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Interaction of aminooxyacetic acid and ethacrynic acid with intense sound at the level of the cochlea
Hearing
Research, 6 ( 1982)
129
129- 140
Eisevier Biomedical Press
Interaction of aminooxyacetic acid and ethacrynic acid with intense sound at the level of the cochlea * Dennis L. I&id** Louisiana State Universi~
and Richard P. Bobbin
Medieaf Center. Kresge Hearing
of Otorhinolaryngology,
If00
Research L&oratory
of the South, Department
Florida Ave., Bldg. 147, New Orleans, L.A 70119, U.S.A.
(Received 6 April 1981; accepted 15 July 198I)
Results of previous investigations of the interaction of intense sound and drugs have, in general, failed to show a protective effect mediated by pre-admi~stration with a drug having transient ototoxic effects. The present investigation was designed to further evaluate a protective effect found previously at the anatomical level and explained with an electrochemical theory of noise damage. The alternating current (a.c.) potential and compound eighth nerve action potential (CAP) amplitude were monitored in aminooxyacetic acid (AOAA)- or ethacrynic acid (PA)-treated guinea pigs exposed to either moderate or high levels of intense sound and compared to changes observed in the same potentials in animals exposed to the intense sounds alone. Results showed protective effects only in the moderate-intense sound-exposure groups, with changes in sensitivity and voltage on the Linear part of the input-output curve of the a.e. cochlear potential found to be the only conditions where differences occurred. These results were difficult to interpret in terms of a protective effect and point to the need for obtaining additional data before an electrochemical mechanism is shown to play a role in the effect of intense sound on the cochlea. Key words: aminooxyacetic acid; ethacrynic acid; intense sound; cochlear potential.
Intraduction It has been suggested that the reduction of hair-cell scarring seen in a group of animals treated with aminooxyacetic acid (AOAA) followed by intense sound as compared with the scarring observed in a group treated with saline followed by intense sound [Z] was neither the result of hypothermia nor the elimination of the facilitation of the middle-ear transfer mechanism [3]. Instead, the conclusions seemed to support a metabolic theory which purports that a reduction in the endocochlear potential (EP) mitigates damaging effects of intense sound [2]. It should be noted, however, that only the light-microscopic surface preparation view of hair cells was monitored. Other effects of intense sound, such as ultrastructural * Data taken from dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy by D.L. Kisiel. l * Present address: The Cleveland Hearing and Speech Center, Cleveland, Ohio, U.S.A. 0378-5955/82/~-~/~2.7~
8 1982 Ehevier Biomedical Press
130
changes, reductions in the alternating current (a.c.) cochlear potential and the compound eighth nerve action potential (CAP), or changes in behavioral thresholds were not examined. Moreover, reduction in hair-cell scarring observed with AOAA pre-treatment may have been an irrelevant finding because of a low correlation between hair-cell scarring and cochlear functioning [ 131. Since the cause of the reduced hair-cell scarring with AOAA pre-treatment is uncertain, it was considered necessary to evaluate the effect of AOAA as well as that of an additional drug, ethacrynic acid (EA) which causes a greater reduction in EP than does AOAA [ 1,181, by employing a possibly more valid and sensitive index of cochlear functioning than behavioral thresholds or hair-cell scarring. It is noted that electrophysiological measures are considered to be the most valid and sensitive indices of damage caused by intense sound [ 13). Therefore, this study proposed to investigate the electrophysiological effects of intense sound on the cochlea by examining changes in the a.c. cochlear potential and the CAP induced by intensesound exposure.
Materials and Methods Subjecrs
Male and female pigmented guinea pigs weighing between 200 and 500 g were used. Animals were not accepted into the study unless they had normal bilateral Preyer reflexes and translucent eardrums upon otoscopic examination Surgical preparation
Atropine methyl nitrate (Atro Dote”, Hart-Delta, 2 mg/kg, i.m.) was administered 30 min prior to anesthesia delivery to reduce salivation and mucosecretions during the surgical procedure. Guinea pigs were anesthetized with sodium pentobarbital (Nembutal”, Abbott, 25 mg/kg, i.p.). Additional quantities were adminis’tered intravenously throughout the experiment. Rectal temperature was monitored every 15 min by a thermocouple and maintained between 38.5O and 40°C with the aid of a heating pad. The external jugular vein divascannulated for drug injection. A post-auricular surgical approach was used to expose the round window [27]. A silver wire electrode insuIated with Teflon, except at the balled tip, was used to monitor both the CAP and the a-c. cochlear potential from the round window. A needle electrode inserted into the, contralateral superficial masseter muscle served as the ground electrode. Upon completion of the electrophysiological recordings described in the ‘Experimental protocol’ section, the electrode was removed from the middle ear cavity and the hole in the bulla closed with dental acrylic. Antibiotic therapy consisting of administration of oxytetracycline (Liquamycina, Pfizer, 50 mg/kg, i.m.) was carried out every 24 h for 48 h post-surgery.
131
Experimental
protocol
Znstrumentation
Upon completion of the surgical procedure, animals were placed in an electrically shielded sound-proof (IAC) room. Tone bursts of 4, 5 and 6 kHz, 10 ms in duration, and 1 ms rise/fall time, delivered to the ear at the rate of one per second, were used to elicit the CAP since the maximum depression of the CAP has been noted to occur from the center frequency of a noise band to one-half octave above the exposure frequency [21]. Similar tone bursts of 3,4,5 and 6 kHz were used to elicit a.c. cochlear potentials. A 3 kHz stimulation tone was added to the protocol since the frequency of maximum depression of the ac. cochlear potential may appear below the exposure frequency [23]. Sounds were delivered to the external auditory meatus through a hollow ear bar, closed sound system. Both spectra and SPL at maximum intensity of the stimuli were measured near the eardrum in decibels (re 0.0002 pbar) using a condenser microphone (Brtiel and Kjaer, model 4133) and sound level meter. The second harmonic of the stimulating frequencies 3,4,5 and 6 kHz were down 10, 14, 18 and 15 dB from the fundamental, respectively. The second harmonic of the 4 kHz exposure frequency was down 22 dB from the fundamental. a.c. cochlear potentials and the CAP were selectively filtered out of the round window recordings by using an appropriate band-pass characteristic. The a.c. cochlear potential and the CAP were monitored simultaneously on the display unit of an oscilloscope and were photographed on 35-mm film from the display unit of the oscilloscope. Definitions
of electrophysiological
measures
Measurements of the amplitude in microvolts (pV) were made for the following points of the CAP waveform: N,, peak of the first negative wave of the CAP; P,, peak of the first positive deflection following N,; CAP amplitudes were defined as: N,P,, N, amplitude relative to P,. In addition to CAP magnitude, a.c. cochlear potential amplitude was obtained by stimulating the ear with intensities ranging from 40 to 140 dB SPL (re 20 PPa) in 5 dB steps. The following terminology was used for the dependent and pseudo-independent variables [7] which were evaluated in a descriptive manner. Changes in the dB SPL which elicit visual detection threshold values for the a.c. cochlear potential and CAP were referred to as shifts in sensitivity. Changes in the intensity level of the stimulation tone which elicited the maximum a.c. cochlear potential and CAP amplitudes were referred to as shifts in dB SPL for CAP or a.~. cochlear potential max. Changes in maximum amplitude of both the a.c. cochlear potential and CAP were referred to as changes in maximum voltage. To quantitatively analyze the data we chose data points which were on the linear portion of all the input-output curves with the exception of the CAP curves obtained after maximum exposure which had several values at the noise level of the recording system (20 PV). The data points were arbitrarily chosen as the output (ruv)
