Department of Otology and Laryngology, Harvard Medical School, Boston 02115; and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114
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ABSTRACT |
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Kujawa, Sharon G. and M. Charles Liberman. Conditioning-related protection from acoustic injury: effects of chronic deefferentation and sham surgery. J. Neurophysiol. 78: 3095-3106, 1997. The inner ear can be made less vulnerable to acoustic injury by a "conditioning" treatment involving exposure to a moderate-level acoustic stimulus before the acoustic overexposure. The present study was designed to explore the role of the olivocochlear (OC) system in this "protection." Guinea pigs were divided into a number of groups: some (trauma-only) were exposed to a traumatic noise for 4 h at 109 dB SPL; others (condition/trauma) were conditioned by daily exposure to the same noise at 85 dB SPL before the traumatic exposure. In OC-intact animals, the condition/trauma group showed significantly less permanent threshold shift (PTS) than the trauma-only group as measured via compound action potentials and distortion-product otoacoustic emissions (DPOAEs). Other animals with identical noise-exposure regimens underwent deefferentation surgery before the start of conditioning: the OC bundle (OCB) was cut in the brain stem, either at the midline (cutting the crossed OCB to both ears) or at the sulcus limitans (cutting all OC fibers to 1 side). Lesion success was quantified by measuring OC fascicles to the outer hair cell region in each ear. The results from the surgical groups showed that total loss of the OCB significantly increased the noise-induced PTS, whereas loss of the COCB only did not; that the conditioning exposure in deefferented animals increased, rather than decreased, the PTS from the traumatic exposure; and that animals undergoing sham surgery (brain stem cuts that failed to transect the OCB) appeared protected whether or not they received the conditioning noise exposure. The latter result suggests that conditioning-related protection may arise from a generalized stress response, which can be elicited by noise exposure, brain surgery, or a variety of other means. The former results make an OC role in the conditioning process, per se, difficult to assess, given the large effects of OC activity on general acoustic vulnerability.
Activation of the olivocochlear (OC) efferent system in anesthetized animals has been shown in a number of laboratories to minimize the acute and temporary threshold shifts (TTSs) seen after acoustic overstimulation (Rajan 1991 Experimental groups and experimental design
Experimental animals were albino guinea pigs of either sex. Animals entered the surgery/conditioning/exposure protocol weighing between 325 and 350 g. Animals were assigned randomly to one of eight groups, as described below. Exactly 39 days after entering the protocol, the auditory function of each animal was tested via measurement of compound action potentials (CAPs) and ear-canal distortion-product otoacoustic emissions (DPOAEs) in a terminal experiment (Fig. 1, Final Test), which was followed immediately by fixation and harvesting of the cochleas and brain stem. The timing, for the different groups, of the surgical treatments (if any), conditioning noise exposure (if any) and traumatic noise exposure (if any) are schematized in Fig. 1. The number of ears in each group is given at the left. The timing of experimental procedures was designed so that the animals would all be the same age at the time of acoustic overexposure and final physiological testing. All procedures were approved by the Animal Care and Use Committee of the Massachusetts Eye and Ear Infirmary.
Deefferentation surgery
Animals to undergo chronic lesions of the OCB were anesthetized with pentobarbital sodium (17.5 mg/kg ip) and ketamine/xylazine (3.5 mg/kg im). After surgical levels of anesthesia were achieved, the skin and muscle layers over the occiput were reflected, and a small piece of the skull overlying the central portion of cerebellar cortex was removed with rongeurs. The cerebellum was elevated gently, revealing the floor of the IVth ventricle, and an anterior-to-posterior cut was made in the dorsal surface of the brain stem with a small sickle-shaped knife. In some animals (Fig. 1, Midline OCB), the cut was positioned so as to sever the crossed component of the OCB to both ears. For other animals, the lesion was positioned at one side of the dorsal brain stem (Fig. 1, Unilateral OCB), to sever the entire OCB to one side (Liberman 1990 Pretest
As illustrated in Fig. 1, all animals except those in the unilateral OCB lesion group were pretested on day 8 of the protocol, to ensure that no animal with a preexisting threshold shift was entered into the protocol. Animals to be pretested were sedated with pentobarbital (25 mg/kg ip) to allow insertion of the acoustic assembly into the ear canal. The pretest comprised the measurement of growth functions for the DPOAEs at 2f1-f2 in each ear. Pairs of equilevel primaries were used, with f2 (the higher of the 2 primary tones) set at each of eight logarithmically spaced values between 2.78 and 12.14 kHz (f2:f1 = 1.2). (Details of the DPOAE measurement techniques are described below: see Final testing of CAP and DPOAEs). Animals passed the pretest only if the 2f1-f2 DPOAEs to 40 dB SPL primaries were present (3 dB above the surrounding noise floor) at all eight f2 test frequencies.
