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.

Introduction

Aminoglycoside antibiotics have long been known to have ototoxic effects. These antibiotics, including gentamicin, tobramycin, kanamycin, streptomycin, amikacin, and neomycin, are associated with hearing loss and vestibular dysfunction due to hair cell loss. Some of the aminoglycoside antibiotics, such as gentamicin, have a more vestibulotoxic profile, while others, such as kanamycin, have a more cochleotoxic profile. For those aminoglycosides that are cochleotoxic, their effects target the high frequency portion of the cochlea, and in mammals, they affect outer hair cells while sparing inner hair cells. In current practice, gentamicin remains the most commonly prescribed intravenous aminoglycoside antibiotic and is sometimes associated with vestibular impairment. Hair cell loss after aminoglycoside ototoxicity is permanent, and our interventions for vestibular and hearing dysfunction after aminoglycoside ototoxicity are limited. Despite the various studies that have examined the protective effects of antioxidant therapies and other strategies against aminoglycoside ototoxicity, none of these proposed agents is an accepted therapy for hearing loss prevention (Bock et al., 1983, Campbell et al., 2007, Jiang et al., 2005, Kawamoto et al., 2004, Rybak and Whitworth, 2005).

Aminoglycoside antibiotics continue to be popular in their use as an ototoxic agent in animal research to study the process of hair cell survival, death and regeneration. Many prior studies have demonstrated the predictable effects of aminoglycoside antibiotics administered both in vivo and in vitro with experimental animals (Gratacap et al., 1985, Richardson and Russell, 1991, Stone et al., 1996, Weisleder and Rubel, 1993). Currently, aminoglycosides are used in research to eradicate hair cells in studies of hair cell regeneration and animal models of cochlear implantation (Ryugo et al., 2010, Shepherd et al., 1994, Warchol, 2010). In many of these studies, the goal of aminoglycoside administration is to eradicate hair cells and subsequently test an intervention targeting the remaining supporting cells or spiral ganglion cells. However, when aminoglycoside antibiotics are administered in vivo, complete loss of hair cells is not achieved, and low frequency hearing remains. In the case of mice, aminoglycosides are particularly ineffective in eliminating hair cells (Wu et al., 2001). As our research community expands the use of mice in studies of deafness, combination drug therapy to achieve hair cell loss has become routine. In recent work, loop diuretics have been used to augment the ototoxic effect of aminoglycoside antibiotics and eliminate hair cells in mice as well as in other mammals (Agterberg et al., 2008, Oesterle et al., 2008, Taylor et al., 2008, Xu et al., 1993).

Morphologic changes in the mouse cochlea that occur after the combined use of these two ototoxins have not been formally studied. Here, we compare the effect of aminoglycoside alone with the effect of aminoglycoside in combination with furosemide to 1. Characterize the difference in residual hearing function between these two regimens 2. Characterize the difference in hair cell susceptibility and other sensory and non-sensory cell susceptibility and 3. Determine whether there is an inflammatory response in the cochlea after cell damage from either aminoglycoside or aminoglycoside + loop diuretic.