From the concepts of well known ototoxic effects of loop diuretics
and hypoxic state, twenty anesthetized guinea pigs with diuretics and/or hypoxia
were evaluated with click-evoked otoacoustic emissions (CEOAEs) by timesequential
monitoring. Furosemide 25 mg/kg was infused intravenously and
CEOAEs were measured before and 5, 10, 20, 30, 45 and 60 minutes after
injection in 8 guinea pigs. Both echo responses and reproducibilities were
decreased significantly at 10 minutes after injection and showed minimal level at
20 minutes. Recovery of CEOAEs was noted by spectral analysis at 30 minutes
after injection, and full recovery of CEOAEs was evident after then. At 60
minutes after injection, hypoxia of 20 seconds by turning off the artificial
respirator was added. The CEOAEs showed statistically significant decreases
compared with those of prehypoxic state (p<O.OI). The CEOAEs of 8 guinea pigs
with injection of furosemide. 50 mg/kg showed decreased responses and no
fr
recoveries for the 60 minutes observation periods. Findings from this study
support the clinical utility of CEOAEs as a screening test for early detection of
various ototoxicities

Chang, Sun O

Jung, Ha Won

SNU Open Repository and Archive

The Effects of Furosemide and Hypoxia on C l  ick-Evoked Otoacoust ic Emissions in Anesthetized Guinea Pigsr Sun 0 Chang and Ha Won Jung =Abstract  =From the concepts of well known ototoxic effects of loop diuretics and hypoxic state, twenty anesthetized guinea pigs with diuretics and/or hypoxia were evaluated with click-evoked otoacoustic emissions (CEOAEs) by time- sequential monitoring. Furosemide 2 5  mg/kg was infused intravenously and CEOAEs were measured before and 5, 10, 20, 30, 45 and 60 minutes after injection in 8 guinea pigs. Both echo responses and reproducibilities were decreased significantly a t  10 minutes after injection and showed minimal level a t  2 0  minutes. Recovery of CEOAEs was noted by spectral analysis a t  30 minutes after injection, and full recovery of CEOAEs was evident after then. At 60 minutes after injection, hypoxia of 2 0  seconds by turning off the artificial respirator was added. The CEOAEs showed statistically significant decreases compared with those of prehypoxic state (p~0 .01) .  The CEOAEs of 8 guinea pigs with injection of furosemide. 50 mg/kg showed decreased responses and no recoveries for the 60 minutes observation periods. Findings from this study support the clinical utility of CEOAEs as a screening test for early detection of various ototoxicities. Key Wo r d s : Fur-osemide, H~po.\ ill, C1ic.k. E\lokrtl otorrc.oustic. en~issiorz, Gziit~ea pig of sound waves that were measured by a INTRODUCTION microphone inserted into the external ear. The presence of the EOAE provides evidence of a The evoked otoacoustic emission (EOAE) is functioning cochlea (Collet 1978; Stevens 1988). a reflection of the sound waves to a specific Thus it can be used as a functional cochleogram acoustic stimulus. It is generated from the that represents the physiology of the peripheral cochlea and emitted via ossicles and the sound processing mechanism (Kemp et a / .  tympanic membrane retrogradely. In 1978, 1986). Kemp reported the presence of faint reflections The EOAE test can be performed without anesthesia in human subjects. What should be Kec,ei\.ccl March 1993, and in final form June 1993. considered first on measuring the click evoked tThis st~lili  was silplx)rtc~l h i  gut11 #9?