Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10347
RESEARCH ARTICLE
Growth factors have a protective effect on neomycin-induced hair cell loss Xiangxin Lou*, Huihua Yuan, Jing Xie, Xianliu Wang, Liangliang Yang and Yanzhong Zhang Department of Bioengineering, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China
Abstract We have demonstrated that selected growth factors are involved in regulating survival and proliferation of progenitor cells derived from the neonatal rat organ of Corti (OC). The protective and regenerative effects of these defined growth factors on the injured organ of Corti were therefore investigated. The organ of Corti dissected from the Wistar rat pups (P3-P5) was split into apical, middle, and basal parts, explanted and cultured with or without neomycin and growth factors. Insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF) protected the inner hair cells (IHCs) and outer hair cells (OHCs) from neomycin ototoxicity. Using EGF, IGF-1, and FGF-2 alone induced no protective effect on the survival of auditory hair cells. Combining 2 growth factors (EGF þ IGF-1, EGF þ FGF-2, or IGF-1 þ FGF-2) gave statistically protective effects. Similarly, combining all three growth factors effectively protected auditory hair cells from the ototoxic insult. None of the growth factors induced regeneration of hair cells in the explants injured with neomycin. Thus various combinations of the three defined factors (IGF-1, FGF-2, and EGF) can protect the auditory hair cells from the neomycin-induced ototoxic damage, but no regeneration was seen. This offers a possible novel approach to the treatment of hearing loss. Keywords: growth factors; hair cells; neomycin; ototoxicity; protection
Introduction Auditory hair cells are essential for hearing function and their loss results in hearing disorders and deafness. Unlike the sensory epithelia in birds and other nonmammalian vertebrates, the organ of Corti does not spontaneously regenerate hair cells to replace those lost in mammalian, sensorineural hearing loss is often progressive and irreversible in mammalian (Chardin and Romand, 1995; Taylor and Forge, 2005; Rubel et al., 2013). The biochemical and molecular mechanism of aminoglycoside antibotic have been extensively studied, at least three different routes of cell death are currently recognized: necrosis, apoptosis, and autophagy (Debnath et al., 2005). Among them, apoptosis was a major pathway of hair cell death after aminoglycoside antibiotics treatment of the inner ear (Lang and Liu, 1997). On this basis, three major agents have been investigated to prevent hair cell loss: antioxidants, inhibitors of intracellular stress pathways, and neurotrans-
mission blockers. Intracellular stress pathways are activated in the organ of Corti after ototoxic drug exposure (Wu et al., 2002). Neurotrophic factors are also important in the development of auditory system (Pirvola and Ylikoski, 2003; Perde-Schrepler et al., 2012), but the specific factors that regulate the proliferation of cells in the sensory epithelia of the inner ear have not been conclusively identified (Di Domenico et al., 2011; Perde-Schrepler et al., 2012). Several growth factors (e.g., FGF-2, FGF-4, FGF-6, FGF-7, IGF-1, IGF-2, and EGF) can promote cell cycle and mitosis in developing inner ear, and have the potential to rescue hearing loss and facilitate hair cell regeneration in vivo and in vitro (Malgrange et al., 1999). It has been shown that both EGF and transforming growth factor-a (TGF-a) are mitosis-promoting factors for inner ear supporting cells, which contribute to hair cell proliferation and regeneration in avian utricles (Zine and de Ribaupierre, 1998; Oesterle et al., 2000). Regeneration of lost hair cells has also been found in rat utricular after treatment with FGF-2 and IGF-1
Corresponding author: e-mail: [email protected]
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(Zheng et al., 1997). Both EGF and TGF-a stimulated the hair cell replacement after aminoglycoside ototoxic trauma in rat cochlear organotypic culture (Zine and de Ribaupierre, 1998). Growth factors may be involved in protection and regeneration of the mammalian inner ear, such as FGF-2 alone (Zhai et al., 2002), combination retinoic acid (RA) and TGF-a (Malgrange et al., 1998), or combination RA and FGF-2 (Low et al., 1996). We previously isolated and cultured auditory stem/ progenitor cells obtained from the organ of Corti of young rats (Lou et al., 2007; 2013), and found defined growth factors (IGF-1, FGF-2, and EGF) promote their self-renewal and proliferation. These growth factors to some extent can prevent hair cells lost from damage and even induce hair cell regeneration after neomycin-induced ototoxic damage. We have now used cochlear organotypic culture to examine the protective and regenerative effects of IGF-1, FGF-2, EGF alone, or mixtures on auditory hair cells damaged by neomycin toxicity. Materials and methods
Preparation of organotypic cultures Organ of Corti epithelial sheets were dissected from postnatal day 3–5 (P3-P5) Wistar rats in buffered saline glucose (BSG) under sterile conditions, based on a previous method r (Mazurek et al., 2003; 2012). After removing the modiolus and stria vascularis, the specimens were divided into apical, middle, and basal parts. The explants were cultured in 4-well microtiter plates (500 mL/well) in Dulbecco’s modified Eagle’s medium and Ham’s F12 (1:1) (DMEM/F12; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 0.6% glucose, 2 mL/mL insulin-transferrin-Na-Selenit-Mix, and 100 U/mL penicillin (all from Invitrogen). Cultures were maintained at 37 C in 5% CO2 atmosphere. Culture medium was exchanged everyday during the culture period, and each organ was cultured individually. The Animal Research Committee of the Donghua University approved all experimental protocols, which were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
(Figure 1A). To examine the regeneration effects of the growth factors (Figure 1B), organ of Corti explants were treated with 0.1 mM neomycin 48 h after 24 h culture in normal medium and subsequently incubated for 4 and 7 days in normal medium supplemented with a combination of IGF-1 25 ng/mL (Malgrange et al., 2002), FGF-2 10 ng/mL (Zheng et al., 1997), and EGF 20 ng/mL (Romand and Chardin, 1999). Two groups were used as control: the first, white control group without neomycin treatment; the second was not given growth factors but treated with neomycin as before. Apical, middle, and basal parts of the explants were analyzed in this report.
Tissue processing Cultures were rinsed with phosphate buffered saline (PBS; Invitrogen) and fixed 30 min in 4% paraformaldehyde in 0.1 M PBS (pH 7.4, Sigma). After two washes with PBS, explants were permeabilized with 0.2% Triton X-100 in PBS for 30 min. For staining, explants were incubated in buffered solution containing TRITC (tetramethyl rhodamine isothiocyanate)-labeled phalloidin (Invitrogen) for 30 min at room temperature. After rinsing in PBS, fragments were incubated in anti-fading for cell count (Sigma) on microscope.
