Biomedical subjects

Y Raphael

Publications and source records attributed to Y Raphael.

At least 55 records · Page 3Linked to original sources

PubMed1996

Re-innervation patterns of chick auditory sensory epithelium after acoustic overstimulation.

There is evidence from several studies showing that sensory cells which are destroyed by trauma in the chick auditory epithelium are replaced by new cells. The fate of neurons that innervate the injured and degenerating sensory cells in the lesion, and the temporal sequence of re-innervation of regenerated hair cells are not well understood. This study examined efferent terminals in the chick auditory sensory epithelium following acoustic overstimulation using synapsin-specific immunocytochemistry. Chicks were exposed to an octave band noise (1.5 kHz center frequency, 116 dB SPL, 16 h) and killed on each day from 0 to 9 days postexposure. In the proximal half of control whole mounts of the basilar papillae, synapsin-specific immunoreactivity stained efferent terminals throughout the abneural portion of the sensory epithelium (the short hair cell region). In this area, the labeling appeared as 2-3 bouton-shaped clusters along the abneural edge of each hair cell. After acoustic overstimulation, a lesion was observed at the abneural edge of the papilla where many short hair cells were lost. The center of the lesion was located at 40% distance from the proximal end of each traumatized papilla. Synapsin-specific labeling was not found in sites where expanded supporting cells had replaced missing hair cells. Hair cells which survived the trauma exhibited a shrunken apical area, and synapsin-labeled boutons were observed near their basal domains. New hair cells, which first appeared in the papilla 4 days after trauma, were not initially associated with synapsin-labeled boutons. Regenerated hair cells first displayed contacts with synapsin-labeled boutons 7 days after trauma. Nine days after acoustic overstimulation, most new hair cells appeared to be associated with synapsin-labeled boutons which resembled the normal horseshoe configuration of efferent terminals. The data suggest that direct contact with functional efferent synapses is not necessary for the generation and differentiation of new hair cells.

Acoustic Stimulation↗

PubMed1995

Monoclonal antibody induced hearing loss.

Monoclonal antibodies KHRI-3 and KHRI-5 identify antigens expressed on inner ear supporting cells and auditory hair cells respectively. To determine if these antibodies affect inner ear function groups of syngeneic Balb/c mice were inoculated with hybridomas KHRI-3, KHRI-5 and other Ig-secreting hybridomas. Hybridomas UM-A9, UM-7F11, the non-secreting SP2/0 myeloma and mice with no hybridoma were used as controls. Animals were tested for auditory brainstem responses (ABR) for frequencies of 4, 8, 16 and 24 kHz, before the inoculation of the hybridomas and at intervals of 6 to 10 days thereafter or daily once tumors became palpable. In normal mice there were no changes in ABR thresholds over the course of the experiment. Other control animals showed little change in ABR even when the growth of the hybridoma or myeloma tumors were far advanced. Of the KHRI-5 hybridoma bearing animals only one of seven animals exhibited threshold shifts greater than 15 dB. In contrast, most mice bearing the KHRI-3 hybridoma exhibited high frequency threshold shifts of 40-50 dB that coincided temporally with the growth of the hybridoma, the presence of circulating KHRI-3 antibody, and greatly increased immunoglobulin titers. Ears from KHRI-3-bearing mice that developed high frequency hearing loss also had a novel type of lesion in the basal turn of the cochlea that was characterized by loss of outer hair cells and absence of typical supporting cell scars. Such changes were not found in control hybridoma-bearing mice. These findings suggest that KHRI-3 antibody has an effect on hearing that is secondary to damage to the organ of Corti and loss of outer hair cells. Our results have important implications for antibody-mediated mechanisms of hearing loss and provide an animal model in which to study this phenomenon.

Acoustic Stimulation↗

PubMed1995

Tectorial membrane regeneration in acoustically damaged birds: an immunocytochemical technique.

