The Laryngoscope C 2015 The American Laryngological, V
Rhinological and Otological Society, Inc.
Melatonin Protects Inner Ear Against Radiation Damage in Rats Isil Karaer, MD; Gokce Simsek, MD; Mehmet Gul, PhD; Leyla Bahar, PhD; Simay G€ urocak, MD; Hakan Parlakpinar, MD; Ayse Nuransoy, MD Objectives/Hypothesis: To examine the effects of N-acetyl-5-methoxytryptamine (melatonin) on radiation-induced inner ear damage. Study Design: An experimental animal model. Methods: Forty rats were randomized into five groups, as follows: 1) melatonin and then radiotherapy group (n 5 8), which received intraperitoneal (i.p.) melatonin (5 mg/kg) followed by irradiation 30 minutes later; 2) radiotherapy and then melatonin group (n 5 8), which received irradiation with i.p. melatonin (5 mg/kg) 30 minutes later; 3) melatonin group (n 5 8), which received i.p. melatonin (5 mg/kg); 4) radiotherapy group (n 5 8), which underwent only irradiation; 5) and the control group (n 5 8), which received i.p. 0.9% NaCl. The medications and irradiation were administered for 5 days. All rats underwent the distortion product otoacoustic emission (DPOAE) test before and 10 days after the experiment. The middle ears of the rats were excised, and assessment of tissue alterations in the organs of Corti, spiral ganglions, and stria vascularis were compared among the groups. Results: In the radiotherapy group, the DPOAE amplitudes at frequencies of 4000 to 6000 Hz were significantly decreased when compared with the controls. The DPOAE amplitudes both in the melatonin and then radiotherapy group and the radiotherapy and then melatonin group exhibited better values than they did in the radiotherapy group. Histopathological evidence of damage to the organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in both these two groups when compared to the radiotherapy group. Conclusion: These results indicate that melatonin may have significant ameliorative effects on cochlear damage secondary to ionizing radiation. Key Words: Inner ear, melatonin, otoacoustic emission, radiotherapy. Laryngoscope, 125:E345–E349, 2015
INTRODUCTION Radiotherapy is the most commonly used treatment modality in head and neck cancers and brain tumors.1 However, radiation treatment has serious otological side effects, including Eustachian tube dysfunction, otitis media with effusion, chronic otitis media, and sensorineural hearing loss.2 Radiotherapy-dependent hearing loss is dosedependent and has been reported as a result of transient alterations in the endolymph and perilymph caused by dysfunction in the stria vascularis.3,4 Ionizing radiation directly targets nuclear and mitochondrial deoxyribonucleic acid (DNA) and causes indirect cell damage via the excessive production of reactive oxygen species (ROS).5,6
From the ENT Department, Malatya State Hospital (I.K); the Inonu University Faculty of Medicine (A.N.); the Department of Histology and Embriyology (M.G.); the Department of Radiation Oncology (S.G.), the on€ u University Faculty of MediDepartment of Pharmacology (H.P.), In€ cine, Malatya; ENT Department (G.S.), Kirikkale University, Faculty of Medicine, Kirikkale; and the Vocational School of Health Services (L.B.), Mersin University, Mersin, Turkey Editor’s Note: This Manuscript was accepted for publication April 20, 2015. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Dr. Isil Karaer, MD, Malatya State Hospital, Department of Otolaryngology, Malatya, Turkey. E-mail: drisil_cakmak@ yahoo.com DOI: 10.1002/lary.25376
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Melatonin (N-acetyl-5-methoxytryptamine), the chief secretory product of the pineal gland, directly neutralizes a number of toxic reactants and inhibits production of ROS7; thus, it can stabilize cell membranes, making them more resistant to oxidative attack.8 Besides protecting DNA from free radical-mediated damage in situations of high ionizing radiation, melatonin could ameliorate the functional disruption that results from the destruction of DNA by enhancing repair processes.9 Sliwinski et al. examined melatonin’s ability to reduce free radical damage resulting from oxidizing agents, and like many others, reported that melatonin is highly effective in this regard.10 Distortion product otoacoustic emissions (DPOAEs) have provided an objective and noninvasive means of investigating cochlear functions. It is believed that DPOAEs are generated by the electromotor activity of the outer hairy cells of the organs of Corti, and that they are lost in the case of cochlear hearing loss.11 Based on this background, this experimental study aimed to investigate the effects of melatonin on inner ear damage stemming from irradiation by using DPOAE and histological parameters.
