Comparative analysis of combination kanamycin-furosemide versus kanamycin alone in the mouse cochlea

Abstract

Combinations of aminoglycosides and loop diuretics have been known to have a synergistic effect in ototoxic injury. Because murine hair cells are relatively resistant to ototoxicity compared to those of other mammals, investigators have turned to combination therapies to create ototoxic lesions in the mouse inner ear. In this paper, we perform a systematic comparison of hearing thresholds, hair cell damage and monocyte migration into the mouse cochlea after kanamycin versus combined kanamycin/furosemide and explore the pathophysiology of enhanced hair cell loss in aminoglycoside ototoxicity in the presence of loop diuretic. Combined kanamycin-furosemide resulted in elevation of threshold not only in the high frequencies, but across all frequencies with more extensive loss of outer hair cells when compared to kanamycin alone. The stria vascularis was severely atrophied and stellate cells in the spiral limbus were missing in kanamycin-furosemide exposed mice while these changes were not observed in mice receiving kanamycin alone. Monocytes and macrophages were recruited in large numbers to the spiral ligament and spiral ganglion in these mice. Combination therapy resulted in a greater number of macrophages in total, and many more macrophages were present further apically when compared to mice given kanamycin alone. Combined kanamycin-furosemide provides an effective method of addressing the relative resistance to ototoxicity that is observed in most mouse strains. As the mouse becomes increasingly more common in studies of hearing loss, and combination therapies gain popularity, recognition of the overall effects of combined aminoglycoside-loop diuretic therapy will be critical to interpretation of the interventions that follow.

Fig. 1

ABR threshold after saline, kanamycin, and kanamycin-furosemide injection. ABR thresholds were elevated at 16 kHz and higher after kanamycin, while thresholds were elevated at all tested frequencies after kanamycin-furosemide injections. Figure icons represent means and error bars represent standard errors of the mean (SEM). Asterisks demonstrate thresholds at which the difference between kanamycin and kanamycin-furosemide were statistically significantly different as evaluated by one-way ANOVA test with p < 0.05.

Fig. 2

Cytocochleograms after saline, kanamycin, and kanamycin-furosemide injection. A loss of outer hair cells was seen in the extreme base in saline-injected control animals. This degree of high frequency hair cell loss has been reported and is expected in the C57Bl6 mouse strain. Mild to moderate inner hair cell (IHC) damage was seen in high frequencies after kanamycin and kanamycin-furosemide injection. Moderate to severe outer hair cell (OHC) damage was seen in 14 kHz or higher frequency after kanamycin injection, whereas almost all OHC disappeared after kanamycin-furosemide injection. Mild spiral ligament damage was seen in the control mice, and there was no difference among three groups. Three out of 11 mice exhibited spiral limbus damage after kanamycin-furosemide injection. Figure icons represent means, and error bars represent SEM. Asterisks demonstrate regions in which the difference between hair cell survival in kanamycin and kanamycin-furosemide treated groups were statistically significantly different as evaluated by one-way ANOVA with p < 0.05.

Fig. 3

Complete outer hair cell loss was seen after combination kanamycin/furosemide injection. A: After kanamycin alone, outer hair cells (arrowhead) were preserved in lower apical turn. B: After combined kanamycin and furosemide, outer hair cells (black arrowhead) degenerated, pillar cells (white arrowhead) appear somewhat collapsed, but inner hair cells (arrow) were preserved in lower apical turn. C: Normal structures of the organ of Corti (arrowhead) were replaced by a flat epithelium in the lower basal turn after kanamycin and furosemide injection. Scale bar = 50 μm in A applies to A–C.

Fig. 4

Degeneration of the spiral limbus was seen after kanamycin and furosemide injection. A: Saline injection. B: Kanamycin-furosemide injection. Stellate cells (fibrocytes) were damaged in the apical turn of the spiral limbus after kanamycin injection (B, arrowhead). Scale bar = 100 μm in A applies to A–B.

Fig. 5

Stria vascularis in the lower apical turn after kanamycin-furosemide injection. A: Saline injection. B: Kanamycin-furosemide injection. Stria vascularis was shrunken after kanamycin-furosemide injection. Scale bar = 50 μm in A applies to A–B.

Fig. 6

CD45 immunohistochemistry demonstrates migratory mononuclear phagocytes after saline (A), kanamycin (B and D), and kanamycin-furosemide (C and E). Figures A, B, and C show the basal turn; D and E demonstrate the apical turn. High magnification images are shown in A′, B′, C′, D′ and E′. A: After saline, there are few cochlear mononuclear phagocytes that are present in the spiral limbus (arrow in A), in the spiral ligament (see arrow in A′) and in the scala tympani (arrowhead in A′). B: After kanamycin, there are more CD45 positive cells in the spiral ligament, concentrated in the type IV fibrocyte region (arrow in B). C: CD45+ cells after kanamycin-furosemide were significantly increased when compared to kanamycin alone. There are notably more phagocytic cells present in the scala tympani (arrowheads in C). D: In the apical turn, there are few macrophages seen in the kanamycin alone treated ears (D and D′). E: After combination kanamycin-furosemide, there is an abundance of cochlear mononuclear phagocytes extending well into the apex as seen in E and E′. F: The number of CD45+ cells is quantified across the cochlear duct in mice exposed to saline, kanamycin and kanamycin-furosemide in F. (Scale bar in A also applies for figures B, C, D, and E. Scale bar in A′ applies for B′, C′, D′, and E′).