Plate Of Outer Hair Cells Research Articles

Sensorineural hearing loss and ischemic injury: Development of animal models to assess vascular and oxidative effects

Hearing loss may be genetic, associated with aging or exposure to noise or ototoxic substances. Its aetiology can be attributed to vascular injury, trauma, tumours, infections or autoimmune response. All these factors could be related to alterations in cochlear microcirculation resulting in hypoxia, which in turn may damage cochlear hair cells and neurons, leading to deafness. Hypoxia could underlie the aetiology of deafness, but very few data about it are presently available. The aim of this work is to develop animal models of hypoxia and ischemia suitable for study of cochlear vascular damage, characterizing them by electrophysiology and gene/protein expression analyses. The effects of hypoxia in infarction were mimicked in rat by partial permanent occlusion of the left coronary artery, and those of ischemia in thrombosis by complete temporary carotid occlusion. In our models both hypoxia and ischemia caused a small but significant hearing loss, localized at the cochlear apex. A slight induction of the coagulation cascade and of oxidative stress pathways was detected as cell survival mechanism, and cell damages were found on the cuticular plate of outer hair cells only after carotid ischemia. Based on these data, the two developed models appear suitable for in vivo studies of cochlear vascular damage.

Analysis of Cochlear Protein Profiles of Wistar, Sprague−Dawley, and Fischer 344 Rats with Normal Hearing Function

Differences in the expression of cochlear proteins are likely to affect the susceptibility of different animal models to specific types of auditory pathology. However, little is currently known about proteins that are abundantly expressed in inner ear. Identification of these proteins may facilitate the search for biomarkers of susceptibility and intervention targets. To begin to address this issue, we analyzed cochlear protein profiles of three strains of rats, Wistar, Sprague-Dawley, and Fischer 344, using a broad spectrum antibody microarray. Normal hearing function of the animals was ascertained using distortion product otoacoustic emissions (DPOAE). Of 725 proteins screened in whole cochlea, more than 80% were detected in all three strains. However, there were striking differences in the levels at which they occur. Among 213 proteins expressed at levels>or=2 fold of actin, only 7.5% were detected at these levels in all three strains. Myosin light chain kinase (MLCK) was immunolocalized in cuticular plate of outer hair cells (OHC) while mitogen activated protein (MAP) kinase-extracellular-signal regulated kinase1/2 (ERK1/2) was detected as foci in OHC, pillar cells, strial marginal cells, and fibroblasts of spiral ligament. A review of literature indicated that the expression of 7 (44%) of these 16 proteins were detected for the first time in the inner ear, although there were implications of the presence of some of these proteins. One of these abundant, but unstudied, proteins, MAP kinase activated protein kinase2 (MAPKAPK2), shows strong immunolabeling in pillar cells and inner hair cells (IHC). There was moderate MAPKAPK2 labeling in OHC, supporting cells, neurons, and marginal, intermediate, and basal cells. The current study provides the first, large cochlear protein profile of multiple rat strains. The diversity in expression of abundant proteins in these strains may contribute to differences in susceptibility of these strains to aging, noise, or ototoxic drugs.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine β-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen’s cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.

Immunohistochemical Localization of Cyclooxygenase Isoforms in the Organ of Corti and the Spiral Ganglion Cells of Guinea Pig Cochlea

Prostaglandins have been used in experimental models and clinical studies for the therapy of sudden hearing loss and tinnitus with conflicting results. However, little is known about the rate-limiting enzymes of prostaglandin synthesis in the inner ear, the generally constitutively expressed cyclooxygenase 1 (COX-1) and the distress-inducible cycloxygenase 2 (COX-2). To extend our knowledge concerning the physiological expression and localization of these two enzymes, immunohistochemical stainings of the guinea pig cochlea were performed. Light microscopical analysis revealed a homogenous distribution of COX-1 within nearly all cell types of the organ of Corti, but no COX-1 expression in the cuticular plates of pillar cells. COX-2 was found to be expressed in all cell types, with much stronger expression in Hensen cells, neighboring Deiters cells and cuticular plates of outer hair cells. Both COX-1 and COX-2 immunoreactions were also found in the spiral ganglion. We conclude that both COX subtypes are expressed in the guinea pig cochlea under physiological conditions. The prominent expression of the distress-inducible COX-2 isoform in cell types under mechanical stress during noise reception might support the hypothesis of a cytoprotective function of COX products in hearing and in cellular stress situations like intense noise exposure.

Degenerative changes in the organ of Corti and lateral cochlear wall in experimental endolymphatic hydrops and human Menière's disease.

