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Biomedical subjects

Y Raphael

Publications and source records attributed to Y Raphael.

At least 37 records · Page 2Linked to original sources
PubMed1999

The influence of interleukin-1 receptor antagonist transgene on spiral ganglion neurons.

The cytokine interleukin-1beta (IL-1) has been shown to induce the secretion of NGF and GDNF in several types of neuronal populations. IL-1 has also been shown to mediate immune response following trauma or presence of foreign antigens. We investigated the influence of an IL-1 antagonist on the survival of spiral ganglion neurons in inner ears in which hair cells have been eliminated. We used a replication-deficient adenoviral vector containing the human IL-1 receptor antagonist (IL-1ra) cDNA. Guinea pigs were bilaterally deafened with ototoxic drugs. One week later their left cochleae were inoculated with the IL-1ra vector, designated Ad.IL-1ra. The vector was delivered by injection through the cochlear round window. IL-1ra protein levels within the perilymph of Ad.IL-1ra-injected animals were measured with ELISA and found to be significantly elevated compared to our controls. Spiral ganglion cell counts in experimental ears revealed a lower density of neurons after Ad.IL-1ra inoculation. Taken together, the data suggest that the Ad.IL-1ra-infected cochlear cells synthesized the transgenic human IL-1ra protein, which was then secreted by the cells into the perilymph, resulting in an accelerated neuronal degeneration in hair cell-depleted ears.

Animals↗
PubMed1999

Cochlear gene transfer: round window versus cochleostomy inoculation.

Two possible approaches for cochlear gene transfer have been inoculation via the round window membrane and through a cochleostomy. The aim of this study was to determine which of the two is more effective. Using both approaches, normal-hearing and deafened guinea pigs were inoculated with adenovirus carrying the reporter gene lacZ. After 5 days, the animals were killed and the cochlear tissue was stained with X-gal. The distribution and intensity of staining was estimated by a score system developed to compare gene transfer results between animals. We found that gene transfer via the cochleostomy resulted in a better distribution throughout the cochlea and in higher staining intensity, due to more efficient transfection. Auditory brainstem response (ABR) results showed that neither virus inoculation through a cochleostomy nor through the round window membrane had a significant effect on the click-ABR threshold measured on day 5 following virus injection. Gene transfer via both approaches was also found to be more effective in deafened animals than in hearing animals.

Adenoviridae↗
PubMed1999

Morphological and physiological effects of long duration infusion of strychnine into the organ of Corti.

Acute strychnine administration has long been used as a method to eliminate the effects of efferent activity. It has been shown that long after termination of chronic strychnine infusion into the cochlea, the ear becomes more susceptible to acoustic trauma suggesting that chronic strychnine infusion results in long lasting or permanent disruption of efferent function. Much research has been directed towards the functional significance of the olivocochlear system. However, there is little information concerning the effect of long duration inactivation of the medial olivocochlear system in an awake behaving animal. This study was designed to determine the structural and functional consequences of inactivation of the efferents by chronic infusion of strychnine into the cochlear perilymph of guinea pigs for two weeks via an osmotic pump. Physiological evaluations showed that the strychnine infusion eliminated the efferent induced reduction of the cochlear whole-nerve action potential three weeks after cessation of strychnine infusion. Contralateral efferent function remained unaltered. Histological evaluation at the light and electron microscopic levels revealed disoriented efferent synapses under the outer hair cells.

Acoustic Stimulation↗
PubMed1998

Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene.

The shaker-2 mouse mutation, the homolog of human DFNB3, causes deafness and circling behavior. A bacterial artificial chromosome (BAC) transgene from the shaker-2 critical region corrected the vestibular defects, deafness, and inner ear morphology of shaker-2 mice. An unconventional myosin gene, Myo15, was discovered by DNA sequencing of this BAC. Shaker-2 mice were found to have an amino acid substitution at a highly conserved position within the motor domain of this myosin. Auditory hair cells of shaker-2 mice have very short stereocilia and a long actin-containing protrusion extending from their basal end. This histopathology suggests that Myo15 is necessary for actin organization in the hair cells of the cochlea.

Amino Acid Sequence↗
PubMed1998

Role of glutathione in protection against noise-induced hearing loss.

