Abstract Otology 2000 D11-1

Alan F. Ryan MD, K. Pak MD, A. Battaglia MD, Stefan Dazert MD, D. Dulon MD

Div. of Otolaryngology UCSD School of Medicine USA-La Jolla CA

Damage to hair cells is a leading cause of hearing and balance disorders. New strategies for the prevention and treatment of hair cell damage have emerged from the growing fields of molecular and pharmacological otology. The intracellular pathways involved in hair cell damage and death are being elucidated. In addition, receptors that promote cell survival have recently been discovered on hair cells. This information has provided new opportunities for pharmacological intervention to prevent hair cell loss. For example, we have found that inhibitors of G-protein signaling and apoptosis will protect hair cells from damage due to ototoxicity. Also, activation of cell surface receptors by FGF-2 and TGFa is protective of hair cells. Another source of potential new treatments has emerged from molecular genetic studies. Genes that produce inherited forms of hearing loss are being identified at an accelerating rate. Understanding of these molecular defects allows genetic counseling and provides additional potential modes of therapy. Manipulation of the genetic response of air cells using gene therapy is an additional avenue for treatment. For example, we have found that incorporation of a bacterial antibiotic resistance gene into hair cells protects them from aminoglycoside ototoxicity. Finally, molecular biological techniques have been developed with which to pursue new avenues of research. They permit the separation of events involved in hair cell death, such as loss of the stereociliary array and death of the cell body. They also allow the study of transfer of cells between sensory epithelia. (Supported by grant DC00139 from the NIH/NIDCD and by the Research Service of the VA.)

Abstract Otology 2000 D11-2

Timo Stöver Dr.med., Masao Yagi Dr.med., Thomas Lenarz MD, Yehoash Raphael PhD

Department of Otolaryngology Kresge Hearing Research Institute The University of Michigan USA-Ann Arbor, MI

In vivo intracochlear gene transfer has been shown to be a feasible tool for inner ear gene therapy in the guinea pig. By transfecting cochlear cells with gene sequences that encode neurotrophic factors and thereby inducing the expression of neurotrophic factors in cochlear tissue, viral-mediated gene transfer provides an alternative to direct drug application (Yagi et al., 1999). Interestingly, we observed protective transgene effects in both ears, following viral vector inoculation via a cochleostomy into one ear. Local cochlear drug delivery, via osmotic pumps, has also been described to cause drug related effects in the contralateral ear (LePrell et al., 1999). The mechanism of the contralateral effect is unclear. Hypotheses to explain the contralateral cochlear effects following local cochlear manipulation included blood mediated transport, bone marrow mediated diffusion or brain fluid mediated transfer into the untreated cochlea (Lalwani et al., 1998). To directly test these hypotheses, we used adenoviral vectors containing the reporter gene b-galactosidase. We determined that inoculation of 15-25 microliters or more in guinea pig ears results in reproducible contralateral transfection with the reporter gene. Preliminary data indicate that the contralateral effect is based on transport via the perilymph and CSF. This work was supported by NIH NIDCD Grant P01 DC00078. TS is a scholar of the Alexander-von Humboldt-Foundation.

Abstract Otology 2000 D11-3

Yehoash Raphael PhD

Kresge Hearing Research Institute The Univ.of Michigan Medical School MSRB III Room-9303 USA-Ann Arbor, MI

Approaching the last year of this millenium, treatment for genetic and environmentally induced inner ear (IE) disease is rapidly advancing. Since loss of hair cells (HCs) in mammals is irreversible, their protection and rescue are of clinical importance. Neurotrophic factors (NFs) have been shown to protect the structure and function of cochlear and vestibular epithelia from trauma. We have used an adenoviral vector expressing the human GDNF gene in guinea pig IEs for protection and rescue of HCs and cochlear function. The viral vector was inoculated into the scala tympani of the left cochlea through the round window. Controls included the unoperated (right) ears, and additional operated ears, inoculated with a lacZ-expressing vector or artificial perilymph. ABRs were measured to assess function, before experiments begun, and at sacrifice. Four days post injection, animals were traumatized with noise (4 kHz octave band noise, 115 dB SPL, 5 hours) or aminoglycosides and sacrificed 7 days later. In both noise and drug groups, GDNF over-expression protected HCs and cochlear function. Inoculation with control solutions alone demonstrated that gene transfer by itself is not toxic to the IE. These data demonstrate the feasibility for gene therapy for protection and rescue of HCs after noise and drug trauma. Current experiments are aimed at extending gene-based therapy to treating genetic IE disease. Supported by Amgen Inc. and NIH NIDCD Grants P01 DC00078 and RO1 DC 01634.

Abstract Otology 2000 D11-4

Thomas J. McDonald M.D., M.S., F.A.C.S.

Department of Otolaryngology Head and Neck Surgery Mayo Clinic & Mayo Foundation USA-Rochester, MN

Bilateral fluctuating sensorineural hearing loss or fluctuating hearing loss in the only hearing ear are two situations that truly constitute a medical emergency. Eighteen patients having either of these problems are presented in the preliminary report and the early results of treatment with Methotrexate are reviewed. Criteria for inclusion in the study were patients having truly idiopathic hearing loss which had responded to corticosteroids. All patients received the following studies: ophthalmologic examination, CBC, SGOT, creatinine levels, FTA-ABS, head imaging, sedimentation rates, and antiphospholipid antibody titers (aPL). Each patient was started on 7.5 mg. of Methotrexate weekly and monitored monthly for evidence of liver toxicity. The dose of corticosteroids was tapered and ultimately discontinued in each patient. Hearing results were very encouraging. There were no side effects. The rationale for using Methotrexate as an alternate treatment to corticosteroids in patients with autoimmune disease is presented and the possibility of this disease being a complication of aPL are discussed.

