The Laryngoscope C 2015 The American Laryngological, V
Rhinological and Otological Society, Inc.
Dexamethasone Prevents Hearing Loss by Restoring Glucocorticoid Receptor Expression in the Guinea Pig Cochlea Ulf-R€ udiger Heinrich, PhD; Sebastian Strieth, MD; Irene Schmidtmann, PhD; Roland Stauber, PhD; Kai Helling, MD Objectives/Hypothesis: Dexamethasone is widely used in the treatment of various inner ear diseases. However, knowledge about its direct impact on glucocorticoid receptor (GR) expression is still limited. Study Design: Prospective animal study in male guinea pigs. Methods: A therapeutic concentration of dexamethasone (8mg/mL) or a physiological concentration of NaCl (0.9% solution) were intratympanically injected into the ears of guinea pigs (n 5 10 in each case) 14 hours prior to 90 dB noise exposure (1 hour). Eighteen ears were exposed to noise only. Seven untreated ears were used as controls. Auditory brainstem responses were recorded prior to noise exposure or treatment and 2 hours thereafter. The cochleae were removed from the bullae, transferred to fixative, and embedded in paraffin. GR expression was identified immunohistochemically in the cochlea. Local staining intensities were quantified for seven regions by a computer. Results: Dexamethasone application significantly lowered noise-induced hearing loss. Statistically significant alterations in the average GR expression levels were identified exclusively in the spiral ligament. Comparing GR expression at the level of individual ear, numerous highly significant local associations were identified in the other six cochlear regions. Conclusions: The intratympanic application of dexamethasone is suitable for supporting cochlear homeostasis under stress conditions. The lateral wall, mainly responsible for potassium recycling, seems to be the main target in glucocorticoid therapy. Key Words: Glucocorticoids, cochlea, noise exposure, hearing impairment. Level of Evidence: NA Laryngoscope, 126:E29–E34, 2016
INTRODUCTION Glucocorticoids (GC), including the synthetic dexamethasone (DEX), are routinely used in the treatment of various inner ear pathologies such as sudden sensorineural hearing loss, acute tinnitus, or Menie`re’s disease.1,2 In addition, the protective effect of DEX against noise-induced hearing loss was demonstrated in experimental animal models and in human therapy.3,4 The success of GC therapy is primarily based on its influence on cellular regulators of cochlear homeostasis, especially on the glucocorticoid receptors (GRs). The GRs can act in two different ways, in a genomic and a nongenomic way. In the classical genomic pathways, GCs bind to GRs in the cytoplasm and induce a conformation change within the GRs, allowing their
From the Department of Otorhinolaryngology, Head and Neck Surgery (U.-R.H., S.S., R.S., K.H.) and Institute for Medical Statistics, Epidemiology and Informatics (I.S.), University Medical Center of the Johannes Gutenberg–University, Mainz, Germany. Editor’s Note: This Manuscript was accepted for publication March 30, 2015. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Kai Helling, MD, Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg–University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail: [email protected] DOI: 10.1002/lary.25345
Laryngoscope 126: January 2016
release from the heat shock protein 90 complex. Thereafter, GRs are translocated to the nucleus and bind to specific DNA sequences. This will lead either to stimulation or alternatively to inhibition of target gene transcripts.1 In the nongenomic pathway, the mechanisms are faster. It is assumed that DEX acts on GRs attached to membranes or localized within the cytosol. For the inner ear, it was recently postulated that these mechanisms are responsible for the anti-inflammatory actions of GCs.1,5 In the human inner ear, the highest concentration of GRs was measured by enzyme-linked immunosorbent assay in the spiral ligament.6 This finding was confirmed for rat inner ear tissues using the same analytical approach.7 Immunohistochemical investigations of murine inner ears revealed the strongest GR expression in the type III fibrocytes of the spiral ligament.8 In the rat animal model, strong immunostaining intensities for GR were detected in the stria vascularis and in outer and inner hair cells.5 However, in contrast to the studies described above, Terakado et al. reported reduced GR staining intensities in the spiral ligament, the spiral limbus, the spiral ganglion cells, the cochlear nerve, and Reissner’s membrane.5 In summary, based on the widespread distribution of GR in the vertebrate inner ear, cell type-specific GC-dependent processes must be expected, especially in connection with noise-induced hearing loss. Heinrich et al.: Cochlear Glucocorticoid Receptor Expression
E29
TABLE I. Experimental Groups. Applied Substance
Duration of Substance Impact
Noise Exposure
Analyzed Ears Immunohistochemically
Analyzed Ears by ABR
none
0 hours
0 dB
7
7
DEX NaCl
14 hours 14 hours
90 dB, 1 hour 90 dB, 1 hour
6 6
10 10
none
0 hours
90 dB, 1 hour
4
18
ABR 5 auditory brainstem response; DEX 5 dexamethasone.
