JOURNAL OF THE

NEUROLOGICAL SCIENCES

ELSEVIER Journal of the Neurological Sciences 131(1995) 21-29

Effect of hypoxemia and ethacrynic acid on ABR and distortion product emission thresholds *

Sharon Freeman a, Kalman Goitein b, Joseph Attias ‘, Miriam Furst d, Haim Sohmer a a Department of Physiology, Hebrew University-Hadassnh Medical School, P.O.B. 12272, Jerusalem91120, Israel

b Pediatric Intensive Care Unit, Hadassah University Ho.spitaI, Jerusalem, Israei ’ Institute for Noise Hazardr Research and Evoked Potentials, IDF Medical Corps, Tel AviE, Israel

’ Department of Electrical Engineering Systems, Tel Aviv University, Tel Aviv, Israel

Received 27 September 1994; revised 12 January 1995; accepted 19 January 1995

Abstract

Various studies have shown that induction of hypoxemia in animals such that arterial blood oxygen tensions reach 20-30 mm Hg is accompanied by reversible threshold elevations of the auditory nerve-brain-stem evoked response (ABR). In this state, the endocochlear potential (EP) is depressed, causing a smaller potential difference across the hair cells and/or reduced activity of the cochlear amplifier of the outer hair cells. In order to test these possibilities, ABR threshold (an expression of the overall sensitivity of the cochlea) and changes in threshold of the cubic (2f, - f,) distortion product emissions (DPE) (an expression of activity of the cochlear amplifier) were measured in the same cats while the EP was depressed by hypoxemia or by ethacrynic acid. During the episodes of hypoxemia, DPE thresholds were elevated by 10 dB while ABR thresholds were elevated by 22.8 dB. Therefore, it seems that a normal EP is necessary both for normal cochlear transduction (inner hair cells) and for normal cochlear amplification (outer hair cells). The human fetus in utero is relatively hypoxic and there is evidence that its auditory threshold is also similarly elevated. Therefore the threshold elevation in the fetus in utero, estimated to be about 20 dB, is a consequence of both reduced transduction current through the inner hair cells (about 10 dB) and an additional 10 dB reduction in the activity of the cochlear amplifier of the outer hair cells.

Keywords: Threshold; ABR; Emission; Ethacrynic acid; Hypoxia; Distortion products; Cochlear amplifier

1. Introduction

Hypoxia induces a reversible sensorineural hearing loss as has been shown in adult cats and rats (Lawrence et al., 1975; Sohmer et al., 1989,1986a,1986b; Hildesheimer et al., 1987; Sohmer and Freeman, 1991) and in kittens, neonatal guinea pigs and goat (Gycowicz et al., 1988; Sohmer and Freeman, 1991). The degree of controlled hypoxemia (i.e. hypoxia while maintaining normal pH, arterial carbon dioxide tension and arterial blood pressure) required to induce this auditory threshold elevation (as indicated by auditory nerve- brain-stem evoked response-ABR) was an arterial oxy- gen tension (P,O,) of 20-30 mm Hg, equivalent to arterial blood oxygen saturation (S,O,) of about 25-

* A preliminary version of this article was presented at the Inter- national Congress of Audiology, Halifax, 1994.

0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0022-510X(95)00038-0

45%. This was induced by respirating the animal with hypoxic gas mixtures containing about 6-8% oxygen (Sohmer et al., 1986a,1989; Sohmer and Freeman, 1991).

This ABR threshold elevation is probably the result of suppression of the endocochlear potential (EP) since it has been shown that similar degrees of hypoxemia cause a reduction of this potential (Gafni and Sohmer, 1976; Rebillard and Lavigne-Rebillard, 1992). It is un- likely that the ABR threshold elevation during such levels of hypoxemia is due to a direct effect on nerve fibers since somatosensory and visual evoked potentials are unaffected under the same conditions (Sohmer et al., 1986a).

