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Endocrinology Vol. 147, No. 9 4430-4437
Copyright © 2006 by The Endocrine Society

Glucocorticoid Receptor and Nuclear Factor-{kappa}B Interactions in Restraint Stress-Mediated Protection against Acoustic Trauma

Yeasmin Tahera1, Inna Meltser1, Peter Johansson1, Anita C. Hansson and Barbara Canlon

Department of Physiology and Pharmacology (Y.T., I.M., P.J., B.C.), Karolinska Institutet, 171 77 Stockholm, Sweden; and National Institute on Alcohol Abuse and Alcoholism (A.C.H.), National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Barbara Canlon, Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail: barbara.canlon{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of glucocorticoid receptors (GRs) in the protective effect of restraint stress (RS) before acoustic trauma was studied in spiral ganglion neurons of CBA mice. RS increased corticosterone and protected against elevated auditory brain stem thresholds caused by acoustic trauma. This protection was inhibited by the pretreatment with a corticosterone synthesis inhibitor, metyrapone (MET), and a GR antagonist (RU486). RS followed by acoustic trauma caused an immediate increase in corticosterone that triggered nuclear translocation of GR, without a change in the expression of GR protein. RU486 + MET before RS and acoustic trauma caused an immediate increase in GR mRNA followed by increased GR protein expression (24 h after trauma). GR signaling was further characterized by analyzing nuclear factor-{kappa}B (NF{kappa}B) nuclear translocation and protein expression. NF{kappa}B nuclear translocation was reduced after acoustic trauma or pretreatment with RU486 + MET before RS and acoustic trauma. On the contrary, RS protected against the trauma-induced NF{kappa}B reduction of its nuclear translocation in inhibitory-{kappa}B (I{kappa}B)-dependent manner. RU486 + MET caused a simultaneous decreased I{kappa}B expression and NF{kappa}B nuclear translocation, demonstrating an interference with the I{kappa}B-mediated activation of NF{kappa}B. In summary, RS protects the cochlea from acoustic trauma by increasing corticosterone and activating GRs. These results emphasis how GR activity modulates hearing sensitivity and its importance for the rationale use of glucocorticoids in inner ear diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE AUDITORY SYSTEM is particularly sensitive to physical and psychological stress (1, 2). The different stressors that affect the auditory system include restraint stress (RS), heat shock, and acoustic trauma. RS and acoustic trauma have been found to activate the hypothalamic-pituitary-adrenal axis resulting in the release of glucocorticoids (3, 4, 5, 6). The stress caused by RS or heat shock has been shown to protect against subsequent acoustic trauma (1, 7, 8). These studies have indirectly implied the involvement of glucocorticoid receptors in this protection. However, the direct activation and underlying mechanisms of glucocorticoid receptor (GR) signaling has not been demonstrated. The understanding of glucocorticoid signaling is of particular importance because of the widespread clinical use of steroids for treating hearing disorders (9, 10, 11, 12). There is clear evidence that sensitivity to glucocorticoid therapy demonstrates a large variation among individuals (13, 14). This variation to glucocorticoid therapy extends from no response to hypersensitive responses. Possible causes for this may include availability of receptors or ligand, ligand binding, or the alteration of GR signaling pathways.

One of the most promising targets for manipulating the sensitivity to glucocorticoid treatment is nuclear factor-{kappa}B (NF{kappa}B). This transcription factor is regulated directly or indirectly by glucocorticoids, depending on the tissue being investigated. NF{kappa}B has been shown as a proinflammatory factor in the immune system (15, 16). In the central nervous system, it has been demonstrated to protect against long-term depression (17), oxidative stress (18), and N-methyl-D-aspartate excitotoxicity (19). In the peripheral auditory system, NF{kappa}B has been shown to protect against aminoglycoside toxicity (20) as well as acoustic trauma (6).

Acoustic trauma alters the GR signaling pathways in the organ of Corti (21), spiral ligament (22), and spiral ganglion neurons (6), all of which have a relatively high expression of GR (23, 24, 25). Restraint stress has been shown to protect against the damaging effects of acoustic trauma and is most likely mediated by corticosterone. It is our working hypothesis that corticosterone-induced changes of GR and NF{kappa}B transcriptional activity leads to this protection. By pharmacologically interrupting corticosterone synthesis and blocking GR, we directly show the inversion of the effect of restraint stress on the physiological and biochemical consequences of acoustic trauma. To test our hypothesis, we analyzed the expression of GR and NF{kappa}B in spiral ganglion neurons after the combined treatment of restraint stress and acoustic trauma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
A total of 146 CBA male mice (B&K Universal AB, Sollentuna, Sweden), aged 10–12 wk (25–29 g) without any evidence of middle ear pathology were used in this study. Before the experimental start, the animals were allowed to acclimatize to the animal facility for at least 2 wk after delivery. The animals were housed in groups of five animals per cage on an artificial light/dark cycle (12/12 h, lights on at 0700 h), with free access to food and water. The cage was bedded with sawdust and contained environmental enrichment (a paper nest and shredded paper). The Ethical Committee at the Karolinska Institute approved the care and use of animals in this experiment.

