Effect of anesthetic agents and middle ear pressure application on distortion product otoacoustic emissions in the gerbil

ELSEVIER

Hearing Research 112 (1997) 167-174

Effect of anesthetic agents and middle ear pressure application on distortion product otoacoustic emissions in the gerbil Yulian

Zheng

a,,, Kenji

Ohyama

b, K o j i

Hozawa

a, H i r o s h i

Wada

c, T o m o n o r i

Takasaka

~

a Department of Otolaryngology, Tohoku University School of Medicine, 1-1 Seiryo-machi. Aoba-ku, Sendai 980-77, Japan b Department of Otolaryngology, Tohoku Rosai Hospital, Sendai 980, Japan c Department of Mechanical Engineering, Tohoku University, Sendai 980, Japan Received 28 July 1996; revised 26 June 1997; accepted 4 July 1997

Abstract

The functional status of the middle ear system has a crucial importance in the measurements of distortion product otoacoustic emissions (DPOAEs), because each emission signal has to be detected indirectly in the external canal. It was observed that DPOAEs were scarcely detectable in the gerbil anesthetized with pentobarbital. On the other hand, when ketamine was used as an anesthetic, the DPOAE levels were generally high. The differences in the effects of these anesthetic agents on the DPOAEs became less clear when the tympanic bulla was opened. This strongly suggests that the effects might be due to a modification of the middle ear pressure. This study was designed to elucidate the mechanisms of the effects of these anesthetics on the DPOAEs. Comparing the effects of pentobarbital and those of pressure application to the middle ear on the frequency characteristics of DPOAEs, the following conclusions emerged: (1) pentobarbital administration causes negative middle ear pressure in the gerbil; (2) the generated pressure strongly reduces DPOAE conduction through the middle ear; and thus (3) proper selection of anesthetic agents is very important in gerbil experiments that involve OAE measurements.

Keywords: Distortion product otoacoustic emission; Anesthetic; Middle ear pressure; Gerbil

1. Introduction

Distortion product otoacoustic emissions (DPOAEs) are generated in the cochlea by the non-linear interaction of two primary tones externally introduced through the middle ear. They have to pass through the middle ear again before being detected by a microphone in the ear canal. Therefore, the middle ear transfer function should play an important role in determining the frequency characteristics of D P O A E levels. Based on theoretic considerations, K e m p et al. (1986) have shown that the forward and reverse transfer properties of the middle ear m a y have a significant influence on the variations of O A E levels. O h y a m a et al. (1992) reported that alterations of the middle ear environment,

* Corresponding author. Fax: +81 (22) 717-7307.

0378-5955197l$17.00 © 1997 Elsevier Science B.V. All rights reserved PIIS0378-5955(97)001 18-4

such as compliance changes produced by a hole made in the bulla, significantly affected D P O A E levels. Some clinical studies have shown that pathological middle ear pressure, which is commonly observed in young children, disturbs the O A E measurement (Prellner et al., 1992) and that the pressure problems can be more troublesome than noise contamination in such patients (Lonsbury-Martin et al., 1994). In gerbils, D P O A E levels are generally very high. During D P O A E measurements in the gerbil, however, it was noticed that pentobarbital-anesthetized animals often showed p o o r results, i.e. D P O A E levels were undetectable except in a very limited frequency range. Using different anesthetics made little difference in D P O A E measurements when the tympanic bulla was opened. The aim of the present study was to elucidate the mechanisms of the different effects of two widely used anesthetics, pentobarbital and ketamine, on D P O A E s in gerbils.

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Y. Zheng et aL /Hearing Research 112 (1997) 16~174