A low amplitude EP is always accompanied by audi- tory threshold elevations. For example, EP reductions following administration of drugs like loop diuretics, which suppress activity of Na+-K+ ATPase pumps in the stria vascularis, cause an elevation in auditory

22 S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29

threshold (Kusakari et al., 1978; Bosher, 1980). This is also accompanied by a reduction in the cochlear action potential, the source of wave 1 of the ABR (Aran and Charlet de Savage, 1977). Furthermore during the de- velopment of auditory function in altricious animals, there is a substantial increase in EP amplitude concur- rent with a large improvement in ABR threshold dur- ing the same period of time (Bosher and Warren, 1971; Jewett and Romano, 1972; Fernandez and Hinojosa, 1974; Walsh et al., 1986; Rybak et al., 1992). In addi- tion, direct manipulation of the EP by passing current is accompanied by changes in firing rate of auditory nerve fibers (Vossieck et al., 1991).

The EP together with the intracellular membrane potential is the main driving force for ion transport across the hair bearing surface of the hair cell mem- brane during transduction in the inner ear. Ion concen- tration also plays a part (Davis, 1958). Thus a smaller EP can lead to a threshold elevation by two different, though not conflicting mechanisms. A smaller potential gradient across the inner hair cells (IHC) would lead to smaller transduction currents during conductance in- creases induced by the acoustic stimulus (Johnstone and Sellick, 1972; Russel, 1983). Hypoxia is also accom- panied by depression of otoacoustic emissions (Rebi- llard and Lavigne-Rebillard, 1992) which are consid- ered to be expressions of an active cochlear amplifier (Davis, 1983), second filter (Evans, 1975) related to the outer hair cells (OHC). This provides mechanical feed- back to the basilar membrane, enhancing its displace- ment and thereby further augmenting the direct trans- duction through the IHC.

Low intensity cubic 2f, - f, distortion product otoa- caustic emissions (DPEs) have also been shown to be affected by EP magnitude changes induced in other ways. Furosemide (30 and 50 mg/kg i.v.) was shown to reversibly reduce evoked otoacoustic emissions in guinea pigs (Ueda et al., 1992). Reduction of the EP by anoxia or ethacrynic acid (EA) injection caused these DPEs to disappear in rabbits (Whitehead et al., 1992) and led to the conclusion that these DPEs were depen- dent on a normal amplitude EP (Ueda et al., 1992; Whitehead et al., 1992). Furthermore, the depression of the EP by furosemide is accompanied by a reduction of an additional manifestation of cochlear sensitivity, namely in the magnitude of basilar membrane vibra- tions (Ruggero and Rich, 1991). Therefore it is likely that the cochlear amplifier is also dependent on the magnitude of this potential difference (Whitehead et al., 1992; Mills et al., 1993) and that the activity of this amplifier is also depressed in hypoxia.

In order to further study these 2 mechanisms (de- creased transduction by inner hair cells and/or de- creased cochlear amplification by outer hair cells) and to evaluate their relative contributions to ABR thresh- old elevation, experiments were conducted on anes-

thetized, paralyzed, ventilated cats. The animals were subjected to controlled hypoxemia which depresses the EP. Recordings (ABR and DPE) were conducted dur- ing the initial control state, during the experimental manipulation and during recovery. A limited number of experiments were conducted using an additional manipulation which depresses the EP-administration of ethacrynic acid (EA). During both manipulations, the magnitude of their effect on the DPE was com- pared to that on the ABR.

2. Methods

This study was carried out in 9 adult cats (average body weight 3 kg, range 2.25-3.75 kg), 6 of which were subjected to hypoxemia and 3 to i.v. injections of ethacrynic acid (EA). All of the animals were anes- thetized with sodium pentobarbital, with an initial i.p. injection of 40 mg/kg. Cannulae were inserted into the femoral vein for administering additional sodium pentobarbital (when required, as signified by an in- crease in blood pressure and heart rate) and other drugs (for example, EA), into the femoral artery, for monitoring blood pressure and blood gases and into the trachea for ventilation. The head was firmly held so that it would not move during the experiment.

The animal was paralysed by administering gal- lamine (1 ml/kg i.v.), and artificially respirated. After at least 15 min of respiration with room air, blood samples were taken for blood gas analysis to ensure adequate ventilation and that pH was normal.

Debris in the external ear canal was gently removed. The probe of a commercial otoacoustic distortion prod- uct analyzer (Otodynamics Ltd., model IL092) was inserted into the ear canal using a sponge cuff supplied with the equipment and held in position by a clamp. The position of the probe was adjusted until there was a satisfactory flat ear canal response, which was moni- tored throughout the experiment to ensure there was no probe slippage.