132
at 110 dB SPL for the CAP and at 90 dB SPL for the a.c. cochlear potential. The former measure was referred to as the CAP-110 and the latter as U.C. cochlear potential-90. Analysis of variance and Duncan’s test for multiple comparisons were used to test for significant differences at these points [ 121. Drugs and intense-sound-exposure
administration
A base-line recording of all amplitude and latency functions was obtained immediately prior to drug administration for all animals to assess the integrity of the middle and inner ears of the experimental animals and to ensure proper placement of the round window electrode and ear bar. Animals were not accepted into the study if their maximum a.c. cochlear potential output at 3 kHz was less than 1800 pV. Following base-line recordings of electrophysiological potentials, dosages of saline, AOAA or EA were administered to all animals depending upon treatment group. Guinea pigs were randomly assigned to one of ten treatment groups: AOAA + 0 group administered AOAA; group administered EA; EA+O the saline-treated group with the short-term post-treatment reSA(EA) + 0 cording schedule identical to the corresponding EA-treated group’s schedule; the saline-treated group with the short-term post-treatment reSA(AOAA) + 0 cording schedule identical to the corresponding AOAA-treated group’s schedule. AOAA + 10 and two groups exposed to different levels * of intense sound and AOAA + 25 administered AOAA (AOAA-treated-intense-sound-exposure groups); EA $I 10 and EA+25
two groups administered
SA + 10 and SA-I-25
exposed to different levels * of intense EA (EA-treated-intense-sound-exposure
sound and groups);
two groups exposed to different levels * of intense sound and administered saline (saline - treated - intense - sound - exposure groups). EA (Sodium Edecrin@; Merck, Sharp and Dohme, 10 mg/ml, 50 mg/kg, iv.) was administered over a 1 min period to the EA + 0, EA + 10 and the EA + 25 groups. The 1 mm period was chosen ,to ensure rapid uptake of the drug but prevent a massive cardiac reaction caused by a more rapid injection rate. Numerous investigators have shown this dosage of EA to cause the EP to reach negative values comparable to those observed in early anoxic stages [ 181. AOAA (Sigma, 4 mg/ml, 20 mg/kg, i.v.) was administered over a 1 mm period to the AOAA + 0, AOAA + 10 and the AOAA + 25 groups. Saline, an equal volume/kg, was administered intravenously to the SA(EA) + 0, SA(AOAA) + 0, SA + 10 and SA + 25 groups. l
See p. 137 for definition of levels.
133
45 min after AOAA administration and 15 min after EA or saline administration, the drug-treated-intense-sound-exposure groups were exposed to a 4-kHz tone for 30 min using the same instrumentation employed to elicit the electrophysiological potentials. The frequency of the exposure stimulus and duration of exposure were chosen to duplicate the corresponding measures in an earlier study [2]. The particular temporal relationships between drug administration and intense-sound exposure were selected in order that the peak effect of the drugs occurred during the period of intense-sound exposure [l]. Two different intensity levels of the exposure stimulus were used to provide further information concerning possible reduction in a.c. cochlear potential and CAP losses for the AOAA-or EA-treated groups by quantifying these losses in terms of sound attenuation in dB of the exposure stimulus. In addition, different mechanisms of the effect of intense sound on the cochlea may be disclosed by using SPLs known to affect differentially the a.c. cochlear potential and the CAP. Exposure stimulus levels were detemined in relation to maximum output capabilities of each animal’s cochlea. This method of ‘tailoring’ the exposure signal intensity level has, been used successfully by Price [23] and others [21] to reduce the variability seen in the a.c. co&ear potential obtained from animals exposed to high-intensity acoustic stimuli. For example, if the intensity of the signal is at a sound level 5 dB above that necessary to elicit the maximum a.c. cochlear potential, the experimental condition would be called ‘Max + 5’. Utilizing this paradigm, the two intensity levels of the CkHz tone-exposure stimulus chosen for this investigation were ‘Max + 10 and ‘Max + 25’ and labelled Drug + 10 and Drug + 25, respectively. The individual drug-treated-intense-sound-exposure groups were thus labelled as follows: AOAA -t 10; AOAA + 25; SA f 10; SA + 25; EA -t 10; and EA + 25. Potentials were monitored immediately after the intense-sound exposure in all drug-treated-intense-sound-exposure groups (short-term post-treatment recordings). The short-term post-treatment recording of the AOAA- and SA(AOAA)-treated groups’ potentials were taken at the same time as the corresponding recordings in the AOAA-treated-intense-sound-expos~e group or, I h and 15 min post-drug administration. The short-term post-treatment recording of the EA- and SA(EA)treated groups’ potentials were taken at the same time as the corresponding recordings in’ the EA-treated-intense-sound-exposure group or 45 min post-drug administration. The dissimilar recording times were related to the different periods of peak effect of EA and AOAA. Immediately after the short-term post-treatmet recording, the wound was closed as described above and the animal was removed from the sound suite and placed on a heating pad. Rectal temperature was monitored every 15 min until the animal’s righting reflex returned. At this time, the guinea pig was returned to animal care facilities. At 48 h post-base-line recording, the animal was returned to the sound suite for long-term post-treatment recordings. Pre-operative medication, anesthesia and recording protocol were identical to the conditions obtained at base line.
134
Results
While there are numerous group-paired comparisons that can be made, the following discussion will be confined to those comparisons that are germane to the experimental hypothesis. The relationships between groups at the short-term posttreatment recording interval will not be discussed since the effect of the drugs on the cochlear potentials at this interval were still apparent, thus masking out any possible protective effect from intense sound. At the long-term post-treatment recording interval (third recording), the effects of AOAA and EA on the measures discussed were similar to the comparable effects of the control vehicle, saline. Therefore, any possible protective effect afforded the animal during the intense-sound exposure MAX
MAX + lW&
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00
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.
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Fig. I. Effect of combinations of saline, AOAA. EA and intense sound on the input-output functions of the a.c. cochlear potential (CM) and the CAP evoked by 4-kHz tone bursts. The curves were obtained at the long-term post-treatment recording after the administration of saline alone (0). saline plus the intense tone (0) or drug plus intense tone (X). The effects of both the Max+ IO dB and Max+25 dB intense tone exposure are shown. Only data points above the noise level of the recording system (20 uV) were used in calculating the values in order IO reduce distortion of the curves. Drug curves represent mean data (n = 5) taken from 5 animals and saline alone curves (n = 10) from 10 animals, except ,x=4 in EA + 25 and SA+ 25, each of which had one animal with all values at noise level.
