Cisplatin Ototoxicity in the Sprague Dawley Rat Evaluated by Distortion Product Otoacoustic Emissions:Ototoxicidad por Cisplatino en la rata Sprague Dawley evaluada mediante productos de distorsión de las emisiones otoacústicas

Original Article Audiology 2001; 40:253–264

Stavros Hatzopoulos* M. Di Stefano* K. C. M. Campbell † D. Falgione ‡ D. Ricci § M. Rosignoli* M. Finesso $ A. Albertin* M. Previati § S. Capitani § A. Martini* *Department of Audiology and Centre of Bioacoustics, University of Ferrara, Italy † Department of Surgery, Audiology, Southern Illinois University School of Medicine, Springfield, Illinois, USA ‡ Department of Biology, Section of General Physiology, University of Ferrara, Italy § Department of Morphology and Embryology, Section of Human Anatomy, University of Ferrara, Italy $ Fidia Research Laboratories, Abano Terme, Padova, Italy

Key Words Distortion product otoacoustic emissions, cisplatin, ototoxicity

Cisplatin Ototoxicity in the Sprague Dawley Rat Evaluated by Distortion Product Otoacoustic Emissions Ototoxicidad por Cisplatino en la rata Sprague Dawley evaluada mediante productos de distorsión de las emisiones otoacústicas Abstract

Sumario

The present study has evaluated the use of distortion product otoacoustic emission (DPOAE) responses in the detection of cisplatin-induced ototoxicity in a Sprague Dawley rat animal model. The cisplatin was administered as a 16 mg/kg, dose introduced by a slow 30-min intraperitoneal infusion. Data from three DPgram protocols, DPOAE input–output responses at 8 kHz, and auditory brainstem responses (ABRs) at 8, 12 and 16 kHz were collected before and 72 h after treatment. The post-treatment ABRs at 16 kHz showed the greatest mean threshold shift of 33.6 dB. The post-treatment DP-gram data showed significant reduction of the signal to noise ratios in the majority of the frequencies tested, across all tested protocols. The data suggest that the most sensitive DPOAE procedure for the early detection of the cisplatininduced ototoxic damage is the DPOAE I/O protocol. Morphological analyses indicated that the inner hair cells remained intact, while several types of alterations were observed in the arrangement of the stereocilia in the outer hair cells.

Este estudio evaluó el uso de los productos de distorsión de las emisiones otoacústicas (DPOAE) en la detección de daño por ototoxicidad inducida por Cisplatino en el modelo animal, rata Sprague Dawley. Se administró Cisplatino a 16 mg/kg por infusión intraperitoneal lenta durante 30 minutos. Se colectaron resultados de tres protocolos de DPgramas, respuestas de entrada/salida (I/O) a 8 kHz y respuestas de tallo cerebral (ABRs) a 8, 12 y 16 kHz; antes y 72 hrs. después del tratamiento. Las respuestas ABR a 16 kHz post-tratamiento mostraron el mayor cambio de umbral, en promedio de 33.6 dB. El DPgrama post-tratamiento mostró una reducción significativa de la diferencia señal/ruido en la mayoría de las frecuencias examinadas, de todos los protocolos realizados. Los datos sugieren que el procedimiento más sensible para detectar el daño por ototoxicidad inducida por Cisplatino es la función I/O DPOAE. El análisis morfológico indicó que las células ciliadas internas quedaron intactas, mientras que varios tipos de alteraciones se observaron en la disposición de los estereocilios de las células ciliadas externas.

Introduction When the cochlea is stimulated simultaneously with two pure tones at frequencies f1 and f2 (with f2 > f1), it generates additional frequency components called intermodulation distortion tones. These are not present in the original stimuli and are viewed as products of two-tone interactions caused by the non-linearity of the cochlea.1 The frequencies of these distortion tones or products are called primaries f1 and f2. For two given primaries, numerous families of distortion products (distortion product otoacoustic emissions (DPOAEs)) exist, but in the mammalian ear the cubic distortion product 2f1–f2 is the most prominent one.1,2 There is evidence that the 2f1–f2 product is generated by the outer hair cells Received: February 19, 2001 Accepted: May 19, 2001

(OHCs),2–5 and that it is sensitive even to small cochlear alterations.6, 7 The methods available for DPOAE testing combine stimulus and frequency parameters. In the DP-gram modality, the DPOAE responses are recorded using fixed intensities of the tonal stimuli (L1, L2) and a fixed frequency ratio (f2/f1) at increasing f2 frequency values (1.5, 2.0, 2.5, 3.0 kHz). In the input–output curve (I/O) modality, the DPOAE responses are recorded at a fixed f2 frequency and frequency ratio, while the level of the tonal stimuli (L1 and L2) is increased in systematic steps (30, 35, 40 dB SPL). For the I/O curve estimation the primary levels (L1 and L2) can be equal, but often in clinical practice unequal levels are employed (e.g. L2L1 + 8 dB SPL). Stavros Hatzopoulos University of Ferrara Department of Audiology and Centre of Bioacoustics 203 Corso Giovecca, Ferrara 44100, Italy