A novel immunocytochemical method was used to determine whether the sound-damaged adult quail ear can repair its tectorial membrane (TM) and to compare the repair in quail to that in chicks. Birds were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 h. The chicks were grouped based on recovery duration (0 and 7 days), while the quail were divided into 0-, 7-, and 14-day recovered groups. At the end of the recovery period, the animals were sacrificed, and their basilar papillae labeled with a TM-specific monoclonal primary antibody solution followed by a diaminobenzidine process. Examinations under a stereoscope revealed that a patch lesion devoid of TM was located on all 0-day recovered papillae. Seven days later, a honeycomb-patterned layer was observed covering the lesion. In 14-day recovered quail ears, the honeycomb layer appeared similar to that seen at 7 days post-exposure. These observations indicated that both chicks and quail were able to repair their TM within 7 days following exposure to intense sound.

Acoustic Stimulation↗

PubMed1995

Comparative analysis of patch lesions in the chick inner ear following acoustic trauma: optical versus scanning electron microscopy.

Neonatal chicks were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 hours. Seven days following overstimulation, the birds were sacrificed. Their basilar papillae were removed, fixed in 4% paraformaldehyde, and processed in two steps. First, the ears were immunostained with a supernatant of mouse anti-tectorial membrane antibodies, followed by a diaminobenzidine process. Examinations of the papillae under an optical stereo microscope revealed a patch site with a partially regenerated tectorial membrane (referred to as the honeycomb). After the optical studies, the same ears were post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and processed for scanning electron microscopy (SEM). SEM examinations demonstrated a honeycomb-covered patch lesion in the papilla. Patch lesion perimeters were traced from both the optical and SEM images, and patch areas were calculated. Also, papilla height was measured at the midpoint of the inner ear in both groups. These calculations showed that the patch area and papilla height had shrunk by approximately 37% and 33%, respectively, following the SEM methodology. The decrease in these dimensions may be attributed to several steps required for the SEM specimen preparation, such as critical point drying.

Animals↗

PubMed1994

F-actin, tubulin and spectrin in the organ of Corti: comparative distribution in different cell types and mammalian species.

Laser scanning confocal microscopy was used to determine the distribution of actin, spectrin and tubulin in whole mounts of the organ of Corti of guinea pig, monkey, rat and chinchilla. Actin, spectrin and tubulin were localized in all cell types in the auditory epithelium. No specialized cytoskeletal organization of tubulin was detected in the cytoplasmic domain of hair cells. The only specialized organization of actin and spectrin in the cytoplasmic domain was the infra-cuticular network, found exclusively in apical guinea pig outer hair cells. In contrast, the lateral wall of inner and outer hair cells contained a homogeneous distribution of label specific for actin and spectrin. The label intensity was similar in the base and the apex of the cochlea. These results indicate that the distribution of spectrin and actin in the auditory epithelium is similar to that in other epithelial cells, suggesting that actin and spectrin participate in the formation of cellular shape and possibly in docking molecules to the membrane.

Actins↗

PubMed1994

Cell cycle of transdifferentiating supporting cells in the basilar papilla.

Mitosis of supporting cells has been shown to contribute to the cellular repopulation of the basilar papilla after acoustic trauma. In the present work we report data obtained with light and transmission electron microscopy after acoustic trauma in chicks. We report changes that occur in cell shape, surface morphology, intercellular junctions, nuclear shape and location, and cytoplasmic organization of supporting cells after trauma. The findings strongly suggest that supporting cells transdifferentiate and that the proliferative pattern is similar to interkinetic nuclear migration, as previously shown in the developing neural tube and basilar papilla. S-phase nuclei were positioned adjacent to the basement membrane, suggesting that interaction with the extracellular matrix may occur during the cell cycle. Supporting cells divided with the long axis of the spindle parallel to the reticular lamina and displayed no signs of intercellular communication during mitosis. This suggested to us that the fate of the progeny cells is determined prior to mitosis and that the progeny may be of identical phenotypic fate. Dividing cells had a smooth apical surface. The smooth surface may provide a marker to help identify dividing cells with scanning electron microscope analysis.

Animals↗

PubMed1994

Return of auditory function following structural regeneration after acoustic trauma: behavioral measures from quail.