135 TABLE I EFFECT OF COMBINATIONS OF SALINE, AOAA, EA AND INTENSE SOUND ON THE a.c. COCHLEAR POTENTIALS AND CAP EVOKED BY TONE BURSTS OF DIFFERENT FREQUENCIES MONITORED AT THE LONG-TERM POST-TREATMENT RECORDING Tone burst frequency: 3kHz a.~. cochlearpo~enlial-90” SA+O 2032* 908= SA+ IO AOAA+ 10 1516k EAt 10 16422 SA+25 2122 AOAA+25 366= EA+25 244= CAP-110= SA+O SA+ 10 AOAACIO EA+ 10 SA+25 AOAA + 25 I%+25
20 104 108 97 51 37 31
4kHz
5 kHz
6 kHz
1126-120 448k60 1022*96 948-c41 171 -t41 216zt24 167* 19
1 l&k24 584*51 860268 838255 126-c29 195223 1282 16
455rt 7 251 k31 494 * 50 428*23 64k 14 118-tl4 592 7
400*13 2691 9 272=35 294228 96-c I5 82= 5 82212
369* 11 188” 12 229230 210224 85~ 9 so* 5 82111
2282 5 110-c 8 162220 18lkl9 48* 12 542 3 68k 9
’ Shown are the means (i;v) -t SE with n = 5 for each mean except for SA f 0 where n = 10.
would be realized at this interval. The two control groups (SA(EA) + 0, SA(AOAA) + 0) were virtually identical for all measures at every recording interval, therefore the data were pooled in Fig. 1 and Table I and called ‘SA + 0’. Comparison of effect of infense sound on a.c. cochlear potential in groups SA i- 10, EA + 10 and AOAA f 10 at the third recording interval Shifts in sensitivity for the SA + 10 group, while small compared to the corresponding shifts in the SA + 25 group, were larger than the corresponding shifts in the AOAA i 10 and EA + IO groups (see Fig. 1 for 4 kHz). The changes in sensitivity for the SA f 10 group ranged from 7 to 10 dB, while the corresponding changes for the AOAA + 10 and EA + 10 groups ranged from 2 to 8 dB in the former group and from 0 to 3 dB in the latter group. Shifts of dB SPL for a.c. cochlear potential max were similar for all three groups. There were no appreciable differences in a.c. cochlear potential maximum voltage at 48 h post-treatment with the exception of 4 kHz where the reduction in maximum voltage for the AOAA + 10 group appeared greater than the corresponding reduction for the SA -f- 10 group. The a.c. co&ear potential-90 value for the SA + 10 group was significantly (P < 0.05) smaller than the SA + 0 group at every frequency (Table I). Except for 5 kHz (EA + 10 vs. SA(EA) + 0, P = 0.019; AOAA + 10 vs. SA(EA) + 0, P = O-022), at no other frequency was the a.c. co&ear potential-90 for the AOAA + 10 or the EA + 10 groups significantly different from the SA + 0 groups (P > 0.05). The a.c.
136
potential-90 value for the SA + 10 group was significantly smaller than the AOAA i- 10 and the EA + 10 group values at 3 kHz (P < 0.02) and at 4 kHz (P
Comparison of effect of intense sound an a.~. cochfear potential in groups SA + 25, EA + 25 and A UAA -t- 25 at the third recording interval Shifts in sensitivity from base-line to 48 h post-treatment were generally equivalent as were shifts in dB SPL for a.c. cochlear potential max and reductions in a.c. cochlear potential maximum voltage (see Fig. 1 for 4 kHz). The a.c. cochlear potential-90 value was reduced to a greater extent in SA 4 25, AOAA + 25 and EA + 25 groups compared to SA + 10, AOAA + 10 and EA 4 10 groups for each frequency (Table I). At no frequency did this measure in either AOAA + 25 or EA + 25 groups differ significantly (P > 0.05) from that of the SA + 25 groups (Table I). Comparison of effect of intense sound on CAP measures in groups SA + IO, EA + 10 and AOAA + JO at the third recording interval There were no marked differences in sensitivity from base line to 48 h for the SA + 10, EA + 10 and AOAA + 10 groups (see Fig. 1 for 4 kHz). In addition, the shifts in dB SPL for CAP max were no larger for the SA + 10 group than for the corresponding shifts for the AOAA -t- 10 and EA + 10 groups at any frequency. Reductions in CAP maximum voltage from base-line to 48 h were similar for all groups at all frequency-measure combinations except at 4 kHz (AOAA and EA) and 6 kHz (EA only) where SA + 10 losses of maximum voltage were greater than analogous losses in drug-intense-sound-exposure groups, The CAP- 110 value was reduced in SA + 10 group compared to the SA + 0 group at the P = 0.20 level for 4 kHz, at the P = 0.06 level for 5 kHz, and at the P = 0.17 level for 6 kHz (Table I). Also, no significant difference ( P > 0.05) was found between the CAP- 110 values for the SA -t- 0, SA + 10, EA t 10 and AOAA + 10 groups within each frequency. Comparison of effect of intense sound on CAP measures in groups SA + 25, EA + 25 and AOAA + 25 at the third recording interval Shifts in sensitivity for the AOAA + 25 and EA -t 25 groups were comparable to the shifts in sensitivity for the SA,+ 25 group (see Fig. 1 for 4 kHz). Shifts in dB SPL for CAP max and reductions in maximum voltage for the SA + 25 group were not appreciably different from the AOAA -t 25 group or the EA + 25 group at any frequency. The CAP-l 10 value was reduced to a significantly (P< 0.05) greater extent in the SA + 25 group when compared to the SA + 0 group for each frequency (Table I), However, the CAP-I 10 value did not significantly differ (P > 0.05) between the SA + 25, AOAA + 25 and EA + 25 groups.
