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American Journal of OtolaryngologyHead and Neck Medicine and Surgery 34 (2013) 16 21 www.elsevier.com/locate/amjoto

The protective role of caffeic acid phenethyl ester against streptomycin ototoxicity
Salih Bakr, MD a,, Musa zbay, MD a , Ramazan Gn, MD a , Ediz Yorganclar, MD a , Vefa Kn, MD a , Ayenur Kele, MD b , Abdurrahman Abakay, MD c , Osman Gkalp, MD d , smail Topu, MD a
a

Department of ENT & Head and Neck Surgery, Dicle University Medical College, Diyarbakir, Turkey b Department of Pathology, Dicle University Medical College, Diyarbakir, Turkey c Department of Chest Diseases, Dicle University Medical College, Diyarbakir, Turkey d Department of Pharmacology, Dicle University Medical College, Diyarbakir, Turkey Received 5 March 2012

Abstract

Objective: The aim of this experimental study was to investigate the efficacy of caffeic acid phenethyl ester (CAPE) in the prevention of streptomycin-induced ototoxicity. Materials and Methods: Thirty-two adult Wistar albino rats were divided into 4 groups: control (n = 8), streptomycin (n = 8), CAPE (n = 8), and streptomycin + CAPE (n = 8). Rats were tested with distortion product otoacoustic emissions (DPOAEs) before drug administration. The animals in all groups were killed under general anesthesia on the 45th day following last DPOAE measurements. Hearing results were analyzed statistically to determine differences in amplitudes of DPOAE. Also, the cochleas of each rat were evaluated by histopathological and immunohistochemical examination. Results: Significant difference was not observed in cochlear hair cells in the control and CAPE groups. In the streptomycin group, severe degeneration of hair cells and increased apoptotic cells were observed. In the streptomycin + CAPE group, although some deteriorations were observed, hair cells were mostly preserved. The DPgram of the streptomycin and streptomycin + CAPE groups was significantly deteriorated (P b .05). The analysis of the DPgram results revealed statistically significant differences between the groups of streptomycin and streptomycin + CAPE (P b .05). Conclusions: Caffeic acid phenethyl ester treatment attenuated hair cells injury in the inner ear, possibly via its antioxidant effect. Prophylactic administration of CAPE for streptomycin ototoxicity ameliorated hearing deterioration in rats. 2013 Elsevier Inc. All rights reserved.

1. Introduction Class of aminoglycoside antibiotic is the first and the most common agent implicated as having ototoxic adverse effects [1]. Although the true incidence is difficult to determine, the reported incidence of significant ototoxicity from aminoglycosides varies widely in the literature and ranges from 2% to 5% [2]. Because of well-known
Corresponding author. Department of ENT & Head and Neck Surgery, Dicle University Medical College and Department of Physiology, Dicle University Medical College, 21280, Diyarbakir, Turkey. Tel.: +90 412 2488001/4492; fax: +90 412 248 8440. E-mail address: drsalihbakir@gmail.com (S. Bakr). 0196-0709/$ see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2012.07.003

cochleotoxic or vestibulotoxic adverse effects, in recent decades, especially in industrialized countries, the use of aminoglycosides has been relatively limited, several are only used topically, and many aminoglycoside drugs are no longer in use [1,3]. In developed countries, class of aminoglycoside antibiotic currently tends to be reserved for clinical use only in cases of resistant tuberculosis because of the need for life-saving treatment [2]. On the other hand, aminoglycosides are one of the most commonly prescribed antibiotics in other parts of the world, mostly in developing countries and underdeveloped countries, because of regional and economic reasons. Economically, aminoglycoside drugs are very inexpensive to produce, which is of greatest importance to powerless countries [1].

