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Mechanisms underlying noise-induced hearing loss
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. 3, No. 1 2006
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Hearing disorders MECHANISMS
Mechanisms underlying noise-induced hearing loss Ulf-Ru¨diger Heinrich*, Ralph Feltens Department of Otolaryngology – Head and Neck Surgery, Johannes Gutenberg University Medical School, 55101 Mainz, Germany
Noise-induced hearing loss (NIHL) is the worldwide leading occupational disease and presents an impor-
Section Editor: Richard Smith – University of Iowa, Iowa City, USA
tant socio-economic factor. Despite numerous identified
details
about
its
etiology,
the
underlying
mechanisms, which induce NIHL, have been only partially identified. In the present article, we shall discuss possible mechanisms focusing on failure in cellular calcium regulation, leading to a rise in mitochondrial NO production and reactive oxygen species formation. In cases where radical production is significantly elevated, pathological concentrations lead to alterations of cell physiological conditions and finally contribute to NIHL. A more detailed knowledge about the induction of free radical production and its regulation at the cellular or organelle level will be a basic requirement for new therapeutic strategies. Introduction: Noise-induced hearing loss in developed countries Noise-induced hearing loss (NIHL) is a widespread disease in developed countries resulting in high costs to society [1]. Depending on the kind of noise exposure, duration and intensity, temporary (TTS) and permanent threshold shifts (PTS) can occur. In various animal models different stimuli were used to identify the underlying mechanisms of NIHL, and the induced alterations were recorded by biochemical, physiological and microscopic methods [2–5]. Further indirect information about NIHL-dependent physiological *Corresponding author: U.-R. Heinrich ([email protected]) 1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2006.02.003
changes was obtained by application of protective substances before and after noise exposure [6,7]. A timetable of morphological alterations had been published several years ago, revealing a degradation pattern in the organ of Corti after noise exposure to 3.85 kHz at a sound pressure level (SPL) of 120 dB for 22.5 min [2]. Since these early findings, numerous additional observations have been made that shed more light on the underlying mechanisms of NIHL. It is now commonly accepted that mechanical damage as well as metabolic disturbances induced by intense sound exposure lead to noise-induced hearing loss. In the present article, we focus our attention on early and possibly harmful calcium- and NO-dependent events in the most vulnerable cell type in the organ of Corti, the outer hair cell [8]. Additionally, we discuss the deleterious effects that injured outer hair cells might have on other cell types.
Cellular structural damage and NIHL Damaged cellular structures in response to intensive noise exposure were identified particularly in the organ of Corti. The organ of Corti consists of supporting cells as well as two types of sensory cells, the inner and outer hair cells. The inner hair cells transfer noise-induced signals via afferent neurons to the brain, whereas the vulnerable outer hair cells with their high intracellular cytoplasmic pressure act as a cochlear amplifier by enhancing the basilar membrane movements [9]. Both types of hair cells possess a bundle of sensory hairs (also named stereocilia or, more correctly, stereovilli). Sound 131
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stimulation leads to deflection of the hair bundles, thereby opening nonspecific ion channels, causing membrane depolarization, neurotransmitter release and generating action potentials in attached cochlear nerves. These transduction channels have been localized in the tips and shafts of the stereocilia, and their gating mechanism is regulated mechanically by tip links [10]. At the subcellular level, broken tip links were detected after acoustical overstimulation, which resulted in reduced hair bundle stiffness, disrupted mechanoelectric transduction and temporary noise-induced hearing loss [11]. Evidence for reestablished signal transduction was obtained upon regeneration of the tip links in cultured explants 6 h after the stimulus [11], or in vivo after 24 h [12] or 120 h [13]. The time course of tip link regeneration suggests that this process might underlie recovery from temporary threshold shift induced by noise exposure [11]. Besides injury to tip links, additional molecular deterioration with subsequent repair has been implied in contributing to temporary hearing loss after acoustic overstimulation, such as destruction of filamentous proteins in the actin core or rootlet of the stereovilli or through the activity of contractile proteins surrounding the rootlets [14]. Regarding the repair processes after mechanical damage to hair bundles, it is of interest to note that a constitutive and complete renewal of their actin-filament core takes place about every 48 h [15]. Thus, repair of damaged microvilli might occur automatically within a relatively short timeframe to avoid any longer lasting dysfunction in signal transduction. A reduction in outer hair cell stiffness and cell length was reported after intense noise exposure, with recovery taking place over a 2-week period. These findings were seen as an indication for cellular repair mechanisms taking place during that time [8]. The short outer hair cells of the high-frequency region were found to be more vulnerable to sound stimulation than the taller receptor cells in the low-frequency area [8]. Furthermore, exposure to moderate levels of noise causes a buckling of supporting cells, which results in a temporary loss of contact between the outer hair cell stereovilli and the tectorial membrane [5]. Interestingly, this mechanical uncoupling was found to be directly correlated with a TTS and thus might not just represent an accidental damage, but rather constitute a protective response, preventing longer lasting hair bundle deflection and excessive depolarization of outer hair cell membranes. It was postulated that the mechanisms leading to TTS are fundamentally different from those resulting in PTS, depending on functioning recovery or repair processes within the cochlea [5]. In contrast to TTS, PTS is observed under conditions causing irreparable damage to cells [2]. To obtain a clearer picture about the events through which a temporary injury becomes permanent, physiological alterations have to 132
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Vol. 3, No. 1 2006
be taken into account such as changes in calcium homeostasis and its role in the production of free radicals.
