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Dexamethasone treatment of naïve organ of Corti explants alters the expression pattern of apoptosis-related genes
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Dexamethasone treatment of naïve organ of Corti explants alters the expression pattern of apoptosis-related genes Kimberly N. Hoang a , Christine T. Dinh a , Esperanza Bas a,b , Shibing Chen a , Adrien A. Eshraghi a , Thomas R. Van De Water a,⁎ a
Cochlear Implant Research Program, University of Miami Ear Institute, Department of Otolaryngology, University of Miami, Miller School of Medicine, Miami, FL, USA b Department of Otolaryngology, University of Valencia, Valencia, Spain
A R T I C LE I N FO
AB S T R A C T
Article history:
Background: Dexamethasone treatment of organ of Corti explants challenged with an
Accepted 26 August 2009
ototoxic level of an inflammatory cytokine modulates NFκB signaling and the expression
Available online 9 September 2009
levels of both pro-and anti-apoptosis-related genes. It is not known if naïve organ of Corti explants will respond in a similar manner to treatment with a corticosteroid. This study
Keywords:
examines the response of naïve organ of Corti explants to treatment with dexamethasone.
Dexamethasone
Methods: Three-day-old rat organ of Corti explants were cultured for 1, 2, or 4 days. Four-day
Otoprotection
in vitro cultures were fixed, stained with FITC-phalloidin and hair cells were counted. ELISA
Gene expression
was performed on 2-day cultures to determine the levels of phosphorylated nuclear factor
Apoptosis
kappa B protein. One- and 2-day cultures were studied with real-time RT-PCR for expression
Hair cell
levels of β-actin, Bax, Bcl-xl, Bcl-2 and TNFR1 genes with mean fold changes determined with
Organ of Corti
the 2−ΔΔCt method. All mean fold changes in gene and protein expression were analyzed by the Kruskal–Wallis non-parametric test. Results: There were no significant differences in hair cell counts between naïve explants and explants treated with dexamethasone. Dexamethasone treatment of naïve explants resulted in a significant increase (p < 0.01) in the level of phosphorylated-nuclear factor kappa B protein. Bax expression was significantly decreased (p < 0.01) in the dexamethasone-treated explants compared to untreated-naïve explants at 1 and 2 days. TNFR1 expression was significantly reduced in dexamethasonetreated explants at 1 (p < 0.01) and 2 days (p = 0.001). Both Bcl-2 and Bcl-xl expression levels were significantly increased in dexamethasone-treated cultures compared to naïve-cultures at 2 days in vitro (p < 0.001). Dexamethasone-treated explants showed a significant decrease in the Bax/Bcl-2 ratio at both 1 (p = 0.004) and 2 days (p < 0.001) in vitro. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Cochlear implants are used to successfully treat hearingimpaired individuals suffering from profound to total senso-
rineural hearing loss (Balkany et al., 2002). However, a patient's residual hearing may be lost during and following cochlear implant surgery due to a number of factors which include, but not limited to: (1) mechanical damage caused during insertion
⁎ Corresponding author. Cochlear Implant Research Program University of Miami Ear Institute 1600 NW 10th Avenue, RMSB 3160 Miami, FL 33136-1015, USA. Fax: +1 305 243 5552. E-mail address: [email protected] (T.R. Van De Water). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.08.097
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of the electrode array; (2) vibration damage generated during drilling of the calvaria required for placement of the device; (3) disturbance of cochlear fluid homeostasis; and (4) an inflammatory response caused by the presence of a foreign body within the scala tympani of the inner ear (Kiefer et al., 2004; Gstoettner et al., 2006). Current trends in cochlear implantation research are aimed at developing strategies that will prevent or lessen the level of electrode trauma-induced hearing loss during and after implantation. A protective effect by a glucocorticoid against noiseinduced hearing loss was demonstrated in a study where methyl prednisone was administered to mice before and after noise exposure (Henry, 1992). Subsequent experiments have provided support for protection of hearing by glucocorticoids administered either during or immediately after insults caused by: (1) ischemia/reperfusion; (2) mechanical trauma; (3) ototoxic medications; and (4) noise (Himeno et al., 2002; Park et al., 2004; Takemura et al., 2004; Yildirim et al., 2005; Tabuchi et al., 2006; Tahera et al., 2006; Hill et al., 2008; Zhou et al., 2008). Because of these observations cited above, glucocorticoids have become a mainstay treatment for the management of inner ear diseases such as; (1) sudden idiopathic sensorineural hearing loss; (2) hearing loss related to
Ménière's disease; and (3) autoimmune inner ear disease (Haynes et al., 1981; Garcia-Purrinos et al., 2005; Ghosh and Jackson, 2005). In addition, animal studies have shown that local delivery of dexamethasone (DXM), a synthetic corticosteroid, conserves hearing and protects hair cells in an animal model of electrode insertion trauma-induced hearing loss (Eshraghi et al., 2007; Vivero et al., 2008). Tumor necrosis factor alpha (TNFα), a pro-inflammatory cytokine, is released following a vibration generated trauma to the tissues of the inner ear (Zou et al., 2005) and a series of in vitro studies have demonstrated that TNFα is ototoxic to auditory hair cells (HCs) and that DXM treatment of TNFα-challenged organ of Corti cultures protects these auditory HCs from TNFα-induced apoptosis (Dinh et al., 2008a, 2008b; Haake et al., 2009). This knowledge lends support to the rationale that glucocorticoid therapy protects the inner ear against trauma-induced hearing loss such as occurs in an animal model of cochlear implant surgery (Eshraghi et al., 2007; Vivero et al., 2008). The present study builds upon results from the previously described experiments where organ of Corti explants were protected against TNFα ototoxicity by DXM treatment. It explores the role of DXM treatment of auditory HCs in the absence of a trauma-related stimulus (e.g. TNFα). Studies
Fig. 1 – The density and the surface morphology of auditory hair cells in naïve organ of Corti explants were unaffected by treatment with dexamethasone. (A) Counts of intact outer hair cells (OHCs), inner hair cells (IHCs), and total hair cells (HCs) from P3 rat organ of Corti explants after 4 days in vitro under two culture conditions: (1) naïve explants with no treatment; and (2) naïve explants treated with DXM (70 μg/ml) are presented as mean values ± SD (error bars) (n = 17 explants/culture condition). Hair cells were counted/415 μm of explant basilar membrane from basal, middle and apical segments of each explant. (B and C) Images of the basal turn areas from FITC-phalloidin stained organ of Corti explants, after 4 days in vitro. (B) A naïve explant showing a typical organization of HCs, i.e. 1 row of IHCs (arrow) and 3 rows of OHCs (bracket). (C) A dexamethasone (DXM)-treated (70 μg/ml of media) explant revealing a similar morphological pattern of the explant's auditory HCs, with no evidence of damage to either the hair cells or their stereocilliary bundles. Scale bar in B = 50 μm for B and C. NS = not significant (p > 0.05).
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examining the effects of glucocorticoids on undamaged HCs have been scarce because most in vitro and in vivo experiments have involved glucocorticoid treatment in the presence of or shortly after an exposure to a traumatic insult of several different types (Himeno et al., 2002; Takemura et al., 2004; Tabuchi et al., 2006; Vivero et al., 2008; Haake et al., 2009). However, recent studies investigating pretreatment of tissues with DXM have demonstrated promising results of protection from injury and initiation of repair in several cell types (Sun et al., 2008; Rafacho et al., 2009; Song et al., 2009). The present experiment measures changes in protein levels for an activated signal molecule, i.e. NFκB, that is known to participate in DXM otoprotection of auditory HCs against TNFα-induced apoptosis (Haake et al., 2009) and the changes in expression levels of specific pro-and anti-apoptotic genes that occur in these TNFαchallenged explants (Dinh et al., 2008a) in naïve, undamaged organ of Corti explants following DXM treatment. Together these results will provide evidence for or against DXM treatment of auditory HCs prior to a planned trauma event such as insertion of a cochlear implant electrode array into the scala tympani of an animal model or of a patient.
2.
Results
2.1.
Hair cell (HC) counts
Inner hair cell (IHC) and outer hair cell (OHC) counts were obtained from the basal, middle and apical turn segments of FITC-phalloidin stained naïve and DXM-treated organ of Corti explants, after 4 days in vitro. These data are presented as mean values in the histogram figure presented in Fig. 1A. Analysis of these mean HC count values showed that there were no significant differences between the naïve explants and the DXM (70 mg/ml) treated organ of Corti explants for all three categories of hair cell counts (i.e. total HCs, IHCs, OHCs; p > 0.05 for all). Photomicrographs from FITC-phalloidin stained surface preparations of the basal turn segments of organ of Corti explants that represent each of the two studied groups, i.e. naïve, untreated-control explants and DXM-treated explants, are presented in Figs. 1B and C, respectively. In the naïve, untreated-control explant (48 h in vitro), a single row of IHCs and three well-organized rows of OHCs were visualized (Fig. 1B) while an explant treated with DXM (70 μg/ml) for 48 h exhibited a nearly identical morphological pattern of auditory HC distribution (Fig. 1C).
2.2.
Protein expression
ELISA studies were performed for the naïve, untreated-control explants and the DXM-treated explants, determining the levels of p-NFκB p65 (Ser 536) protein expression (Fig. 2). There was no significant change in protein expression levels for p-NFκB p65 in the apical region of either DXM treated explants or naïve explants. In contrast, the absorbance value was significantly higher in the middle + basal turn segment specimens from the DXM treated explants compared to the values obtained from these specimens from the naïve, untreated-control cultures (p < 0.01).