137
Discussion
While there is ample documentation of the deleterious effect of intense sound on the cochlea, numerous issues such as the effect of high-intensity stimuli on cochlear vasculature and the authenticity of the mechanical theory remain controversial [2,14,25,28]. In addition, previous studies in the literature attempting to delineate the effect(s) of intense sound on the cochlea using an intense-sound-drug interaction paradigm have been fraught with ambiguities due to varying drug dosages, infusion rates, constancy of infusion, and the period of time between acoustic and drug trauma and histological examination [26]. In a study designed to evaluate a theory of the effect of intense sound on the cochlea, it was shown that pre-treatment with AOAA caused a reduction in the hair-cell scarring observed in ears exposed to injurious sound levels alone [il. The authors attributed the protective action of AOAA to the drug-related reduction of EP purportedly reducing current flow and prevention of depletion of energy stores (ATP). On the other hand, others [26] failed to reveal any interaction between intense-sound exposure and EA, a drug which also reduces the EP. The latter investigation was designed to maximize any possible potentiation effect of the combination of drug and intense sound, rather than to examine a possible protective effect afforded the ear by pre-treatment with the diuretic. The current investigation, as reported in a preliminary report [3], showed few appreciable protective effects afforded the cochlea by pre-administration of either AOAA or EA. Comparisons of dB SPL for CAP and a.c. cochlear potential max and CAP and a.c. cochlear potential maximum voltage for the SA + 10, EA + 10, AOAA + 10, SA + 25, EA + 25 and AOAA + 25 groups showed no marked differences. On the other hand, shifts in a.c. cochlear potential-90 and a.c. cochlear potential sensitivity at 48 h were larger for the SA + 10 group than for the corresponding changes in the AOAA + 10 and EA + 10 groups. These differences in shifts in a.c. cochlear potential-90 and a.c. cochlear potential sensitivity are difficult to interpret in that they may be due to either a permanent middle or inner ear conductive loss, the interference effect acting in the cochlea, or a temporary reversible fatigue condition [7,10,29,30]. As discussed by Durrant [l 11,it is impossible on an a priori basis to determine the contributions of each component to the composite shift. Since the a.c. cochlear potential is generally considered a precursor for normal amplitude CAP, changes in the former measure are generally considered to be reflected in the CAP potentials [7]. The fact that there were no marked differences in sensitivity for the CAP measures in the SA + 10 group further clouds the interpretation of changes in the a.c. cochlear potential. If the shifts in the a.c. cochlear potentials are interpreted to mean that pretreatment with AOAA or EA has significantly reduced the effect of intense sound on cochlear potentials, one interpretation may be that change(s) in the EP produced by the drugs accounted for the results [2]. In other words, the drug-induced reduction in the EP may have reduced the voltage which drove abnormally excessive and toxic amounts of depolarizing current (potassium or calcium ion) [8,16,19] from Scala media into the hair cell during sound exposure. The decrease in Scala media
138
potassium ion monitored by Snow et al. [24] and Melichar et al. [20] during intense sound exposure may be taken as evidence for the occurrence of excessive current flow. Additional evidence for an excessive current flow may be found in the data of Bohne [4,5] who has proposed that intense sound creates openings in the reticular lamina through which s&ala media fluid, endolymph, is allowed to pass and surround cells of the organ of Corti where it acts to distroy them. Bohne [6] has shown that an artificial endolymph containing high potassium, low sodium ion concentrations destroyed hair cells when placed in Scala tympani and therefore in contact with the base of the hair cells. One interpretation of these artificial endolymph studies is that the high potassium ion solution caused excessive depolarization of the hair cells which resulted in cell death. The question of interest here is whether cell death is caused by excessive depolarization due to the entrance of ions from Scala media directly into the hair cell membrane without a requirement for openings in the reticular lamina. Attributing excitable tissue death to excessive depolarization is not a novel theory and has been proposed as the basis for the neurotoxicity of the excitatory amino acids [22]. However, further evidence must be obtained before excessive depolarization together with EP involvement, the electrochemical theory proposed earlier [2], is accepted as having a role in some forms of noise damage in the cochlea. Another plausible explanation of the effect of EA or AOAA may be that they produced a temporary reduction of protein synthesis and secretion since EA has been shown to inhibit these and other cellular functions of adenylcyclase 1151.Related alternative theories involving a variety of cochlear metabolites, such as lipids, proteins, sugars, and glycoproteins, are too numerous to be discussed. On the other hand, if the changes in a.c. cochlear potentials do not reflect a reduced effect of intense sound, then there are a number of possibilities for the conflict between results reported in this paper and the previous one [2] where a reduced scarring was reported. First, body temperature was controlled in the present investigation, thus eliminating a possible mechanism for protection from intensesound exposure. Another possible explanation is linked to the time(s) of monitoring of the effects of intense-sound treatment; immediately after and 48 h post-treatment in the present investigation and 21 days post-treatment in the previous study [2]. A third argument is based on the inadequacies of the round window recording site, since the voltage from the second cochlear turn at suprat~eshold levels is attenuated relative to the local activity viewed by the round window electrode [9]. Another is related to the surface preparation of the organ of Corti. Others [ 171 have demonstrated alterations in hair-cell organelles not evident in surface preparations. In summary, the present investigation found an interaction between AOAA, EA, and intense sound in terms of a-c. cochlear potential-90 and a.c. co&ear potential sensitivity. These results raise questions regarding the relationship between hair-cell function and the a.c. cochlear potentials. It seems that these questions will have to be answered and additional data obtained before an electro-chemical mechanism is proven to play a role in noise damage.
139
Acknowledgments This research was supported in part by a grant from the Deafness Research Foundation. Laboratory facilities were provided by a grant from The Kresge Foundation. Thanks are due to Dr. E. Moore who provided the initial arrangements and guidance for this study and whose support was maintained throughout. The
authors also wish to thank Drs. Beasley, Irwin, Kahane, Wark, Berlin and Tilley for their support in various stages of this project. Drs. Cullen and Hughes, and Mr. Stephen Winbery have contributed to this work and are thanked for their efforts.
References 1 Bobbin, R.P. and Gondra, M.I. (1973): Effect of intravenous aminooxyacetic acid on guinea pig cochlear potentials, Neuropharmacology 12, 1005- 1007. 2 Bobbin, R.P., Guth, M.Si and Mines, A.B. (1976): An examination of an electrochemical mechanism for noise-induced hair cell loss: Noise with aminooxyacetic acid (AOAA). Trans. Am. Acad. Ophthalmol. Otolaryngol. 82, 299-304. 3 Bobbin, R.P. and Kisiel, D.L. (1980): Interaction of intense sound with two drugs which reduce the endocochlear potential. In: Noise as a Public Health Problem, pp. 666-670.. Editors: J.V. Tobias, G.Jansen and W.D. Ward. The American Speech-Language-Hearing Association, Rockvilie, Md. 4 Bohne, B.A. (1971): Scar formation in the inner ear following acoustical injury: sequence of changes from early signs of damage to healed lesion. Ph.D. dissertation, Washington University, St. Louis, MO. 5 Bohne, B.A. (1974): Mechanism or continuing degeneration in the organ of Corti. J. Acoust. Sot. Am. 55, S77-S78. 6 Bohne, B.A. (1976): Mechanisms of noise damage in the inner ear. In: Effects of Noise on Hearing, pp. 41-67. Editors: D. Henderson, R.P. Hamemik, D.S. Dosanjh and J.H. Mills. Raven Press, New York. 7 Benitez, L.D., Eldredge, D.H. and Templer, J.W. (1972): Temporary threshold shifts in chinchillas: Electrophysiological correlates. J. Acoust. Sot. Am. 52, 1I 15-l 123. 8 Corey, D.P. and Hudspeth, A.J. (1979): Ionic basis of the receptor potential in a vertebrate hair cell. Nature (London) 281,675-677. 9 Dallas, P. (1973): The Auditory Periphery: Biophysics and Physiology. Academic Press, New York. 10 Davis, H. et al. (1953): Acoustic trauma in the guinea pig. J. Acoust. Sot. Am. 25, 1180-l 189. I I Durrant, J.D. (1976): Effects of noise on cochlear potentials. In: Effects of Noise on Hearing, pp. 179-197. Editors: D. Henderson, R.P. Hamernik, D.S. Dosanjh and J.H. Mills. Raven Press, New York. 12 Edwards, A.L. (I 964): Experimental Design in Psychological Research. Holt, Rinehart and Winston, New York. I3 Eldredge, D.H.. Mills, J.H. and Bohne, B.A. (I 973): Anatomical, behavioral, and electrophysiological observation on chinchillas after long exposures to noise. Adv. Otorhinolaryngol. 20, 64-81. I4 Hawkins, J.E. Jr., Johnsson, L.G. and Preston, R.G. (1972):Cochlear microvasculature in normal and damaged ears. Laryngoscope 82, 1091-I 104. I5 Kerr, T.P. and Schacht, J. (1975): Adenyl cyclase in the guinea pig cochlea. J. Acoust. Sot. Am. 57, S61. 16 Leng, G. (1980): Davis theory: A review, and implications of recent electrophysiological evidence. Hearing Res. 3, 17-25. 17 Lim. D.J. and Melnick, W. (1975): Mode of cochlear damage by excessive noise-an overview. Agard Conference Proceedings, No. 171, Agard, Washington, D.C. I8 Matschinsky, F.M. and Thalmann, R. (1970):Energy metabolism of the co&ear duct. In: Biochemical Mechanisms in Hearing and Deafness, pp. 265-288. Editor: M. Paparella. Charles C. Thomas, Springfield, 111.
