Timo Stöver

Clinical Predictors for Germline Mutations in Head and Neck Paraganglioma Patients: Cost Reduction Strategy in Genetic Diagnostic Process as Fall-Out

Multiple genes and their variants that lend susceptibility to many diseases will play a major role in clinical routine. Genetics-based cost reduction strategies in diagnostic processes are important in the setting of multiple susceptibility genes for a single disease. Head and neck paraganglioma (HNP) is caused by germline mutations of at least three succinate dehydrogenase subunit genes (SDHx). Mutation analysis for all 3 costs approximately US

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation

2,700 per patient. Genetic classification is essential for downstream management of the patient and preemptive management of family members. Utilizing HNP as a model, we wanted to determine predictors to prioritize the most heritable clinical presentations and which gene to begin testing in HNP presentations, to reduce costs of genetic screening. Patients were tested for SDHB, SDHC, and SDHD intragenic mutations and large deletions. Clinical parameters were analyzed as potential predictors for finding germline mutations. Cost reduction was calculated between prioritized gene testing compared with that for all genes. Of 598 patients, 30.6% had SDHx germline mutations: 34.4% in SDHB, 14.2% SDHC, and 51.4% SDHD. Predictors for an SDHx mutation are family history [odds ratio (OR), 37.9], previous pheochromocytoma (OR, 10.9), multiple HNP (OR, 10.6), age <or=40 years (OR, 4.0), and male gender (OR, 3.5). By screening only preselected cases and a stepwise approach, 60% cost reduction can be achieved, with 91.8% sensitivity and 94.5% negative predictive value. Our data give evidence that clinical parameters can predict for mutation and help prioritize gene testing to reduce costs in HNP. Such strategy is cost-saving in the practice of genetics-based personalized health care.

Cochlear gene transfer: round window versus cochleostomy inoculation.

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line‐derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad‐GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad‐GDNF or ES or both. Ad‐GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES‐treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad‐GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES‐treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF‐ and ES‐mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. J. Comp. Neurol. 454:350–360, 2002.

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

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.

Hearing and hair cells are protected by adenoviral gene therapy with TGF-β1 and GDNF

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 μl 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.

Effects of delayed treatment with combined GDNF and continuous electrical stimulation on spiral ganglion cell survival in deafened guinea pigs.

Glial cell line-derived neurotrophic factor (GDNF) overexpression in the inner ear can protect hair cells against degeneration induced by aminoglycoside ototoxicity. The protective efficiency of GDNF increases when it is combined with co-factors such as transforming growth factor beta1 (TGF-beta1), a ubiquitous cytokine. The aim of this study was to determine whether TGF-beta1 receptors are expressed in the inner ear and whether a cocktail of GDNF and TGF-beta1 transgenes provides enhanced protection of the inner ear against ototoxic trauma. Using RT-PCR analysis, we determined that both TGF-beta1 receptors, type 1 and 2 are present in rat cochlea. We co-inoculated two adenoviral vectors, one encoding human TGF-beta1 gene (Ad.TGF-beta1) and the other encoding human GDNF gene (Ad.GDNF) into guinea pig cochleae 4 days prior to injecting an ototoxic dose of aminoglycosides. Inoculated ears had better hearing and fewer missing inner hair cells after exposure to the aminoglycoside ototoxicity, as compared with controls and ears treated only with Ad.GDNF. Cochleae with TGF-beta1 overexpression exhibited fibrosis in the scala tympani regardless of the presence of GDNF. Our results suggest that the adenovirus-mediated overexpression of GDNF and TGF-beta1 can be used in combination to protect cochlear hair cells and hearing from ototoxic trauma.

A true minimally invasive approach for cochlear implantation: high accuracy in cranial base navigation through flat-panel-based volume computed tomography.

Electrical stimulation (ES) of spiral ganglion cells (SGC) via a cochlear implant is the standard treatment for profound sensor neural hearing loss. However, loss of hair cells as the morphological correlate of sensor neural hearing loss leads to deafferentation and death of SGC. Although immediate treatment with ES or glial cell line–derived neurotrophic factor (GDNF) can prevent degeneration of SGC, only few studies address the effectiveness of delayed treatment. We hypothesize that both interventions have a synergistic effect and that even delayed treatment would protect SGC. Therefore, an electrode connected to a pump was implanted into the left cochlea of guinea pigs 3 weeks after deafening. The contralateral untreated cochleae served as deafened intraindividual controls. Four groups were set up. Control animals received intracochlear infusion of artificial perilymph (AP/−). The experimental groups consisted of animals treated with AP in addition to continuous ES (AP/ES) or treated with GDNF alone (GDNF/−) or GDNF combined with continuous ES (GDNF/ES). Acoustically and electrically evoked auditory brain stem responses were recorded. All animals were killed 48 days after deafening; their cochleae were histologically evaluated. Survival of SGC increased significantly in the GDNF/− and AP/ES group compared with the AP/− group. A highly significant increase in SGC density was observed in the GDNF/ES group compared with the control group. Additionally, animals in the GDNF/ES group showed reduced EABR thresholds. Thus, delayed treatment with GDNF and ES can protect SGC from degeneration and may improve the benefits of cochlear implants.