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Fig. 2 – Dexamethasone-treatment of naïve organ of Corti explants initiated a significant up regulation in the protein levels of phosphorylated-nuclear factor kappa B (p-NFκB). Naïve P3 organ of Corti explants were either exposed to or not exposed to DXM (70μg/ml) for 48 h and subsequently divided into two segments: (1) apical turns and (2) middle + basal turns. There was a statistically significant increase (p < 0.01) in p-NFκB protein in the middle + basal turn segments of DXM-treated explants compared to levels of this protein in the untreated naive controls. ELISA was used to determine protein levels of p-NFκB with data presented as mean fold change ± S.E.M. (represented by error bars); **p < 0.01; n = 12 explants/culture condition.
2.3.
Gene expression
Expression levels of genes that encode for one pro- and two anti-apoptotic factors as well as one pro-inflammatory factor were compared between DXM-treated and naïve, untreatedcontrol organ of Corti explants and these changes in expression levels are based upon real time RT-PCR results with normalization of results to 0 h excised control specimens and to β-actin (a housekeeping gene). There were no significant differences between DXM-treated and naïve, untreated-control organ of Corti explant expression levels for either Bcl-2 or Bcl-xl genes (Figs. 3A and B) after 24 h in vitro (p = 0.27 and p = 0.60, respectively). However, after 48 h in vitro a substantial up regulation of the expression levels of both the Bcl-2 and the Bcl-xl genes was observed compared to the expression levels for these genes in naïve, untreatedcontrol specimens at this time point (p < 0.001 for both genes). A significant down regulation in Bax gene expression was observed in DXM-treated cultures at both 24 and 48 h in vitro time points compared with the Bax expression levels observed in naïve, untreated-control explants (Fig. 4A). Mean fold change of Bax in the DXM-treated explants decreased as time in vitro progressed, i.e. from <0.8 mean fold reduction at 24 h to a reduction of <0.6 after 48 h in vitro (p = 0.002 and p < 0.01, respectively; Fig. 4A) compared to no significant change (p > 0.05) in the naïve, untreated control explant values which remained at 1.0. As seen in Fig. 4B, DXM-treated organ of Corti explants down regulated TNFR1 gene expression levels to a mean fold change of 0.8 when compared to naïve, untreated control explants (1.0) after 24 h in vitro (p < 0.01). Gene expression
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level changes of TNFR1 in the DXM-treated cultures were further reduced at the 48-h in vitro time point to a mean fold change of 0.6 which achieved a high level of significance compared to the values of the naïve, untreated control explants at that time point (Fig. 4B; p = 0.001). Modulation of both the Bcl-2 and the Bax genes by DXM-treatment were evident when the mean fold change of the Bax/Bcl-2 ratios were examined for explants treated with DXM and were compared to the results from naïve, untreated-control explants (Fig. 5). The DXMtreated cultures showed a significant decrease in the Bax/Bcl-2 ratio at both the 24-h and 48-h in vitro time points (p = 0.004 and p < 0.001, respectively) compared to the lack of any significant change that was observed for the naïve, untreated control organ of Corti specimens for both the 24- and 48-h time points.
Fig. 4 – Treatment of naïve organ of Corti explants with dexamethasone caused a significant down regulation of a pro-apoptosis related-gene and a pro-inflammatory-related gene. (A) Bax gene expression level was significantly down regulated in DXM-treated explants compared to expression levels of this gene in naïve, untreated-controls after 24 h in vitro (p = 0.002). There is a further decline in Bax expression in the DXM-treated cultures after 48 h in vitro (p < 0.01). (B) TNFR1 expression levels showed a statistically significant reduction in the DXM-treated explants at both 24 h (p < 0.01) and 48 h (p = 0.001) in vitro when compared to the gene expression levels of naïve, untreated-controls. Error bars represent mean fold change ± S.E.M. **p < 0.01; n = 10 explants/culture condition time point.
Fig. 3 – Treatment of naïve organ of Corti explants with dexamethasone caused a significant up regulation of two anti-apoptosis related-genes. (A) Bcl-2, there was no statistically significant difference in the levels of Bcl-2 gene expression between DXM-treated and naïve, untreated-control cultures after 24 h in vitro (p = 0.27), but expression levels of this gene were significantly increased at 48 h in vitro by DXM-treatment (p < 0.001). (B) Bcl-xl, no significant change was noted in Bcl-xl gene expression levels between the two culture conditions after 24 h in vitro (p = 0.60), however, there was a significant up regulation of Bcl-xl expression after 48 h of DXM-treatment, resulting in a mean fold change of greater than 2 (p<0.001). Error bars represent ±S.E.M.; ***p<0.001. (n =10 explants/culture condition time point).
3.
Discussion
There have been no reported adverse effects on residual hearing after local and/or systemic treatment of various inner ear diseases with corticosteroids (e.g. DXM) (Chandrasekhar, 2001; Himeno et al., 2002; Silverstein et al., 1996). Hill et al. (2008) reported that intratympanic administration of DXM to the middle ear cavity in mice resulted in significant protection against cisplatin-induced hearing loss in a frequencydependent manner and was without systemic side-effects. The results presented in the current paper show that both naïve and DXMb-treated organ of Corti explants possessed nearly identical patterns of auditory HC cell distribution after
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Fig. 5 – Dexamethasone treatment of naïve organ of Corti explants lowered the Bax/Bcl-2 ratio to favor cell survival. DXM treatment of naïve organ of Corti cultures caused a significant reduction in the mean fold change in the Bax/Bcl-2 ratio compared to this ratio in naïve, untreated-control explants at both the 24-h and 48-h time points. At 24 h, this Bax/Bcl-2 ratio is significantly reduced (p = 0.004) in the DXM-treated explants with an even more significant reduction in this ratio after 48 h of DXM-treatment (p < 0.001). Error bars represent ± S.E.M. of the mean fold change. **p < 0.01; n = 10 explants/culture condition time point.
4 days in vitro with no statistical difference in HC counts between these two culture conditions. The Bcl-2 family is comprised of proteins that either promote (e.g. Bax, Bid) or inhibit (e.g. Bcl-2, Bcl-xl) apoptosis (Youle and Strasser, 2008). TNFα has been shown to activate apoptosis of auditory HCs in organ of Corti (OC) explants by increasing Bax and TNFR1 expression levels and decreasing Bcl-xl and Bcl-2 expression levels (Dinh et al., 2008a, 2008b). The results from these same in vitro studies have demonstrated that simultaneous DXM-treatment of TNFα-challenged OC explants protects the explant's HCs from TNFα-induced cell death by reversing the TNFα-induced changes in gene expression. It was not known if DXM-treatment of naïve explants would parallel these changes in gene expression in the absence of a TNFα-challenge. The results reported in the present study support and extend the observations of DXM's otoprotective action of the modulation of the expression levels of apoptosisrelated genes showing that a damaged stimulus is not required for the initiation of these DXM-initiated changes in gene expression. Studies involving several different cell lines have implicated an elevated Bax/Bcl-2 ratio as a relative indicator of cell survival with an elevated ratio associated with apoptosis (Oltvai et al., 1993; Perlman et al., 1999; Wiren et al., 2006; John et al., 2007; Park et al., 2007). Treatment of the naïve explants with DXM in the present study resulted in a substantial decrease in the Bax/Bcl-2 ratio which would favor HC survival if these sensory cells were to be challenged by a trauma-related stimulus, e.g. cochlear implantation . DXM pretreatment has been shown to prevent apoptosis and cell injury as well as induce cell proliferation in a number of organ systems (Sasson
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and Amsterdam, 2003; Sun et al., 2008; Tenenbaum et al., 2008; Rafacho et al., 2009; Song et al., 2009). Pretreatment with DXM has been shown to suppress chemokines and inflammatory cell infiltration in the liver cells of animal models of liver damage by reducing the level of oxidative stress injury (Hsieh et al., 2006; Lee et al., 2006). The results of the present study show that DXM-treatment of naïve organ of Corti explants reduces the expression levels of Bax and TNFR1, i.e. known proapoptotic and pro-inflammatory genes respectively, which would reduce the cellular injury that can result from exposure to an excessive level of oxidative stress. Glucocorticoid receptor distribution in the cochlea is concentrated in the cells of the spiral ganglion, spiral ligament and are also present in the organ of Corti (Rarey and Curtis, 1996; Terunuma et al., 2003; Canlon et al., 2007). NFκB p65 is a transcription factor in the NFκB family that is highly expressed in spiral ganglion neurons of the cochlea and is activated in spiral ganglion cells following exposure to a traumatizing level of noise (Masuda et al., 2005; Tahera et al., 2006; Canlon et al., 2007). Blocking of NFκB with an inhibitor following acoustic overstimulation significantly increases the level of resultant hearing loss, demonstrating the role that this transcription factor plays as a protective response to limit noise-induced hearing loss (Tahera et al., 2006). In vitro studies in an auditory receptorderived cell line and in organ of Corti explants have also demonstrated that NFκB is protective against aminoglycosideinduced ototoxicity and promotes auditory HC health and survival in culture, respectively (Jiang et al., 2005; Nagy et al., 2005). DXM-treatment of naïve organ of Corti explants in the present study has been shown to increase p-NFκB levels in the middle-and-basal turn segments of the treated organ of Corti explants relative to the expression level of this transcription factor in naïve untreated-control explants. This increase in NFκB in the naïve OC explants may be important because a recent in vitro study has shown that blocking NFκB in TNFαchallenged OC explants prevents DXM-treatment from protecting the explant's HCs against TNFα-induced apoptosis (Haake et al., 2009). Local DXM pretreatment in various in vivo studies have shown significant protective effects on the preservation of hearing thresholds following both noiseinduced and cisplatin-induced traumas (Takemura et al., 2004; Tahera et al., 2006; Hill et al., 2008). It has also recently been demonstrated in vivo that short-term preoperative delivery of DXM via the round window membrane has a significant protective effect upon conservation of hearing in a guinea pig model of cochlear implantation (James et al., 2008; Chang et al., 2009). DXM has already been demonstrated to be a successful rescue therapy for sensory HCs in an inflammatory cytokine model of trauma-induced HC death in vitro (Dinh et al., 2008a; Haake et al., 2009). Based on the results of the present study, the gene and protein expression results suggest that DXM pretreatment can protect an OC explant's HCs by modulating expression levels of NFκB and of apoptosis-related genes to favor the survival of auditory HCs. Glucorticoid treatment of animals can also suppress chemokines expression and the inflammatory process (Hsieh et al., 2006) so these activities need to also be kept in mind when considering the action of DXM on the naïve OC explants. The results of the present study support local
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pretreatment of the cochlear duct in an animal model of cochlear implantation prior to a planned trauma (e.g. round window membrane dexamethasone prior to the insertion of an electrode array into the scala tympani; Chang et al., 2009) and suggest that the effective conservation of hearing against trauma-induced loss is due in least in part to DXM's protection of auditory hair cells.