Pharmacological manipulations
Animals were given pharmacological agents to deplete the function of corticosterone. The glucocorticoid synthesis inhibitor metyrapone [2-methyl-1, 2-di-3-pyridyl-1-propanone (MET)] (Sigma, St. Louis, MO) dissolved in deionized water was used in a dose of 200 mg/kg and injected ip. The glucocorticoid receptor antagonist RU486 (provided by Roussel Uclaf, Romain Ville, France) dissolved in vegetable oil was used in a dose of 100 mg/kg and injected sc. After several pilot studies, this dose was shown to give a distinct and reliable physiological response. Vehicle treatment consisted of deionized water injected ip, simulating MET injection, and vegetable oil injected sc, simulating RU486 injection. All injections were given in a constant volume of 0.1 ml each.

Experimental design
The animals were divided into five groups: 1) controls (n = 32) received the vehicle only (control); 2) RU486 + MET (n = 28); 3) vehicle and acoustic trauma (trauma, n = 20); 4) vehicle + RS followed by acoustic trauma (RS + trauma, n = 30); and 5) RU486 + MET followed by RS and acoustic trauma (RU486 + MET + RS + trauma, n = 28). Another eight animals were used to separately test the effect of MET and RU486 on restraint stress (n = 4 in each group).

One day before the experimental start, each animal was subjected to auditory brain stem response (ABR) measurements to establish thresholds. The experimental design is visualized in Fig. 1Go. On the day of the experiment, each animal received injections of either vehicle or drugs between 1000 and 1100 h to avoid the circadian variation of corticosterone (filled arrow, Fig. 1Go). After injection the animals were put back into their home cages for 1.5 h and then subjected to restraint stress for 4 h. The mice in the control group were placed in their home cage for 4 h. Blood samples were taken at the same time of day (empty arrows, Fig. 1Go). The samples were put in heparinized tubes, and plasma was separated by spinning at 6000 rpm at room temperature and then stored at –20 C. After blood sampling, the animals were subjected to the acoustic trauma for 45 min. Blood samples were collected immediately after and 2 h after acoustic trauma (empty arrows, Fig. 1Go) using the same procedure described above. Twenty-four hours after noise treatment, the ABRs were remeasured. For in situ hybridization, the animals were killed by cervical dislocation immediately after noise treatment and cochlea removed and prepared as described below. For Western blot another group of animals were killed both immediate after and 24 h after acoustic trauma.


Figure 1
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  		FIG. 1. Schematic diagram indicating the time of manipulation of the different experimental groups.

 
ABR
Auditory sensitivity was assessed with ABR thresholds as previously described (26) for the frequencies of 8, 12.5, 16, and 20 kHz after being anesthetized with an ip injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). The body temperature of the animals was maintained at 38 (0) C using light heating and cotton coverings. ABR thresholds were recorded with sc stainless-steel electrodes. The potential difference between an electrode on the vertex and an electrode on the mastoid was measured, whereas the lower back served as ground. The stimulus signals were generated through equipment controlled by computer and delivered by an electrostatic speaker (EC-1; Tucker-Davis Technologies, Gainesville, FL). The acoustic signal was delivered through the system inserted into the external auditory meatus. The evoked responses were amplified 100,000 times and averaged. The stimuli were presented at a repetition rate of 20/sec. At first the stimuli were presented at intensity well above threshold and then decreased in 10-dB steps until threshold was found and then in 5-dB steps until the ABR wave 1 disappeared. Threshold was defined as the lowest intensity at which a visible ABR wave was seen in two averaged runs.

Restraint protocol
Animals were placed into 50-ml conical tubes with ventilation holes made throughout the tube. The mice were placed into the tube and were not able to turn in any direction. The mice were maintained in these tubes for 4 h in a soundproof booth without any access to food or water.

Acoustic trauma
Animals were placed individually into small wire mesh cages (10 cm3) and then put inside an open field acoustic chamber (225 x 120 x 100 cm). During the exposure the animals did not have any access to food or water. Free-field broadband noise at 6–12 kHz was used for 45 min at intensity of 100 dB SPL. The stimulus was generated by a noise generator (model 33120 A; Hewlett Packard, Portland. OR), and delivered to the upper corners of with four microphones. Calibration of the sound-exposure levels was performed with a 12.5-mm condenser microphone (model 2213; Bruel and Kjaer, Stockholm, Sweden). The acoustic trauma used is this study caused a temporary change in auditory threshold shifts. This type of trauma does not lead to any morphological changes such as hair cell loss or degeneration of spiral ganglion neurons, and complete recovery of ABR thresholds occurs by 48 h.