MATERIALS AND METHODS Care of Animals and Treatment This study was carried out in the experimental research laboratory of the Inonu University Faculty of Medicine, Malatya,
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Turkey. It was approved by the university ethics committee and complied with the guidelines for the care and use of experimental animals. Forty female Wistar albino rats, each weighing between 250 and 300 g, were used in this study. They were maintained according to the standard guidelines. Animals were ranked by weight at the beginning of the study to ensure similar starting body masses between groups. All animals were housed in plastic cages under standard environmental conditions, as follows: 12hour light and 12-hour dark cycle photoperiods, 208C constant temperature, and a humidity range of 40% to 60%. The rats had free access to standard dry pellets and tap water ad libitum until the end of the study. The external ears and tympanic membranes of all rats were examined. Cerumen in the external ear canal was removed. Rats that showed any signs of otitis, opacification, and/or tympanic membrane perforation were excluded from the study.
Drug Preparation and Treatment The 40 rats were randomized (using random number tables) into five groups, as follows: 1) the melatonin plus radiotherapy group (n 5 8), 2) the radiotherapy plus melatonin group (n 5 8), 3) the melatonin group (n 5 8), 4) the radiotherapy group (n 5 8), and 5) the control group (n 5 8). In the melatonin and then radiotherapy group, 5 mg/kg of melatonin (Sigma Chemical, St. Louis, MO) was administered intraperitoneally (i.p.) to the rats 30 minutes before irradiation. The rats in the radiotherapy and then melatonin group were treated with irradiation, followed by melatonin i.p. (5 mg/kg) 30 minutes before. In the melatonin group, 5 mg/kg of melatonin was injected i.p. In the radiotherapy group; rats underwent irradiation treatment without any medication. In the control group, rats received 0.9% NaCl i.p. The rats in the melatonin and then radiotherapy group, radiotherapy and then melatonin group, and radiotherapy group underwent irradiation for 5 days. The rats in the control group and the melatonin group received no irradiation. The drug doses were chosen based on previous biological studies related to the antioxidant effects of melatonin in an experimental rat model.12 All injections were administered under general anesthesia.
Irradiation The rats were irradiated with Cobalt-60 (60Co) photons. The source of radiation was a Theratron 780 C 60Co machine (Theratronics, Ottawa, Ontario, Canada), and the photons were delivered from anterior–posterior parallel fields at an source-to-axis distance of 80 cm, with the bodies of the rats positioned radially and their heads placed centrally so as to rule out the influence of a possible dose loss at the periphery. All of the rats were anesthetized i.p. with 20 to 30 mg/kg of ketamine hydrochloride (Rotex, Trittau, Germany) before irradiation, and 33 Gy to the total cranium in five fractions of 6.6 Gy/day for 5 successive days were applied. The calculated (a/b 5 3.5) biological effective dose of fractionated irradiation was equal to 60 Gy conventional fractionation. In the radiotherapy group, one animal died within 72 hours of undergoing irradiation. Animals were returned to their home cages following irradiation. In the radiotherapy group, the melatonin and then radiotherapy group, and the radiotherapy and then melatonin group, radiotherapy was given in five fractions over 4 consecutive days based on previous studies.13 The radiation dose was chosen based on previous research on cochlear tissue, which demonstrated that this dose causes inner ear damage.14
Distortion Product Otoacoustic Emissions The rats in all experimental groups were anesthetized with 10 mg/kg of xylazine (Alfazyne; Alfasan International B.V., Woerden, The Netherlands) and 60 mg/kg of ketamine (Ketalar; Eczacıbas¸ı Parke-Davis, Istanbul, Turkey) to record the baseline
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DPOAE data at the beginning of the study and collect the final DPOAE measurements carried out 10 days after the experiment. Otodynamics ILO-288 Echoport equipment (Otodynamics Ltd., London, UK) was used to measure DPOAE. The animals were examined under anesthesia and were confirmed to have normal external auditory canals and tympanic membranes before the baseline and final audiometric measurements. During the tests, the room temperature was maintained at 218C. When under general anesthesia, the rats were warmed up using an electrical heater to stabilize their normal body functions. The procedure for recording DPOAEs in Wistar Albino rats has been described elsewhere. Briefly, a special probe neonatal type eartips (Medelec, UK) was introduced into the external auditory canal. Then, sound stimuli were delivered to the animals via a intraauricular headset (Medelec, UK). The sound stimulus that composed DPOAE consisted of two simultaneous permanent pure tones at different frequencies. The stimulus parameters L1 5 L2 5 80 dB SPL with an f1/f2 ratio of 1.22 were used, and the amplitude of the DPOAE signal was recorded. Distortion product otoacoustic emission assessment was carried out prior to irradiation, and amplitude values of 1000, 2000, 3000, 4000, and 6000 Hz were detected in all groups. A complete DPOAE testing of each animal lasted approximately 20 to 30 minutes.