A potential mechanism of hearing loss due to acoustic overstimulation is the generation of reactive oxygen species (ROS). ROS not removed by antioxidant defenses could be expected to cause significant damage to the sensory cells of the cochlea. We studied the influence of the antioxidant glutathione (GSH) on noise-induced hearing loss by using l-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of GSH synthesis, and 2-oxothiazolidine-4-carboxylate (OTC), a cysteine prodrug, which promotes rapid restoration of GSH when GSH is acutely depleted. Pigmented female guinea pigs were exposed to broadband noise (102 dB SPL, 3 h/day, 5 days) while receiving daily injections of BSO, OTC, or saline. By weeks 2 and 3 after noise exposure, BSO-treated animals showed significantly greater threshold shifts above 12 kHz than saline-treated subjects, whereas OTC-treated animals showed significantly smaller threshold shifts at 12 kHz than controls. Histologically assessed noise-induced damage to the organ of Corti, predominantly basal turn row 1 outer hair cells, was most pronounced in BSO-treated animals. High performance liquid chromatographic analysis showed that OTC significantly increased cysteine levels, but not GSH levels, in the cochlea. These findings show that GSH inhibition increases the susceptibility of the cochlea to noise-induced damage and that replenishing GSH, presumably by enhancing availability of cysteine, attenuates noise-induced cochlear damage.

Animals↗
PubMed1998

Interactive effects of aging with noise induced hearing loss.

Aging is associated with a down-regulation in metabolism, which may underlie an increased sensitivity to stress agents and a decreased repair of tissues following stress. In the auditory system this could lead to increased sensitivity to noise induced hearing loss (NIHL) with age. This study examines the effects of high intensity noise exposure (0.5-40 kHz noise at 108 dB SPL for 45 min) on young and old normal (CBA/Ca) mice and young premature presbycusis (C57BL/6) mice. Tone evoked auditory brainstem response (ABR) audiograms were obtained before and after the exposure. The animals were sacrificed and cytocochleograms were performed. The ABR threshold shifts and hair cell losses which followed noise exposure increased with increasing age in the normal (CBA/Ca) mice. Subjects which showed early presbycusis (C57BL/6) associated with vascular pathology, showed an increased sensitivity to noise induced hearing loss over normal subjects. Interestingly, in some cases the physiological loss in C57BL/6 mice was not associated with hair cell loss. These findings support the view that aging with or without hearing loss increased the sensitivity of the ear to NIHL. However, it seems that other factors may contribute to NIHL in the animals with premature presbycusis.

Aging↗
PubMed1997

Human autoantibodies and monoclonal antibody KHRI-3 bind to a phylogenetically conserved inner-ear-supporting cell antigen.

Autoimmunity is thought to be one cause of sensorineural hearing loss (SNHL). Sera from patients with rapidly progressive hearing loss have been shown to contain antibodies to a 68-kD protein in heterologous inner-ear tissue. Using guinea pig inner-ear tissue as the antigenic substrate and either Western blot or immunofluorescence (IF) or both, we tested sera from 74 patients suspected to have autoimmune hearing loss for inner-ear antibodies. Sera from 73 patients were tested by Western blot, and sera from 36 were tested by IF. Thirty-seven of 73 (51%) had antibody to a 68-70-kD protein by Western blot. Sera positive by IF stained supporting cells with a staining pattern like that previously observed with the KHRI-3 monoclonal antibody. There was concordance between Western blot and IF assays. Of 36 patients tested by both assays, 29/31 (94%) that were positive in Western blot were also positive by IF, three were negative by both tests, and two each were positive by one assay but negative by the other. Absorption of patient sera with human inner-ear tissue removed antibody reactivity to the guinea pig supporting cells, indicating that the antigen detected by the autoantibody is also present in the human inner ear. Absorption with an equal volume of white or red blood cells from the tissue donor did not remove the antibody reactivity to inner ear, showing that the absorption by inner-ear tissue is specific. Sera from three patients positive in both assays also stained a 68-70-kD inner-ear protein immunoprecipitated by the KHRI-3 monoclonal antibody, indicating that the monoclonal and human antibodies recognize the same antigen. The results support the hypothesis that patients with autoimmune sensorineural hearing loss produce autoantibodies to an inner-ear supporting cell antigen that is phylogenetically conserved and defined by the murine monoclonal antibody KHRI-3. Since KHRI-3 can induce hearing loss after infusion into the inner ear, it is likely that autoantibodies with the same antigenic target are also pathogenic in humans.