Alterations in calcium homeostasis, a possible precondition for NIHL In outer hair cells, the opening of nonspecific ion channels at the apical cell side during noise-induced perilymph movement causes membrane depolarization and calcium influx through voltage-sensitive L-type channels, resulting in activation of large-conductance, calcium-activated potassium channels [16,17]. Such a postulated rise in cytoplasmic Ca2+ concentration after acoustic stimulation was identified in isolated outer hair cells [18] as well as under in vivo conditions [19]. The calcium increase correlated with a contraction of the organ of Corti and with a decrease of the amplitude of the cochlear potential. As organ functionality was completely recovered in 30–45 min in several cases after the end of noise stimulation, the temporary calcium rise after moderate noise stimulation is probably connected to TTS [19]. Nevertheless, depending on the time and intensity of noise exposure, longer lasting depolarization in the case of acoustic overstimulation will lead to an increased calcium concentration in outer hair cells via L-type calcium channels located in the lateral cell membrane [20]. To generally prevent any possible calcium overloading, outer hair cells possess numerous calcium-binding proteins such as calbindin [21], calsequestrin [21] and oncomodulin [22], which all act as calcium buffers. Besides this, the efficiency of the Ca2+-extrusion systems and the mitochondrial Ca2+-buffering capacity also play an important role in outer hair cell calcium homeostasis. Indirect evidence for outer hair cell protection against harmful Ca2+ influx was obtained by application of the calcium channel blocker Diltiazem before and after acute noise trauma, which reduced the degree of hair cell destruction significantly [23]. Furthermore, without application of protective substances the detectable calcium level in the few apparently intact outer hair cells remaining 60 h after acute noise trauma was comparable to unexposed control hair cells [24]. However, in cellular debris of destroyed outer hair cells after acute noise trauma, high amounts of loosely bound calcium can be seen in the residual cytoplasm (Fig. 1). Therefore, it might be assumed that the substantial influx of calcium during severe noise exposure generates calcium concentrations that most outer hair cells cannot tolerate.
The possible role of mitochondria-produced free radicals in NIHL Besides calcium binding proteins, mitochondria are known to participate in cytoplasmic calcium sequestration and cellular signal transduction. In outer hair cells, mitochondria were found to be located along the subsurface cisterns underneath the basolateral cell membrane (Fig. 2) and in the
Vol. 3, No. 1 2006
Figure 1. Numerous calcium particles can be identified in cellular debris of outer hair cells after acute noise trauma in the absence of protective substances, revealing the calcium accumulation in this cell type. cd ohc: cellular debris of outer hair cell. Scale bar = 1 mm.