DEX-dependent effects were preferentially observed within culture systems. A noise-induced downregulation of GR mRNA was found in the dissected lateral wall of the guinea pig cochlea using semiquantitative reverse transcription polymerase chain reaction (PCR).9 In addition, a systemic DEX treatment resulted in the upregulation of GR mRNA and GR protein expression in the cochlear soft tissues, including the basilar membrane and the lateral wall, using quantitative real-time PCR and Western blots.10 To obtain more information about the correlation between hearing loss and GR expression after intratympanic application of DEX in an animal model, the present study was performed. The findings are discussed in respect to the protective effects of intratympanic DEX application.
MATERIALS AND METHODS Animals All experiments were conducted in accordance with the German Prevention of Cruelty to Animals Act and were approved by the supervising authorities. In total, 45 healthy guinea pigs weighing 200 to 400 g with good Preyer‘s reflexes and no evidence of middle ear disease were used for the study (2-week-old pigmented guinea pigs, (BFA strain, established by the former Bundesforschungsanstalt, T€ ubingen, Germany), Charles River Laboratories, Sulzfeld, Germany). Animals were kept on a 12:12hour light:dark cycle in the university’s animal facility. The guinea pigs were anaesthetized with intraperitoneal injections of esketamin hydrochloride (Ketanest; Pfizer, Karlsruhe, Germany; 175 mg/ kg body weight) and xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany; 10 mg/kg body weight) before recording the acoustic-evoked potentials or before any substance application.
application and 2 hours later. The stimulation, calibration of noise exposure, and data management were coordinated using the Nicolet Spirit system (Nicolet Biomedical, Madison, WI) as described recently.11,12
Fixation, Embedding Procedure, and Immunohistochemistry Two hours after noise stimulation, the animals were sacrificed, The cochleae of the left ear were immediately removed from the bullae and opened at the base and the apex for fixation by a solution that consisted of 0.2 % picric acid, 4 % paraformaldehyde, and 0.1 % glutardialdehyde for one day at 48C. The control ears and the ears exposed by noise at 90 dB were treated in the same way. After decalcification with ethylenediaminetetraacetic acid for 3 weeks at 48C, the cochleae were dehydrated by an increasing ethanol series followed by xylene. Specimens were embedded in paraffin. Using a microtome (Leica RM 2165; Leica Microsystems GmbH, Wetzlar, Germany), 3-mm sections were prepared, mounted onto Superfrost glass slides, and deparaffinized by xylene and a decreasing alcohol series. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2/methanol. After preincubation with 10% normal serum and 1% bovine albumin in phosphate-buffered saline (PBS) for 20 minutes to avoid unspecific binding, primary antibodies (anti-GC receptor antibody, rabbit polyclonal antibody raised against a peptide mapping at the C-terminus of GR a of human origin, GR [p-29]: sc-1002, diluted 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were overlaid overnight at 48C. Slides were consecutively incubated with biotinylated secondary antibody (1:250; Dako, Hamburg, Germany) for 30 minutes, streptavidin peroxidase (1:200; Dako) for 30 minutes, and finally DAB/H2O2 (1.85 mM) for 1 minute. All washing procedures were performed in PBS; dilutions of antibodies were prepared in 1% bovine albumin in PBS at room temperature. For all stainings, negative controls were included (i.e., no primary antibody). Counterstaining of the section was performed by hemalaune/PBS for 1 minute.