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19 McGlone, F.P., Russell, I.J. and Sand, 0. (1979): Measurement of calcium ion concentration in the lateral line cupulae of Xenopus laevis. J. Exp. Biol. 83. 123- 130. 20 Melichar, I., Syka, J. and Ulehlova, L. (1980): Recovery of the endocochlear potential and the K + concentrations in the cochlear fluids after acoustic trauma. Hearing Res. 2. 55-63. 21 Mitchell, C., Brummett, R. and Vernon, J. (1977): Frequency effects of temporary N, depression following acoustic overload, Arch. Otolaryngol. 103, I 17- 123. 22 Olney, J.W. (1978): Neurotoxicity of excitatory amino acids. In: Kainic Acid as a Tool in Neurobiology, pp. 95-122. Editors: E.G. McGeer, J.W. Olney and P.L. McGeer. Raven Press, New York. 23 Price, G.R. (1968): Functional changes in the ear produced by high intensity sound. I. 5.0 kHz stimulation. J. Acoust. Sot. Am. 44, 1541- 1545. 24 Snow, J., Nakashima, T., Suga, F., Leonard, J.E., Sullivan, M.J. and Meiring, N.L. (1971): Pathophysiologic response of the inner ear to intense sound. Ann. Otol. Rhinol. Laryngol. 80. 871-880. 25 Spoendlin, H. (1976): Anatomical changes following noise exposure. In: Effect of Noise on Hearing, pp. 69-89. Editors: D. Henderson, R.P. Hamernik, D.S. Dosanjh and J.H. Mills. Raven Press, New York. 26 Vernon, J., Brummett, R. and Brown, R. (1977): Noise trauma induced in the presence of loopinhibiting diuretics. Trans. Am. Acad. Ophthalmol. Otolaryngol. 84, 407-419. 27 Vernon, J. and Meikle, M.B. (1974): Electrophysiology of the cochlea. In: Bioelectric Recording Techniques, pp. 3-60. Editors: R. Thompson and M. Patterson. Academic Press, New York. 28 Ward, W.D. and Duvall, A.J. III (1971): Behavioral and ultra-structural correlates of acoustic trauma. Ann. Otol. Rhinol. Laryngol. 80. 881-896. 29 Wever, E.G., Bray, C.W. and Lawrence, M. (1940): The interference of tones in the cochlea. J. Acoust. Sot. Am. 12, 268-280. 30 Wever, E.G. and Lawrence, M. (1941): Tonal interference in relation to tonal injury. J. Exp. Psychol. 29. 283-295.
Research, 6 ( 1982)
129
129- 140
Eisevier Biomedical Press
Interaction of aminooxyacetic acid and ethacrynic acid with intense sound at the level of the cochlea * Dennis L. I&id** Louisiana State Universi~
and Richard P. Bobbin
Medieaf Center. Kresge Hearing
of Otorhinolaryngology,
If00
Research L&oratory
of the South, Department
Florida Ave., Bldg. 147, New Orleans, L.A 70119, U.S.A.
(Received 6 April 1981; accepted 15 July 198I)
Results of previous investigations of the interaction of intense sound and drugs have, in general, failed to show a protective effect mediated by pre-admi~stration with a drug having transient ototoxic effects. The present investigation was designed to further evaluate a protective effect found previously at the anatomical level and explained with an electrochemical theory of noise damage. The alternating current (a.c.) potential and compound eighth nerve action potential (CAP) amplitude were monitored in aminooxyacetic acid (AOAA)- or ethacrynic acid (PA)-treated guinea pigs exposed to either moderate or high levels of intense sound and compared to changes observed in the same potentials in animals exposed to the intense sounds alone. Results showed protective effects only in the moderate-intense sound-exposure groups, with changes in sensitivity and voltage on the Linear part of the input-output curve of the a.e. cochlear potential found to be the only conditions where differences occurred. These results were difficult to interpret in terms of a protective effect and point to the need for obtaining additional data before an electrochemical mechanism is shown to play a role in the effect of intense sound on the cochlea. Key words: aminooxyacetic acid; ethacrynic acid; intense sound; cochlear potential.
Intraduction It has been suggested that the reduction of hair-cell scarring seen in a group of animals treated with aminooxyacetic acid (AOAA) followed by intense sound as compared with the scarring observed in a group treated with saline followed by intense sound [Z] was neither the result of hypothermia nor the elimination of the facilitation of the middle-ear transfer mechanism [3]. Instead, the conclusions seemed to support a metabolic theory which purports that a reduction in the endocochlear potential (EP) mitigates damaging effects of intense sound [2]. It should be noted, however, that only the light-microscopic surface preparation view of hair cells was monitored. Other effects of intense sound, such as ultrastructural * Data taken from dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy by D.L. Kisiel. l * Present address: The Cleveland Hearing and Speech Center, Cleveland, Ohio, U.S.A. 0378-5955/82/~-~/~2.7~
8 1982 Ehevier Biomedical Press
130
changes, reductions in the alternating current (a.c.) cochlear potential and the compound eighth nerve action potential (CAP), or changes in behavioral thresholds were not examined. Moreover, reduction in hair-cell scarring observed with AOAA pre-treatment may have been an irrelevant finding because of a low correlation between hair-cell scarring and cochlear functioning [ 131. Since the cause of the reduced hair-cell scarring with AOAA pre-treatment is uncertain, it was considered necessary to evaluate the effect of AOAA as well as that of an additional drug, ethacrynic acid (EA) which causes a greater reduction in EP than does AOAA [ 1,181, by employing a possibly more valid and sensitive index of cochlear functioning than behavioral thresholds or hair-cell scarring. It is noted that electrophysiological measures are considered to be the most valid and sensitive indices of damage caused by intense sound [ 13). Therefore, this study proposed to investigate the electrophysiological effects of intense sound on the cochlea by examining changes in the a.c. cochlear potential and the CAP induced by intensesound exposure.
Materials and Methods Subjecrs
Male and female pigmented guinea pigs weighing between 200 and 500 g were used. Animals were not accepted into the study unless they had normal bilateral Preyer reflexes and translucent eardrums upon otoscopic examination Surgical preparation
Atropine methyl nitrate (Atro Dote”, Hart-Delta, 2 mg/kg, i.m.) was administered 30 min prior to anesthesia delivery to reduce salivation and mucosecretions during the surgical procedure. Guinea pigs were anesthetized with sodium pentobarbital (Nembutal”, Abbott, 25 mg/kg, i.p.). Additional quantities were adminis’tered intravenously throughout the experiment. Rectal temperature was monitored every 15 min by a thermocouple and maintained between 38.5O and 40°C with the aid of a heating pad. The external jugular vein divascannulated for drug injection. A post-auricular surgical approach was used to expose the round window [27]. A silver wire electrode insuIated with Teflon, except at the balled tip, was used to monitor both the CAP and the a-c. cochlear potential from the round window. A needle electrode inserted into the, contralateral superficial masseter muscle served as the ground electrode. Upon completion of the electrophysiological recordings described in the ‘Experimental protocol’ section, the electrode was removed from the middle ear cavity and the hole in the bulla closed with dental acrylic. Antibiotic therapy consisting of administration of oxytetracycline (Liquamycina, Pfizer, 50 mg/kg, i.m.) was carried out every 24 h for 48 h post-surgery.