infranuclear region near the synapses (not shown). This specific spatial distribution pattern of the organelles, especially their proximity to the calcium channels of the basolateral membrane, supports the idea that mitochondria are directly involved in cellular Ca2+ signaling in outer hair cells. Mitochondria are capable of capturing and storing a substantial fraction of the Ca2+ flowing into the cell through influx channels or release channels which are located in membranes of intracellular compartments. Furthermore, mitochondria are able to generate microdomains of low Ca2+ near the mouth of these channels, thereby influencing Ca2+-dependent processes by specifically controlling the concentration of this cation [25,26]. The primary role of mitochondrial Ca2+ is the stimulation of oxidative phosphorylation. In addition, the locally limited calcium flow activates enzymes such as the mitochondrial nitric oxide synthase (mtNOS) directly within the organelles and modulates further Ca2+-dependent mitochondrial functions such as activation of N-acetylglutamine synthetase and additional processes [27,28]. Calciuminduced stimulation of mtNOS and increased nitric oxide (NO) production can be seen as part of a feedback loop that
Drug Discovery Today: Disease Mechanisms | Hearing disorders
Figure 2. Electron microscopic image of outer hair cells demonstrating the spatial distribution of mitochondria beneath the basolateral membrane. m: mitochondrion. Scale bar = 2 mm.
prevents calcium overloading in mitochondria and allows its release from the organelles, preserving membrane potential [29]. It is now accepted that Ca2+ is primarily a global positive effector of mitochondrial function, and thus any pertubation in mitochondrial or cytosolic Ca2+ homeostasis will have profound implications for the cell [28]. As was shown recently, elevation of the intracellular and especially the intramitochondrial calcium level is responsible for activation of reactive oxygen species (ROS)-generating enzymes and formation of free radicals by the mitochondrial respiratory chain [28,30]. These ROS include the superoxide anion O2, the hydroxyl radical OH and hydrogen peroxide H2O2. Superoxide anions can react with excessive amounts of NO forming peroxynitrite, ONOO, which is known to be highly toxic. As moderate concentrations of superoxide and hydrogen peroxide produced by mitochondria were found to participate in the regulation of a large variety of physiologic processes [31,32], the amount of the produced radicals might be the most important factor in determining the route towards physiological or, alternatively, pathophysiological responses. www.drugdiscoverytoday.com
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A direct correlation between Ca2+ concentration and free radical production was shown by in vitro experiments where the application of the calcium chelator bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA AM) and agents that block calcium release from the endoplasmic reticulum and influx through voltage-dependent channels prevented mitochondrial ROS accumulation [33]. The direct stimulation of mitochondrial NO and ROS generation by Ca2+ was postulated in detail recently [28], assuming that Ca2+ might stimulate mitochondrial ROS generation by interacting with different parts of the respiratory chain. First, Ca2+ stimulation of the tricarboxylic acid (TCA) cycle enhances electron flow into the respiratory chain, enhancing ROS output. Second, Ca2+ activates NO production by mtNOS, which will inhibit complex IV of the respiratory chain and thus also leads to an enhanced ROS generation. This process is considered to be a redox signaling box, converting an NO signal into an ROS signal [28]. Besides this, high intramitochondrial Ca2+ concentrations will trigger the opening of permeability transition pores (PT pores) and facilitate cytochrome C release, which is known to be involved in apoptosis induction. Taking these lines of evidence together, it can be postulated that moderate noise exposure is causally connected to physiological calcium-activated and mitochondrion-dependent processes in outer hair cells. Intensive noise exposure results in prolonged deflection of hair cell bundles, continued membrane depolarization and intracellular and intramitochondrial accumulation of Ca2+. Under these conditions, the high cellular Ca2+-binding capacity of outer hair cells becomes saturated after several hours, and calcium overload in mitochondria will finally lead to high NO and ROS production. Cell damage, as it has been identified by others [2– 5,34], occurs, possibly involving apoptotic processes. Additionally, the damaging effects of increased ROS production are probably aggravated by the fact that the intracellular concentration of the important antioxidant glutathione was found to be especially low in outer hair cells [35].
Spread of noise-induced damage into the surrounding tissue After noise damage has been inflicted on outer hair cells, different mechanisms might be involved to signal the occurrence of hair cell damage to supporting cells. First, it was demonstrated recently in a culture preparation of the rat cochlea, that mechanical manipulation elicited an intercellular Ca2+ wave that spread with a constant speed from the site of damage to the surrounding supporting cells [36]. ATP release from damaged hair cells was required for the propagation of this wave [36]. This finding is in line with the fact that elevated levels of intramitochondrial Ca2+ caused a faster respiratory chain activity and higher ATP output, which might contribute to the spread of the Ca2+ wave. This calcium wave might also induce altered gene expression in supporting 134
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Vol. 3, No. 1 2006
cells, resulting in an increased NO- and/or ROS-production. For example, an increase of endothelial nitric oxide synthase (eNOS) expression and loosely bound calcium was identified 60 h after acute noise trauma in different cell types of the reticular lamina [24], probably representing such an induced physiological alteration. Second, NO radicals and ROS released from damaged outer hair cells might induce degenerative processes in other cell types. Using specific dyes for NO, 4,5-diaminofluoresceine diacetate, and for ROS, dihydrorhodamine 1,2,3, an intense fluorescence labeling was observed in inner and outer hair cells after intense noise exposure [37]. Besides this, an increase in NO concentration was also measured in the perilymph [37,38]. Although the identification of increased NO levels in various cell types and in the perilymph fluid is no sufficient evidence for a destructive process, it can be considered to be part of a TTS-dependent physiological alteration [38] or even a protection mechanism [24].