A sweep of cubic (2f, - f2) distortion product emis- sions (DPE) was made for a range of frequencies from f, = 3500 Hz up to 7000 Hz (f/f1 = 1.22) at various intensity levels (Ll = L2) of the probe frequencies from 50 dB SPL up to 80 dB SPL, and a frequency of f, was chosen that gave clear and consistent DPEs at the lower intensities, such that f, was slightly greater than 4 kHz (the center frequency of the click transduced by the TDH-39 earphone used to elicit ABR in the other ear). It is thought that cubic DPEs of lower intensity are generated at a cochlear site that is maximally sensitive to the primary tones f, and f, (Martin et al., 1987), probably at a site closer to f, (Kimberley et al., 1993), and hence this DPE should have its origin at approximately the same place along the basilar mem-

S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29 23

brane as the click-elicited ABR (in the other ear). Whitehead et al. (1992) differentiate between at least two kinds of DPE components (in rabbits). The lower intensity (induced by stimuli < 60-70 dB SPL) compo- nents were shown by them as being a true measure of active cochlear amplification and as such very vulnera- ble to physiological insult. They showed that the higher intensity (> 60-70 dB SPL) components were much less vulnerable, and were probably less indicative of active processes. The lower intensities were used in this study.

Growth (input-output) functions were then recorded so that the threshold of the DPE could be evaluated. A near-linear growth function was expected, which disap- peared at very low levels into the noise floor. In order to improve the signal to noise ratio, several (3-4) epochs of the intensity function were averaged. Two DPE response measures were analyzed: threshold, which was defined as the intensity of f2 which gave a DPE with an amplitude 2 dB above the noise floor (Lenoir and Puel, 1987; Lonsbury-Martin et al., 1987; Henley et al., 1989; Norton et al., 1991); and sensitivity, which was defined as the intensity of f, (Ll = L2) which gave a DPE amplitude of 0 dB SPL (see Fig. 1).

ABR was recorded in the other ear using a Micro- shev 4000 Evoked Potential System. It was not possible to measure both ABR and DPE in the same ear, for technical reasons. However, the factors which pro- duced EP depression (hypoxia and EA) were induced systemically, so they affected both ears simultaneously. Furthermore, Kimberley et al. (1993) report very little intra-subject left v. right ear differences for DPEs measured in man. As the DPEs were probably gener- ated at a cochlear site similar to that of ABR in the opposite ear, comparison between the two parameters was considered valid. The ABR recording needle elec- trodes were placed subdermally at the vertex and at the pinna, with a ground electrode placed in a hind- paw. Alternating polarity clicks at a rate of 20/s were presented to the ear using an earphone clamped in close proximity to the pinna. The responses were digi- tally filtered (200-2000 Hz), amplified and averaged (N = 128 at high intensities and 256 near threshold), and displayed vertex positive up. Initially the response to a click with an intensity of 120 dB SPL (approxi- mately 70 dB above the subjective threshold of normal hearing listeners, i.e. nHL) was recorded. The intensity of the click was reduced until ABR threshold was reached (in steps of 5 dB). Repeated recordings were made at each intensity. Threshold was defined as the lowest intensity where consistent responses could still be recorded.

Throughout the experiment, temperature was moni- tored with a rectal probe (Yellow Springs Instrument Co.) and kept at 37” C, using heating pads. Blood pressure was kept above 80 mm Hg, with the addition

a) ASR d6 SPL

120

100

80

60

50

T-+ 40

35

b) DPE

Stimulus (F2, dB SPL)

Fig. 1. Typical recording of ABR (a) and DPE (b) during the control period, in one cat. “T” marks the threshold of both the ABR and DPE whilst “S” shows the sensitivity of the DPE. The solid region at the bottom represents the mean noise floor while the stippled region is 1 SD.

of fluids at the beginning of the experiment, or using dopamine i.v. during the episodes of hypoxemia. Fur- thermore, blood samples- were taken for blood gas analysis at regular intervals, and sodium bicarbonate was administered as required, to maintain pH at around 7.4.

