Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo

Abstract

Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy.In bullfrogs, GTTR uptake was prominent in mature hair cells, but not in immature hair cells. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla. GTTR was associated with the hair bundle; and, in guinea pigs and mice, somatic GTTR fluorescence was initially diffuse before punctate (endosomal) fluorescence could be observed. A baso-apical gradient of intracellular GTTR uptake in guinea pig cochleae could only be detected at early time points (<3 h). In 21−28 day mice, cochlear GTTR uptake was greatly reduced compared to guinea pigs, 6-day-old mice, or mice treated with ethacrynic acid. In mice, GTTR was also rapidly taken up, and retained, in the kidney, dorsal root and trigeminal ganglia. In linguinal and vibrissal tissues rapid GTTR uptake cleared over a period of several days.The preferential uptake of GTTR by mature saccular, and proximal hair cells resembles the pattern of aminoglycoside-induced hair cell death in bullfrogs and chicks. Differences in the degree of GTTR uptake in hair cells of different species suggests variation in serum levels, clearance rates from serum, and/or the developmental and functional integrity of the blood-labyrinth barrier. GTTR uptake by hair cells in vivo suggests that GTTR has potential to elucidate aminoglycoside transport mechanisms into the inner ear, and as a bio-tracer for in vivo pharmacokinetic studies.

A systemic gentamicin pathway across the stria vascularis

“…1G). Puncta of autofluorescence at emission wavelengths similar to Texas Red was observed in the supporting cells surrounding the IHCs, as previously described (Dai et al, 2006), in both GTTR-and hTR-treated tissues (Fig. 1G,H,J,K).…”

“…Three hours after subcutaneous injection of GTTR, the distribution of GTTR fluorescence in cochlear hair cells, strial and kidney tissues were similar to those previously described (Dai et al, 2006). In the stria vascularis, GTTR fluorescence was most prominent in the tissues associated with the strial capillaries, with less intense, diffuse fluorescence in the tissues between the capillaries (Fig.…”

“…Co-administration of GTTR and ethacrynic acid (a loop diuretic that disrupts the strial BLB), results in enhanced uptake of GTTR by both strial tissues and hair cells (Dai et al, 2006), confirming previous reports that ethacrynic acid synergistically enhances aminoglycoside uptake and ototoxicity (Brummett, 1981;Hayashida et al, 1989;Mathog and Capps, 1977;Rybak, 1982;Tran Ba Huy et al, 1983a;Yamane et al, 1988). Thus, the strial BLB is an important barrier for preventing the entry of aminoglycosides into the cochlea and subsequent ototoxicity.…”

“…However, after maturation of the BLB, murine hair cells no longer take up AM1-43 (Meyers et al, 2003). In contrast, GTTR is taken up by hair cells in adult murine cochleae over time (Dai et al, 2006). Thus GTTR, unlike AM1-43, is able to cross the functionally mature BLB, like other aminoglycosides.…”

“…Neonatal murine cochlear hair cells rapidly take up systemically-administered aminoglycosides, including gentamicin-conjugated Texas Red (GTTR), and also AM1-43 (Bernard, 1981;Dai et al, 2006;Henry et al, 1981;Lenoir et al, 1983;Meyers et al, 2003;Pujol, 1986), prior to the development of a functional blood-labyrinth barrier (BLB) in the stria vascularis and endocochlear potential that occurs between post-natal days 12-15 (Ehret, 1976;Shnerson and Pujol, 1981). However, after maturation of the BLB, murine hair cells no longer take up AM1-43 (Meyers et al, 2003).…”