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In addition, aminoglycosides are often prescribed indispensably as a first-line therapy in multidrug-resistant bacteria in many types of infections especially tuberculosis disease (TB), which is still a very serious problem worldwide. The World Health Organization declared that TB is the second leading cause of death from a single infectious agent in the world and continues to be one of the world's major infectious diseases [4]. Despite the incidence of TB stabilizing in recent years, an estimated one-third of the world's population remain infected, resulting in over 9 million cases of currently active disease and approximately 1.3 million deaths per year [4]. Tuberculosis cases are still more common in Asian and African (85% of the cases) countries [5]. Therefore, the prescription of aminoglycoside is probably rather high in these countries. The objective of this work was to assess the caffeic acid phenethyl ester (CAPE) with antioxidant properties in the prevention or attenuation of ototoxicity caused by long-term aminoglycoside administration in a rat model. Caffeic acid phenethyl ester is one of the major components of honeybee propolis extracts. It has been used in traditional medicine for many years as a pharmacologically active and safe compound with anti-inflammatory, antiviral, antimitogenic, anticarcinogenic, immunomodulatory, and antioxidant effects [6-9]. It has been already shown that CAPE administration has protective effects against oxidative damage caused by different agents in various tissues such as kidney, liver, heart, lung, red blood cell, brain, and neural structures in rat models [6,7,10-14]. But, to our knowledge, the efficacy of CAPE against the toxic effects of aminoglycoside in the inner ear has not been investigated yet. In the present study, we examined the auditory and histopathological changes in cochlea exposed to aminoglycoside with/without CAPE. 2. Materials and methods All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council [15]. The study was also approved by the Ethical Committee of our institution under permit 2011/27. 2.1. Experimental design Thirty-two male adult Wistar albino rats weighing between 185 and 295 g were used in this study. They were maintained according to the standard guidelines. All animals were housed reasonably in cages under standard environmental conditions (room temperature between 22C and 24C and 50% relative humidity within a 12-hour light/12hour dark cycle photoperiod). All the animals had free access to water and conventional laboratory diet until before sacrifice. Animals were ranked by weight at the beginning of the study to ensure similar starting body masses between

groups. They were divided into 4 experimental groups: control group (n = 8), streptomycin-treated group (n = 8), CAPE-treated group (n = 8), and streptomycin + CAPE treated group (n = 8). Saline (2.5 mL/kg, intramuscularly) was used in the control group. In the other groups; streptomycin, CAPE, and streptomycin + CAPE were used for 45 days until surgery. Streptomycin was administered via the intramuscular way (dose of 20 mg/[kg d]). Caffeic acid phenethyl ester was purchased from SIGMA (Sigma-Aldrich Co LLC, St Louis, MO) and intraperitoneally injected once a day at a dose of 10 mol/kg. According to a previous report, at a concentration of 10 mol/L, CAPE completely inhibits the production of reactive oxygen species [8]. Another report suggested that the antioxidant activity of CAPE is dose dependent [9]. Because of these reasons, throughout the entire study without interruption, 10 mol/L CAPE was administered every day. The first dose of CAPE was given 24 hours before streptomycin administration and continued until sacrifice. 2.2. Anesthesia Rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar, Pfizer, Istanbul, Turkey) 60 mg/kg and 2% xylazine hydrochloride (Rompun, Bayer, Istanbul, Turkey) 10 mg/kg by intramuscular injection before evaluating the hearing and before sacrifice. 2.3. Hearing assessment To test the integrity of the hair cells, all animals were tested with distortion product otoacoustic emissions (DPOAEs) under anesthesia in a quiet room (less than 50 dB background noise). Before the DPOAE was measured, otoscopy was performed to confirm that the external auditory canal and tympanic membrane were normal. Only rats with normal ear canal and tympanic membrane and with initial otoacoustic examination showing normal responses were included in this study. The OAE recordings were elicited from the right and left ear of each animal using a standard commercial ILO-96 OAE apparatus cochlear emission analyzer (Otodynamics Ltd, London, UK). The data were processed and evaluated with otoacoustic emission (OAE) software (EZ Screen 2 Otodynamics OAE Screening and Data Management Software, Hatfield, UK). Each DPOAE test required about 3 minutes to perform. The DPOAEs were recorded before drug administration at the first day and before the animals were killed. Following anesthesia, the primary tones were introduced into the animals outer ear canal through an inserted earphone using a plastic adapter that sealed the probe in the outer ear canal. Equilevel primary tones f1 (65 dB) and f2 (55 dB) were fixed at f1/f2 = 1.22, and DPOAEs were measured at 5 different frequencies ranging from 2000 to 8000 Hz (2002, 3003, 4004, 6006, and 8008 Hz). The DPOAEs were determined as DPgrams. The baseline hearing status of all animals was determined with DPgram, and the signal-to-noise ratio (SNR) was found. For