Conclusion The earliest morphological correlates of TTS are broken tip links and their reorganization, the induction of repair mechanisms in hair bundles and the uncoupling and reestablishment of contacts between the stereovilli and the tectorial membrane. Prolonged noise stimulation leads to disturbances in outer hair cell calcium homeostasis and to an imbalance of mitochondrial Ca2+ concentrations, two alterations that must be considered as key elements in NIHL. Mitochondrial matrix Ca2+ overload results in enhanced generation and release of ROS and ATP from outer hair cells, which in turn might disturb physiological pathways in other cell types in the cochlea. Therefore, future therapeutic strategies have to take free radical production and regulation at the mitochondrial level into account.
References 1 Lynch, E.D. and Kil, J. (2005) Compounds for the prevention and treatment of noise-induced hearing loss. Drugs Discov. Today 10, 1291– 1298 2 Fredelius, L. (1988) Time sequence of degeneration pattern of the organ of Corti after acoustic overstimulation. Acta Otolaryngol. 106, 373–385 3 Saunders, J.C. et al. (1991) The structural and functional consequences of acoustic injury in the cochlea and peripheral auditory system: a five year update. J. Acoust. Soc. Am. 90, 136–146 4 Kopke, R. et al. (1999) A radical demise. Toxins and trauma share common pathways in hair cell death. Ann. N. Y. Acad. Sci. 884, 171–191 5 Nordmann, A.S. et al. (2000) Histopathological differences between temporary and permanent threshold shift. Hear Res. 139, 13–30 6 Pujol, R. and Puel, J.L. (1999) Excitotoxicity, synaptic repair, and functional recovery in the mammalian cochlea: a review of recent findings. Ann. N. Y. Acad. Sci. 884, 249–254 7 Duan, M.L. et al. (2002) Protection and treatment of sensorineural hearing disorders caused by exogenous factors: experimental findings and potential clinical application. Hear Res. 169, 169–178 8 Chan, E. et al. (1998) Acoustic trauma causes reversible stiffness changes in auditory sensory cells. Neuroscience 83, 961–968 9 Holley, M.C. (2005) The auditory system, hearing loss and potential targets for drug development. Drug Discov. Today 10, 1269–1282
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Kachar, B. et al. (2000) High-resolution structure of hair-cell tip links. Proc. Natl. Acad. Sci. U S A 97, 13336–13341 Zhao, Y-D. et al. (1996) Regeneration of broken tip links and restoration of mechanical transduction in hair cells. Proc. Natl. Acad. Sci. U S A 94, 15469– 15474 Husbands, J.M. et al. (1999) Tip link integrity on chick tall hair cell stereocilia following intense sound exposure. Hear Res. 135, 135–145 Kurian, R. et al. (2003) Tip link loss and recovery on chick short hair cells following intense exposure to sound. Hear Res. 181, 40–50 Duncan, R.K. and Saunders, J.C. (2000) Stereocilium injury mediates hair bundle stiffness loss and recovery following intense water-jet stimulation. J. Comp. Physiol. A 186, 1095–1106 Schneider, M.E. et al. (2002) Rapid renewal of auditory hair bundles. Nature 418, 837–838 Lewis, R.S. and Hudspeth, A.J. (1983) Voltage- and ion-dependent conductances in solitary vertebrate hair cells. Nature 304, 538–541 Art, J.J. and Fettiplace, R. (1987) Variation of membrane properties in hair cells isolated from the turtle cochlea. J. Physiol. 385, 207–242 Fridberger, A. and Ulfendahl, M. (1996) Acute mechanical overstimulation of isolated outer hair cells causes changes in intracellular calcium levels without shape changes. Acta Otolaryngol. 116, 17–24 Fridberger, A. et al. (1998) Acoustic overstimulation increases outer hair cell Ca2+ concentrations and causes dynamic contractions of the hearing organ. Proc. Natl. Acad. Sci. U S A 95, 7127–7132 Oshima, T. et al. (1996) Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines. Am. J. Physiol. 271 (3 Pt. 1), C044–C949 Slepecky, N.B. and Ulfendahl, M. (1993) Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti. Hear Res. 70, 73–84 Sakaguchi, N. et al. (1998) Oncomodulin is expressed exclusively by outer hair cells in the organ of Corti. J. Histochem. Cytochem. 46, 29–39 Heinrich, U-R. et al. (1999) Ultrastructural evidence for protection of the outer hair cells of the inner ear during intense noise exposure by application of the organic calcium channel blocker Diltiazem. ORL 61, 321–327