The pathogenesis of sensorineural hearing loss in Menière's disease and experimental endolymphatic hydrops is not fully understood. At the light microscopic level, there is poor correlation between the histopathology and loss of sensitivity and speech discrimination. The results of electron microscopic investigation of histopathology and alterations in immunoreactivity in the organ of Corti and lateral cochlear wall in the hydropic guinea pig are presented. Loss of outer and inner hair cells and spiral ganglion cells, particularly in the apical turn was evident by light microscopy. By electron microscopy, further evidence of degeneration was detected in the cuticular plate of outer hair cells, neural endings of both inner and outer hair cells, myelinated dendritic fibers, spiral ganglion cells, and types I and II fibrocytes of the lateral cochlear wall. There was a marked decrease in immunoreactivity for a variety of enzymes, calcium binding proteins, structural proteins, and integral membrane proteins of gap junctions, particularly among type I and type II fibrocytes of the lateral cochlear wall. The evidence suggests that dysfunction and degeneration of hair cells, afferent neurons and fibrocytes of the lateral cochlear wall are involved in the pathogenesis of hearing loss in endolymphatic hydrops.

Ultrastructure of the horseshoe Bat's organ of corti. II. Transmission electron microscopy

The fine structure of the organ of Corti was investigated in the echolocating horseshoe bat (Rhinolophus rouxi) by transmission electron microscopy. Particular emphasis was placed on the receptor cells and their supporting cells. The receptor cells, inner hair cells (IHC) and outer hair cells (OHC), possess the typical mammalian shape, but OHCs are extremely short (length: 12-15 microns in the basal turn and up to 28-30 microns in the apical turn). The afferent innervation of both types of receptor cells and the efferent innervation of the IHC system conform to the general mammalian scheme; however, confirming earlier reports, an efferent innervation to the OHCs is absent. Throughout the cochlea, IHCs and OHCs possess a single layer of subsurface cisternae. Above the level of the nucleus of the OHCs, the arrangements of the subsurface cisternae and their connection to the lateral cell membrane via pillars are highly regular, whereas in IHCs, the cisternae are of irregular shape and the pillar system is much less distinct. In the basal turn of the cochlea, the attachment sites of the OHCs to the supporting cells possess specialized features: (a) in the reticular lamina, the contact sites of the cuticular plates of OHCs with the outer pillar cells and the Deiters cell phalanges are of exaggerated length, and (b) the cup formation of the Deiters cell body, which houses the bottom of the OHC, has a specialized shape and is packed with electron-dense material and microtubules. The results are discussed in relation to cochlear ultrastructure in other mammals and in the context of active processes in cochlear mechanics.

Actin-binding and microtubule-associated proteins in the organ of Corti

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.

Qualitative assessment of postnatal maturation of the organ of Corti in two rat strains

The radial width of three rows of cuticular plates of outer hair cells (width of the OHC triad), the distribution of inner hair cells along the organ of Corti, and postnatal maturation of the middle ear were examined in rats of the Srague-Dawley and Lewis strains aged 0–24 days and two months. The width of the OHC triad is a good indication of the course of maturation of the reticular lamina and the organ of Corti in different regions of the cochlea. The width of the OHC triad in adult rats decreases continuously from the apex toward the base, while in newborn pups the trend is just the reverse. Thus, there is a region of the cochlea in which the width of the OHC triad is the same, or changes insignificantly, from birth to adulthood. This particular region (where the process of postnatal maturation of the organ of Corti begins?) corresponds to the region of maximum density of inner hair cells and tonotopically to the region of ‘best hearing’. The width of the OHC triad will reach its adult values earlier in the basal than in the apical regions. The time-course of middle ear maturation correlates well with the time-course of maturation of the reticular lamina. The parameters under study are in many respects strain-specific.

Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells

The distribution of actin, alpha-actinin, fimbrin, tropomyosin and tubulin in the apical region of inner and outer hair cells was studied by immunofluorescent localization of antibodies to these proteins. The macromolecular distribution of actin and alpha-actinin was studied using post-embedding immunoelectron microscopic techniques. Actin is present in the stereocilia and cuticular plate of both inner and outer hair cells. Antibodies to actin were localized with fluorescence and colloidal gold. Colloidal gold particles were distributed uniformly over the stereocilia, stereocilia rootlets and cuticular plate. Fimbrin is present in the stereocilia and the cuticular plate. Immunofluorescent label was more intense over the cuticular plate of outer hair cells than over the cuticular plate of inner hair cells. Alpha-actinin is present in the cuticular plate only. At the ultrastructural level, antibodies to alpha-actinin were labeled throughout the cuticular plate, with larger accumulations of colloidal gold over the electron dense bodies in the cuticular plate, as well as over the electron dense region at the junctional complex. There was no label over the electron dense portion of the stereocilia rootlets. Tropomyosin is observed in the area of the stereocilia rootlets by immunofluorescent techniques, but like fimbrin, the antigenic sites of tropomyosin did not withstand processing for ultrastructural localization. Tubulin is not present in the apical region of inner or outer hair cells, although its presence could be documented in the hair cell body and in the supporting cells.