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Y Raphael

Publications and source records attributed to Y Raphael.

At least 19 recordsLinked to original sources
PubMed2001

The cell adhesion molecule BEN defines a prosensory patch in the developing avian otocyst.

The distribution of the cell adhesion molecule BEN in the developing chick inner ear is described. BEN is first detected in the otic placode at stage 11. As the placode begins to invaginate, BEN becomes concentrated in a ventromedial region extending from the anterior to the posterior end of the otic pit. BEN expression levels increase in this region as the pit closes to form the otocyst, and distinct boundaries become defined along the dorsal and ventral edges of the ventromedial band of BEN expression. BEN expression also becomes concentrated dorsally within the otic epithelium as the pit closes and is observed in the condensing otic ganglion. By stage 22, the ventromedial band of BEN expression splits into two distinct regions, a small caudal patch within which the posterior crista will develop, and a larger anterior patch. By stage 26, this larger anterior patch of cells expressing BEN becomes subdivided into five separate areas corresponding to the regions within which the anterior crista, the lateral crista, the utricle, the saccule, and both the basilar papilla and lagenar macula form. Hair cells only develop within these regions defined by BEN distribution. The data suggest that the ventromedial patch of BEN expression observed from stage 11 onwards defines a single sensory competent zone from which all sensory organs of the inner ear develop. BEN immunoreactivity in the inner ear declines after stage 38. In response to noise exposure, upregulation of BEN expression is mainly detected in regions of the posthatch papilla where the damage is severe and regenerating hair cells are not observed. The regenerating hair and supporting cells do not express BEN, highlighting a molecular difference between the processes of development and regeneration.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed2001

Surface modification of neural recording electrodes with conducting polymer/biomolecule blends.

The interface between micromachined neural microelectrodes and neural tissue plays an important role in chronic in vivo recording. Electrochemical polymerization was used to optimize the surface of the metal electrode sites. Electrically conductive polymers (polypyrrole) combined with biomolecules having cell adhesion functionality were deposited with great precision onto microelectrode sites of neural probes. The biomolecules used were a silk-like polymer having fibronectin fragments (SLPF) and nonapeptide CDPGYIGSR. The existence of protein polymers and peptides in the coatings was confirmed by reflective microfocusing Fourier transform infrared spectroscopy (FTIR). The morphology of the coating was rough and fuzzy, providing a high density of bioactive sites for interaction with neural cells. This high interfacial area also helped to lower the impedance of the electrode site and, consequently, to improve the signal transport. Impedance spectroscopy showed a lowered magnitude and phase of impedance around the biologically relevant frequency of 1 kHz. Cyclic voltammetry demonstrated the intrinsic redox reaction of the doped polypyrrole and the increased charge capacity of the coated electrodes. Rat glial cells and human neuroblastoma cells were seeded and cultured on neural probes with coated and uncoated electrodes. Glial cells appeared to attach better to polypyrrole/SLPF-coated electrodes than to uncoated gold electrodes. Neuroblastoma cells grew preferentially on and around the polypyrrole/CDPGYIGSR-coated electrode sites while the polypyrrole/CH(3)COO(-)-coated sites on the same probe did not show a preferential attraction to the cells. These results indicate that we can adjust the chemical composition, morphology, electronic transport, and bioactivity of polymer coatings on electrode surfaces on a multichannel micromachined neural probe by controlling electrochemical deposition conditions.

Animals↗
PubMed2001

The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice.