Conditioning and traumatization
For both conditioning and traumatization, the stimulus was an octave band of noise (2.0-4.0 kHz) delivered in the free-field to unanesthetized and unrestrained animals suspended in cages (1 animal per cage division) within a small reverberant sound-exposure box (Liberman and Gao 1995 Final testing of CAP and DPOAEs
For the final test, animals were anesthetized with sodium pentobarbital (25 mg/kg ip) and fentanyl and droperidol (0.2 and 10 mg/kg im, respectively). Surgical levels of anesthesia were maintained with booster injection of pentobarbital (1/2 of original dose every 6 h) and fentanyl and droperidol (1/3 of original dose every 2 h). Surgical preparation for the terminal experiment involved insertion of a tracheostomy tube, exposing the bullas bilaterally, and severing the ear canals near the tympanic ring. The bullas were opened by shaving the bone with a scalpel blade.
Histological preparation and analysis of deefferentation
After the final test, the cochleas of each animal were harvested for processing as plastic-embedded surface preparations. Before removal from the skull, the ears were fixed by intralabyrinthine perfusion of a buffered solution of paraformaldehyde and glutaraldehyde. After overnight postfixation, the cochleas were osmicated, dehydrated, and infused with epoxy resins. After the epoxy polymerized, the cochleas were dissected into a series of pieces, each containing ~1 mm of the organ of Corti, re-embedded in plastic, thinned with sanding disks, and mounted on microscope slides.
Histological assessment
The cochlear material allowed assessment of the degree of deefferentation in the animals with brain stem lesions and also allowed assessment of the nature and extent of the hair cell and neuronal lesions.
DEGREE OF DEEFFERENTATION.
In a previous study ofchronic OCB lesions (Liberman and Gao 1995
DEGREE AND EXTENT OF COCHLEAR DAMAGE.
Although cochleas from all animals with chronic surgery were processed histologically, cytocochleograms were obtained for only a subset of the animals: i.e., one cochlea from each of the midline lesion cases (whether successful or unsuccessful). Although the extent and severity of the hair cell losses varied from case to case (as did the degree of PTS as described later), the general pattern was the same.
Physiological assessment
CAP THRESHOLDS.
Animals without chronic surgery. The conditioning exposure in our experimental paradigm was designed to be one that, by itself, does not cause cochlear damage. Indeed, as indicated in Fig. 4, average CAP thresholds for animals that received only the conditioning exposure (group C: no brain stem surgery and no traumatic exposure) were slightly lower than CAP thresholds for the control group, which received neither brain stem surgery, conditioning nor traumatic exposure. Note that these condition-only animals were tested 6 days after the end of the conditioning period, i.e., at exactly the time when the condition-trauma animals would have been exposed to the high-level noise. The possible significance of this response enhancement due to conditioning is discussed elsewhere (Kujawa and Liberman 1996
DPOAE THRESHOLDS.
For all of the ears in the present study, measures of DPOAE growth functions at 2f1-f2 also were performed as part of the final test. These growth functions, obtained at a number of f2 frequencies, were transformed into iso-response contours, analogous to the CAP iso-response contours analyzed earlier, and those DPOAE contours were averaged within animal groups as for the CAP. A consideration of the detailed relationship between changes in DPOAE response and changes in CAP response is beyond the scope of the present study. Nevertheless, it clear from the data in Fig. 9 that for the groups of animals illustrated here (the same as those illustrated in Fig. 6) the same conclusions are reached as those arising from a consideration of the CAP. Specifically, it is clear from DPOAEs as well as CAPs that successful deefferentation increases the vulnerability to the acoustic overexposure and that the conditioning exposure in deefferented animals slightly increases, rather than diminishes, the PTS from the acoustic overexposure.