4.4. ELISA-phosphorylated-nuclear factor kappa B (p-NFκB) p65 (Ser536)
All of the neonate rats utilized in this study were treated in accordance with the University of Miami Animal Care and Use Committee, study protocol #08-099 and in compliance with the published Guide for the Care and Use of Laboratory Animals of the National Institutes of Health No. 80-23. Organ of Corti (OC) explants were obtained from 3-day-old (P3) rats of the Sprague–Dawley strain of laboratory rats (Charles River Laboratories, Inc., Wilmington, MA, USA). Every effort was made to limit suffering and to keep the number of animals used in this study to a minimum.
Twenty-four OC explants were cultured under the same two conditions described previously for 48 h and then divided into two sections: (1) apical turns and (2) middle + basal turns. Two independent experiments were carried out. Six specimens sections from each group were pooled and respectively placed in 100 μl of chilled RIPA-buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Inc., Waltham, MA) and lysed with a Microson XL-2000 Sonifier (Misonix, Inc., Farmingdale, NY). Following centrifugation at 10,000×g for 10 min, the proteins present in the supernatants were quantified by the Bradford's method (Bio-Rad Laboratories, Hercules, CA). Subsequently, 30 μg of protein were placed in each well, with 4 repetitions of each sample. Phosphorylated nuclear factor kappaB p65 (Ser536) proteins (p-NFκB) were detected using the PathScan phospho-NFκB p65 (Ser536) Sandwich ELISA Kit (Cell Signaling, Inc., Danvers, MA) as described in the manufacturer's protocol and quantified at 450 nm wave length light using the ELx800 Absorbance Microplate Reader (Biotek, Inc., Winooski, VT). The p-NFκB protein is the activated form of NFκB.
4.2.
4.5.
4.
Experimental procedures
4.1.
Animals
Organ of Corti explants
One-hundred eighteen OC explants were dissected from the cochleae of sixty-nine 3-day-old (P-3) rats and placed in culture media consisting of modified Eagle's medium (Gibco, Grand Island Biologicals, Grand Island, NY, USA; www. invitrogen.com) enriched with a 1% concentration of N-1 supplement (100 × concentrate; Sigma-Aldrich, Saint Louis, MO, USA; www.sigma-aldrich.com) and glucose (adjust final conc. to 6 g/l). Explants were cultured at 37 °C in a 98% humidified atmosphere with 5% CO2 in room air. Explants were distributed into various sets for HC counts, gene expression studies and protein studies. Except for the HC counts, explants were divided into two groups and then cultured in either: (1) naïve, untreated-control; or (2) naïve, DXM-treated (70 μg/ml; Boston Scientific Corporation, Natick, MA, USA; www.bostonscientific.com) and harvested at specified time points (as listed below).
4.3.
Hair cell counts
Thirty-four OC explants were divided and cultured under the two conditions described above. After 4 days in vitro, they were then fixed, permeabilized and stained with fluorscein isothiocyanate (FITC)-labeled phalloidin. Explants were sequentially mounted on a slide with gelatin, cover slipped and examined under fluorescent illumination using a 40× lens (Zeiss Axiovert 200 microscope; Carl Zeiss Microscopy, Gottingen, Germany). Total HCs were counted for a 415-μm length of the basal segment of the basilar membrane of each explant and categorized as either inner HCs (IHC) or outer HCs (OHC). A countable HC was identified as one that possessed both an intact stereocilliary bundle and a cuticular plate. The investigator performing the HC counts was blinded to the identity of the stained explant specimens until all of the HC counts were completed.
Gene expression
Sixty OC explants were likewise cultured under the same two conditions for 0, 24 and 48 h in vitro (n = 10 explants/culture condition time point). Total cellular RNA was isolated using the Qiagen RNEasy® Protect Mini Kit (Qiagen Sciences, Inc., Germantown, MD, USA; www1.qiagen.com). The animal tissue protocol included in the kit was used with modifications: (1) stabilized tissue was disrupted in a 1.5-ml centrifuge tube with an RNase-free pestle; (2) tissue was homogenized with an RNase-free syringe; (3) DNase solution was added to degrade residual DNA and thus enhance RNA purity; and (4) before RNA extraction, RNA spin columns were centrifuged at full speed (14,000×g ) for 1 min to minimize contamination. cDNA synthesis was performed using the Bio-Rad iScript cDNA synthesis kit and its protocol (Bio-Rad Laboratories, Inc., Hercules, CA, USA; www.bio-rad.com). Samples were then placed in an Eppendorf Mastercycler® thermal cycler (Eppendorf Inc., Westbury, NY, USA, www.eppendorfna.com). Purified RNA was stored at −20 °C.
4.6.
Quantitative RT-PCR
Two-step real-time RT-PCR was completed with the following primers: β-actin (housekeeping gene), Bax, Bcl-xl, Bcl-2 and TNFR1; these were synthesized by Sigma-Genosys (SigmaGenosys, The Woodlands, TX, USA; www.sigma-genosys. com). Amplification products were detected using the MyiQ Single-Color Real-Time PCR Detection System from Bio-Rad and IQ SYBR Green Supermix. These following primer sequences were utilized: β-actin forward (fwd), 5′-CGTTGACATCCGTAAAGACC-3′; β-actin reverse (rev), 5′-AGCCACCAATCCACACAGAG-3′ (173 bp) (Nudel et al., 1983); TNFR1 fwd, 5′-GAACACCGTGTGTAACTGCC-3′; TNFR1 rev, 5′-ATTCCTTCACCCTCCACCTC-3′ (301 bp) (Raina and Jeejeebhoy, 2004); Bax fwd, 5′-CTGCAGAGGATGATTGCTGA-3′; Bax rev, 5′-GATC-
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AGCTCGGGCACTTTAG-3′ (174 bp); Bcl-2 fwd, 5′-GCTACGAGTGGGATACTGG-3′; Bcl-2 rev, 5′-GTGTGCAGATGCCGGTTCA-3′ (472 bp); Bcl-xl fwd, 5′-AGGATACAGCTGGAGTCAG-3′; and Bcl-xl rev, 5′-TCTCCTTGTCTACGCTTTCC-3′ (417 bp) (Valks et al., 2003). For primer TNFR1, real-time PCR was carried out for 40 cycles at 94 °C, 59 °C and 72 °C for 1 min each; β-actin, 94 °C, 55 °C and 72 °C for 1 min, 1 min, and 1.5 min, respectively; Bax, Bcl-2 and Bcl-xl, 95 °C, 61 °C and 72 °C for 50 s each. The 2−ΔΔCt method was applied to assess for relative changes in the mRNA levels of genes promoting or inhibiting apoptosis.
4.7.
Data analysis
One-way analysis of variance (ANOVA) was used to analyze HC counts. Analyses of the mean fold changes for both gene expression and protein expression studies were performed by the Kruskal–Wallis non-parametric test with a p value of < 0.05 considered statistically significant. Relative changes in mRNA expression are presented in the bar graphs as mean values ± S.E.M. Values were normalized to 0 h excised control explants and to β-actin, a housekeeping gene.
Acknowledgments Research supported by grants from Advanced Bionics Corporation, Valencia, CA and MED-EL, Medical Electronics Corporation, Innsbruck, Austria to TRV.
REFERENCES
Balkany, T.J., Hodges, A.V., Eshraghi, A.A., Butts, S., Bricker, K., Lingvai, J., Polak, M., King, J., 2002. Cochlear implants in children—a review. Acta Otolaryngol. 122, 356–362. Canlon, B., Meltser, I., Johansson, P., Tahera, Y., 2007. Glucocorticoid receptors modulate auditory sensitivity to acoustic trauma. Hear. Res. 226, 61–69. Chandrasekhar, S.S., 2001. Intratympanic dexamethasone for sudden sensorineural hearing loss: clinical and laboratory evaluation. Otol. Neurotol. 22, 18–23. Chang, A., Eastwood, H., Sly, D., James, D., Richardson, R., O'Leary, S., 2009. Factors influencing the efficacy of round window dexamethasone protection of residual hearing post-cochlear implant surgery. Hear. Res. 255, 67–72. Dinh, C.T., Haake, S., Chen, S., Hoang, K., Nong, E., Eshraghi, A.E., Balkany, T.J., Van De Water, T.R., 2008a. Dexamethasone protects organ of Corti explants against tumor necrosis factor-alpha-induced loss of auditory hair cells and alters the expression levels of apoptosis-related genes. Neuroscience 157, 405–413. Dinh, C., Hoang, K., Haake, S., Chen, S., Angeli, S., Nong, E., Eshraghi, A., Balkany, T.J., Van De Water, T.R., 2008b. Biopolymer-released dexamethasone prevents tumor necrosis factor α-induced loss of auditory hair cells in vitro: implications toward the development of a drug-eluting cochlear implant electrode array. Otol. Neurotol. 29, 1012–1019. Eshraghi, A.A., Adil, E., He, J., Graves, R., Balkany, T.J., Van De Water, T.R., 2007. Local dexamethasone therapy conserves hearing in an animal model of electrode insertion trauma-induced hearing loss. Otol. Neurotol. 28, 842–849. Garcia-Purrinos, F.J., Ferri, E., Rosell, A., Calvo, J., 2005. Combined intratympanic and intravenous dexamethasone to control vertigo in Meniere disease. Acta Otorinolaryngol. Esp. 56, 74–77.
