PMC

Gentamicin in the capillary lumen (1) is translocated into endothelial cells (E), passaged into intermediate cells via gap junctions (2), and then cleared from endothelial cells and/or intermediate cells into the intra-strial space against the intra-strial electrical gradient (3). (4) Gentamicin is then translocated across the basolateral membrane of marginal cells, and (5) cleared across the lumenal/apical membrane of marginal cell into endolymph.

Fluorescence intensity isopleths (contour lines) for MDCK cells exposed to either 5 and 0.5 μg/mL GTTR for 30 seconds at room temperature. Each isopleth indicates the pinhole size and laser power settings required to reach a mean pixel intensity. The lower isopleth reflects the pinhole setting (x-axis) and laser power (y-axis) required to give a mean pixel intensity of MDCK cells treated with 5 μg/mL. The upper isopleths reflects the confocal settings required MDCK cells treated with 0.5 μg/mL. Typical confocal settings for kidney or strial tissues are also plotted.

(A) Normalized data for gentamicin antagonism of GTTR fluorescence in vivo were fitted to the Gaddum equation () by a non-linear, least squares fit. The curvefit for marginal cells is displaced to the right of the curvefit for proximal tubules, corresponding to the higher concentrations of gentamicin required to reduce GTTR fluorescence. (B) Normalized data for kanamycin antagonism of GTTR fluorescence in vivo were fitted to the Gaddum equation by a non-linear, least squares fit. The curvefit for marginal cells is similar to proximal tubules. (C) Normalized data for aminoglycoside antagonism of GTTR fluorescence in MDCK cells in vitro were fitted to the Gaddum equation by a non-linear, least squares fit. The curvefits were responsive to increasing gentamicin, but not kanamycin, concentrations.

After a 30 second exposure to GTTR prior to fixation, GTTR fluorescence was diffusely distributed throughout the cytoplasm and associated with intra-nuclear structures (▶). (A-E) Increasing molar ratios of gentamicin:GTTR (>10:1) significantly decreased GTTR fluorescence (*** = p<0.005; error bars = standard error of the mean [s.e.m.]). (F-J) Increasing molar ratios of kanamycin:GTTR were less effective in reducing GTTR fluorescence in MDCK cells, with a statistical drop in GTTR fluorescence occurring only at the highest dose of kanamycin:GTTR (1000:1; * = p <0.05; error bars = s.e.m.). Images acquired and post-processed identically.

Animals treated with 2 mg/kg GTTR alone for 30 minutes displayed GTTR fluorescence most intensely in marginal cells (A), with significantly weaker fluorescence in the intra-strial tissues (E, I; *** = p<0.05; error bars = s.e.m.). Co-administration of GTTR with unconjugated gentamicin at 10:1 (B, F) and 100:1 (C, G) molar dilutions of gentamicin:GTTR did not significantly (p >0.25) affect the distribution or intensity of GTTR fluorescence within the marginal cells or intra-strial tissues. (D, H, I) 400:1 molar ratios of gentamicin:GTTR significantly decreased the intensity of GTTR fluorescence in marginal cells and intra-strial tissues (*** = p<0.005; error bars = s.e.m.). All tissues from basal coil of cochlea. Images acquired and post-processed identically.

(1) Aminoglycosides are known to enter hair cells via apical endocytosis or permeation of the mechanotransduction channels on the apical surface of hair cells, and presumably from endolymph in vivo. (2) Systemically-administered aminoglycosides could enter the positively charged endolymph (+80 mV) via a trans-strial trafficking route from strial capillaries to marginal cells, followed by clearance into endolymph; or (3) by traversing the blood-labyrinth barrier into perilymph and thence (4) into endolymph via transcytosis across the epithelial perilymph/endolymph barrier, or (5) trafficking of aminoglycosides from the perilymph domain via gap junctions in fibrocytes to the stria vascularis, and trans-strial trafficking to marginal cells and clearance into endolymph. Alternatively, (6) aminoglycosides in the perilymph of the scala tympani may enter hair cell directly across their basolateral membranes. Diagram is not to scale).

Animals treated with 2 mg/kg GTTR alone for 30 minutes displayed GTTR fluorescence in marginal cells (A), and in the intra-strial tissues (E), with the most intense fluorescence in endothelial cells lining the strial capillaries (E). Co-administration of GTTR with 100:1 molar dilutions of kanamycin:GTTR did not significantly affect the distribution or intensity of GTTR fluorescence within the marginal cells or intra-strial tissues (B, F, I; p >0.05; error bars = s.e.m.). Higher molar ratios of kanamycin:GTTR (400:1, C, G; and 500:1, D, D′, H, I) significantly decreased the intensity of GTTR fluorescence in marginal cells and intra-strial tissues (* = p<0.05; *** = p<0.005, error bars = s.e.m.). All tissues from basal coil of cochlea. Images acquired and post-processed identically.

(A, F) Intra-peritoneal injection of GTTR alone for 30 minutes resulted in cytoplasmic and intense punctate fluorescence in proximal tubule cells (p), with weaker diffuse cytoplasmic fluorescence in distal tubule cells (d). (B-E) Simultaneous injection of unconjugated gentamicin with GTTR significantly reduced cytoplasmic GTTR fluorescence in proximal (and distal tubule cells) proportional to the increased dose of gentamicin (*** = p<0.005; error bars = s.e.m.). GTTR fluorescence at the brush border (▶) of, and punctate GTTR fluorescence in, proximal tubule cells appeared diminished in intensity with increasing molar ratios of gentamicin:GTTR (>10:1). (G-J) Animals simultaneously dosed with kanamycin revealed only a small significant decrease in cytoplasmic (or punctate) GTTR fluorescence intensity in proximal tubule cells at very high kanamycin:GTTR molar ratios (>400:1; H, I, J; * = p<0.05; error bars = s.e.m.). Images acquired and post-processed identically.