OC-mediated protection from permanent acoustic injury
Most of the existing literature investigating the role of the OCB in protecting the ear from acoustic injury has been on acute, TTS-producing exposures in anesthetized animals, comparing the TTS seen with and without activation of the OCB, whether by electric shocks or addition of contralateral sound (e.g., Puel et al. 1988 Conditioning-mediated protection: an OC role or a generalized stress response?
The protective effect of repeated daily exposure to moderate-level sounds now has been demonstrated in a number of species (including rabbit, guinea pig, chinchilla, gerbil, and rat), using a number of different exposure paradigms (for review, see Canlon 1996
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Reiter and Liberman 1995
). The role of olivocochlear activation in protecting the ear in awake animals (or humans) from the permanent effects of acoustic overexposure is much less well studied, and the existing literature is somewhat contradictory (Handrock and Zeisberg 1982
; Liberman and Gao 1995
; Trahiotis and Elliott 1970
; Zheng et al. 1997a
). Nevertheless, a number of studies have suggested that chronically deefferented animals can show greater permanent threshold shifts (PTSs) than identically exposed control animals (Handrock and Zeisberg 1982
; Liberman and Gao 1995
; Zheng et al. 1997a
) .
; Subramaniam et al. 1996
). These protective effects of acoustic-exposure history have been studied in a number of different laboratories, with a number of different paradigms; however, the protocols fall into two main classes. In one type of experiment, animals are conditioned by daily exposure (for 10-21 days) to a moderate-level acoustic stimulus, which, by itself, causes no damage or minimal damage to the auditory periphery. Then, after a variable rest period (with no experimental sound exposure), the animals are given a traumatic exposure of shorter duration to an acoustic stimulus; usually, the same spectrum as the conditioning stimulus is applied at a higher sound pressure (e.g., Canlon and Fransson 1995
; Canlon et al. 1988
). The protection observed is that the condition/trauma animals show less PTS (and/or cochlear damage) than a trauma-only group exposed to the same traumatic exposure without the conditioning exposures. In a second type of experiment, there is only one type of exposure stimulus, usually a mildly traumatic one, which is delivered in daily doses (e.g., 6 h on, 18 h off) with some threshold measure obtained immediately before and immediately after the daily noise dose (e.g., Subramaniam et al. 1991a
,b
). The toughening observed in this paradigm is that thresholds measured each day, immediately postexposure, improve as the number of daily doses increases. However, in most such studies, the thresholds measured each day immediately before the exposure progressively deteriorate, and the animals demonstrate PTS (and/or permanent cochlear damage) weeks after the daily exposures are terminated (Boettcher et al. 1992
; Subramaniam et al. 1991b
). Thus the toughening consists of an observed decrease in a compound threshold shift (CTS) consisting of a small, increasing PTS and a larger, decreasing TTS component.
; Ryan et al. 1994
). One recent study has examined the role of the OC system in protection by comparing the behavior of normal and chronically deefferented animals in a hybrid paradigm involving repeated exposure to a mildly traumatic stimulus followed by a single exposure to a highly traumatic stimulus (Zheng et al. 1997a
). The study's conclusions were limited by the fact that only three successfully deefferented animals survived. The conclusions regarding an OC role in toughening were mixed. First, they showed that the decrease in CTS during the repeated exposure disappeared at some test frequencies, but not others, in the deefferented animals. Second they showed that PTS after the final exposure was greater in the deefferented animals; however, this latter result speaks to the issue of a protective role for the OC system in acoustic trauma, in general, rather than to the issue of an OC role in toughening, per se.
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 1.
Schematic illustrating the timing of experimental manipulations for different groups. Number of ears in each group is indicated at the left; data were obtained from both ears in all cases. Shorthand used to describe the groups is given to the right of the colon within each name-box. Initial letters (C, T, or CT) indicate the noise-exposure protocol (condition-only, trauma-only, or condition/trauma, respectively). Letters within the brackets indicate the surgical procedure and the success or failure of the OCB cut: X+ and X for successful and unsuccessful cuts, respectively (either midline or unilateral); O+ and O
for opposite (control) sides of successful and unsuccessful unilateral lesions, respectively. Asterisks indicate either + or
. Thus for example, the CT[X*] group consists of those ears from animals with intended OC cuts (successful or not) in the condition/trauma group, whereas T[O
] comprises those ears contralateral to the brain stem cut in those cases for which the cut was unsuccessful from animals in the trauma-only noise-exposure group.