Animals↗
PubMed1997

Effects of chronic high-rate electrical stimulation on the cochlea and eighth nerve in the deafened guinea pig.

This study was undertaken to examine the effects of chronic high-rate stimulation on the eighth nerve and cochlea. Fifty-four male pigmented guinea pigs were deafened and implanted with single ball electrodes in scala tympani. Four groups of animals received chronic electrical stimulation at a level of 5 microCol/cm2/ph for 1000 h as follows: Group A: 1000 Hz, 100 microseconds/ph duration, 100 microA peak; Group B: 250 Hz, 100 microseconds/ph duration, 100 microA peak; Group C: 2750 Hz, 36 microseconds/ph duration, 250 microA peak; Group D: 250 Hz, 400 microseconds/ph duration, 25 microA peak. Also, two control groups received 20 min stimulation during weekly electrically evoked auditory brainstem response (eABR) measurement (Group E) and about 5 s stimulation (Group F) during a brief eABR 3 day postimplantation and at perfusion. On Day 50, animals were perfused, midmodiolar sections cut and a quantitative assessment of spiral ganglion cells (SGC) performed. All stimulated subjects showed a similar decrease in eABR thresholds and dynamic range over time. No stimulation conditions induced pathology. All stimulation conditions enhanced survival of SGCs compared to unimplanted ears and implanted non-stimulated ears (Group F). There were no statistically significant differences in SGC survival between any stimulated groups, including Group E stimulated once a week. In conclusion, high-rate stimulation, under the conditions of this study, provides no additional risks and the same benefits to SGC survival as low-rate stimulation.

Animals↗
PubMed1997

Organization of cell junctions and cytoskeleton in the reticular lamina in normal and ototoxically damaged organ of Corti.

The reticular lamina creates an ion barrier, withstands mechanical stress in the organ of Corti and is able to maintain its integrity during and after severe hair cell loss. Tight junctions maintain the ionic gradient whereas adherens junctions and the cytoskeleton are responsible for the integrity and mechanical resistance of tissues. In this study we used immunofluorescence and electron microscopy to examine the distribution of proteins of tight junctions (cingulin), adherens junctions (E-cadherin, alpha- and beta-catenin) and the cytoskeleton (actin, cytokeratin and tubulin) in whole-mounts of the normal and ototoxically damaged organ of Corti. In normal ears the proteins of adherens junctions were found in all cell types of the reticular lamina. We now demonstrate that all cells forming the reticular lamina partially overlap each other organizing extensive cell contacts with a complex three-dimensional shape. During scar formation, the tight junctions as well as adherens junctions between hair and supporting cells appeared in two distinct focal planes, which could help to preserve the ionic barrier and tissue integrity during hair cell degeneration. During scar formation all cytoskeletal structures in the reticular lamina were reorganized in a specific spatio-temporal pattern. We present a three-dimensional model of cell contact organization in the reticular lamina of normal ears and during scar formation.

Actins↗
PubMed1997

Further evidence for supporting cell conversion in the damaged avian basilar papilla.

Two lines of evidence suggested that a process other than supporting cell divisions may give rise to new hair cells in the bird inner ear injured by either noise or ototoxic drugs. This process, supporting cell conversion, occurs when non-dividing supporting cells transdifferentiate into hair cells. First, noise-exposed chicks received zero, one or two daily i.p. injections of cytosine arabinoside (a DNA synthesis blocker), as well as two daily intraperitoneal injections of bromodeoxyuridine, for four days. Following sacrifice, the papillae were processed for bromodeoxyuridine immunocytochemistry. All the ears demonstrated dividing cells, but increasing the number of cytosine arabinoside injections decreased the number of labeled cells. Indeed, two cytosine arabinoside injections per day nearly completely blocked supporting cell divisions in the short hair cell region within the sound-induced lesion. This suggested that unpaired, immature cells observed in a similar region with scanning electron microscopy, despite the presence of cytosine arabinoside, may have been products of supporting cell conversion. In the second experiment, birds were treated with gentamicin for three days. Upon sacrifice at 6 days post-treatment, papillae were processed for light and transmission electron microscopy. Several unusual cells were observed with phenotypic features of both hair cells and supporting cells. The peculiar cells may be in a transition from the supporting cell phenotype to that of a hair cell.