Intratympanic Application Under microscopic control, 0.1 mL of DEX (8 mg/mL Fortecortin; Merck, Darmstadt, Germany) or 0.9% saline solution (NaCl) was injected through the anterior parts of the tympanic membrane into the middle ear (10 ears in each case) 14 hours prior to noise exposure. Twenty-five animals received no injection, eighteen were exposed to 90 dB without any additional substance application, and seven animals were used as controls. The guinea pigs were randomly assigned to the different experimental groups (Table I).
Auditory Brainstem Response Auditory brainstem responses (ABRs) were recorded prior to noise exposure and 2 hours afterward or prior to substance
Laryngoscope 126: January 2016
E30
Quantification of Immunocytochemical Staining Images were taken from paraffin sections using a color video camera (Sony 3-CCD DXC-390P; Sony Corp., Tokyo, Japan) connected to a Zeiss microscope (Axiovert 200; Carl Zeiss Microscopy GmbH, Jena, Germany). All images of a given experimental group of animals whose immunoreactions were to be compared were opened in Photoshop (version 7; Adobe Systems, San Jose, CA) and stored as a single image file. Areas with the same color (tolerance level of 8) were selected with the cursor of the Magic Wand tool and quantified using the Histogram command from the image menu. The resulting staining intensities were multiplied with the number of immunostained pixels (representing the area of immunostaining) and expressed
Heinrich et al.: Cochlear Glucocorticoid Receptor Expression
indication of differences between the 90-dB group and the NaCl-treated group (P 5.2380).
Cellular Differences in Basal GR Expression
Fig. 1. Hearing loss is shown in decibels sound pressure level (mean 6 standard deviation) by the determination of auditory brainstem responses in noise-exposed ears only and after application of NaCl or dexamethasone (DEX) given 14 hours prior to sound stimulation (**P
Rhinological and Otological Society, Inc.
Dexamethasone Prevents Hearing Loss by Restoring Glucocorticoid Receptor Expression in the Guinea Pig Cochlea Ulf-R€ udiger Heinrich, PhD; Sebastian Strieth, MD; Irene Schmidtmann, PhD; Roland Stauber, PhD; Kai Helling, MD Objectives/Hypothesis: Dexamethasone is widely used in the treatment of various inner ear diseases. However, knowledge about its direct impact on glucocorticoid receptor (GR) expression is still limited. Study Design: Prospective animal study in male guinea pigs. Methods: A therapeutic concentration of dexamethasone (8mg/mL) or a physiological concentration of NaCl (0.9% solution) were intratympanically injected into the ears of guinea pigs (n 5 10 in each case) 14 hours prior to 90 dB noise exposure (1 hour). Eighteen ears were exposed to noise only. Seven untreated ears were used as controls. Auditory brainstem responses were recorded prior to noise exposure or treatment and 2 hours thereafter. The cochleae were removed from the bullae, transferred to fixative, and embedded in paraffin. GR expression was identified immunohistochemically in the cochlea. Local staining intensities were quantified for seven regions by a computer. Results: Dexamethasone application significantly lowered noise-induced hearing loss. Statistically significant alterations in the average GR expression levels were identified exclusively in the spiral ligament. Comparing GR expression at the level of individual ear, numerous highly significant local associations were identified in the other six cochlear regions. Conclusions: The intratympanic application of dexamethasone is suitable for supporting cochlear homeostasis under stress conditions. The lateral wall, mainly responsible for potassium recycling, seems to be the main target in glucocorticoid therapy. Key Words: Glucocorticoids, cochlea, noise exposure, hearing impairment. Level of Evidence: NA Laryngoscope, 126:E29–E34, 2016