). The side of the cut (right vs. left) was randomized among the animals in this group. In some animals, the landmarks on the brain stem surface were not sufficiently clear (due to bleeding), and no brain stem cuts were made; in others, the brain stem cuts did not section the intended pathway (see further text): both of the latter groups constitute the sham surgery groups.
). The acoustic stimulus was generated by a custom-made white-noise generator, filtered (Brickwall Filter with a 60 dB/octave slope), amplified (Crown power amp), and delivered (JBL compression driver) through an exponential horn fitted securely to a hole in the top of the reverberant box.
) .
20 and
10 dB SPL, depending on frequency.
) and the success of the cut within the brain.
). In each animal, the measurements were made over one complete dissected piece of the organ of Corti (corresponding to ~1 mm of cochlear length). In each case, the piece was chosen from the cochlear region located ~25% of the distance from the cochlear base, where the volume of efferent fascicles is greatest in control animals. Results from a previous study (Liberman and Gao 1995
) show that, in cases of incomplete OC lesion, the degree of deefferentation is uniform across the entire cochlea; thus a single sample (placed at the point of maximal normal innervation density) can estimate effectively the overall success of the lesion.
for technical details). These analyses of hair cell loss also were performed by an observer blind to the physiological results or animal groupings.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
), it was shown that the degree of deefferentation could be estimated effectively by measuring the summed diameters of all the fiber fascicles crossing to the OHCs through the tunnel of Corti. This metric of OC innervation is highly reproducible in normal ears: as shown in Fig. 2 (A and B), the normal values for the 12-kHz region of the cochlea (where the density of MOC innervation is greatest) are clustered tightly between values of 130 and 160 µm/mm.
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FIG. 2.
Assessment of the success of the deefferentation surgery by measuring olivocochlear (OC) fascicles in the tunnel of Corti after unilateral lesions (A) or midline lesions (B). Each point represents the average summed diameter of OC fascicles (in micrometers) seen per millimeter of cochlear length, as averaged over a ~1-mm piece of the organ of Corti. Control data (from ears with no brain stem lesions) are shown in both panels. Cochlear location is converted to frequency via a cochlear map based on single-fiber labeling in the auditory nerve (Tsuji and Liberman 1997 ). A: cases are classified as successful or unsuccessful based on an independent assessment of acetylcholinesterase (AChE)-stained brain stem (see text for further details). B: brain stems were not processed in these cases, thus successful (X+) vs. unsuccessful (X
) cases are differentiated based on the medial olivocochlear (MOC) fascicle analysis only.
). In making the division, we chose to be conservative and thus may have classified a few ears with partial loss of MOC fibers as unsuccessful lesions. Brain stems were not harvested from these animals, thus no independent anatomic measure of lesion success is available. Existing anatomic information on the origins and courses of the MOC innervation of OHCs suggests that a completely successful midline lesion (i.e., one that completely transects the crossed component of the OCB) should leave the LOC efferent innervation largely intact to both ears and should eliminate roughly two-thirds of the MOC innervation to OHCs. Consistent with this view, the most complete of the midline cases (Fig. 2B, X+) produces only a two-thirds reduction in MOC density.1
). A qualitative analysis of stereocilia damage suggests that there was little damage to the IHCs, whereas there was widespread damage to the OHC stereocilia extending apical and basal to the lesion focus as defined by the cytocochleogram (plotting hair cell loss only).
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FIG. 3.
Average pattern of hair cell loss for ears exposed to the traumatic stimulus. Values displayed here represent the average cytocochleogram for a sample of 21 ears including 1 of the 2 ears (selected at random) from each of the animals with the midline OC lesion (whether from the condition/trauma or the trauma-only group). This average cytocochleogram plots the percent of cells remaining in each of the 4 rows of sensory cells, as indicated in the key. For each individual cytocochleogram, all hair cells were scored, and then the average hair cell population computed over consecutive bins corresponding to 1% of total cochlear length. All the individual cytocochleograms then were averaged to produce the data shown.
