Yehoash Raphael

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

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.

Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.

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.

Structure and innervation of the cochlea.

The role of the cochlea is to transduce complex sound waves into electrical neural activity in the auditory nerve. Hair cells of the organ of Corti are the sensory cells of hearing. The inner hair cells perform the transduction and initiate the depolarization of the spiral ganglion neurons. The outer hair cells are accessory sensory cells that enhance the sensitivity and selectivity of the cochlea. Neural feedback loops that bring efferent signals to the outer hair cells assist in sharpening and amplifying the signals. The stria vascularis generates the endocochlear potential and maintains the ionic composition of the endolymph, the fluid in which the apical surface of the hair cells is bathed. The mechanical characteristics of the basilar membrane and its related structures further enhance the frequency selectivity of the auditory transduction mechanism. The tectorial membrane is an extracellular matrix, which provides mass loading on top of the organ of Corti, facilitating deflection of the stereocilia. This review deals with the structure of the normal mature mammalian cochlea and includes recent data on the molecular organization of the main cell types within the cochlea.

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

SummaryAcoustic 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.

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.

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-Cs 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.

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.

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 patients 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.