Учебники / Auditory Trauma, Protection, and Repair Fay 2008

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of glutamate in the VIIIth nerve dendrites occurs because of the inability of the IHC/VIIIth nerve synapse to recycle glutamate (see Wangemann, Chapter 3). Consequently, glutamate that accumulates in the dendritic terminal creates the condition of excitotoxicity, characterized by swelling of the postsynaptic cell bodies and dendrites (Kandel et al. 2000; Pujol et al. 1990). Interestingly, these excitotoxic reactions at the synapse are repairable and may contribute only to the temporary threshold shift (TTS) component of the hearing loss (Zheng et al. 1997b).

5. Cochlear Vascular System

The role of the cochlear vascular system in NIHL is complicated. Under normal homeostatic conditions, cochlear blood flow (CBF) is controlled by a combination of factors, including systemic changes (Miller and Dengerink 1988), sympathetic influence over the cochlear vasculature (Laurikainen et al. 1994, 1997), and local autoregulation (Miller et al. 1995; Konishi et al. 1998). The degree to which each of these factors affects CBF under normal conditions and under traumatic conditions is currently unclear. As detailed in a review by Miller and Dengerink (1988), CBF was once thought to be a passive response to systemic blood flow in the body. Clearly, CBF is influenced strongly by systemic changes in the body, but the cochlea has its own mechanisms of altering blood flow that enable it to modulate or fine tune the blood supply with which it is provided. . The relative contribution of the two local elements of sympathetic nervous innervation and autoregulation is not completely clear. Cochlear sympathetic influence on blood flow is mediated heavily by a bilateral innervation from the stellate ganglion (Laurikainen et al. 1993,1997), as well as a secondary influence from unilateral–ipsilateral innervation from the superior cervical ganglion (Ren et al. 1993).

Autoregulation refers to the cochlear vasculature’s local intrinsic factors that can alter blood blow. The cochlear vasculature is very sensitive to the carbon dioxide content of the blood (Kallinen et al. 1991; Ugnell et al. 2000), but a number of additional factors have been implicated in local autoregulation of CBF, including: nitric oxide (Fessenden and Schacht 1998), prostaglandins (Nagahara et al. 1988), and tropomyosin (Konishi et al. 1998).

The vascular system’s response to a potentially traumatic noise varies with the type of noise. For example, the trauma associated with a high-level impulse noise can be an instantaneous mechanical failure that occurs before the vascular system reacts; by contrast, long-duration noise exposure can modulate blood flow in patterns that vary with the intensity and duration of the exposure (Perlman and Kimura 1962; Thorne and Nuttall 1987; Yamane et al. 1995; Lamm and Arnold 2000; Miller et al. 2003). With a continuous noise exposure, there may be first an increase in blood and then a decrease or active blocking of cochlear vessels (Fig. 7.7). What remains unclear is the extent to which CBF

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Figure 7.7. A capillary from spiral ligament 30 min after traumatic noise exposure. Note the accumulation of red blood cells blocking blood flow.

changes during and after noise might be influencing the cochlear pathology, or, conversely, whether the CBF changes are the result of noise-induced cochlear pathology (Miller et al. 2003). The actual influence of cochlear blood flow on the magnitude of NIHL is made more difficult to determine because of the complexity of the network of vessels supplying the cochlea. However, it is clear that reducing O2 by breathing low levels of CO (Chen and Fechter 1999) during an exposure increases the hearing loss. In addition, interruption of the sympathetic nervous innervation of the cochlea reduces susceptibility to noise, although the effect is fairly small (Borg 1982; Hildesheimer et al. 1991, 2002). The possible influence of changes in CBF on noise-induced cochlear pathology is discussed further in Section 10.

6. Acoustic Characteristics of Noise and Patterns of HL and Cochlear Pathology

The patterns of hearing loss and cochlear pathology are related to both the response of the basilar membrane and the acoustic characteristics of the noise. In a classic study, Davis and colleagues showed that short duration (1–20 min) higher-frequency exposures (i.e., 4 kHz) produced a temporary hearing loss

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(TTS) that was focused at 1/2 to 1 octave above 4 kHz (Davis et al. 1950). By contrast, low-frequency exposures (i.e., 0.5-kHz tones) produced a hearing loss that spanned the 500–8000 Hz range.