Histopathological Studies For light microscopic evaluation, tissue specimens were fixed in phosphate-buffered 10% formaldehyde solution at 48C for 72 hours. For decalcification, specimens were stored in 10% formic acid solution (renewed every other day) at 48C for 8 days. The cochleas were separated from the temporal bones and divided into two from the cochlear modiolus. All specimens were dehydrated and embedded in paraffin wax. Paraffin-embedded specimens were cut into 6-mm thick sections, mounted on slides, and stained with hematoxylin–eosin. The sections of tympanic bullas were examined with a Leica DFC280 light microscope and analyzed using a Leica Q Win Plus V3 Image Analysis System (Leica Micros Imaging Solutions Ltd., Cambridge, UK). Assessment of tissue alterations in the organs of Corti, spiral ganglions, and stria vascularis of sections for each specimen was conducted by an experienced histologist who was unaware of the groups. Changes (hydropic and vacuolar degeneration and loss of outer and inner hair cells) in the organs of Corti and in the stria vascularis (edema, vacuolization, and loss of cells) were noted. The changes in the stria vascularis and organs of Corti were scored as absent (0), mild (1), moderate (2) or severe (3). The percentage of degenerated cells in the spiral ganglion was evaluated (Table I).
Statistical Analysis Data analyses were performed using a statistical software package (SPSS 15.0; SPSS Inc., Chicago IL). The values were expressed as mean 6 standard deviation whenever appropriate. The normality of the distributions was tested using the Kolmogorov–Smirnov test. The DPOAE 2f1-f2 recordings were analyzed for the signal-to-noise ratios, which reflected the hearing levels. Right ear values were evaluated in the same group for each frequency. The pre- and postirradiation measurements were analyzed using the Kruskal-Wallis variance analysis test. When a difference was found, the Mann–Whitney U test with Bonferroni correction was used to find the frequency involved. For all comparisons, the statistical significance was determined as P < 0.05.
RESULTS The DPOAE responses for 1000, 2000, and 3000 Hz frequencies in radiotheraphy and control groups showed Karaer et al.: Melatonin Protects Inner Ear Against Radiation
TABLE I. Comparison of Histologic Evaluation of Bilateral Inner Ear Damage Between Groups. Group C n 5 16
Group M n 5 16
Group RT n 5 14
Group M 1 RT n 5 16
Group RT 1 M n 5 16
Edema in Spiral Ganglion Absent (0) Mild (1)
0 0
0 0
0 3
0 12
0 10
Moderate (2)
0
0
7
4
6
0
4
0
0
Severe (3) 0 Epithelial Degeneration in Stria Vascularis Absent (0)
0
0
0
0
0
Mild (1) Moderate (2)
0 0
0 0
3 7
12 4
8 8
Severe (3)
0
0
4
0
0
Edema in Stria Vascularis Absent (0)
0
0
0
0
0
Mild (1)
0
0
0
11
10
Moderate (2) Severe (3)
0 0
0 0
8 6
5 0
6 0
Degenerated Changes in the Organ of Corti Absent (0) Mild (1)
0 0
0 0
0 7
0 7
0 8
Moderate (2)
0
0
7
7
8
Severe (3)
0
0
0
0
0
C 5 control; M 5 melatonin; M 1 RT 5 melatonin and then radiotherapy; RT 5radiotherapy; RT 1 M 5 radiotherapy and then melatonin.
no statistically significant difference (P > 0.05). However, radiotherapy group exhibited significantly decreased DPOAE responses at frequencies of 4000 and 6000 Hz when compared to group C (P < 0.001, P < 0.001, respectively) (Fig. 1). In contrast, DPOAE responses for 4000 and 6000 Hz frequencies both in the melatonin and then radiotherapy group and the radiotherapy and then melatonin group were significantly higher than in the radiotherapy group (P < 0.001, P < 0.001 for the melatonin and then radiotherapy group; P 5 0.015, P 5 0.01 for the radiotherapy and then melatonin group). However, DPOAE responses for 4000 and 6000 Hz in the radiotherapy and then melatonin group were significantly
Fig. 1. The DPOAE responses in group C, group M, and group RT. DPOAE were decreased at 4000 and 6000 Hz in group RT (P < 0.001 and P < 0.001). C 5 control; DPOAE 5 distortion product otoacoustic emission; M 5 melatonin; RT 5radiotherapy.
