Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,对照组（16只）导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异（P〉0．05）;鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异（P〉0．05）,8kHz较术前增高（P〈0．05）,16kHz、20kHz较术前明显增高（P〈0．01）,术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01）,但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。     

Simultaneous Measurements of Ossicular Velocity and Intracochlear Pressure Leading to the Cochlear Input Impedance in Gerbil

Recent measurements of three-dimensional stapes motion in gerbil indicated that the piston component of stapes motion was the primary contributor to intracochlear pressure. In order to make a detailed correlation between stapes piston motion and intracochlear pressure behind the stapes, simultaneous pressure and motion measurements were undertaken. We found that the scala vestibuli pressure followed the piston component of the stapes velocity with high fidelity, reinforcing our previous finding that the piston motion of the stapes was the main stimulus to the cochlea. The present data allowed us to calculate cochlear input impedance and power flow into the cochlea. Both the amplitude and phase of the impedance were quite flat with frequency from 3 kHz to at least 30 kHz, with a phase that was primarily resistive. With constant stimulus pressure in the ear canal the intracochlear pressure at the stapes has been previously shown to be approximately flat with frequency through a wide range, and coupling that result with the present findings indicates that the power that flows into the cochlea is quite flat from about 3 to 30 kHz. The observed wide-band intracochlear pressure and power flow are consistent with the wide-band audiogram of the gerbil.     

Multifrequency admittancemetry in Ménière's Disease: a preliminary study for a new diagnostic test.

OBJECTIVE: To assess the qualitative and quantitative aspects of multifrequency tympanograms in patients with Ménière's disease. STUDY DESIGN: Prospective study of adults. SETTING: University medical center. PATIENTS: Forty patients with Ménière's disease outside of episodes and 24 normal-hearing subjects with no otologic history. INTERVENTIONS: Resonance frequency sweep and tympanograms of admittance, conductance, and susceptance at 2 kHz and at resonance frequency were performed in each ear. MAIN OUTCOME MEASURES: A qualitative assessment was performed to find reproducible and comparable tympanograms (Student's t test) as a prerequisite for quantitative assessment. Four measurement criteria were compared between the groups to establish a diagnostic test (chi Pearson test). A p value less than 0.05 was considered statistically significant. RESULTS: Resonance frequency was decreased in Ménière's disease. Only conductance tympanograms at 2 kHz had a constant pattern. Width of conductance tympanograms at 2 kHz was increased in symptomatic Ménière's disease compared with normal ears. Using conductance width at 2 kHz as a diagnostic test with a threshold of 235 daPa, we found a statistically significant difference between symptomatic ears of patients with Ménière's disease and ears of normal subjects (p < 10). More than 95% of normal subjects' ears had a negative test; 56.5% of affected ears of patients with Ménière's disease and 45.8% of nonaffected ears of patients with Ménière's disease had a positive test. CONCLUSION: Width of conductance at 2 kHz seems to be a sensitive, simple diagnostic test in Ménière's disease. It would also be of great interest in studying Ménière's disease bilaterality.     

Stapes Vibration in the Chinchilla Middle Ear: Relation to Behavioral and Auditory-Nerve Thresholds

The vibratory responses to tones of the stapes and incus were measured in the middle ears of deeply anesthetized chinchillas using a wide-band acoustic-stimulus system and a laser velocimeter coupled to a microscope. With the laser beam at an angle of about 40 ° relative to the axis of stapes piston-like motion, the sensitivity-vs.-frequency curves of vibrations at the head of the stapes and the incus lenticular process were very similar to each other but larger, in the range 15–30 kHz, than the vibrations of the incus just peripheral to the pedicle. With the laser beam aligned with the axis of piston-like stapes motion, vibrations of the incus just peripheral to its pedicle were very similar to the vibrations of the lenticular process or the stapes head measured at the 40 ° angle. Thus, the pedicle prevents transmission to the stapes of components of incus vibration not aligned with the axis of stapes piston-like motion. The mean magnitude curve of stapes velocities is fairly flat over a wide frequency range, with a mean value of about 0.19 mm^.(s Pa⁻¹), has a high-frequency cutoff of 25 kHz (measured at −3 dB re the mean value), and decreases with a slope of about −60 dB/octave at higher frequencies. According to our measurements, the chinchilla middle ear transmits acoustic signals into the cochlea at frequencies exceeding both the bandwidth of responses of auditory-nerve fibers and the upper cutoff of hearing. The phase lags of stapes velocity relative to ear-canal pressure increase approximately linearly, with slopes equivalent to pure delays of about 57–76 μs.     