Hair cell counts and quantitation At least 6 cochleae were used for each test, from which several tens of fragments were analyzed. Hair cells were counted on a Leica DMIL fluorescence microscope (Leica
Growth factors Neonatal organ of Corti explants were maintained for 24 h in culture in normal medium followed by 48 h incubation with 0.1 mM neomycin. To test possible protective effects of growth factors: IGF-1 (Cat. No. 133-FB; Sigma, St. Louis, MO), EGF-2 (Cat. No. F5392; Sigma), and EGF (Cat. No. E4127; Sigma) alone or mixtures were added to the culture when neomycin was added, and incubated for 48 h 66
Figure 1 Schematic of experimental paradigms used in the study. A: EGF, IGF-1, and FGF-2 alone or mixtures were used to test the potential protective effects on auditory hair cells damaged by neomycin treatment. B: Combination of EGF, IGF-1, and FGF-2 were used to detect the regeneration effects of growth factors after neomycin-induced auditory hair loss. Ctr: Control; Nm: Neomycin.
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Microsystems, Wetzlar, Germany). Quantitative analysis of inner hair cells (IHCs) and outer hair cells (OHCs) was performed on the explants divided in segments of 100 mm. The number of hair cells was counted over a distance of three times in the one IHCs row and the three OHCs rows by microscopy. Three to four organ of Corti explants per experimental paradigm were studied in each individual experiment. Three separate repetitions of the experiment were conducted for those growth factors demonstrating a protective effect.
Statistical analysis Means standard errors of the means (SEM) were calculated for the numbers of hair cells counted in the three cochlear parts. The effect of growth factors on the neomycin-induced hair cell loss was tested by 2-way or oneway analysis of variance (ANOVA) followed by TukeyKramer tests. The codes of the grouping variable (controls, neomycin, neomycin þ GFs) were selected altogether and in pairs. P < 0.05 was considered statistically significant. All statistical test and graphics were made by the smart application of Origin Software 8.0. Results Epifluorescence micrographs show representative examples of OC explants treated with phalloidin-stained surface
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preparations of control organotypic cultures, which has good preservation of the organization and mosaic of both IHCs and OHCs up to 72 h after explantation (Figures 2A, 2B, and 2C). Since the principal target for antibiotic neomycin in the inner ear is auditory hair cells, after treatment with 0.1 mM neomycin for 48 h, selective loss of both IHC and OHC was observed (Figures 2D, 2E, and 2F). Hair cells in the apical turn of explants showed more resistance to neomycin ototoxicity (Figure 2D); however, the basal turn was more vulnerable to aminoglycoside and significant hair cells loss was examined in both IHC and OHC regions (Figure 2F). Missing hair cells in these explants appear as black holes in the phalloidin-TRITC stained reticular lamina of these fragments (Figure 2F). The effects of EGF, FGF-2, IGF-1 alone, and mixtures (EGF þ IGF-1, EGF þ FGF-2, IGF-1 þ FGF-2, or EGF þ IGF-1 þ FGF-2) were tested in the explants simultaneously co-treated with neomycin for 48 h. Pronounced protective effects on auditory hair cells was seen when EGF, FGF-2, and IGF-1 were co-treated with neomycin in culture, and significant decrease of hair cell loss was noted (Figures 2G, 2H, and 2I). The number of IHCs and OHCs counted in the three cochlear parts after 48 h treatment with neomycin or neomycin with one, two, or three GFs in comparison to the controls are shown in Figure 3. Neomycin (0.1 mM) severely damaged the IHCs and OHCs with the damage being most pronounced in the basal cochlear part. IGF-1 alone did not protect the hair cells from neomycin-induced hair cell loss.
Figure 2 Representative images from the apical, middle, and basal segments of the organ of Corti, showing the outline of hair cells stained with Phalloidin-TRITC. Ctr: Segments of the organ of Corti after 72 h of incubation in vitro demonstrate three orderly rows of outer hair cells and a single row of inner hair cells (A, B, and C); Nm-GFs: Explants treated with 0.1 mM neomycin for 48 h showed significant loss of hair cells (D, E, and F); Nm þ GFs: 0.1 mM neomycin plus 20 ng/mL EGF, 10 ng/mL FGF-2, and 25 ng/mL IGF-1 for 48 h resulted in significant decrease in hair cells loss compared with neomycin treatment only (G, H, and I). Scare bar ¼ 20 mm.
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Figure 3 Number of IHCs and OHCs measured in the apical, middle, and basal cochlear part cultured without neomycin, with neomycin or with neomycin and 1, 2, or 3 growth factors. Hair cells were counted under the epifluorescent microscope. Values are expressed as mean SEM.