Corticosterone assay
The serum corticosterone level was determined by corticosterone ELISA kit (Assay Designs Correlate-EIA, Assay Designs, Inc., catalog no. N 900-097). The assay sensitivity was 5 pg/ml.

Glucocorticoid receptor mRNA in situ hybridization
In situ hybridization was performed to examine whether GR mRNA expression changes in spiral ganglion locally. To avoid any RNA degradation, the bony shell around the cochlea was rapidly removed, and the sample was then frozen without being decalcified. Temporal bones were isolated and immediately dissected in ice-cold PBS. The bony wall surrounding the cochlea was removed, and the remaining soft tissue was attached to a piece of cork with Cryomount (HistoLab, Gothenburg, Sweden) and frozen in liquid isopentane (–40 C).

Sections (14 µm) were taken at –30 C, mounted on slides (Superfrost Slides Plus, Menzel GmbH, Frelburg, Germany), and stored at –80 C until fixation. RNA probe synthesis and in situ hybridization were performed as described before (27). The GR riboprobes, both sense and antisense, were generated from 673 bp EcoRI-PstI rat GR cDNA fragment (position 1691–2364 bp, corresponding to the 3'portion of the coding region), subcloned into the vector pSP64 (Promega, Madison, WI). The hybridized sections were exposed to BAS-5000 Phosphor imager tritium plates (Fuji, Tokyo, Japan). Phosphor imager-generated digital images were analyzed using AIS Image Analysis software (Imaging Research Inc., St. Catharines, Ontario, Canada). All slides were counterstained with cresyl violet, and measurements were made on sections that had a clear signal and, according to the cresyl violet staining, were in the middle part of cochlea showing all three turns. Image values were obtained from the whole section using the same square of the same size for all sections. Based on the known radioactivity in the 14C standards, image values were converted to nanocuries per gram after sense signals were subtracted from each value.

Tissue collection and protein isolation
Mice were killed by transcardiac perfusion with ice-cold PBS with heparin, and cochleae were immediately removed and placed into ice-cold solution containing Complete protease inhibitor (Roche Diagnostics, Mannheim, Germany), 1 mM dithiothreitol, and 1 mM Na3VO4. Cochleae were perfused through the round window, the bony shell was removed, and stria together with the tectorial membrane and organ of Corti was separated from the modiolus. Collected tissue was preserved in –70 C. Tissue was lysed for 40 min on ice in lysis buffer containing (in millimoles): 20 Tris-HCl (pH 7.0), 1 EDTA, 1 EGTA, 150 NaCl, 2,5 sodium pyrophosphate, 1 sodium glycerophosphate, 1 dithiothreitol, 1 Na3VO4, 1% (vol/vol) Triton X-100, and Complete protease inhibitor (Roche Diagnostics). The mixture was centrifuged at 15,000 x g for 5 min at 4 C to give lysates. All lysates were frozen and stored at –80 C until assayed. Protein concentration in lysates was determined according to Bradford method (28), using BSA as a standard.

Western blot analysis
Lysates containing 8–10 µg total protein per sample were resuspended in Laemmli sample buffer, heated for 5 min at 95 C, and separated by SDS-PAGE (29) using a 4% spacer and a 12% (wt/vol) separating polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) by semidry electroblotting. For detection, PVDF membranes blocked within 1 h in TBST [TBS (20 mM Tris, 150 mM NaCl (pH 7.6), containing 0.1% (vol/vol) Tween 20] supplemented with 5% nonfat dry milk before overnight incubation with the indicated primary antibodies in TBST. After three 15-min washing steps using TBST, the PVDF membranes were incubated for 1 h with peroxidase-conjugated antirabbit or antimouse IgG antibodies (Pierce, Rockford, IL). Thereafter, the PVDF membranes were washed three times for 10 min and two times for 1 min with TBST or TBS, respectively, to remove residual antibody. The PVDF membranes were developed with an enhanced chemiluminescence Western blot detection kit (Pierce SuperSignal West Dura) and exposed to Lumi-Film chemiluminescent detection films (Roche Diagnostics) for 20 sec to 20 min. To measure the relative density of immunoreactive bands, images were scanned and analyzed by Tina software (Raytest, Isotopen Messgeräte GmbH, Munich, Germany).