The experiment was terminated by administering a lethal dose of barbiturate. Within a few minutes of the injection, the arterial blood pressure dropped to close to zero and both the ABR and DPE disappeared. The care and use of the animals in this study was approved by the University Animal Care and Use Committee.

2. I. Hypoxemia

Initially, the animals were ventilated with room air, and control recordings of ABR and DPE were con- ducted. Hypoxemia was then induced in 6 animals by ventilation with a gas mixture of approximately 7.5% oxygen in nitrogen. A DPE growth function was recorded 10 min after the start of ventilation with the gas mixture, together with sampling of arterial blood

24 S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29

for blood gas analysis. Sodium bicarbonate was admin- istered to correct for pH if required. Immediately afterwards, ABR threshold was evaluated, followed by a second recording of the DPE growth function (ap- proximately 20 min after the start of ventilation with the gas mixture). It took about 10 min to determine ABR threshold, whereas only l-2 min for the DPE recording. At this stage, ventilation was returned to room air, and the procedure for recording DPEs and ABR repeated 10 min into recovery, including taking a sample for blood gas analysis and correction of pH, if needed.

If both the ABR and the DPE growth function showed recovery to near control values, ventilation with an hypoxic gas mixture, either with the same, or a slightly more severe, degree of hypoxia was carried out, followed by recovery, as described above. In some of the initial experiments, no great change was seen in either recording. In this case, ventilation of the animal was reduced to cause a greater degree of hypoxemia, without the intermediate recovery stage. Up to three episodes of hypoxemia were possible in each animal before the preparation was no longer viable. The dura- tion of severe hypoxemia was limited, to prevent dete- rioration of the preparation, which would then not give a true reflection of the effect of hypoxemia alone.

2.2. Ethacrynic acid

Following control recordings of ABR and DPE thresholds, ethacrynic acid (EA, sodium edecrin, Merck) was administered i.v.: 16 mg/kg in the first cat; 10 mg/kg in the second, and 7.5 mg/kg in the third.

ABR at 120 dB SPL and growth functions of the DPE were measured every 2 min following EA injec- tion, until there was little or no ABR at this high (120 dB SPL) intensity (approximately 20’). In order to see if there would be recovery, ABR threshold was mea- sured 10 min later, together with a further measure- ment of the DPE both immediately prior to and follow- ing ABR recording. Thereafter, recordings were made every 30-60 min up to at least 4.5 h following the injection of EA.

2.3. Analysis of results

For each experimental state, values of DPE thresh- old, DPE sensitivity, ABR threshold, the latency and amplitude of wave 1 of ABR, and the brain-stem transmission time (BTT, between waves 1 and 4 in the cat) were evaluated, and compared to the values in the control state.

In the hypoxemia experiments, the changes in DPE threshold were compared to the changes in ABR threshold both for the control-hypoxic states and for hypoxic-recovery states.

Mean f SD were calculated and control state and experimental state means were compared using Stu- dent’s t-test. A significance criteria of p = 0.05 was used.

3. Results

The average (k SD) frequencies of f,, f, and 2f, - f, used to elicit the DPEs were 4.32 (k 0.44),5.27 (& 0.54) and 3.37 (fO.34) kHz respectively. DPE noise floor levels were all between - 5 and - 15 dB SPL, similar to those reported by Schmiedt (19861, Whitehead et al. (1992) and Mills et al. (19931, and did not change during each individual experiment. One exception had a noise floor level of 10 dB SPL. This however was stable throughout the experiment and therefore the results were considered suitable for inclusion in this study. Control DPE thresholds ranged between 28 and 60 dB SPL (average 43.5 + 10.3 dB SPL). These pa- rameters are similar to those reported in the review by Probst et al. (1991) on otoacoustic emissions. A large variability in DPE recordings from cats of unknown auditory history has also been reported by Schmiedt (1986).

Fig. 1 shows a typical recording of the ABR and DPE in one cat during the control period, demonstrat- ing determination of ABR threshold, DPE threshold and DPE sensitivity. In general, the changes in DPE sensitivity were parallel to the changes in DPE thresh- old, as would be expected (see Fig. 2).