).
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FIG. 4.
Average compound action potential (CAP) thresholds for animals in the control and condition-only [C] group, demonstrating that the conditioning exposure does not raise cochlear thresholds. In this and all subsequent figures, CAP threshold is defined as the sound pressure required to produce a 10-µV response. Error bars correspond to standard errors of the mean. Numbers of ears in each group are shown in the legend.
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FIG. 5.
Average CAP thresholds for animals exposed to the traumatic stimulus, either without (A) or with (B) midline deefferentation. A: data from animals without any brain stem cuts, demonstrating the protective effect of conditioning exposures. Animals in the trauma-only group [T] show more permanent threshold shift (PTS) than animals in the condition/trauma group [CT]. Control data are the same as those shown in Fig. 5. B: data from all animals with midline lesions are plotted in black, grouped according to the success of the lesion and the noise-exposure history. Data from A are reproduced in gray to facilitate comparison of threshold shifts. Error bars correspond to standard errors of the mean. Numbers of ears in each group are shown in the legend.
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FIG. 8.
Measure of the effect of conditioning on the PTS from the traumatic exposure for normal (gray squares) vs. deefferented (black squares) ears, demonstrating that, whereas conditioning provides protection in normal ears, the same conditioning exposure increased the PTS in deefferented, traumatized animals. The normal data in this figure represent the difference between the trauma only (T) and condition/trauma (CT) groups, as shown in Fig. 5A; the deefferented data represent the difference between the analogous exposure groups from the successful unilateral deefferentations (T[X+] - CT[X+]), as shown in Fig. 6A.
) and T(X
)]. To facilitate comparison, the data from no-surgery, condition/trauma (CT), trauma only (T), and control groups from Fig. 5A are superimposed (lines, no symbols). As for Fig. 5A, the PTS for any group is the difference between their mean threshold values and the control data shown by the thick gray line. The data in Fig. 5B suggest three main conclusions for animals in the midline-lesion group. First, for either noise-exposure treatment, there is little systematic difference in CAP thresholds between the deefferented (open triangles and open circles) and nondeefferented (filled triangles and filled circles) cases.2 Second, the conditioning-related protection largely has disappeared in this group of animals: there is no systematic difference in PTS between the condition/trauma group (black triangles, filled or unfilled) and the trauma-only group (black circles, filled or unfilled). Third, all chronic-surgery animals, whether conditioned or not and whether successfully deefferented or not, show CAP thresholds similar to the condition/trauma group of animals without chronic brain stem surgery: i.e., all animals that underwent the chronic surgery appear "protected" from the traumatic exposure.
] and [O
]).
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FIG. 6.
Average CAP thresholds for all animals exposed to the traumatic stimulus after unilateral brain stem lesion. Ears are divided into 8 groups according to whether they were ipsilateral ([X]: A) or contralateral ([O]: B) to the OC bundle (OCB) cut. Within each panel, they are further divided according to the noise-exposure history, condition/trauma (CT) vs. trauma only (T), and according to the success ([+]) or failure ([ ]) of the lesions. Data from successful deefferentations are shown as open symbols. Data from A are reproduced in gray to facilitate comparison of threshold shifts. Error bars correspond to standard errors of the mean. Numbers of ears in each group are shown in the legend.
]) and the condition/trauma group (CT[X+] vs. CT[X
]). The magnitude of this OC-mediated effect on PTS is illustrated in Fig. 7, in which the difference between the CAP mean threshold curves are plotted. The PTS difference between the successful and unsuccessful deefferentations peaks at ~20 dB for the condition/trauma group and 15 dB for the trauma-only group. The maximum OC-mediated protection appears at test frequencies of ~8 kHz. The apparent "anti-protective" effect seen at high frequencies among the trauma-only ears arises from a bilateral threshold elevation at high frequencies: see curves labeled T[X
] and T[O
] in Fig. 6. It could be a statistical anomaly due to the small sample size (only 3 animals in this group).
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FIG. 7.
