The Study On Gentamicin Entry Into The Inner Ear Following Intratympanic Injection And Impact On Inner Ear

Objective:1). To explore the distribution of gentamicin when applied to the RWM compared with the stapes footplate of guinea pigs.2) To clarify the entry of gentamicin into the inner ear.3) To explore how gentamicin affect hearing after local administration.Methods:1) Part 1:Purified Gentamicin-Texas Red (GTTR) (0.2mg/ml) was administered to the RWM of guinea pigs for 1 hour. Perilymph was then collected using a protocol in which ten samples, each of approximately 1μ1, were taken sequentially from the lateral semi-circular canal into capillary tubes. GTTR concentration of the samples was determined by plate reader. These data were interpreted using a simulation program that incorporates all the major fluid and tissue compartments of the cochlea and vestibular systems. The proportion of GTTR entering by the two routes was quantified.2) Part 2,3:Four microliters of GT or GTTR was soaked into 2-mm disks of absorbable collagen sponge. The sponge was placed onto the stapes footplate or RWM under the operating microscope, respectively. At 7 days after treatment, animals were sacrificed then perfused the inner ears with 4% paraformaldehyde. The entire inner ear was immersed in the same fixative solution overnight at 4℃. The vestibular end-organs and basilar membrane of cochlea were harvested in PBS. Immunofluorescence staining was conducted with anti-gentamicin、 Alexa Fluor(?) 488 Phalloidin and DAPI. Confocal fluorescence microscopy was used to determine the intensity and localization of GTTR and GT. The intensity of GTTR fluorescence within the cells was quantified using the Image-J software. Measurements of gentamicin-positive cells were statistically analyzed using SPSS 19.0. Hearing was assessed from 8 kHz to 32 kHz by auditory brainstem response for GT administration group.3) Part 4:Clinical Application:A modified low-dose (30mg/ml) intratympanic approach was used in 17 patients with intractable vertigo and the clinical outcomes were followed.Results:1) Drugs entry into the inner ear occurs both through the RWM (59.8%) and a site near the stapes (40.2%) following local drugs applications.2) The substantial difference between the drugs raises the possibility that the rates of entry by each route are substance-dependent.3) Application of GTTR to the RWM resulted in statistically significant intensity of GTTR fluorescence of compared to the same dosage of GTTR applied to the stapes.4) The results of hearing threshold of ABR revealed that the ears under GT administration of RWM group were higher than the preoperative results in frequency of 8 kHz to 32 kHz. Stapes group showed no significant changes.5) Hearing loss following intratympanic gentamicin injection was not significant. Vestibulotoxicity was assessed by changes in threshold and P1 latency of cVEMP test. It was demonstrated by statistically significant difference between preinjection (threshold:82.5±5.59 dBnHL, P1 latency:16.64±2.38 ms) and postinjection (threshold:89±4.90 dBnHL, P1 latency:18.52±1.62ms, P<0.05).Conclusion:1) This study confirms that for local applications of drugs entry into the ear occurs both through the RW membrane and directly into the vestibule at a site near the stapes.2) The rates of entry by each route are substance-dependent..3) Animals receiving gentamicin applied to the RWM exhibited markedly higher levels of hearing loss between 8 and 32 kHz than those receiving gentamicin near stapes or saline controls.4) Intratympanic injection of gentamicin was effective in controlling vertigo. No statistical differences were noted in hearing loss postinjection compared to preinjection. Vestibulotoxicity was demonstrated by the results of cVEMP test.