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Ghosh, A., Jackson, R., 2005. Best evidence topic report. Steroids in sudden sensorineural hearing loss. Emerg. Med. J. 22, 732–733. Gstoettner, W.K., Helbig, S., Maier, N., Kiefer, J., Radeloff, A., Adunka, O.F., 2006. Ipsilateral electric acoustic stimulation of the auditory system: results of long-term hearing preservation. Audiol. Neurotol. 11, 49–56. Haake, S., Dinh, C.T., Chen, S., Eshraghi, A.A., Van De Water, T.R., 2009. Dexamethasone protects auditory hair cells against TNFalpha-initiated apoptosis via activation of PI3K/Akt and NFkB signaling. Hear. Res. 255, 22–32. Haynes, B.F., Pikus, A., Kaiser-Kupfer, M., Fauci, A., 1981. Successful treatment of sudden hearing loss in Cogan's syndrome with glucocorticoids. Arthritis Rheum. 24, 501–503. Henry, K.R., 1992. Noise-induced auditory loss: influence of genotype, naloxone and methyl-prednisolone. Acta Otolaryngol. 112, 599–603. Hill, G.W., Morest, K., Parham, K., 2008. Cisplatin-induced ototoxicity: effect of intratympanic dexamethasone injections. Otol. Neurotol. 29, 1005–1011. Himeno, C., Komeda, M., Izumikawa, M., Takemura, K., Yagi, M., Weiping, Y., Doi, T., Kuriyama, H., Miller, J.M., Yamashita, T., 2002. Intra-cochlear administration of dexamethasone attenuates aminoglycoside ototoxicity in the guinea pig. Hear. Res. 167, 61–70. Hsieh, C.S., Wang, P.W., Lee, S.Y., Huang, C.C., Chang, N.K., Chen, C.M., Wu, C.L., Wang, H.C., Chuang, J.H., 2006. Glucocorticoid pretreatment suppresses chemokine expression and inflammatory cell infiltration in cholestatic rats receiving biliary intervention. J. Pediatric Surg. 41, 1669–1675. James, D.P., Eastwood, H., Richardson, R.T., O'Leary, S.J., 2008. Effects of round window dexamethasone on residual hearing in a guinea pig model of cochlear implantation. Audiol. Neurotol. 13, 86–96. Jiang, H., Sha, S.H., Schacht, J., 2005. NFκB pathway protects cochlear hair cells from aminoglycoside-induced ototoxicity. J. Neurol. Res. 79, 644–651. John, T., Muller, R.D., Oberholzer, A., Zreigat, H., Kohl, B., Ertel, W., Hostmann, A., Tschoeke, S.K., Schulze-Tanzil, G., 2007. Interleukin-10 modulates pro-apoptotic effects of TNF-alpha in human articular chondrocytes in vitro. Cytokine 40, 226–234. Kiefer, J., Gstoettner, W., Baumgartner, W., Pok, S.M., Tillein, J., Ye, Q., von Ilberg, C., 2004. Conservation of low-frequency hearing in cochlear implantation. Acta Otolaryngol 124, 272–280. Lee, C.W., Chuang, J.H., Wang, P.W., Chang, N.K., Wang, H.C., Huang, C.C., Tiao, M.M., Lo, S.K., 2006. Effect of glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with endotoxin cholangitis. World J. Surg. 30, 2217–2226. Masuda, M., Nagashima, R., Kanzaki, S., Fujioka, M., Ogita, K., Ogawa, K., 2005. Nuclear factor-kappa B nuclear translocation in the cochlea of mice following acoustic overstimulation. Brain Res. 88, 585–589. Nagy, I., Monge, A., Albinger-Hegyi, A., Schmid, S., Bodmer, D., 2005. NF-κB is required for survival of immature auditory hair cells in vitro. J. Assoc. Res. Otolaryngol. 6, 260–268. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z., Yaffe, D., 1983. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 11, 1759–1771. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Park, S.K., Choi, D., Russell, P., John, E.O., Jung, T.T., 2004. Protective effect of corticosteroid against the ototoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells. Laryngoscope 114, 768–771. Park, C., Moon, D.O., Rhu, C.H., Choi, B.T., Lee, W.H., Kim, G.-Y., Choi, Y.H., 2007. Beta-sitosterol induced anti-proliferation and apoptosis of human leukemic U937 cells through activation of caspase-3 and induction of Bax/Bcl-2 ratio. Biol. Pharm. Bull. 30, 1317–1323.
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Perlman, H., Zhang, X., Chen, M.W., Walsh, K., Buttyan, R., 1999. An elevated Bax/Bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death Differ. 6, 48–54. Rafacho, A., Cestari, T.M., Taboga, S.R., Boschero, A.C., Bosqueiro, J.R., 2009. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am. J. Physiol. Endocrinol. Metab. 296, E681–689. Raina, N., Jeejeebhoy, K.N., 2004. Effect of low-protein diet and protein supplementation on the expressions of TNF-alpha, TNFR-1, and TNFR-II in organs and muscle of LPS-injected rats. Am. J. Physiol. Endocrinol. Metab. 286, E481–487. Rarey, K.E., Curtis, L.M., 1996. Receptors for glucocorticoids in the human inner ear. Otolaryngol. Head Neck Surg. 115, 38–41. Sasson, R., Amsterdam, A., 2003. Pleiotropic anti-apoptotic activity of glucocorticoids in ovarian follicular cells. Biochem. Pharmacol. 66, 1393–1401. Silverstein H., Choo D., Rosenberg S.I., Kuhn J., Seidman M., Stein I., 1996. Intratypanic steroid treatment of inner ear disease and tinnitus (preliminary report). Ear Nose Throat J. 75, 468-71,474,476. Song, I.H., Caplan, A.I., Dennis, J.E., 2009. In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo. J. Orthop. Res. 27, 916–921. Sun, J., Guo, W., Ben, Y., 2008. Preventive effects of curcumin and dexamethasone on lung transplantation-associated lung injury in rats. Crit. Care Med. 236, 1205–1213. Tabuchi, K., Oikawa, K., Murashita, H., Hoshino, T., Tsuji, S., Hara, A., 2006. Protective effects of corticosteroids on ischemia-reperfusion injury of outer hair cells. Laryngoscope 116, 627–629. Tahera, Y., Meltser, I., Johansson, P., Brian, Z., Stierna, P., Hansson, A.C., Canlon, B., 2006. NF-kappa B mediated glucocorticoid response in the inner ear after acoustic trauma. J. Neurosci. Res. 83, 1066–1076. Takemura, K., Komeda, M., Yagi, M., Chiemi, H., Izumikawa, M., Doi, T., Kuriyama, H., Miller, J.M., Yamashita, T., 2004. Direct
inner ear infusion of dexamethasone attenuates noise-induced trauma in guinea pig. Hear. Res. 196, 58–68. Tenenbaum, T., Matalon, D., Adam, R., Seibt, A., Wewer, C., Schwerk, C., Galla, H.J., Schroten, H., 2008. Dexamethasone prevents alteration of tight junction-associated proteins and barrier function in porcine choroid plexus epithelial cells after infection with Streptococcus suis in vitro. Brain Res. 1229, 1–17. Terunuma, T., Kawauchi, S., Kaji, M., Takahashi, S., Hara, A., 2003. Effect of acoustic stress on glucocorticoid receptor mRNA in the cochlea of the guinea pig. Brain Res. Mol. Brain Res. 120, 65–72. Valks, D.M., Kemp, T.J., Clerk, A., 2003. Regulation of Bcl-xl expression by H2O2 in cardiac myocytes. J. Biol. Chem. 278, 25542–25547. Vivero, R.J., Joseph, D.E., Angeli, S., He, J., Chen, S., Eshraghi, A.A., Balkany, T.J., Van De Water, T.R., 2008. Dexamethasone base conserves hearing from electrode trauma-induced hearing loss. Laryngoscope 118, 2028–2035. Wiren, K.M., Toombs, A.R., Semirale, A.A., Zhang, X., 2006. Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio. Bone 38, 637–651. Yildirim, A., Coban, L., Satar, B., Yetiser, S., Kunt, T., 2005. Effect of intratympanic dexamethasone on noise-induced temporary threshold shift. Laryngoscope 115, 1219–1222. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59. Zhou, Y., Zheng, H., Shen, X., Zhang, Q., Yang, M., 2008. Intratympanic administration of methyl prednisone reduces impact of experimental intensive impulse noise trauma on hearing. Acta Otolaryngol. 24, 1–6. Zou, J., Pyykkö, I., Sutinen, P., Toppila, E., 2005. Vibration induced hearing loss in guinea pig cochlea: expression of TNFα and VEGF. Hear. Res. 202, 13–20.
