An in vitro model system to study gene therapy in the human inner ear.

The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10(5) and 10(8) viral particles/ml directly to the cultures for 4-24 h and examined the tissue 12-96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.

Time course of hearing loss in an animal model of pneumococcal meningitis.

A leading cause of morbidity from bacterial meningitis is an irreversible, usually profound sensorineural hearing loss, with an incidence as high as 30% in some studies. Bacterial meningitis remains the most common cause of acquired postnatal sensorineural deafness. Although several clinical studies have examined the long-term outcome of hearing in meningitis, few studies have examined the time course of hearing loss during the acute course of the disease. We have developed an animal model of meningogenic hearing loss in the rat and have plotted the time course of that hearing loss. Serial auditory brain stem responses (ABRs) were measured in rats inoculated in the cisterna magna (subarachnoid space) with Streptococcus pneumoniae (10(5) to 10(7) colony-forming units). All rats injected developed meningitis as evidenced by increased cerebrospinal fluid (CSF) white cell counts and positive CSF cultures. Serial ABR measurements taken 6, 12, 15, 18, 21, and 24 hours after inoculation demonstrated significant threshold shifts and eventual loss of the ABR waveform as compared with measurements in control rats injected with sterile culture medium. Hearing loss began approximately 12 to 15 hours after inoculation and progressed to complete loss by 24 hours (17 of 18 animals). No correlation was found between the magnitude of hearing loss and CSF white cell count or bacterial titer. Temporal bone histology of rats with meningitis shows a dense inflammatory cell infiltrate throughout the subarachnoid space. Labyrinthine inflammatory cells were confined to the scala tympani. The cochlear aqueduct is the proposed route of infection from the meninges to the labyrinth (scala tympani). Endolymphatic hydrops was also noted throughout the cochlea. These experiments both establish a reproducible animal model of meningogenic hearing loss and support the hypothesis that this hearing loss is progressive rather than abrupt in onset and is related to the duration of untreated infection. CSF inflammatory cells appear to enter the cochlea through the cochlear aqueduct. This reliable animal model will enable future studies directed toward further understanding the pathogenesis and pathophysiology of this hearing loss.

Intact canal wall mastoidectomy with tympanoplasty for cholesteatoma in children.

OBJECTIVE/HYPOTHESIS: Cases of cholesteatoma in pediatric patients were reviewed to determine which factors influence the outcome of surgical treatment. Cholesteatoma is considered a more aggressive disease in children than in adults. The outcomes of intact canal wall (ICW) mastoidectomy and canal wall down (CWD) mastoidectomy were assessed, as comparisons of different surgical technique. STUDY DESIGN: A retrospective analysis of all cases of pediatric cholesteatoma treated at a single institution by the senior author (P.R.L.) over a period of 11 years was conducted. METHODS: Patient information was collected from an otology database, patient records, and audiology files. RESULTS: Sixty-six patients, aged 10 months to 18 years, were treated and followed for an average of 37.7 months (range 12.2 months to 12.5 y). ICW mastoidectomy with tympanoplasty was the primary surgical treatment in 41 patients. Nineteen percent had residual disease at a planned second stage surgery and 22% developed recurrent cholesteatoma for a total recidivism rate of 41%. A SRT of less than 30 dB HL was achieved in 75% of these patients. Seventeen patients underwent CWD mastoidectomy with tympanoplasty initially. Two patients (12%) had residual cholesteatoma found at a planned second state procedure, and no recurrent cholesteatoma was encountered. Seventy-two percent maintained a SRT of less than 30 dB HL. CONCLUSIONS: These results support the continued use of ICW mastoidectomy with tympanoplasty for pediatric cholesteatoma. If planned second stage surgery is necessary, the long-term results of an ear with useful hearing and few problems with chronic medical care are gratifying. For reasons of anatomy or in an only hearing ear, CWD mastoidectomy with tympanoplasty provides a safe ear and good hearing results. Mastoid cavity care must be maintained indefinitely in many cases.

Thermal imaging of the temporal bone in CO2 laser surgery: an experimental model.

The unique properties of lasers create an enormous potential for specific treatment of chronic ear disease. Despite the widespread acceptance and use of the laser, however, a complete understanding of the time- and space-dependent temperature distribution in otic capsule bone immediately after pulsed laser exposure has not been elucidated. Using a liquid nitrogen-cooled mercury-cadmium telluride infrared detector, the temperature distribution in human cadaveric otic capsule bone was determined immediately after pulsed (100 msec) carbon dioxide laser exposure (0.3 to 4.0 W; 200 microm spot diameter). The time- and space-dependent temperature increases and thermal diffusion were determined as a function of the laser power density and were found to vary linearly.

Effects of unilateral cochlea removal on anteroventral cochlear nucleus neurons in developing gerbils.

Afferent regulation of neurons in the cochlear nucleus as a function of age was investigated at the light microscope level. Unilateral cochlea removal was performed on Mongolian gerbils of three age groups: 1, 8, and 20 weeks postnatal. Animals survived for either 2 days or 2 weeks. An additional group of neonatally operated animals had a prolonged survival of 9 weeks. The number of neurons in anteroventral cochlear nucleus (AVCN) was counted, and cross-sectional area measurements of large spherical cells in AVCN were made. In animals 1 week old at the time of surgery, there was a 35% reduction in neuron number in AVCN after 2 days, a 58% reduction after 2 weeks, and a 59% reduction 9 weeks after inner ear destruction. However, in animals 20 weeks old at the time of surgery, there was no cell loss in AVCN either 2 days or 2 weeks after surgery. Animals in each age group showed a reduction in cross-sectional area of large spherical cells in AVCN after cochlea ablation. The gerbils that underwent cochlea removal at 8 and 20 weeks showed an average decrease of 14-18%. This effect was seen as early as 2 days after cochlea removal. Animals that underwent cochlea removal at 1 week exhibited the greatest change; a 25% decrease at 2 days progressed to 38% at 2 weeks following cochlea removal. No appreciable further changes were seen at 9 weeks after neonatal cochlea removal. The results indicate greater susceptibility of 1-week-old gerbil cochlear nucleus neurons to peripheral loss than found in older animals.(ABSTRACT TRUNCATED AT 250 WORDS)