Measures of the protection afforded by an intact OCB, expressed as the difference in average CAP thresholds between successful [X+] and unsuccessful [X ] deefferentations for both the condition/trauma (CT) groups and the trauma only (T) group. Mean CAP data for each of the four groups used to compute these 2 difference functions are shown in Fig. 6A.
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FIG. 9.
Average distortion-product otoacoustic emission (DPOAE) thresholds for the all animals exposed to the traumatic stimulus after unilateral brain stem lesion, i.e., the same 8 groups of animals shown in Fig. 6. DPOAE threshold is derived from growth functions for 2f1-f2 and is defined as the sound pressure of the equilevel primaries required to produce a DP of 5 dB SPL. Error bars correspond to standard errors of the mean.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Rajan 1988
; Reiter and Liberman 1995
). It is now abundantly clear that OC activation can decrease TTS, however, the strength of that protective effect varies with exposure frequency: it is most robust for exposures that involve cochlear frequency regions near 15 kHz (Rajan 1995
; Reiter and Liberman 1995
). A recent study from our laboratory suggested that these protective effects were related causally to the slow effects of OC stimulation (Reiter and Liberman 1995
); these effects are thought to involve slow increase in intracellular Ca2+ in the OHCs (Sridhar et al. 1995
, 1997
).
) showed that cutting the OCB in guinea pig (by cutting the inferior vestibular nerve) greatly increased the PTS seen after an exposure to a 4-kHz octave band noise at 125 dB SPL for 30 min, although there was no effect on the TTS from the same noise at 120 dB for 5 min. The PTS increased from a mean of 16 dB to a mean of 40 dB in deefferented animals. Two studies of unilateral deefferentation in chinchillas (Zheng et al. 1997a
,b
), performed by cutting the inferior vestibular nerve, also showed a dramatic increase in PTS after exposure to an octave band of noise centered at 4 kHz presented at 95 dB for 48 h, although the number of successful deefferentations was extremely small (see further). Previous work in our laboratory showed that lesions to the OCB at the midline IVth ventricle produce modest increases in vulnerability of guinea pigs to a noise band centered at 10 kHz presented at 112 dB SPL for 2 h (Liberman and Gao 1995
).
) and PTST(X+) > PTST(X
). Furthermore, a comparison of the data in Fig. 6, A and B, shows that for both noise-exposure groups, the successful unilaterally deefferented ears showed significantly more PTS than the opposite control sides: i.e., PTSCT(X+) > PTSCT(O+) and PTST(X+) > PTST(O+). Indeed, the presence of an asymmetrical pattern of PTS in a particular animal (higher thresholds on the cut side) was an excellent physiological predictor of the success of the unilateral lesion (as verified histologically). The results from the midline lesions were much less dramatic. Indeed, even those animals with successful midline cut (CT[X+] and T[X+]) showed no more PTS than their counterparts with an intact (CT[X
] and T[X
]) OC system.
; Robertson et al. 1987
). Consistent with this prediction is the observation, shown in Fig. 2, that the maximum loss of OHC efferents in our midline lesion cases was roughly two-thirds. In contrast, a cut at the side of the brain stem or in the inferior vestibular nerve should interrupt completely both the MOC and LOC systems to one ear because all fibers are intermingled in a reasonably compact bundle at these loci (Arnesen and Osen 1984
; Terayama et al. 1969
).
). The idea that the contralaterally responsive (contra) OC neurons contribute a larger protective effect than the ipsilaterally responsive (ipsi) fibers does not directly follow from any known aspect of the physiology or anatomy of the MOC system. For example, existing evidence (Brown 1989
; Liberman 1988
) suggests that the sound-evoked discharge patterns of contra and ipsi fibers are very similar and that there are no fundamental differences in the nature or extent of their cochlear projections, except that the COCB (ipsi units) projections, as a whole, are slightly skewed toward basal cochlear regions compared with the uncrossed OCB (UOCB) (contra units) projections (Guinan et al. 1984
). Nevertheless, the suggestion that the contralaterally responsive UOCB has a larger protective role than the ipsilaterally responsive COCB has some precedent in the literature from OC-mediated protection from TTS. For example, Rajan and colleagues have shown repeatedly that activation of the UOCB via contralateral sound decreases ipsilateral TTS, whereas cutting the COCB, which should interrupt all sound-evoked activation of the ipsilateral OC reflex, does not increase the TTS from a monaural exposure (Rajan 1991
). Of course, other interpretations of the latter result (and the present result) include the hypothesis that some threshold level of OC activation is necessary to provide protection and that activation of both contra and ipsi MOC reflexes are necessary to cross that threshold.
