition of 5 mM tetraethylammonium (TEA) further blocked the remaining current leaving a very fast inactivating transient outward current. Therefore, at least three different types of K+ current were identified in Deiters' cells, such as fast activating and fast inactivating current, fast activating slow inactivating current, and very fast inactivating transient outward current. Physiological role of them needs to be established.

Development of Vestibular Organ and Cochlea / 대한이비인후과학회지

Inner ear is an important organ that is responsible for balance and hearing. It is composed of two complicated major subunits, vestibular organ and cochlea. During development, both functional units require complex genetic interactions to achieve proper patterning and morphology as its purpose. This review comprises three parts. In the first part, general information about technical tools in studying mouse ear development is introduced. In the second part, it is shown how semicircular canals are formed and what genetic interaction is involved in canal formation. In the last part, genetic interactions required for proper development of cochlea are elaborated, focusing on the length of cochlea and morphology of organ of Corti. The study about development of mutant mice provides us useful information about genetic interaction involved in the formation of structures for hearing and balance. The data acquired in the development field could provide a breakthrough to hair cell regeneration and stem cell therapy fields in stuck, which are directly applicable to human.

Analysis of the cytoprotective effect of amifostine on the irradiated inner ear of guinea pigs: an experimental study / Análise do efeito citoprotetor da amifostina na orelha interna irradiada de cobaias: estudo experimental

Radiation can cause damage to the inner ear, from a simple hearing loss all the way to profound deafness. Amifostine is a cytoprotective substance extensively used during radio-chemotherapy for malignant tumors. AIM: the objective of the present investigation was to establish the antioxidant and radioprotective effects of amifostine on the organ of Corti of albino guinea pigs irradiated in the head and neck region. MATERIALS AND METHODS: An experimental study conducted on four groups of guinea pigs were used; One group received only amifostine, one group was submitted to a single dose of 350 cGy and the other two were similarly irradiated but received amifostine doses of 100 or 200 mg/kg. All animals were slaughtered 30 days after the experiment, their bullae were removed and the damaged outer hair cells were counted. RESULT: The extent of injury was lower in the outer hair cells of the two groups treated with amifostine compared to the group that was only irradiated. There was no difference between the group treated with 100 and 200 mg/kg of amifostine. The group that received only amifostine had no cochlear damage. CONCLUSION: Amifostine is an effective cytoprotective substance in the Organ of Corti of irradiated guinea pigs.

A radiação pode causar lesão na orelha interna podendo provocar surdez sensório-neural e inclusive levar à anacusia. A amifostina é uma substância citoprotetora seletiva de tecidos sadios, amplamente utilizada durante a radio e quimioterapia de tumores malignos. OBJETIVO: O objetivo deste estudo experimental foi verificar se existe efeito antioxidante e radioprotetor da amifostina no órgão de Corti de cobaias albinas irradiadas em região de cabeça e pescoço. MATERIAL E MÉTODO: O estudo realizado envolveu quatro grupos de animais: um grupo foi submetido à irradiação em dose única de 350cGy. Dois grupos receberam a mesma dose de radiação, porém receberam doses de 100 e 200mg/kg de amifostina, 30 minutos antes da irradiação. Um grupo recebeu apenas amifostina, na dose de 200mg/Kg. Todas as cobaias foram sacrificadas 30 dias após o experimento e suas bulas retiradas para estudo em microscópio de varredura. RESULTADO: O grau de lesão das células ciliadas externas foi menor nos dois grupos que receberam a amifostina que no grupo apenas irradiado. Não foi encontrada diferença de proteção entre os grupos que receberam doses de 100 e 200mg/kg de amifostina. Não houve lesão no grupo que recebeu apenas amifostina. CONCLUSÃO: Amifostina mostrou ser um radioprotetor do órgão de Corti de cobaias albinas irradiadas.