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higher than in the melatonin and then radiotherapy group (P 5 0.004 and P 5 0.02, respectively) (Fig. 2). Histological examination of the control and melatonin groups showed normal histological appearance of the organs of Corti, spiral ganglions, and stria vascularis. The sections from the radiotherapy group showed histopathological alterations such as hydropic and vacuolar degeneration and loss of cells (both inner and outer hair cells and pillar cells) in the organs of Corti; deformation of basilar membrane in the organs of Corti; fibrinoid material deposition in the scala tympani and scala
Fig. 2. The DPOAE responses in group RT, group M and then RT, and group RT and then M. There were statistically significant increased at 4000 and 6000 Hz of DPOAE values for melatonin treatment and better DPOAE values in group RT and then M (P 5 0.004 and P 5 0.021). C 5 control; DPOAE 5 distortion product otoacoustic emission; M 5 melatonin; RT 5radiotherapy.
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Fig. 3. (a) Histopathological examination in radiotherapy group as material deposition in the scala tympani and scala media. (b) Cytoplasmic and nuclear condensation, neuron loss, and edema in the spiral ganglions. (c) Edema, epithelial degeneration, vacuolization, and loss of cells in the stria vascularis. (d,e,f) Organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in melatonin administration before and after radiotheraphy. Hematoxylin–eosin, 3 40.
media (Fig. 3a); cytoplasmic and nuclear condensation; neuron loss; edema in the spiral ganglions (Fig. 3b); and edema, epithelial degeneration, vacuolization, and loss of cells in the stria vascularis (Fig. 3c). Histopathological evidence from the organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in both the melatonin and then radiotherapy group and the radiotherapy and then melatonin group (Fig. 3d,e,f).
DISCUSSION In this study, we showed that administration of melatonin after or even before ionizing radiation could prevent radiotherapy-dependent hearing loss in higher frequencies in an animal model. The prevention of irradiation-dependent hearing loss was greater in the radiotherapy and then melatonin group than in the melatonin and then radiotherapy group. Furthermore, the histopathological findings confirmed that administration of melatonin was protective of cells in the spiral ganglions, stria vascularis, and organs of Corti. In the present study, we used Cobalt 60 for irradiation. Previous studies showed that treatment with Co-60 results in better performance when compared with other treatment methods.15 The Co-60 provides a strong option for improving the quality of life for patients with cancer. Indeed, its low cost, uncomplicated maintenance, and relative ease of availability may provide a viable treatment option.16 Laryngoscope 125: October 2015
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In this study, 60 Gy radiotherapy was given to the rats. Previous studies found that the cochlear damage occur in various irradiation dosages. The histopathological findings in the ear after irradiation were well summarized by Linskey and Johnstone.3 They noted that two principal sites of damage are the hair cells of the cochlea and the stria vascularis. The researchers found that the outer hair cells are more sensitive than the inner hair cells for gamma irradiation damage.17 Bohne et al.18 found that the damage in the organ of Corti occur in 31% of animals at 30 to 40 Gy compared to 62% of animals at 60 to 90 Gy. Winther19–21 exposed guinea pigs to single doses of photon radiation ranging from 10 to 70 Gy and identified no hair cell changes with doses 20 Gy. Outer hair cell damage began to occur at 40 Gy, and vestibular sensory epithelial cells were damaged with single photon doses of 70 Gy.20,21 Similarly, Nagel and Schafer22 exposed guinea pigs to single-dose photon radiation of 35 to 70 Gy. They noted no hair cell damage in short follow-up for doses 35 Gy. Only outer hair cells were affected at 40 to 45 Gy, whereas inner hair cells began to be affected at 50 Gy. It is known that gamma knife surgery produces tissue damage through vascular injury.23 After gamma knife surgery, the blood vessels exhibit narrowing caused by endothelial proliferation, which tend to occur within days after irradiation and lead to ischemia and necrosis.24 In a clinical study, the researchers showed that ischemia and necrosis caused by irradiation damage can occur with Karaer et al.: Melatonin Protects Inner Ear Against Radiation