EGF and FGF-2 given alone had a small but significant protective effect on the IHCs (Figure 3A) (ANOVA, P < 0.05 and 0.001 vs. controls) and OHCs (Figure 3B) (ANOVA, P < 0.01 and 0.00001). Post-hoc analysis (Tukey-Kramer tests) only showed a reduced OHC loss in the middle cochlear part treated with neomycin and EGF or FGF-2 (P < 0.05 and 0.001). After treatment with neomycin and FGF-2, the number of apical IHCs and OHCs was significantly higher than in the controls (P < 0.01 and 0.001). The combination of two and three GFs was more effective in preventing the hair cells from neomycin-induced damage than the addition of one GF. The IHC loss was reduced in the middle and basal part by 68
the combination of EGF þ IGF-1 as well as EGF þ FGF-2 (P < 0.001) (Figure 3C). Combination of EGF þ IGF-1 reduced the IHC loss in the middle part (P < 0.01) (Figure 3C). OHC loss was reduced by all three combinations of two GFs in the apical, middle, and basal cochlear part (EGF þ IGF-1, apical OHCs: P < 0.001; others: P < 0.00001) (Figure 3D). Combination of all three GFs reduced neomycininduced IHC (Figure 3E) loss in the apical (P < 0.001) and basal part (P < 0.00001), and the OHC (Figure 3F) loss in all cochlear parts (P < 0.00001). To find the most effective combination of the GFs because the neomycin-induced damage differed in the three series, Cell Biol Int 39 (2015) 65–73 © 2014 International Federation for Cell Biology
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the IHCs and OHCs numbers of the groups treated with neomycin and GFs were calculated as percent of the corresponding neomycin group without GFs. The results of the basal cochlear part, which was mostly affected by neomycin, are shown in Figure 4. IHCs with IGF-1, FGF-2, or EGF alone for 48 h showed survivals of 104 27, 148 26, and 132 37% compared to the neomycin treatment, the OHCs with IGF-1, EGF, or FGF-2 alone for 48 h showed survivals of 115 16, 89 11, and 93 13% (Figures 4A and 4B). EGF and FGF-2 had a potent effect on hair cell survival, but the potential could only be found on OHCs in the middle turn of explants. With four combinations, FGF-2 þ IGF-1, EGF þ IGF-1, EGF þ FGF-2, or IGF-1 þ EGF þ FGF-2, IHCs had increased survival rates
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of 118 23, 181 47, 177 34, and 189 17%, respectively; the survival rate of OHCs also showed significant enhancement, 181 24, 221 56, 202 24, and 180 14%, respectively (Figures 4A and 4B). EGF þ IGF-1 þ FGF-2 mixture had an additive effect on the survival of hair cells, while the mixture of FGF-2 þ IGF-1 had no significant effect on IHCs in basal turn of the explants (Figure 4A). Although it has been claimed that EGF, IGF-1, and FGF-2 induces regeneration of hair cells in vitro after neomycin treatment in rats, this was not confirmed in the present study. It is well established that EGF, IGF-1, and FGF-2 alone or mixture can induce cell proliferate and differentiate, but this is less obvious for hair cell; no regenerative effects of growth factors on auditory hair cell loss were observed in organ of Corti cultured 4 or 7 days after neomycin treatment (Figure 5). Treatment of the OC explants with the combination of three GFs for 4 and 7 days following the exposure to neomycin for 48 h did not induce an increase of the IHC or OHC number (Figure 6). In contrast, there was a very severe hair cell loss in all cochlear parts of the experimental groups (with the OHC loss even proceeding between day 7 and day 10 of culture). There was no obvious difference of number of OHCs and IHCs between EGF, IGF-1, FGF-2 combination added culture and basic culture without growth factors (Figure 6). However, we did observe that the selected growth factors provided a relative protection of hair cells after antibiotic treatment. This suggests that EGF, IGF-1, and FGF-2 might present a protective effect instead of proliferative effects on hair cells against neomycin ototoxicity. Discussion
Figure 4 IHCs and OHCs (% of neomycin without GFs) counted in the basal cochlear part of the groups treated with neomycin and 1, 2, or 3 GFs. */** P < 0.05/0.01 vs. FGF-2 þ IGF-1; o/oo/ooo P < 0.01/ 0.001/0.0001 vs. IGF-1; þ/þþ P < 0.05/0.0001 vs. EGF; # P < 0.0001 vs. FGF-2.
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Loss of hair cells from the organ of Corti leads to irreversible hearing impairment in mammals. The mechanism for hair cell death is not well understood, and currently there is no efficient therapy available to prevent hair cell loss and promote hair cell regeneration. Alternative potential strategies to reduce the incidence of acquired hearing loss are thus to protect hair cells from death or maintain the survival of inner ear cells damaged by ototoxic antibiotics. Some trophic factors can prevent apoptotic cell death and enhance the regenerative proliferation in vitro in damaged vestibular organs, whereas many other growth factors have no protective effect (Zheng et al., 1997; Kopke et al., 2001). Our previous study demonstrated the efficacy of growth factors to support the inner ear cells survival and proliferation in vitro (Lou et al., 2007). These results provide support for the validity of our culture system in which we tested the potential of EGF, IGF-1, and FGF-2 to promote auditory hair cells survival and regeneration in organ of Corti explants. The cultured inner ear epithelial sheets here contain only supporting cells and hair cells, along with the 69
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Figure 5 Photograph of phalloidin-labeled organs of Corti. No regenerative effects were found 4 and 7 days of incubation with or without three GFs (20 ng/mL EGF, 10 ng/mL FGF-2, and 25 ng/mL IGF-1) after neomycin-induced hair cells loss. Control incubations in the absence of neomycin maintained a normal appearance at 10 days (A, B, and C). 4 days incubated with (G, H, and I) or without three GFs (D, E, and F) after 0.1 mM neomycin treatment 48 h and 7 days incubated with (M, N, and O) or without three GFs (J, K, and L) after 0.1 mM neomycin treatment 48 h, respectively, no significant regenerative effects were found in all the segments. Scare bar ¼ 20 mm.
phalloidin-TRITC staining; this is a convenient method to evaluate possible effects of growth factors on protection and regeneration of hair cells. Several growth factors co-treatments with neomycin have significant effects on reduction of hair cell loss. Among
them, EGF is effective in supporting the survival of OHCs, in accordance with previously described EGF involvement in auditory hair cell repair in neonatal organ of Corti explants (Kopke et al., 2001). EGF receptor transcripts are upregulated in the postnatal 3 days rat cochlear epithelia treated
Figure 6 Number of IHCs and OHCs measured in the apical, middle and basal part of the organ of Corti explants, which had been treated with neomycin for 48 h and subsequent treatment with three GFs (IGF, EGF and FGF) for 4 (left) and 7 days (right), respectively.