INTRODUCTION Glucocorticoids (GC), including the synthetic dexamethasone (DEX), are routinely used in the treatment of various inner ear pathologies such as sudden sensorineural hearing loss, acute tinnitus, or Menie`re’s disease.1,2 In addition, the protective effect of DEX against noise-induced hearing loss was demonstrated in experimental animal models and in human therapy.3,4 The success of GC therapy is primarily based on its influence on cellular regulators of cochlear homeostasis, especially on the glucocorticoid receptors (GRs). The GRs can act in two different ways, in a genomic and a nongenomic way. In the classical genomic pathways, GCs bind to GRs in the cytoplasm and induce a conformation change within the GRs, allowing their
From the Department of Otorhinolaryngology, Head and Neck Surgery (U.-R.H., S.S., R.S., K.H.) and Institute for Medical Statistics, Epidemiology and Informatics (I.S.), University Medical Center of the Johannes Gutenberg–University, Mainz, Germany. Editor’s Note: This Manuscript was accepted for publication March 30, 2015. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Kai Helling, MD, Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg–University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail: [email protected] DOI: 10.1002/lary.25345
Laryngoscope 126: January 2016
release from the heat shock protein 90 complex. Thereafter, GRs are translocated to the nucleus and bind to specific DNA sequences. This will lead either to stimulation or alternatively to inhibition of target gene transcripts.1 In the nongenomic pathway, the mechanisms are faster. It is assumed that DEX acts on GRs attached to membranes or localized within the cytosol. For the inner ear, it was recently postulated that these mechanisms are responsible for the anti-inflammatory actions of GCs.1,5 In the human inner ear, the highest concentration of GRs was measured by enzyme-linked immunosorbent assay in the spiral ligament.6 This finding was confirmed for rat inner ear tissues using the same analytical approach.7 Immunohistochemical investigations of murine inner ears revealed the strongest GR expression in the type III fibrocytes of the spiral ligament.8 In the rat animal model, strong immunostaining intensities for GR were detected in the stria vascularis and in outer and inner hair cells.5 However, in contrast to the studies described above, Terakado et al. reported reduced GR staining intensities in the spiral ligament, the spiral limbus, the spiral ganglion cells, the cochlear nerve, and Reissner’s membrane.5 In summary, based on the widespread distribution of GR in the vertebrate inner ear, cell type-specific GC-dependent processes must be expected, especially in connection with noise-induced hearing loss. Heinrich et al.: Cochlear Glucocorticoid Receptor Expression
E29
TABLE I. Experimental Groups. Applied Substance
Duration of Substance Impact
Noise Exposure
Analyzed Ears Immunohistochemically
Analyzed Ears by ABR
none
0 hours
0 dB
7
7
DEX NaCl
14 hours 14 hours
90 dB, 1 hour 90 dB, 1 hour
6 6
10 10
none
0 hours
90 dB, 1 hour
4
18
ABR 5 auditory brainstem response; DEX 5 dexamethasone.
DEX-dependent effects were preferentially observed within culture systems. A noise-induced downregulation of GR mRNA was found in the dissected lateral wall of the guinea pig cochlea using semiquantitative reverse transcription polymerase chain reaction (PCR).9 In addition, a systemic DEX treatment resulted in the upregulation of GR mRNA and GR protein expression in the cochlear soft tissues, including the basilar membrane and the lateral wall, using quantitative real-time PCR and Western blots.10 To obtain more information about the correlation between hearing loss and GR expression after intratympanic application of DEX in an animal model, the present study was performed. The findings are discussed in respect to the protective effects of intratympanic DEX application.
