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Gentamicin alters Akt-expression and its activation in the guinea pig cochlea
Neuroscience 311 (2015) 490–498
GENTAMICIN ALTERS Akt-EXPRESSION AND ITS ACTIVATION IN THE GUINEA PIG COCHLEA U.-R. HEINRICH, a S. STRIETH, a I. SCHMIDTMANN, b H. LI c AND K. HELLING a*
Key words: hearing loss, immunohistochemistry, inner ear, organ of Corti, cytoprotection.
a
Department of Otolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg – University Mainz, Germany b Institute for Medical Statistics, Epidemiology and Informatics (IMBEI), University Medical Center of the Johannes Gutenberg – University Mainz, Germany
INTRODUCTION Intratympanic application of gentamicin is a successful medical approach in the treatment of vertigo spells in Me´nie`re’s disease (Blakley, 2000; Lange et al., 2004; Helling et al., 2007). However, in addition to its therapeutic effects in eliminating the function of vestibular hair cells, gentamicin also induced unwanted morphological damage and physiological alterations in the cochlea (Hotta et al., 1998; Aran et al., 1999; Heinrich et al., 2006). When analyzing the underlying gentamicininduced biochemical mechanisms, both apoptotic cascades and protective pathways were identified in the cochlea (Chung et al., 2006; Rybak and Ramkumar, 2007; Caelers et al., 2010). After gentamicin entrance into the outer hair cells through the mechano-electrical transducer channels, the formation of an aminoglycoside-iron complex particularly increases cellular reactive oxygen species (ROS)-production (Rybak and Ramkumar, 2007), In addition, the activation of small GTPases, such as Ras and Rho/Rac/Cdc42, as well as the c-Jun-N-terminal kinase (JNK) was demonstrated. These are the main players in pre-programed cell death, which leads finally to apoptosis (Wang et al., 2003; Chung et al., 2006; Jeong et al., 2010). Currently, different experimental approaches to otoprotection which focus on upstream and downstream biochemical methods are under investigation (Rybak and Whitworth, 2005; Schacht et al., 2012) On the one hand, it has to be mentioned that the expression of endothelial nitric oxide synthase (eNOS) was increased after the application of gentamicin in the reticular lamina, i.e. at the apical side of the organ of Corti (Heinrich et al., 2006). Furthermore, when organ cultures were analyzed, an up-regulation of nitric oxide (NO) production was identified in the lateral wall (Heinrich et al., 2008). Both processes are said to contribute to gentamicin-induced cochlear damage. On the other hand, it was also demonstrated that gentamicin is able to promote hair cell survival through the H-Ras/Raf/MEK/Erk and the phosphatidylinositol-3 kinase (PI3K) pathways via its downstream targets, protein kinase C (PKC) and Akt (Battaglia et al., 2003; Chung et al., 2006). Based on these findings, the activation state of Akt was declared as an indicator of
c
Department of Pharmacology, University Medical Center of the Johannes Gutenberg – University Mainz, Germany
Abstract—Gentamicin treatment induces hair cell death or survival in the inner ear. Besides the well-known toxic effects, the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway was found to be involved in cell protection. After gentamicin application, the spatiotemporal expression patterns of Akt and its activated form (p-Akt) were determined in male guinea pigs. A single dose of 0.1 mL gentamicin (4 mg/ear/animal) was intratympanically injected. The auditory brainstem responses (ABRs) were recorded prior to application and 1, 2 and 7 days afterward. At these three time points the cochleae (n = 10 in each case) were removed, transferred to fixative and embedded in paraffin. Seven ears were used as untreated controls. Gentamicin, Akt and p-Akt were identified immunohistochemically in various regions of the cochlea and their staining intensities were quantified on sections using digital image analysis. The application of gentamicin resulted in hearing loss with a concomitant up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells and an additional activation in spiral ganglion cells. At the level of individual ears, clear intracellular correlations were found between Akt- and p-Akt-expression in the stria vascularis and interdental cells and, to a minor extent, in the spiral ligament and the organ of Corti. Furthermore, statistical evidence for the connection between gentamicin up-take and hearing loss was detected. The increase in Akt- and p-Akt-expression in the organ of Corti and spiral ganglion cells indicates a selected response of the cochlea against gentamicin toxicity. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg – University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany. Tel: +49-6131-177361; fax: +49-6131176637. E-mail address: [email protected] (K. Helling). Abbreviations: ABR, auditory brainstem response; AU, arbitrary units; JNK, c-Jun-N-terminal kinase; PI3K, phosphatidylinositol-3 kinase; ROS, reactive oxygen species; SD, standard deviation. http://dx.doi.org/10.1016/j.neuroscience.2015.10.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 490
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the extension of the PI3K survival pathway (Caelers et al., 2010). Physiological stimuli are able to induce Akt-kinase activity by up to 40-fold (Datta et al., 1999). Full activation of Akt requires its translocation from the cytoplasm to the cell membrane and its phosphorylation at the two residues, at its catalytic domain (Thr-308), and at the C terminus (Ser-473). Afterward, activated Akt, i.e. p-Akt, can detach from the plasma membrane and is able to phosphorylate substrates in the cytosol and nucleus. Then the activity of Akt can influence different cell pathways, such as the two main components of the intrinsic cell- death machinery, the pro-apoptotic protein BAD (member of the Bcl-family) and caspases. Their actions are inhibited in the cytoplasm after phosphorylation by Akt. Akt can inhibit transcription factors of the forkhead family in the nucleus, thus avoiding the initiation of apoptosis. Furthermore, Akt phosphorylates IjB kinase (IKK) in the cytoplasm, which in turn activates the anti-apoptotic transcription factor NFjb in the nucleus (Rathmell et al., 2003). It was also shown that Akt promotes glycolytic metabolism (Van der Wheehle et al., 2004), stimulates the transcription of glucose transporter genes and the translocation of glucose transporters and additional nutrient transporters to the plasma membrane (Rathmell et al., 2003). All these Akt-regulated pathways control important processes of cell survival and energy balance. Generally, it is assumed that, along its downstream pathways, Akt phosphorylates more than one substrate at a time, so several effects may occur in parallel (Kim et al., 2001). In the un-stimulated cochlea, Akt was identified in all cell types of the organ of Corti, in the stria vascularis and spiral ligament as well as in the interdental cells of the limbus, in the spiral ganglion cell and in the nerve fibers of the osseous spiral lamina (Selivanova et al., 2007). In addition, p-Akt was localized in the organ of Corti, spiral ganglion cells and lateral wall in guinea pigs (Hess et al., 2006; Selivanova et al., 2007). After moderate noise exposure for one hour, a fast down-regulation of Akt and p-Akt was detected in numerous cochlear regions (Selivanova et al., 2007). With respect to its widespread distribution in the mammalian cochlea and its fast response to external stimuli resulting in the activation of survival pathways, Akt has received expanded attention as an important anti-apoptotic protein. In order to obtain more information about the spatiotemporal alterations in Akt-expression after gentamicin application, the expression levels of Akt and p-Akt were quantified one, two and seven days after the application of gentamicin by immunohistochemistry. In addition, the ratio between Akt and p-Akt-expression was determined for the different cochlear regions in order to identify intracellular and intercellular correlations. The findings are discussed with respect to possible survival processes in the cochlea.