Mice present an ideal model for inner ear gene therapy because their genome is being rapidly sequenced, their generation time is relatively short, and they serve as a valuable model for human hereditary inner ear disease. However, the small size of the mouse inner ear poses a particular challenge for surgical procedures. We have developed a new approach for viral inoculation into the mature mouse inner ear, using a replication-deficient adenovirus expressing the bacterial gene lacZ. We administered the virus through the posterior semicircular canal (canalostomy) and into the cochlea (cochleostomy). Both approaches caused lacZ to be expressed in cells lining the perilymphatic space. One canalostomy case showed gene expression in sensory cells of the crista ampullaris, whereas the cochleostomy group showed gene expression in the sensory cells in the organ of Corti and saccule. Functional tests after the surgery showed that the canalostomy preserved hearing, whereas the cochleostomy did not. Any vestibular function transiently lost after the canalostomy was recovered. Our findings indicate that inoculation of adenovirus vectors into the mouse inner ear through the semicircular canal has the potential to efficiently introduce transgenes to the vestibular system and the cochlea without compromising hearing.

Adenoviridae↗
PubMed2001

Severe vestibular and auditory impairment in three alleles of Ames waltzer (av) mice.

The genetic and physiological characterization of circling, hearing-impaired mouse mutants has greatly facilitated our understanding of non-syndromic sensorineural deafness, the most common form of hereditary human hearing loss. Here we report the first phenotypic characterization of three alleles of Ames waltzer (av). Neither electrical potentials (auditory brainstem response) nor behavioral responses to sound could be evoked in any of the three alleles at any age or frequency. However, the endocochlear potential was found to be normal, indicating that the primary pathology is not in the stria vascularis. To determine the earliest changes and help identify the primary causes of deafness in av, we performed morphological studies in 15-16 day old mutants, just prior to the maturation of the cochlea. Although av(2J) is slightly more affected than the other two alleles, our studies show a high similarity between all three alleles. The first detectable changes are observed in the stereocilia and cytoplasm of hair cells, and in the cellular shape and microvilli of supporting cells. These changes are followed by degeneration of the cochlear and vestibular neuroepithelium.

Alleles↗
PubMed2001

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat.

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Aminoglycosides↗
PubMed2001

[Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector].

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.

Adenoviruses, Human↗
PubMed2000

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Alleles↗
PubMed2000

Spiral ganglion neurons are protected from degeneration by GDNF gene therapy.

Perceptual benefits from the cochlear prosthesis are related to the quantity and quality of the patient's auditory nerve population. Multiple neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have been shown to have important roles in the survival of inner ear auditory neurons, including protection of deafferented spiral ganglion cells (SGCs). In this study, GDNF gene therapy was tested for its ability to enhance survival of SGCs after aminoglycoside/diuretic-induced insult that eliminated the inner hair cells. The GDNF transgene was delivered by adenoviral vectors. Similar vectors with a reporter gene (lacZ) insert served as controls. Four or seven days after bilateral deafening, 5 microl of an adenoviral suspension (Ad-GDNF or Ad-lacZ) or an artificial perilymph was injected into the left scala tympani of guinea pigs. Animals were sacrificed 28 days after deafening and their inner ears prepared for SGC counts. Adenoviral-mediated GDNF transgene expression enhanced SGC survival in the left (viral-treated) deafened ears. This observation suggests that GDNF is one of the survival factors in the inner ear and may help maintain the auditory neurons after insult. Application of GDNF and other survival factors via gene therapy has great potential for inducing survival of auditory neurons following hair cell loss.

Animals↗
PubMed2000

Octylcyanoacrylate: a new medical-grade adhesive for otologic surgery.

