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.

Protection against Noise-Induced Hearing Loss in Young CBA/J Mice by Low-Dose Kanamycin

“…Since OHC function and survival represent the most salient common target of both noise and KM, we suggest that preservation of OHC function and survival constitutes the most likely locus of KM-related protective mechanisms and that KM engages protective cascades within OHCs. Recent studies in young mice (Wu et al 2001;Wang and Steyger 2009) indicate that significant levels of KM would have been present in both cochlear fluids and hair cells at the time of our noise exposure. Aminoglycosides may enter hair cells from either endolymph or perilymph and may require 24 h to be cleared (Wang and Steyger 2009;Li and Steyger 2009).…”

“…Recent studies in young mice (Wu et al 2001;Wang and Steyger 2009) indicate that significant levels of KM would have been present in both cochlear fluids and hair cells at the time of our noise exposure. Aminoglycosides may enter hair cells from either endolymph or perilymph and may require 24 h to be cleared (Wang and Steyger 2009;Li and Steyger 2009). Thus, significant levels of KM were likely maintained and probably increased over the course of treatment.…”

“…The laboratory mouse is a well-established model for human auditory conditions, as it possesses similar cochlear anatomy and physiology and similar patterns of age-related, noise-induced, and ototoxicity-related hearing loss (Ohlemiller 2006;Wang and Steyger 2009;Wu et al 2001;Henry and McGinn 1992). Among inbred strains, CBA/J mice are frequently used as a "good hearing" strain that is resistant to ageassociated pathology.…”

Transient ischemia/hypoxia enhances gentamicin ototoxicity via caspase-dependent cell death pathway

“…26,27 In brief, 2.2 ml of GM sulfate (Sigma, St Louis, MO, USA; 50 mg/ml in 100 mM K 2 CO 3 at pH 9.0) and 0.6 ml of succinimidyl esters of TR (Molecular Probes; 2 mg/ml in dimethyl formamide) were mixed and agitated at 4 1C overnight to produce a GM-Texas Red conjugate (GMTR). A high molar ratio (B150:1) of GM to TR was used to prevent overlabeling GM molecule with more than one TR molecule and ensure the polycationic nature of conjugated GMTR.…”

“…A high molar ratio (B150:1) of GM to TR was used to prevent overlabeling GM molecule with more than one TR molecule and ensure the polycationic nature of conjugated GMTR. 27 The left ear of the animal underwent transient cochlear ischemia of 30 min as described above and the right ear served as control. Immediately after the surgery, GM/GMTR (125 or 250 mg/kg) was injected subcutaneously.…”