Antibodies used for Western blot were: anti-GR rabbit polyclonal, 2.5 µg/ml (catalog no. PA1–511A; Affinity Bioreagents, Golden, CO), anti-NF{kappa}B p65, 1:1,000 (catalog no. 3034; Cell Signaling Inc., Beverly, MA), antiinhibitory-{kappa}B (I{kappa}B)-{alpha} 1:1,000 (catalog no. 9242; Cell Signaling), and antiglyceraldehyde-3-phosphate dehydrogenase 1:10,000 (catalog no. ab9484; Abcam Ltd., Cambridge, UK).

Immunocytochemistry and quantification
Immediately after acoustic trauma, the animals were killed; cochleae were removed and perfused with 4% paraformaldehyde for 1 h. After fixation the cochleae were decalcified (2% EDTA in 0.5% paraformaldehyde) for 4 d and then placed in 10% sucrose for 12–24 h (4 C), followed by 20% sucrose for 24 h for cryoprotection. Specimens were quickly deep frozen and stored at –70 C. Serial midmodiolar sections (14 µm thickness) were cut on a cryostat (HM 500M, Zeiss, Göttingen, Germany) at –24 C. The sections were rinsed in PBS for 30 min before permeabilization with 0.3% Triton X-100 in PBS and then rinsed twice in PBS for 5 min each. Endogenous peroxidase was removed by using 3% H2O2 in methanol for 60 min. After rinsing twice in PBS for 5 min each, the slides were blocked with 1.5% goat serum in PBS for 30 min. Then polyclonal rabbit anti-GR antibody (catalog no. PA1-511A, Affinity Bioreagents) at a concentration of 5 µg/ml and NF{kappa}B (catalog no. SC 7151, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) at a concentration of 1 µg/ml were used on the specimens overnight in refrigerator. After several pilot studies, this concentration was found to be optimal for cochlear tissues. After rinsing twice in PBS, the secondary antibody, antirabbit-IgG (Vector ABC kit; Vector Laboratories, Inc., Burlingame, CA), was added followed by an immunoperoxidase reaction (VectaStain ABC kit and diaminobenzidine substrate kit; Vector). A negative control was treated exactly the same way except without the use of primary antibody.

Cochlear sections were analyzed in a Zeiss Axiovert microscope at x40 magnification. The total number of spiral ganglion neurons in the basal, middle, and apical turns was counted from six sections from each animal, and then the positively stained nuclei (darker than background level) were recorded. For the qualitative analysis of the immunoreactivity, images of the spiral ganglion neurons were obtained using fixed microscope settings (Image Pro Plus; Kannikestraede, Copenhagen, Denmark).

Statistical analysis
Data are presented as a mean values ± SEM. Then comparisons of means were performed with either a Student’s t test or a one-way ANOVA with Tukey correction. The software SigmaStat (version 2.03; Systat Inc., Richmond, CA) was used for statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RS protects against elevated auditory thresholds caused by acoustic trauma
The effect of RS after acoustic trauma on auditory thresholds was evaluated by measuring ABR threshold shifts. Posttrauma measurement revealed that RS reduced auditory threshold shifts in all frequencies, compared with the trauma group and the RU486 + MET + RS + trauma group (Fig. 2Go). The trauma group demonstrated a threshold shift between 21 and 30 dB in different frequencies when measured 24 h after trauma. RS significantly reduced threshold shifts by 8–19 dB across all frequencies, compared with the trauma group. Treatment with RU486 + MET before RS abolished this restraint-induced reduction of the threshold shift. In this group the threshold shifts ranged between 13 and 33 dB across all frequencies. This increase is significant in all frequencies, compared with RS + trauma group except for frequency 8 kHz. There was no significant difference between the trauma group and the RU486 + MET + RS + trauma group (Fig. 2Go). No effect of RU486 + MET were found on preexposure ABR thresholds, compared with the control group (data not shown).


Figure 2
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  		FIG. 2. Auditory brainstem response threshold shift values 24 h after acoustic trauma. The RS + trauma group (shaded bar, n = 9) showed significantly lower threshold shifts in all frequencies, compared with trauma group (open bar, n = 6). RU486 + MET treatment before RS (black bar, n = 6) caused a threshold shift elevation that was similar to the trauma group, except at 8 kHz. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

 
RS elevates plasma corticosterone
After 1.5 h vehicle injection (control), the corticosterone values were 90 ± 5 ng/ml, and after RU486 + MET, the value was 30 ± 6 ng/ml (Fig. 3Go), as previously shown (6). The effect of the pharmacological treatment resulted in a significant decrease in circulating corticosterone levels at this time point. We also determined corticosterone levels at 5.5 h after injection because this is the time-matched control for the RS group (injection is 1.5 h before the 4-h RS). At 5.5 h after injection, the control was 28 ± 5 ng/ml, showing the return to the basal levels. In contrast, 5.5 h after RU486 + MET treatment, corticosterone was elevated up to 135 ± 20 mg/ml. This increase is most likely due to the long-lasting effect of RU486 (90 h), compared with that of MET (4 h). In a previous study (30), we have shown that RS alone causes a significant (6.3 times) corticosterone elevation (176 ± 21 ng/ml), compared with a time-matched control (5.5 h). The effect of RS in the RU486 + MET-treated group also resulted in a corticosterone increase to 212 ± 32 ng/ml. Thus, in the RU486 + MET group, RS increased corticosterone only 1.6 times, compared with without RS.