MATERIALS AND METHODS Animals All experiments were conducted in accordance with the German Prevention of Cruelty to Animals Act and were approved by the supervising authorities. In total, 45 healthy guinea pigs weighing 200 to 400 g with good Preyer‘s reflexes and no evidence of middle ear disease were used for the study (2-week-old pigmented guinea pigs, (BFA strain, established by the former Bundesforschungsanstalt, T€ ubingen, Germany), Charles River Laboratories, Sulzfeld, Germany). Animals were kept on a 12:12hour light:dark cycle in the university’s animal facility. The guinea pigs were anaesthetized with intraperitoneal injections of esketamin hydrochloride (Ketanest; Pfizer, Karlsruhe, Germany; 175 mg/ kg body weight) and xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany; 10 mg/kg body weight) before recording the acoustic-evoked potentials or before any substance application.
application and 2 hours later. The stimulation, calibration of noise exposure, and data management were coordinated using the Nicolet Spirit system (Nicolet Biomedical, Madison, WI) as described recently.11,12
Fixation, Embedding Procedure, and Immunohistochemistry Two hours after noise stimulation, the animals were sacrificed, The cochleae of the left ear were immediately removed from the bullae and opened at the base and the apex for fixation by a solution that consisted of 0.2 % picric acid, 4 % paraformaldehyde, and 0.1 % glutardialdehyde for one day at 48C. The control ears and the ears exposed by noise at 90 dB were treated in the same way. After decalcification with ethylenediaminetetraacetic acid for 3 weeks at 48C, the cochleae were dehydrated by an increasing ethanol series followed by xylene. Specimens were embedded in paraffin. Using a microtome (Leica RM 2165; Leica Microsystems GmbH, Wetzlar, Germany), 3-mm sections were prepared, mounted onto Superfrost glass slides, and deparaffinized by xylene and a decreasing alcohol series. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2/methanol. After preincubation with 10% normal serum and 1% bovine albumin in phosphate-buffered saline (PBS) for 20 minutes to avoid unspecific binding, primary antibodies (anti-GC receptor antibody, rabbit polyclonal antibody raised against a peptide mapping at the C-terminus of GR a of human origin, GR [p-29]: sc-1002, diluted 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were overlaid overnight at 48C. Slides were consecutively incubated with biotinylated secondary antibody (1:250; Dako, Hamburg, Germany) for 30 minutes, streptavidin peroxidase (1:200; Dako) for 30 minutes, and finally DAB/H2O2 (1.85 mM) for 1 minute. All washing procedures were performed in PBS; dilutions of antibodies were prepared in 1% bovine albumin in PBS at room temperature. For all stainings, negative controls were included (i.e., no primary antibody). Counterstaining of the section was performed by hemalaune/PBS for 1 minute.
Intratympanic Application Under microscopic control, 0.1 mL of DEX (8 mg/mL Fortecortin; Merck, Darmstadt, Germany) or 0.9% saline solution (NaCl) was injected through the anterior parts of the tympanic membrane into the middle ear (10 ears in each case) 14 hours prior to noise exposure. Twenty-five animals received no injection, eighteen were exposed to 90 dB without any additional substance application, and seven animals were used as controls. The guinea pigs were randomly assigned to the different experimental groups (Table I).
Auditory Brainstem Response Auditory brainstem responses (ABRs) were recorded prior to noise exposure and 2 hours afterward or prior to substance
Laryngoscope 126: January 2016
E30
Quantification of Immunocytochemical Staining Images were taken from paraffin sections using a color video camera (Sony 3-CCD DXC-390P; Sony Corp., Tokyo, Japan) connected to a Zeiss microscope (Axiovert 200; Carl Zeiss Microscopy GmbH, Jena, Germany). All images of a given experimental group of animals whose immunoreactions were to be compared were opened in Photoshop (version 7; Adobe Systems, San Jose, CA) and stored as a single image file. Areas with the same color (tolerance level of 8) were selected with the cursor of the Magic Wand tool and quantified using the Histogram command from the image menu. The resulting staining intensities were multiplied with the number of immunostained pixels (representing the area of immunostaining) and expressed
Heinrich et al.: Cochlear Glucocorticoid Receptor Expression
indication of differences between the 90-dB group and the NaCl-treated group (P 5.2380).
Cellular Differences in Basal GR Expression
Fig. 1. Hearing loss is shown in decibels sound pressure level (mean 6 standard deviation) by the determination of auditory brainstem responses in noise-exposed ears only and after application of NaCl or dexamethasone (DEX) given 14 hours prior to sound stimulation (**P