2. Materials and methods

3. Results

Twelve healthy Mongolian gerbils (Meriones unguiculatus) 60 100 g in body weight and 3-5 months of age with clean external ears and no signs of middle ear infection were chosen for this investigation. All animals used in this study were obtained from the Institute for Experimental Animals of the T o h o k u University School of Medicine. The animals were initially anesthetized with ketamine hydrochloride (100 mg/kg, i.m.) 30 min after a single injection of atropine sulfate (0.2 mg, i.m.), and were artificially ventilated by means of a tracheal cannula introduced by tracheotomy. Rectal temperature was maintained at 37°C by an automatically regulated warmer during the experiments. The pinna and cartilaginous portion of the ear canal were removed to gain clear access to the meatus. Skin and soft tissues covering the bulla were removed to open the bulla at once as required. Then the probe was carefully inserted into the external ear canal. D P O A E levels at 2fl-f2 were measured using a measuring system from Etymotic Research (earphone: ER2; microphone: ER-10B; IBM PC based DSP board: Ariel DSP16+; software: C U B D I S , v2.4). Equilevel primaries (L1 = L2 = 65 dB SPL) at a frequency ratio of f2/ fl = 1.2 were used. D P O A E measurements were carried out at 129 frequency combinations from 16 to 0.5 kHz of f2. An initial measurement was made with the bulla in a closed condition. For the purpose of this investigation, routine use of muscle relaxant was avoided during the experiments. The sound pressure level at the probe tip was calibrated before each measurement session using two earphones (see the two curves of the left panels of Figs. 3-5). All the calibration data were recorded to be analyzed later. The experiments were performed as follows: (1) investigation of ketamine effects on D P O A E s in comparison with pentobarbital effects in four gerbils, (2) observation of the effects of middle ear pressure application on D P O A E s in five gerbils, and (3) testing the influence of a muscle relaxant on D P O A E s in three gerbils. Ketamine was always given first before pentobarbital. To provide pressure to the middle ear, a short capillary needle (length: 0.5 cm, inner diameter: 0.45 mm) was inserted through a small hole made in the wall of the bulla and the hole was sealed with dental cement. This needle was connected to a pressure transducer through a plastic tube, Then the middle ear pressure was adjusted manually and was monitored by a pressure amplifier recording system (Krone, PA501; Secon/c, SS-250F). Absence of air leaks was carefully checked before measuring D P O A E s ; if the system was leaky, tl~e whole process was repeated.

3.1. Pentobarbital and ketamine effects

The D P - g r a m was greatly influenced by pentobarbital anesthesia. Typical examples are shown in Fig. 1. There were commonly three patterns of DP-gram in gerbils anesthetized with pentobarbital ( 4 0 4 5 mg/kg, i.p.) with closed bulla: (1) no detectable D P O A E s in the whole frequency range (Fig. l a), (2) measurable D P O A E s only in the mid and high frequency regions (Fig. lb), (3) normal D P O A E measurement in the frequency range above 0.8 kHz (Fig. lc). The third pattern was rarely seen, however. If the middle ear cavity was opened, however, D P O A E s could be detected at high levels throughout the frequency range irrespective of

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Y. Zheng et al. / Hearing Research 112 (1997) 167-174

canal, the frequency response showed a large difference before and after the administration of pentobarbital. This indicates that a significant change of the middle ear dynamics occurred shortly after pentobarbital injection. In the frequency region of 4-6 kHz, the frequency response curve showed a marked dip after the injection of pentobarbital. It became deeper and moved slightly towards higher frequencies. At the same time a peak on the DP-gram was observed in a frequency region essentially identical to the frequency where a dip on the corresponding frequency-response curve appeared. All animals examined showed a similar trend.

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what type of DP-gram was previously evident. The results indicate that cochlear function is not affected by pentobarbital administration. The generation of abnormal middle ear pressure seemed to be the crucial problem. It is most likely that negative pressure was present in the middle ear because inward displacements of the tympanic membrane were always observed with an operating microscope during the pentobarbital anesthesia. To confirm the effects of pentobarbital on DPOAEs, the effects of ketamine were investigated. When ketamine was used in the closed bulla condition, DPOAEs were always detected at high levels across the frequency range above 0.8 kHz and D P O A E detection was quite stable throughout the anesthesia, as shown in Fig. 2. However, when pentobarbital was given, the D P O A E level was quickly decreased, especially in the low frequency region below 3 kHz. The panels of Fig. 3 show the time course of the effects of pentobarbital on the DP-gram (right panel) and the corresponding frequency-response curves taken from the ear canal calibration data (left panel). At different frequencies, the effects of pentobarbital on DPOAEs were different. D P O A E levels dropped in the low frequency region initially. High frequency reduction followed, and finally, the middle frequency region was decreased, especially for the 4-6 kHz DPOAEs. In most cases, complete reduction of DPOAEs occurred within 40 min after pentobarbital injection. Changes of the middle ear dynamics were indirectly reflected in the ear canal calibration data obtained at the beginning of each session. As shown in Fig. 3 (left panel), although the electrical driving signal was always identical and all data were collected from the same ear