Cisplatin is a very powerful antineoplastic drug used in the treatment of various types of cancer and presents multiple dose-dependent side-effects,8 including nephrotoxicity and ototoxicity. While in clinical practice the incidence of nephrotoxicity is decreased by use of diuretic agents and hydration,9 the ototoxicity and the subsequent hearing loss are unaffected by these treatments.10 Various studies have shown that the hearing loss induced affects mainly the high frequencies, both in humans11 and in animals.8,10,12–14 It has been demonstrated that there is a high correlation between lesions in the inner ear and cisplatin dosage.15 The mechanism by which cisplatin damages the cochlea is not fully understood, but data from histological analyses have suggested that: (1) the OHCs of the organ of Corti and the stria vascularis are involved;12,15–20 and (2) there is a decrease in the level of cochlear glutathione (GSH) and formation of reactive oxygen species.21–24 To assess the ototoxicity of cisplatin in an animal model, it is common to use electrophysiological techniques such as electrocochleography (ECochG)14,25,26 and auditory brainstem responses (ABRs).19,21 These techniques record global responses from the cochlea or from the afferent auditory nerve fibres, but do not allow a direct evaluation of the subtle alterations of the functional status of the OHCs, which occur after the administration of cisplatin. For the latter it is assumed that the DPOAEs, which are considered to be responses mediated by the OHCs, might provide a useful tool for detecting any induced functional changes of the cochlear structure. The main objective of this investigation was the employment of a robust animal (rat) model in the early detection of cisplatin ototoxicity. Analytically:

2. Although the cubic distortion products have been employed as a method for the early detection of ototoxicity in various animal models,27,30,31 until now no study has compared DP-grams and I/O functions to determine which is the more sensitive and specific method for using DPOAEs in the early detection of ototoxicity. The emphasis of the present work is placed mainly on a DPOAE analysis, since previous papers by some of the authors19 have investigated in detail the ABR alterations after cisplatin administration. Materials and methods Chemicals The cisplatin solution (Platinex, concentration 0.5 mg/ml in normal saline) was purchased from BristolMyers Squibb, Italy. For the animal anaesthesia, an equalvolume combination of ketamine hydrochloride (Ketavet, 100 mg/ml), xylazine (Rompun, 20 mg/ml) and saline was used in dosages of 1 ml/kg of body weight. The anaesthetic was administered in two consecutive phases. In phase one, the animal received an intraperitoneal dose, and upon the first signs of muscular relaxation an additional half-dose volume was administered subcutaneously (phase 2). Animals Twenty male SD rats (weight 145–170 g) obtained from Charles River Italy were divided into two groups. Group G1 (15 rats) was treated with 16.0 mg/kg of cisplatin. Group G2 (five rats) received an equivalent volume of saline and was used as a control. The cisplatin was administered by an intraperitoneal slow infusion (postanaesthesia) of about 30 min using a micro-pump from Harvard Apparatus. The ABR and DPOAE responses were recorded during anaesthesia, before and 72 h after the cisplatin administration. The animals were treated according to the Italian guidelines DL 116/92 with reference to EEC directive n. 86-609.

1. We have chosen to evaluate DPOAE-based procedures (ABRs were considered as the gold standard) of early cisplatin ototoxicity detection, with a rat model because the rat is currently the most commonly used animal for studying cisplatin ototoxicity. Such an animal model demonstrates marked hearing loss after the administration of high dosages of cisplatin with a very low mortality rate.19,27 In addition, its high resistance to middle ear infections and lower sensitivity to multiple doses of anaesthesia28 favour the choice of the rat model for an ototoxic study, rather than other models using the guinea pig or the chinchilla. A number of American studies19,21,29 have used the Wistar rat strain for the early detection of cisplatin ototoxicity. Because the Sprague Dawley (SD) strain is very popular in European biomedical research and in order to provide comparable ototoxicity data between rat strains, we have investigated the performance of an (SD) rat model using a cisplatin ototoxicity dosage of 16 mg/kg infused over 30 min.

Distortion product otoacoustic emissions recordings The distortion product otoacoustic emissions were recorded in a sound-treated cabin with the VIRTUAL 330 equipment. The frequency bandwidth of the DPOAE responses was set to 4.0–8.0 kHz (referenced to f2) and 12 points were sampled per octave. The primary tone ratio f2/f1 was set to 1.21. Each record was the average of 32 responses with a noise tolerance of 10 dB SPL. The responses were evoked by a DPOAE protocol using unequal primary tone stimulus intensities, i.e. L1 > L2. Such protocols are generally considered a better choice for the identification of cochlear dysfunction.32–36 The protocols used were: high level (L175 and L265);