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with neomycin, which enhances the processes of mammalian neonatal hair cell replacement after hair cell loss induced by neomycin toxicity (Zine et al., 2000). Since hair cells response to growth factor is regulated by the availability of their ligands and specific growth factor receptors on the cell membrane, the protective effects by EGF might due to the upregulation of EGF receptors on the surface of the hair cells. Several members of the FGF family and their corresponding FGF receptors are involved in the development and regeneration of hair cells. FGF-2 is one of the most well known mitogenic and protective substances working in the inner ear, which has been proposed to fulfill a variety of functions during vertebrate inner ear development and within the mature sensory organ (Corwin et al., 1996). FGF2 protected cochlear hair cells in organotypic cultures from ototoxic damage in vitro in rats, possibly acting on FGF receptor 3 which is expressed in hair cells (Low et al., 1996), and FGF-2 protects auditory hair cells from noise damage in guinea pigs in vivo (Zhai et al., 2002; Carnicero et al., 2004). Therefore, introduction of FGF-2 into the inner ear may be taken as a potential mechanism to protect hair cells. Consistent with previous research, the present studies suggest that FGF-2 plays a role in stimulating hair cell survival within the organ of Corti. However, it remains unclear how FGF-2 prevents damage to inner ear sensory epithelia in mammals. FGF-2 may fulfill a protective function through initiate repair mechanisms during hair cell loss, which may include the reinitiation of cellular transdifferentiation processes. With IGF-1 treatment alone, there were no significant differences between damaged and IGF-1 supplemented groups, demonstrating that exogenous IGF-1 may not be involved in the maintenance of the inner ear following damage. Although our results show that IGF-1 alone does not protect hair cells in mammals, sustained delivery of IGF-1 (1.0 mg/mL) into the cochlear fluid can increase survival of OHCs (Angunsri et al., 2011). The physiology significance of IGF-1 (100–500 ng/mL) mediated cell death protection has also been shown in auditory sensory cells in vitro culture (Oesterle et al., 1997; Malgrange et al., 2002). These experiments demonstrate that IGF-1 is a candidate trophic factor keeping hair cell survival at pharmacological concentration, and perhaps IGF-1 protects the inner ear from damage under certain conditions. Addition of IGF-1 to explants cultures of normal auditory epithelia from postnatal 3–5 days rats inner ear did not induce proliferation within the sensory epithelia (data not shown). Since the cultured inner ear epithelial sheets contain supporting cells and hair cells within the organ of Corti, the survival promoting effect of the growth factors can result either from a direct interaction of growth factor ligands with their corresponding receptors present on OHCs themselves, Cell Biol Int 39 (2015) 65–73 © 2014 International Federation for Cell Biology
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or might be mediated through an effect on non-sensory cells of the organ of Corti, which would result in a release of a secondary trophic factors. Since FGF-2 (Pirvola et al., 1995) and EGF (Zine et al., 2000) receptors have been localized not only onto the hair cells, but also in supporting cells, the survival promoting effects of FGF-2 and EGF are probably indirect and mediated by non-sensory cells. In these adult organ of Corti explants, IHCs have a level of independence on exogenous trophic support under these in vitro conditions. IHCs either may not require trophic support or alternatively the needed growth factors are produced within the explants by its corresponding supporting cells. Several growth factors are associated with the regeneration process: insulin, IGF-1, and fibroblast growth factors are important for avian inner ear regeneration (Oesterle et al., 1997), whereas EGF, transforming growth factor a (TGF-a), insulin, FGF-2 are important for regeneration in the mammalian labyrinth (Zheng et al., 1997; Staecker and Van de Water, 1998; Staecker et al., 2001). Regeneration of mammalian auditory hair cells is possible during the early neonatal period and may exist to a very limited degree at later time (Zine et al., 2000); however, our findings are in contrast to these studies. Exogenously added combinations of EGF, IGF1, and FGF-2 4 and 7 days after neomycin treatment did not result in hair cell regeneration in organtypic culture. The interpretation of the discrepancy between these results is that our cultured tissues were treated with a low concentration of aminoglycoside (0.1 mM) compared to the (Zine et al., 2000). With intensive damage, the immature explants might be triggered to return to an earlier development stage and induce regeneration by new cells proliferate or other supporting cells transdifferentiate. Our findings are harmonious with other, which reported that the addition of FGF-1 to dissociated explants of isolated rat utricular (Zheng et al., 1997), and the avian inner ear sensory epithelia (Oesterle et al., 2000) elicited no detectable effects on cell proliferation. Oesterle et al. also showed that FGF-2 may be involved in inhibiting cell proliferation or stimulating cell differentiation in avian inner ear sensory epithelia. It remains unclear how and to what extent growth factors inducing proliferation and regeneration to damaged inner ear epithelia in mammals. There are no conclusive evidence that supports the participation of regenerative cell proliferation as a major participant in the replacement of lost hair cells from either the auditory or vestibular receptors of mammals. Although our observations of the protective effects of EGF, IGF-1, and FGF-2 are consistent with previous reports (Zheng et al., 1997; Zine and de Ribaupierre, 1998), the results of combination of two or three members of EGF, IGF-1, and FGF-2 are novel. Among the growth factors, combination FGF and IGF-1, combination EGF, IGF-1, and FGF-2 two groups have noticeable effects on reducing hair cells loss from neomycin ototoxicity. Several growth factors 71
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have additive protective effective on auditory hair cells after aminoglycoside antibiotics treatment. One of the possible explanations of the protective effect of selected growth factors is that several growth factor work together to activate more than one signal to induce protection, and individual signaling pathways interact with each other, forming complex signaling networks that respond to stimulate the survival of hair cells from neomycin ototoxicity. Mixtures of several growth factors might to some extent promote cooperation between growth factors and their receptors. It is possible that multiple growth factors may contribute together to protection of hair cells in a sequencing manner or at multiple steps. Acknowledgement and funding The work was supported by the Fundamental Research Funds for the Central Universities (Grant No. 2232013D313) and Donghua University Scientific Research Foundation for Youths (Grant No.13D210502). Authors’ contributions XXL, JX, XLW, and LLY carried out the organ of Corti dissection, culture, and data analysis. JX, XLW, and LLY carried out the immunoassay. ZZY were involved in the design and coordination of this project as well as manuscript writing. All authors reviewed and approved the final manuscript. Conflict of interests The authors declare that they have no competing interests. References Angunsri N, Taura A, Nakagawa T, Hayashi Y, Kitajiri S-i, Omi E, Ishikawa K, Ito J (2011) Insulin-like growth factor 1 protects vestibular hair cells from aminoglycosides. Neuroreport 22: 38–43. Carnicero E, Zelarayan LC, Ruttiger L, Knipper M, Alvarez Y, Alonso MT, Schimmang T (2004) Differential roles of fibroblast growth factor-2 during development and maintenace of auditory sensory epithelia. J Neurosci Res 77: 787–97. Chardin S, Romand R (1995) Regeneration and mammalian auditory hair-cells. Science 267: 707–9. Corwin JT, Warchol ME, Saffer LD, Finley JE, Gu RD, Lambert PR (1996) Growth factors as potential drugs for the sensory epithelia of the ear. Ciba Found Symp 196: 167–82. Debnath J, Baehrecke EH, Kroemer G (2005) Does autophagy contribute to cell death? Autophagy 1: 66–74. Di Domenico M, Ricciardi C, Martone T, Mazzarella N, Cassandro C, Chiarella G, D’Angelo L, Cassandro E (2011) Towards gene therapy for deafness. J Cell Physiol 226: 2494–9.