Drug delivery to the cochlea using PLGA nanoparticles

OBJECTIVES: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea. STUDY DESIGN: An experimental study. METHODS: We examined the distribution of rhodamine, a fluorescent dye, in the cochlea, liver, and kidney of guinea pigs. Intravenous injection of rhodamine or rhodamine-encapsulated PLGA nanoparticles was used to target the fluorescent dye systemically to the liver, kidney, and cochlea, and these molecules were applied locally to the round window membrane (RWM) of the cochlea. The localization of rhodamine fluorescence in each region was quantitatively analyzed. RESULTS: After systemic application of rhodamine nanoparticles, fluorescence was identified in the liver, kidney, and cochlea. The systemic application of nanoparticles had a significant effect on targeted and sustained delivery of rhodamine to the liver but not the kidney or cochlea. Rhodamine nanoparticles placed on the RWM were identified in the scala tympani as nanoparticles, indicating that the PLGA nanoparticles can permeate through the RWM. Furthermore, the local application of rhodamine nanoparticles to the RWM was more effective in targeted delivery to the cochlea than systemic application. CONCLUSIONS: These findings indicate that PLGA nanoparticles can be an useful drug carrier to the cochlea via local application.     

Three-dimensional stapes footplate motion in human temporal bones

The literature provides conflicting information on whether the motion of the stapes footplate is piston-like or some other type of motion, such as rotational or rocking. Examination of the three-dimensional (3D) motion of the stapes footplate appears to be an excellent way to understand this complicated motion. Five microsphere reflective targets were placed on the stapes footplate in ten fresh human cadaver temporal bone preparations, and their vibration measured through an extended facial recess approach using a laser Doppler vibrometer. The five target sites on the stapes footplate were center, anterior, posterior, superior and inferior. The stimulus was a sound input of 80-120 dB SPL at the tympanic membrane over a frequency range of 0.1 to 10 kHz. The 3D motion of the stapes footplate was calculated using the velocity amplitude and phase obtained for each target. For frequencies up to 1.0 kHz the vibration of the stapes footplate was primarily piston-like; this motion became complex at higher frequencies, with rotary motion along both the long and short axis of the footplate. When the cochlea was drained, stapes footplate motion became essentially piston-like for all frequencies.     

Holographic observation of the tympanic membrane vibration after stapes fixation

Sclerosis of auditory ossicle was experimentally induced by fixation of the stapes, and its effect on the tympanic membrane vibration was examined using fresh 10 canine temporal bones by means of holographic interferometry. By changing the sound pressure (from 90 to 110dB) and the frequency (from 250Hz to 8kHz) in steps, vibration of the membrane was induced in a free field. At each frequency, the vibration of the membrane was photographed by the time averaged method using He-Ne laser. A small hole was created in the tympanic bulla and the stapes was fixed with an adhesive (Alon Alpha), followed by closure of the hole. Before stapes fixation, the posterior part of the membrane showed a simple vibration pattern, forming cocentric interference circles at frequencies from 250Hz to 2kHz. At 3kHz, segmental vibration began to be noted, and complex segmental vibration was seen at frequencies 4.5 and 6kHz. The vibration after stapes fixation became simpler at frequencies over 3kHz, accompanied by decrease in the number of segmental vibrations. Vibration amplitude in the posterior part of the membrane decreased from 250Hz to 3kHz, while it increased at frequencies over 4kHz. In the anterior part of the membrane, the amplitude decreased at frequencies under 2kHz, remained unchanged at 3kHz and increased at frequencies over 4kHz after stapes fixation. In the tip of the manubrium mallei, the amplitude decreased at frequencies under 3kHz and increased at frequencies over 4kHz. Thus, a shift of peak vibration to high sound region was observed after stapes fixation at every part of tympanic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Round window electrode insertion potentiates retention in the scala tympani

Abstract Conclusions: The round window membrane (RWM)-intentioned approach is superior to the traditional bony cochleostomy (BC) approach in obtaining electrode placement within the scala tympani (ST). Objective: Cochlear implant outcome is influenced by several factors, including optimal placement and retention of the electrode array within the ST. The present study aimed to assess whether the RWM route is superior to a traditional BC for placement and retention of the electrode array in the ST. Methods: This was a prospective consecutive non-randomized comparison study. All patients were implanted with the Advanced Bionics 1J electrode array. The RWM approach (n = 32) was compared with a traditional BC group (n = 33). The outcome measure was the electrode position as judged within the scalar chambers at four points along the basal turn using postoperative computed tomography (CT). Results: When the mean position scores were compared, the RWM-intentioned group had significantly more electrodes directed towards the ST compartment than the BC group (p < 0.001). The RWM electrodes achieved 94% ST retention compared with 64% for the BC group (p < 0.05). All electrodes stayed in the ST in the RWM group, whereas in the BC group 9% crossed from the ST to the scala vestibuli.     