-132 fmtll SCOLII N111011a1 OtOacOustic is  adequate Ilnlvenity Hospital fitting of the probe in the external ear canal. '.1%r;q%L-z C,l~! \ r ;Hq 0 ] ~ l ? ! T 4 ~ ~ 4 :  %?!2,%5\$! Addit ional changes in acoustic damping (electric impedance) of earprobe cause different characteristics of emissions (Zwicker 1990). Measuring of CEOAEs in guinea pigs was thought to be a difficult problem because of their short latency and duration. The authors' data on the profiles of the CEOAE findings in normal guinea pigs(1992) showed stable findings for test-retest reliability, even though the thresholds were higher than human ones. And the predominant response was shifted to higher frequencies according to the spectral analyses. Loop diuretics cause interstitial edema of stria vascularis and secondary hair cel l  changes. These changes are reversible and occur in high dose intoxication (Quick and Duvall 1970; Kohonen and Janhidinen 1970). EOAEs could be  involved with reduction or elimination of its amplitude (Anderson and Kemp 1979; Anderson 1980; Kemp and Brown 1984; Wilson and Evans 1983). Hypoxia also reduces the amplitude of OAE in laboratory animals (Kim 1980; Schmidt and Adams 1981; Lonsbury- Martin et a/. 1987). Whether the ingestion of loop diuretics and the application of hypoxia have synergistic effects on the reduction of OAEs is not clear. This study was planned to evaluate the influence of furosemide and superimposed hypoxia on the profiles of the CEOAE. The hypoxic state was arti f icial ly created for evaluation of a synergistic insult to inner ear having al tered physiology by furosemide infusion. The primary intention of this study was to elucidate the efficacy of the CEOAE test as a surveillance index for detecting the effects of ototoxic drugs and toxic environments. animal which failed to show CEOAEs in a screening test was excluded from this study. Anesthesia was init iated with an intraperitoneal injection of sodium pentobarbital (Nembutal )  30 mg lkg .  The animal was tracheotomized in supine posit ion. After intramuscular injection of Myoblock (1 .25 mglkg) ,  respiration was controlled with an artificial ventilator (Narishige) at the rate of 60 timeslminute and 6-8 ml of tidal volume. Body temperature was maintained by convection heat from the surgical light. The total time of the experiment did not exceed 2 hours, so booster injections of the anesthetic agent were not necessary. After the experiment, the animal was sacrified immediately. , Measurement of the CEOAE This experiment was conducted using the IL088 Otodynamic Analyzer developed by Bray and Kemp (1987). The click stimuli elicited by 80 psec electr ical  pulses were used with a frequency range of 0-6 kHz and an interstimulus repetition rate of 20 msec. Figure 1 shows the system block diagram used in this study. The infant-style ear probe containing an earphone and a microphone was connected to the amplifier and the averaging computer system. Tight fitting of the probe on the EAC is a very important step on measuring the CEOAE (Zwicker 1990). We performed this experiment in a noise shielded booth with the animal deeply anesthetized to minimize external and internal noise. The artificial ventilator was also placed out of the booth to minimize noise. Threshold for the noise level was set on the ILO 88 Otodynamic analyser hardware configuration MATERIALS AND METHODS S e l e c t i o n  a,nd m a n i p u l a t i o n  o f  t e s t  animals Twenty albino guinea pigs weighing 250 to 400 gm and having normal Preyer reflexes were used. The tympanic membrane was assessed to be normal under microscopic examination. Any -4 IBM PCiXTiAT [ Standard IBM PC bus I Storage media generator processor earphone microphone F i g. 1. System block diagram used in this study IBM PC and the CEOAE was measured using the standard setting of the IL088 (260 repetition, window from 2 .5  msec to 20 msec) .  The amplitude of echo response above 5 dB and the reproducibility above 50% were regarded as positive responses (Kemp 1990). In jec t ion  o f  fu rosemide  and i nduc t i on  of hypox ia  After the preparation of the animal, a small probe was fitted into the external ear canal of the guinea pig. Measurement of the CEOAE was started from 0 dB test gain (84 dB peak SPL). Sequential measurement of the CEOAE was done by ascending and descending the test gain by 5 dB steps and eventually determining the minimal threshold of the click that suffices the reference value of a posit ive CEOAE. Variable parameters manifested on the CEOAE at a given threshold level were compared with those in a different ototoxic environmental