After measuring baseline behavioral audiograms, three of four behaviorally trained quail and fifteen untrained cohorts were exposed to a 1.5-kHz octave-band noise at 116-dB SPL for 4 h. The trained birds were tested daily following the exposure and showed a steady recovery of absolute sensitivity with a return to normal absolute thresholds by post-exposure days 8-10. Thirteen untrained cohorts were sacrificed after various survival times to evaluate the structural condition of the ear. The cohorts all showed regeneration of sensory cells similar to that seen in chicks. The effects of repeated acoustic trauma on recovery of sensitivity were evaluated by re-exposing the three trained birds and two untrained cohorts 106 days after the first exposure. One of the trained birds was exposed a third time, 113 days following the second exposure. The findings demonstrate that, following acoustic trauma, normal sensitivity returns prior to complete structural regeneration of the sensory epithelium and that repeated acoustic trauma may increase the time course of recovery of normal hearing sensitivity.

Animals↗

PubMed1994

Return of auditory function following structural regeneration after acoustic trauma: behavioral measures from quail.

Acoustic Stimulation↗

PubMed1994

Scar formation in the vestibular sensory epithelium after aminoglycoside toxicity.

Hair cell degeneration and the repair process due to differing types of trauma have been studied extensively in the organ of Corti. It has been determined that, during scar formation, after differing types of trauma to the auditory sensory system, the reticular lamina is maintained with adherens junctions and tight junctions. We investigated the repair process within the vestibular epithelium. Hair cell degeneration was induced by the unilateral application of streptomycin to the inner ears of guinea pigs. Whole mount preparations of all five vestibular organs were processed and examined by fluorescence, light and electron microscopy. Scar formation was seen as early as 4 days post-treatment with streptomycin and was noted to coincide with hair cell degeneration. Neighboring supporting cells swelled and filled the space beneath the degenerating hair cell. Between three and five supporting cells participate in the reparative process. The distribution of cytokeratin is also altered during scar formation. The area once occupied by the hair cell becomes filled with cytokeratin-rich processes of supporting cells. It appears that differing numbers of supporting cells are involved in the reparative process within the vestibular sensory epithelium as compared to the auditory system. The reticular lamina remains intact at all times. This may possibly prevent mixing of fluids between different compartments in the inner ear and dysfunction of the vestibular sensory organs.

Actins↗

PubMed1994

Intercellular contacts between chick stereocilia after acoustic overstimulation.

The goal of this study was to analyze the distribution of actin and the shape of stereocilia of chick hair cells that survive acoustic trauma. Chicks were exposed to intense octave band noise for 4 h. They were killed either immediately after the exposure, after 6 or after 72 h. The basilar papillae were examined using scanning electron microscopy and fluorescence microscopy, with phalloidin as an actin-specific probe. Injured hair cells which survived the trauma displayed disorganized stereocilia bundles, elongated stereocilia, and supernumerary stereocilia bundles. Tips of stereocilia in the damaged region of the basilar papilla appeared to be in contact with tips of stereocilia of neighboring hair cells. These contacts may represent 'stress links' which appear in traumatized hair cells. These results show that substantial changes in stereocilia occur within hours of exposure to intense noise. We speculate that surviving hair cells may play a role in the process of repair of the basilar papilla after noise trauma and that the changes in stereocilia structure described here are related to this role.

Acoustic Stimulation↗

PubMed1994

Distribution of cytokeratins in the vestibular epithelium of the guinea pig.

Cytokeratin expression in the vestibular labyrinth of the guinea pig was investigated with immunofluorescence and immunoperoxidase staining on surface preparations of the vestibular epithelium. Phalloidin, an F-actin-specific probe, was used to distinguish between hair cells and supporting cells. Cytokeratin expression was not found in the cytoplasmic domain of hair cells of the crista ampullaris, utricle, or saccule. Cytokeratin expression was abundant in supporting cells of the vestibular sensory epithelium. Electron microscopy revealed the presence of desmosomes, which are associated with cytokeratins, within type 2 hair cells of the vestibular epithelium. It appears that cytokeratins are absent within the cytoplasmic domain of hair cells, but are present in association with intercellular junctions. The functional significance of this unique pattern of cytokeratin expression within the vestibular epithelium is discussed.