EXPERIMENTAL PROCEDURES Subjects Thirty-seven pigmented guinea pigs (tricolor, Charles River, Sulzfeld, Germany) weighing 200–250 g with
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good Preyer’s reflexes and no evidence of middle-ear disease were used in this study. Young animals were used in order to minimize age-related effects or hormonal fluctuations that occur in older animals. All experiments were conducted in accordance with the German Prevention of Cruelty to Animals Act and were approved by the supervising authorities. Animals were kept on a 12:12-h light:dark cycle in our local animal facility at University Medical Center, Mainz. Auditory brainstem responses (ABRs) Before recording the acoustic evoked potentials, the guinea pigs were anesthetized with intraperitoneal injections of esketamin hydrochloride (Ketanest; 175 mg/kg body weight) and xylazine hydrochloride (Rompun; 10 mg/kg body weight). Subdermal needle electrodes (Biomedical, Madison, WI, USA) were placed at the scalp vertex (inverting), the right mastoid (non-inverting) and lower back (ground) to record ABRs. ABRs were recorded in a sound-attenuated room after click stimuli (duration 100 s, 300 averaged stimuli) beginning from 90 dB (SPL) to 10 dB (SPL). The stimulus presentation and data management were coordinated using the Spirit-evoked potential system (Biomedical, Madison, WI, USA). The hearing threshold was determined by discrimination of wave Jewett I, III, and V. ABRs were determined before the injection of gentamicin and on the first, second and seventh day after application. Gentamicin application After anesthesia, a volume of 0.1 mL (4 mg/ear/animal) gentamicin (Ratiopharm, Ulm, Germany) was injected through the anterior region of the tympanic membrane into both ears of the experimental animals (n = 30) under microscopic control. After gentamicin treatment, 10 animals were killed after one day, 10 animals after two days and 10 animals after seven days. Seven untreated animals served as controls. Cochlea preparation Animals were killed by pentobarbital – sodium (NarcorenÒ, Hallbergmoos, Germany; 448 mg/kg body weight) on the first, second and seventh day after gentamicin injection. Both bullae were completely removed and transferred into a solution that consisted of 0.2% picric acid, 4% para-formaldehyde and 0.1% glutardialdehyde. After decalcification with EDTA (20% solution adjusted to pH 7.4) for three weeks at 4 °C, the cochleae were dehydrated by an increasing ethanol series followed by xylene. Specimens were embedded in paraffin. Five-lm sections were prepared using a microtome (Leica RM 2165) mounted onto superfrost glass slides and deparaffinated by xylene and a decreasing alcohol series. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2/methanol. After pre-incubation with 10% normal serum and 1% bovine serum albumin in PBS for 20 min to avoid unspecific binding, the primary rabbit polyclonal anti-Akt
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antibody (Cell Signaling #9272, Danvers, MA, USA, diluted 1:100), rabbit polyclonal anti-p-Akt (Cell Signaling #9275, phosphorylated at Thr308, Danvers, MA, USA, diluted 1:100) and the mouse polyclonal antigentamicin antibody (Fitzgerald Industries, MA, USA, diluted 1:450), respectively, were overlaid overnight at 4 °C. Slides were then consecutively incubated with biotinylated secondary antibody (1:250, DAKO, Hamburg, Germany) for 30 min, streptavidin peroxidase (1:200, DAKO) for 30 min and finally Diaminobenzidine/ H2O2 (1.85 mM) for 1 min. All washing procedures were performed in PBS; dilutions of antibodies were prepared in PBS containing 1% bovine serum albumin at room temperature. For negative controls, the primary antibodies were omitted. All assessments were performed blinded with respect to treatments. Quantification of immunocytochemical staining Paraffin sections were analyzed in detail in six cochlear regions: nerve fibers, organ of Corti, spiral ganglion cells, stria vascularis, spiral ligament and interdental cells of the Limbus (Fig. 5a). Images were taken from the sections using a high-density 1/300 type, three-chip Exwave HAD CCD red-green-blue (RGB) color video camera (Sony 3-CCD DXC-390P) connected to a ZEISS microscope (Axiovert 200) equipped with a halogen light source using a 40x objective. The illumination intensity of the specimens was assured by precise voltage control. The CCD camera settings (gain and sensitivity) were kept constant for all analyses. All images were analyzed in Photoshop (version 7; Adobe Systems, San Jose, CA, USA) and stored in a single file. In all regions, the immunostaining intensities were determined as described recently (Heinrich et al., 2010). In brief, corresponding images of control specimens and images of gentamicin-treated animals were stored in one single image file to compare the different sample levels. Areas with the same brown intensities (tolerance level 15 of the computer program) were selected with the Magic Wand tool and quantified using the Histogram command from the Image menu. Firstly, the background
staining values (values without any cellular structure) were subtracted from the immunostaining intensities, resulting in the ‘‘net staining intensity”. Secondly, the areas of the stained tissue (given in pixel) were compared to the size of the whole analyzed section (given in pixel) and determined as a ‘‘percentage of the immunostained area”. The ‘‘net staining intensity” was multiplied by the ‘‘percentage of the immunostained area” and is then expressed as arbitrary units (AU) for the different cell types. In order to minimize insufficient data quantification, we analyzed a couple of sections from each ear. As not all slides could be antibody-stained in one pass, slides of various experimental groups were mixed during each labeling procedure (Heinrich et al., 2010). Nevertheless, the repetition of labeling for each ear resulted in comparable values with respect to cellular staining intensities. Generally, the experimental approach can be considered as semi-quantitative, allowing a relative grading of staining intensities. Statistical approaches Alterations in Akt and pAkt-immunostaining intensity were analyzed both in controls and after the application of gentamicin using a linear mixed model. In this model, gentamicin, cochlea turn and the interaction between gentamicin and cochlea turn were considered as fixed effects. In addition, a random animal effect was included in the model, thereby taking repeated measurements on each animal into account. We adjusted for multiple comparisons within each cell type and each parameter (Akt, pAkt, Akt–pAkt-ratio, hearing threshold, gentamicin concentration) by using the Tukey–Kramer method. The mean values and standard deviation (SD) are presented in the graphs below and the adjusted p-values are indicated by one, two or three asterisks representing p < 0.05, p < 0.01 and p < 0.001, respectively. Statistical analysis was performed using PROC MIXED from SAS 9.3.
RESULTS ABRs ABRs were recorded prior to and one, two and seven days after gentamicin application (Fig. 1). Gentamicin induced a mean hearing threshold shift of 11.57 ± 10.7 dB after one day, of 18.5 ± 15.7 dB after two days, and of 20.9 ± 21.9 dB after seven days compared with the individual pre-treatment hearing levels. Hearing thresholds were worse at every time point after gentamicin application compared with pre-treatment levels [p = .0214 (control vs. day 1), p < .001 (control vs. day 2 and 7)]. There were no statistical differences between the 1st and the 2nd day (p = .075), between the 1st and 7th day (p = .2209) and between the 2nd and the 7th day (p = .9550).
Fig. 1. Hearing thresholds measured by the determination of ABRs (ten animals per group) prior to and one, two, and seven days after the application of gentamicin in dB (SPL) (mean ± SD). (*p < 0.05; *** p < 0.001).
Localization of Akt At the light microscopic level, a distinct Akt-immunoreaction was identified in un-treated animals
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in all cell types of the organ of Corti (Fig. 2a), in spiral ganglion cells (Fig. 2c), in the stria vascularis and in the spiral ligament (not shown). The reaction was detectable in the cytoplasm and in the cell nuclei (Fig. 2a, c). Seven days after gentamicin application, the staining intensity was much stronger in all cells of the organ of Corti (Fig. 2b) and in the spiral ganglion cells (Fig. 2d). The intensity of the Akt immune reaction in the stria vascularis and spiral ligament remained unaltered at all time points comparable to controls (not shown). Localization of p-Akt Slight staining intensities of p-Akt were identified by light microscopic analysis in the untreated organ of Corti
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(Fig. 3a). After gentamicin application, an increased immunoreaction was found only in two cochlear cell types, the Deiters’ cells and pillar cells (Fig. 3b). In spiral ganglion cells, a clearly increased staining intensity was seen after seven days (Fig. 3d) compared with the moderate staining reaction in untreated controls (Fig. 3c). Localization of gentamicin Intense widespread gentamicin immunostaining intensities were identified by microscopic analysis in the cochlea at the three time points after application. In the organ of Corti, gentamicin was found to be located within supporting cells as well as in inner and outer hair
Fig. 2. Examples of Akt immunostaining intensities in the cochlea of the guinea pig in the second cochlear turn. In untreated animals, a basal Akt immunoreaction was found in all regions of the organ of Corti (a). Seven days after the application of gentamicin, the staining intensity of Akt was clearly increased (b). In spiral ganglion cells, a faint staining of Akt was visible (c) which was more intense seven days after gentamicin treatment (d). DC Deiters’ cells, HE Hensen cells, IHC inner hair cells, OHC outer hair cells, PC pillar cells; Scale bar = 30 lm.
Fig. 3. p-Akt specific staining intensities in the second cochlear turn. In controls, a basal faint p-Akt immunoreaction was found the organ of Corti (a). Seven days after the application of gentamicin, the staining intensity of p-Akt was clearly increased, especially in Deiters’ cells and pillar cells (b). In spiral ganglion cells, a slight p-Akt immunoreaction was visible (c) which was more intense seven days after gentamicin treatment (d). DC Deiters’ cells, PC pillar cells; Scale bar = 30 lm.
