Research paperProphylactic and therapeutic functions of drug combinations against noise-induced hearing loss
Introduction
Noise-induced hearing loss (NIHL) is the single predominant health hazard posed by occupational and recreational settings (Bohnker et al., 2002, Henderson et al., 2003, Seixas et al., 2005). Although promising approaches have been identified for reducing NIHL mainly based on free radical pathways (Campbell et al., 2007, Kopke et al., 2005, Le Prell and Bao, 2012, Lynch and Kil, 2005), currently no effective pharmacologic agents are approved by FDA to diminish permanent threshold shifts (PTS). Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL.
One major mechanism underlying NIHL is mitochondrial free radical formation due to noise-induced intense metabolic activity in the cochlea (Darrat et al., 2007, Henderson et al., 2006, Le Prell et al., 2003). The involvement of this pathway in NIHL is strongly supported by three main lines of evidence: (1) Noise-induced increase of free radicals is observed in stria vascularis, outer hair cells (OHCs), supporting cells of the organ of Corti, and spiral ganglion (Ohinata et al., 2000, Ohlemiller, 2006, Yamane et al., 1995). This elevation may continue up to at least 14 days post-exposure (Yamashita et al., 2004); (2) Depletion of endogenous antioxidants such as superoxide dismutase and glutathione peroxidase results in increased susceptibility to NIHL (McFadden et al., 2001, Ohlemiller et al., 1999, Ohlemiller et al., 2000a, Ohlemiller et al., 2000b); (3) Enhancement of antioxidants attenuates NIHL (Lynch et al., 2004, McFadden et al., 2005, Ohinata et al., 2003). Thus, it is not surprising that attempts to prevent NIHL with antioxidants have become the focus of much research (Bielefeld et al., 2007, Hight et al., 2003, McFadden et al., 2005, Seidman et al., 1993, Yamashita et al., 2005). Nevertheless, most interventions with single agents are only partially effective in preventing NIHL. A few studies have sought to intervene at multiple sites in free radical pathways, or in combination with other pathways. Synergic effects have been observed in some, but not all, studies (Le Prell et al., 2007, Yamasoba et al., 1999).
Two additional pathways that have emerged as major contributors to NIHL involve calcium and glucocorticoid (GC) signaling. Disturbance in calcium homeostasis has long been suspected to contribute to trauma-induced neuronal injury (Hansen et al., 2003, Nikonenko et al., 2005, Park et al., 2008, Werling et al., 2007). Calcium homeostasis in the cochlea is maintained in part by several types of calcium channels, which include voltage-gated calcium channels (VGCCs) (Adamson et al., 2002, Errington et al., 2005, Fuchs, 2002, Rodriguez-Contreras and Yamoah, 2001, Schnee and Ricci, 2003). VGCCs can be divided into two groups: high-voltage-activated (L-type) and low-voltage-activated calcium channels (T-type) (Lacinova et al., 2000, Perez-Reyes, 1998, Triggle, 2006, Yunker and McEnery, 2003). Blockers of L-type channels have been found to attenuate NIHL in some studies (Heinrich et al., 1997, Uemaetomari et al., 2009), but not others (Boettcher, 1996, Boettcher et al., 1998, Ison et al., 1997). We reported that NIHL can be reduced by the administration of anticonvulsant drugs blocking T-type calcium channels, applied either before or after noise exposure, and these channels are present in the organ of Corti and spiral ganglion neurons (SGNs) (Shen et al., 2007). Inhibition of T-type calcium channels also protects neurons after stroke (Nikonenko et al., 2005). Thus, it is possible that anticonvulsant drugs blocking T-type calcium channels can prevent injury-induced alterations of calcium homeostasis that contribute to NIHL.
Another major molecular mechanism in NIHL involves glucocorticoid signaling. Synthetic GCs are already used clinically to treat hearing loss in a variety of cochlear disorders such as autoimmune inner ear disease, tinnitus and Meniére's disease (Dodson et al., 2004, MacArthur et al., 2008, Trune and Canlon, 2012). Extensive evidence also suggests an important role of GC pathways in NIHL. First, glucocorticoid receptors (GRs) are present in the cells of the organ of Corti, spiral ligament, spiral limbus, and SGNs (Shimazaki et al., 2002, ten Cate et al., 1993, Zuo et al., 1995). Second, stressful ‘preconditioning’ events such as restraint, heat stress, and even low-level sound—all of which are likely to engage GC signaling—have been found to be protective against NIHL in animals (Paz et al., 2004, Wang and Liberman, 2002, Yoshida et al., 1999). Third, because the noise exposure itself is a stressful event, pretreatment with blockers of GC signaling accordingly makes animals more susceptible to NIHL (Tahera et al., 2006a). Fourth, synthetic GCs such as dexamethasone and methylprednisolone can protect against NIHL (Canlon et al., 2007, Sendowski et al., 2006, Tabuchi et al., 2006, Tahera et al., 2006b, Tahera et al., 2006c). Fifth, although GCs can bind to both GR and mineralocorticoid receptors, antagonists against mineralocorticoid receptors have no effect on NIHL (Tahera et al., 2006a). Finally, a series of studies have systematically revealed the role of GR signaling pathways in NIHL (Canlon et al., 2007, Lang et al., 2006, Peppi et al., 2011, Tahera et al., 2006b, Tahera et al., 2006c, Shen et al., 2011).
Interventions based on synthetic GC drugs or anticonvulsants blocking T-type calcium channels show limited success in preventing NIHL (Canlon et al., 2007, Shen et al., 2007). Since these agents are from completely different drug families, and likely act on different molecular pathways underlying NIHL, identifying optimal combinations of these that may act in a synergistic manner seemed to us a logical next step. Here we describe methods for identifying and quantifying synergistic drug interactions against NIHL in a mouse model.
Section snippets
Animals
All animal procedures were approved by the Animal Studies Committee at Washington University in St. Louis. The study included a total 297 C57BL/6J mice aged two months (150 males and 147 females), purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed three to five per cage in a noise-controlled environment on a 12 h light/dark cycle with light onset at 6:00 a.m. Except as noted (Fig. 5) all experimental groups contained equal numbers of male and female animals.
Drug application
Pre-exposure application of anticonvulsant drugs
Ethosuximide doses of 0, 60, 90, 130, 190, or 260 mg/kg were used to determine the ED50 of this drug in preventing NIHL. Fig. 1A shows ABR threshold shifts two weeks after noise exposure in animals given different doses of ethosuximide. Three-way ANOVA analysis indicated a significant main effect of drug concentration (F5 = 24.85, p < 2.0 × 10−16), but not gender (F1 = 0.21, p = 0.65). However, the ethosuximide dose–response pattern was complicated. At 60 mg/kg, ethosuximide significantly
Discussion
Building on previous studies, we tested a combination therapy against NIHL that included anticonvulsants ethosuximide and zonisamide, and synthetic GCs dexamethasone and methylprednisolone. ED50s for these drugs could be determined in most cases. A synergistic effect was observed for the combination of methylprednisolone and zonisamide. Three issues raised by this study are discussed below.
Acknowledgments
We thank Drs. Barbara Bohne and Colleen Garbe Le Prell for their thoughtful suggestions, and Drs. Yixin Chen and Yi Mao for their help of computational simulations. The project was supported by grants to J.B. from the National Institute of Health (DC010489 and DC011793), and the National Organization for Hearing Research Foundation.
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