3.1. Hypoxemia

It was possible to achieve 15 different episodes of hypoxemia in these 6 cats (mean k SD PaO, = 22 + 2 mm Hg; SaO, = 38 f lo%, compared with control val- ues of 87 f 13 mm Hg and 96 k 3%, respectively). There was no clear connection between the PaO, or SaO, measured in the arterial blood and the elevation in DPE thresholds for the range of hypoxemia tested. However, ABR threshold became more elevated with a greater degree of hypoxemia, as found in an earlier study (Sohmer et al., 1989).

Fig. 2 shows the relationship between the ABR thresholds and DPE thresholds and sensitivities, during one complete experiment, consisting of the initial con- trol (CONT.) period, three different episodes of hypox- emia (HYP. 1, 2 and 31, and subsequent recoveries (REC.). In the course of this single experiment one can see the types of ABR and DPE threshold changes which took place during all the experiments. The changes in the DPE thresholds and sensitivities were very similar in general. The error bars mark the ranges of the DPE thresholds and sensitivities during the control and recovery periods. The arrow on the DPE

S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29 25

40’ I CONTR. HYP. 1 REC. HYP.2 REC. HYP.3 REC.

Fig. 2. The changes in ABR (black squares) and DPE (white squares) thresholds during the course of one complete hypoxemia experiment. The hourglass symbols show the changes in DPE sensitivity, which can be seen to closely follow the changes in DPE threshold. CONTR. = control period; HIP. 1, 2, 3 = first, second and third hypoxemic episodes; REC. = intermediate periods of recovery. The error bars show the range of values measured for the DPE threshold and sensitivity during the control and recovery periods, whilst the arrows signify that the DPE was not detectable above the noise floor at that particular maximal intensity of f, (the value plotted). Note that hypoxemia consistently induced a greater ABR threshold shift.

points during the first and third hypoxemia indicates that the DPE had disappeared into the noise floor at that particular maximal intensity of f2, even though the ABR was still present, though with an elevated thresh- old. Note that the reproducibility of the ABR thresh- old is f5 dB, and of the DPE threshold is of a similar magnitude, such that a threshold shift 2 10 dB can be considered to be a true change in threshold.

It can be seen that during each of the three hypox- emias, the ABR threshold shift exceeded that of the DPE. Furthermore, there is an almost complete return of the ABR threshold to its control value after cessa- tion of the hypoxia (recovery to room air). In addition, it was still possible to record a clear DPE during recovery. This enabled us to expose the animal to additional hypoxemic episodes. During all the three hypoxemic episodes, and the recovery periods follow- ing the first and third hypoxemia, there were large shifts in the ABR and DPE thresholds (2 10 dB). However, following the second hypoxemia, the ABR almost completely recovered independently of the DPE, which remained slightly elevated.

When the differences in thresholds between the control and hypoxic states were compared (i.e. control threshold-hypoxic threshold) for all the experiments, it was found that during several hypoxemic episodes there were small threshold changes while in others, the threshold shifts were larger. Nevertheless, the hypox- emia induced threshold shift of ABR (22.8 f 17.1 dB)

was significantly (p < 0.025) greater than that of the DPE (10.0 + 7.9 dB) for all 15 episodes of hypoxemia. The magnitude of the ABR threshold shift and the DPE threshold shift in the individual hypoxic episodes were not significantly correlated (linear regression analysis) with each other.

Overall there was no significant change in ABR wave 1 (Wl) latency during the hypoxemia (control: 0.97 f 0.24 msec; hypoxemia: 0.92 f 0.20 msec) indicat- ing that the hypoxemia caused a peripheral sensorineu- ral hearing loss (Sohmer et al., 1986a). The wave l-wave 4 interpeak interval was also unchanged on

A) 130

301 0 60 120 180 240 3

TIME AFTER ETHACRYNIC ACII) (min.) NJ

- ABR --B DPE

Fig. 3. ABR (black squares) and DPE (white squares) threshold changes following ethacrynic acid injection in three cats: A, 16 mg/kg; B, 10 mg/kg; and C, 7.5 mg/kg. Note that in (A), both ABR and DPE show partial recovery. However, in (B) DPE threshold recovers completely, with only partial recovery of ABR, whilst in (C) ABR threshold almost fully recovers with no recovery of the DPE. The arrows signify that the DPE was not detectable above the noise floor at that particular maximal intensity of f, (value plotted). Note that generally the changes in ABR threshold exceeded those of DPE.