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Fig. 4. Gentamicin specific staining intensities in the second cochlear turn. In the organ of Corti, gentamicin was identified in supporting cells and in inner and outer hair cells (a). In the lateral wall, a clear gentamicin immunoreaction was seen in the spiral ligament, whereas only a faint reaction was found in the stria vascularis (b). IHC inner hair cell, OHC outer hair cells, SL spiral ligament, SV stria vascularis; Scale bar = 30 lm.
cells (Fig. 4a). Within the lateral wall, clear staining reactions were seen in the spiral ligament, whereas the stria vascularis was nearly free from gentamicin (Fig. 4b). Basal Akt-expression and activation In order to judge the importance of the alterations in Akt- and p-Akt-expression in context with their spatiotemporal distribution patterns, the basal cellular expression levels were determined by quantitative immunohistochemistry for the six analyzed regions. All staining intensities are expressed in AU, mentioning mean values and standard deviation (mean ± SD). For a better understanding of the inner ear anatomy, the areas and important cell types are presented in a corresponding graph (Fig. 5a). In control ears, the highest Akt-expression was found in the stria vascularis (4659 ± 2608 AU; Fig. 5b). A clear expression was also seen in the interdental cells (2456 ± 1158 AU) and the spiral ligament (2463 ± 1554 AU). Low expression levels were identified in spiral ganglion cells (661 ± 659 SD), nerve fibers (543 ± 408 AU) and in the organ of Corti (1440 ± 921 AU; Fig 5b). When focusing on the p-Akt-expression, the most intense signals were localized in the organ of Corti (1235 ± 1246 AU), the spiral ganglion cells (1011 ± 930 AU) and the stria vascularis (872 ± 966 AU; Fig. 5c). Minor amounts of p-Akt staining reaction were found in the nerve fibers (257 ± 349 AU), interdental cells (346 ± 465 AU) and spiral ligament (294 ± 315 AU; Fig. 5c). Because of high SDs in most of the six areas, the Akt/p-Akt ratios were determined at the level of the individual ear. Clear differences became evident (Fig. 5d). The highest Akt/p-Akt ratios were found in the spiral ligament (20.8) and in interdental cells (20.3). In all other regions only minor ratios were identified for nerve fibers (1.9), the organ of Corti (1.3) and the stria vascularis (2.9). In the spiral ganglion cells, the p-Akt-expression was 2.8 times higher than the Akt-expression. Thus, the findings demonstrated clear cellular differences between Akt-expression and its activation in untreated control ears. Gentamicin dependence on Akt and p-Akt In order to obtain detailed information about spatial- and temporal-dependent alterations in the Akt and p-Akt staining intensities after gentamicin application, the expression patterns were determined at the different
time points (one, two and seven days) for the various cochlear regions and compared with controls. As no differences between the cochlear turns were found, data were pooled for this calculation. The evaluation revealed clear differences only for two cochlear regions – the organ of Corti and the spiral ganglion cells (Fig. 6). In the organ of Corti, Akt-expression was upregulated one, two and seven days after application of gentamicin (controls vs. gentamicin: 1 day p < .0001; 2 days p = .0027; 7 days p = .0003; controls 1440 ± 921 AU; 1 day 5512 ± 3788 AU; 2 days 4768 ± 2206 AU; 7 days 5463 ± 1847 AU). There was only a slight increase in p-Akt immunostaining intensity for the organ of Corti, however, lacking statistical significance (controls 1235 ± 1246 AU; 1 day 2007 ± 1580 AU; 2 days 1661 ± 1292 AU; 7 days 2688 ± 2073 AU). Due to the small areas in sections, the above-mentioned microscopically observed intense immunostaining in pillar cells and Deiters’ cells (Fig. 3b) have no consequence when analyzing the organ of Corti in its entirety. For all three time points, a clear up-regulation of both Akt-expression (controls vs. gentamicin: 1 day p < .001; 2 days p = .0027; 7 days p = .0003; controls 661 ± 659 AU; 1 day 7911 ± 2616 AU; 2 days 5662 ± 1947 AU; 7 days 6546 ± 1823 AU) and p-Aktexpression (controls vs. gentamicin: 1 day p = .0014, 2 days p = .00891, 7 days p = .0057; controls 1011 ± 930 AU; 1 day 5181 ± 1278 AU; 2 days 3658 ± 2523 AU; 7 days 3903 ± 1908 AU) was found in the spiral ganglion cells. There was no up-regulation of Akt and p-Akt in any other analyzed regions of the cochlea (not shown). Intracellular Akt/p-Akt ratios At the cellular level, clear correlations were found between Akt- and p-Akt-expression in the stria vascularis and the interdental cells (Table 1). Less pronounced relations were identified in the spiral ligament and the organ of Corti, whereas no correlations were detected in nerve fibers and spiral ganglion cells (Table 1). These findings undoubtedly point to specific gentamicin-dependent relations between Akt-expression and its activation in areas involved in potassium release into the Scala media. In addition, a correlation between gentamicin up-take and Akt-expression was only identified for the organ of Corti (Table 1). Furthermore, clear correlations between gentamicin up-take and hearing loss were detected for the stria vascularis, the organ of Corti and the spiral ganglion cells and a less
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Intercellular Akt/p-Akt ratios At the level of the cochlea, clear intercellular correlations were found between the Akt/p-Akt ratios. There were highly significant correlations between the interdental cells and the spiral ligament, between the interdental cells and nerve fibers and between the nerve fibers and the spiral ligament (Table 2). It has to be mentioned that these three areas have no direct anatomical contact with each other.
DISCUSSION Application of gentamicin resulted in a very restricted response on Akt-expression and activation. Many intercellular Akt/p-Akt ratios remained stable despite treatment. Hearing loss was concomitant with an up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells. Additionally, an activation of Akt was located in spiral ganglion cells. Clear intracellular correlations were found between Akt-expression and its activation in the stria vascularis and interdental cells when the three gentamicin-treated groups were pooled. In addition, correlations were identified between gentamicin uptake and hearing loss, but no correlations were detected when comparing Akt-expression and hearing loss, p-Akt-expression and hearing loss and p-Akt-expression and gentamicin up-take. Analyzing the intracellular Akt/p-Akt ratios in untreated ears, the most prominent values were identified in interdental cells and the spiral ligament, whereas the ratio was inverted in spiral ganglion cells. Despite gentamicin application, highly significant intercellular Akt/p-Akt ratios were detected when comparing interdental cells and the spiral ligament, the interdental cells and nerve fibers and the nerve fibers and the spiral ligament. Basal expression levels
Fig. 5. Distribution of Akt and p-Akt immunoreactions in the cochlea of the guinea pig. (a) Schematic drawing of the three fluid-filled compartments (Scm Scala media, Sct Scala tympani, Scv Scala vestibule) and the most important regions and cell types. (b) Basal Akt-expression in the cochlea given by arbitrary units (AU; mean ± SD) demonstrates the most prominent staining intensity in the stria vascularis. (c) Basal p-Akt staining intensities in the cochlea, revealing the highest signals in the spiral ganglion cells, organ of Corti and stria vascularis. (d) Akt/p-Akt ratios in the six analyzed cochlear regions at the level of the individual ear, demonstrating a higher Akt content in five out of six regions, the spiral ganglion being the exception. DC Deiters’ cells, HE Hensen dells, ID interdental cells, IHC inner hair cells, NF nerve fibers, OC organ of Corti, OHC outer hair cell, PC pillar cells, LI limbus, Scm Scala media, Scv Scala vestibule, Sct Scala tympani, SGC spiral ganglion cells, SL spiral ligament, SV stria vascularis.
pronounced correlation for the interdental cells (Table 1). There were no correlations found when comparing gentamicin and p-Akt, Akt and hearing loss and p-Akt and hearing loss (not shown).
In untreated control ears, the basic Akt/p-Akt ratios considerably varied between the different cell types. In addition, high fluctuations in immunostaining intensities were detected in each analyzed region visualized by the high SDs in the corresponding graphs. Analyzing the intensities at the level of the individual ear, prominent Akt/p-Akt ratios were found in the spiral ligament and interdental cells. These areas belong to the routes involved in potassium recycling which enables normal cochlear signal transduction (Spicer and Schulte, 1991, 1996). Akt-expression In this study, it was demonstrated that the application of gentamicin resulted in the up-regulation of Akt-expression in the organ of Corti and in spiral ganglion cells. There are only a restricted number of articles in the literature that report on an up-regulation of Akt. In a cell model of murine macrophages, it was shown by the cDNA microarray technique that H2O2 treatment caused the up-regulation of genes involved in stress, survival and apoptosis, including Akt, and led at
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Fig. 6. Akt and p-Akt specific staining intensities are presented in arbitrary units (AU; mean ± SD) in the organ of Corti (OC) and in spiral ganglion cells (SGC). Values are given for controls (C), after 1 day (1 d), 2 days (2 d) and 7 days (7 d) after the application of gentamicin. The descriptive p values were obtained by comparing control animals and gentamicin-treated animals in the different cochlea turns and statistic significance was marked by asterisks (**p < 0.01, ***p < 0.001).