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Heinrich, U-R. et al. (2005) Endothelial nitric oxide synthase upregulation in the guinea pig organ of Corti after acute noise trauma. Brain Res. 1047, 85–96 Bootman, M.D. et al. (2001) The organization and functions of local Ca2+ signals. J. Cell Sci. 114, 2213–2222 Neher, E. (1998) Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20, 389–399 Brookes, P.S. (2004) Mitochondrial nitric oxide synthase. Mitochondrion 3, 187–204 Brookes, P.S. et al. (2004) Calcium, ATP, and ROS: a mitochondrial love– hate triangle. Am. J. Physiol. Cell Physiol. 287, C817–C833 Ghafourifar, P. and Richter, C. (1997) Nitric oxide synthase activity in mitochondria. FEBS Lett. 418, 291–296 Tan, S. et al. (1998) The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol. 141, 1423–1432 Brookes, P.S. et al. (2002) Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species. Free Radic. Biol. Med. 33, 755–764 Dro¨ge, W. (2002) Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 Keller, J.N. et al. (1998) Increased sensitivity to mitochondrial toxininduced apoptosis in neural cells expressing mutant presenilin-1 is linked to perturbed calcium homeostasis and enhanced oxyradical production. J. Neurosci. 18, 4439–4450 Shi, X. and Nuttall, A.L. (2003) Upregulated iNOS and oxidative damage to the cochlear stria vascularis due to noise stress. Brain Res. 967, 1–10 Usami, S. et al. (1996) Differential cellular distribution of glutathione – an endogenous antioxidant – in the guinea pig inner ear. Brain Res. 743, 337– 340 Gale, J.E. et al. (2004) A mechanism for sensing noise damage in the inner ear. Curr. Biol. 14, 526–529 Shi, X. et al. (2002) The electrochemical and fluorescence detection of nitric oxide in the cochlea and its increase following loud sound. Hear Res. 164, 49–58 Chen, Y-S. et al. (2005) Involvement of nitric oxide generation in noise-induced temporary threshold shift in guinea pigs. Hear Res. 203, 94–100
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135
DRUG DISCOVERY
TODAY
Vol. 3, No. 1 2006
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Hearing disorders MECHANISMS
Mechanisms underlying noise-induced hearing loss Ulf-Ru¨diger Heinrich*, Ralph Feltens Department of Otolaryngology – Head and Neck Surgery, Johannes Gutenberg University Medical School, 55101 Mainz, Germany
Noise-induced hearing loss (NIHL) is the worldwide leading occupational disease and presents an impor-
Section Editor: Richard Smith – University of Iowa, Iowa City, USA
tant socio-economic factor. Despite numerous identified
details
about
its
etiology,
the
underlying
mechanisms, which induce NIHL, have been only partially identified. In the present article, we shall discuss possible mechanisms focusing on failure in cellular calcium regulation, leading to a rise in mitochondrial NO production and reactive oxygen species formation. In cases where radical production is significantly elevated, pathological concentrations lead to alterations of cell physiological conditions and finally contribute to NIHL. A more detailed knowledge about the induction of free radical production and its regulation at the cellular or organelle level will be a basic requirement for new therapeutic strategies. Introduction: Noise-induced hearing loss in developed countries Noise-induced hearing loss (NIHL) is a widespread disease in developed countries resulting in high costs to society [1]. Depending on the kind of noise exposure, duration and intensity, temporary (TTS) and permanent threshold shifts (PTS) can occur. In various animal models different stimuli were used to identify the underlying mechanisms of NIHL, and the induced alterations were recorded by biochemical, physiological and microscopic methods [2–5]. Further indirect information about NIHL-dependent physiological *Corresponding author: U.-R. Heinrich ([email protected]) 1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2006.02.003
changes was obtained by application of protective substances before and after noise exposure [6,7]. A timetable of morphological alterations had been published several years ago, revealing a degradation pattern in the organ of Corti after noise exposure to 3.85 kHz at a sound pressure level (SPL) of 120 dB for 22.5 min [2]. Since these early findings, numerous additional observations have been made that shed more light on the underlying mechanisms of NIHL. It is now commonly accepted that mechanical damage as well as metabolic disturbances induced by intense sound exposure lead to noise-induced hearing loss. In the present article, we focus our attention on early and possibly harmful calcium- and NO-dependent events in the most vulnerable cell type in the organ of Corti, the outer hair cell [8]. Additionally, we discuss the deleterious effects that injured outer hair cells might have on other cell types.
