Uptake And Impacts Of Gentamicin To The Auditory And Vestibular Nuclei After Intratympanic Administration

Objects 1) To explore the uptake of gentamicin into the central vestibular nuclei in the brainstem after intratympanic administration.2) To explore the uptake of gentamicin into the central auditory nuclei in brainstem after intratympanic administration.3) To explore the impacts of gentamicin on these immunolabeled nuclei.Methods 1) Adult guinea pigs were used in this study. Gentamicin was administrated transtympanically to the guinea pigs to establish an animal model. Retrograde axonal transport of HRP via the oval window was conducted to identify the vestibular efferent neurons.2) Guinea pigs were sacrificed at 24 hour,3 day,7 day,14 day and 30 day respectively. For animals to undertake immunostaining,4% paraformaldehyde was used as the fixative for transcardial perfusion; while for animals to undertake transmission electron microscope, a mixed liquor of paraformaldehyde and glutaraldehyde were utilized. Brainstem and vestibular ganglion were harvested and followed by cryosections. Immunofluorescent staining was conducted with anti-gentamicin、anti-beta-Ⅲ-tubulin and DAPI.3) Toluidine blue staining was used to identify the relative location of different nuclei. Three-labeled immunofluorescent staining was used to explore the uptake of gentamicin in nuclei. Transmission electron microscope was used to explore the ultrastructural changes happened to these nuclei. Measurements of gentamicin-positive cells were statistically analyzed using SPSS 19.0 (SPSS, Inc., Chicago, IL). One-way analysis of variance (ANOVA) was performed and followed by the least-significant differences (LSD test) to test the differences between groups of different survival time point. Comparison of cell counts between the two laterals of each group was performed using the paired-sample t test. A level of P value< 0.05 was judged to be statistically significant.Results 1) Gentamicin presents in the bilateral vestibular efferent neurons, superior olive complex as well as ipsilateral cochlear nuclei. Within the vestibular efferent neurons、superior olive complex, gentamicin is predominantly labeled in the cytoplasm and processes of neurons, while within the cochlear nuclei positive gentamicin labeling presents mainly around the neurons but not in the neuronal cytoplasm. The Scarpa’s ganglion was demonstrated containing no gentamicin at 3 d after the injection. Neither were any subgroups of the vestibular nuclear complex (data not shown).2) Gentamicin in the L-EVE was first demonstrated at 24 h after the drug administration. Then the number of neurons containing gentamicin in the L-EVE increases to the peak at 3 d, remains on the equal level at 7 d, and then decreases at 14 d. At 30 d, there were very weak gentamicin and rare gentamicin-positive neurons in the L-EVE.3) Nerve fibers within the dorsal cochlear nucleus、the root of the 8th nerve、as well as the descending pathway of SOC were immunostained with gentamicin.4) At 3 day after the administration, electron transmission microscope revealed evident vacuole within the afferents and efferents of typeⅠ&Ⅱvestibular hair cells.5) At 3 day after the administration,brisement of myelin sheath presents over the ipsilateral EVE、SOC and CN. Vacuoles were demonstrated in the neurites of the efferent neurons but not in the cochlear nucleus. At 30 day after administration, layers of myelin sheath shows intact and in regularity within the EVE、SOC and CN. Besides, Swelling neurons, dark cells and/or vacuole in mitochondria were demonstrated within the EVE and SOC nuclei, while astroglia cell swelling is the mainly presentation within the CN.Conclusion 1) After transtympanic administration, gentamicin is not only uptaken by the peripherial cochlear and vestibular end organs, but also by the corresponding auditory and vestibular nuclei within the brainstem. Retrograde axonal transport of gentamicin is responsible for this distribution of gentamicin in the bilateral EVE and SOC, and antereograde transport to the ipsilateral cochlear nucleus.3) Intratympanic adminsitration of gentamicin results in reversible brisement of myelin sheath in the EVE、SOC and CN. Several neurons were demonstrated with severe swelling even apoptosis within the EVE and SOC nuclei over time.