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Control

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CAPE Streptomycin Streptomycin + CAPE

DPOAE Amplitude levels (dB SPL)

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24

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0 2000 3000 4000 Frequency (Hz) 6000 8000

Fig. 1. DPgrams from rats treated with saline, streptomycin, CAPE, and streptomycin plus CAPE. Determination of DPOAEs on day 0 (DP = distortion product; SPL = sound pressure level).

each animal, SNRs at 5 frequencies were recorded. Data were collected separately for each rat, and the results were statistically analyzed. 2.4. Operation procedure All procedures were performed under clean but nonsterile conditions. After being anesthetized, they were decapitated. After fixation by intralabyrinthine perfusion of 4% paraformaldehyde (pH 7.4), the cochleas were removed. The skull bone of the subjects was cut in the midline, and each temporal bone was dissected by scalpel and a pair of scissors. Using the hands and having the external auditory canal as a guide, the bulla was localized with the thumbs. Their temporal bones containing the tympanic bulla were separated from other structures to reveal the cochlea by holding it with one hand and with a hemostatic clamp. The bulla was opened by holding it with one hand; and with a hemostatic clamp, an opening on the posterior air sinus (mastoid) was made. Then, positioning the clamp in the external auditory canal, in a single movement, all bone parts of the leaflet were broken, exposing the cochlea. In the end, the cochleas were dissected and removed en bloc. Then, the cochleas were fixed into a 10% formaldehyde solution and stored for immunohistochemical study. 2.5. Tissue preparation The cochleas of each rat were fixed for 24 hours in 10% formaldehyde solution and subjected to decalcification for 3 weeks in 10% ethylenediamine tetraacetic acid solution. After the fixation and decalcification processes, the cochleas were washed with tap water for 24 hours, were dehydrated by reaction with graded alcohol series, and were transparented and blocked after infiltration with paraffin. Each paraffin-embedded specimen was sectioned with a microtome (Leica RM 2125, Leica Microsystems, Nussloch GmbH, Germany) at a thickness of 5 m. Each section was stained with hematoxylin and eosin (H&E) solution and observed with a Nikon ECLIPSE 80i (Japan) microscope.

On the other hand, the remaining tissue sections were deparaffinized and washed after reaction with alcohol series and after incubation with 0.1% to 1% H2O2, washed with phosphate-buffered saline (PBS), and dyed with avidinbiotin. The sections were conducted with 10% normal bovine serum. Then, caspase-3 rat polyclonal IgG primary antibody was diluted with 1:400 normal bovine serum. The sections were incubated with caspase-3 during the night. Phosphate-buffered saline was dripped onto negative control sections. Next day, the sections were washed with PBS and incubated firstly with secondary antibody (biotin bovine antirat) and then horseradish peroxidase and, after washing with PBS, conducted with diaminobenzidine chromogen. The sections washed with distilled water were dyed by hemotoxylin. Then, the sections were washed with distilled water until blueness disappeared, and closed after reaction with alcohol and xylene. The sections were evaluated according to the intensity of caspase-3 immunoreaction under light microscope (Nikon ECLIPSE 80i). 2.6. Statistical analysis Statistical evaluation was carried out using SPSS 15.0 version for Windows (SPSS Inc, Chicago, IL). Results were analyzed statistically by Kruskal-Wallis to determine differences in amplitudes of DPOAEs and corresponding noise floor differences and thresholds for each frequency. The histopathological variations of streptomycin, CAPE, and streptomycin with CAPE and without medication (control) were evaluated and compared with each other by MannWhitney U test. Statistically significant level was accepted as P value less than .05.

3. Results 3.1. Hearing results In the DPgrams, for all sessions, the emission amplitude levels were greater than the noise floor throughout the
Control CAPE Streptomycin Streptomycin + CAPE

DPOAE Amplitude levels (dB SPL)

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24

16

0 2000 3000 4000 Frequency (Hz) 6000 8000

Fig. 2. DPgrams from rats treated with saline, streptomycin, CAPE, and streptomycin plus CAPE. Determination of DPOAEs on day 45.