For experimental purposes, there are three methodological techniques for changing middle ear pressure: first, by changing the pressure in the closed outer ear canal as described by some authors (Naeve et al., 1992; Veuillet et al., 1992); second, by varying the atmospheric pressure in a pressure chamber as described by Hauser et al. (1993) and Richter et al. (1994); third, through the tympanic bulla directly (Ohmura et al., 1987). The latter method was chosen for this study because it is more representative of what occurs pathophysiologically in the clinical case. The effects of middle ear pressure changes on DPOAEs recorded from one animal are shown in Fig. 4. When a negative pressure of 50-100 mm H 2 0 was applied in the middle ear, the obtained results were quite similar to the pentobarbital effects described above, although some differences were observed between animals. The DP-gram (right panel) and the corresponding frequency-response curves (left panel) were almost identical in shape to those of pentobarbital, as shown in Fig. 4b,c. Similarly, in the frequency region of 4-6 kHz, there was also a peak response in D P O A E levels (right panel) and a dip in the frequency-response levels (left panel). However, different results were observed under positive pressure (see Fig. 4d,e). No marked peak in D P O A E levels and no dip in the frequency response levels could be observed in the former region. Thus, it is clear from the experimental results that pentobarbital simulates precisely the negative middle ear pressure effect in gerbils. D P O A E levels showed frequency-specific changes, with the primary effect caused by increased pressure on frequencies below 3 kHz. As the applied negative pressure was increased, there was a depression of D P O A E levels initially in low, then in high, and finally in the middle frequency regions. Under positive pressure, the D P O A E levels decreased gradually from low toward high frequencies. In the high frequency region above 6 kHz, the D P O A E levels remained high even when the positive pressure was raised above 200 mm H20.

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3.3. Effect of muscle relaxant In animal studies, muscle relaxant is frequently used along with anesthetic agents to achieve immobility. Investigating the effect of muscle relaxants on DPOAEs is also important and might be helpful for understanding the pentobarbital-related mechanisms that cause negative middle ear pressure.

The effects of suxamethonium chloride (depolarizing muscle relaxant) on DPOAEs were additionally investigated. Fig. 5 shows the DP-gram and corresponding ear canal calibration data before and following i.m. injection of suxamethonium chloride (5 mg) in a ketamine-anesthetized animal. Level reductions of DPOAEs were observed after suxamethonium administration. The frequency pattern of the suxamethonium-

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Fig. 4. Effect of middle ear pressure changes on DPOAEs. Left panel: Corresponding frequency response curve. DPOAEs were influenced differently by negative and positive pressure. When the middle ear underwent applied negative pressure, the pattern of reduced DPOAEs resembled that observed with pentobarbitaL Under positive pressure, the DPOAE level dropped gradually from low to high frequency. For frequencies > 6 kHz, DPOAE levels remained high, even when the pressure was increased to 200 mm H20. r e l a t e d r e d u c t i o n o f D P O A E s was s i m i l a r to t h a t p r o duced by app!ied negative middle ear pressure, although the effect w a s s m a l l e r t h a n t h a t in t h e p e n t o b a r b i t a l c o n d i t i o n at this d o s a g e level.

4. Discussion A u d i t o r y e l e c t r o p h y s i o l o g i c a l e x p e r i m e n t s o n animals often require an anesthetized and immobile prep-

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aration. Because m a n y of the experiments are carried out under an opened bulla condition, the influence of anesthetic agents on middle ear conduction is not normally noticed. It was observed in the present experiments that pentobarbital causes negative pressure in the middle ear in gerbils. This can be supported by the following arguments: (1) pentobarbital-related reduction in D P O A E levels can be recovered by ventilation of the middle ear cavity, and (2) the level-reducing effects of pentobarbital on D P O A E s closely resemble the effects of applying negative pressure in the middle ear cavity. According to the present findings, it might be necessary to reconsider some previous experimental results from gerbils with a closed bulla. The effects of anesthesia on cochlear potentials, audi-

tory brainstem responses, and late auditory evoked responses are well documented. Fullerton et al. (1987) reported that anesthesia with barbiturate produced a large decrease in the amplitude of the low frequency component of the brainstem response in the cat. Smith and Mills (1991) obtained different results in gerbils. Ketamine anesthesia induced only minor decreases in the amplitude of the low frequency component of the brainstem response. In that report, the authors suggest that the difference in anesthetics (barbiturate versus ketamine) or the level of anesthesia was probably responsible for the difference between the results of other authors and their findings in the gerbil• According to the present findings, differences might have resulted from the dissimilar effects of pentobarbital and ketamine on