131
Experimental
protocol
Znstrumentation
Upon completion of the surgical procedure, animals were placed in an electrically shielded sound-proof (IAC) room. Tone bursts of 4, 5 and 6 kHz, 10 ms in duration, and 1 ms rise/fall time, delivered to the ear at the rate of one per second, were used to elicit the CAP since the maximum depression of the CAP has been noted to occur from the center frequency of a noise band to one-half octave above the exposure frequency [21]. Similar tone bursts of 3,4,5 and 6 kHz were used to elicit a.c. cochlear potentials. A 3 kHz stimulation tone was added to the protocol since the frequency of maximum depression of the ac. cochlear potential may appear below the exposure frequency [23]. Sounds were delivered to the external auditory meatus through a hollow ear bar, closed sound system. Both spectra and SPL at maximum intensity of the stimuli were measured near the eardrum in decibels (re 0.0002 pbar) using a condenser microphone (Brtiel and Kjaer, model 4133) and sound level meter. The second harmonic of the stimulating frequencies 3,4,5 and 6 kHz were down 10, 14, 18 and 15 dB from the fundamental, respectively. The second harmonic of the 4 kHz exposure frequency was down 22 dB from the fundamental. a.c. cochlear potentials and the CAP were selectively filtered out of the round window recordings by using an appropriate band-pass characteristic. The a.c. cochlear potential and the CAP were monitored simultaneously on the display unit of an oscilloscope and were photographed on 35-mm film from the display unit of the oscilloscope. Definitions
of electrophysiological
measures
Measurements of the amplitude in microvolts (pV) were made for the following points of the CAP waveform: N,, peak of the first negative wave of the CAP; P,, peak of the first positive deflection following N,; CAP amplitudes were defined as: N,P,, N, amplitude relative to P,. In addition to CAP magnitude, a.c. cochlear potential amplitude was obtained by stimulating the ear with intensities ranging from 40 to 140 dB SPL (re 20 PPa) in 5 dB steps. The following terminology was used for the dependent and pseudo-independent variables [7] which were evaluated in a descriptive manner. Changes in the dB SPL which elicit visual detection threshold values for the a.c. cochlear potential and CAP were referred to as shifts in sensitivity. Changes in the intensity level of the stimulation tone which elicited the maximum a.c. cochlear potential and CAP amplitudes were referred to as shifts in dB SPL for CAP or a.~. cochlear potential max. Changes in maximum amplitude of both the a.c. cochlear potential and CAP were referred to as changes in maximum voltage. To quantitatively analyze the data we chose data points which were on the linear portion of all the input-output curves with the exception of the CAP curves obtained after maximum exposure which had several values at the noise level of the recording system (20 PV). The data points were arbitrarily chosen as the output (ruv)
132
at 110 dB SPL for the CAP and at 90 dB SPL for the a.c. cochlear potential. The former measure was referred to as the CAP-110 and the latter as U.C. cochlear potential-90. Analysis of variance and Duncan’s test for multiple comparisons were used to test for significant differences at these points [ 121. Drugs and intense-sound-exposure
administration
A base-line recording of all amplitude and latency functions was obtained immediately prior to drug administration for all animals to assess the integrity of the middle and inner ears of the experimental animals and to ensure proper placement of the round window electrode and ear bar. Animals were not accepted into the study if their maximum a.c. cochlear potential output at 3 kHz was less than 1800 pV. Following base-line recordings of electrophysiological potentials, dosages of saline, AOAA or EA were administered to all animals depending upon treatment group. Guinea pigs were randomly assigned to one of ten treatment groups: AOAA + 0 group administered AOAA; group administered EA; EA+O the saline-treated group with the short-term post-treatment reSA(EA) + 0 cording schedule identical to the corresponding EA-treated group’s schedule; the saline-treated group with the short-term post-treatment reSA(AOAA) + 0 cording schedule identical to the corresponding AOAA-treated group’s schedule. AOAA + 10 and two groups exposed to different levels * of intense sound and AOAA + 25 administered AOAA (AOAA-treated-intense-sound-exposure groups); EA $I 10 and EA+25
two groups administered
SA + 10 and SA-I-25
exposed to different levels * of intense EA (EA-treated-intense-sound-exposure
sound and groups);
two groups exposed to different levels * of intense sound and administered saline (saline - treated - intense - sound - exposure groups). EA (Sodium Edecrin@; Merck, Sharp and Dohme, 10 mg/ml, 50 mg/kg, iv.) was administered over a 1 min period to the EA + 0, EA + 10 and the EA + 25 groups. The 1 mm period was chosen ,to ensure rapid uptake of the drug but prevent a massive cardiac reaction caused by a more rapid injection rate. Numerous investigators have shown this dosage of EA to cause the EP to reach negative values comparable to those observed in early anoxic stages [ 181. AOAA (Sigma, 4 mg/ml, 20 mg/kg, i.v.) was administered over a 1 mm period to the AOAA + 0, AOAA + 10 and the AOAA + 25 groups. Saline, an equal volume/kg, was administered intravenously to the SA(EA) + 0, SA(AOAA) + 0, SA + 10 and SA + 25 groups. l
See p. 137 for definition of levels.
133
45 min after AOAA administration and 15 min after EA or saline administration, the drug-treated-intense-sound-exposure groups were exposed to a 4-kHz tone for 30 min using the same instrumentation employed to elicit the electrophysiological potentials. The frequency of the exposure stimulus and duration of exposure were chosen to duplicate the corresponding measures in an earlier study [2]. The particular temporal relationships between drug administration and intense-sound exposure were selected in order that the peak effect of the drugs occurred during the period of intense-sound exposure [l]. Two different intensity levels of the exposure stimulus were used to provide further information concerning possible reduction in a.c. cochlear potential and CAP losses for the AOAA-or EA-treated groups by quantifying these losses in terms of sound attenuation in dB of the exposure stimulus. In addition, different mechanisms of the effect of intense sound on the cochlea may be disclosed by using SPLs known to affect differentially the a.c. cochlear potential and the CAP. Exposure stimulus levels were detemined in relation to maximum output capabilities of each animal’s cochlea. This method of ‘tailoring’ the exposure signal intensity level has, been used successfully by Price [23] and others [21] to reduce the variability seen in the a.c. co&ear potential obtained from animals exposed to high-intensity acoustic stimuli. For example, if the intensity of the signal is at a sound level 5 dB above that necessary to elicit the maximum a.c. cochlear potential, the experimental condition would be called ‘Max + 5’. Utilizing this paradigm, the two intensity levels of the CkHz tone-exposure stimulus chosen for this investigation were ‘Max + 10 and ‘Max + 25’ and labelled Drug + 10 and Drug + 25, respectively. The individual drug-treated-intense-sound-exposure groups were thus labelled as follows: AOAA -t 10; AOAA + 25; SA f 10; SA + 25; EA -t 10; and EA + 25. Potentials were monitored immediately after the intense-sound exposure in all drug-treated-intense-sound-exposure groups (short-term post-treatment recordings). The short-term post-treatment recording of the AOAA- and SA(AOAA)-treated groups’ potentials were taken at the same time as the corresponding recordings in the AOAA-treated-intense-sound-expos~e group or, I h and 15 min post-drug administration. The short-term post-treatment recording of the EA- and SA(EA)treated groups’ potentials were taken at the same time as the corresponding recordings in’ the EA-treated-intense-sound-exposure group or 45 min post-drug administration. The dissimilar recording times were related to the different periods of peak effect of EA and AOAA. Immediately after the short-term post-treatmet recording, the wound was closed as described above and the animal was removed from the sound suite and placed on a heating pad. Rectal temperature was monitored every 15 min until the animal’s righting reflex returned. At this time, the guinea pig was returned to animal care facilities. At 48 h post-base-line recording, the animal was returned to the sound suite for long-term post-treatment recordings. Pre-operative medication, anesthesia and recording protocol were identical to the conditions obtained at base line.