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RESEARCH ARTICLE
Growth factors have a protective effect on neomycin-induced hair cell loss Xiangxin Lou*, Huihua Yuan, Jing Xie, Xianliu Wang, Liangliang Yang and Yanzhong Zhang Department of Bioengineering, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China
Abstract We have demonstrated that selected growth factors are involved in regulating survival and proliferation of progenitor cells derived from the neonatal rat organ of Corti (OC). The protective and regenerative effects of these defined growth factors on the injured organ of Corti were therefore investigated. The organ of Corti dissected from the Wistar rat pups (P3-P5) was split into apical, middle, and basal parts, explanted and cultured with or without neomycin and growth factors. Insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF) protected the inner hair cells (IHCs) and outer hair cells (OHCs) from neomycin ototoxicity. Using EGF, IGF-1, and FGF-2 alone induced no protective effect on the survival of auditory hair cells. Combining 2 growth factors (EGF þ IGF-1, EGF þ FGF-2, or IGF-1 þ FGF-2) gave statistically protective effects. Similarly, combining all three growth factors effectively protected auditory hair cells from the ototoxic insult. None of the growth factors induced regeneration of hair cells in the explants injured with neomycin. Thus various combinations of the three defined factors (IGF-1, FGF-2, and EGF) can protect the auditory hair cells from the neomycin-induced ototoxic damage, but no regeneration was seen. This offers a possible novel approach to the treatment of hearing loss. Keywords: growth factors; hair cells; neomycin; ototoxicity; protection
Introduction Auditory hair cells are essential for hearing function and their loss results in hearing disorders and deafness. Unlike the sensory epithelia in birds and other nonmammalian vertebrates, the organ of Corti does not spontaneously regenerate hair cells to replace those lost in mammalian, sensorineural hearing loss is often progressive and irreversible in mammalian (Chardin and Romand, 1995; Taylor and Forge, 2005; Rubel et al., 2013). The biochemical and molecular mechanism of aminoglycoside antibotic have been extensively studied, at least three different routes of cell death are currently recognized: necrosis, apoptosis, and autophagy (Debnath et al., 2005). Among them, apoptosis was a major pathway of hair cell death after aminoglycoside antibiotics treatment of the inner ear (Lang and Liu, 1997). On this basis, three major agents have been investigated to prevent hair cell loss: antioxidants, inhibitors of intracellular stress pathways, and neurotrans-
mission blockers. Intracellular stress pathways are activated in the organ of Corti after ototoxic drug exposure (Wu et al., 2002). Neurotrophic factors are also important in the development of auditory system (Pirvola and Ylikoski, 2003; Perde-Schrepler et al., 2012), but the specific factors that regulate the proliferation of cells in the sensory epithelia of the inner ear have not been conclusively identified (Di Domenico et al., 2011; Perde-Schrepler et al., 2012). Several growth factors (e.g., FGF-2, FGF-4, FGF-6, FGF-7, IGF-1, IGF-2, and EGF) can promote cell cycle and mitosis in developing inner ear, and have the potential to rescue hearing loss and facilitate hair cell regeneration in vivo and in vitro (Malgrange et al., 1999). It has been shown that both EGF and transforming growth factor-a (TGF-a) are mitosis-promoting factors for inner ear supporting cells, which contribute to hair cell proliferation and regeneration in avian utricles (Zine and de Ribaupierre, 1998; Oesterle et al., 2000). Regeneration of lost hair cells has also been found in rat utricular after treatment with FGF-2 and IGF-1
Corresponding author: e-mail: [email protected]
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(Zheng et al., 1997). Both EGF and TGF-a stimulated the hair cell replacement after aminoglycoside ototoxic trauma in rat cochlear organotypic culture (Zine and de Ribaupierre, 1998). Growth factors may be involved in protection and regeneration of the mammalian inner ear, such as FGF-2 alone (Zhai et al., 2002), combination retinoic acid (RA) and TGF-a (Malgrange et al., 1998), or combination RA and FGF-2 (Low et al., 1996). We previously isolated and cultured auditory stem/ progenitor cells obtained from the organ of Corti of young rats (Lou et al., 2007; 2013), and found defined growth factors (IGF-1, FGF-2, and EGF) promote their self-renewal and proliferation. These growth factors to some extent can prevent hair cells lost from damage and even induce hair cell regeneration after neomycin-induced ototoxic damage. We have now used cochlear organotypic culture to examine the protective and regenerative effects of IGF-1, FGF-2, EGF alone, or mixtures on auditory hair cells damaged by neomycin toxicity. Materials and methods
Preparation of organotypic cultures Organ of Corti epithelial sheets were dissected from postnatal day 3–5 (P3-P5) Wistar rats in buffered saline glucose (BSG) under sterile conditions, based on a previous method r (Mazurek et al., 2003; 2012). After removing the modiolus and stria vascularis, the specimens were divided into apical, middle, and basal parts. The explants were cultured in 4-well microtiter plates (500 mL/well) in Dulbecco’s modified Eagle’s medium and Ham’s F12 (1:1) (DMEM/F12; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 0.6% glucose, 2 mL/mL insulin-transferrin-Na-Selenit-Mix, and 100 U/mL penicillin (all from Invitrogen). Cultures were maintained at 37 C in 5% CO2 atmosphere. Culture medium was exchanged everyday during the culture period, and each organ was cultured individually. The Animal Research Committee of the Donghua University approved all experimental protocols, which were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
(Figure 1A). To examine the regeneration effects of the growth factors (Figure 1B), organ of Corti explants were treated with 0.1 mM neomycin 48 h after 24 h culture in normal medium and subsequently incubated for 4 and 7 days in normal medium supplemented with a combination of IGF-1 25 ng/mL (Malgrange et al., 2002), FGF-2 10 ng/mL (Zheng et al., 1997), and EGF 20 ng/mL (Romand and Chardin, 1999). Two groups were used as control: the first, white control group without neomycin treatment; the second was not given growth factors but treated with neomycin as before. Apical, middle, and basal parts of the explants were analyzed in this report.
Tissue processing Cultures were rinsed with phosphate buffered saline (PBS; Invitrogen) and fixed 30 min in 4% paraformaldehyde in 0.1 M PBS (pH 7.4, Sigma). After two washes with PBS, explants were permeabilized with 0.2% Triton X-100 in PBS for 30 min. For staining, explants were incubated in buffered solution containing TRITC (tetramethyl rhodamine isothiocyanate)-labeled phalloidin (Invitrogen) for 30 min at room temperature. After rinsing in PBS, fragments were incubated in anti-fading for cell count (Sigma) on microscope.