Large-scale epidemiological studies of NIHL in workplace or laboratory studies with white noise both show a hearing loss centered at 4 kHz. The 4-kHz notch is a hallmark of NIHL and is the consequence of both acoustic transformations and biochemical factors. When broad-band, high-level noise enters the pinna and external meatus, the spectrum of the noise is changed to a band-passed noise tuned to approximately 3 kHz. The transformation is the result of quarter wave resonance response of the pinna and external meatus [resonant frequency = (speed of sound)/(4 × length of the EAM)]. As the 3-kHz band of noise stimulates the cochlea, the displacement of the traveling wave frequency occurs not at the normal 3 kHz but at a place 1/2 to 1 octave above the center frequency (Sellick et al. 1982). Consequently, a flat spectrum noise primarily stresses the 4-kHz region of the cochlea. Consequently, a broad-band noise produces a highfrequency “notch” audiogram because of the mechanical transformation at the EAM and basilar membrane.

7. High-Level Transients

Exposure to impulse noise (gunfire or any other explosive event) or highlevel impact noise (two hard objects hitting together forcefully) can lead to direct and pervasive mechanical failure in the cochlea (see Henderson and Hamernik 1986 for review). Figure 7.8 shows a scanning electron microscope view of a chinchilla cochlea 30 minutes after exposure to impulse noise at 155 dB pSPL. Several points are interesting. First, the organ of Corti is ripped from the basilar membrane to a relatively large extent (arrows). The sensory cells of the detached organ of Corti will not recover and the tissue will be digested by surrounding tissue. Second, at a point basal to the detached organ of Corti, there is a cleft between the first and second row of OHCs. This cleft will allow endolymph to enter the organ of Corti, creating large osmotic and ionic changes across the OHC membranes which cause additional cell death. These mechanical failures as seen in Fig. 7.8 happen because of the extreme acceleration and displacement associated with impulse noise.

Impulse and impact noise can have another, more subtle, but also traumatic, effect on the cochlea. Noise exposures that are a combination of moderate levels of impact/impulse noise and continuous noise are much more traumatic to the ear than the simple additive effects of either noise alone (see review by Henderson and Hamernik 1986). There are examples of combination exposures in working populations. For example, construction workers exposed to a combination of continuous noise and impact noise develop larger hearing losses

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Figure 7.8. Scanning electron microscopic view of partially dissected cochlea showing OHC separated from the basilar membrane. Right view shoes the region adjacent the dissociated OHC. Note the split between the rows of OHCs, which provides an open channel for endolymph to enter the organ of Corti. Also, IHCs appear normal.

than would be expected on the basis of their A-weighted noise dose (Sweeney et al. 2005).

8. Temporary Effects of Noise

After a damaging noise is turned off, hearing recovers either to preexposure levels or to some permanent threshold shift (PTS). The pathology associated with PTS has been well studied, and key features have been described in this chapter. The pathological changes associated with noise-induced temporary threshold shift (TTS) are not as well understood. Possible pathologies underlying TTS include a detachment of the tectorial membrane from the tips of the stereocilia (Nordmann et al. 2000), excitotoxicity of the VIIIth nerve synapses at the IHC (see Section 4), and partial depolymerization of actin in the supporting cells (Fig. 7.9). The biological basis of TTS is an interesting open question. Studies that correlate TTS with PTS do not show strong correlations, suggesting that the fundamental pathologies of TTS are different from those of PTS (Ward 1966).

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Figure 7.9. Section of organ of Corti showing the tops of pillar cells and an OHC separated from the Deiters’ cup. Notice that the normally cylindrical OHC has shrunk and is more oval shaped. The OHC nucleus is shrunken and going through apoptosis. The animal was exposed to impulse noise at 155 dBpSPL. (From Henderson et al. 2006)

9. Dynamics of Cochlear Pathology

We have known for some time that the cochlear pathology, especially hair cell loss, continues to increase for approximately 30 days after an exposure (Hamernik et al. 1984; Bohne 1999). Recent studies by Hu et al. (2002) have shown that after a high-level, short-duration exposure, there can be a small focal lesion that primarily involves OHCs. In the next few days, the lesion continues to expand, primarily in the basal direction and with cells dying by both necrosis and apoptosis (Fig. 7.10).

The detailed anatomical studies of the growth of the cochlear pathology have used primarily high-level, short-duration exposures. In an interesting study of the relationship between long-term TTS or PTS (Bohne and Clark 1982), chinchillas were exposed to noise for 24 hours a day for up to 6 months. The animals developed a stable level of threshold shift by 24 hours and this level did not significantly change for up to 6 months; consequently the change in hearing sensitivity is referred to as asymptotic threshold shift (ATS). When the chinchillas were removed after only several days of exposure, hearing completely recovered and there was essentially no permanent hearing loss or cochlear pathology. However, if the subjects were exposed for 6 months, the recovery of hearing sensitivity was minimal and there was a large hair cell loss. Mills and collegues (1981) systematically studied the ATS phenomenon and found that the level of ATS at any frequency was determined by the spectral level at that frequency; and for noise exposures at the threshold for creating hearing loss, the