fractioned radiation doses 50 Gy or greater.25 Wackym et al.26 proposed that limiting the dose of radiation to < 4 Gy to the cochlea would likely reduce vascular injury to the stria vascularis and improve hearing outcomes. However, a typical gamma knife surgery dose for vestibular schwannoma is between 12 and 13 Gy, which is approximately equivalent to a fractioned dose of 48 to 50 Gy.26 Therefore, it is likely that small cochlear vasculature, such as in the stria vascularis, may be affected by a single fraction of gamma knife surgery. In a clinical study, Massager et al.27 reported in a series of 82 patients with vestibular schwannoma treated with a marginal dose of 12 Gy (median radiation dose delivered to cochlea 4.15 Gy). They found that a statistically significant association between higher cochlear volume radiation dose and deterioration of hearing was reported in these patients. Likewise, Lasak et al.28 reported 33 patients with vestibular schwannoma undergoing gamma knife surgery. Eighteen of their patients received cochlear volume doses greater than 4.75 Gy, and they found that these higher radiation doses were associated with hearing loss. In the present study, we found that the most prominent hearing loss occurred at frequencies of 4000 to 6000 Hz. In agreement with this study, Akmansu et al. found that the most prominent irradiation-dependent hearing loss occurred at 3000 to 6000 Hz.29 The loss of hearing at higher frequencies that was previously reported by several investigators indicates a greater susceptibility of the basal turn of the cochlea to radiation damage because this is responsible for the detecting higher frequencies.6 To the best of our knowledge, this is the first experimental study showing possible beneficial effects of melatonin on radiation-dependent inner ear damage. In this study, we found that melatonin was protective of cells in spiral ganglions, stria vascularis, and organs of Corti in rats administered a dose of irradiation of 60 Gy. When melatonin was administered after irradiation, the prevention ability was greater. In previously articles, melatonin was effective agent against damage produced by irradiation in several organs, including the liver, lung, lymphatic tissue, and lens.30–33 Melatonin was put forward as a potent free radical scavenger and an antiapoptotic agent in utricular hair cells.34 The studies observed that melatonin attenuated hairy cell loss caused by exposure to an ototoxic level of gentamicin.35,36
CONCLUSION In conclusion, these are pioneering results for studies that will be performed with melatonin to protect from radiation toxicity in the inner ear. It would be worthwhile to study the effects of melatonin supplements in radiation-treated cancer patients in the hope of reducing radiation-induced toxicity.
BIBLIOGRAPHY 1. Barbara A, Jereczek F, Andrzej Z, Milani F, Orecchia R. Radiotherapyinduced ear toxicity. Cancer Treat Rev 2003;29:417–430. 2. Thibadoux GM, Pereira WV, Hodges JM, Aur RJ. Effects of cranial irradiation on hearing in children with acute lymphocytic leukemia. J Pediatr 1980;96:403–406. 3. Linskey ME, Johnstone PA. Radiation tolerance of normal temporal bone structures: implications for gamma knife stereotactic radiosurgery. Int J Radiat Oncol Biol Phys 2003;57:196–200.
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4. Bhide SA, Harrington KJ, Nutting CM. Otological toxicity after postoperative radiotherapy for parotid tumours. Clin Oncol 2007;19:77–82. 5. Simsek G, Gurocak S, Karadag N, Karabulut AB, Demirtas E, Karatas E, Pepele E. Protective effects of resveratrol on salivary gland damage induced by total body irradiation in rats. Laryngoscope 2012;122:2743–2748. 6. Low WK, Tan MG, Sun L, Chua AW, Goh LK, Wang DY. Dose-dependant radiation-induced apoptosis in a cochlear cell-line. Apoptosis 2006;11: 2127–2136. 7. Vijayalaxmi, Reiter RJ, Tan DX, Herman TS, Thomas CR Jr. Melatonin as a radioprotective agent: a review. Int J Radiat Oncol Biol Phys 2004;59: 639–653. 8. Reiter RJ, Tan DX, Osuna C, Gitto E. Actions of melatonin in the reduction of oxidative stress. A review. J Biomed Sci 2000;7:444–458. 9. Reiter JR, Korkmaz A, Ma S, Rosales-Corral S, Tan DX. Melatonin protection from chronic, low-level ionizing radiation. Mutat Res 2012; 751:7–14. 