Table 1. Relation between Akt- and p-Akt-expression in the six analyzed cochlear regions summarizing the values for the different gentamicin treatments (1 d, 2 d, 7 d). The significant correlations are highlighted in by gray color
Table 2. Correlations of the Akt/p-Akt ratios between interdental cell, spiral ligament and nerve fibers ID vs. SL
ID vs. NF
NF vs. SL
r = 0.47288 p = 0.0083 n = 30
r = 0.52280 p = 0.0030 n = 30
r = 0.69461 p < 0.0001 n = 30
the same time to the down-regulation of genes promoting growth and cell cycle (Zhang et al., 2005). Based on known physiological cascades, it can be assumed that in the present study the increased expression of Akt might be cytoprotective for the cochlea, preventing the release of cytochrome C from mitochondria and thus contributing
to the inactivation of the forkhead transcription factors (Kim and Chung, 2002; Pauley et al., 2006; Jackson et al., 2010). p-Akt-expression It is widely known that the application of gentamicin commonly results in an increased production of free radicals in the inner ear (Takumida et al., 1999; Heinrich et al., 2006; Choung et al., 2009). One of these free oxygen radicals, H2O2, was found to elevate Akt-activity in different cell cultures, e.g. HeLa cells, lung carcinoma A549 cells, T cell lymphocytes and others, in a timeand dose-dependent manner (Wang et al., 2000). Such
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increased levels of H2O2 might also be responsible for the p-Akt-expression detected in this study. Surprisingly, this activation was only identified in spiral ganglion cells. Based on numerous published findings, it is generally accepted that the application of gentamicin often results in apoptosis, leading finally to cell death (Wang et al., 2003; Chung et al., 2006; Jeong et al., 2010). Apoptotic processes are mediated by ROS and the JNK (Jeong et al., 2010). For the cell types in the rat liver, it was also demonstrated that Akt inactivated the pro-apoptotic molecule BAD (Harada et al., 2004) and blocked a cytochrome C-mediated caspase 9 activation (Li et al., 2005). Therefore, these cytoprotective processes might also occur in spiral ganglion cells after p-Akt-expression, initiated possibly via H2O2-dependent steps in a still unidentified pathway. Time-dependent effects In this study, an up-regulation of Akt-expression in the organ of Corti and in the spiral ganglion cells and an increase in p-Akt-expression in spiral ganglion cells was already found 1 d after gentamicin application by immunohistochemistry. The expression patterns remained comparably high over the next 6 days. A comparably fast gentamicin-dependent response with respect to protein expression profiles was also reported by others. When analyzing cultures from the organ of Corti of Sprague–Dawley rats by DNA microarray technology and the dChip software package, 12 genes were identified which differ in their expression patterns when comparing gentamicin-treated animals and controls at a 4-h time-point (Nagy et al., 2004). Among other things, a down-regulation was found for genes encoding proteins of the mitochondrial electron transport chain, for the delta subunit of the F1F0 ATPase, for a subunit of the NMDA receptor and for apoptose-related genes (Nagy et al., 2004). Using cultures of the organ of Corti of perinatal mice, the messenger RNA level of more than three thousand genes was significantly changed within hair cells after 3 h (Tao and Segil, 2015). All these data reveal that gentamicin treatment induces numerous processes of cell destruction and of protection at the same time. Cell type-specific effects In this study, four cell type-specific effects of Akt-expression and -activation have to be distinguished. Firstly, in unstimulated control ears the basic Akt/p-Akt ratio differs widely between the various cell types, demonstrating a natural heterogeneous expression pattern. Secondly, analyzing the various experimental groups in their entirety, an up-regulation of Akt-expression was identified only in the organ of Corti and in the spiral ganglion cells and an increase in p-Akt-expression was restricted to spiral ganglion cells. Thus, only a very limited number of cell types were affected by gentamicin. Thirdly, when analyzing cellular response at the level of the individual ear, high correlations between Akt and p-Akt were detected in the stria vascularis and interdental cells, the two cochlear
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regions involved in potassium release into the endolymph. Fourthly, correlations were found between interdental cells and spiral ligament, between the interdental cells and nerve fibers and between the spiral ligament and nerve fibers, revealing highly significant stable conditions within the cochlea in respect to the Akt/p-Akt ratios. Thus, it can be speculated that the selfprotecting mechanisms of the cochlea against gentamicin are regulated primarily in the sensory parts of the cochlea, the organ of Corti harboring the hair cells and the spiral ganglion cells. The Akt/p-Akt ratio seems to play no important role in the ion-transporting regions such as the stria vascularis, the spiral ligament or the interdental cells in the Limbus. The specific increase in AKT-expression might visualize an activation of a cell-specific protection under stress conditions.
CONCLUSION Based on the observation that the influence of gentamicin on protein kinase B (Akt)-expression and its activation (p-Akt) were exclusively found in the organ of Corti and spiral ganglion cells, it is evident that protection mechanisms occurred preferentially in these two regions. Acknowledgment—The authors thank Mrs K. Benz for her invaluable technical assistance.
REFERENCES Aran JM, Erre JP, Lima da Costa D, Debbarh I, Dulon D (1999) Acute and chronic effects of aminoglycosides on cochlear hair cells. Ann NY Acad Sci 884:60–68. Battaglia A, Pak K, Brors D, Bodmer D, Frangos JA, Ryan AF (2003) Involvement of Ras activation in toxic hair cell damage of the mammalian cochlea. Neuroscience 122:1025–1035. Blakley BW (2000) Update on intratympanic gentamicin for Meniere’s disease. Laryngoscope 110:236–240. Caelers A, Radojevic V, Traenkle J, Brand Y, Bodmer D (2010) Stress and survival pathways in the mammalian cochlea. Audiol Neurotol 15:282–290. Choung YH, Taura A, Pak K, Choi SJ, Masuda M, Ryan AF (2009) Generation of highly-reactive oxygen species is closely related to hair cell damage in rat organ of Corti treated with gentamicin. Neuroscience 161:214–226. Chung WH, Pak K, Lin B, Webster N, Ryan AF (2006) A PI3K pathway mediates hair cell survival and opposes gentamicin toxicity in neonatal rat organ of Corti. J Assoc Res Otolaryngol 7:373–382. Datta SR, Brunet A, Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13:2905–2927. Harada N, Hatano E, Koizumi N, Nitta T, Yoshida M, Yamamoto N, Brenner DA, Yamaoka Y (2004) Akt activation protects rat liver from ischemia/reperfusion injury. J Surg Res 121:159–170. Heinrich UR, Selivanova O, Brieger J, Mann W (2006) Endothelial nitric oxide synthase upregulation in the cochlea of the guinea pig after intratympanic gentamicin injection. Eur Arch Otorhinolaryngol 263:62–68. Heinrich UR, Helling K, Sifferath M, Brieger J, Li H, Schmidtmann I, Mann WJ (2008) Gentamicin increases NO production and induces hearing loss in guinea pigs. Laryngoscope 118: 1438–1442. Heinrich UR, Selivanova O, Schmidtmann I, Feltens R, Brieger J, Mann WJ (2010) Noise exposure alters cyclooxygenase 1 (COX1) und 5-lipoxygenase (5-LO) expression in the guinea pig cochlea. Acta Otolaryngol 130:358–365.
