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The Journal of Laryngology & Otology (2016), 130, 225–234. © JLO (1984) Limited, 2016 doi:10.1017/S0022215115003473

Comparison of the protective effects of intratympanic dexamethasone and methylprednisolone against cisplatininduced ototoxicity ˘ AN1, S G GÜRGEN2, E ESEN1, S GENÇ1, A SELÇUK1 H E ÖZEL1, F ÖZDOG 1

Department of Otolaryngology, Kocaeli Derince Research and Training Hospital, Kocaeli, and 2Department of Histology and Embryology, Celal Bayar University School of Vocational Health Service, Manisa, Turkey

Abstract Objective: This study aimed to compare the efficacies of intratympanic dexamethasone and methylprednisolone in preventing in cisplatin-induced ototoxicity in rats. Methods: Experimental groups of rats (n = 8 each) received intratympanic isotonic saline, intraperitoneal cisplatin and intratympanic isotonic saline, intraperitoneal cisplatin and intratympanic dexamethasone, or intraperitoneal cisplatin and intratympanic methylprednisolone. Distortion product otoacoustic emission thresholds were compared on days 0 and 10 in all rats, and correlations between drug effects and changes in cochlear histology were evaluated. Results: Distortion product otoacoustic emission thresholds were comparable in groups III and IV ( p > 0.05). Significant protection against cisplatin-induced ototoxicity was seen in groups III and IV compared with group II ( p < 0.05). Dexamethasone and, to a lesser extent, methylprednisolone protected against cellular apoptosis in cisplatin-induced ototoxicity. Conclusion: Dexamethasone (and possibly methylprednisolone) may be clinically useful as an intratympanic chemopreventive agent to treat cisplatin ototoxicity. Future clinical studies should investigate the use of dexamethasone for this purpose in adult patients. Key words: Cisplatin; Chemically-Induced Disorders; Hearing Loss; Sensorineural; Dexamethasone; Methylprednisolone; Model; Animal

Introduction Cisplatin (cis-diamminedichloroplatinum(II)) is a highly effective anticancer drug that is widely used to treat various cancer types including sarcomas, carcinomas, lymphomas and germ cell tumours. However, this drug has a number of side effects that can limit its use, such as nephrotoxicity, neurotoxicity, ototoxicity and myelotoxicity; it also causes electrolyte disturbance, nausea and vomiting. Ototoxicity may be severe and no effective preventive treatment is currently available for this side effect. If this problem cannot be overcome, then cisplatin therapy may need to be proscribed, thus adversely affecting antineoplastic treatment. Cisplatininduced ototoxicity is characterised by irreversible, progressive, bilateral, high-frequency and sensorineural hearing loss.1–3 Cisplatin primarily damages outer hair cells in the cochlear base. Thus, distortion product otoacoustic emissions (DPOAE) testing is an appropriate way of detecting cisplatin-induced ototoxicity.4,5 Accepted for publication 23 September 2015

Many studies have previously demonstrated that intratympanic glucocorticoid administration can improve cisplatin-induced ototoxicity in young people. However, most diseases that require cisplatin treatment are seen in adults. Therefore, young animals may not be a good model for studying cisplatin ototoxicity and developing strategies for its prevention in adult humans. Indeed, the ototoxicity incidence after cisplatin treatment is greater in older cancer patients.6 The main goals of this study were to assess and compare the efficacies of intratympanic dexamethasone and intratympanic methylprednisolone in protecting against cisplatin-induced ototoxicity in middle-aged rats and to correlate drug effects with changes in cochlear histology.

Materials and methods This study was approved by the Animal Experiments Local Ethics Committee (protocol number 2/4-2014),