HYPOTHESIS: The adhesive octylcyanoacrylate is not associated with significant inner ear toxicity in a guinea pig model. BACKGROUND: Many cyanoacrylate adhesives have been investigated for use in otologic surgery, but variable ototoxicity has been reported. Octylcyanoacrylate is a medical-grade adhesive with many properties that make it ideal for use in the ear. It is free of contaminants; it forms a strong, flexible bond; and it inhibits the growth of gram-positive organisms in culture. This is the first study to assess the ototoxicity of this new adhesive. METHODS: Fourteen adult guinea pigs were used. Preoperative auditory brainstem responses (ABRs) were determined. Bilateral antrotomies were performed, and the ears were randomized to adhesive and control (saline) groups. In the adhesive ears. 0.5 or 0.1 mL of octylcyanoacrylate was instilled into the middle ear. Eight weeks later, postoperative ABRs were determined, the animals were killed, and the temporal bones were removed. Middle ear changes were noted, and the ossicular chain was assessed. Cochlear hair cell analyses were performed. Histologic assessment of the middle ear mucosa was performed. RESULTS: There was a higher incidence of conductive hearing loss in the adhesive group secondary to fixation of the ossicular chain, but there was no significant difference in bone conduction thresholds. The median postoperative bone conduction thresholds (dB peak sound pressure level) was 15.0 in the control group and 17.5 in the adhesive group, p = 0.89. There was also no significant difference in inner hair cell counts (0.4% vs. 0.5% median hair cell loss, p = 0.72) or outer hair cell counts (3.7% vs. 3.0% median hair cell loss, p = 0.23) for the adhesive and control groups, respectively. Histopathologic analysis of the middle ear mucosa demonstrated variable mild to moderate foreign body reaction with no evidence of mucosal ulceration or necrosis. CONCLUSIONS: A large amount of octylcyanoacrylate placed in the middle ear of the guinea pig did not cause any morphologic or functional evidence of inner ear toxicity. This new adhesive is a promising tool for otologic surgery.

Animals↗
PubMed2000

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice.

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions "cytocauds." The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Actin Cytoskeleton↗
PubMed2000

Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer.

Cochlear gene transfer is a promising new approach for inner ear therapy. Previous studies have demonstrated hair cell protection with cochlear gene transfer not only in the inoculated, but also in the uninoculated ear. To characterize the kinetics of viral spread, we investigated the extent of transgene expression in the contralateral (uninoculated) cochlea after unilateral adenoviral cochlear gene transfer. We used a lacZ reporter gene vector, and demonstrated spread of the adenovirus into the cerebrospinal fluid (CSF) after cochlear inoculation of 25 microl viral vector. Direct virus application into the CSF resulted in transduction of both cochleae, whereas virus inoculation into the bloodstream did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virus-mediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Our results showed a functional communication between the CSF and the perilymphatic space of the inner ear, that is not only of importance for otological gene transfer, but also for CNS gene transfer. Gene Therapy (2000) 7, 377-383.

Adenoviridae↗
PubMed2000

Effect of transgenic GDNF expression on gentamicin-induced cochlear and vestibular toxicity.

Gentamicin administration often results in cochlear and/or vestibular hair cell loss and hearing and balance impairment. It has been demonstrated that adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) can protect cochlear hair cells against ototoxic injury. In this study, we evaluated the protective effects of adenovirus-mediated overexpression of GDNF against gentamicin ototoxicity. An adenovirus vector expressing the human GDNF gene (Ad.GDNF) was administered into the scala vestibuli as a rescue agent at the same time as gentamicin, or as a protective agent, 7 days before gentamicin administration. Animals in the Rescue group displayed hearing thresholds that were significantly better than those measured in the Gentamicin or Ad.LacZ/Gentamicin groups. In the Protection group, Ad.GDNF afforded significant preservation of utricular hair cells. The data demonstrated protection of the inner ear structure, and rescue of the inner ear structure and function against ototoxic insults. These experiments suggest that inner ear gene therapy may be developed as a clinical tool for protecting the ear against environmentally induced insults.

Adenoviridae↗
PubMed1999

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.

Animals↗
PubMed1999

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

Adenoviridae↗
PubMed1999

Hair cell protection from aminoglycoside ototoxicity by adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor.