Animals↗

PubMed1993

Reorganization of the chick basilar papilla after acoustic trauma.

The auditory epithelium in birds and mammals consists of a postmitotic population of hair cells and supporting cells. Unlike mammals, birds can regenerate their auditory epithelia after trauma. Recent evidence indicates that supporting cells undergo mitosis after acoustic trauma, suggesting that supporting cells may transdifferentiate into hair cells. The goals of this study were to 1) characterize the responses of hair cells and supporting cells to acoustic trauma, and 2) determine whether hair cell loss is a prerequisite for generation of new hair cells. Chicks were exposed to an octave-band noise and their inner ears assayed with fluorescence or scanning electron microscopy. In one area of the basilar papilla, defined as the center of the lesion, extensive hair cell degeneration occurred. Expanded supporting cells obliterated degenerating hair cells and invaded spaces normally occupied by hair cells. Aggregates of DNA were found within the basilar papilla, suggesting that hair cell death and disintegration may occur within the epithelium. The epithelial sheet appeared structurally confluent at all times examined. Supporting cells exhibited altered apical contour in distal regions of the basilar papilla, where hair cell damage was mild or inconspicuous. Four days after noise exposure, newly generated hair cells were found in the center of the lesion and in the distal areas, where no hair cell loss could be detected. The results suggest that supporting cells may play an important role in maintenance and repair of the traumatized basilar papilla and raise the possibility that production of new hair cells is not dependent on hair cell loss in the immediate vicinity.

Animals↗

PubMed1993

3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure.

Fluorescence microscopy can be a useful tool in the early detection of pathological changes in the stereocilia of outer hair cells which have undergone acoustic overstimulation. Fluorescent phalloidin, a highly specific F-actin stain, can be used to label F-actin in stereocilia. In this study, phalloidin label is used to determine quantitative changes of F-actin in the stereocilia of guinea pigs exposed to loud noise (117 dB; octave band noise, centered at 1 kHz; 4 h). Reliably determining three-dimensional (3-D) structural changes in stereocilia is a challenging problem in optical microscopy since stereocilia diameter is close to the optical resolution limit. In order to alleviate the problem, a computational 3-D microscopy technique is used (Avinash et al., 1992). Whole-mounts of the cochlear second and third turns were examined in a Leitz Orthoplan microscope through a Leitz Plan Apo objective lens (100 x; 1.32 N.A.; 170/0.17). Images were acquired with a charge-coupled device camera where the focus was shifted in 0.2 microns steps using a piezoelectric translator. Images were processed with the appropriate point spread function of the optical system. Analysis of control cochleas indicate that our technique can resolve single stereocilia and distinguish between various intensities of label along each stereocilia. In noise-exposed cochleas, our data show length and intensity changes in the phalloidin label. These results suggest that both depolymerization and polymerization of F-actin can occur in stereocilia of outer hair cells after acoustic overstimulation. Our findings demonstrate the applicability of computational 3-D microscopy to quantitative and qualitative analysis of stereocilia.

Actins↗

PubMed1993

Structure of the reticular lamina and repair after noise injury.

Laser Scanning Confocal Microscopy (LSCM) and specific labeling techniques were employed to examine the distributing of F-actin and microtubules in the reticular lamina of the guinea pig and monkey organ of Corti. Actin specific label was found in the circumferential belt of adherens junction at the borders between cells in the reticular lamina, and in the cuticular plate of hair cells. The distribution of actin in the adherens junction belt was asymmetric. Actin label was not found in the fonticulus, where the microtubule organizing center resides. Actin free areas were also found between the junctional actin and the cuticular plate. Microtubule specific label was very intense in supporting cells. In normal hair cells, the spatial distribution of tubulin at the reticular lamina is mutually exclusive with that of actin. After noise exposure, a belt of actin was found in the central portion of degenerating outer hair cells, possibly representing a constricted circumferential junction. Expanded supporting cells replaced degenerating hair cells and maintained the confluence of the reticular lamina during the dynamic process of scar formation. A complex network of actin-rich cables appeared at sites of degenerating inner hair cells, suggesting that more than two supporting cells are involved in scar formation for inner hair cells. LSCM proved an attractive method for analysis of the organ of Corti since preparation of the tissue is relatively rapid, preparation artefacts are minimized, different markers in the same specimen may be co-localized and out-of focus fluorescence blurring is eliminated.