setting. The animals were divided into four groups (Table 1); First group: Respiration of four guinea pigs was halted by turning off the respirator for 100 seconds and the CEOAE was measured immediately after the cessation of artificial respiration. Second group: The left internal jugular vein of eight guinea pigs was exposed surgically and 25 mglkg of furosemide was infused. The CEOAE was checked 5, 10, 20, 30, 45, and 60 minutes after the infusion of furosemide. These guinea pigs were exposed to 20 seconds of additional hypoxia, sixty minutes after the infusion of furosemide, and the CEOAE was checked again. Third group: Furosemide at a dosage of 50 mglkg was infused into the eight guinea pigs as in the secoi:d group. The CEOAE was checked sequentially 5, 10, 20, 30, 45, and 60 minutes after the infusion. Stat is t ica l  analysis Statistical analysis was performed by the SAS program. The clinical significance of the experiment was tested with the Wilcox signed rank test within p value of 0.01 RESULTS The CEOAE f indings under the hypox ic  env i ronment The threshold level of the CEOAE was checked in the first group (four guinea pigs). After identifying the threshold, the artificial ventilator was disconnected for 100 seconds and the CEOAE at the preset threshold level was checked right after the hypoxic event. The hypoxic state for 100 seconds proved to have no statistically significant impact on the profiles of the CEOAE. One example is displayed in Figure 2. Both amplitude of echo response and reproducibility did not show any significant change. The CEOAE f i nd ings  af ter  exposure to f u rosem i de. The ampli tude of echo responses and reproducibility of the second group (eight guinea p igs )  which were infused with 25 mg lkg  furosemide showed decreased parameters from 10 minutes to 45 minutes (p<0 .01) .  The amplitude and the reproducibility of the CEOAE were decreased from 10 minutes after infusion, to their lowest value at 20 minutes after infusion, and recovered to pre-infusion levels by 45 and 60 minutes after infusion (Fig. 3). Panel A in Fig. Table 1. Groups of experimental animals (N=20 guinea pigs) - - - -- - - -. - - .- Group No. of animals Dosage of furosemide Hypoxia exposure 100 sec 20 sec (-1 Am: 13.4 dB, R: 75% Am: 13.6 dB. R. 73% 0 5 mPa 1 20 msec Fig. 2. Time domain responses of CEOAEs to 20 dB test gain (104 dB pSPL) click stimuli before (A) and after (B) the hypoxic exposure for 100 seconds in a guinea pig. Am: Amplitude, R :  Reproducibility 3 shows the changes in the amplitude of echo response of 8 guinea pigs and panel B shows the changes of the reproducibility. Panel C plots the changes in both measures,with the horizontal plane indicating the amplitude of echo response and the vert ical  plane indicat ing the reproducibility. The echo response and the reproducibility in the CEOAE of the 8 guinea pigs which were infused with 50 mglkg furosemide (the third group) showed statistically significant decrease right after the exposure. There was no evidence of recovery of the echo response and the reproducibility after 60 minutes had passed (p<0.01). Fig. 4 depicts the CEOAE findings of the third group A d d i t i v e  e f f e c t  o f  h y p o x i a  o n  t h e  furosemide in fus ion The variables in the CEOAE were normal at 60 minutes after the exposure to the furosemide at 25 mglkg. Additional exposure to the hypoxia for 20 seconds after recovery from the furosemide infusion resulted in dysmotility of cardiac contraction during the hypoxic event. Profiles of the CEOAE immediately after the combined exposure revealed a statistically signif icant addit ive decrease in the echo response and the reproducibility than those caused by the separate exposures (p<0.01). Figure 4 displays the results of the additive insult of hypoxia in animals receiving furosemide (25 mglkg). DISCUSSION In this study, a deep anesthesia was maintained with the artificial ventilator via the tracheotomy tube to minimize noise from the test animal. Deep anesthesia was also necessary