134
Results
While there are numerous group-paired comparisons that can be made, the following discussion will be confined to those comparisons that are germane to the experimental hypothesis. The relationships between groups at the short-term posttreatment recording interval will not be discussed since the effect of the drugs on the cochlear potentials at this interval were still apparent, thus masking out any possible protective effect from intense sound. At the long-term post-treatment recording interval (third recording), the effects of AOAA and EA on the measures discussed were similar to the comparable effects of the control vehicle, saline. Therefore, any possible protective effect afforded the animal during the intense-sound exposure MAX
MAX + lW&
lam
,!
0 0 0 0 0
CM
00
100
0
90
0
O -
.
t
s”D
Od””
x
0”
l
00” qG X .
.
70
.
90
.
t
110 1%
SbGTGizQ dB SPL
3
0
0
T
!io
*#
0
1
110 l#I
00
0
f
o”xx
70
0 0
0
0
r
EA
00
200
al
2568
AOAA
EA
AOAA
2mo
l
I
-9
1
1
I
,
L ae”
0"
O bp= oo” Xb
50
70
90
110 1
Fig. I. Effect of combinations of saline, AOAA. EA and intense sound on the input-output functions of the a.c. cochlear potential (CM) and the CAP evoked by 4-kHz tone bursts. The curves were obtained at the long-term post-treatment recording after the administration of saline alone (0). saline plus the intense tone (0) or drug plus intense tone (X). The effects of both the Max+ IO dB and Max+25 dB intense tone exposure are shown. Only data points above the noise level of the recording system (20 uV) were used in calculating the values in order IO reduce distortion of the curves. Drug curves represent mean data (n = 5) taken from 5 animals and saline alone curves (n = 10) from 10 animals, except ,x=4 in EA + 25 and SA+ 25, each of which had one animal with all values at noise level.
135 TABLE I EFFECT OF COMBINATIONS OF SALINE, AOAA, EA AND INTENSE SOUND ON THE a.c. COCHLEAR POTENTIALS AND CAP EVOKED BY TONE BURSTS OF DIFFERENT FREQUENCIES MONITORED AT THE LONG-TERM POST-TREATMENT RECORDING Tone burst frequency: 3kHz a.~. cochlearpo~enlial-90” SA+O 2032* 908= SA+ IO AOAA+ 10 1516k EAt 10 16422 SA+25 2122 AOAA+25 366= EA+25 244= CAP-110= SA+O SA+ 10 AOAACIO EA+ 10 SA+25 AOAA + 25 I%+25
20 104 108 97 51 37 31
4kHz
5 kHz
6 kHz
1126-120 448k60 1022*96 948-c41 171 -t41 216zt24 167* 19
1 l&k24 584*51 860268 838255 126-c29 195223 1282 16
455rt 7 251 k31 494 * 50 428*23 64k 14 118-tl4 592 7
400*13 2691 9 272=35 294228 96-c I5 82= 5 82212
369* 11 188” 12 229230 210224 85~ 9 so* 5 82111
2282 5 110-c 8 162220 18lkl9 48* 12 542 3 68k 9
’ Shown are the means (i;v) -t SE with n = 5 for each mean except for SA f 0 where n = 10.
would be realized at this interval. The two control groups (SA(EA) + 0, SA(AOAA) + 0) were virtually identical for all measures at every recording interval, therefore the data were pooled in Fig. 1 and Table I and called ‘SA + 0’. Comparison of effect of infense sound on a.c. cochlear potential in groups SA i- 10, EA + 10 and AOAA f 10 at the third recording interval Shifts in sensitivity for the SA + 10 group, while small compared to the corresponding shifts in the SA + 25 group, were larger than the corresponding shifts in the AOAA i 10 and EA + IO groups (see Fig. 1 for 4 kHz). The changes in sensitivity for the SA f 10 group ranged from 7 to 10 dB, while the corresponding changes for the AOAA + 10 and EA + 10 groups ranged from 2 to 8 dB in the former group and from 0 to 3 dB in the latter group. Shifts of dB SPL for a.c. cochlear potential max were similar for all three groups. There were no appreciable differences in a.c. cochlear potential maximum voltage at 48 h post-treatment with the exception of 4 kHz where the reduction in maximum voltage for the AOAA + 10 group appeared greater than the corresponding reduction for the SA -f- 10 group. The a.c. co&ear potential-90 value for the SA + 10 group was significantly (P < 0.05) smaller than the SA + 0 group at every frequency (Table I). Except for 5 kHz (EA + 10 vs. SA(EA) + 0, P = 0.019; AOAA + 10 vs. SA(EA) + 0, P = O-022), at no other frequency was the a.c. co&ear potential-90 for the AOAA + 10 or the EA + 10 groups significantly different from the SA + 0 groups (P > 0.05). The a.c.
136
potential-90 value for the SA + 10 group was significantly smaller than the AOAA i- 10 and the EA + 10 group values at 3 kHz (P < 0.02) and at 4 kHz (P
Comparison of effect of intense sound an a.~. cochfear potential in groups SA + 25, EA + 25 and A UAA -t- 25 at the third recording interval Shifts in sensitivity from base-line to 48 h post-treatment were generally equivalent as were shifts in dB SPL for a.c. cochlear potential max and reductions in a.c. cochlear potential maximum voltage (see Fig. 1 for 4 kHz). The a.c. cochlear potential-90 value was reduced to a greater extent in SA 4 25, AOAA + 25 and EA + 25 groups compared to SA + 10, AOAA + 10 and EA 4 10 groups for each frequency (Table I). At no frequency did this measure in either AOAA + 25 or EA + 25 groups differ significantly (P > 0.05) from that of the SA + 25 groups (Table I). Comparison of effect of intense sound on CAP measures in groups SA + IO, EA + 10 and AOAA + JO at the third recording interval There were no marked differences in sensitivity from base line to 48 h for the SA + 10, EA + 10 and AOAA + 10 groups (see Fig. 1 for 4 kHz). In addition, the shifts in dB SPL for CAP max were no larger for the SA + 10 group than for the corresponding shifts for the AOAA -t- 10 and EA + 10 groups at any frequency. Reductions in CAP maximum voltage from base-line to 48 h were similar for all groups at all frequency-measure combinations except at 4 kHz (AOAA and EA) and 6 kHz (EA only) where SA + 10 losses of maximum voltage were greater than analogous losses in drug-intense-sound-exposure groups, The CAP- 110 value was reduced in SA + 10 group compared to the SA + 0 group at the P = 0.20 level for 4 kHz, at the P = 0.06 level for 5 kHz, and at the P = 0.17 level for 6 kHz (Table I). Also, no significant difference ( P > 0.05) was found between the CAP- 110 values for the SA -t- 0, SA + 10, EA t 10 and AOAA + 10 groups within each frequency. Comparison of effect of intense sound on CAP measures in groups SA + 25, EA + 25 and AOAA + 25 at the third recording interval Shifts in sensitivity for the AOAA + 25 and EA -t 25 groups were comparable to the shifts in sensitivity for the SA,+ 25 group (see Fig. 1 for 4 kHz). Shifts in dB SPL for CAP max and reductions in maximum voltage for the SA + 25 group were not appreciably different from the AOAA -t 25 group or the EA + 25 group at any frequency. The CAP-l 10 value was reduced to a significantly (P< 0.05) greater extent in the SA + 25 group when compared to the SA + 0 group for each frequency (Table I), However, the CAP-I 10 value did not significantly differ (P > 0.05) between the SA + 25, AOAA + 25 and EA + 25 groups.