Actins↗

PubMed1992

Evidence for supporting cell mitosis in response to acoustic trauma in the avian inner ear.

Acoustic overstimulation can lead to sensory cell (hair cell) loss in the auditory epithelium. Damaged hair cells in the organ of Corti (the mammalian auditory end-organ) degenerate and are replaced by non-sensory cells (supporting cells) which construct an irreversible scar. In birds, however, auditory hair cells which are damaged by acoustic trauma or ototoxic drugs may be replaced by new hair cells. As first step in determining the mechanism of hair cell regeneration, we developed an assay for cell divisions in the auditory epithelium after acoustic trauma. The results of these experiments demonstrate that supporting cells in damaged regions of the auditory epithelium incorporate the DNA-specific marker bromodeoxyuridine as early as one day after noise exposure. We provide direct evidence that following acoustic insult to the avian inner ear, supporting cells which reside within the sensory epithelium divide near the luminal surface and repopulate the epithelium. These results suggest that supporting cells participate in scar formation during hair cell degeneration, and produce new cells for regeneration.

Animals↗

PubMed1992

Early microfilament reorganization in injured auditory epithelia.

Microfilaments (MFs) play an important role in wound healing and other regenerative events. The purpose of this study was to characterize changes in the distribution of MFs in traumatized auditory epithelia and compare these changes between avian (regenerating) and mammalian (nonregenerating) ears. Chicks and guinea pigs were acoustically overstimulated and their auditory epithelia analyzed fluorescence microscopy with phalloidin as a MF-specific marker. Immediately or several hours after overstimulation, we observed a substantial reduction of MFs in stereocilia and the cuticular plate. The circumferential belt of MF which is associated with the adherens junctional complex was constricted in damaged hair cells (HCs) as early as 1 day after the exposure. Concomitant with the junctional constriction, the apical surface area of supporting cells was increased relative to normal, whereas the surface area of HCs was decreased. We conclude the changes in the amount and distribution of MFs which characterize early responses to acoustic damage are similar in avian (regenerating) and mammalian (nonregenerating) auditory epithelia. We hypothesize that changes in MF-mediated tensile forces trigger the process of tissue repair in auditory epithelia in response to insult. In mammals the reorganization of MFs may help maintain the integrity of the reticular lamina and thereby prevent further damage. In contrast, early changes in MFs in chicks may play a role in regulating regenerative tissue responses.

Acoustic Stimulation↗

PubMed1991

The sensory epithelium and its innervation in the mole rat cochlea.

The mole rat (Spalax ehrenbergi) burrows throughout its life in subterranean tunnels. Several structural and functional features which adapt the mole rat to its habitat have been discerned. The goal of this study was to elucidate the structural basis for adaptation of the auditory end-organ to an environment where low-frequency acoustical signals prevail. For this purpose, cochleae of adult mole rats were studied with light and electron microscopy. Inner hair cells throughout the cochlea, and outer hair cells in the basal (high-frequency) portion of the cochlea, were similar in structure to those seen in other mammals. In contrast, outer hair cells in the apical (low-frequency) portion displayed unique structural features. These features resembled the structure of inner hair cells or immature outer hair cells. The innervation of outer hair cells was most uncommon, in that classical medial efferent innervation was not found throughout the cochlear spiral.

Animals↗

PubMed1991

Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration.

Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of microfilaments, intermediate filaments, and tight junction-specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub-apical region of the dying hair cell. This region becomes cytokeratin-positive. The two supporting cells meet at the mid-line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens-type junction, but desmosomes are absent.

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