Figure 3
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  		FIG. 3. Left, Plasma corticosterone measured from the control at 1.5 h injection (striped bar, n = 9) showed 9-fold increase of corticosterone over the basal level (horizontal line). RU486 + MET significantly decreased the level at this time point (hatched bar, n = 7). After 5.5 h of injection, the vehicle-injected control returns almost to basal line, but RU486 + MET group showed significant elevation. When RS was applied, both groups showed an elevation of corticosterone level (n = 7 in each group; right). Immediately after acoustic trauma, the trauma group (white bar, n = 6) showed an elevation of corticosterone; however, the level was significantly higher when RS was applied before trauma with (n = 6) or without RU486 + MET (n = 9). By 2 h after acoustic trauma, the corticosterone levels returned to near basal levels. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

 
We determined the separate effect of RU486 or MET followed by RS on corticosterone concentration. When MET was combined with RS, the corticosterone was elevated up to 115 ± 27 ng/ml. This value is significantly lower than the combined drug treatment (RU486 + MET) followed by RS. When RU486 was combined with RS, the corticosterone was elevated up to 480 ± 28 ng/ml.

The immediate post- (0 h) effects of acoustic trauma (trauma) caused an elevation of corticosterone (96.1 ± 21.4 ng/ml). Further increases in corticosterone were found after RS + trauma (242 ± 37 ng/ml). Pretreatment with RU486 + MET followed by RS caused elevated corticosterone immediately after trauma (275 ± 35 ng/ml). By 2 h after acoustic trauma, corticosterone returned to basal levels in all groups (Fig. 3Go).

RS increases GR nuclear translocation in the spiral ganglion neurons
It is well accepted that the first step of corticosterone-mediated activation of GR is their translocation into the nucleus (31). We evaluated nuclear translocation of GR immediate after trauma to determine whether corticosterone activates GR in the spiral ganglia neurons. Nuclear translocation was evaluated on cochlear sections immunostained with anti-GR antibodies. The basal level of neurons with GR translocated into the nuclei was approximately one third of the total spiral ganglia neurons in the control group (Fig. 4Go). RU486 + MET (alone) had no effect on GR nuclear translocation, as shown previously (30). The trauma group showed 29% of neurons with GR-positive nuclei, which was not different from the control group (Fig. 4Go). RS followed by trauma significantly increased nuclear translocation (45%), compared with the trauma without RS. Only 20% of spiral ganglia neurons showed nuclear translocation in the RU486 + MET + RS + trauma group, and this reduction was significant, compared with the RS + trauma group (Fig. 4Go). Representative photomicrographs taken from the base of the cochlea shows GR nuclear translocation. After RS + trauma, the number of dark nuclei increased, whereas after the RU486 + MET + RS + trauma treatment, there was a marked decrease, compared with the RS + trauma group (Fig. 4Go).


Figure 4
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  		FIG. 4. Quantification of GR nuclear translocation in the spiral ganglion neurons. Nuclear-stained neurons are presented as percent of total spiral ganglion neurons. RU486 + MET or acoustic trauma did not show any significant difference with control. RS + trauma group (n = 3) demonstrated a significantly higher number of nuclear-translocated GRs, compared with trauma (n = 3). RU486 + MET + RS + trauma group (n = 5) showed a reduced number, compared with the RS + trauma group. *, P < 0.05 by Student’s t test. Representative photomicrograph taken from the base of the cochlea illustrating the GR nuclear translocation in the spiral ganglia neurons. Six sections from each cochlea were analyzed. RS + trauma group demonstrates a greater number of darkly stained nuclei, an effect that was reduced after RU486 + MET + RS + trauma.