137
Discussion
While there is ample documentation of the deleterious effect of intense sound on the cochlea, numerous issues such as the effect of high-intensity stimuli on cochlear vasculature and the authenticity of the mechanical theory remain controversial [2,14,25,28]. In addition, previous studies in the literature attempting to delineate the effect(s) of intense sound on the cochlea using an intense-sound-drug interaction paradigm have been fraught with ambiguities due to varying drug dosages, infusion rates, constancy of infusion, and the period of time between acoustic and drug trauma and histological examination [26]. In a study designed to evaluate a theory of the effect of intense sound on the cochlea, it was shown that pre-treatment with AOAA caused a reduction in the hair-cell scarring observed in ears exposed to injurious sound levels alone [il. The authors attributed the protective action of AOAA to the drug-related reduction of EP purportedly reducing current flow and prevention of depletion of energy stores (ATP). On the other hand, others [26] failed to reveal any interaction between intense-sound exposure and EA, a drug which also reduces the EP. The latter investigation was designed to maximize any possible potentiation effect of the combination of drug and intense sound, rather than to examine a possible protective effect afforded the ear by pre-treatment with the diuretic. The current investigation, as reported in a preliminary report [3], showed few appreciable protective effects afforded the cochlea by pre-administration of either AOAA or EA. Comparisons of dB SPL for CAP and a.c. cochlear potential max and CAP and a.c. cochlear potential maximum voltage for the SA + 10, EA + 10, AOAA + 10, SA + 25, EA + 25 and AOAA + 25 groups showed no marked differences. On the other hand, shifts in a.c. cochlear potential-90 and a.c. cochlear potential sensitivity at 48 h were larger for the SA + 10 group than for the corresponding changes in the AOAA + 10 and EA + 10 groups. These differences in shifts in a.c. cochlear potential-90 and a.c. cochlear potential sensitivity are difficult to interpret in that they may be due to either a permanent middle or inner ear conductive loss, the interference effect acting in the cochlea, or a temporary reversible fatigue condition [7,10,29,30]. As discussed by Durrant [l 11,it is impossible on an a priori basis to determine the contributions of each component to the composite shift. Since the a.c. cochlear potential is generally considered a precursor for normal amplitude CAP, changes in the former measure are generally considered to be reflected in the CAP potentials [7]. The fact that there were no marked differences in sensitivity for the CAP measures in the SA + 10 group further clouds the interpretation of changes in the a.c. cochlear potential. If the shifts in the a.c. cochlear potentials are interpreted to mean that pretreatment with AOAA or EA has significantly reduced the effect of intense sound on cochlear potentials, one interpretation may be that change(s) in the EP produced by the drugs accounted for the results [2]. In other words, the drug-induced reduction in the EP may have reduced the voltage which drove abnormally excessive and toxic amounts of depolarizing current (potassium or calcium ion) [8,16,19] from Scala media into the hair cell during sound exposure. The decrease in Scala media
138
potassium ion monitored by Snow et al. [24] and Melichar et al. [20] during intense sound exposure may be taken as evidence for the occurrence of excessive current flow. Additional evidence for an excessive current flow may be found in the data of Bohne [4,5] who has proposed that intense sound creates openings in the reticular lamina through which s&ala media fluid, endolymph, is allowed to pass and surround cells of the organ of Corti where it acts to distroy them. Bohne [6] has shown that an artificial endolymph containing high potassium, low sodium ion concentrations destroyed hair cells when placed in Scala tympani and therefore in contact with the base of the hair cells. One interpretation of these artificial endolymph studies is that the high potassium ion solution caused excessive depolarization of the hair cells which resulted in cell death. The question of interest here is whether cell death is caused by excessive depolarization due to the entrance of ions from Scala media directly into the hair cell membrane without a requirement for openings in the reticular lamina. Attributing excitable tissue death to excessive depolarization is not a novel theory and has been proposed as the basis for the neurotoxicity of the excitatory amino acids [22]. However, further evidence must be obtained before excessive depolarization together with EP involvement, the electrochemical theory proposed earlier [2], is accepted as having a role in some forms of noise damage in the cochlea. Another plausible explanation of the effect of EA or AOAA may be that they produced a temporary reduction of protein synthesis and secretion since EA has been shown to inhibit these and other cellular functions of adenylcyclase 1151.Related alternative theories involving a variety of cochlear metabolites, such as lipids, proteins, sugars, and glycoproteins, are too numerous to be discussed. On the other hand, if the changes in a.c. cochlear potentials do not reflect a reduced effect of intense sound, then there are a number of possibilities for the conflict between results reported in this paper and the previous one [2] where a reduced scarring was reported. First, body temperature was controlled in the present investigation, thus eliminating a possible mechanism for protection from intensesound exposure. Another possible explanation is linked to the time(s) of monitoring of the effects of intense-sound treatment; immediately after and 48 h post-treatment in the present investigation and 21 days post-treatment in the previous study [2]. A third argument is based on the inadequacies of the round window recording site, since the voltage from the second cochlear turn at suprat~eshold levels is attenuated relative to the local activity viewed by the round window electrode [9]. Another is related to the surface preparation of the organ of Corti. Others [ 171 have demonstrated alterations in hair-cell organelles not evident in surface preparations. In summary, the present investigation found an interaction between AOAA, EA, and intense sound in terms of a-c. cochlear potential-90 and a.c. co&ear potential sensitivity. These results raise questions regarding the relationship between hair-cell function and the a.c. cochlear potentials. It seems that these questions will have to be answered and additional data obtained before an electro-chemical mechanism is proven to play a role in noise damage.
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Acknowledgments This research was supported in part by a grant from the Deafness Research Foundation. Laboratory facilities were provided by a grant from The Kresge Foundation. Thanks are due to Dr. E. Moore who provided the initial arrangements and guidance for this study and whose support was maintained throughout. The
authors also wish to thank Drs. Beasley, Irwin, Kahane, Wark, Berlin and Tilley for their support in various stages of this project. Drs. Cullen and Hughes, and Mr. Stephen Winbery have contributed to this work and are thanked for their efforts.
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