to maintain access with the intravenous drug- administration route. We used the small s ized probe t ip  commercialized by Otodynamics Ltd. This probe tip had the merit of superior detection capability of high frequency emission above 3 kHz and Echo res nse Reproducibilrty Reproducib~llty ( d B 7  ("1.1 ("/.I 100 100- o ~relnjectm 0 5 rnln A 1Orn1n 80 80. + a rnm 15 I XI rnm 0 @ 45mn 60 60. A 60rn1n 10 40 40 - Echo response (dB) Fig. 3. Click evoked otoacoustic emissions in furosemide 25 mg/kg group (8 guinea pigs) to the given threshold level of click stimuli. Panel A: time domain changes of echo amplitude, B: time domain changes of reproducibility, C: amplitude and reproducibility Echo res nse Reproducibility Reprod rc~Sility ( d a y  ("/.I (<.Lo ) 29 1 1~~ g,iyK!an min o h  40- -40- 0 20 40 @I Mln O 20 40 6I Mln O 5 10 15 X) Echo response (dB) 80 Fig. 4. Click evoked otoacoustic emissions in furosemide 50 mglkg group (8 guinea pigs) to the given threshold level of click stimuli. Panel A:  time domain changes of echo amplitude, B: time domain changes of reproducibility, C: amplitude and reproducibiliity. A 10 rnin + m min Preinjection A 60min hypoxia, 60 min I 0 0 5 10 15 20 Echo response (dB) 1 30 m ~ n  0 60' 0 45min 40. 20 0 subjects, but they have homogenous features for the same subjects that were tested at different times under the same stimulus intensity (Kemp et a / .  1986). Positive echo responses were regarded as funct ioning outer hair cel ls ,  especially within middle frequency region (Collet et a/. 1989). The CEOAE has a benefit of early detection capability of cochlear lesion, e.g any disease process, change due to exposure to noise, and exposure to ototoxic drugs. Temporary threshold shift after exposure to noise has been recorded objectively with the CEOAE in human subjects - 2 0  F ig.  5. Click evoked otoacoustic emissions in (Zurek 1985; Kemp et a/ .  1989). With ototoxic furosemide 25 mglkg group with hypoxia for 20 drugs, the initial decrease and the gradual seconds at the end of 60 minutes' observation period (8 guinea pigs, same group as Figure 3) recovery of the echo response of the CEOAE A - .  to the threshold level of click stimuli. have variable time intervals according to the kind of drug, that had already been previously capability of eliminating the distortion of the response by the stimulus itself (Kemp et a/ .  1986). The fitting procedure must not be so complete as that of the tympanometry test, but the noise should be restricted to an adequate level for a significant test. In this study, malleable silicon rubber sealing was applied around the tip to promote the correct fitting to the ear canal. The tip of the probe must be directed to the tympanic membrane without being blunted against the canal walls (Kemp 1986; Kim 1980; Bonfils and Uziel 1989; Bonfils et a/. 1989). The profiles of the CEOAE vary with different observed in patas monkeys in which loop diuretics had been infused (Zurek 1985; Kemp et a/. 1 989). The ototoxic effect of furosemide and hypoxia had been a proven cochlear manipulation method by many authors, but the effects on CEOAEs were not common. So these were selected as the test materials for evaluation of the ototoxic effect manifested in the changes of the CEOAE. Hypoxic state made with cessation of re,spiration for a period of 100 seconds in 4 guinea pigs had no statistically significant influence on the CEOAE. For the grljnea pigs that were infused with 50 mglkg of fuljsemide, the variable parameters of the CEOAE were decreased immediately after the infusion and there was no evidence of recovery even after 60 minutes had passed. The other group exposed to furosemide at 25 mglkg concentration had decreased parameters of the CEOAE and the lowest value at 20 minutes after the infusion. They began to recover from 30 minutes after the infusion and normalized to the previous level around 45 minutes after the infusion. These were tested with the Wilcoxon signed rank test and were statistically significant (p < 0.01). The CEOAE profiles to an additive toxicity of the hypoxia on the loop diuretics could be deduced from the fact that additional exposure of the hypoxia immediately after the recovery from the ototoxic loop diuretic insult resulted in a more statistically significant decrease of the variables of the CEOAE than that of separate exposure to the ototoxic drug.  