Age impact on the eferent system actitivies in cochlear mechanical properties in normal hearing individuals / Influência da idade na atividade do sistema eferente nas propriedades mecânicas da cóclea de ouvintes normais

The medial olivocochlear tract has efferent control over the outer hair cells, regulating the slow contractions and damping the fast ones. Using ipsilateral, contralateral or bilateral otoacoustic emissions amplitude studies, it is possible to estimate the conditions of this tract, since the effect resulting from the reduction/suppression of these emissions indicate the tract's functioning. Aging implies an activity reduction in the central auditory system, because of the degeneration of the structures involved in hearing skills. AIM: our goal was to investigate the effects of age on the activities of this tract on the cochlea, through the analysis of the emissions' amplitude with contralateral acoustic stimulation. MATERIALS AND METHODS: Our series was made up of 75 individuals grouped according to age. The methodology was conventional, with a linear click and a white noise. CASE STUDY: the analysis considered the response from both ears and the comparison between the groups. RESULTS: the results show a statistically significant difference between the emissions' response with and without contralateral acoustic stimulation in the individuals between 20 and 39 years of age. The emissions reduction/suppression effect reduced with age (fourth decade). CONCLUSION: aging impairs the tract effectiveness.

O trato olivococlear medial realiza o controle eferente das células ciliadas externas, regulando as contrações lentas e atenuando as rápidas. Com a pesquisa da amplitude das emissões otoacústicas sem e com estimulação acústica contra, ipsi ou bilateralmente, é possível estimar as condições desse trato, uma vez que o efeito resultante de redução/supressão das emissões indica seu funcionamento. O envelhecimento implica em diminuição da atividade do sistema auditivo central, em função da degeneração das estruturas envolvidas nas habilidades auditivas. OBJETIVO: O objetivo foi investigar o efeito da idade na atividade do trato sobre a cóclea, com a análise da amplitude das emissões com estimulação acústica contralateral. MATERIAL E MÉTODO: A casuística foi composta por 75 indivíduos agrupados conforme a idade. A metodologia foi o modo convencional, com clique linear e o ruído branco. ESTUDO DE CASO: A análise considerou a resposta das orelhas e a comparação entre os grupos. RESULTADOS: Os resultados revelam diferenças estatisticamente significantes entre o response das emissões sem e com estimulação acústica contralateral, nos indivíduos (20 a 39 anos). O efeito redução/supressão das emissões diminui com a idade (quarta década). CONCLUSÃO: O envelhecimento prejudica a efetividade da atividade do trato.

Outward Rectifying Current in Isolated Deiters' Cells from Guinea Pig Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The Deiters' cell is one of the supporting cells in the organ of Corti and is known to possibly regulate the signal transduction pathway in the organ of Corti. The signal transduction process can be modulated by ATP and acetylcholine, the so-called neurotransmitters, in Deiters' cells. Intracellular Ca2+ concentration can be also increased by these neurotransmitters and the control mechanism on the organ of Corti is highly suggested in Deiters' cells. Potassium ion (K+) is known to be important both in hair cells and supporting cells. Through K+ channel, the membrane potential may be controlled and the signal transduction pathway can be regulated. Furthermore, the motility of outer hair cell and the signal transduction from the apical stereocilia are considered to be regulated by this channel. The aim of this study is to record the K+ current in the isolated Deiters' cells from guinea pig cochlea. MATERIALS AND METHODS: Deiters' cells were isolated from the organ of Corti of guinea pig by using collagenase and a pipet. A whole cell patch clamp was performed under the inverted microscope and the current was measured with List-7 amplifier and pClamp 8.0.2 software. RESULTS: The resting membrane potential was -15.02+/-2.66 mV (n=6). When the cell membrane was hyperpolarized into -110 mV from the -40 mV holding potential, the peak current was -227+/-39.9 pA (n=15). After having depolarized to the maximum, (50 mV), the peak current was 7123+/-737 pA, and the reversal potentials of different external K+ concentration changed in the K+-dependent manner. About 80% of this current was inhibited by TEA. When K+ was substituted by Cs+, the peak current was 1788+/-231 pA at 50 mV step pulse. Activation curve of this outward current showed two different Vh (half activation voltage) and K (slope factor). CONCLUSION: Outward rectifying K+ channels exist in Deiters' cells and they can be inhibited by TEA and permeable to Cs+. More than two types of K+ current can exist and they may play a role in the recovery of membrane potential after depolarization,