Hair cell counts and quantitation At least 6 cochleae were used for each test, from which several tens of fragments were analyzed. Hair cells were counted on a Leica DMIL fluorescence microscope (Leica
Growth factors Neonatal organ of Corti explants were maintained for 24 h in culture in normal medium followed by 48 h incubation with 0.1 mM neomycin. To test possible protective effects of growth factors: IGF-1 (Cat. No. 133-FB; Sigma, St. Louis, MO), EGF-2 (Cat. No. F5392; Sigma), and EGF (Cat. No. E4127; Sigma) alone or mixtures were added to the culture when neomycin was added, and incubated for 48 h 66
Figure 1 Schematic of experimental paradigms used in the study. A: EGF, IGF-1, and FGF-2 alone or mixtures were used to test the potential protective effects on auditory hair cells damaged by neomycin treatment. B: Combination of EGF, IGF-1, and FGF-2 were used to detect the regeneration effects of growth factors after neomycin-induced auditory hair loss. Ctr: Control; Nm: Neomycin.
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Microsystems, Wetzlar, Germany). Quantitative analysis of inner hair cells (IHCs) and outer hair cells (OHCs) was performed on the explants divided in segments of 100 mm. The number of hair cells was counted over a distance of three times in the one IHCs row and the three OHCs rows by microscopy. Three to four organ of Corti explants per experimental paradigm were studied in each individual experiment. Three separate repetitions of the experiment were conducted for those growth factors demonstrating a protective effect.
Statistical analysis Means standard errors of the means (SEM) were calculated for the numbers of hair cells counted in the three cochlear parts. The effect of growth factors on the neomycin-induced hair cell loss was tested by 2-way or oneway analysis of variance (ANOVA) followed by TukeyKramer tests. The codes of the grouping variable (controls, neomycin, neomycin þ GFs) were selected altogether and in pairs. P < 0.05 was considered statistically significant. All statistical test and graphics were made by the smart application of Origin Software 8.0. Results Epifluorescence micrographs show representative examples of OC explants treated with phalloidin-stained surface
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preparations of control organotypic cultures, which has good preservation of the organization and mosaic of both IHCs and OHCs up to 72 h after explantation (Figures 2A, 2B, and 2C). Since the principal target for antibiotic neomycin in the inner ear is auditory hair cells, after treatment with 0.1 mM neomycin for 48 h, selective loss of both IHC and OHC was observed (Figures 2D, 2E, and 2F). Hair cells in the apical turn of explants showed more resistance to neomycin ototoxicity (Figure 2D); however, the basal turn was more vulnerable to aminoglycoside and significant hair cells loss was examined in both IHC and OHC regions (Figure 2F). Missing hair cells in these explants appear as black holes in the phalloidin-TRITC stained reticular lamina of these fragments (Figure 2F). The effects of EGF, FGF-2, IGF-1 alone, and mixtures (EGF þ IGF-1, EGF þ FGF-2, IGF-1 þ FGF-2, or EGF þ IGF-1 þ FGF-2) were tested in the explants simultaneously co-treated with neomycin for 48 h. Pronounced protective effects on auditory hair cells was seen when EGF, FGF-2, and IGF-1 were co-treated with neomycin in culture, and significant decrease of hair cell loss was noted (Figures 2G, 2H, and 2I). The number of IHCs and OHCs counted in the three cochlear parts after 48 h treatment with neomycin or neomycin with one, two, or three GFs in comparison to the controls are shown in Figure 3. Neomycin (0.1 mM) severely damaged the IHCs and OHCs with the damage being most pronounced in the basal cochlear part. IGF-1 alone did not protect the hair cells from neomycin-induced hair cell loss.
Figure 2 Representative images from the apical, middle, and basal segments of the organ of Corti, showing the outline of hair cells stained with Phalloidin-TRITC. Ctr: Segments of the organ of Corti after 72 h of incubation in vitro demonstrate three orderly rows of outer hair cells and a single row of inner hair cells (A, B, and C); Nm-GFs: Explants treated with 0.1 mM neomycin for 48 h showed significant loss of hair cells (D, E, and F); Nm þ GFs: 0.1 mM neomycin plus 20 ng/mL EGF, 10 ng/mL FGF-2, and 25 ng/mL IGF-1 for 48 h resulted in significant decrease in hair cells loss compared with neomycin treatment only (G, H, and I). Scare bar ¼ 20 mm.
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Figure 3 Number of IHCs and OHCs measured in the apical, middle, and basal cochlear part cultured without neomycin, with neomycin or with neomycin and 1, 2, or 3 growth factors. Hair cells were counted under the epifluorescent microscope. Values are expressed as mean SEM.
EGF and FGF-2 given alone had a small but significant protective effect on the IHCs (Figure 3A) (ANOVA, P < 0.05 and 0.001 vs. controls) and OHCs (Figure 3B) (ANOVA, P < 0.01 and 0.00001). Post-hoc analysis (Tukey-Kramer tests) only showed a reduced OHC loss in the middle cochlear part treated with neomycin and EGF or FGF-2 (P < 0.05 and 0.001). After treatment with neomycin and FGF-2, the number of apical IHCs and OHCs was significantly higher than in the controls (P < 0.01 and 0.001). The combination of two and three GFs was more effective in preventing the hair cells from neomycin-induced damage than the addition of one GF. The IHC loss was reduced in the middle and basal part by 68
the combination of EGF þ IGF-1 as well as EGF þ FGF-2 (P < 0.001) (Figure 3C). Combination of EGF þ IGF-1 reduced the IHC loss in the middle part (P < 0.01) (Figure 3C). OHC loss was reduced by all three combinations of two GFs in the apical, middle, and basal cochlear part (EGF þ IGF-1, apical OHCs: P < 0.001; others: P < 0.00001) (Figure 3D). Combination of all three GFs reduced neomycininduced IHC (Figure 3E) loss in the apical (P < 0.001) and basal part (P < 0.00001), and the OHC (Figure 3F) loss in all cochlear parts (P < 0.00001). To find the most effective combination of the GFs because the neomycin-induced damage differed in the three series, Cell Biol Int 39 (2015) 65–73 © 2014 International Federation for Cell Biology