Aminoglycosides are commonly used antimicrobial drugs that often have ototoxic side effects. The ototoxicity often involves permanent loss of cochlear hair cells (HCs). Neurotrophic factors have been shown to protect a variety of tissues, including HCs, from toxic trauma. To determine if glial cell line-derived neurotrophic factor (GDNF) can protect cochlear HCs from trauma, we inoculated an adenoviral vector encoding the human GDNF gene into guinea pig cochleae via the round window membrane 4 days prior to injection of aminoglycosides. Control groups showed little or no negative influence of the viral inoculation on cochlear structure and function. In contrast, ears that were inoculated with the GDNF vector had better hearing and fewer missing HCs after exposure to the ototoxins, as compared with controls. Our results demonstrate the feasibility of gene therapy for cochlear application and suggest that virus-mediated overexpression of GDNF may be developed as a valuable prevention against trauma-induced HC death.

Adenoviridae↗
PubMed1999

KHRI-3 monoclonal antibody-induced damage to the inner ear: antibody staining of nascent scars.

Intracochlear infusion of the KHRI-3 monoclonal antibody results in in vivo binding to guinea pig inner ear supporting cells, loss of hair cells and hearing loss. To further characterize the basis for KHRI-3-induced hearing loss, antibody was produced in a bioreactor in serum-free medium, affinity purified, and compared to conventionally prepared antibody by infusion into the scala tympani using mini-osmotic pumps. In vivo antibody binding was observed in 10 of 11 guinea pigs. A previously unreported pattern of KHRI-3 antibody binding to cells involved in scar formation was noted in five guinea pigs. All but one of the KHRI-3-infused animals demonstrated a hearing loss of > 10 dB in the treated ear. In five of 11 animals the threshold shift was 30 dB or more, and all had hair cell losses. In one guinea pig infused with 2 mg/ml of antibody, the organ of Corti was absent in the basal turn of the infused ear. This ear had a 45-50 dB threshold shift but, curiously, no detectable antibody binding in the residual organ of Corti. Organ of Corti tissue was fragile in antibody-infused ears. Breaks within the outer hair cell region occurred in 5/11 infused ears. The contralateral ears were normal except for one noise-exposed animal that demonstrated hair cell loss in the uninfused ear. Three animals were exposed to 6 kHz noise (108 dB) for 30 min on day 7. Antibody access to the organ of Corti may be increased in animals exposed to noise, since the strongest in vivo binding was observed in noise-exposed animals. Loss of integrity of the organ of Corti seems to be the primary mechanism of inner ear damage by KHRI-3 antibody. The binding of KHRI-3 antibody in new scars suggests a role of the antigen in scar formation. Antibodies with binding properties similar to KHRI-3 have been detected in 51% of patients diagnosed with autoimmune sensorineural hearing loss; thus, it seems likely that such autoantibodies also may have pathologic effects resulting in hearing loss in humans.

Animals↗
PubMed1999

Application of a platinum replica method to the study of the cytoskeleton of isolated hair cells, supporting cells and whole mounts of the organ of Corti.

We adapted a method of platinum replica to study the cytoskeleton of isolated cells of the guinea pig organ of Corti. This technique combined high image resolution with the ability to visualize the three-dimensional organization of the cytoskeleton of a whole cell. The procedure includes: isolation of hair cells and supporting cells using collagenase digestion, attachment of the cells to a coverslip, detergent extraction, chemical fixation, critical point drying, platinum/carbon coating, and transmission electron microscopy analysis. By using the method of platinum replica, we confirmed the existence of structural domains in the cortical lattice of outer hair cells. Based on the analysis of the partly destroyed cortical lattice, we propose that circumferential filaments are underlined with a thin flexible network. In addition, we established that the base of each stereocilium had a cone-like expansion of actin filaments and was surrounded by a thin bundle of filaments. We also produced replicas of the protrusion of the cuticular plate into the cytoplasm (infracuticular network) and the reticular lamina cytoskeleton. Our data indicated that the platinum replica method is useful for studying structural interactions among different cytoskeletal elements in the reticular lamina, as well as the cortex of outer hair cells and the cytoskeleton of supporting cells.

Animals↗