10. Sliwinski T, Rozej W, Morawiec-Bajda A, Morawiec Z, Reiter RJ, Blasiak J. Protective action of melatonin against DNA damage–chemical inactivation verses base-excision repair. Mutat Res 2007;634:220–227. 11. Probst R. Otoacoustic emissions: an overview. Adv Otorhinolaryngol 1990; 44:1–91. 12. Koc M, Buyukokuroglu ME, Taysi S. The effect of melatonin on peripheral blood cells during total body irradiation in rats. Biol Pharm Bull 2002; 25:656–657. 13. Kucuktulu E. Protective Effect of melatonin against radiation induced nephrotoxicity in rats. Asian Pac J Cancer Prev 2012;13:4101–4105. 14. Gamble JE, Peterson EA, Chandler JR. Radiation effects on the inner ear. Arch Otolaryngol 1968;88:156–161. 15. Van Dyk J, Battista JJ. Cobalt-60: an old modality, a renewed challenge. Curr Oncol 1996;3:8–17. 16. Marcu L, Bezak E, Allen BJ. Palliative radiotheraphy. In:Marcu L, Bezak E, Allen BJ,eds. Biomedical Physics in Radiotheraphy for Cancer. Australia: CSIRO Publishing; 2012:372–376. 17. Kim CS, Shin SO. Ultrastructural changes in the cochlea of the guinea pig after fast neutron irradiation. Otolaryngol Head Neck Surg 1994;110:419–427. 18. Bohne BA, Marks JE, Glasgow GP. Delayed effects of ionizing radiation on the ear. Laryngoscope 1985;95:818–828. 19. Winther FO. Early degenerative changes in the inner ear sensory cells of the guinea pig following local x-ray irradiation. Acta Otolaryngol 1969; 67:262–268. 20. Winther FO. X-ray irradiation of the inner ear of the guinea pig: early changes in the cochlea. Acta Otolaryngol 1969;68:98–117. 21. Winther FO. X-ray irradiation of the inner ear of the guinea pig: an electron micrographic study of the degenerating outer hair cells of the organ of Corti. Acta Otolaryngol (Stockh) 1970;69:61–76. 22. Nagel D, Schafer J. Changes in cochlear microphonic response after X-ray irradiation of the inner ear of the guinea pig. Arch Otorhinolaryngol 1984;241:17–21. 23. Adams RD. The neuropathology of radiosurgery. Steriotact Funct Neurosurg 1991;57:82–86. 24. Burger PC, Boyko OB. Pathology of central nervous system radiation injury. In: Gutin PH, Leibel SA, Sheline GE, eds. Radiation Injury in the Nervous System. New York, NY: Raven Press; 1991: 191–208. 25. Sheline GE, Wara WM, Smith V. Therapeutic irradiation and brain injury. Int J Radiat Oncol Biol Phys 1980;6:1215–1228. 26. Wackym PA, Runge-Samuelson CL, Nash JJ, Poetker DM, Albano K, Bovi J. Gamma knife surgery of vestibular schwannomas: volumetric dosimetry correlations to hearing loss suggest stria vascularis devascularization as the mechanism of early hearing loss. Otol Neurotol 2010;31:1480–1487. 27. Massager N, Nissim O, Delbrouck C, et al. Irradiation of cochlear structures during vestibular schwannoma radiosurgery and associated hearing outcome. J Neurosurg 2007;107:733–739. 28. Lasak JM, Klish D, Kryzer TC, Hearn C, Gorecki JP, Rine GP. Gamma knife radiosurgery for vestibular schwannoma: early hearing outcomes and evaluation of the cochlear dose. Otol Neurotol 2008;29; 1179–1186. 29. Akmansu H, Eryilmaz A, Korkmaz H, et al. Ultrastructural and electrophysiologic changes of rat cochlea after irradiation. Laryngoscope 2004; 114:1276–1280. 30. Shirazi A, Haddadi GH, Asadi-Amoli F, Sakhaee S, Ghazi-Khansari MM, Avand A. Radioprotective effect of melatonin in reducing oxidative stress in rat lenses. Cell J 2011;13:79–82. 31. Koc M, Taysi S, Buyukokuroglu ME, Bakan N. Melatonin protects rat liver against irradiation-induced oxidative injury. J Radiat Res 2003;44: 211–215. 32. Jang SS, Kim HG, Lee JS, et al. Melatonin reduces X-ray radiationinduced lung injury in mice by modulating oxidative stress and cytokine expression. Int J Radiat Biol 2013;89:97–105. 33. Sharma S, Haldar C, Chaube SK. Effect of exogenous melatonin on X-ray induced cellular toxicity in lymphatic tissue of Indian tropical male squirrel, Funambulus pennanti. Int J Radiat Biol 2008;84:363–374. 34. Kim JB, Jung JY, Ahn JC, Rhee CK, Hwang HJ. Antioxidant and antiapoptotic effect of melatonin on the vestibular hair cells of rat utricles. Clin Exp Otorhinolaryngol 2009;2:6–12. 35. Ye LF, Tao ZZ, Hua QQ, et al. Protective effect of melatonin against gentamicin ototoxicity. J Laryngol Otol 2009;123:598–602. 36. Bas E, Van De Water TR, Gupta C, et al. Efficacy of three drugs for protecting against gentamicin-induced hair cell and hearing losses. Br J Pharmacol 2012;166:1888–1904.
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Rhinological and Otological Society, Inc.
