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Trafficking of systemic fluorescent gentamicin into the cochlea and hair cells.

ABSTRACT: Aminoglycosides enter inner ear hair cells across their apical membranes via endocytosis, or through the mechanoelectrical transduction channels in vitro, suggesting that these drugs enter cochlear hair cells from endolymph to exert their cytotoxic effect. We used zebrafish to determine if fluorescently tagged gentamicin (GTTR) also enters hair cells via apically located calcium-sensitive cation channels and the cytotoxicity of GTTR to hair cells. We then examined the serum kinetics of GTTR following systemic injection in mice and which murine cochlear sites preferentially loaded with systemically administered GTTR over time by confocal microscopy. GTTR is taken up by, and is toxic to, wild-type zebrafish neuromast hair cells. Neuromast hair cell uptake of GTTR is attenuated by high concentrations of extracellular calcium or unconjugated gentamicin and is blocked in mariner mutant zebrafish, suggestive of entry via the apical mechanotransduction channel. In murine cochleae, GTTR is preferentially taken up by the stria vascularis compared to the spiral ligament, peaking 3 h after intra-peritoneal injection, following GTTR kinetics in serum. Strial marginal cells display greater intensity of GTTR fluorescence compared to intermediate and basal cells. Immunofluorescent detection of gentamicin in the cochlea also revealed widespread cellular labeling throughout the cochlea, with preferential labeling of marginal cells. Only GTTR fluorescence displayed increasing cytoplasmic intensity with increasing concentration, unlike the cytoplasmic intensity of fluorescence from immunolabeled gentamicin. These data suggest that systemically administered aminoglycosides are trafficked from strial capillaries into marginal cells and clear into endolymph. If so, this will facilitate electrophoretically driven aminoglycoside entry into hair cells from endolymph. Trans-strial trafficking of aminoglycosides from strial capillaries to marginal cells will be dependent on as-yet-unidentified mechanisms that convey these drugs across the intra-strial electrical barrier and into marginal cells.

SUBMITTER: Wang Q

PROVIDER: S-EPMC2674203 | biostudies-other | 2009 Jun

REPOSITORIES: biostudies-other

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Project description:ObjectiveIn addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours.ResultsLow intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs' sensory cells. Control vestibular tissues exposed to Dulbecco's phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence.ConclusionsAll peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells.

Project description:Reactive oxygen species are important elements in ototoxic damage to hair cells (HCs), appearing early in the damage process. Higher levels of natural antioxidants are positively correlated with resistance to ototoxins and many studies have shown that exogenous antioxidants can protect HCs from damage. While a very wide variety of antioxidants with different characteristics and intracellular targets exist, most ototoxicity studies have focused upon one or a few well-characterized compounds. Relatively little research has attempted to determine the comparative efficacy of large variety of different antioxidants. This has been in part due to the lack of translation between cell culture and in vivo measures of efficacy. To circumvent this limitation, we used an in vitro assay based on micro-explants from the basal and middle turns of the neonatal mouse organ of Corti to screen a commercial redox library of diverse antioxidant compounds for their ability to protect mammalian HCs from a high dose of the ototoxic antibiotic gentamicin. The library included several antioxidants that have previously been studied as potential treatments for HC damage, as well as many antioxidants that have never been applied to ototoxicity. The micro-explants were treated with 200 μM gentamicin alone, gentamicin plus one of three dosages of a redox compound, the highest dosage of compound alone, or were untreated. HC counts were determined before the gentamicin insult and at 1, 2, and 3 days afterward to evaluate the HC survival. From a total of 81 antioxidant compounds, 13 exhibited significant protection of HCs. These included members of a variety of antioxidant classes with several novel antioxidants, not previously tested on HCs, appearing to alleviate the damaging gentamicin effect. Some compounds previously shown to be protective of HCs were correspondingly protective in this in vitro screen, while others were not. Finally, one of the three pro-oxidant compounds included in the library as well as six antioxidants exhibited evidence of toxicity in the absence of gentamicin. The results demonstrate the wide variability in the ability of antioxidants to protect HCs from high-dose gentamicin damage, and identify promising candidate leads for further study as potential drug targets.Highlights• A medium-throughput assay based on micro-explants of the organ of Corti was developed to screen mammalian cochlear hair cells for protection from damage by ototoxins.• Eighty one antioxidants and 3 pro-oxidants were evaluated for hair cell protection from high-dose gentamicin.• Thirteen antioxidants were significantly protective, while 6 proved to be damaging.• The use of a common assay permitted an evaluation of the relative capacity of different antioxidants for the protection of hair cells.

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Trafficking of systemic fluorescent gentamicin into the cochlea and hair cells.

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