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Helling K, Scho¨nfeld U, Clarke AH (2007) Treatment of Me´nie`re’s disease by low-dosage intratympanic gentamicin application: effect on otolith function. Laryngoscope 117:2244–2250. Hess A, Labbe` D, Watanabe KI, Bloch W, Michel O (2006) Evidence for an Akt-kinase/NO/cGMP pathway in the cochlea of guinea pigs. Eur Arch Otorhinolaryngol 263:75–78. Hotta S, Takahashi M, Itoh T, Yamamura K (1998) Experimental study on the effects of gentamicin injection on the guinea-pig: electrophysiological studies. Acta Otolaryngol 118:362–368. Jackson BC, Carpenter C, Nebert DW, Vasiliou V (2010) Update of human and mouse forkhead box (FOX) gene families. Hum Genomics 4:345–352. Jeong SW, Kim LS, Hur D, Bae WY, Kim JR, Lee JH (2010) Gentamicin-induced spiral ganglion cell death: apoptosis mediated by ROS and the JNK signaling pathway. Acta Otolaryngol 130:670–678. Kim AH, Khursigara G, Sun X, Franke TF, Chao MV (2001) Akt phorphorylates and negatively regulates apoptosis signalregulating kinase 1. Mol Cell Biol 21:893–901. Kim D, Chung J (2002) Akt: versatile mediator of cell survival and beyond. J Biochem Mol Biol 35:106–115. Lange G, Maurer J, Mann W (2004) Long-term results after interval therapy with intratympanic gentamicin for Menie`re’s disease. Laryngoscope 114:102–105. Li XK, Man K, Ng KT, Sun CK, Lo CM, Fan ST (2005) The influence of phosphatidylinositol 3-kinase/Akt pathway on the ischemic injury during rat liver graft preservation. Am J Transplant 5:1264–1275. Nagy I, Bodmer M, Brors D, Bodmer D (2004) Early gene expression in the organ of Corti exposed to gentamicin. Hear Res 195:1–8. Pauley S, Lai E, Fritzsch B (2006) Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev Dyn 235:2470–2482. Rathmell JC, Fox CJ, Plas DR, Hammerman PS, Cinalli RM, Thompson CB (2003) Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factorindependent survival. Mol Cell Biol 23:7315–7328. Rybak LP, Ramkumar V (2007) Ototoxicity. Kidney Int 72:931–935.
Rybak LP, Whitworth CA (2005) Ototoxicity: therapeutic options. Drug Discov Today 10:1313–1321. Schacht J, Talaska AE, Rybak LP (2012) Cisplatin and aminoglycoside antibiotics: hearing loss and its prevention. Anat Rec 295:1837–1850. Selivanova O, Brieger J, Heinrich UR, Mann W (2007) Akt and JNK are regulated in response to moderate noise exposure in the cochlea of the guinea pigs. ORL J Otorhinolaryngol Relat Spe 69:277–282. Spicer SS, Schulte BA (1991) Differentiation of inner ear fibrocytes according to their ion transport related activity. Hear Res 56:53–64. Spicer SS, Schulte BA (1996) The fine structure of spiral ligament cells relates to ion return to the stria and varies with placefrequency. Hear Res 100:80–100. Takumida M, Popa R, Anniko M (1999) Free radicals in the guinea pig inner ear following gentamicin exposure. ORL J Otorhinolaryngol Relat Spe 61:63–70. Tao L, Segil N (2015) Early transcriptional response to aminoglycoside antibiotic suggests alternate pathways leading to apoptosis in sensory hair cells in the mouse inner ear. Front Cell Neurosci 9:190. http://dx.doi.org/10.3389/fncel.2015.00190. Van der Wheele DJ, Zhou R, Rudin CM (2004) Akt up-regulation increases resistance to mirotubule-directed chemotherapeutic agents through mammalian target of rapamycin. Mol Cancer Ther 3:1605–1613. Wang X, McCullough KD, Franke TF, Holbrooks NJ (2000) Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 275:14624–14631. Wang J, Van de Water TR, Bonny C, De Ribaupierre F, Puel JL, Zine A (2003) A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J Neurosci 23:8596–8607. Zhang Y, Fong CC, Wong MS, Tzang CH, Lai WP, Fong WF, Sui SF, Yang M (2005) Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress. Apoptosis 10:545–556.
(Accepted 27 October 2015) (Available online 31 October 2015)
GENTAMICIN ALTERS Akt-EXPRESSION AND ITS ACTIVATION IN THE GUINEA PIG COCHLEA U.-R. HEINRICH, a S. STRIETH, a I. SCHMIDTMANN, b H. LI c AND K. HELLING a*
Key words: hearing loss, immunohistochemistry, inner ear, organ of Corti, cytoprotection.
a
Department of Otolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg – University Mainz, Germany b Institute for Medical Statistics, Epidemiology and Informatics (IMBEI), University Medical Center of the Johannes Gutenberg – University Mainz, Germany
INTRODUCTION Intratympanic application of gentamicin is a successful medical approach in the treatment of vertigo spells in Me´nie`re’s disease (Blakley, 2000; Lange et al., 2004; Helling et al., 2007). However, in addition to its therapeutic effects in eliminating the function of vestibular hair cells, gentamicin also induced unwanted morphological damage and physiological alterations in the cochlea (Hotta et al., 1998; Aran et al., 1999; Heinrich et al., 2006). When analyzing the underlying gentamicininduced biochemical mechanisms, both apoptotic cascades and protective pathways were identified in the cochlea (Chung et al., 2006; Rybak and Ramkumar, 2007; Caelers et al., 2010). After gentamicin entrance into the outer hair cells through the mechano-electrical transducer channels, the formation of an aminoglycoside-iron complex particularly increases cellular reactive oxygen species (ROS)-production (Rybak and Ramkumar, 2007), In addition, the activation of small GTPases, such as Ras and Rho/Rac/Cdc42, as well as the c-Jun-N-terminal kinase (JNK) was demonstrated. These are the main players in pre-programed cell death, which leads finally to apoptosis (Wang et al., 2003; Chung et al., 2006; Jeong et al., 2010). Currently, different experimental approaches to otoprotection which focus on upstream and downstream biochemical methods are under investigation (Rybak and Whitworth, 2005; Schacht et al., 2012) On the one hand, it has to be mentioned that the expression of endothelial nitric oxide synthase (eNOS) was increased after the application of gentamicin in the reticular lamina, i.e. at the apical side of the organ of Corti (Heinrich et al., 2006). Furthermore, when organ cultures were analyzed, an up-regulation of nitric oxide (NO) production was identified in the lateral wall (Heinrich et al., 2008). Both processes are said to contribute to gentamicin-induced cochlear damage. On the other hand, it was also demonstrated that gentamicin is able to promote hair cell survival through the H-Ras/Raf/MEK/Erk and the phosphatidylinositol-3 kinase (PI3K) pathways via its downstream targets, protein kinase C (PKC) and Akt (Battaglia et al., 2003; Chung et al., 2006). Based on these findings, the activation state of Akt was declared as an indicator of
c
Department of Pharmacology, University Medical Center of the Johannes Gutenberg – University Mainz, Germany
Abstract—Gentamicin treatment induces hair cell death or survival in the inner ear. Besides the well-known toxic effects, the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway was found to be involved in cell protection. After gentamicin application, the spatiotemporal expression patterns of Akt and its activated form (p-Akt) were determined in male guinea pigs. A single dose of 0.1 mL gentamicin (4 mg/ear/animal) was intratympanically injected. The auditory brainstem responses (ABRs) were recorded prior to application and 1, 2 and 7 days afterward. At these three time points the cochleae (n = 10 in each case) were removed, transferred to fixative and embedded in paraffin. Seven ears were used as untreated controls. Gentamicin, Akt and p-Akt were identified immunohistochemically in various regions of the cochlea and their staining intensities were quantified on sections using digital image analysis. The application of gentamicin resulted in hearing loss with a concomitant up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells and an additional activation in spiral ganglion cells. At the level of individual ears, clear intracellular correlations were found between Akt- and p-Akt-expression in the stria vascularis and interdental cells and, to a minor extent, in the spiral ligament and the organ of Corti. Furthermore, statistical evidence for the connection between gentamicin up-take and hearing loss was detected. The increase in Akt- and p-Akt-expression in the organ of Corti and spiral ganglion cells indicates a selected response of the cochlea against gentamicin toxicity. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg – University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany. Tel: +49-6131-177361; fax: +49-6131176637. E-mail address: [email protected] (K. Helling). Abbreviations: ABR, auditory brainstem response; AU, arbitrary units; JNK, c-Jun-N-terminal kinase; PI3K, phosphatidylinositol-3 kinase; ROS, reactive oxygen species; SD, standard deviation. http://dx.doi.org/10.1016/j.neuroscience.2015.10.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 490
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the extension of the PI3K survival pathway (Caelers et al., 2010). Physiological stimuli are able to induce Akt-kinase activity by up to 40-fold (Datta et al., 1999). Full activation of Akt requires its translocation from the cytoplasm to the cell membrane and its phosphorylation at the two residues, at its catalytic domain (Thr-308), and at the C terminus (Ser-473). Afterward, activated Akt, i.e. p-Akt, can detach from the plasma membrane and is able to phosphorylate substrates in the cytosol and nucleus. Then the activity of Akt can influence different cell pathways, such as the two main components of the intrinsic cell- death machinery, the pro-apoptotic protein BAD (member of the Bcl-family) and caspases. Their actions are inhibited in the cytoplasm after phosphorylation by Akt. Akt can inhibit transcription factors of the forkhead family in the nucleus, thus avoiding the initiation of apoptosis. Furthermore, Akt phosphorylates IjB kinase (IKK) in the cytoplasm, which in turn activates the anti-apoptotic transcription factor NFjb in the nucleus (Rathmell et al., 2003). It was also shown that Akt promotes glycolytic metabolism (Van der Wheehle et al., 2004), stimulates the transcription of glucose transporter genes and the translocation of glucose transporters and additional nutrient transporters to the plasma membrane (Rathmell et al., 2003). All these Akt-regulated pathways control important processes of cell survival and energy balance. Generally, it is assumed that, along its downstream pathways, Akt phosphorylates more than one substrate at a time, so several effects may occur in parallel (Kim et al., 2001). In the un-stimulated cochlea, Akt was identified in all cell types of the organ of Corti, in the stria vascularis and spiral ligament as well as in the interdental cells of the limbus, in the spiral ganglion cell and in the nerve fibers of the osseous spiral lamina (Selivanova et al., 2007). In addition, p-Akt was localized in the organ of Corti, spiral ganglion cells and lateral wall in guinea pigs (Hess et al., 2006; Selivanova et al., 2007). After moderate noise exposure for one hour, a fast down-regulation of Akt and p-Akt was detected in numerous cochlear regions (Selivanova et al., 2007). With respect to its widespread distribution in the mammalian cochlea and its fast response to external stimuli resulting in the activation of survival pathways, Akt has received expanded attention as an important anti-apoptotic protein. In order to obtain more information about the spatiotemporal alterations in Akt-expression after gentamicin application, the expression levels of Akt and p-Akt were quantified one, two and seven days after the application of gentamicin by immunohistochemistry. In addition, the ratio between Akt and p-Akt-expression was determined for the different cochlear regions in order to identify intracellular and intercellular correlations. The findings are discussed with respect to possible survival processes in the cochlea.