Cellular structural damage and NIHL Damaged cellular structures in response to intensive noise exposure were identified particularly in the organ of Corti. The organ of Corti consists of supporting cells as well as two types of sensory cells, the inner and outer hair cells. The inner hair cells transfer noise-induced signals via afferent neurons to the brain, whereas the vulnerable outer hair cells with their high intracellular cytoplasmic pressure act as a cochlear amplifier by enhancing the basilar membrane movements [9]. Both types of hair cells possess a bundle of sensory hairs (also named stereocilia or, more correctly, stereovilli). Sound 131
Drug Discovery Today: Disease Mechanisms | Hearing disorders
stimulation leads to deflection of the hair bundles, thereby opening nonspecific ion channels, causing membrane depolarization, neurotransmitter release and generating action potentials in attached cochlear nerves. These transduction channels have been localized in the tips and shafts of the stereocilia, and their gating mechanism is regulated mechanically by tip links [10]. At the subcellular level, broken tip links were detected after acoustical overstimulation, which resulted in reduced hair bundle stiffness, disrupted mechanoelectric transduction and temporary noise-induced hearing loss [11]. Evidence for reestablished signal transduction was obtained upon regeneration of the tip links in cultured explants 6 h after the stimulus [11], or in vivo after 24 h [12] or 120 h [13]. The time course of tip link regeneration suggests that this process might underlie recovery from temporary threshold shift induced by noise exposure [11]. Besides injury to tip links, additional molecular deterioration with subsequent repair has been implied in contributing to temporary hearing loss after acoustic overstimulation, such as destruction of filamentous proteins in the actin core or rootlet of the stereovilli or through the activity of contractile proteins surrounding the rootlets [14]. Regarding the repair processes after mechanical damage to hair bundles, it is of interest to note that a constitutive and complete renewal of their actin-filament core takes place about every 48 h [15]. Thus, repair of damaged microvilli might occur automatically within a relatively short timeframe to avoid any longer lasting dysfunction in signal transduction. A reduction in outer hair cell stiffness and cell length was reported after intense noise exposure, with recovery taking place over a 2-week period. These findings were seen as an indication for cellular repair mechanisms taking place during that time [8]. The short outer hair cells of the high-frequency region were found to be more vulnerable to sound stimulation than the taller receptor cells in the low-frequency area [8]. Furthermore, exposure to moderate levels of noise causes a buckling of supporting cells, which results in a temporary loss of contact between the outer hair cell stereovilli and the tectorial membrane [5]. Interestingly, this mechanical uncoupling was found to be directly correlated with a TTS and thus might not just represent an accidental damage, but rather constitute a protective response, preventing longer lasting hair bundle deflection and excessive depolarization of outer hair cell membranes. It was postulated that the mechanisms leading to TTS are fundamentally different from those resulting in PTS, depending on functioning recovery or repair processes within the cochlea [5]. In contrast to TTS, PTS is observed under conditions causing irreparable damage to cells [2]. To obtain a clearer picture about the events through which a temporary injury becomes permanent, physiological alterations have to 132
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Vol. 3, No. 1 2006
be taken into account such as changes in calcium homeostasis and its role in the production of free radicals.