Melatonin Protects Inner Ear Against Radiation Damage in Rats Isil Karaer, MD; Gokce Simsek, MD; Mehmet Gul, PhD; Leyla Bahar, PhD; Simay G€ urocak, MD; Hakan Parlakpinar, MD; Ayse Nuransoy, MD Objectives/Hypothesis: To examine the effects of N-acetyl-5-methoxytryptamine (melatonin) on radiation-induced inner ear damage. Study Design: An experimental animal model. Methods: Forty rats were randomized into five groups, as follows: 1) melatonin and then radiotherapy group (n 5 8), which received intraperitoneal (i.p.) melatonin (5 mg/kg) followed by irradiation 30 minutes later; 2) radiotherapy and then melatonin group (n 5 8), which received irradiation with i.p. melatonin (5 mg/kg) 30 minutes later; 3) melatonin group (n 5 8), which received i.p. melatonin (5 mg/kg); 4) radiotherapy group (n 5 8), which underwent only irradiation; 5) and the control group (n 5 8), which received i.p. 0.9% NaCl. The medications and irradiation were administered for 5 days. All rats underwent the distortion product otoacoustic emission (DPOAE) test before and 10 days after the experiment. The middle ears of the rats were excised, and assessment of tissue alterations in the organs of Corti, spiral ganglions, and stria vascularis were compared among the groups. Results: In the radiotherapy group, the DPOAE amplitudes at frequencies of 4000 to 6000 Hz were significantly decreased when compared with the controls. The DPOAE amplitudes both in the melatonin and then radiotherapy group and the radiotherapy and then melatonin group exhibited better values than they did in the radiotherapy group. Histopathological evidence of damage to the organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in both these two groups when compared to the radiotherapy group. Conclusion: These results indicate that melatonin may have significant ameliorative effects on cochlear damage secondary to ionizing radiation. Key Words: Inner ear, melatonin, otoacoustic emission, radiotherapy. Laryngoscope, 125:E345–E349, 2015
INTRODUCTION Radiotherapy is the most commonly used treatment modality in head and neck cancers and brain tumors.1 However, radiation treatment has serious otological side effects, including Eustachian tube dysfunction, otitis media with effusion, chronic otitis media, and sensorineural hearing loss.2 Radiotherapy-dependent hearing loss is dosedependent and has been reported as a result of transient alterations in the endolymph and perilymph caused by dysfunction in the stria vascularis.3,4 Ionizing radiation directly targets nuclear and mitochondrial deoxyribonucleic acid (DNA) and causes indirect cell damage via the excessive production of reactive oxygen species (ROS).5,6
From the ENT Department, Malatya State Hospital (I.K); the Inonu University Faculty of Medicine (A.N.); the Department of Histology and Embriyology (M.G.); the Department of Radiation Oncology (S.G.), the on€ u University Faculty of MediDepartment of Pharmacology (H.P.), In€ cine, Malatya; ENT Department (G.S.), Kirikkale University, Faculty of Medicine, Kirikkale; and the Vocational School of Health Services (L.B.), Mersin University, Mersin, Turkey Editor’s Note: This Manuscript was accepted for publication April 20, 2015. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Dr. Isil Karaer, MD, Malatya State Hospital, Department of Otolaryngology, Malatya, Turkey. E-mail: drisil_cakmak@ yahoo.com DOI: 10.1002/lary.25376
Laryngoscope 125: October 2015
Melatonin (N-acetyl-5-methoxytryptamine), the chief secretory product of the pineal gland, directly neutralizes a number of toxic reactants and inhibits production of ROS7; thus, it can stabilize cell membranes, making them more resistant to oxidative attack.8 Besides protecting DNA from free radical-mediated damage in situations of high ionizing radiation, melatonin could ameliorate the functional disruption that results from the destruction of DNA by enhancing repair processes.9 Sliwinski et al. examined melatonin’s ability to reduce free radical damage resulting from oxidizing agents, and like many others, reported that melatonin is highly effective in this regard.10 Distortion product otoacoustic emissions (DPOAEs) have provided an objective and noninvasive means of investigating cochlear functions. It is believed that DPOAEs are generated by the electromotor activity of the outer hairy cells of the organs of Corti, and that they are lost in the case of cochlear hearing loss.11 Based on this background, this experimental study aimed to investigate the effects of melatonin on inner ear damage stemming from irradiation by using DPOAE and histological parameters.
MATERIALS AND METHODS Care of Animals and Treatment This study was carried out in the experimental research laboratory of the Inonu University Faculty of Medicine, Malatya,
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Turkey. It was approved by the university ethics committee and complied with the guidelines for the care and use of experimental animals. Forty female Wistar albino rats, each weighing between 250 and 300 g, were used in this study. They were maintained according to the standard guidelines. Animals were ranked by weight at the beginning of the study to ensure similar starting body masses between groups. All animals were housed in plastic cages under standard environmental conditions, as follows: 12hour light and 12-hour dark cycle photoperiods, 208C constant temperature, and a humidity range of 40% to 60%. The rats had free access to standard dry pellets and tap water ad libitum until the end of the study. The external ears and tympanic membranes of all rats were examined. Cerumen in the external ear canal was removed. Rats that showed any signs of otitis, opacification, and/or tympanic membrane perforation were excluded from the study.