EXPERIMENTAL PROCEDURES Subjects Thirty-seven pigmented guinea pigs (tricolor, Charles River, Sulzfeld, Germany) weighing 200–250 g with
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good Preyer’s reflexes and no evidence of middle-ear disease were used in this study. Young animals were used in order to minimize age-related effects or hormonal fluctuations that occur in older animals. All experiments were conducted in accordance with the German Prevention of Cruelty to Animals Act and were approved by the supervising authorities. Animals were kept on a 12:12-h light:dark cycle in our local animal facility at University Medical Center, Mainz. Auditory brainstem responses (ABRs) Before recording the acoustic evoked potentials, the guinea pigs were anesthetized with intraperitoneal injections of esketamin hydrochloride (Ketanest; 175 mg/kg body weight) and xylazine hydrochloride (Rompun; 10 mg/kg body weight). Subdermal needle electrodes (Biomedical, Madison, WI, USA) were placed at the scalp vertex (inverting), the right mastoid (non-inverting) and lower back (ground) to record ABRs. ABRs were recorded in a sound-attenuated room after click stimuli (duration 100 s, 300 averaged stimuli) beginning from 90 dB (SPL) to 10 dB (SPL). The stimulus presentation and data management were coordinated using the Spirit-evoked potential system (Biomedical, Madison, WI, USA). The hearing threshold was determined by discrimination of wave Jewett I, III, and V. ABRs were determined before the injection of gentamicin and on the first, second and seventh day after application. Gentamicin application After anesthesia, a volume of 0.1 mL (4 mg/ear/animal) gentamicin (Ratiopharm, Ulm, Germany) was injected through the anterior region of the tympanic membrane into both ears of the experimental animals (n = 30) under microscopic control. After gentamicin treatment, 10 animals were killed after one day, 10 animals after two days and 10 animals after seven days. Seven untreated animals served as controls. Cochlea preparation Animals were killed by pentobarbital – sodium (NarcorenÒ, Hallbergmoos, Germany; 448 mg/kg body weight) on the first, second and seventh day after gentamicin injection. Both bullae were completely removed and transferred into a solution that consisted of 0.2% picric acid, 4% para-formaldehyde and 0.1% glutardialdehyde. After decalcification with EDTA (20% solution adjusted to pH 7.4) for three weeks at 4 °C, the cochleae were dehydrated by an increasing ethanol series followed by xylene. Specimens were embedded in paraffin. Five-lm sections were prepared using a microtome (Leica RM 2165) mounted onto superfrost glass slides and deparaffinated by xylene and a decreasing alcohol series. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2/methanol. After pre-incubation with 10% normal serum and 1% bovine serum albumin in PBS for 20 min to avoid unspecific binding, the primary rabbit polyclonal anti-Akt
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antibody (Cell Signaling #9272, Danvers, MA, USA, diluted 1:100), rabbit polyclonal anti-p-Akt (Cell Signaling #9275, phosphorylated at Thr308, Danvers, MA, USA, diluted 1:100) and the mouse polyclonal antigentamicin antibody (Fitzgerald Industries, MA, USA, diluted 1:450), respectively, were overlaid overnight at 4 °C. Slides were then consecutively incubated with biotinylated secondary antibody (1:250, DAKO, Hamburg, Germany) for 30 min, streptavidin peroxidase (1:200, DAKO) for 30 min and finally Diaminobenzidine/ H2O2 (1.85 mM) for 1 min. All washing procedures were performed in PBS; dilutions of antibodies were prepared in PBS containing 1% bovine serum albumin at room temperature. For negative controls, the primary antibodies were omitted. All assessments were performed blinded with respect to treatments. Quantification of immunocytochemical staining Paraffin sections were analyzed in detail in six cochlear regions: nerve fibers, organ of Corti, spiral ganglion cells, stria vascularis, spiral ligament and interdental cells of the Limbus (Fig. 5a). Images were taken from the sections using a high-density 1/300 type, three-chip Exwave HAD CCD red-green-blue (RGB) color video camera (Sony 3-CCD DXC-390P) connected to a ZEISS microscope (Axiovert 200) equipped with a halogen light source using a 40x objective. The illumination intensity of the specimens was assured by precise voltage control. The CCD camera settings (gain and sensitivity) were kept constant for all analyses. All images were analyzed in Photoshop (version 7; Adobe Systems, San Jose, CA, USA) and stored in a single file. In all regions, the immunostaining intensities were determined as described recently (Heinrich et al., 2010). In brief, corresponding images of control specimens and images of gentamicin-treated animals were stored in one single image file to compare the different sample levels. Areas with the same brown intensities (tolerance level 15 of the computer program) were selected with the Magic Wand tool and quantified using the Histogram command from the Image menu. Firstly, the background
staining values (values without any cellular structure) were subtracted from the immunostaining intensities, resulting in the ‘‘net staining intensity”. Secondly, the areas of the stained tissue (given in pixel) were compared to the size of the whole analyzed section (given in pixel) and determined as a ‘‘percentage of the immunostained area”. The ‘‘net staining intensity” was multiplied by the ‘‘percentage of the immunostained area” and is then expressed as arbitrary units (AU) for the different cell types. In order to minimize insufficient data quantification, we analyzed a couple of sections from each ear. As not all slides could be antibody-stained in one pass, slides of various experimental groups were mixed during each labeling procedure (Heinrich et al., 2010). Nevertheless, the repetition of labeling for each ear resulted in comparable values with respect to cellular staining intensities. Generally, the experimental approach can be considered as semi-quantitative, allowing a relative grading of staining intensities. Statistical approaches Alterations in Akt and pAkt-immunostaining intensity were analyzed both in controls and after the application of gentamicin using a linear mixed model. In this model, gentamicin, cochlea turn and the interaction between gentamicin and cochlea turn were considered as fixed effects. In addition, a random animal effect was included in the model, thereby taking repeated measurements on each animal into account. We adjusted for multiple comparisons within each cell type and each parameter (Akt, pAkt, Akt–pAkt-ratio, hearing threshold, gentamicin concentration) by using the Tukey–Kramer method. The mean values and standard deviation (SD) are presented in the graphs below and the adjusted p-values are indicated by one, two or three asterisks representing p < 0.05, p < 0.01 and p < 0.001, respectively. Statistical analysis was performed using PROC MIXED from SAS 9.3.