Organotypic Culture of Organ of Corti with Floating Drop Method from Newborn Rat / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Organotypic culture of organ of Corti maintains the basic organization of the spiral lamina and can conserve several factors responsible for the neuronal growth of the nervous components. The explant culture technique has been widely used in organ culture system, however, the floating drop method using collagen gel was also developed as a simple and reliable method. In order to study the effect of growth factors on the regenerative and protective ability of cochlear hair cells, we first had to establish an in vitro model of the inner ear. MATERIALS AND METHODS: Organ of Corti was obtained from newborn rats and cultured with the floating drop method using collagen gel. Immunohistochemical staining was used to visualize the stereocilia and scanning electron microscopic study was also carried out. RESULTS: Explants were maintained up to 10 days without contamination. Morphologically, immunofluorescent staining with phalloidin showed well preserved outer and inner hair cells with stereocilia on the second day of culture. On the tenth day of culture, the staining result showed inner and outer hair cells, although the stereocilia were poorly stained. In scanning electron microscopic examination, an explant on the tenth day of culture showed preserved outer and inner hair cells and stereocilia, although damaged hair cells and stereocilia were also observed. CONCLUSION: The floating drop method was an appropriate method for maintaining the organ of Corti in vitro with the advantage being the easiness in its manual manipulation.

In Vitro Cultivation of Organ of Corti from Neonatal Rat / 听力学及言语疾病杂志

Objective To establish reliable tissue culture methods of organ of Corti in neonatal Wistar Rat.Methods The organ of corti was aquired from Wistar Rat with the method of microdissection.The tissues were cultured by two different methods.Results Twenty-four hours after the tissue cultivation,new epithelia cells and fibroblast were found around the tissue.Inner hair cell、outer hair cell and supporting cell grew well.The tissue was stained by Acridine orange and Prodium iodine.It became green,meaning the tissue had good activity.Conclusion The culture of organ of corti we introduced is an a ideal method for otology research.

A SEM STUDY OF STRUCTURE IN THE RABBIT COCHLEAE / 解剖学报

Five inner ears were removed from five rabbits under aneasthesia. The hard, thick bones of outer and middle ears were trimmed off, and the auditory ossicles were taken away. The cochleae were fixed with 2％ glutaraldehyde in phosphate buffer for eight hours, then they were decalcified in 10％ EDTA-2Na solution for ten days, and the softened shells of the cochleae were stripped off, and post-fixed in 1％ OsO_4 for an hour, dried at critical point, coated with gold, and observed under SEM.The rabbit cochlea shows two and a half turns, the modiolus is about 3.5mm high, and the organ of Corti lies on the basilar membrane of the spiral lamina. The outer hair cells usually form three parallel rows on the middle and basal turns but occasionally four rows on the top half turn. The inner hair cells form only a single row. Every bunch of the outer hairs consists of about 93-114 stereocilia which together show a "W" shape. Every bunch of inner hairs has about 50 cilia which together show an arch shape. The cilia on the top turn are longer than those on the base. The length and width of the inner and outer hair cells and four other kinds of cells were measured according to photos, and the numbers of these kinds of cells were estimated.Other three cochleae fixed at the time of 1, 2 or 4 hours after death were also observed. In the cochlea fixed four hours after death, the majority of the auditory hairs were found fallen down, which means the fixation of cochlea should be carried out within less than four hours after death.Some of the structural similarities and differences of cochleae among rabbit,human being and guinea pig were also compared in this paper.