; Smith and Rasmussen 1963
). The LOC system should be relatively unaffected by the midline cuts but should be completely eliminated by a successful cut at the side of the brain stem or in the internal auditory meatus. We only assessed the tunnel-crossing fascicles, which consist, mainly or exclusively, of MOC neurons to OHCs; however, a number of lines of evidence suggest that the LOC system is equally affected. First, in cats with unilateral OCB lesions in the brain stem, we have shown, via electron-microscopy of the cochlea, that the efferent system in the inner hair cell area, (i.e., the inner spiral bundle) is missing in ears judged successfully deefferented based on tunnel-crossing MOC fascicles (Liberman 1990
). Second, the absence of an inner spiral bundle also has been noted via AChE staining of chinchilla cochleas deefferented by cutting the vestibular nerve (Zheng et al. 1997b
) and with anti-neurofilament immunohistochemistry in guinea pigs deefferented with a surgical procedure identical to that used in the present study (unpublished observations). There are no other data relevant to a possible role of the LOC system in protecting the ear from acoustic overstimulation because nothing is known about the functional effects of activating this system on the inner ear. The fact that the LOC system does not directly target the OHCs, at least not in the basal half of the cochlea, is not consistent with the notion that its activation could decrease PTS in the present study, because the DPOAE data (Fig. 9) strongly suggest that the protection involves decreasing damage to the OHCs. Nevertheless, the possibility that some of the protective effects seen here are mediated by the LOC system is impossible to rule out.
). For example, for a white-noise elicitor presented at levels up to 100 dB SPL (i.e., 30 dB re acoustic reflex threshold), there was no measurable attenuation at frequencies >2 kHz and mean attenuation for frequencies <2.5 kHz was 1 dB. Second, a study of the acoustic injury in gerbils showed that surgical removal of the MEM tendons did not change the mean PTS in animals exposed, awake and unrestrained, to a two-octave noise band (1.4-5.6 kHz) at 110 dB for 1 h (Ryan et al. 1994
). Further argument for a MOC role rather than a MEM role is the observation that the magnitude of the protective effects versus frequency (i.e., Fig. 7) is reminiscent of the density distribution of MOC terminals on OHCs as a function of cochlear location: both show a pronounced peak in the upper basal turn at cochlear regions corresponding to the 6- to 12-kHz regions (Liberman et al. 1990
).
). The paradigm we have chosen is a variant of the condition/trauma paradigm, in which the chronic conditioning stimulus, delivered 6 h/day for 10 days, does not by itself produce any permanent threshold elevations, at least in normal animals (Kujawa and Liberman 1996
). Yet when this conditioning exposure is presented in advance of a traumatic exposure, it can reduce significantly the PTS from the latter. A form of protection is also demonstrable in a very different paradigm, the repeated-exposure paradigm, in which animals are exposed on a daily basis to a mildly traumatic stimulus. In the repeated-exposure paradigm, protection is seen as a daily decrease in the acute TS measured immediately after the end of each day's exposure. However, as the daily exposures continue, the animals develop a slowly growing residual TS, as seen in from the deterioration of thresholds measured before each daily exposure, which ultimately becomes a PTS, as documented many days post exposure (Boettcher et al. 1982). Thus the protection measured in the repeated-exposure paradigm appears to be a compound threshold shift, consisting of relatively large TTS and a smaller accumulating PTS.
). Cells with damaged stereocilia would be expected to have decreased ion fluxes during the daily noise exposure and, as such, might undergo progressively less TTS each day if TTS involves acute changes in hair cell homeostasis brought on by abnormally high potassium fluxes through the transduction channels. Furthermore, the practical value of protecting the ear by inducing a mild PTS is questionable.