RESULTS ABRs ABRs were recorded prior to and one, two and seven days after gentamicin application (Fig. 1). Gentamicin induced a mean hearing threshold shift of 11.57 ± 10.7 dB after one day, of 18.5 ± 15.7 dB after two days, and of 20.9 ± 21.9 dB after seven days compared with the individual pre-treatment hearing levels. Hearing thresholds were worse at every time point after gentamicin application compared with pre-treatment levels [p = .0214 (control vs. day 1), p < .001 (control vs. day 2 and 7)]. There were no statistical differences between the 1st and the 2nd day (p = .075), between the 1st and 7th day (p = .2209) and between the 2nd and the 7th day (p = .9550).
Fig. 1. Hearing thresholds measured by the determination of ABRs (ten animals per group) prior to and one, two, and seven days after the application of gentamicin in dB (SPL) (mean ± SD). (*p < 0.05; *** p < 0.001).
Localization of Akt At the light microscopic level, a distinct Akt-immunoreaction was identified in un-treated animals
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in all cell types of the organ of Corti (Fig. 2a), in spiral ganglion cells (Fig. 2c), in the stria vascularis and in the spiral ligament (not shown). The reaction was detectable in the cytoplasm and in the cell nuclei (Fig. 2a, c). Seven days after gentamicin application, the staining intensity was much stronger in all cells of the organ of Corti (Fig. 2b) and in the spiral ganglion cells (Fig. 2d). The intensity of the Akt immune reaction in the stria vascularis and spiral ligament remained unaltered at all time points comparable to controls (not shown). Localization of p-Akt Slight staining intensities of p-Akt were identified by light microscopic analysis in the untreated organ of Corti
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(Fig. 3a). After gentamicin application, an increased immunoreaction was found only in two cochlear cell types, the Deiters’ cells and pillar cells (Fig. 3b). In spiral ganglion cells, a clearly increased staining intensity was seen after seven days (Fig. 3d) compared with the moderate staining reaction in untreated controls (Fig. 3c). Localization of gentamicin Intense widespread gentamicin immunostaining intensities were identified by microscopic analysis in the cochlea at the three time points after application. In the organ of Corti, gentamicin was found to be located within supporting cells as well as in inner and outer hair
Fig. 2. Examples of Akt immunostaining intensities in the cochlea of the guinea pig in the second cochlear turn. In untreated animals, a basal Akt immunoreaction was found in all regions of the organ of Corti (a). Seven days after the application of gentamicin, the staining intensity of Akt was clearly increased (b). In spiral ganglion cells, a faint staining of Akt was visible (c) which was more intense seven days after gentamicin treatment (d). DC Deiters’ cells, HE Hensen cells, IHC inner hair cells, OHC outer hair cells, PC pillar cells; Scale bar = 30 lm.
Fig. 3. p-Akt specific staining intensities in the second cochlear turn. In controls, a basal faint p-Akt immunoreaction was found the organ of Corti (a). Seven days after the application of gentamicin, the staining intensity of p-Akt was clearly increased, especially in Deiters’ cells and pillar cells (b). In spiral ganglion cells, a slight p-Akt immunoreaction was visible (c) which was more intense seven days after gentamicin treatment (d). DC Deiters’ cells, PC pillar cells; Scale bar = 30 lm.
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Fig. 4. Gentamicin specific staining intensities in the second cochlear turn. In the organ of Corti, gentamicin was identified in supporting cells and in inner and outer hair cells (a). In the lateral wall, a clear gentamicin immunoreaction was seen in the spiral ligament, whereas only a faint reaction was found in the stria vascularis (b). IHC inner hair cell, OHC outer hair cells, SL spiral ligament, SV stria vascularis; Scale bar = 30 lm.
cells (Fig. 4a). Within the lateral wall, clear staining reactions were seen in the spiral ligament, whereas the stria vascularis was nearly free from gentamicin (Fig. 4b). Basal Akt-expression and activation In order to judge the importance of the alterations in Akt- and p-Akt-expression in context with their spatiotemporal distribution patterns, the basal cellular expression levels were determined by quantitative immunohistochemistry for the six analyzed regions. All staining intensities are expressed in AU, mentioning mean values and standard deviation (mean ± SD). For a better understanding of the inner ear anatomy, the areas and important cell types are presented in a corresponding graph (Fig. 5a). In control ears, the highest Akt-expression was found in the stria vascularis (4659 ± 2608 AU; Fig. 5b). A clear expression was also seen in the interdental cells (2456 ± 1158 AU) and the spiral ligament (2463 ± 1554 AU). Low expression levels were identified in spiral ganglion cells (661 ± 659 SD), nerve fibers (543 ± 408 AU) and in the organ of Corti (1440 ± 921 AU; Fig 5b). When focusing on the p-Akt-expression, the most intense signals were localized in the organ of Corti (1235 ± 1246 AU), the spiral ganglion cells (1011 ± 930 AU) and the stria vascularis (872 ± 966 AU; Fig. 5c). Minor amounts of p-Akt staining reaction were found in the nerve fibers (257 ± 349 AU), interdental cells (346 ± 465 AU) and spiral ligament (294 ± 315 AU; Fig. 5c). Because of high SDs in most of the six areas, the Akt/p-Akt ratios were determined at the level of the individual ear. Clear differences became evident (Fig. 5d). The highest Akt/p-Akt ratios were found in the spiral ligament (20.8) and in interdental cells (20.3). In all other regions only minor ratios were identified for nerve fibers (1.9), the organ of Corti (1.3) and the stria vascularis (2.9). In the spiral ganglion cells, the p-Akt-expression was 2.8 times higher than the Akt-expression. Thus, the findings demonstrated clear cellular differences between Akt-expression and its activation in untreated control ears. Gentamicin dependence on Akt and p-Akt In order to obtain detailed information about spatial- and temporal-dependent alterations in the Akt and p-Akt staining intensities after gentamicin application, the expression patterns were determined at the different
time points (one, two and seven days) for the various cochlear regions and compared with controls. As no differences between the cochlear turns were found, data were pooled for this calculation. The evaluation revealed clear differences only for two cochlear regions – the organ of Corti and the spiral ganglion cells (Fig. 6). In the organ of Corti, Akt-expression was upregulated one, two and seven days after application of gentamicin (controls vs. gentamicin: 1 day p < .0001; 2 days p = .0027; 7 days p = .0003; controls 1440 ± 921 AU; 1 day 5512 ± 3788 AU; 2 days 4768 ± 2206 AU; 7 days 5463 ± 1847 AU). There was only a slight increase in p-Akt immunostaining intensity for the organ of Corti, however, lacking statistical significance (controls 1235 ± 1246 AU; 1 day 2007 ± 1580 AU; 2 days 1661 ± 1292 AU; 7 days 2688 ± 2073 AU). Due to the small areas in sections, the above-mentioned microscopically observed intense immunostaining in pillar cells and Deiters’ cells (Fig. 3b) have no consequence when analyzing the organ of Corti in its entirety. For all three time points, a clear up-regulation of both Akt-expression (controls vs. gentamicin: 1 day p < .001; 2 days p = .0027; 7 days p = .0003; controls 661 ± 659 AU; 1 day 7911 ± 2616 AU; 2 days 5662 ± 1947 AU; 7 days 6546 ± 1823 AU) and p-Aktexpression (controls vs. gentamicin: 1 day p = .0014, 2 days p = .00891, 7 days p = .0057; controls 1011 ± 930 AU; 1 day 5181 ± 1278 AU; 2 days 3658 ± 2523 AU; 7 days 3903 ± 1908 AU) was found in the spiral ganglion cells. There was no up-regulation of Akt and p-Akt in any other analyzed regions of the cochlea (not shown). Intracellular Akt/p-Akt ratios At the cellular level, clear correlations were found between Akt- and p-Akt-expression in the stria vascularis and the interdental cells (Table 1). Less pronounced relations were identified in the spiral ligament and the organ of Corti, whereas no correlations were detected in nerve fibers and spiral ganglion cells (Table 1). These findings undoubtedly point to specific gentamicin-dependent relations between Akt-expression and its activation in areas involved in potassium release into the Scala media. In addition, a correlation between gentamicin up-take and Akt-expression was only identified for the organ of Corti (Table 1). Furthermore, clear correlations between gentamicin up-take and hearing loss were detected for the stria vascularis, the organ of Corti and the spiral ganglion cells and a less
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Intercellular Akt/p-Akt ratios At the level of the cochlea, clear intercellular correlations were found between the Akt/p-Akt ratios. There were highly significant correlations between the interdental cells and the spiral ligament, between the interdental cells and nerve fibers and between the nerve fibers and the spiral ligament (Table 2). It has to be mentioned that these three areas have no direct anatomical contact with each other.