 
Inhibition of GR signaling increases its mRNA and protein expression in the spiral ganglion neurons
To determine whether the GR nuclear translocation after RS + trauma (with or without RU486 + MET) is altering GR transcription, we evaluated GR mRNA expression in the spiral ganglia neurons immediately after trauma. Quantification of in situ hybridization showed a moderate GR mRNA expression in the control group, and RS alone caused a slight decrease (data not shown). RU 486 + MET (alone) caused an elevation (2.75 ± 0.11 nCi/g) of GR mRNA expression, compared with the control (2.35 ± 0.08 nCi/g). Treatment with RU486 + MET before RS significantly elevated the GR mRNA expression immediately after trauma, compared with the RS + trauma (Fig. 5AGo). GR mRNA expression was detected on cochlear sections by in situ hybridization with 35S-labeled oligoprobes. Representative radioimages of cochlear sections immediately after acoustic trauma are shown (Fig. 5Go, B and C). The RU486 + MET + RS + trauma group show an increased signal, compared with the RS + trauma group. A low-magnification micrograph of a cresyl violet-stained cochlear section (i.e. without decalcification, as described in Materials and Methods) shows that the in situ samples contained primarily auditory nerve and spiral ganglion bundles (Fig. 5DGo). The arrows in this figure are pointing to the spiral ganglion neurons. To help with orientation, a micrograph showing an intact cochlear section is shown (Fig. 5EGo) at the same magnification as Fig. 5DGo. In this figure the auditory nerve is through the middle of the section, and the spiral ganglion neurons are shown with arrows.


Figure 5
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  		FIG. 5. In situ hybridization of cochlear glucocorticoid receptor mRNA immediately after acoustic trauma. A, Quantification showed a statistically significant elevation of GR mRNA in the spiral ganglia neurons in RU486 + MET + RS + trauma group, compared with RS + trauma group (n = 4) *, P < 0.05 by Student’s t test. B and C, Photomicrograph showing GR mRNA expression in cochlear sections. The corresponding cresyl violet-stained section through modiolus (D) with arrows showing the spiral ganglion neurons. A micrograph showing an intact cochlear section is shown for orientation purposes (Fig. 5EGo). In these figures (D and E), the auditory nerve is through the middle of the section, and the spiral ganglion neurons are shown with arrows.

 
To determine whether GR mRNA changes affect GR protein expression, modiolus protein extract was analyzed by Western blot (Fig. 6Go). We previously found that RU486 + MET has no effect on GR protein expression 5.5 h after injection (30), compared with the time-matched control (Fig. 6Go, before trauma). Immediately after trauma, there was no difference in GR protein expression between the RS + trauma and RU486 + MET + RS + trauma groups (Fig. 6Go, RS + trauma, 0 h after). Quantification of the OD of bands showing GR protein expression 24 h after acoustic trauma was also made. At this time point, a significant elevation was found in the RU486 + MET + RS + trauma group, compared with the RS + trauma group (Fig. 6Go, RS + trauma, 24 h after).


Figure 6
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  		FIG. 6. Western blot analysis of GR protein expression. No difference between the control (n = 9) and the RU486 + MET (alone) (n = 9) were found at the pretrauma time point (5.5 h after injection) or when determined 0 h after trauma. Representative bands of GR before trauma (upper left panel) and 0 h after trauma (middle left panel). Right panel shows loading controls. Representative bands of GR Western blot 24 h after acoustic trauma (lower left panel). Quantitative analysis revealed a statistically significant elevation of GR protein in the RU486 + MET + RS group 24 h after acoustic trauma, compared with RS group (n = 9). **, P < 0.01 by Student’s t test.

 
RS prevents I{kappa}B-dependent down-regulation of NF{kappa}B nuclear translocation in the spiral ganglia neurons
To evaluate whether GR activation affects NF{kappa}B nuclear expression (32), we analyzed p65 subunit nuclear translocation immediate after trauma by immunocytochemistry. In the control group, the relative number of neurons with NF{kappa}B p65 translocated into the nuclei was approximately 56% of the total spiral ganglia neurons (Fig. 7AGo). RU486 + MET (alone) significantly decreased the nuclear translocation of NF{kappa}B (32%), compared with the control (30). Immediately after acoustic trauma, a significant decrease of NF{kappa}B nuclear translocation was found (31%), compared with the control group. RS prevented the trauma-induced decrease in NF{kappa}B nuclear translocation (RS + trauma). RU486 + MET treatment abolished the effect of RS (RS + trauma) on NF{kappa}B nuclear translocation (Fig. 7AGo). Representative photomicrographs taking from the basal turn of cochlea illustrate NF{kappa}B immunoreactivity in the spiral ganglia neurons (Fig. 7BGo). The photomicrographs show that RS + trauma increased NF{kappa}B nuclear translocation as demonstrated by the vast number of spiral ganglia neurons with darkly stained nuclei (Fig. 7BGo). On the contrary, RU486 + MET treatment decreased NF{kappa}B nuclear translocation after RS + trauma (Fig. 7BGo).


