Experimental Studies Of The Cell Biologic Effects Of Intratympanic Gentamicin Injection On The Inner Ear Cells

PART I The Distribution of Gentamicin in Inner Ear afterIntratympanic Gentamicin InjectionDistribution of gentamicin in Inner Ear after a single dose administration Purpose Uptake and accumulation of gentamicin by cells in the guinea pig inner ear after a single dose intratympanic application were investigated using a fluorescent probe --gentamicin-Texas-red conjunction (GTTR).Methods Adult guinea pigs (n=80) were administered a single dose of GTTR to the middle ear cavity through the intact membrane and survived for 12h,24h,48h,3d,4d,7d,14d and 28d. Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear and vestibular cells after staining with phalloidin -alexa-488. TR and DMSO were injected into the tympanum as control.Results Soon after local drug administration, there was diffuse staining all tissue in the labyrinth. At later time the outer hair cells and sensory cells of vestibular organ were staining more densely than other cells in the inner ear. The peak level of fluorescent density was reached 3 days after local injection. The GTTR was seen in the infracuticular zone. This protocol produces no damage to the inner hair cells. Conclusion GTTR was a potential fluorescent probe to investigate the pharmacokinetics and mechanisms of gentamicin accumulation in local application over time. Distribution of gentamicin in inner ear after different protocol applications Purpose The uptake and accumulation of gentamicin in the guinea pig inner ear after different protocol treatments was compared using GTTR intratypanic injection. Methods Intratympanic injection of GTTR was applied to adult guinea pigs (n=32) using different protocol: in group A and B only one dose of GTTR was applied. In group C GTTR were injected once a day for four consecutive days and in Group D GTTR was given once a week for a total of four treatments. Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear and vestibular cells after staining with phalloidin -alexa-488.Results After local drug administration using different protocols, the fluorescent was observed in the labyrinth especially in the outer hair cells and in the sensory cells of the vestibular. In Group C and Group D more GTTR accumulation was observed than that in Group A and B. But there was still density fluorescent 28 days after a singledose. The fluorescent of GTTR was also seen in the infracuticular zone.Conclusion Gentamicin accumulation was varied in local application using differentprotocols.PART II Damage of Inner Ear Cells after I ntratympanicGentamicin Injection using Different ProtocolsPurpose To compare the damage of inner ear cells after intratympanic gentamicininjection using different protocols.Methods Intratympanic injections of gentamicin were applied to adult guinea pigs(n=32) using different protocol administration: In Group A and B only one dose ofgentamicin was applied. In Group C gentamicin was injected one once a day for fourconsecutive days and in Group D gentamicin was given once a week for a total of fourinjection. Electrical scanning microscopy and Laser confocal microscopy were usedto observe the damage of gentamicin to the cochlear and vestibular cells.Results Damage can be seen in all of the four groups. In Group C and Group D thedamage were more severe than that in Group A and B. But there is less damage to theinner hair cells in Group A and B.Conclusion Damage was varied in local application using different protocols. Thedamage using a single lower dose to the inner hair cells was seen less severely thanother protocols in this study.PART III Uptake and accumulation of gentamicin By mouse hair cells in vitroPurpose To investigate the uptake and accumulation of gentamicin by mouse haircells in vitro.Methods The cochlear explants were prepared from the microdissected neonatalmouse cochlea. Cochlear explants were cultured with GTTR for different time. Laserconfocal microscopy was used to observe the distribution of GTTR in the cochlearand vestibular cells after labeling with phalloidin -alexa-488.Results Soon after culture, there was diffuse staining all tissue cells in the explants.At later time the outer hair cells were more staining than other cells in the explants.The peak level of fluorescent density was reached at 24 hours culture. The GTTR wasseen in the infracuticular zone of the hair cells.Conclusion GTTR and cochlea explants were useful methods to investigate thepharmacokinetics and mechanisms of gentamicin accumulation over time.