Studies On The Distributation Of Gentamicin And Degeneration In The Peripheral Vestibular System Following Intratympanic Injection

PART ONE Temporal and spatial gentamicin distribution in the peripheral vestibular System after Intratympanic Injection I Distribution of gentamicin on peripheral vestibular end organ after a single dose administrationPurpose uptake and accumulation of gentamicin by cells in the guinea pig peripheral vestibular end organ after a single dose intratympanic application were investigated using a fluorescent probe-gentamicin-Texas-red conjunction (GTTR).Methods Adult guinea pigs were sacrificed and fixed at12h,24h,3d,7d,14d and28d after administered with a single dose of GTTR(n=35,0.1mg/ml) or TR (n=10,0.065mg/ml) through tympanic membrane. Confocal fluorescence microscopy was used to determine the intensity and localization of GTTR at vestibular end organ.Results GTTR was first observed accumulated at the cuticular and the infra-cuticular regions of hair cells, with weak diffuse cytoplasmic labeling in the cell body at12hour after local injection. With the time course more and more GTTR was accumulated and which reached its peak density level at3to7days and lasting until four weeks. In addition, the transitional cells and planum semilunatum cells also displayed GTTR labeling in the supra-nuclear regions shortly after intra-tympanic injection, and retained this distribution for up to4weeks. GTTR was seldom identified in the supporting cells and the dark cells. Among the vestibular end organs, the saccule accumulated more GTTR than the utricle.Conclusion GTTR reached high peak density level between3to7days and lasting for4weeks after a single dose administration. The saccule accumulated more GTTR than the utricle. â…¡ Dosage depended Gentamicin Distributation on peripheral vestibular organ after Intratympanic InjectionPurpose To clarified gentamicin uptake and accumulate at the sensory hair cell and non-sensory cell of crista ampullaris using a fluorescent probe-gentamicin-Texas-red conjunction (GTTR).Methods Different concentration of GTTR (0.1,0.4,0.8mg/ml)) were applied to the adult guinea pigs (n=9) through tympanic membrane. The animals were killed and fixed at7days. Confocal fluorescence microscopy was used to obtain the intensity and localization of GTTR at section of crista ampullaris.Results GTTR has a concentration gradient accumulation from the central zone type â…  hair cell to peripheral zone type â…  hair cell and then type â…¡ hair cell in the ampullar sensory epithelium and the difference was statistically significant. With the concentration increased more and more diffuse cytoplasmic labeling in the cell body at type-â…  hair cell. On the contrary, there was not obviously diffuse cytoplasmic labeling appeared at type â…¡ hair cell, transitional cells and Planum semilunatum cells.Conclusion GTTR has a concentration gradient accumulation from the central zone type â…  hair cell to peripheral zone type â…  hair cell and then type â…¡ hair cell in the ampullar sensory epithelium. â…¢ Gentamicin Distributation on peripheral vestibular organ after Intratympanic InjectionPurpose Using gentamicin antibody to clarify gentamicin uptake and accumulate at peripheral vestibular organ and campared to the GTTR distribution.Methods Adult guinea pigs were sacrificed at7d and28d after administered with a single dose of gentamicin (n=10,5mg/ml) and Sodium Chloride through tympanic membrance. Confocal fluorescence microscopy was used to determine the intensity and localization of gentamicin.Results gentamicin has the same concentration gradient accumulation from the Central zone type â…  hair cell to peripheral zone type â…  hair cell and then type â…¡ hair cell in the ampullar sensory epithelium and with obviouly statistics difference among them (P<0.05). Type-â…  hair cell with diffused cytoplasmic labeling with the dosage increased. There was not obviously gentamicin labeling appeared at transitional cells and planum semilunatum cells and dark cell and supporting cells.Conclusion GTTR has almost the same distribution as gentamicin antibody at sensory epithelium. GTTR was more sensitive than gentamicin antibody. PART TWO Studies on the degeneration in the peripheral vestibular end organ after Intratympanic InjectionPurpose To clarifiy the degneration and the distribution relationship of gentamicin on peripheral vestibular end organ after Intratympanic InjectionMethods Different concentration of gentamicin (15,30mg/ml) were applied to adult guinea pigs (n=20) through tympanic menbrance, which were sacrificed and fixed at7d and28d. Anti-caretinin and anti-tenscin was used as marker of type I hair cell on crista ampullaris. Phalloidin-TRIRC marked stereocilium on macula. Laser confocal microscopy was used to observe the degeneration of the peripheral vestibular organ.Results The calyces of calretinin positive hair cells were partially or totally retracted without nucleus missing at dosage of15mg/ml at7d. It is the same as the crista ampullaris obtained at7d (30mg/ml) and28d (15mg/ml) that all calyces of calretinin positive hair cells were totally retracted as a ball at basement membrane and the nucleus of hair cell decreased in the centre part. The animal sacrificed at28d (30mg/ml) not only calretinin positive hair cells dispeared but also tenscin positive hair cells were damaged at the peripherin part. Conclusion The degenerationt of crista ampullaris was corresponding to gentamicin distributation. On the contrary there was a negtative relationship between gentamicin distributation and degeneration on the macula. PART THREE Uptake and accumulation of gentamicin by adult guinea pigs peripheral vestibular end organ in vitroPurpose To investigate contributation of the cupula and otolith on gentamicin uptake and accumulation by adult guinea pigs peripheral vestibular end organ.Methods The crista ampullaris and utricle explants were prepared and removed the the cupula and otolith. Cochlear explants were cultured with GTTR for different time (30min,2hour) Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear and vestibular cells after labeling with phalloidin-FITC and anti-calretinin.Results In the control group the stereocilium of the utricle was concinnous at30min and2hours and became disorganiztion, lodging and missing after culture24hour. The neucles of the har cell at crista ampullaris was regular until2hour and became disorganization at24hour group. In GTTR administrated group which was seen first localized at striola of utricle and Type-I hair cell at centre part of crista ampullarisConclusion the movement cupula and otolith was not the majority affect factor of the difference of gentamicin distributation of the different type of cell.