Figure 7
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  		FIG. 7. A, Quantification of NF{kappa}B nuclear translocation in the spiral ganglion neurons. Values are presented as percent nuclear stained neurons of the total spiral ganglion neurons. RU486 + MET or acoustic trauma significantly decreased the nuclear translocation of NF{kappa}B in the spiral ganglion neurons, compared with control. However, a significantly higher number was found in the RS + trauma group, compared with trauma. RU486 + MET + RS + trauma group (n = 5) showed a significant lower number, compared with RS + trauma group (n = 3). *, P < 0.05; **, P < 0.01 by Student’s t test. B, Representative photomicrograph taken from the base of the cochlea illustrating the NF{kappa}B nuclear translocation in the spiral ganglia neurons.

 
To evaluate whether the NF{kappa}B nuclear translocation is related to its protein expression, Western blot analysis was performed on protein extracted from the modiolus. We previously found that RU486 + MET (alone) decreased NF{kappa}B expression (1.45 ± 0.07 x 103 OD units) (30), compared with the control (1.9 ± 0.02 x 103 OD units) at 5.5 h after injection. The RU486 + MET + RS + trauma showed elevated NF{kappa}B protein expression, compared all other groups (Fig. 8AGo). The trauma or RS + trauma groups did not show any significant changes, compared with the control group (Fig. 8AGo) in the expression of NF{kappa}B. These data indicate that the NF{kappa}B nuclear translocation (Fig. 7Go) is not directly related to NF{kappa}B expression in spiral ganglion neurons.


Figure 8
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  		FIG. 8. Western blot analysis of NF{kappa}B and I{kappa}B protein expression. A, Quantitative analysis of NF{kappa}B protein revealed a statistically significant elevation in the RU486 + MET + RS + trauma group immediately after, compared with all other groups. B, The significant elevation of I{kappa}B expression was found in trauma group, compared with all other groups. RU486 + MET treatment resulted in a decrease of I{kappa}B expression in RU486 + MET only group, compared with control. I{kappa}B expression was also down-regulated by RU486 + MET treatment after RS + trauma (RU486 + MET + RS + trauma vs. RS + trauma). *, P < 0.05, **, P < 0.01, ***, 0.001 by Tukey test. Representative bands of loading controls, NF{kappa}B, and I{kappa}B. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

 
To determine whether the observed NF{kappa}B nuclear translocation is regulated through the I{kappa}B-dependent pathway, I{kappa}B {alpha} protein expression was analyzed by Western blot. RU486 + MET (alone) had a significant decrease of I{kappa}B expression (0.63 ± 0.01 x 103 OD units) (30), compared with the control (1.68 ± 0.04 x 103 OD units) at 5.5 h after injection. Immediately after acoustic trauma, I{kappa}B protein was significantly increased in the trauma group, compared with all other groups (Fig. 8BGo). Here we also found that the RS + trauma group did not differ from the control group, as was found for NF{kappa}B nuclear translocation shown in Fig. 7Go. The RU486 + MET + RS + trauma group showed decreased I{kappa}B expression, compared with the RS + trauma group.

Summary of results
A summary of the findings on how restraint stress regulates GR signaling in the spiral ganglion neurons is shown (Table 1Go). The different groups presented in this table are in relation to the control group.


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  		TABLE 1. RS-induced regulation of GR signaling (0 h after acoustic trauma)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Restraint stress was found to protect the functional response of the cochlea from a subsequent acoustic trauma by activating GR and by preventing the trauma-induced down-regulation of NF{kappa}B. The restraint-induced GR activation, initiated by elevated corticosterone, resulted in decreased auditory brain stem thresholds. The combined use of the GC synthesis inhibitor (MET) and receptor antagonist (RU486) before RS abolished the protective effect of restraint on hearing. The combined use of RU486 and MET has previously been shown to have physiological effect in other systems (33). Corticosterone elevation immediately after acoustic trauma was the trigger for the subsequent nuclear translocation of GR in the spiral ganglion neurons. An increase of GR mRNA transcription and the subsequent increase of GR protein expression after 24 h are direct evidence of GR inactivation by RU486 + MET followed by RS and acoustic trauma. The maximal changes in GR protein expression have previously been found at 18–24 h after glucocorticoid administration (34), implying an autoregulation of GR (35). The elevation of GR mRNA by RU486 administration seen here has been previously described in the spiral ganglion neurons (6) and the hippocampus (36). However, RU486 + MET without subsequent challenge (RS or acoustic trauma) did not affect GR activity (nuclear translocation) or protein expression, implying that interrupting GR signaling prevents the RS-induced activation.

GR is known to regulate other transcription factors such as activator protein-1 and NF{kappa}B in a tissue-specific manner (37). The cascade of NF{kappa}B-mediated gene regulation starts with its nuclear translocation. In our study we found significantly lower nuclear translocation of NF{kappa}B immediately after acoustic trauma and RU486 + MET treatment with or without after RS and trauma. Although RS combined with acoustic trauma did not alter the nuclear transport of NF{kappa}B, it prevented trauma-induced decrease of NF{kappa}B translocation into the nuclei. Previously we showed an activation of NF{kappa}B in spiral ganglia neurons at 4 h after acoustic trauma (6). The experimental paradigms in these two studies differed in both the treatment and time points in which the tissue was collected. Thus, NF{kappa}B nuclear translocation in spiral ganglia neurons is sensitive to the time point at which it is analyzed.