Animals↗
PubMed1997

Viral-mediated gene transfer in the cochlea.

Gene transfer is an exciting new tool in medical therapy and scientific investigation, but only very recently has it begun to be developed in the auditory system. This paper describes in vivo and ex vivo experiments using an adenoviral vector (Ad. RSVntlacZ), which is a replication-deficient virus based on a human adenoviral (serotype 5) genomic backbone. The in vivo experiments demonstrate successful gene transfer into multiple types of cochlear cells. We observed a relatively efficient transduction, several weeks of sustained transgene expression and an absence of major lethal cytotoxicity in spiral ganglion and epithelial cells of the cochlea in adult animals. The ex vivo experiments were performed using fibroblasts transduced in vitro with Ad. RSVntlacZ. Two weeks after inoculation of the fibroblasts into the perilymph, we observed transplanted fibroblasts, which were adherent to the lining of the perilymphatic spaces, and were expressing the lacZ transgene. We speculate that, as the genetic basis of degenerative cochlear diseases is characterized on a mutational level, transgene expression will allow us to test hypotheses regarding the effects of specific genes on cochlear cell biology. Gene transfer will not only increase our understanding of the pathophysiology of hearing loss, but also may provide gene therapy for disease.

Adenoviridae↗
PubMed1997

Neurotrophins can enhance spiral ganglion cell survival after inner hair cell loss.

Following destruction of sensory cells of the organ of Corti, spiral ganglion cells (SGC) in the guinea pig degenerate. Chronic electrical stimulation via cochlear prostheses can enhance their survival, with the effect blocked by stopping the electrically elicited action potentials with tetrodotoxin. Blocking action potentials in the normal hearing ear with tetrodotoxin, however, does not cause degeneration. This suggests that in the pathological ear VIII N activity acts as a survival factor, while in the normal ear there are other survival factors that maintain SGCs. We examined neurotrophins, as survival factors in the deafened ear. Two weeks of treatment with BDNF (brain derived neurotrophic factor) administered chronically via a mini-osmotic pump into scala tympani at 50 ng/ml, provided a statistically significant enhanced SGC survival over untreated deafened ears or deafened ears treated with artificial perilymph. Neurotrophin 3 provided some enhanced survival, but this was not statistically significant over untreated deafened ears. These observations suggest there are survival factors in the inner ear, including those coupled to direct activation of the auditory nerve fibers, that may serve to maintain the auditory nerve. These factors may be applied following deafness to maintain and enhance neural populations and to increase benefits to the profoundly deaf receiving cochlear implants.

Animals↗
PubMed1997

Presynaptic terminals in hyaline cells of normal and overstimulated chick inner ears.

Hyaline cells are non-sensory epithelial cells of the vibrating part of the basilar membrane of chicks; they receive an extensive efferent innervation. Although these anatomical features suggest roles in auditory transduction, very little is known about the function of these cells. One possible way to understand function is by lesion experiments. We used synapsin-specific antibodies to study changes that occur in the pattern of efferent innervation in hyaline cells after lesion of the sensory epithelium induced by acoustic overstimulation. We found only small changes in hyaline cells after such trauma. These included a small increase in size and a small decrease in density of nerve terminals on hyaline cells. This suggests that hyaline cells and their nerve terminals are less susceptible to acoustic trauma than hair cells. Using neurofilament-specific antibodies we found little or no trauma-induced change in the density of nerve fibres that cross the basilar papilla and reach the hyaline cell region. This finding suggested that trauma to the hair cells does not necessarily lead to changes in the efferent fibres that cross the papilla and extend into the hyaline cell region. Using the trauma and the morphological parameters studied here, it appears that a moderate lesion in the hair cell region in the avian inner ear does not influence the hyaline cells or their innervation.

Acoustic Stimulation↗
PubMed1996

Gentamicin distribution in the basilar papilla: possible association with regenerated hair cell orientation.