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.     

Basilar Membrane Vibrations Near the Round Window of the Gerbil Cochlea

Using a laser velocimeter, responses to tones were measured at a basilar membrane site located about 1.2 mm from the extreme basal end of the gerbil cochlea. In two exceptional cochleae in which function was only moderately disrupted by surgical preparations, basilar membrane responses had characteristic frequencies (CFs) of 34–37 kHz and exhibited a CF-specific compressive nonlinearity: Sensitivity near the CF decreased systematically and the response peaks shifted toward lower frequencies with increasing stimulus level. Response phases also changed with increases in stimulus level, exhibiting small relative lags and leads at frequencies just lower and higher than CF, respectively. Basilar membrane responses to low-level CF tones exceeded the magnitude of stapes vibrations by 54–56 dB. Response phases led stapes vibrations by about 90° at low stimulus frequencies; at higher frequencies, basilar membrane responses increasingly lagged stapes vibration, accumulating 1.5 periods of phase lag at CF. Postmortem, nonlinearities were abolished and responses peaked at ~0.5 octave below CF, with phases which lagged and led in vivo responses at frequencies lower and higher than CF, respectively. In conclusion, basilar membrane responses near the round window of the gerbil cochlea closely resemble those for other basal cochlear sites in gerbil and other species.     

Assessment of stapes mobility by use of a newly developed piezoelectric ceramic device. A preliminary experiment in dogs

A piezoelectric device was developed for assessment of stapes mobility during middle ear surgery. The device comprises a pair of ceramic bimorph elements: one for activation of the stapes and the other to pick up the vibration as an electric output, which varies in accordance with the stapes mobility, ie, the inverse of the cochlear input impedance (Zsc). The device is compact and easily manipulated even in the narrow surgical field of the ear. However, the measuring range is restricted to between 1 and 10 kHz. Measurement of Zsc was conducted with this device in 5 ears of 5 dogs. The mean magnitude of Zsc increased with frequency in the range from 1 to 10 kHz: 0.95 megohm at 1 kHz and 8.8 megohms at 10 kHz. After fixation of the stapes with dental cement, the magnitude increased to more than 10 megohms, except at 1 kHz. The results suggest that the device is useful in detecting decreases in stapes mobility in patients with chronic otitis media.     

Stapedial reflex and ears with high static acoustic admittance

PURPOSE: To evaluate modified acoustic reflex diagnostic protocols for a group of individuals (n = 9) with high peak compensated static acoustic admittance (Y(tm)) tympanograms. METHOD: A modified procedure designed to improve acoustic stapedius reflex threshold (ASRT) measurements in individuals with high-admittance tympanograms was employed in both an experimental and a control group. ASRTs were measured at 0.5, 1.0, and 2.0 kHz, ipsilateral and contralateral. Measurements were obtained within each condition for 7 ear canal pressures that were set to 0, +/-50, +/-100, and +/-150 daPa (relative to tympanometric peak pressure [TPP]). RESULTS: Though measuring ASRTs at -50 daPa (relative to TPP) in the high-admittance and normal groups did not result in significantly better thresholds than at TPP, the absent reflex rate was reduced when the ear canal pressure was changed by -50 daPa during ASRT measurements. CONCLUSIONS: Based on this sample, it is suggested that a patient presenting with high peak compensated static acoustic admittance (peak Y(tm) > or = 2.1 mmho) undergo ASRT evaluation with the ear canal pressure set to -50 daPa (relative to TPP).     

Morphological changes in the round window membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla

ObjectiveThe round window membrane (RWM) encloses the round window, the opening into the scala tympani (ST) from the middle ear. During the course of acute otitis media (AOM), structural changes of the RWM can occur that potentially affect sound transmission into and out of the cochlea. The relationship between such structural changes and conductive hearing loss during AOM has remained unclear. The focus of the current study was to compare the thickness distribution across the RWM surface between normal ears and those with AOM in the chinchilla. We assessed the occurrence of AOM-associated histological changes in this membrane compared to uninfected control animals after AOM of two relatively short durations.Material and methodsAOM was induced by transbullar injection of the nontypeable Haemophilus influenzae strain 86-028NP into two groups of adult chinchillas (n = 3 each). Bullae were obtained from the two infected groups, at 4 days or 8 days post challenge. Structures and thickness of these RWMs were compared between the two infected treatment groups and to RWMs from uninfected control animals (n = 3) at seven different RWM locations.ResultsRWM thickness in infected chinchillas increased significantly at locations along the central line on the 4th day post bacterial challenge compared to values found for uninfected control animals. Lymphocyte infiltration and edema were the primary contributors to these thickness increases. No significant further increases in RWM thickness were observed when RWMs from chinchillas ears infected for 4 and 8 days were compared. Thickness and structural changes at the RWM lateral and medial areas were less visually obvious and not statistically significant among the three treatment groups. These latter RWM regions clearly were less affected during AOM than the central areas.ConclusionsThis histological study establishes that H. influenzae-induced AOM causes significant acute changes in chinchilla RWM structure that are characterized by region-specific increases in thickness. Our new morphological findings comparing normal and diseased chinchilla RWMs identify yet another biomechanical mechanism by which nontypeable H. influenzae may contribute to hearing loss in AOM.     

On a novel type of neuron with proposed mechanoreceptor function in the human round window membrane--an immunohistochemical study.

An immunohistochemical study was performed on surgically obtained human fresh cochlear tissue, using synaptophysin antibodies. After immediate aldehyde fixation and decalcification in Na-EDTA serial cryosections were made of the cochlea including the round window membrane (RWM). Apart from highly specific immunostaining of spiral ganglion cells and unmyelinated nerve fibers an immunoreactive neuroreceptor could be demonstrated at the postero-medial insertion of the RWM. The perikaryon showed intense synaptophysin immunoreativity with a distal process projecting into the fibrous stroma of the RWM displaying structural specializations suggestive of a mechanoreceptor function. It is speculated whether the neuroreceptor may be involved in the proprioception and/or mechanoreception of tensile forces generated within the lamina propria during displacement of the yielding RWM in the bony labyrinth. Such a function could be important for the regulation of perilymph pressure.     

Effect of middle ear fluid on sound transmission and auditory brainstem response in guinea pigs

Combined measurements of middle ear transfer function and auditory brainstem response (ABR) in live guinea pigs with middle ear effusion (MEE) are reported in this paper. The MEE model was created by injecting saline into the middle ear cavity. Vibrations of the tympanic membrane (TM), the tip of the incus, and the round window membrane (RWM) were measured with a laser vibrometer at frequencies of 0.2-40?kHz when the middle ear fluid increased from 0 to 0.2?ml (i.e., full fill of the cavity). The click and pure tone ABRs were recorded as the middle ear fluid increased. Fluid introduction reduced mobility of the TM, incus and RWM mainly at high frequencies (f?>?1?kHz). The magnitude of this reduction was related to the volume of fluid. The displacement transmission ratio of the TM to incus varied with frequency and fluid level. The volume displacement ratio of the oval window to round window was approximately 1.0 over most frequencies. Elevation of ABR thresholds and prolongation of ABR latencies were observed as fluid level increased. Reduction of TM displacement correlated well with elevation of ABR threshold at 0.5-8?kHz. Alterations in the ratio of ossicular displacements before and after fluid induction are consistent with fluid-induced changes in complex ossicular motions.     

Effects of a perilymphatic fistula on the passive vibration response of the basilar membrane

In this study, a three-dimensional finite-element model of the passive human cochlea was created. Dynamic behavior of the basilar membrane caused by the vibration of the stapes footplate was analyzed considering a fluid-structure interaction with the cochlear fluid. Next, the effects of a perilymphatic fistula (PLF) on the vibration of the cochlea were examined by making a small hole on the wall of the cochlea model. Even if a PLF existed in the scala vestibuli, a traveling wave was generated on the basilar membrane. When a PLF existed at the basal end of the cochlea, the shape of the traveling wave envelope showed no remarkable change, but the maximum amplitude became smaller at the entire frequency range from 0.5 to 5kHz and decreased with decreasing frequency. In contrast, when a PLF existed at the second turn of the cochlea, the traveling wave envelope showed a notch at the position of the PLF and the maximum amplitude also became smaller. This model assists in elucidating the mechanisms of hearing loss due to a PLF from the view of dynamics.     

Stapesfixation bei Kindern.