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Research Report
Dexamethasone treatment of naïve organ of Corti explants alters the expression pattern of apoptosis-related genes Kimberly N. Hoang a , Christine T. Dinh a , Esperanza Bas a,b , Shibing Chen a , Adrien A. Eshraghi a , Thomas R. Van De Water a,⁎ a
Cochlear Implant Research Program, University of Miami Ear Institute, Department of Otolaryngology, University of Miami, Miller School of Medicine, Miami, FL, USA b Department of Otolaryngology, University of Valencia, Valencia, Spain
A R T I C LE I N FO
AB S T R A C T
Article history:
Background: Dexamethasone treatment of organ of Corti explants challenged with an
Accepted 26 August 2009
ototoxic level of an inflammatory cytokine modulates NFκB signaling and the expression
Available online 9 September 2009
levels of both pro-and anti-apoptosis-related genes. It is not known if naïve organ of Corti explants will respond in a similar manner to treatment with a corticosteroid. This study
Keywords:
examines the response of naïve organ of Corti explants to treatment with dexamethasone.
Dexamethasone
Methods: Three-day-old rat organ of Corti explants were cultured for 1, 2, or 4 days. Four-day
Otoprotection
in vitro cultures were fixed, stained with FITC-phalloidin and hair cells were counted. ELISA
Gene expression
was performed on 2-day cultures to determine the levels of phosphorylated nuclear factor
Apoptosis
kappa B protein. One- and 2-day cultures were studied with real-time RT-PCR for expression
Hair cell
levels of β-actin, Bax, Bcl-xl, Bcl-2 and TNFR1 genes with mean fold changes determined with
Organ of Corti
the 2−ΔΔCt method. All mean fold changes in gene and protein expression were analyzed by the Kruskal–Wallis non-parametric test. Results: There were no significant differences in hair cell counts between naïve explants and explants treated with dexamethasone. Dexamethasone treatment of naïve explants resulted in a significant increase (p < 0.01) in the level of phosphorylated-nuclear factor kappa B protein. Bax expression was significantly decreased (p < 0.01) in the dexamethasone-treated explants compared to untreated-naïve explants at 1 and 2 days. TNFR1 expression was significantly reduced in dexamethasonetreated explants at 1 (p < 0.01) and 2 days (p = 0.001). Both Bcl-2 and Bcl-xl expression levels were significantly increased in dexamethasone-treated cultures compared to naïve-cultures at 2 days in vitro (p < 0.001). Dexamethasone-treated explants showed a significant decrease in the Bax/Bcl-2 ratio at both 1 (p = 0.004) and 2 days (p < 0.001) in vitro. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Cochlear implants are used to successfully treat hearingimpaired individuals suffering from profound to total senso-
rineural hearing loss (Balkany et al., 2002). However, a patient's residual hearing may be lost during and following cochlear implant surgery due to a number of factors which include, but not limited to: (1) mechanical damage caused during insertion
⁎ Corresponding author. Cochlear Implant Research Program University of Miami Ear Institute 1600 NW 10th Avenue, RMSB 3160 Miami, FL 33136-1015, USA. Fax: +1 305 243 5552. E-mail address: [email protected] (T.R. Van De Water). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.08.097
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of the electrode array; (2) vibration damage generated during drilling of the calvaria required for placement of the device; (3) disturbance of cochlear fluid homeostasis; and (4) an inflammatory response caused by the presence of a foreign body within the scala tympani of the inner ear (Kiefer et al., 2004; Gstoettner et al., 2006). Current trends in cochlear implantation research are aimed at developing strategies that will prevent or lessen the level of electrode trauma-induced hearing loss during and after implantation. A protective effect by a glucocorticoid against noiseinduced hearing loss was demonstrated in a study where methyl prednisone was administered to mice before and after noise exposure (Henry, 1992). Subsequent experiments have provided support for protection of hearing by glucocorticoids administered either during or immediately after insults caused by: (1) ischemia/reperfusion; (2) mechanical trauma; (3) ototoxic medications; and (4) noise (Himeno et al., 2002; Park et al., 2004; Takemura et al., 2004; Yildirim et al., 2005; Tabuchi et al., 2006; Tahera et al., 2006; Hill et al., 2008; Zhou et al., 2008). Because of these observations cited above, glucocorticoids have become a mainstay treatment for the management of inner ear diseases such as; (1) sudden idiopathic sensorineural hearing loss; (2) hearing loss related to
Ménière's disease; and (3) autoimmune inner ear disease (Haynes et al., 1981; Garcia-Purrinos et al., 2005; Ghosh and Jackson, 2005). In addition, animal studies have shown that local delivery of dexamethasone (DXM), a synthetic corticosteroid, conserves hearing and protects hair cells in an animal model of electrode insertion trauma-induced hearing loss (Eshraghi et al., 2007; Vivero et al., 2008). Tumor necrosis factor alpha (TNFα), a pro-inflammatory cytokine, is released following a vibration generated trauma to the tissues of the inner ear (Zou et al., 2005) and a series of in vitro studies have demonstrated that TNFα is ototoxic to auditory hair cells (HCs) and that DXM treatment of TNFα-challenged organ of Corti cultures protects these auditory HCs from TNFα-induced apoptosis (Dinh et al., 2008a, 2008b; Haake et al., 2009). This knowledge lends support to the rationale that glucocorticoid therapy protects the inner ear against trauma-induced hearing loss such as occurs in an animal model of cochlear implant surgery (Eshraghi et al., 2007; Vivero et al., 2008). The present study builds upon results from the previously described experiments where organ of Corti explants were protected against TNFα ototoxicity by DXM treatment. It explores the role of DXM treatment of auditory HCs in the absence of a trauma-related stimulus (e.g. TNFα). Studies
Fig. 1 – The density and the surface morphology of auditory hair cells in naïve organ of Corti explants were unaffected by treatment with dexamethasone. (A) Counts of intact outer hair cells (OHCs), inner hair cells (IHCs), and total hair cells (HCs) from P3 rat organ of Corti explants after 4 days in vitro under two culture conditions: (1) naïve explants with no treatment; and (2) naïve explants treated with DXM (70 μg/ml) are presented as mean values ± SD (error bars) (n = 17 explants/culture condition). Hair cells were counted/415 μm of explant basilar membrane from basal, middle and apical segments of each explant. (B and C) Images of the basal turn areas from FITC-phalloidin stained organ of Corti explants, after 4 days in vitro. (B) A naïve explant showing a typical organization of HCs, i.e. 1 row of IHCs (arrow) and 3 rows of OHCs (bracket). (C) A dexamethasone (DXM)-treated (70 μg/ml of media) explant revealing a similar morphological pattern of the explant's auditory HCs, with no evidence of damage to either the hair cells or their stereocilliary bundles. Scale bar in B = 50 μm for B and C. NS = not significant (p > 0.05).
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examining the effects of glucocorticoids on undamaged HCs have been scarce because most in vitro and in vivo experiments have involved glucocorticoid treatment in the presence of or shortly after an exposure to a traumatic insult of several different types (Himeno et al., 2002; Takemura et al., 2004; Tabuchi et al., 2006; Vivero et al., 2008; Haake et al., 2009). However, recent studies investigating pretreatment of tissues with DXM have demonstrated promising results of protection from injury and initiation of repair in several cell types (Sun et al., 2008; Rafacho et al., 2009; Song et al., 2009). The present experiment measures changes in protein levels for an activated signal molecule, i.e. NFκB, that is known to participate in DXM otoprotection of auditory HCs against TNFα-induced apoptosis (Haake et al., 2009) and the changes in expression levels of specific pro-and anti-apoptotic genes that occur in these TNFαchallenged explants (Dinh et al., 2008a) in naïve, undamaged organ of Corti explants following DXM treatment. Together these results will provide evidence for or against DXM treatment of auditory HCs prior to a planned trauma event such as insertion of a cochlear implant electrode array into the scala tympani of an animal model or of a patient.
2.
Results
2.1.
Hair cell (HC) counts
Inner hair cell (IHC) and outer hair cell (OHC) counts were obtained from the basal, middle and apical turn segments of FITC-phalloidin stained naïve and DXM-treated organ of Corti explants, after 4 days in vitro. These data are presented as mean values in the histogram figure presented in Fig. 1A. Analysis of these mean HC count values showed that there were no significant differences between the naïve explants and the DXM (70 mg/ml) treated organ of Corti explants for all three categories of hair cell counts (i.e. total HCs, IHCs, OHCs; p > 0.05 for all). Photomicrographs from FITC-phalloidin stained surface preparations of the basal turn segments of organ of Corti explants that represent each of the two studied groups, i.e. naïve, untreated-control explants and DXM-treated explants, are presented in Figs. 1B and C, respectively. In the naïve, untreated-control explant (48 h in vitro), a single row of IHCs and three well-organized rows of OHCs were visualized (Fig. 1B) while an explant treated with DXM (70 μg/ml) for 48 h exhibited a nearly identical morphological pattern of auditory HC distribution (Fig. 1C).
2.2.
Protein expression
ELISA studies were performed for the naïve, untreated-control explants and the DXM-treated explants, determining the levels of p-NFκB p65 (Ser 536) protein expression (Fig. 2). There was no significant change in protein expression levels for p-NFκB p65 in the apical region of either DXM treated explants or naïve explants. In contrast, the absorbance value was significantly higher in the middle + basal turn segment specimens from the DXM treated explants compared to the values obtained from these specimens from the naïve, untreated-control cultures (p < 0.01).