Abstract Otology 2000 D11-5

Run Sheng Ruan MD, Seng Kee Leong MD, Kian Hian Yeoh MD

Department of Otolaryngology National University of Singapore SGP-Singapur

The aim of this study is to determine whether neurotrophic factors, like brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) would protect auditory hair cells from ototoxicity of aminoglycoside antibiotic. Twenty seven Wistar guinea pigs were divided into 3 groups of 9 animals each. BDNF, NT-3 at a concentration of 100 µg/ml were delivered into the right scala tympani of guinea pig cochlea through a cannula-osmotic pump device. Artificial perilymph (AP) was used as control. Right after implantation of the device, each animal was given 5 successive doses of kanamycin at 400 mg/kg body weight. At 15, 30 and 60 days after infusion, surviving inner and outer hair cells were counted at each turn of every cochlea with a Philips 515 scanning electron microscope. Multiple comparison tests were carried out among the groups, using ANOVA and Dunnett T3 /Tukey HSD. Protective effects of NT-3 on hair cells were observed at 30 and 60 days after kanamycin injection. BDNF had no protective effect on hair cells at 15 and 60 days, but some at 30 days. This study suggests that NT-3 and BDNF may protect against cochlea hair cell damage caused by kanamycin treatment. Possible mechanisms for the otoprotective effects were discussed. No single mechanism postulated can explain fully the results seen in this study. It is possible that the mechanisms act in concert to produce the observed effects, or there are as yet undiscovered mechanisms or secondary messengers responsible for the otoprotective effects.

Abstract Otology 2000 D11-6

Steven J.H. Bom MD, M.H. Kemperman MD, Y.J.M. De Kok MD, P.L.M. Huygen MD, W.I.M. Verhagen PD, F.P.M. Cremers MD, Cor W.R.J. Cremers MD

ORL Department University Hospital Nijmegen NL-Nijmegen

We examined auditory and vestibulo-ocular functions in a Dutch family with hereditary sensorineural hearing impairment (SNHI), caused by a 208C>T mutation in the COCH gene located in chromosome 14q12-q13 (DFNA9), which showed an autosomal dominant pattern of inheritance with full penetrance. We were able to evaluate fifteen of the sixteen genetically affected persons. They all developed hearing and vestibular impairment symptoms in the fourth to fifth decade and, in many cases, also cardiovascular disease. We derived the gross characteristics for the hearing impairment in this trait from linear regression analysis of individual longitudinal data (n=11) of age corrected hearing thresholds and scatterplots of all data available. At the low frequencies (0.25-2 kHz), it started at the age of about 40 and showed an average annual progression of approximately 3 dB. In two exceptional cases, annual progression attained levels of up to 24 dB. At the high frequencies (4-8 kHz), the average threshold increased from about 50 dB at the age of 35 to about 120 dB at the age of 75 (which amounts to 1.8 dB annual threshold increase). All affected individuals tested showed normal ocular motor functions. The patients aged over 46 years showed absence and in one case severe impairment of the vestibulo-ocular reflex and enhancement of the cervico-ocular reflex, whereas those aged 40-46 years showed either severe bilateral vestibular hyporeflexia or unilateral caloric areflexia. These findings suggest a gradual development of vestibular areflexia.

Abstract Otology 2000 D11-7

A.M. Meyer zum Gottesberge MD, Heidi Felix Ph.D., H. Weiher PhD

Department of Otorhinolaryngology University of Duesseldorf D-Düsseldorf

The Mpv17 transgenic mouse strain, generated by insertion of defective retrovirus into the germline, is characterized by early onset focal segmental glomerulosclerosis, concomitant hypertension and impairment of the inner ear function. In an effort to identify the role of the Mpv17 proteine in the inner ear, a comparative study was performed using histological, immunohistological, histochemical, and electron microscopical approaches. Time dependent degeneration of the stria vascularis and of the spiral ligament, the organ of Corti, and cochlear neurons were revealed. The changes in the stria vascularis range from thickening and splitting of the basement membrane of the capillary wall to the complete desintegration of strial epithelium and its atrophy. Morphological studies of kidneys and inner ears in transgenic MPV17-negative mice reveled similar histopathologic findings to patients with clinical Alport syndome, an inherited disorder characterized by progressive nephritis and neurosensory deafness. Our results indicate that beside primary mutations of several type IV collagens known for Alport syndrome, also the loss of Mpv17 gene function is somehow responsible for the renal- and otopathology. The primary cause of the disease in mutants is a loss of function of Mpv17 protein, a peroxisomal gene product, involved in reactive oxygenmetabolism. As recently found, Mpv17 gene play a crucial role in the regulation of matrix metalloproteinase-2 (MMP-2) a member of large subfamily of proteinases which has a major role in physiological control of extracellular matrix components. Both locations of pathology the inner ear and the kidney, show increased MMP-2 expression in the absence of the Mpv17 gene product. Thus, it seems conceivable, that MMP-2mediates the molecular mechanisms involved in the pathology of both organs. (supported by DFG ME 890-1, 890-2)