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medium level (L165 and L257) and low level (L155 and L249 dB SPL) . The I/O data were collected at f28.0 kHz, which is the highest frequency allowed by the VIRTUAL 330 software. The responses were evoked by stimuli of 30–75 dB SPL in 5-dB increments, using an asymmetrical stimulus intensity scheme of L2L18 dB SPL. A DPOAE response was considered to be present when the DPOAE amplitude was at least 3 dB SPL above the noise level. To identify the stimulus level corresponding to the DPOAE threshold in the I/O measurements, we used the following criterion,37 according to which three consecutive DPOAE responses (corresponding to three consecutive stimulus levels) must be in an increasing sequence (i.e. the corresponding DPOAE amplitudes must be also be increasing) before the primary-tone level which elicited the third DPOAE response is taken as the detection threshold. The noise floor level in the VIRTUAL 330 is measured at 70–80 Hz below and above the DP 2f1 –f2 frequency. In order to record a DPOAE response from the anaesthetised rat, the animal was placed on a stereotaxic device supporting a plastic funnel (total length: 22 mm; large inner diameter, 4.5 mm; small inner diameter 2.5 mm) which was inserted into the rat’s external acoustic meatus. Once inserted inside the funnel, the DPOAE probe was positioned firmly by multiple layers of parafilm. Auditory brainstem responses The ABRs were recorded by three stainless steel needle electrodes placed subdermally over the vertex (positive), ipsilateral mastoid (negative) and dorsum area (common/reference). The responses were generated by tone-pip stimuli with a decreasing intensity from 100 to 40 dB SPL in steps of 10 dB, at 8, 12 and 16 kHz (10-ms plateau, 1.0ms rise–fall time for 8-kHz burst and 0.5-ms rise–fall time for 12- and 16-kHz bursts), amplified 20,000 times and filtered from 20 to 5,000 Hz. The stimulus level was measured in free field 4 cm from the sound transducer by a probe microphone (Brùel and Kjaer 4186). This procedure resulted in a reliable calibration for all test frequencies up to 16 kHz. The stimuli were presented at the rate of 11/s using a Motorola tweeter (SH # 13245) placed 4 cm from the tested (right) ear. Each recording was the average of 250–1000 individual responses. All responses were recorded by an EWP Biolab apparatus inside a soundtreated cabin. The criterion for the presence of an ABR was a visual identification of wave III, according to Bourre et al.38 Several recordings were acquired at threshold level. No responses were present below the stimulus level of 40 dB SPL (pretreated animals), which was considered to be the threshold level for our experimental setup. Latencies were estimated for each ABR wave I and wave III. Cisplatin Ototoxicity in the Sprague Dawley Rat Evaluated by DPOAE

Throughout the recordings, the body temperature of each animal was maintained at a 37 ± 0.5°C by a Harvard Apparatus homeothermic blanket. Morphological analysis After the electrophysiological and acoustical measurements, a random number of 10 animals (4 controls and 6 treated) was chosen for an exploratory scanning electron microscopy (SEM) histological investigation. Each rat was perfused intra-aortically, under deep anaesthesia, with 2% glutharaldehyde in 0.1 M sodium cacodylate– HCl buffer, pH 7.2, for 1 h. The temporal bones were removed from the animals by exposing the otic capsule. The cochleas were fixed by local perfusion through the opened round and oval windows and holes were drilled at the cochlear apex. For the post-fixation a 0.5% aqueous solution of osmium tetroxide was used for 1 h and washed again in 0.1 M sodium cacodylate–HCl buffer, pH 7.2. The specimens were dehydrated in a graded series of ethanol and subjected to critical-point drying (Balzers CPD 030). The dried samples were mounted on alumen studs, sputter-coated with gold (EDWARDS Sputter coating S 150) and then examined by SEM (Cambridge S-360, Cambridge Technology Inc., Cambridge, MA, USA). Since the ABR and the DPOAE equipment tested relatively low frequencies, in terms of the hearing acuity range of the rat,39 the morphological observations refer to structural data from the middle cochlear coil (turn). Statistical methods The DPOAE 2f1–f2 recordings were analysed as signalto-noise ratios. The rationale for such a choice is that between the pre- and post-recording sessions, the DPOAE amplitude and the noise floor level values varied. It was decided to use as a point of reference the noise level during each recording, so that the resulting signal-tonoise ratio could reflect in a more efficient way the induced alterations in the DPOAE response. The ABRs were mainly analysed in terms of threshold shift and changes in the latency of waves I and III. For the DP-gram and ABR measurements, an analysis of covariance (ANCOVA) was performed with weight as a covariate. The DPOAE and ABR post-treatment data sets were compared to their corresponding pretreatment data sets using a Wilcoxon matched-pair test for dependent samples. The data of the DPOAE I/O curves and the ABR latencies, by stimulus level, were fitted to linear regression models (assuming that the probability of type I error was not high) in order to define possible changes between the slopes of pre- and post-treatment data. The statistical significance of the changes in the regression slopes of the ABR latencies was evaluated by a Wilcoxon matched-pair test. Hatzopoulos/Di Stefano/Campbell/Falgione/Ricci/ Rosignoli/Finesso/Albertin/Previati/Capitani/Martini

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The statistical analysis was carried out using SPSS version 6.1 software. The criterion for statistical significance for all measures was p