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Fig. 2 – Dexamethasone-treatment of naïve organ of Corti explants initiated a significant up regulation in the protein levels of phosphorylated-nuclear factor kappa B (p-NFκB). Naïve P3 organ of Corti explants were either exposed to or not exposed to DXM (70μg/ml) for 48 h and subsequently divided into two segments: (1) apical turns and (2) middle + basal turns. There was a statistically significant increase (p < 0.01) in p-NFκB protein in the middle + basal turn segments of DXM-treated explants compared to levels of this protein in the untreated naive controls. ELISA was used to determine protein levels of p-NFκB with data presented as mean fold change ± S.E.M. (represented by error bars); **p < 0.01; n = 12 explants/culture condition.
2.3.
Gene expression
Expression levels of genes that encode for one pro- and two anti-apoptotic factors as well as one pro-inflammatory factor were compared between DXM-treated and naïve, untreatedcontrol organ of Corti explants and these changes in expression levels are based upon real time RT-PCR results with normalization of results to 0 h excised control specimens and to β-actin (a housekeeping gene). There were no significant differences between DXM-treated and naïve, untreated-control organ of Corti explant expression levels for either Bcl-2 or Bcl-xl genes (Figs. 3A and B) after 24 h in vitro (p = 0.27 and p = 0.60, respectively). However, after 48 h in vitro a substantial up regulation of the expression levels of both the Bcl-2 and the Bcl-xl genes was observed compared to the expression levels for these genes in naïve, untreatedcontrol specimens at this time point (p < 0.001 for both genes). A significant down regulation in Bax gene expression was observed in DXM-treated cultures at both 24 and 48 h in vitro time points compared with the Bax expression levels observed in naïve, untreated-control explants (Fig. 4A). Mean fold change of Bax in the DXM-treated explants decreased as time in vitro progressed, i.e. from <0.8 mean fold reduction at 24 h to a reduction of <0.6 after 48 h in vitro (p = 0.002 and p < 0.01, respectively; Fig. 4A) compared to no significant change (p > 0.05) in the naïve, untreated control explant values which remained at 1.0. As seen in Fig. 4B, DXM-treated organ of Corti explants down regulated TNFR1 gene expression levels to a mean fold change of 0.8 when compared to naïve, untreated control explants (1.0) after 24 h in vitro (p < 0.01). Gene expression
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level changes of TNFR1 in the DXM-treated cultures were further reduced at the 48-h in vitro time point to a mean fold change of 0.6 which achieved a high level of significance compared to the values of the naïve, untreated control explants at that time point (Fig. 4B; p = 0.001). Modulation of both the Bcl-2 and the Bax genes by DXM-treatment were evident when the mean fold change of the Bax/Bcl-2 ratios were examined for explants treated with DXM and were compared to the results from naïve, untreated-control explants (Fig. 5). The DXMtreated cultures showed a significant decrease in the Bax/Bcl-2 ratio at both the 24-h and 48-h in vitro time points (p = 0.004 and p < 0.001, respectively) compared to the lack of any significant change that was observed for the naïve, untreated control organ of Corti specimens for both the 24- and 48-h time points.
Fig. 4 – Treatment of naïve organ of Corti explants with dexamethasone caused a significant down regulation of a pro-apoptosis related-gene and a pro-inflammatory-related gene. (A) Bax gene expression level was significantly down regulated in DXM-treated explants compared to expression levels of this gene in naïve, untreated-controls after 24 h in vitro (p = 0.002). There is a further decline in Bax expression in the DXM-treated cultures after 48 h in vitro (p < 0.01). (B) TNFR1 expression levels showed a statistically significant reduction in the DXM-treated explants at both 24 h (p < 0.01) and 48 h (p = 0.001) in vitro when compared to the gene expression levels of naïve, untreated-controls. Error bars represent mean fold change ± S.E.M. **p < 0.01; n = 10 explants/culture condition time point.
Fig. 3 – Treatment of naïve organ of Corti explants with dexamethasone caused a significant up regulation of two anti-apoptosis related-genes. (A) Bcl-2, there was no statistically significant difference in the levels of Bcl-2 gene expression between DXM-treated and naïve, untreated-control cultures after 24 h in vitro (p = 0.27), but expression levels of this gene were significantly increased at 48 h in vitro by DXM-treatment (p < 0.001). (B) Bcl-xl, no significant change was noted in Bcl-xl gene expression levels between the two culture conditions after 24 h in vitro (p = 0.60), however, there was a significant up regulation of Bcl-xl expression after 48 h of DXM-treatment, resulting in a mean fold change of greater than 2 (p<0.001). Error bars represent ±S.E.M.; ***p<0.001. (n =10 explants/culture condition time point).
3.
Discussion
There have been no reported adverse effects on residual hearing after local and/or systemic treatment of various inner ear diseases with corticosteroids (e.g. DXM) (Chandrasekhar, 2001; Himeno et al., 2002; Silverstein et al., 1996). Hill et al. (2008) reported that intratympanic administration of DXM to the middle ear cavity in mice resulted in significant protection against cisplatin-induced hearing loss in a frequencydependent manner and was without systemic side-effects. The results presented in the current paper show that both naïve and DXMb-treated organ of Corti explants possessed nearly identical patterns of auditory HC cell distribution after
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Fig. 5 – Dexamethasone treatment of naïve organ of Corti explants lowered the Bax/Bcl-2 ratio to favor cell survival. DXM treatment of naïve organ of Corti cultures caused a significant reduction in the mean fold change in the Bax/Bcl-2 ratio compared to this ratio in naïve, untreated-control explants at both the 24-h and 48-h time points. At 24 h, this Bax/Bcl-2 ratio is significantly reduced (p = 0.004) in the DXM-treated explants with an even more significant reduction in this ratio after 48 h of DXM-treatment (p < 0.001). Error bars represent ± S.E.M. of the mean fold change. **p < 0.01; n = 10 explants/culture condition time point.
4 days in vitro with no statistical difference in HC counts between these two culture conditions. The Bcl-2 family is comprised of proteins that either promote (e.g. Bax, Bid) or inhibit (e.g. Bcl-2, Bcl-xl) apoptosis (Youle and Strasser, 2008). TNFα has been shown to activate apoptosis of auditory HCs in organ of Corti (OC) explants by increasing Bax and TNFR1 expression levels and decreasing Bcl-xl and Bcl-2 expression levels (Dinh et al., 2008a, 2008b). The results from these same in vitro studies have demonstrated that simultaneous DXM-treatment of TNFα-challenged OC explants protects the explant's HCs from TNFα-induced cell death by reversing the TNFα-induced changes in gene expression. It was not known if DXM-treatment of naïve explants would parallel these changes in gene expression in the absence of a TNFα-challenge. The results reported in the present study support and extend the observations of DXM's otoprotective action of the modulation of the expression levels of apoptosisrelated genes showing that a damaged stimulus is not required for the initiation of these DXM-initiated changes in gene expression. Studies involving several different cell lines have implicated an elevated Bax/Bcl-2 ratio as a relative indicator of cell survival with an elevated ratio associated with apoptosis (Oltvai et al., 1993; Perlman et al., 1999; Wiren et al., 2006; John et al., 2007; Park et al., 2007). Treatment of the naïve explants with DXM in the present study resulted in a substantial decrease in the Bax/Bcl-2 ratio which would favor HC survival if these sensory cells were to be challenged by a trauma-related stimulus, e.g. cochlear implantation . DXM pretreatment has been shown to prevent apoptosis and cell injury as well as induce cell proliferation in a number of organ systems (Sasson
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and Amsterdam, 2003; Sun et al., 2008; Tenenbaum et al., 2008; Rafacho et al., 2009; Song et al., 2009). Pretreatment with DXM has been shown to suppress chemokines and inflammatory cell infiltration in the liver cells of animal models of liver damage by reducing the level of oxidative stress injury (Hsieh et al., 2006; Lee et al., 2006). The results of the present study show that DXM-treatment of naïve organ of Corti explants reduces the expression levels of Bax and TNFR1, i.e. known proapoptotic and pro-inflammatory genes respectively, which would reduce the cellular injury that can result from exposure to an excessive level of oxidative stress. Glucocorticoid receptor distribution in the cochlea is concentrated in the cells of the spiral ganglion, spiral ligament and are also present in the organ of Corti (Rarey and Curtis, 1996; Terunuma et al., 2003; Canlon et al., 2007). NFκB p65 is a transcription factor in the NFκB family that is highly expressed in spiral ganglion neurons of the cochlea and is activated in spiral ganglion cells following exposure to a traumatizing level of noise (Masuda et al., 2005; Tahera et al., 2006; Canlon et al., 2007). Blocking of NFκB with an inhibitor following acoustic overstimulation significantly increases the level of resultant hearing loss, demonstrating the role that this transcription factor plays as a protective response to limit noise-induced hearing loss (Tahera et al., 2006). In vitro studies in an auditory receptorderived cell line and in organ of Corti explants have also demonstrated that NFκB is protective against aminoglycosideinduced ototoxicity and promotes auditory HC health and survival in culture, respectively (Jiang et al., 2005; Nagy et al., 2005). DXM-treatment of naïve organ of Corti explants in the present study has been shown to increase p-NFκB levels in the middle-and-basal turn segments of the treated organ of Corti explants relative to the expression level of this transcription factor in naïve untreated-control explants. This increase in NFκB in the naïve OC explants may be important because a recent in vitro study has shown that blocking NFκB in TNFαchallenged OC explants prevents DXM-treatment from protecting the explant's HCs against TNFα-induced apoptosis (Haake et al., 2009). Local DXM pretreatment in various in vivo studies have shown significant protective effects on the preservation of hearing thresholds following both noiseinduced and cisplatin-induced traumas (Takemura et al., 2004; Tahera et al., 2006; Hill et al., 2008). It has also recently been demonstrated in vivo that short-term preoperative delivery of DXM via the round window membrane has a significant protective effect upon conservation of hearing in a guinea pig model of cochlear implantation (James et al., 2008; Chang et al., 2009). DXM has already been demonstrated to be a successful rescue therapy for sensory HCs in an inflammatory cytokine model of trauma-induced HC death in vitro (Dinh et al., 2008a; Haake et al., 2009). Based on the results of the present study, the gene and protein expression results suggest that DXM pretreatment can protect an OC explant's HCs by modulating expression levels of NFκB and of apoptosis-related genes to favor the survival of auditory HCs. Glucorticoid treatment of animals can also suppress chemokines expression and the inflammatory process (Hsieh et al., 2006) so these activities need to also be kept in mind when considering the action of DXM on the naïve OC explants. The results of the present study support local
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pretreatment of the cochlear duct in an animal model of cochlear implantation prior to a planned trauma (e.g. round window membrane dexamethasone prior to the insertion of an electrode array into the scala tympani; Chang et al., 2009) and suggest that the effective conservation of hearing against trauma-induced loss is due in least in part to DXM's protection of auditory hair cells.