BACKGROUND AND OBJECTIVE: The audiometric results after stapes surgery in children with isolated stapes footplate fixation were reason enough to analyze intraoperative findings and surgical procedures. PATIENTS/METHODS: A total of 438 stapes surgeries were performed in the years 1985-2000, and a retrospective analysis was made of the anamnestic data, intraoperative findings and audiometric data from 12 children with isolated fixation of the stapes footplate. The average age of the children when operated was 10.2 years (the youngest was 7 and the eldest 13). A loss of hearing had set in 5 years earlier. RESULTS: Taking the anamnestic data and the intraoperative into account, otosclerosis was found in 5 children. One child had a narrow oval window niche, in a further six children a congenital fixation of the stapes footplate was evident. Only 58% (n=7) of the 12 children operated showed acceptable postoperative hearing results. The best hearing results were found in children with juvenile otosclerosis. In three of six children with congenital stapes fixation, a worsening of both bone and air conduction was evident. The average conductive hearing loss was 30 dB at 1, 2 and 4 kHz. CONCLUSIONS: When explorative tympanotomy is indicated, a thin-layer CT scan of the temporal bone should be made to assess the risk of a liquor pressure labyrinth developing.     

Sequential motion of the ossicular chain measured by laser Doppler vibrometry

Objective: In order to help a surgeon make the best decision, a more objective method of measuring ossicular motion is required.

Methods: A laser Doppler vibrometer was mounted on a surgical microscope. To measure ossicular chain vibrations, eight patients with cochlear implants were investigated. To assess the motions of the ossicular chain, velocities at five points were measured with tonal stimuli of 1 and 3?kHz, which yielded reproducible results. The sequential amplitude change at each point was calculated with phase shifting from the tonal stimulus. Motion of the ossicular chain was visualized from the averaged results using the graphics application.

Results: The head of the malleus and the body of the incus showed synchronized movement as one unit. In contrast, the stapes (incudostapedial joint and posterior crus) moved synchronously in opposite phase to the malleus and incus. The amplitudes at 1?kHz were almost twice those at 3?kHz.

Conclusions: Our results show that the malleus and incus unit and the stapes move with a phase difference.     

The frequency selectivity of the normal and pathological human cochlea

Summary Human AP tuning curves (tone on tone simultaneous and forward masking curves) were measured during transtympanic electrocochleography. For subjects with near normal thresholds, average Q_{10 dB} values (simultaneous masking) were 2.3 at 2 kHz, 3.6 at 4 kHz, and 4.7 at 8 kHz. Patients with threshold elevations of more than 40 dB, resulting from sensorineural hearing loss of cochlear origin, had tuning curves less sharply tuned by a factor of 2–3, with Q_{10 dB} values of 1–2 at 2 kHz and at 4 kHz, and 1–2.3 at 8 kHz. AP tuning curves and single fibre tuning curves (frequency threshold curves) were measured in normal guinea pigs; cochlear fibre tuning is sharper than AP tuning (simultaneous masking) by a factor of 1.8 (2–20 kHz). Assuming that this factor can be applied to the human cochlea, estimates of normal human cochlear fibre q_{10 db} values are 4.2 at 2 kHz, 6.5 at 4 kHz, and 8.5 at 8 kHz.This study was supported by INSERM grant no.: 79.1.418.6Presented at the 17th Workshop on Inner Ear Biology in Stockholm, June 23–25, 1980     

Marker entry into vestibular perilymph via the stapes following applications to the round window niche of guinea pigs

It has been widely believed that drug entry from the middle ear into perilymph occurs primarily via the round window (RW) membrane. Entry into scala vestibuli (SV) was thought to be dominated by local, inter-scala communication between scala tympani (ST) and SV through permeable tissues such as the spiral ligament. In the present study, the distribution of the ionic marker trimethylphenylammonium (TMPA) was compared following intracochlear injections or applications to the RW niche, with or without occlusion of the RW membrane or stapes area. Perilymph TMPA concentrations were monitored either in real time with TMPA-selective microelectrodes sealed into ST and SV, or by the collection of sequential perilymph samples from the lateral semi-circular canal. Local inter-scala communication of TMPA was confirmed by measuring SV and ST concentrations following direct injections into perilymph of ST. Application of TMPA to the RW niche also showed a predominant entry into ST, with distribution to SV presumed to occur secondarily. When the RW membrane was occluded by a silicone plug, RW niche irrigation produced higher concentrations in SV compared to ST, confirming direct TMPA entry into the vestibule in the region of the stapes. The proportion of TMPA entering by the two routes was quantified by perilymph sampling from the lateral semi-circular canal. The TMPA levels of initial samples (originating from the vestibule) were markedly lower when the stapes area was occluded with silicone. These data were interpreted using a simulation program that incorporates all the major fluid and tissue compartments of the cochlea and vestibular systems. From this analysis it was estimated that 65% of total TMPA entered through the RW membrane and 35% entered the vestibule directly in the vicinity of the stapes. Direct entry of drugs into the vestibule is relevant to inner ear fluid pharmacokinetics and to the growing field of intratympanic drug delivery.