26 S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29

average (control: 2.57 + 0.24 msec; hypoxemia: 2.55 f 0.18 msec; no significant difference in Student’s paired t-test).

The average improvement in threshold during re- covery (the hypoxic threshold-recovery threshold) was greater for the ABR (18.3 dB) than for the DPE (7.7 dB). The threshold following recovery compared to that at the very beginning of the experiment (e.g. from Fig. 2, Rec. following Hyp. 2-Contr.) was greater for ABR (7.9 dB) than for DPE (2.8 dB) while when the post-hypoxia recovery threshold was compared to that just preceding that hypoxia (e.g. from Fig. 2, Rec. following Hyp. 2-Rec. following Hyp.l), the ABR threshold was also greater (5.4 dB) than that of the DPE (2.5 dB). None of these differences were signifi- cant.

3.2. Ethacrynic acid

Fig. 3 shows the changes in ABR and DPE thresh- olds with time in the 3 animals injected with different doses of EA (A, 16 mg/kg; B, 10 mg/kg; and C, 7.5 mg/kg). EA administration was followed by elevations of both ABR and DPE thresholds.

About 20 min after injection, the DPE threshold increased until it was not possible to detect a DPE in 2 of the cats, whilst in the third cat, this stage of DPE disappearance took place later (35 min). In two of the animals (A and C in Fig. 3), there was no recovery of the lower intensity component (< 65 dB SPL) of the DPE, whilst in the third animal (B in Fig. 3) there was almost total recovery of the DPE to its control values within 4.5 h of the EA injection.

At the same time, in all three animals ABR thresh- old, which had increased to 120 dB SPL or higher by the end of the first 20 min, rapidly recovered to an intermediate value of approximately 85 dB SPL (30-45 dB above the control threshold) 30 min after the injec- tion. Here, too, the threshold changes of ABR and DPE were not correlated. In two of the cats (A and B in Fig. 3) there was no further recovery of the ABR threshold, even 5 h after the injection, whilst in the third cat (C in Fig. 3) ABR threshold recovered to within 5 dB of its control value towards the end of the 5-h period. However this animal showed no recovery of the DPE. Thus, it can be seen that during several time periods following EA intoxication, the threshold changes of ABR exceeded those of DPE. Furthermore, an ABR, though with elevated threshold, could still be recorded when the low level DPE, reflecting the active cochlear amplifier, was no longer present. There was some change in the latency of ABR wave 1, beginning 20 min after the EA injection, with a slow, but incom- plete, recovery (control: 0.83 k 0.10 msec; EA k 60’: 1.12 + 0.15 msec; EA + 280’: 0.91 + 0.17 msec). There were no significant changes in the l-4 interpeak inter-

val (control: 2.54 f 0.15 msec; EA f 60’: 2.50 + 0.04 msec; EA + 280’: 2.54 + 0.07 msec). This increase in Wl latency probably reflects changes that took place in the cochlea during this time. EA is thought to induce hearing loss of a sensorineural nature by decreasing the EP. It also causes morphological changes in the cochlea, which may be temporary or permanent, in- cluding edema in the stria vascularis (Bosher, 1980) and possibly outer hair cell degeneration (Mathog et al., 1970; Forge and Brown, 1982). In these studies such changes were not seen immediately after injection when the EP changes were already apparent.

4. Discussion

This study has shown that both DPE (assumed to be a measure of cochlear amplifier activity) and ABR (reflecting overall cochlear sensitivity) are depressed by hypoxemia and EA, the ABR more so, and that these changes in threshold are not very well correlated with each other. The time courses of the effects of the variables induced in this and other studies (hypoxemia, anoxia, EA, furosemide) on, for example, basilar mem- brane mechanics, the cochlear microphonic potentials, DPE, spontaneous activity in auditory nerve fibers and the compound cochlear action potential are similar to the effect of these manipulations on the endocochlear potential (EP). All of these are dependent on the magnitude of the EP. This is considered evidence that the primary site of their effect is the stria vascularis, which generates the EP, and not the OHC (Sewell, 1984; Ruggero and Rich, 1991; Whitehead et al., 1992; Mills et al., 1993). More direct corroboration for this conclusion comes from consideration of the lower con- centration of furosemide needed to produce a shorter latency decline of EP when applied intravenously (be- tter access to the stria) compared to that required when applied directly to the Scala tympani (better access to the hair cells) (Sewell, 1984; Ruggero and Rich, 1991).