4.4. ELISA-phosphorylated-nuclear factor kappa B (p-NFκB) p65 (Ser536)
All of the neonate rats utilized in this study were treated in accordance with the University of Miami Animal Care and Use Committee, study protocol #08-099 and in compliance with the published Guide for the Care and Use of Laboratory Animals of the National Institutes of Health No. 80-23. Organ of Corti (OC) explants were obtained from 3-day-old (P3) rats of the Sprague–Dawley strain of laboratory rats (Charles River Laboratories, Inc., Wilmington, MA, USA). Every effort was made to limit suffering and to keep the number of animals used in this study to a minimum.
Twenty-four OC explants were cultured under the same two conditions described previously for 48 h and then divided into two sections: (1) apical turns and (2) middle + basal turns. Two independent experiments were carried out. Six specimens sections from each group were pooled and respectively placed in 100 μl of chilled RIPA-buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Inc., Waltham, MA) and lysed with a Microson XL-2000 Sonifier (Misonix, Inc., Farmingdale, NY). Following centrifugation at 10,000×g for 10 min, the proteins present in the supernatants were quantified by the Bradford's method (Bio-Rad Laboratories, Hercules, CA). Subsequently, 30 μg of protein were placed in each well, with 4 repetitions of each sample. Phosphorylated nuclear factor kappaB p65 (Ser536) proteins (p-NFκB) were detected using the PathScan phospho-NFκB p65 (Ser536) Sandwich ELISA Kit (Cell Signaling, Inc., Danvers, MA) as described in the manufacturer's protocol and quantified at 450 nm wave length light using the ELx800 Absorbance Microplate Reader (Biotek, Inc., Winooski, VT). The p-NFκB protein is the activated form of NFκB.
4.2.
4.5.
4.
Experimental procedures
4.1.
Animals
Organ of Corti explants
One-hundred eighteen OC explants were dissected from the cochleae of sixty-nine 3-day-old (P-3) rats and placed in culture media consisting of modified Eagle's medium (Gibco, Grand Island Biologicals, Grand Island, NY, USA; www. invitrogen.com) enriched with a 1% concentration of N-1 supplement (100 × concentrate; Sigma-Aldrich, Saint Louis, MO, USA; www.sigma-aldrich.com) and glucose (adjust final conc. to 6 g/l). Explants were cultured at 37 °C in a 98% humidified atmosphere with 5% CO2 in room air. Explants were distributed into various sets for HC counts, gene expression studies and protein studies. Except for the HC counts, explants were divided into two groups and then cultured in either: (1) naïve, untreated-control; or (2) naïve, DXM-treated (70 μg/ml; Boston Scientific Corporation, Natick, MA, USA; www.bostonscientific.com) and harvested at specified time points (as listed below).
4.3.
Hair cell counts
Thirty-four OC explants were divided and cultured under the two conditions described above. After 4 days in vitro, they were then fixed, permeabilized and stained with fluorscein isothiocyanate (FITC)-labeled phalloidin. Explants were sequentially mounted on a slide with gelatin, cover slipped and examined under fluorescent illumination using a 40× lens (Zeiss Axiovert 200 microscope; Carl Zeiss Microscopy, Gottingen, Germany). Total HCs were counted for a 415-μm length of the basal segment of the basilar membrane of each explant and categorized as either inner HCs (IHC) or outer HCs (OHC). A countable HC was identified as one that possessed both an intact stereocilliary bundle and a cuticular plate. The investigator performing the HC counts was blinded to the identity of the stained explant specimens until all of the HC counts were completed.
Gene expression
Sixty OC explants were likewise cultured under the same two conditions for 0, 24 and 48 h in vitro (n = 10 explants/culture condition time point). Total cellular RNA was isolated using the Qiagen RNEasy® Protect Mini Kit (Qiagen Sciences, Inc., Germantown, MD, USA; www1.qiagen.com). The animal tissue protocol included in the kit was used with modifications: (1) stabilized tissue was disrupted in a 1.5-ml centrifuge tube with an RNase-free pestle; (2) tissue was homogenized with an RNase-free syringe; (3) DNase solution was added to degrade residual DNA and thus enhance RNA purity; and (4) before RNA extraction, RNA spin columns were centrifuged at full speed (14,000×g ) for 1 min to minimize contamination. cDNA synthesis was performed using the Bio-Rad iScript cDNA synthesis kit and its protocol (Bio-Rad Laboratories, Inc., Hercules, CA, USA; www.bio-rad.com). Samples were then placed in an Eppendorf Mastercycler® thermal cycler (Eppendorf Inc., Westbury, NY, USA, www.eppendorfna.com). Purified RNA was stored at −20 °C.
4.6.
Quantitative RT-PCR
Two-step real-time RT-PCR was completed with the following primers: β-actin (housekeeping gene), Bax, Bcl-xl, Bcl-2 and TNFR1; these were synthesized by Sigma-Genosys (SigmaGenosys, The Woodlands, TX, USA; www.sigma-genosys. com). Amplification products were detected using the MyiQ Single-Color Real-Time PCR Detection System from Bio-Rad and IQ SYBR Green Supermix. These following primer sequences were utilized: β-actin forward (fwd), 5′-CGTTGACATCCGTAAAGACC-3′; β-actin reverse (rev), 5′-AGCCACCAATCCACACAGAG-3′ (173 bp) (Nudel et al., 1983); TNFR1 fwd, 5′-GAACACCGTGTGTAACTGCC-3′; TNFR1 rev, 5′-ATTCCTTCACCCTCCACCTC-3′ (301 bp) (Raina and Jeejeebhoy, 2004); Bax fwd, 5′-CTGCAGAGGATGATTGCTGA-3′; Bax rev, 5′-GATC-
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AGCTCGGGCACTTTAG-3′ (174 bp); Bcl-2 fwd, 5′-GCTACGAGTGGGATACTGG-3′; Bcl-2 rev, 5′-GTGTGCAGATGCCGGTTCA-3′ (472 bp); Bcl-xl fwd, 5′-AGGATACAGCTGGAGTCAG-3′; and Bcl-xl rev, 5′-TCTCCTTGTCTACGCTTTCC-3′ (417 bp) (Valks et al., 2003). For primer TNFR1, real-time PCR was carried out for 40 cycles at 94 °C, 59 °C and 72 °C for 1 min each; β-actin, 94 °C, 55 °C and 72 °C for 1 min, 1 min, and 1.5 min, respectively; Bax, Bcl-2 and Bcl-xl, 95 °C, 61 °C and 72 °C for 50 s each. The 2−ΔΔCt method was applied to assess for relative changes in the mRNA levels of genes promoting or inhibiting apoptosis.
4.7.
Data analysis
One-way analysis of variance (ANOVA) was used to analyze HC counts. Analyses of the mean fold changes for both gene expression and protein expression studies were performed by the Kruskal–Wallis non-parametric test with a p value of < 0.05 considered statistically significant. Relative changes in mRNA expression are presented in the bar graphs as mean values ± S.E.M. Values were normalized to 0 h excised control explants and to β-actin, a housekeeping gene.
Acknowledgments Research supported by grants from Advanced Bionics Corporation, Valencia, CA and MED-EL, Medical Electronics Corporation, Innsbruck, Austria to TRV.
REFERENCES
Balkany, T.J., Hodges, A.V., Eshraghi, A.A., Butts, S., Bricker, K., Lingvai, J., Polak, M., King, J., 2002. Cochlear implants in children—a review. Acta Otolaryngol. 122, 356–362. Canlon, B., Meltser, I., Johansson, P., Tahera, Y., 2007. Glucocorticoid receptors modulate auditory sensitivity to acoustic trauma. Hear. Res. 226, 61–69. Chandrasekhar, S.S., 2001. Intratympanic dexamethasone for sudden sensorineural hearing loss: clinical and laboratory evaluation. Otol. Neurotol. 22, 18–23. Chang, A., Eastwood, H., Sly, D., James, D., Richardson, R., O'Leary, S., 2009. Factors influencing the efficacy of round window dexamethasone protection of residual hearing post-cochlear implant surgery. Hear. Res. 255, 67–72. Dinh, C.T., Haake, S., Chen, S., Hoang, K., Nong, E., Eshraghi, A.E., Balkany, T.J., Van De Water, T.R., 2008a. Dexamethasone protects organ of Corti explants against tumor necrosis factor-alpha-induced loss of auditory hair cells and alters the expression levels of apoptosis-related genes. Neuroscience 157, 405–413. Dinh, C., Hoang, K., Haake, S., Chen, S., Angeli, S., Nong, E., Eshraghi, A., Balkany, T.J., Van De Water, T.R., 2008b. Biopolymer-released dexamethasone prevents tumor necrosis factor α-induced loss of auditory hair cells in vitro: implications toward the development of a drug-eluting cochlear implant electrode array. Otol. Neurotol. 29, 1012–1019. Eshraghi, A.A., Adil, E., He, J., Graves, R., Balkany, T.J., Van De Water, T.R., 2007. Local dexamethasone therapy conserves hearing in an animal model of electrode insertion trauma-induced hearing loss. Otol. Neurotol. 28, 842–849. Garcia-Purrinos, F.J., Ferri, E., Rosell, A., Calvo, J., 2005. Combined intratympanic and intravenous dexamethasone to control vertigo in Meniere disease. Acta Otorinolaryngol. Esp. 56, 74–77.