Alterations in calcium homeostasis, a possible precondition for NIHL In outer hair cells, the opening of nonspecific ion channels at the apical cell side during noise-induced perilymph movement causes membrane depolarization and calcium influx through voltage-sensitive L-type channels, resulting in activation of large-conductance, calcium-activated potassium channels [16,17]. Such a postulated rise in cytoplasmic Ca2+ concentration after acoustic stimulation was identified in isolated outer hair cells [18] as well as under in vivo conditions [19]. The calcium increase correlated with a contraction of the organ of Corti and with a decrease of the amplitude of the cochlear potential. As organ functionality was completely recovered in 30–45 min in several cases after the end of noise stimulation, the temporary calcium rise after moderate noise stimulation is probably connected to TTS [19]. Nevertheless, depending on the time and intensity of noise exposure, longer lasting depolarization in the case of acoustic overstimulation will lead to an increased calcium concentration in outer hair cells via L-type calcium channels located in the lateral cell membrane [20]. To generally prevent any possible calcium overloading, outer hair cells possess numerous calcium-binding proteins such as calbindin [21], calsequestrin [21] and oncomodulin [22], which all act as calcium buffers. Besides this, the efficiency of the Ca2+-extrusion systems and the mitochondrial Ca2+-buffering capacity also play an important role in outer hair cell calcium homeostasis. Indirect evidence for outer hair cell protection against harmful Ca2+ influx was obtained by application of the calcium channel blocker Diltiazem before and after acute noise trauma, which reduced the degree of hair cell destruction significantly [23]. Furthermore, without application of protective substances the detectable calcium level in the few apparently intact outer hair cells remaining 60 h after acute noise trauma was comparable to unexposed control hair cells [24]. However, in cellular debris of destroyed outer hair cells after acute noise trauma, high amounts of loosely bound calcium can be seen in the residual cytoplasm (Fig. 1). Therefore, it might be assumed that the substantial influx of calcium during severe noise exposure generates calcium concentrations that most outer hair cells cannot tolerate.
The possible role of mitochondria-produced free radicals in NIHL Besides calcium binding proteins, mitochondria are known to participate in cytoplasmic calcium sequestration and cellular signal transduction. In outer hair cells, mitochondria were found to be located along the subsurface cisterns underneath the basolateral cell membrane (Fig. 2) and in the
Vol. 3, No. 1 2006
Figure 1. Numerous calcium particles can be identified in cellular debris of outer hair cells after acute noise trauma in the absence of protective substances, revealing the calcium accumulation in this cell type. cd ohc: cellular debris of outer hair cell. Scale bar = 1 mm.
infranuclear region near the synapses (not shown). This specific spatial distribution pattern of the organelles, especially their proximity to the calcium channels of the basolateral membrane, supports the idea that mitochondria are directly involved in cellular Ca2+ signaling in outer hair cells. Mitochondria are capable of capturing and storing a substantial fraction of the Ca2+ flowing into the cell through influx channels or release channels which are located in membranes of intracellular compartments. Furthermore, mitochondria are able to generate microdomains of low Ca2+ near the mouth of these channels, thereby influencing Ca2+-dependent processes by specifically controlling the concentration of this cation [25,26]. The primary role of mitochondrial Ca2+ is the stimulation of oxidative phosphorylation. In addition, the locally limited calcium flow activates enzymes such as the mitochondrial nitric oxide synthase (mtNOS) directly within the organelles and modulates further Ca2+-dependent mitochondrial functions such as activation of N-acetylglutamine synthetase and additional processes [27,28]. Calciuminduced stimulation of mtNOS and increased nitric oxide (NO) production can be seen as part of a feedback loop that
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Figure 2. Electron microscopic image of outer hair cells demonstrating the spatial distribution of mitochondria beneath the basolateral membrane. m: mitochondrion. Scale bar = 2 mm.