I{kappa}B is the main regulator of NF{kappa}B activity (38). We were therefore interested to know whether I{kappa}B is involved in the regulation of NF{kappa}B activity after acoustic trauma with or without RS pretreatment. The up-regulation of I{kappa}B expression after acoustic trauma coincided with a decreased NF{kappa}B nuclear translocation. These results indicate that the I{kappa}B-dependent NF{kappa}B activation occurs in spiral ganglion neurons after acoustic trauma. GR nuclear translocation was not affected by acoustic overexposure, indicating that NF{kappa}B activity is not under direct GR control at this time point. After RS + trauma, no change in I{kappa}B expression or NF{kappa}B nuclear translocation was found when GR was activated. Thus, GR activation by restraint prevents the NF{kappa}B down-regulation immediately after acoustic trauma, demonstrating a positive correlation between GR and NF{kappa}B nuclear translocation. It has been previously demonstrated that simultaneous activation of GR and NF{kappa}B enables them to directly interact with each other, resulting in an increase in their nuclear translocation (32), and RU486 impairs this interaction (39). Because only the RS + trauma group showed activation of both GR and NF{kappa}B, we hypothesized that this interaction can be the basis for the RS-induced protection of hearing.

In the auditory system, RU486 + MET treatment leads to the simultaneous down-regulation of I{kappa}B expression and NF{kappa}B nuclear translocation, suggesting a disruption in the I{kappa}B-regulated NF{kappa}B pathway. However, significantly elevated NF{kappa}B protein expression was found when RS and trauma was applied in presence of RU486 + MET. This finding indicates that NF{kappa}B protein expression is elevated by acoustic trauma when GRs are inactivated. On the other hand, we cannot exclude the direct interaction of RU486 with NF{kappa}B independently of GR because it has been shown in an in vitro study (40). It has been shown previously that glucocorticoid-mediated I{kappa}B synthesis and NF{kappa}B antagonism could be independent events and tissue or cell type specific (41). On the other hand, the decreased I{kappa}B expression in the presence of a GR antagonist in spiral ganglion neurons could be the consequence of the positive regulation of I{kappa}B synthesis by GR, as has been shown in some tissues (42), or increase of its degradation. All these findings suggest that GR regulates the activity of NF{kappa}B in the spiral ganglion neurons in a manner that is complex and time dependent. Finally, further studies are needed to evaluate the GR coregulatory proteins and other interacting factors between GR and NF{kappa}B. It has been shown that NF{kappa}B activation protects neurons by the up-regulation of neurotrophic factors, such as brain-derived neurotrophic factor and neurotrophic factor 3 (19, 43, 44, 45). These neurotrophic factors are present in the cochlea and have been reported to provide protection against acoustic trauma as well as ototoxic agents (46, 47).

In conclusion, this study establishes a relationship between the protective effects of RS, GRs, and NF{kappa}B in the cochlea after acoustic trauma. There are several implications for these findings with regard to general issues concerning acoustic trauma. The first is that changes in the level of plasma corticosterone alters GR sensitivity and modulates the degree of hearing loss induced by acoustic trauma. Such information would be useful for targeting GR and its downstream pathways for the therapeutic intervention in the cochlea against acoustic trauma. The second implication is that the availability of GR plays a role in the overall sensitivity to acoustic trauma. This is important because it is well known that there is a large interindividual variability in noise-induced hearing loss. Our results emphasize how the availability and activity of GRs can modulate hearing sensitivity. These findings help elucidate the mechanisms underlying GR function and are therefore important for the rational use of glucocorticoids in inner ear diseases.


   Acknowledgments
 
We acknowledge Pontus Stierna for providing RU486.


   Footnotes
 
This work was supported by grants from the Swedish Research Council, AMF Trygghetsförsäkring, Tysta Skolan, Royal National Institute for Deaf People, and the Karolinska Institute.

Disclosure Summary: Y.T., I.M., P.J., A.C.H., and B.C. have nothing to declare.

First Published Online June 15, 2006

1 Y.T., I.M., and P.J. contributed equally to this work Back

Abbreviations: ABR, Auditory brain stem response; GR, glucocorticoid receptor; I{kappa}B, inhibitory-{kappa}B; MET, metyrapone; NF{kappa}B, nuclear factor-{kappa}B; PVDF, polyvinylidene difluoride; RS, restraint stress; TBST, TBS and Tween 20.

Received February 27, 2006.

Accepted for publication June 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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