7
Ghosh, A., Jackson, R., 2005. Best evidence topic report. Steroids in sudden sensorineural hearing loss. Emerg. Med. J. 22, 732–733. Gstoettner, W.K., Helbig, S., Maier, N., Kiefer, J., Radeloff, A., Adunka, O.F., 2006. Ipsilateral electric acoustic stimulation of the auditory system: results of long-term hearing preservation. Audiol. Neurotol. 11, 49–56. Haake, S., Dinh, C.T., Chen, S., Eshraghi, A.A., Van De Water, T.R., 2009. Dexamethasone protects auditory hair cells against TNFalpha-initiated apoptosis via activation of PI3K/Akt and NFkB signaling. Hear. Res. 255, 22–32. Haynes, B.F., Pikus, A., Kaiser-Kupfer, M., Fauci, A., 1981. Successful treatment of sudden hearing loss in Cogan's syndrome with glucocorticoids. Arthritis Rheum. 24, 501–503. Henry, K.R., 1992. Noise-induced auditory loss: influence of genotype, naloxone and methyl-prednisolone. Acta Otolaryngol. 112, 599–603. Hill, G.W., Morest, K., Parham, K., 2008. Cisplatin-induced ototoxicity: effect of intratympanic dexamethasone injections. Otol. Neurotol. 29, 1005–1011. Himeno, C., Komeda, M., Izumikawa, M., Takemura, K., Yagi, M., Weiping, Y., Doi, T., Kuriyama, H., Miller, J.M., Yamashita, T., 2002. Intra-cochlear administration of dexamethasone attenuates aminoglycoside ototoxicity in the guinea pig. Hear. Res. 167, 61–70. Hsieh, C.S., Wang, P.W., Lee, S.Y., Huang, C.C., Chang, N.K., Chen, C.M., Wu, C.L., Wang, H.C., Chuang, J.H., 2006. Glucocorticoid pretreatment suppresses chemokine expression and inflammatory cell infiltration in cholestatic rats receiving biliary intervention. J. Pediatric Surg. 41, 1669–1675. James, D.P., Eastwood, H., Richardson, R.T., O'Leary, S.J., 2008. Effects of round window dexamethasone on residual hearing in a guinea pig model of cochlear implantation. Audiol. Neurotol. 13, 86–96. Jiang, H., Sha, S.H., Schacht, J., 2005. NFκB pathway protects cochlear hair cells from aminoglycoside-induced ototoxicity. J. Neurol. Res. 79, 644–651. John, T., Muller, R.D., Oberholzer, A., Zreigat, H., Kohl, B., Ertel, W., Hostmann, A., Tschoeke, S.K., Schulze-Tanzil, G., 2007. Interleukin-10 modulates pro-apoptotic effects of TNF-alpha in human articular chondrocytes in vitro. Cytokine 40, 226–234. Kiefer, J., Gstoettner, W., Baumgartner, W., Pok, S.M., Tillein, J., Ye, Q., von Ilberg, C., 2004. Conservation of low-frequency hearing in cochlear implantation. Acta Otolaryngol 124, 272–280. Lee, C.W., Chuang, J.H., Wang, P.W., Chang, N.K., Wang, H.C., Huang, C.C., Tiao, M.M., Lo, S.K., 2006. Effect of glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with endotoxin cholangitis. World J. Surg. 30, 2217–2226. Masuda, M., Nagashima, R., Kanzaki, S., Fujioka, M., Ogita, K., Ogawa, K., 2005. Nuclear factor-kappa B nuclear translocation in the cochlea of mice following acoustic overstimulation. Brain Res. 88, 585–589. Nagy, I., Monge, A., Albinger-Hegyi, A., Schmid, S., Bodmer, D., 2005. NF-κB is required for survival of immature auditory hair cells in vitro. J. Assoc. Res. Otolaryngol. 6, 260–268. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z., Yaffe, D., 1983. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 11, 1759–1771. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Park, S.K., Choi, D., Russell, P., John, E.O., Jung, T.T., 2004. Protective effect of corticosteroid against the ototoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells. Laryngoscope 114, 768–771. Park, C., Moon, D.O., Rhu, C.H., Choi, B.T., Lee, W.H., Kim, G.-Y., Choi, Y.H., 2007. Beta-sitosterol induced anti-proliferation and apoptosis of human leukemic U937 cells through activation of caspase-3 and induction of Bax/Bcl-2 ratio. Biol. Pharm. Bull. 30, 1317–1323.
8
B RA IN RE S EAR CH 1 30 1 (2 0 0 9) 1–8
Perlman, H., Zhang, X., Chen, M.W., Walsh, K., Buttyan, R., 1999. An elevated Bax/Bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death Differ. 6, 48–54. Rafacho, A., Cestari, T.M., Taboga, S.R., Boschero, A.C., Bosqueiro, J.R., 2009. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am. J. Physiol. Endocrinol. Metab. 296, E681–689. Raina, N., Jeejeebhoy, K.N., 2004. Effect of low-protein diet and protein supplementation on the expressions of TNF-alpha, TNFR-1, and TNFR-II in organs and muscle of LPS-injected rats. Am. J. Physiol. Endocrinol. Metab. 286, E481–487. Rarey, K.E., Curtis, L.M., 1996. Receptors for glucocorticoids in the human inner ear. Otolaryngol. Head Neck Surg. 115, 38–41. Sasson, R., Amsterdam, A., 2003. Pleiotropic anti-apoptotic activity of glucocorticoids in ovarian follicular cells. Biochem. Pharmacol. 66, 1393–1401. Silverstein H., Choo D., Rosenberg S.I., Kuhn J., Seidman M., Stein I., 1996. Intratypanic steroid treatment of inner ear disease and tinnitus (preliminary report). Ear Nose Throat J. 75, 468-71,474,476. Song, I.H., Caplan, A.I., Dennis, J.E., 2009. In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo. J. Orthop. Res. 27, 916–921. Sun, J., Guo, W., Ben, Y., 2008. Preventive effects of curcumin and dexamethasone on lung transplantation-associated lung injury in rats. Crit. Care Med. 236, 1205–1213. Tabuchi, K., Oikawa, K., Murashita, H., Hoshino, T., Tsuji, S., Hara, A., 2006. Protective effects of corticosteroids on ischemia-reperfusion injury of outer hair cells. Laryngoscope 116, 627–629. Tahera, Y., Meltser, I., Johansson, P., Brian, Z., Stierna, P., Hansson, A.C., Canlon, B., 2006. NF-kappa B mediated glucocorticoid response in the inner ear after acoustic trauma. J. Neurosci. Res. 83, 1066–1076. Takemura, K., Komeda, M., Yagi, M., Chiemi, H., Izumikawa, M., Doi, T., Kuriyama, H., Miller, J.M., Yamashita, T., 2004. Direct
inner ear infusion of dexamethasone attenuates noise-induced trauma in guinea pig. Hear. Res. 196, 58–68. Tenenbaum, T., Matalon, D., Adam, R., Seibt, A., Wewer, C., Schwerk, C., Galla, H.J., Schroten, H., 2008. Dexamethasone prevents alteration of tight junction-associated proteins and barrier function in porcine choroid plexus epithelial cells after infection with Streptococcus suis in vitro. Brain Res. 1229, 1–17. Terunuma, T., Kawauchi, S., Kaji, M., Takahashi, S., Hara, A., 2003. Effect of acoustic stress on glucocorticoid receptor mRNA in the cochlea of the guinea pig. Brain Res. Mol. Brain Res. 120, 65–72. Valks, D.M., Kemp, T.J., Clerk, A., 2003. Regulation of Bcl-xl expression by H2O2 in cardiac myocytes. J. Biol. Chem. 278, 25542–25547. Vivero, R.J., Joseph, D.E., Angeli, S., He, J., Chen, S., Eshraghi, A.A., Balkany, T.J., Van De Water, T.R., 2008. Dexamethasone base conserves hearing from electrode trauma-induced hearing loss. Laryngoscope 118, 2028–2035. Wiren, K.M., Toombs, A.R., Semirale, A.A., Zhang, X., 2006. Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio. Bone 38, 637–651. Yildirim, A., Coban, L., Satar, B., Yetiser, S., Kunt, T., 2005. Effect of intratympanic dexamethasone on noise-induced temporary threshold shift. Laryngoscope 115, 1219–1222. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59. Zhou, Y., Zheng, H., Shen, X., Zhang, Q., Yang, M., 2008. Intratympanic administration of methyl prednisone reduces impact of experimental intensive impulse noise trauma on hearing. Acta Otolaryngol. 24, 1–6. Zou, J., Pyykkö, I., Sutinen, P., Toppila, E., 2005. Vibration induced hearing loss in guinea pig cochlea: expression of TNFα and VEGF. Hear. Res. 202, 13–20.