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Fig. 3. CAPE group. Histomorphologic appearance of the hair cells in cochlea (H&E 400).

Fig. 5. Streptomycin group. Caspase-3 positivity in hair cells (arrows) (mmunoperoxidase, 400).

testing frequencies. On day 0, the initial baseline DPOAE measurements presented similar values to those of the groups prior to drug administration (P N .05) (Fig. 1). On the 45th day, according to results of the second DPOAE measurements, a prominent decrease in cochlear activity involving frequencies between 2002 and 8008 Hz was observed (P b .05) in the streptomycin and streptomycin + CAPE groups, whereas the control and CAPE groups revealed no significant differences (P N .05) (Fig. 2). The DPgram of the streptomycin group was significantly deteriorated compare with the other groups (CAPE, control, streptomycin plus CAPE). Also, the DPgram of the streptomycin + CAPE group was deteriorated too; but the analysis of the DPgram results revealed statistically significant differences between the groups of streptomycin and streptomycin with CAPE (P b .05).

3.2. Histopathological results According to our results under light microscopy; H&E staining showed no distinctive difference between the control group and the CAPE group in histopathological examination (Fig. 3). But in streptomycin-treated rats, cochlear hair cells showed severe hair losses (Fig. 4). With immunohistochemical examination, caspase-3 immunoreactivity was not observed in the control group, CAPE group, and streptomycin + CAPE group (not shown), whereas in the streptomycin group, cytoplasmic or nuclear caspase-3 immunoreactivity was observed clearly in hair cells, supporting cells, and basilar membrane (Fig. 5). In the streptomycin + CAPE group with H&E staining, although some deteriorations were observed, hair cells were mostly preserved (Fig. 6). With immunohistochemical examination in the streptomycin + CAPE group, again some deteriora-

Fig. 4. Streptomycin group. Cochlear hair cells showed severe hair losses (H&E 200).

Fig. 6. Streptomycin + CAPE group. Histomorphologic appearance of the hair cells in cochlea (H&E 400).

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Fig. 7. Streptomycin + CAPE group. Some deteriorations were demonstrated in hair cells, but apoptotic cells were not observed with caspase-3 (mmunoperoxidase, 1000).

tions were demonstrated; but apoptotic cells were not observed with caspase-3 (Fig. 7).

4. Discussion Aminoglycosides with prolonged treatment have been shown to lead to permanent damage to sensory cells and supporting cells in the cochlea in humans and animals. Because of lack of regeneration capability of hair cells, this damage results in permanent hearing loss [1,16,17]. No therapy currently exists to attenuate the potential ototoxicity of parenterally aminoglycoside antibiotics. Although many hypotheses have been proposed to explain the ototoxic effects of aminoglycosides, the details of biochemical and molecular mechanisms underlying the destruction of cochlear and vestibular hair cells are poorly understood. The formation of aminoglycoside-induced free oxygen radicals within the inner ear is presumed to be a principal mechanism underlying sensory hair cells death [1]. In animal models, in cases of aminoglycoside ototoxicity, a variety of free radical species were detected in the inner ears, which are believed to initiate apoptotic signaling pathways via oxidative stress [18,19]. Therefore, a number of antifree radical agents have been claimed to have a protective effect against the ototoxicity caused by various aminoglycosides in animals. But, to our knowledge, the possible protective effect of CAPE on aminoglycoside-induced ototoxicity has not yet been investigated so far. Recently, the efficacy of CAPE against cisplatin-induced ototoxicity was investigated [20]. In that research, Kizilay et al [20] reported that prophylactic