DISCUSSION Application of gentamicin resulted in a very restricted response on Akt-expression and activation. Many intercellular Akt/p-Akt ratios remained stable despite treatment. Hearing loss was concomitant with an up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells. Additionally, an activation of Akt was located in spiral ganglion cells. Clear intracellular correlations were found between Akt-expression and its activation in the stria vascularis and interdental cells when the three gentamicin-treated groups were pooled. In addition, correlations were identified between gentamicin uptake and hearing loss, but no correlations were detected when comparing Akt-expression and hearing loss, p-Akt-expression and hearing loss and p-Akt-expression and gentamicin up-take. Analyzing the intracellular Akt/p-Akt ratios in untreated ears, the most prominent values were identified in interdental cells and the spiral ligament, whereas the ratio was inverted in spiral ganglion cells. Despite gentamicin application, highly significant intercellular Akt/p-Akt ratios were detected when comparing interdental cells and the spiral ligament, the interdental cells and nerve fibers and the nerve fibers and the spiral ligament. Basal expression levels
Fig. 5. Distribution of Akt and p-Akt immunoreactions in the cochlea of the guinea pig. (a) Schematic drawing of the three fluid-filled compartments (Scm Scala media, Sct Scala tympani, Scv Scala vestibule) and the most important regions and cell types. (b) Basal Akt-expression in the cochlea given by arbitrary units (AU; mean ± SD) demonstrates the most prominent staining intensity in the stria vascularis. (c) Basal p-Akt staining intensities in the cochlea, revealing the highest signals in the spiral ganglion cells, organ of Corti and stria vascularis. (d) Akt/p-Akt ratios in the six analyzed cochlear regions at the level of the individual ear, demonstrating a higher Akt content in five out of six regions, the spiral ganglion being the exception. DC Deiters’ cells, HE Hensen dells, ID interdental cells, IHC inner hair cells, NF nerve fibers, OC organ of Corti, OHC outer hair cell, PC pillar cells, LI limbus, Scm Scala media, Scv Scala vestibule, Sct Scala tympani, SGC spiral ganglion cells, SL spiral ligament, SV stria vascularis.
pronounced correlation for the interdental cells (Table 1). There were no correlations found when comparing gentamicin and p-Akt, Akt and hearing loss and p-Akt and hearing loss (not shown).
In untreated control ears, the basic Akt/p-Akt ratios considerably varied between the different cell types. In addition, high fluctuations in immunostaining intensities were detected in each analyzed region visualized by the high SDs in the corresponding graphs. Analyzing the intensities at the level of the individual ear, prominent Akt/p-Akt ratios were found in the spiral ligament and interdental cells. These areas belong to the routes involved in potassium recycling which enables normal cochlear signal transduction (Spicer and Schulte, 1991, 1996). Akt-expression In this study, it was demonstrated that the application of gentamicin resulted in the up-regulation of Akt-expression in the organ of Corti and in spiral ganglion cells. There are only a restricted number of articles in the literature that report on an up-regulation of Akt. In a cell model of murine macrophages, it was shown by the cDNA microarray technique that H2O2 treatment caused the up-regulation of genes involved in stress, survival and apoptosis, including Akt, and led at
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Fig. 6. Akt and p-Akt specific staining intensities are presented in arbitrary units (AU; mean ± SD) in the organ of Corti (OC) and in spiral ganglion cells (SGC). Values are given for controls (C), after 1 day (1 d), 2 days (2 d) and 7 days (7 d) after the application of gentamicin. The descriptive p values were obtained by comparing control animals and gentamicin-treated animals in the different cochlea turns and statistic significance was marked by asterisks (**p < 0.01, ***p < 0.001).
Table 1. Relation between Akt- and p-Akt-expression in the six analyzed cochlear regions summarizing the values for the different gentamicin treatments (1 d, 2 d, 7 d). The significant correlations are highlighted in by gray color
Table 2. Correlations of the Akt/p-Akt ratios between interdental cell, spiral ligament and nerve fibers ID vs. SL
ID vs. NF
NF vs. SL
r = 0.47288 p = 0.0083 n = 30
r = 0.52280 p = 0.0030 n = 30
r = 0.69461 p < 0.0001 n = 30
the same time to the down-regulation of genes promoting growth and cell cycle (Zhang et al., 2005). Based on known physiological cascades, it can be assumed that in the present study the increased expression of Akt might be cytoprotective for the cochlea, preventing the release of cytochrome C from mitochondria and thus contributing
to the inactivation of the forkhead transcription factors (Kim and Chung, 2002; Pauley et al., 2006; Jackson et al., 2010). p-Akt-expression It is widely known that the application of gentamicin commonly results in an increased production of free radicals in the inner ear (Takumida et al., 1999; Heinrich et al., 2006; Choung et al., 2009). One of these free oxygen radicals, H2O2, was found to elevate Akt-activity in different cell cultures, e.g. HeLa cells, lung carcinoma A549 cells, T cell lymphocytes and others, in a timeand dose-dependent manner (Wang et al., 2000). Such
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increased levels of H2O2 might also be responsible for the p-Akt-expression detected in this study. Surprisingly, this activation was only identified in spiral ganglion cells. Based on numerous published findings, it is generally accepted that the application of gentamicin often results in apoptosis, leading finally to cell death (Wang et al., 2003; Chung et al., 2006; Jeong et al., 2010). Apoptotic processes are mediated by ROS and the JNK (Jeong et al., 2010). For the cell types in the rat liver, it was also demonstrated that Akt inactivated the pro-apoptotic molecule BAD (Harada et al., 2004) and blocked a cytochrome C-mediated caspase 9 activation (Li et al., 2005). Therefore, these cytoprotective processes might also occur in spiral ganglion cells after p-Akt-expression, initiated possibly via H2O2-dependent steps in a still unidentified pathway. Time-dependent effects In this study, an up-regulation of Akt-expression in the organ of Corti and in the spiral ganglion cells and an increase in p-Akt-expression in spiral ganglion cells was already found 1 d after gentamicin application by immunohistochemistry. The expression patterns remained comparably high over the next 6 days. A comparably fast gentamicin-dependent response with respect to protein expression profiles was also reported by others. When analyzing cultures from the organ of Corti of Sprague–Dawley rats by DNA microarray technology and the dChip software package, 12 genes were identified which differ in their expression patterns when comparing gentamicin-treated animals and controls at a 4-h time-point (Nagy et al., 2004). Among other things, a down-regulation was found for genes encoding proteins of the mitochondrial electron transport chain, for the delta subunit of the F1F0 ATPase, for a subunit of the NMDA receptor and for apoptose-related genes (Nagy et al., 2004). Using cultures of the organ of Corti of perinatal mice, the messenger RNA level of more than three thousand genes was significantly changed within hair cells after 3 h (Tao and Segil, 2015). All these data reveal that gentamicin treatment induces numerous processes of cell destruction and of protection at the same time. Cell type-specific effects In this study, four cell type-specific effects of Akt-expression and -activation have to be distinguished. Firstly, in unstimulated control ears the basic Akt/p-Akt ratio differs widely between the various cell types, demonstrating a natural heterogeneous expression pattern. Secondly, analyzing the various experimental groups in their entirety, an up-regulation of Akt-expression was identified only in the organ of Corti and in the spiral ganglion cells and an increase in p-Akt-expression was restricted to spiral ganglion cells. Thus, only a very limited number of cell types were affected by gentamicin. Thirdly, when analyzing cellular response at the level of the individual ear, high correlations between Akt and p-Akt were detected in the stria vascularis and interdental cells, the two cochlear
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regions involved in potassium release into the endolymph. Fourthly, correlations were found between interdental cells and spiral ligament, between the interdental cells and nerve fibers and between the spiral ligament and nerve fibers, revealing highly significant stable conditions within the cochlea in respect to the Akt/p-Akt ratios. Thus, it can be speculated that the selfprotecting mechanisms of the cochlea against gentamicin are regulated primarily in the sensory parts of the cochlea, the organ of Corti harboring the hair cells and the spiral ganglion cells. The Akt/p-Akt ratio seems to play no important role in the ion-transporting regions such as the stria vascularis, the spiral ligament or the interdental cells in the Limbus. The specific increase in AKT-expression might visualize an activation of a cell-specific protection under stress conditions.
CONCLUSION Based on the observation that the influence of gentamicin on protein kinase B (Akt)-expression and its activation (p-Akt) were exclusively found in the organ of Corti and spiral ganglion cells, it is evident that protection mechanisms occurred preferentially in these two regions. Acknowledgment—The authors thank Mrs K. Benz for her invaluable technical assistance.
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(Accepted 27 October 2015) (Available online 31 October 2015)