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the IHCs and OHCs numbers of the groups treated with neomycin and GFs were calculated as percent of the corresponding neomycin group without GFs. The results of the basal cochlear part, which was mostly affected by neomycin, are shown in Figure 4. IHCs with IGF-1, FGF-2, or EGF alone for 48 h showed survivals of 104 27, 148 26, and 132 37% compared to the neomycin treatment, the OHCs with IGF-1, EGF, or FGF-2 alone for 48 h showed survivals of 115 16, 89 11, and 93 13% (Figures 4A and 4B). EGF and FGF-2 had a potent effect on hair cell survival, but the potential could only be found on OHCs in the middle turn of explants. With four combinations, FGF-2 þ IGF-1, EGF þ IGF-1, EGF þ FGF-2, or IGF-1 þ EGF þ FGF-2, IHCs had increased survival rates
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of 118 23, 181 47, 177 34, and 189 17%, respectively; the survival rate of OHCs also showed significant enhancement, 181 24, 221 56, 202 24, and 180 14%, respectively (Figures 4A and 4B). EGF þ IGF-1 þ FGF-2 mixture had an additive effect on the survival of hair cells, while the mixture of FGF-2 þ IGF-1 had no significant effect on IHCs in basal turn of the explants (Figure 4A). Although it has been claimed that EGF, IGF-1, and FGF-2 induces regeneration of hair cells in vitro after neomycin treatment in rats, this was not confirmed in the present study. It is well established that EGF, IGF-1, and FGF-2 alone or mixture can induce cell proliferate and differentiate, but this is less obvious for hair cell; no regenerative effects of growth factors on auditory hair cell loss were observed in organ of Corti cultured 4 or 7 days after neomycin treatment (Figure 5). Treatment of the OC explants with the combination of three GFs for 4 and 7 days following the exposure to neomycin for 48 h did not induce an increase of the IHC or OHC number (Figure 6). In contrast, there was a very severe hair cell loss in all cochlear parts of the experimental groups (with the OHC loss even proceeding between day 7 and day 10 of culture). There was no obvious difference of number of OHCs and IHCs between EGF, IGF-1, FGF-2 combination added culture and basic culture without growth factors (Figure 6). However, we did observe that the selected growth factors provided a relative protection of hair cells after antibiotic treatment. This suggests that EGF, IGF-1, and FGF-2 might present a protective effect instead of proliferative effects on hair cells against neomycin ototoxicity. Discussion
Figure 4 IHCs and OHCs (% of neomycin without GFs) counted in the basal cochlear part of the groups treated with neomycin and 1, 2, or 3 GFs. */** P < 0.05/0.01 vs. FGF-2 þ IGF-1; o/oo/ooo P < 0.01/ 0.001/0.0001 vs. IGF-1; þ/þþ P < 0.05/0.0001 vs. EGF; # P < 0.0001 vs. FGF-2.
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Loss of hair cells from the organ of Corti leads to irreversible hearing impairment in mammals. The mechanism for hair cell death is not well understood, and currently there is no efficient therapy available to prevent hair cell loss and promote hair cell regeneration. Alternative potential strategies to reduce the incidence of acquired hearing loss are thus to protect hair cells from death or maintain the survival of inner ear cells damaged by ototoxic antibiotics. Some trophic factors can prevent apoptotic cell death and enhance the regenerative proliferation in vitro in damaged vestibular organs, whereas many other growth factors have no protective effect (Zheng et al., 1997; Kopke et al., 2001). Our previous study demonstrated the efficacy of growth factors to support the inner ear cells survival and proliferation in vitro (Lou et al., 2007). These results provide support for the validity of our culture system in which we tested the potential of EGF, IGF-1, and FGF-2 to promote auditory hair cells survival and regeneration in organ of Corti explants. The cultured inner ear epithelial sheets here contain only supporting cells and hair cells, along with the 69
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Figure 5 Photograph of phalloidin-labeled organs of Corti. No regenerative effects were found 4 and 7 days of incubation with or without three GFs (20 ng/mL EGF, 10 ng/mL FGF-2, and 25 ng/mL IGF-1) after neomycin-induced hair cells loss. Control incubations in the absence of neomycin maintained a normal appearance at 10 days (A, B, and C). 4 days incubated with (G, H, and I) or without three GFs (D, E, and F) after 0.1 mM neomycin treatment 48 h and 7 days incubated with (M, N, and O) or without three GFs (J, K, and L) after 0.1 mM neomycin treatment 48 h, respectively, no significant regenerative effects were found in all the segments. Scare bar ¼ 20 mm.
phalloidin-TRITC staining; this is a convenient method to evaluate possible effects of growth factors on protection and regeneration of hair cells. Several growth factors co-treatments with neomycin have significant effects on reduction of hair cell loss. Among
them, EGF is effective in supporting the survival of OHCs, in accordance with previously described EGF involvement in auditory hair cell repair in neonatal organ of Corti explants (Kopke et al., 2001). EGF receptor transcripts are upregulated in the postnatal 3 days rat cochlear epithelia treated
Figure 6 Number of IHCs and OHCs measured in the apical, middle and basal part of the organ of Corti explants, which had been treated with neomycin for 48 h and subsequent treatment with three GFs (IGF, EGF and FGF) for 4 (left) and 7 days (right), respectively.