Gene Expression Profiles of the Rat Cochlea, Cochlear Nucleus, and Inferior Colliculus

Objective: High-precision intraoperative navigation using high-resolution flat-panel volume computed tomography makes feasible the possibility of minimally invasive cochlear implant surgery, including cochleostomy. Conventional cochlear implant surgery is typically performed via mastoidectomy with facial recess to identify and avoid damage to vital anatomic landmarks. To accomplish this procedure via a minimally invasive approach-without performing mastoidectomy-in a precise fashion, image-guided technology is necessary. With such an approach, surgical time and expertise may be reduced, and hearing preservation may be improved. Interventions: Flat-panel volume computed tomography was used to scan 4 human temporal bones. A drilling channel was planned preoperatively from the mastoid surface to the round window niche, providing a margin of safety to all functional important structures (e.g., facial nerve, chorda tympani, incus). Main Outcome Measures: Postoperatively, computed tomographic imaging and conventional surgical exploration of the drilled route to the cochlea were performed. Results: All 4 specimens showed a cochleostomy located at the scala tympani anterior inferior to the round window. The chorda tympani was damaged in 1 specimen-this was preoperatively planned as a narrow facial recess was encountered. Conclusion: Using flat-panel volume computed tomography for image-guided surgical navigation, we were able to perform minimally invasive cochlear implant surgery defined as a narrow, single-channel mastoidotomy with cochleostomy. Although this finding is preliminary, it is technologically achievable.

The long-term effects of modified electrode surfaces and intracochlear corticosteroids on postoperative impedances in cochlear implant patients.

High-throughput DNA microarray technology allows for the assessment of large numbers of genes and can reveal gene expression in a specific region, differential gene expression between regions, as well as changes in gene expression under changing experimental conditions or with a particular disease. The present study used a gene array to profile normal gene expression in the rat whole cochlea, two subregions of the cochlea (modiolar and sensorineural epithelium), and the cochlear nucleus and inferior colliculus of the auditory brainstem. The hippocampus was also assessed as a well-characterized reference tissue. Approximately 40% of the 588 genes on the array showed expression over background. When the criterion for a signal threshold was set conservatively at twice background, the number of genes above the signal threshold ranged from approximately 20% in the cochlea to 30% in the inferior colliculus. While much of the gene expression pattern was expected based on the literature, gene profiles also revealed expression of genes that had not been reported previously. Many genes were expressed in all regions while others were differentially expressed (defined as greater than a twofold difference in expression between regions). A greater number of differentially expressed genes were found when comparing peripheral (cochlear) and central nervous system regions than when comparing the central auditory regions and the hippocampus. Several families of insulin-like growth factor binding proteins, matrix metalloproteinases, and tissue inhibitor of metalloproteinases were among the genes expressed at much higher levels in the cochlea compared with the central nervous system regions.

Technical report: modification of a cochlear implant electrode for drug delivery to the inner ear.

Objective: The objective of this study was to investigate the long-term effect of intraoperative application of steroid suspension and coating of the electrode contacts with a thin film of iridium oxide on the intracochlear impedance development after cochlear implantation and on the impedance difference before and after stimulation. Design: Time-dependent development of intracochlear impedances was investigated in 4 different groups of adult patients up to 4 years after implantation. Additionally, during rehabilitation period just after first fitting, impedances before and after stimulation were measured as to investigate the influence of electrical stimulation on the impedances. Results from standard Nucleus 24 Contour (control), standard Nucleus 24 Contour with intraoperative application of steroids, iridium-coated Nucleus 24 Contour, and iridium-coated Nucleus 24 Contour with intraoperative application of steroids were compared. Results: Steroid application reduced impedances significantly throughout the observation period of up to 4 years after implantation. Iridium oxide coating had no effect. Differences between the groups were mainly found on the basal and middle parts of the cochlea, but not close to the tip of the array, also indicating that postoperative fibrous tissue growth is stronger in the basal region of the cochlea. Group mean values of the stimulation effect were not influenced by the different treatments. Nevertheless, only in both steroid-treated groups a correlation between the impedance before stimulation and the stimulation effect was found. Conclusion: Although the differences between control and steroid-treated groups decrease with time, single intraoperative intracochlear steroid deposition was proven to lower postoperative impedances during first 3 to 4 years after implantation probably because of reduction of fibrous tissue growth.