prevents calcium overloading in mitochondria and allows its release from the organelles, preserving membrane potential [29]. It is now accepted that Ca2+ is primarily a global positive effector of mitochondrial function, and thus any pertubation in mitochondrial or cytosolic Ca2+ homeostasis will have profound implications for the cell [28]. As was shown recently, elevation of the intracellular and especially the intramitochondrial calcium level is responsible for activation of reactive oxygen species (ROS)-generating enzymes and formation of free radicals by the mitochondrial respiratory chain [28,30]. These ROS include the superoxide anion O2, the hydroxyl radical OH and hydrogen peroxide H2O2. Superoxide anions can react with excessive amounts of NO forming peroxynitrite, ONOO, which is known to be highly toxic. As moderate concentrations of superoxide and hydrogen peroxide produced by mitochondria were found to participate in the regulation of a large variety of physiologic processes [31,32], the amount of the produced radicals might be the most important factor in determining the route towards physiological or, alternatively, pathophysiological responses. www.drugdiscoverytoday.com
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A direct correlation between Ca2+ concentration and free radical production was shown by in vitro experiments where the application of the calcium chelator bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA AM) and agents that block calcium release from the endoplasmic reticulum and influx through voltage-dependent channels prevented mitochondrial ROS accumulation [33]. The direct stimulation of mitochondrial NO and ROS generation by Ca2+ was postulated in detail recently [28], assuming that Ca2+ might stimulate mitochondrial ROS generation by interacting with different parts of the respiratory chain. First, Ca2+ stimulation of the tricarboxylic acid (TCA) cycle enhances electron flow into the respiratory chain, enhancing ROS output. Second, Ca2+ activates NO production by mtNOS, which will inhibit complex IV of the respiratory chain and thus also leads to an enhanced ROS generation. This process is considered to be a redox signaling box, converting an NO signal into an ROS signal [28]. Besides this, high intramitochondrial Ca2+ concentrations will trigger the opening of permeability transition pores (PT pores) and facilitate cytochrome C release, which is known to be involved in apoptosis induction. Taking these lines of evidence together, it can be postulated that moderate noise exposure is causally connected to physiological calcium-activated and mitochondrion-dependent processes in outer hair cells. Intensive noise exposure results in prolonged deflection of hair cell bundles, continued membrane depolarization and intracellular and intramitochondrial accumulation of Ca2+. Under these conditions, the high cellular Ca2+-binding capacity of outer hair cells becomes saturated after several hours, and calcium overload in mitochondria will finally lead to high NO and ROS production. Cell damage, as it has been identified by others [2– 5,34], occurs, possibly involving apoptotic processes. Additionally, the damaging effects of increased ROS production are probably aggravated by the fact that the intracellular concentration of the important antioxidant glutathione was found to be especially low in outer hair cells [35].
Spread of noise-induced damage into the surrounding tissue After noise damage has been inflicted on outer hair cells, different mechanisms might be involved to signal the occurrence of hair cell damage to supporting cells. First, it was demonstrated recently in a culture preparation of the rat cochlea, that mechanical manipulation elicited an intercellular Ca2+ wave that spread with a constant speed from the site of damage to the surrounding supporting cells [36]. ATP release from damaged hair cells was required for the propagation of this wave [36]. This finding is in line with the fact that elevated levels of intramitochondrial Ca2+ caused a faster respiratory chain activity and higher ATP output, which might contribute to the spread of the Ca2+ wave. This calcium wave might also induce altered gene expression in supporting 134
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cells, resulting in an increased NO- and/or ROS-production. For example, an increase of endothelial nitric oxide synthase (eNOS) expression and loosely bound calcium was identified 60 h after acute noise trauma in different cell types of the reticular lamina [24], probably representing such an induced physiological alteration. Second, NO radicals and ROS released from damaged outer hair cells might induce degenerative processes in other cell types. Using specific dyes for NO, 4,5-diaminofluoresceine diacetate, and for ROS, dihydrorhodamine 1,2,3, an intense fluorescence labeling was observed in inner and outer hair cells after intense noise exposure [37]. Besides this, an increase in NO concentration was also measured in the perilymph [37,38]. Although the identification of increased NO levels in various cell types and in the perilymph fluid is no sufficient evidence for a destructive process, it can be considered to be part of a TTS-dependent physiological alteration [38] or even a protection mechanism [24].
Conclusion The earliest morphological correlates of TTS are broken tip links and their reorganization, the induction of repair mechanisms in hair bundles and the uncoupling and reestablishment of contacts between the stereovilli and the tectorial membrane. Prolonged noise stimulation leads to disturbances in outer hair cell calcium homeostasis and to an imbalance of mitochondrial Ca2+ concentrations, two alterations that must be considered as key elements in NIHL. Mitochondrial matrix Ca2+ overload results in enhanced generation and release of ROS and ATP from outer hair cells, which in turn might disturb physiological pathways in other cell types in the cochlea. Therefore, future therapeutic strategies have to take free radical production and regulation at the mitochondrial level into account.
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