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with neomycin, which enhances the processes of mammalian neonatal hair cell replacement after hair cell loss induced by neomycin toxicity (Zine et al., 2000). Since hair cells response to growth factor is regulated by the availability of their ligands and specific growth factor receptors on the cell membrane, the protective effects by EGF might due to the upregulation of EGF receptors on the surface of the hair cells. Several members of the FGF family and their corresponding FGF receptors are involved in the development and regeneration of hair cells. FGF-2 is one of the most well known mitogenic and protective substances working in the inner ear, which has been proposed to fulfill a variety of functions during vertebrate inner ear development and within the mature sensory organ (Corwin et al., 1996). FGF2 protected cochlear hair cells in organotypic cultures from ototoxic damage in vitro in rats, possibly acting on FGF receptor 3 which is expressed in hair cells (Low et al., 1996), and FGF-2 protects auditory hair cells from noise damage in guinea pigs in vivo (Zhai et al., 2002; Carnicero et al., 2004). Therefore, introduction of FGF-2 into the inner ear may be taken as a potential mechanism to protect hair cells. Consistent with previous research, the present studies suggest that FGF-2 plays a role in stimulating hair cell survival within the organ of Corti. However, it remains unclear how FGF-2 prevents damage to inner ear sensory epithelia in mammals. FGF-2 may fulfill a protective function through initiate repair mechanisms during hair cell loss, which may include the reinitiation of cellular transdifferentiation processes. With IGF-1 treatment alone, there were no significant differences between damaged and IGF-1 supplemented groups, demonstrating that exogenous IGF-1 may not be involved in the maintenance of the inner ear following damage. Although our results show that IGF-1 alone does not protect hair cells in mammals, sustained delivery of IGF-1 (1.0 mg/mL) into the cochlear fluid can increase survival of OHCs (Angunsri et al., 2011). The physiology significance of IGF-1 (100–500 ng/mL) mediated cell death protection has also been shown in auditory sensory cells in vitro culture (Oesterle et al., 1997; Malgrange et al., 2002). These experiments demonstrate that IGF-1 is a candidate trophic factor keeping hair cell survival at pharmacological concentration, and perhaps IGF-1 protects the inner ear from damage under certain conditions. Addition of IGF-1 to explants cultures of normal auditory epithelia from postnatal 3–5 days rats inner ear did not induce proliferation within the sensory epithelia (data not shown). Since the cultured inner ear epithelial sheets contain supporting cells and hair cells within the organ of Corti, the survival promoting effect of the growth factors can result either from a direct interaction of growth factor ligands with their corresponding receptors present on OHCs themselves, Cell Biol Int 39 (2015) 65–73 © 2014 International Federation for Cell Biology
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or might be mediated through an effect on non-sensory cells of the organ of Corti, which would result in a release of a secondary trophic factors. Since FGF-2 (Pirvola et al., 1995) and EGF (Zine et al., 2000) receptors have been localized not only onto the hair cells, but also in supporting cells, the survival promoting effects of FGF-2 and EGF are probably indirect and mediated by non-sensory cells. In these adult organ of Corti explants, IHCs have a level of independence on exogenous trophic support under these in vitro conditions. IHCs either may not require trophic support or alternatively the needed growth factors are produced within the explants by its corresponding supporting cells. Several growth factors are associated with the regeneration process: insulin, IGF-1, and fibroblast growth factors are important for avian inner ear regeneration (Oesterle et al., 1997), whereas EGF, transforming growth factor a (TGF-a), insulin, FGF-2 are important for regeneration in the mammalian labyrinth (Zheng et al., 1997; Staecker and Van de Water, 1998; Staecker et al., 2001). Regeneration of mammalian auditory hair cells is possible during the early neonatal period and may exist to a very limited degree at later time (Zine et al., 2000); however, our findings are in contrast to these studies. Exogenously added combinations of EGF, IGF1, and FGF-2 4 and 7 days after neomycin treatment did not result in hair cell regeneration in organtypic culture. The interpretation of the discrepancy between these results is that our cultured tissues were treated with a low concentration of aminoglycoside (0.1 mM) compared to the (Zine et al., 2000). With intensive damage, the immature explants might be triggered to return to an earlier development stage and induce regeneration by new cells proliferate or other supporting cells transdifferentiate. Our findings are harmonious with other, which reported that the addition of FGF-1 to dissociated explants of isolated rat utricular (Zheng et al., 1997), and the avian inner ear sensory epithelia (Oesterle et al., 2000) elicited no detectable effects on cell proliferation. Oesterle et al. also showed that FGF-2 may be involved in inhibiting cell proliferation or stimulating cell differentiation in avian inner ear sensory epithelia. It remains unclear how and to what extent growth factors inducing proliferation and regeneration to damaged inner ear epithelia in mammals. There are no conclusive evidence that supports the participation of regenerative cell proliferation as a major participant in the replacement of lost hair cells from either the auditory or vestibular receptors of mammals. Although our observations of the protective effects of EGF, IGF-1, and FGF-2 are consistent with previous reports (Zheng et al., 1997; Zine and de Ribaupierre, 1998), the results of combination of two or three members of EGF, IGF-1, and FGF-2 are novel. Among the growth factors, combination FGF and IGF-1, combination EGF, IGF-1, and FGF-2 two groups have noticeable effects on reducing hair cells loss from neomycin ototoxicity. Several growth factors 71
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have additive protective effective on auditory hair cells after aminoglycoside antibiotics treatment. One of the possible explanations of the protective effect of selected growth factors is that several growth factor work together to activate more than one signal to induce protection, and individual signaling pathways interact with each other, forming complex signaling networks that respond to stimulate the survival of hair cells from neomycin ototoxicity. Mixtures of several growth factors might to some extent promote cooperation between growth factors and their receptors. It is possible that multiple growth factors may contribute together to protection of hair cells in a sequencing manner or at multiple steps. Acknowledgement and funding The work was supported by the Fundamental Research Funds for the Central Universities (Grant No. 2232013D313) and Donghua University Scientific Research Foundation for Youths (Grant No.13D210502). Authors’ contributions XXL, JX, XLW, and LLY carried out the organ of Corti dissection, culture, and data analysis. JX, XLW, and LLY carried out the immunoassay. ZZY were involved in the design and coordination of this project as well as manuscript writing. All authors reviewed and approved the final manuscript. Conflict of interests The authors declare that they have no competing interests. References Angunsri N, Taura A, Nakagawa T, Hayashi Y, Kitajiri S-i, Omi E, Ishikawa K, Ito J (2011) Insulin-like growth factor 1 protects vestibular hair cells from aminoglycosides. Neuroreport 22: 38–43. Carnicero E, Zelarayan LC, Ruttiger L, Knipper M, Alvarez Y, Alonso MT, Schimmang T (2004) Differential roles of fibroblast growth factor-2 during development and maintenace of auditory sensory epithelia. J Neurosci Res 77: 787–97. Chardin S, Romand R (1995) Regeneration and mammalian auditory hair-cells. Science 267: 707–9. Corwin JT, Warchol ME, Saffer LD, Finley JE, Gu RD, Lambert PR (1996) Growth factors as potential drugs for the sensory epithelia of the ear. Ciba Found Symp 196: 167–82. Debnath J, Baehrecke EH, Kroemer G (2005) Does autophagy contribute to cell death? Autophagy 1: 66–74. Di Domenico M, Ricciardi C, Martone T, Mazzarella N, Cassandro C, Chiarella G, D’Angelo L, Cassandro E (2011) Towards gene therapy for deafness. J Cell Physiol 226: 2494–9.
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Zhai SQ, Cheng JC, Wang JL, Yang WY, Gu R, Jiang SC (2002) Protective effect of basic fibroblast growth factor on auditory hair cells after noise exposure. Acta Oto-Laryngol 122: 370–3. Zheng JL, Helbig C, Gao WQ (1997) Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures. J Neurosci 17: 216–26. Zine A, de Ribaupierre F (1998) Replacement of mammalian auditory hair cells. Neuroreport 9: 263–8. Zine A, Nyffeler M, de Ribaupierre F (2000) Spatial expression patterns of epidermal growth factor receptor gene transcripts in the postnatal mammalian cochlea. Hear Res 141: 19–27. Received 14 March 2014; accepted 9 June 2014. Final version published online 4 August 2014.
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