Y. Zheng et al./Hearing Research 112 (1997) 16~174

the middle ear's conduction system. In other species, however, the effect of pentobarbital on middle ear pressure remains unknown. It is difficult to uncover the mechanism whereby pentobarbital causes negative pressure in the gerbil's middle ear. The equalization of pressure between the environment and the middle ear is maintained by the ventilation function of the Eustachian tube and by gas exchange between the middle ear space and the microcirculation in the middle ear mucosa. Under normal physiological conditions, the regulation of the total middle ear pressure is controlled by active opening of the Eustachian tube. One possibility is that pentobarbital may block the activity of the Eustachian tube, thereby preventing communication between the nasopharynx and the middle ear. In the present experiments, D P O A E s were reduced with the same patterns by pentobarbital and suxamethonium chloride. This suggests that an identical mechanism might reduce the D P O A E s in both cases. It is well known that pentobarbital causes dysarthria as a side effect (Robinson et al., 1981), which could prevent proper opening of the Eustachian tube. In the present results, significant changes in middle ear dynamics and D P O A E levels occurred shortly after using pentobarbital, indicating that Eustachian tube dysfunction is not the only cause of the negative pressure. Other factors might include the pressure generating process. The gas composition of the middle ear is fundamentally different from that of air, resembling that of venous blood (Sad6 et al., 1995). Accordingly, a gas exchange process takes place between the middle ear cavity and the blood, and this process m a y be related to the mechanisms involved in the development of negative middle ear pressure (Yee and Cantekin, 1987; Sad~ et al., 1995). However, it is not clear whether the gas exchange process in gerbils is influenced by pentobarbital, since the physiological mechanisms of the drugs used in this study are not so well understood. One factor that can be excluded in the present study is the effect of hypoxia induced by anesthesia, because the animals were artificially ventilated with a respirator after intratracheal intubation. Because either pentobarbital or suxamethonium chloride influences D P O A E s by exerting negative middle ear pressure, one presumes that the post-drug shape of the DP-grams is largely related to the degree of negative pressure exerted in the middle ear. This explains why different types of DP-grams were noted in the various pentobarbital-anesthetized gerbils at the same dosage level. Differences would also result from the depth of anesthesia at the start of the experiments. The results also indicate that the anesthesia sensitivity to pentobarbital varies with the individual animal. The present results show that middle ear pressure has a frequencydependent influence on D P O A E levels, as was described earlier by Hauser et al. (1993) and Richter et al. (1994).

173

Lower frequencies were generally more influenced by middle ear pressure changes than higher frequencies, although the absolute D P O A E levels decreased across the frequencies under increasing middle ear applied pressure. Similar findings at low frequencies were also observed for spontaneous OAEs (Schloth and Zwicker, 1986) and transiently evoked OAEs by other investigators ( K e m p et al., 1990; Naeve et al., 1992; Veuillet et al., 1992). It has been shown in cat and human that the low frequencies are more affected by the elasticity of the tympanic membrane than the high frequencies, which are more dependent on the mass of the vibrating parts of the middle ear (Peake et al., 1992), indicating that the mass of the middle ear was less influenced by pressure changes in the middle ear than the elasticity of the tympanic membrane. In the present experiments, the frequency-response curves in the ear canal showed different behavior under different pressure conditions within the middle ear. O h y a m a et al. (1992) have suggested previously that pressure-induced displacements of the tympanic m e m b r a n e and stapes footplate produce resonance-based frequency changes of the middle ear and stiffness-related changes to the ear drum and ossicles. Such changes in resonance frequency and stiffness are distinct between applied negative and positive middle ear pressure variations, and the effects are reflected in the ear canal calibration data in a complicated manner. Consequently, the ear canal calibration data also provide important information concerning the middle ear condition, and they should be used as a screening test before measuring DPOAEs.

Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research (A) of the Ministry of Education, Science and Culture. The authors would like to thank Dr. K. Esfarjani and B. Bell for critically reading various versions of the manuscript. The helpful comments by reviewers are gratefully acknowledged.

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