; Ryan et al. 1994
). There have been a few studies of the morphological changes seen in ears that have been conditioned but not yet traumatized. Canlon suggested that conditioned ears showed elaboration of membranous tubules/vesicles in the basal pole of OHCs (Canlon et al. 1991
, 1993
); however, no other structures within the cochlear duct have been examined systematically at the ultrastructural level. Our own functional study of conditioned animals, which have not been traumatized, suggested that the conditioning exposure used in this study enhances the amplitudes of the distortion-product (DP) otoacoustic emissions at 2f1-f2 (Kujawa and Liberman 1996
). Given that these DPs, at least at low sound pressure levels, primarily reflect OHC function (Mountain 1980
; Schmiedt 1984
; Siegel et al. 1982
), this observation is also consistent with the hypothesis that changes in the OHCs are induced by the conditioning.
). Each animal was exposed daily to a mildly traumatic stimulus, and DPs were measured before and after each exposure. Then, after the last of these daily exposures, each animal was exposed to the same stimulus at a much higher SPL, and final PTS was measured several days later. In one group of animals, the OCB was cut in the inferior vestibular nerve; however, there were only three successful deefferentations, and only two were carried all the way through the protocol. Nevertheless, the results are of interest, although not very clear cut. During the daily repeated exposure, control animals showed reducing CTS (i.e., protection) at three of the test frequencies, as this group has shown in numerous other studies (Subramaniam et al. 1996
). In contrast, the three deefferented animals showed reducing CTS at one only of the three test frequencies: i.e., less protection, but protection was not abolished, even though the deefferentation was essentially complete. As for the final PTSs after the high-level traumatic exposure, the two deefferented animals that completed this part of the protocol showed significantly higher PTSs than the control animals with the same noise exposure history. However, this difference probably reflects the protective effects of OC activity per se (see further text), rather than a role of the OCB in conditioning-mediated protection. To assess the latter, the experimental design would have to have included a trauma-only group: it did not.
) = PTST(X
) = PTSCT(O
) = PTST(O
) = PTSCT. Analogous results are visible in the midline-lesion group (Fig. 5), except that there is more variability in the PTS at low frequencies. One interpretation of these results is that the changes brought on by conditioning actually are mediated through a more generalized stress response to the noise and that any other treatment (including the chronic brain surgery used in the present study) that elicits the same stress response also will have a protective effect. The stressful effects of noise exposures are well known, and it has been reported recently that emotional stress decreases noise-induced TTS in guinea pigs (Muchnik et al. 1992
). Based on the existing literature on stress and noise, the types of conditioning exposures used in this and other studies could have significant effects on circulating levels of a number of hormones including epinephrine and glucocorticoids (e.g., Rarey et al. 1995
). Similarly, noise exposure is only one of many noxious stimuli that can elicit upregulation of heat-shock proteins (Lim et al. 1993
), and expression of heat-shock proteins can have a protective effect on numerous body functions (e.g., Lindquist and Craig 1988
).
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ACKNOWLEDGEMENTS |
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The assistance of L. W. Dodds in the chronic surgeries and D. F. O'Grady in the acute surgical preparation and deefferentation analysis is gratefully acknowledged as is the work of B. Eisner in the hair cell analysis.
This research was supported by National Institute of Deafness and Other Communication Disorders Grants RO1 DC-000188 and F32 DC-00180.
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FOOTNOTES |
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Present address of S. G. Kujawa: Dept. of OtolaryngologyHead and Neck Surgery, University of Washington School of Medicine and Virginia Merrill Bloedel Hearing Research Center, Seattle, WA 98195.
1
For the midline lesion cases, efferent fascicle density was only measured in one of the two ears. A previous study of this lesion in guinea pigs showed that the degree of deefferentation suggested by this metric was always symmetrical on the two sides of one case (Liberman and Gao 1995
).
2
The true sham surgeries, i.e., those in which there was no brain stem cut made, also have been considered separately from the sham surgeries in which the brain stem lesion was placed incorrectly, or not deep enough, such that no noticeable deefferentation occurred: the true shams are not significantly different from the larger nondeefferented group of animals that underwent the chronic surgery.
Address for reprint requests: M. C. Liberman, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114.
Received 27 June 1997; accepted in final form 25 August 1997.
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REFERENCES |
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