First published online 2 February 2016

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Kocaeli University, Turkey. Adult female Wistar Hannover rats aged 15–17 months and weighing between 420 and 530 g (mean 480 g) were maintained in the central animal laboratory for 10 days under standard laboratory conditions with a 12-hour light–dark cycle at 22 ± 2 °C. These conditions were then maintained throughout the experimental period. Animals had free access to standard laboratory chow and water. Animals with ear disease or the absence of distortion product otoacoustic emission (DPOAE) at any of the ranges test frequencies were not used. Animals were monitored daily for signs of distress, pain and weight loss. This study was performed in two phases. In phase 1, the optimal cisplatin treatment dose was established. For this, three groups of four rats were treated with a single intraperitoneal injection containing 8, 10 or 12 mg/kg cisplatin (1 mg/ml; Cisplatin Ebewe, Liba, Istanbul, Turkey) followed by the intraperitoneal injection (2 ml) of 0.9 per cent saline using an insulin syringe. All animals treated with 12 mg/kg cisplatin and two animals treated with 10 mg/kg cisplatin died on day 10. All animals treated with 8 mg/kg were alive on day 10 and one died on day 13. Based on these results, a single intraperitoneal injection of 8 mg/kg cisplatin was selected as the optimal dose to achieve a low mortality rate in phase 2 of the study. In phase 2, the effects of intratympanic dexamethasone and intratympanic methylprednisolone on cisplatin-induced ototoxicity were determined. The mortality rate in this phase was 20 per cent. Data for all animals that died during phase 1 were removed from the analysis and new animals replaced them in phase 2. Eight animals were assigned randomly into four groups and treated with: intratympanic isotonic saline (saline control group); intraperitoneal cisplatin plus intratympanic isotonic saline (cisplatin group); intraperitoneal cisplatin plus intratympanic dexamethasone (dexamethasone group); and intraperitoneal cisplatin plus intratympanic methylprednisolone (methylprednisolone group). Anaesthesia Animals were sedated by intraperitoneal injection with a ketamine (40 mg/kg) and xylazine (5 mg/kg) cocktail for DPOAE testing and before intratympanic injection. The depth of anaesthesia was determined using the pedal reflex. Drug treatment On days 0, 3, 6 and 9, animals in the saline control and cisplatin groups were intratympanically injected under light microscopy with sufficient isotonic saline to fill the middle-ear cavity using a 28-gauge dental needle. On day 0, 8 mg/kg cisplatin (1 mg/ml) was administered intraperitoneally as a slow infusion into all animals except those in the saline control group, followed by the intraperitoneal administration of a bolus (2 ml) of 0.9 per cent saline via an insulin syringe.

˘ AN, S G GÜRGEN et al. H E ÖZEL, F ÖZDOG

On days 0, 3, 6 and 9, the left tympanic membrane was visualised under light microscopy using an aural speculum and an intratympanic injection of dexamethasone (4 mg/ml; Dekort, Deva Holding, Istanbul, Turkey) or methylprednisolone (40 mg/ml; Prednol, Mustafa Nevzat ˙Ilaç Sanayi, Istanbul, Turkey) was administered slowly via myringotomy into the anteroinferior quadrant of the middle-ear cavity (approximately 0.2 ml) using a 28-gauge dental needle to the relevant glucocorticoid treatment group. Animals were kept in the same position for 20 minutes after every intratympanic injection. Distortion product otoacoustic emission testing Distortion product otoacoustic emission measurements were made under anaesthesia on day 0 (before medication) and day 10 in all animals: testing was performed in a quiet environment using a Madsen Capella DP+TE analyser and software (Otometrics, Miami, Florida, USA). Primary tones were introduced into the outer-ear canal via an insert earphone with a plastic adapter to seal the probe into the outer-ear canal; DPOAE findings are shown as distortion product grams (DP grams). The stimulus consisted of two pure tones (F1 and F2; F2:F1 ratio = 1.22) and DP grams used equilevel primary stimulus levels of 65 dB (L1 = L2); the resulting otoacoustic emissions ranged from approximately 1 to 8 kHz (0.996, 1.416, 2.001, 2.832, 4.003, 4755, 5.654, 6.728 and 7.998 kHz). Distortion product input and output amplitudes were measured by decreasing the primary tones from 75 to 40 dB SPL in 5-dB steps at 2.998, 5.996 and 7.998 kHz. After the final DPOAE test, rats were anaesthetised with ketamine and ether and were then decapitated; the heads were stored for future histological evaluation. Animal care adhered to the criteria of the Ethics Review Committee for Animal Experimentation and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.7,8 Histopathological evaluation Rat heads were fixed in neutral formalin for 24 hours and then placed in ethylene diamine triacetic acid solution for decalcification. After overnight washing under running water, tissues were dehydrated with a graded ethanol series and cleared with xylene. Serial sections (5-μm thick) from the basal turn of cochlea were mounted onto polylysine-coated slides. Terminal deoxynucleotidyl transferase dUTP nick end labelling A terminal deoxynucleotidyl transferase dUTP nick end labelling (‘TUNEL’) assay kit (Merck Millipore, Darmstadt, Germany) was used to detect DNA fragmentation and apoptotic cell death in cochlear sections. Apoptotic scoring was independently performed by two researchers blinded to the experimental groups. The average percentage of TUNEL-positive apoptotic

INTRATYMPANIC DEXAMETHASONE AND METHYLPREDNISOLONE PROTECT AGAINST CISPLATIN-INDUCED OTOTOXICITY

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TABLE I BETWEEN-GROUP COMPARISONS OF DPOAE THRESHOLDS AND DP INPUT AND OUTPUT AMPLITUDES ON DAY 10 Groups∗

p value† for DPOAE

p value† for DP input and output 2998 Hz



I & II II & III

Comparison of the protective effects of intratympanic dexamethasone and methylprednisolone against cisplatin-induced ototoxicity.

This study aimed to compare the efficacies of intratympanic dexamethasone and methylprednisolone in preventing in cisplatin-induced ototoxicity in rat...
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