Hypoxemia reduces the EP apparently by causing a decrease in Nat-K+ ATPase and other active trans- port processes in the stria vascularis (Gafni and Sohmer, 1976; Sohmer et al., 1986b). Rebillard and Lavigne-Rebillard (1992) induced hypoxia in guinea pigs while measuring DPE and the endocochlear po- tential. It is likely that the level of hypoxia was milder than in the present study since it seems that the percent of oxygen in the ventilating gas mixtures was higher (apparent range 4-11%) and for shorter dura- tions (apparent range 1.6-15 min), though they do not present quantitative systemic measures of the level of hypoxia. From their data, one can calculate a mean DPE depression of 5.8 dB, somewhat lower than that found in this study, though not surprising, considering

S. Freeman et al. /Journal of the Neurological Sciences 131 (199.5) 21-29 27

their apparent milder hypoxia. Therefore their results corroborate our findings with respect to the DPE.

In this study, the hypoxemia induced an ABR threshold elevation of 22.8 dB, about 10 dB greater than that of the DPE. Ruggero and Rich (1991) also found that the threshold elevation of the compound action potential of the auditory nerve (equivalent to wave I of the ABR) accompanying the furosemide induced depression of the EP, exceeded the depression of basilar membrane mechanical vibration (an expres- sion of the cochlear amplifier) by 11.5 dB. They there- fore conclude that furosemide affects the threshold of auditory nerve fibers by not only reducing basilar mem- brane mechanical amplification due to a fall in the potential difference across the OHCs but also by re- duction of IHC transduction. Our study supports this conclusion.

With an IHC resting potential of -40 mV (Brow- nell et al., 1985) and an EP of +80 mV, the normal potential difference is 120 mV. In the presence of EA or hypoxemia, this potential difference would be re- duced to about 40 mV (if EP were reduced to 0 mV), i.e. a 10 dB change (assuming linearity). On the other hand, the overall change in sensitivity (ABR) in these experimental conditions was much greater (up to 55 dB; average 20 dB). Therefore, this points to an addi- tional indirect effect of the depression of the EP on cochlear sensitivity, namely via the cochlear amplifier involving the outer hair cells (OHC).

On the other hand, if depression of the cochlear amplifier due to reduction in the EP was the only factor involved in ABR threshold elevation, then it would be expected that experimental manipulations which reduce the EP would be accompanied by ABR and DPE threshold changes of similar magnitudes. This was not found in the present study.

Attempts have been made to model cochlear amplification and DPE behavior under different condi- tions (Furst and Cohen, 1994). This model suggests that the amplification factor of the OHC is dynamically changed as a function of basilar membrane displace- ment, due to mechano-elastic energy transfer. The energy source is related to the EP, and therefore reduction in EP will cause a reduction in the amplifica- tion term. This amplification term determines the de- gree of the cochlear non-linearity, and hence the am- plitude of the DPE. According to this model, DPE level is less sensitive to the changes in the amplification term than the overall cochlear response (ABR). All of these studies based on experimental results and mod- elling indicate that a depression of EP is accompanied by an ABR threshold elevation and a smaller DPE threshold elevation.

The low level DPE is an expression of the active, vulnerable cochlear amplifier which contributes to the overall output of the cochlea. Furthermore, it has been

shown that the time course of the depression of the DPE by various manipulations is the same as the time course of their effects on other expressions of cochlear output - the cochlear microphonic and action poten- tials (Whitehead et al., 1992). Also the DPE saturates at the same stimulus level (about 60 dB SPL) as the saturation of the cochlear amplifier (Davis, 1983; Yates et al., 1992; Whitehead et al., 1992). Furthermore in studies on basilar membrane mechanics, which are an even more direct expression of the cochlear amplifier than the DPE, the time course of depression of basilar membrane mechanics following furosemide (Ruggero and Rich, 1991) was similar to the depression of the DPE (Mills et al., 1993). Therefore it is likely that the magnitude of DPE depression seen in this and other studies is a sign of a similar depression of the activity of the cochlear amplifier. Hence, the finding that hy- poxemia induced a 10 dB depression of the DPE while the ABR threshold was elevated by 20 dB can be taken as evidence that the hypoxia induced depression of the EP led to a 10 dB reduction in activity of the cochlear amplifier (OHC) contribution to cochlear output (ABR), with an additional 10 dB reduction being in- duced in the direct transduction by the IHC.