Gentamicin-specific immunolabeling was examined in the regenerating chick basilar papilla following gentamicin treatment. Surviving hair cells were labeled in the infracuticular area, predominantly in the basal body region. Label intensity in short hair cells (SHCs) was greater than tall hair cells (THCs). Quantitative examination of stereociliary bundle orientation in regenerated hair cells revealed a considerable degree of variability from normal. The orientation of SHC stereocilia was more varied than that of THC. The results suggest an association between the accumulation of gentamicin in the region of the basal body and the degree of disorientation of the cell surface. It is possible that gentamicin accumulation around the basal body compromises the ability of the centrosome to orient hair cells correctly.

Animals↗
PubMed1996

Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.

Loss of ganglion cells is a common and irreversible complication of hair cell loss in the cochlea. Gene transfer could potentially be used to prevent this neuronal degeneration and other pathologies in the cochlea. Human adenoviruses should provide a feasible gene transfer vehicle for transducing the quiescent cochlear neurons and organ of Corti epithelium. We now describe in vivo experiments in which a replication-deficient adenoviral vector, Ad.RSVntlacZ was injected into the perilymphatic fluid of six normal guinea pigs. Postoperative recovery of animals was complete. Inner ear tissues were assessed for histology and for presence of lacZ-positive cells 1 or 2 weeks after the injection. A large number of blue (lacZ-positive) cells were observed in the neural, epithelial and connective tissues of the cochlea. In four ears spiral ganglion cell infection exceeded 50%, throughout the length of the cochlear spiral. No major pathology was detected in the organ of Corti and other cochlear tissues, and no infection was present in the vestibular tissues or the contralateral cochlea. Immunocytochemical assessment of T cells revealed an increased in the number of lymphocytes in the connective tissue lining the perilymphatic spaces. We conclude that efficient gene transfer into multiple types of cochlear cells in vivo can be achieved without major morphological signs of pathology or toxicity.

Adenoviruses, Human↗
PubMed1996

New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear.

Supporting cell mitosis contributes significantly to hair cell regeneration in the acoustically damaged bird inner ear. Yet there may be another mechanism of hair cell replacement: supporting cell conversion. This study used cytosine arabinoside (Ara-C), an inhibitor of DNA synthesis, to better determine whether supporting cells could transdifferentiate into hair cells without cell division. Chicks received Ara-C injections after acoustic overstimulation. Scanning microscopic studies of the basilar papillae revealed several unpaired, immature hair cells. To ensure Ara-C's blockage of DNA synthesis, one group of birds received both Ara-C and bromodeoxyuridine (BrdU), while another group had BrdU only. Immunocytochemical analysis of Ara-C/BrdU and BrdU papillae indicated zero and 16 dividing cells, respectively. This difference confirmed that Ara-C blocked DNA synthesis, arresting supporting cell mitosis. These data strongly suggest that supporting cell can convert into hair cells.

Animals↗
PubMed1996

Identification of genes expressed after noise exposure in the chick basilar papilla.

We used differential display of mRNA, a method based on reverse transcriptase-PCR, to identify genes whose expression increases in response to acoustic trauma in the chick basilar papilla. Identifying these genes would provide insight into processes involved in repair of the damaged epithelium or in hair cell regeneration. We compared mRNA from the basilar papilla of normal chicks, from chicks exposed to an octave band noise (center frequency: 1.5 kHz) presented at 118 dB for 6 h, and from chicks exposed to noise and allowed to recover for 2 days. Thus far, we have identified 70 bands that appear to be differentially displayed on DNA sequencing gels; approximately 40 of these bands have been subcloned and sequenced. DNA sequences were compared with sequences in the GenBank database to identify genes with significant (70-85%) sequence identity to known genes. Chick cDNAs identified included: the parathyroid hormone-related protein, an immediate early gene; the delta-subunit of the neuronal-specific Ca2+/calmodulin-regulated protein kinase II; and the GTP-binding protein CDC42, a member of the ras superfamily of G proteins. A fourth cDNA had 84% sequence identity to an uncharacterized human cDNA (expressed sequence tag), indicating that this is a novel gene. Slot-blot hybridization analysis of these cDNAs probed with labeled DNA generated from mRNA from each experimental group indicated higher levels of mRNA for each of these four genes after noise exposure. These results indicate the potential involvement of both Ca2+/calmodulin-mediated signaling and GTPase cascades in the response to noise damage and during hair cell regeneration in the chick basilar papilla.

Acoustic Stimulation↗