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Figure 7.10. Top row shows the organ of Corti 30 min after a traumatic noise exposure. Bottom row is 2 days later. (B) is focused on the center of the lesion where there are many missing cells and a few cells with condensed nuclei that are going through apoptosis. (A) is at the basal margin of lesion B and most of the OHCs are present, but there are a few shrunken nuclei. (C) is at the apical part of the lesion. Most cells are present, and there are only two shrunken nuclei. Two days later (E), the center of the lesion shows no OHCs. The basal margin of the lesion (D) shows that all OHC are shrunken and will eventually die. The cells at the apical margin of the lesion (F) are stable.

level of ATS grew at the rate of 1.7 dB increase in hearing loss for each dB of increase in the noise level.

For higher-level impact noise as found in factories and construction sites or impulse noise as found in military settings, the relationship between the impulse/impact level and hearing loss has a growth constant of 3–5 dB. In systematic studies of impact noise, Henderson and collegues (1991) found that, for lower level impacts (99–115 dBA), the increase in hearing loss with increase in impact level was about 1.9 dB. However, above 120 dB pSPL, the hearing loss grows at a rate of 3–5 dB for each 1-dB increase in peak level. The data suggest that when the peak level of an exposure exceeds a “critical level” the mode of cochlear damage shifts to a direct mechanical failure, particularly at the points of adhesion between cells in the organ of Corti (as seen in Fig. 7.8). The “critical level” is not fixed but varies with both the species and signature of the impact or impulse noise. Spoendlin (1985) postulated that the “critical level” for guinea pigs was approximately 120 dBA for noise bursts of 100 ms; for chinchillas exposed to impact noise (50–150 ms) the “critical level” is between 119 and 125 dB peak and for impulse noise the “critical level” is between 150 and 155 dB peaks. Given the differences in sensitivity and conductive function, the critical level for humans is probably approximately 10 dB higher (Mills and collegues 1981).

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10. Noise as a Stressor to the Cochlea

The cochlea normally operates at a high level of metabolism (Thalmann et al. 1975). The major demand for energy is at the stria vascularis, which constantly extrudes K+ ions as it maintains the ionic balance and polarity of the endolymph (see review by Wangemann 2002 and Wangemann, Chapter 3). The high energy demands are supported by a large population of mitochondria in marginal, intermediate, and basal cells of stria vascularis.

The mitochondrial electron transport chain has long been recognized as a major source of reactive oxygen species (ROS) inside of cells. Under normal physiological conditions, 98% of molecular oxygen (O2) consumed by the mitochondria is used to promote phosphorylation of ADP to generate ATP. The 1% to 2% of O2 that is not consumed is converted to superoxide (O2·−) or hydrogen peroxide (H2O2) at mitochondrial or extramitochondrial locations (Chance et al. 1979). In a number of pathological processes or in the presence of drugs, toxins, electron chain inhibitors and uncouplers, the mitochondrial generation of ROS can increase several-fold (Turrens et al. 1982). With high-level noise exposure, there is a large increase in cochlear ROS generation because of two factors. First, high-level noise drives cochlear metabolism at a much faster and demanding rate. Therefore, the number of free radicals generated increases. Second, to exacerbate the situation, noise also influences CBF (Miller and Dengerink 1988). When the blood flow is reduced, ischemia develops in the organ of Corti and there is a shortage of O2 for mitochondrial operation leading to an even greater rate of superoxide generation. Conversely, with reperfusion (the return of blood flow to its preischemic level) there is an increased blood flow which also increases availability of O2 to the mitochondria, resulting in another burst of superoxide generation (Halliwell and Gutteridge 1999). Finally, if the cells of the cochlea are damaged, then cellular contents can be spilled into the extracellular matrix. Trace amounts of iron from the cell create the condition for the Fenton reaction which can produce the highly reactive and toxic hydroxyl radical (OH•) from hydrogen peroxide (Beauchamp and Fridovich 1970). Redox homeostasis is discussed in detail in Wangemann, Chapter 3.

Several studies have reported increased activity of reactive oxygen species (ROS; free radicals) following traumatic noise exposure in eluates from the cochlea (Ohlemiller et al. 1999) or localized to marginal cells of the stria vascularis (Yamane et al. 1995). In the organ of Corti, Nicotera et al. (1999) found increased ROS activity around the basal pole of the OHC and along the neural plexus under the IHC (Fig. 7.11) (Henderson et al. 2006). Increased ROS activity continues for several days (Fig. 7.12) after exposure to traumatic noise (Hu et al. 2002; Yamashita et al. 2004). The persistent ROS activity is interesting because hair cells continue to die for days after an exposure (Bohne 1976; Hamernik et al. 1985).