Finally, in this study there were several periods during hypoxemia and following ethacrynic acid injec- tion that the low level DPE, reflecting the active cochlear amplifier, disappeared into the noise floor. At the same time ABR, though with elevated thresholds, could still be recorded. This is evidence that even though the active cochlear amplifier (OHC) was no longer operating, ABR could still be generated, obvi- ously by direct transduction of the IHCs.

It has been shown that the active cochlear amplifier accounts for threshold improvements of 40-60 dB (Evans, 1972; Davis, 1983) (it saturates at these levels) so again the ABR in response to even higher intensity stimuli must involve an additional mechanism, namely direct transduction by the inner hair cells.

These findings that the ABR threshold elevation seen during hypoxemia is due to a depression of the EP which in turn leads to reduction in direct transduc- tion by the inner hair cells and, concurrently, to a reduction in the activity of the outer hair cell-cochlear amplifier, is interesting since it has implications for the hearing ability of the human fetus in utero. The amount of oxygen carried by fetal blood is similar to that which has been shown to cause ABR threshold elevations in cats, rats, guinea pigs and goat. This lower oxygen content of fetal blood is due primarily to the less efficient transfer of oxygen by the placenta compared to the lungs (Long0 and Ching, 1977). Therefore fetal umbilical vein blood carries less oxygen (lower oxygen tension, content and saturation) than neonatal blood (Metcalfe et al., 1967; Battaglia et al., 1968; Itskovitz et al., 1984; Jackson et al., 1987; Carter, 1989). Further-

28 S. Freeman et al. /Journal of the Neurological Sciences 131 (1995) 21-29

more, the fetal circulation leads to the admixture of venous blood from lower parts of the fetus to umbilical vein blood returning from the placenta, further lower- ing the oxygen content of blood delivered to the brain and ear. The presence in the fetus of mechanisms which enhance oxygen carrying capacity in the pres- ence of lower oxygen tensions (e.g. fetal hemoglobin) do not seem to completely compensate for the relative hypoxemia. This became apparent in experiments on newborn guinea pigs and goat when they were less than 12 h old and the fetal compensations were therefore still present. Induction of a level of hypoxemia similar to that present in the fetus (P,O, 20-30 mm Hg) was accompanied by an elevation of ABR threshold by 20 dB (Sohmer and Freeman, 1991). This hypothesis of a fetal hypoxemia induced threshold elevation was cor- roborated by experiments showing that the newborn guinea pig (pulmonary oxygenation) has an auditory threshold improvement compared to that of the same animal a few minutes earlier when it was still a fetus (placental oxygenation) (Sohmer et al., 1994a) and that the human fetus responds to lower intensities of vibro- acoustic stimulation delivered to the maternal ab- domen when the mother is breathing oxygen than when she breathes room air (Sohmer et al., 1994b). It is interesting to point out that in those experiments where the degree of ABR threshold elevation induced by these levels of hypoxemia could be quantified, it was found to be 20 dB (this study; Sohmer et al., 1989,1994a; Sohmer and Freeman, 1991).

In conclusion, the sensorineural hearing loss seen in the human fetus in utero due to its relatively hypox- emit state is probably caused by mechanisms similar to those which lead to a 20 dB ABR threshold elevation in experimental animals made hypoxemic to a similar degree: the hypoxia induces a depression of the EP. This in turn affects the overall sensitivity (ABR) by depressing the activity of the OHC cochlear amplifier about 10 dB and by reducing transduction by the IHCs about 10 dB. At birth, with transition to more efficient pulmonary oxygenation, the EP rapidly reaches its higher, adult magnitude, accompanied by greatly im- proved IHC transduction and OHC amplification, with normal auditory thresholds.

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