administration of CAPE for cisplatin ototoxicity ameliorated hearing deterioration in rats. They evaluated the effect of CAPE on cisplatin-induced ototoxicity with audiologic examination only, whereas in the present study, in addition to audiologic examination, we examined the ultrastructural morphology and histopathological changes in cochlea exposed to aminoglycoside with/without CAPE. According to our results, histopathological examination with H&E staining showed that deterioration was not observed in cochlear hair cells in both the control and CAPE groups. According to this finding, CAPE probably has no harmful effects on hair cells. On the other hand, severe degeneration of hair cells and cochlear nerve fibers was observed in the streptomycin group, similar to previous reports. Schirmer et al [21] reported that the sensory epithelium of the cochlea was severely damaged in streptomycin-treated rats and no hair cells and supporting cells were visible on the basilar membrane. Our histopathological findings with H&E staining were similar to their results. In addition, caspase-3 immunoreactivity was demonstrated in the present study (Fig. 5). The caspase-3 immunoreactivity in the streptomycin group was strongly associated with an increased apoptotic events in the cochlea, which indicates a severe malfunction of the hearing organ. According to the accumulated information obtained from previous reports, excessive accumulation of free radical agents could be the most reasonable cause for the increase in apoptosis. If free radical agents are present at high concentrations, they are thought to act as a cytotoxin and can kill cells [18,19,21]. Then, those dead cells (eg, sensory hair cells) can be demonstrated histopathologically with caspase-3 as in the present study. In the streptomycin group, increased apoptotic cells were demonstrated in the rat cochlea with caspase-3. On the other hand, those dead cells were not observed in the streptomycin + CAPE group. Our histopathological results have shown that hair cells are robust against long-term streptomycin + CAPE administration. Caffeic acid phenethyl ester treatment significantly attenuated tissue injury, possibly via its antioxidant effect. Demonstration of increased apoptotic cells in rat cochlea with caspase-3 indicated a hearing impairment due to longterm streptomycin administration. Our DPOAE results justified this finding. The DPOAE responses are generated when the cochlea is stimulated simultaneously by 2 pure-tone frequencies (f1 and f2). Clinical studies in humans and experimental studies in animal models have established a link between drug-induced changes in DPOAE and changes in the outer hair cells [17]. The DPOAE response was evaluated using the SNR. Loss or impairment of outer hair cell function results in the attenuation of SNR [17]. According to our results, the hearing abilities of streptomycin-treated rats were significantly deteriorated compare with the other groups (control, CAPE, streptomycin plus CAPE) within the tested range of frequencies. The hearing abilities of the streptomycin + CAPEtreated rat group were decreased too, but the analysis of the DPgram results revealed statistically significant

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differences between the groups of streptomycin and streptomycin + CAPE (P b .05). The results of the streptomycin + CAPE group's DPgrams are significantly better than those of the streptomycin group's DPgrams. According to our results, prophylactic administration of CAPE for streptomycin ototoxicity ameliorated hearing deterioration in rats. Caffeic acid phenethyl ester seemed to be beneficial according to the present audiologic and histopathologic results in a streptomycin-induced cochlear damage rat model. Because of its antioxidant property, CAPE might be considered to prevent aminoglycoside ototoxicity. But the current study provides only limited insight regarding the protective effect of CAPE against ototoxicity because of 2 reasons. Firstly, auditory brainstem response thresholds could not be obtained because of technical barriers; this is the biggest limitation of the present study that should be taken into account when interpreting our results. Therefore, it did not allow us to evaluate the magnitude of the audiological effects or effect of CAPE on hearing preservation. On the other hand, there was no possibility to assess the data with electron microscopy, which is another limitation. Further studies using those tools are needed to determine the exact effects of CAPE. We believe further studies such as longer CAPE treatment or higher dosages are needed to investigate the effects of CAPE more clearly in ototoxicity.

5. Conclusion A great effort has been made to develop therapeutic approaches for the prevention of aminoglycoside ototoxicity. Among them, antifree radical agents are becoming increasingly important. Because aminoglycoside drugs induce formation of free oxygen radicals, antioxidant therapies should be considered in the treatment. Caffeic acid phenethyl ester might be a novel agent to protect the inner ear from aminoglycoside-induced oxidative stress. Acknowledgments The authors thank Yilmaz Palanci, assisstant professor, from the Department of Public Health, Dicle University School of Medicine, for his valuable statistical assistance. This experimental study was supported by a grant from the Dicle University Research fund under the project number 10-TF-63. References
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