The significance of the free radical activity in the cochlea raises a fundamental question: Is the free radical activity the consequence of dying cells or is the cell death initiated by increased free radical activity? One approach to this question

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Figure 7.11. OHC region of the cochlea stained with dihydroethidene (DE) 15 min after traumatic exposure. DE reacts with superoxide. Notice the reaction in the OHC. The right panel shows the same of the cochlea, but at 1 h after the exposure. There is no DE reaction, which suggests that superoxide is present during and after an exposure, but that superoxide is not a factor for long after the exposure.

was to stress the cochlea with increased free radical activity by exposing the cochlea to paraquat, an herbicide that reacts with molecular O2 to create O2•− radicals (Nicotera et al. 2004; Bielefeld et al. 2005). Figure 7.13 shows the comparison of cell death patterns induced by paraquat and exposure to noise. There are similarities in the pattern of the two pathologies. Both have damaged OHCs while the IHCs are relatively intact. Additional experiments by Bielefeld et al. (2005) show that paraquat creates a high-frequency based hearing loss. The significance of the paraquat experiments is that the superoxide activity and the activity of other downstream ROS are a sufficient cause to create a pattern of cochlear pathology very similar to the pathology found with noise exposure, but without the mechanical stress associated with noise.

Figure 7.12. Organ of Corti at labeled with dichlorofluorescein at 30 min (A), 2 days (B), and 4 days (C) after noise exposure. Notice the bright reaction at 30 min, but the reaction persists for at least 4 days. (From Henderson et al. 2006.)

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Figure 7.13. The left panel shows the organ of Corti stained with propidium iodide 1 h after paraquat was placed on the round window. The right panel shows the organ of Corti 18 h after exposure to traumatic noise. Notice that both conditions produce a very similar pattern of histology. The IHCs are intact after each treatment. The OHCs are in the process of dying through apoptosis (arrows) and necrosis (arrowheads).

11. Pathways of Sensory Cell Death

In the last few years, Hu et al. (2002) and Nicotera et al. (2003) have reported that noise exposures can produce both necrotic and apoptotic cell death. Both types of cell death are illustrated in Fig. 7.14. The OHC with swollen nuclei are dying by necrosis. The cell membrane has been compromised, Ca2+ and water has leaked into the cell and expanded the cell volume. The cell eventually ruptures and spills its contents into the surrounding area. The trace elements of the cell content will be available to react with H2O2 and create the very reactive and toxic OH• (Ohlemiller et al. 1999). OHCs with condensed nuclei

Figure 7.14. Examples of apoptotic cell death (left) and necrotic cell death (right).

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are dying by apoptosis, in which the proteins of the cell are being disassembled. Apoptosis is an active process and both the cell membrane and mitochondria continue to function. In a normal functioning body, apoptosis is a very useful mechanism for ridding tissues of unwanted cells. For example, in the developing brain, apoptosis is used to reduce drastically the number of neurons in order to maximize the efficiency of the pathways that remain. For this streamlining to be effective, the cells must be eliminated in an organized and controlled way. The cell death pathway of apoptosis allows for controlled cell death that prevents damage to the neighboring cells that survive. In the case of noise trauma, OHC death is problematic because the cells do not regenerate. Therefore, the loss of OHC, even through the controlled death of apoptosis, leaves the cochlea in an impaired state because OHCs are essential for maximal sensitivity and tuning.

Apoptosis is regulated by a family of enzymes called caspases (see also Green, Altschuler, and Miller, Chapter 10). Apoptosis can be initiated by cell death signals from the mitochondria, nucleus, or cell membrane. Caspase-8 is an initiator related to cell death signals from the cell mechanisms; caspase-9 is generated by cell death signals at the mitochondria; caspase-3 is an effector caspase associated with the final stages of apoptosis (see Cohen 1997 for a review of caspase activity in cell death). Figure 7.15 shows a cell labeled with propidium iodide (PI), a stain that is taken up by the nucleus of a dying or fixed cell. Notice that all the darker red shrunken nuclei are also colabeled for caspase-3 (green staining), and the swollen nuclei of necrotic cells do not express caspase-3. Nicotera et al. (2003) reported that after a noise exposure both caspase-8 and -9 were expressed, which implies that apoptosis in hair cells can be triggered into cell death through multiple pathways (Fig. 7.16). There is also

Figure 7.15. Apoptotic cells labeled for caspase-3, an effector caspases at the terminal end of cell death.