Further comparat ive study with conventional acousticophysiological measures is mandatory to determine the underlying processes responsible for the synergistic effect. The CEOAE, as one of the responses of the peripheral sense organ, has a perspective of developing into a functional cochleogram for evaluating cochlear lesions (Kemp et a/. 1986; Bonfils and Uziel 1989; Bonfils et a/ .  1989). Changes in the echo level may not be a sign for differential diagnosis of cochlear pathology, but it remains as a subject of further study. REFERENCES Anderson SD, Kemp DT. The evoked cochlear mechanical response in laboratory primates. Arch Oto-Rhino-Laryngol 1979; 224: 47-54 Anderson SD. Some ECMF: properties in relation to other signals from the auditory periphery. Hear Res 1980; 2:273-96 Bonfils P, Uziel A. Clinical applications of evoked acoustic emissions: Results in normal hearing and hearlng impaired subjects Ann Otol Rhlnol Laryngol 1989; 98:326-3 1 Bonfi ls P, U z ~ e l  A, Narcy P. The propert ies of spontaneous and evoked acoustic emissions in neonates and children; a preliminary report. Arch Otorhinolaryngol 1989; 246:249-51 Bray P, Kemp DT. An advanced cochlear echo technique suitable for infant screening. Br J Audiol 1987; 21:191-204 Chang SO, Jung HW, Chung JW. The properties of click-evoked otoacoustic emissions in guinea pigs and studies of test-retest reliability. J Korean Otolaryngol Soc 1992; 35(4):485-92 Collet L, Gartner M, Moulin A. Evoked otoacoustic emissions and sensorineural hearlng loss. Arch Otolaryngol 1989; 1 15: 1060-62 Kemp DT. Stimulated acoustic emissions from within the human auditory system. J Acoust Soc Am 1978; 64: 1 386-9 1 Kemp DT, Brown AM. Ear canal acoustlc and round window electrical correlates of 2f,-f, distortion generated in the cochlea. Hear Res 1984; 13:39-46 Kemp DT, Bray P, Alexander L. Acoustic emission cochleography-practical aspects. Scand Audiol 1986; 25(suppl):71-95 Kemp DT, Bray P, Ryan S. Otoacoustic emission character~stics relating to individual susceptibility. Assoc Res Otolaryngol 12th Meeting Abstr 1989; p225 Kemp DT, Ryan S, Bray P. A guide to the effective use of otoacoustic emissions. Ear Hear 1990; 1 1 (2):93- 105 Kim DO. Cochlear mechanics : Implications of electrophysiological and acoustical observations. Hear Res 1980; 2:297-317 Kohonen A, Janhidinen T. Experimental deafness caused by ethacrynic acid. Acta Otolaryngol 1970; 70: 187-89 Lonsbury-Martin BL, Martin GK, Probst R, Coats AC. Acoustic distortion products in rabbit ear canal. I .  Basic Features and physiologic vulnerability. Hear Res 1987; 28: 173-89 Quick CA, Duvall AJ. Early changes in the cochlear duct from ethacrynic acid: an electron microscopic evaluation. Laryngoscope 1970; 80:945-65 Schmidt RA, Adams JC. Stimulated otoacoustic emissions in the ear of the gerbil. Hear Res 1981; 5: 295-305 Stevens JC. Click-evoked otoacoustic emissions in normal and hearing-impaired adults. British J Audiol 1988; 22145-9 Wilson JP, Evans EF. Effects of furosemide, flaxedil, noise and tone over-stimulation on the evoked otoacoustic emission in cat. 15th Proc Int Union Physiol Soc 1983; p100 Zwicker E. On the influence of acoustical probe impedance on evoked otoacoustic emissions. Hear Res 1990; 47: 185-90 Zurek PM. Acoustic emission from the ear; A summary of results from humans and animals. J Acoust Soc Am 1985; 78:340-4 

The Effects of Furosemide and Hypoxia on CIick-Evoked Otoacoustic Emissions in Anesthetized Guinea Pigs

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The Effects of Furosemide and Hypoxia on CIick-Evoked Otoacoustic Emissions in Anesthetized Guinea Pigs

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