Drug Preparation and Treatment The 40 rats were randomized (using random number tables) into five groups, as follows: 1) the melatonin plus radiotherapy group (n 5 8), 2) the radiotherapy plus melatonin group (n 5 8), 3) the melatonin group (n 5 8), 4) the radiotherapy group (n 5 8), and 5) the control group (n 5 8). In the melatonin and then radiotherapy group, 5 mg/kg of melatonin (Sigma Chemical, St. Louis, MO) was administered intraperitoneally (i.p.) to the rats 30 minutes before irradiation. The rats in the radiotherapy and then melatonin group were treated with irradiation, followed by melatonin i.p. (5 mg/kg) 30 minutes before. In the melatonin group, 5 mg/kg of melatonin was injected i.p. In the radiotherapy group; rats underwent irradiation treatment without any medication. In the control group, rats received 0.9% NaCl i.p. The rats in the melatonin and then radiotherapy group, radiotherapy and then melatonin group, and radiotherapy group underwent irradiation for 5 days. The rats in the control group and the melatonin group received no irradiation. The drug doses were chosen based on previous biological studies related to the antioxidant effects of melatonin in an experimental rat model.12 All injections were administered under general anesthesia.
Irradiation The rats were irradiated with Cobalt-60 (60Co) photons. The source of radiation was a Theratron 780 C 60Co machine (Theratronics, Ottawa, Ontario, Canada), and the photons were delivered from anterior–posterior parallel fields at an source-to-axis distance of 80 cm, with the bodies of the rats positioned radially and their heads placed centrally so as to rule out the influence of a possible dose loss at the periphery. All of the rats were anesthetized i.p. with 20 to 30 mg/kg of ketamine hydrochloride (Rotex, Trittau, Germany) before irradiation, and 33 Gy to the total cranium in five fractions of 6.6 Gy/day for 5 successive days were applied. The calculated (a/b 5 3.5) biological effective dose of fractionated irradiation was equal to 60 Gy conventional fractionation. In the radiotherapy group, one animal died within 72 hours of undergoing irradiation. Animals were returned to their home cages following irradiation. In the radiotherapy group, the melatonin and then radiotherapy group, and the radiotherapy and then melatonin group, radiotherapy was given in five fractions over 4 consecutive days based on previous studies.13 The radiation dose was chosen based on previous research on cochlear tissue, which demonstrated that this dose causes inner ear damage.14
Distortion Product Otoacoustic Emissions The rats in all experimental groups were anesthetized with 10 mg/kg of xylazine (Alfazyne; Alfasan International B.V., Woerden, The Netherlands) and 60 mg/kg of ketamine (Ketalar; Eczacıbas¸ı Parke-Davis, Istanbul, Turkey) to record the baseline
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DPOAE data at the beginning of the study and collect the final DPOAE measurements carried out 10 days after the experiment. Otodynamics ILO-288 Echoport equipment (Otodynamics Ltd., London, UK) was used to measure DPOAE. The animals were examined under anesthesia and were confirmed to have normal external auditory canals and tympanic membranes before the baseline and final audiometric measurements. During the tests, the room temperature was maintained at 218C. When under general anesthesia, the rats were warmed up using an electrical heater to stabilize their normal body functions. The procedure for recording DPOAEs in Wistar Albino rats has been described elsewhere. Briefly, a special probe neonatal type eartips (Medelec, UK) was introduced into the external auditory canal. Then, sound stimuli were delivered to the animals via a intraauricular headset (Medelec, UK). The sound stimulus that composed DPOAE consisted of two simultaneous permanent pure tones at different frequencies. The stimulus parameters L1 5 L2 5 80 dB SPL with an f1/f2 ratio of 1.22 were used, and the amplitude of the DPOAE signal was recorded. Distortion product otoacoustic emission assessment was carried out prior to irradiation, and amplitude values of 1000, 2000, 3000, 4000, and 6000 Hz were detected in all groups. A complete DPOAE testing of each animal lasted approximately 20 to 30 minutes.
Histopathological Studies For light microscopic evaluation, tissue specimens were fixed in phosphate-buffered 10% formaldehyde solution at 48C for 72 hours. For decalcification, specimens were stored in 10% formic acid solution (renewed every other day) at 48C for 8 days. The cochleas were separated from the temporal bones and divided into two from the cochlear modiolus. All specimens were dehydrated and embedded in paraffin wax. Paraffin-embedded specimens were cut into 6-mm thick sections, mounted on slides, and stained with hematoxylin–eosin. The sections of tympanic bullas were examined with a Leica DFC280 light microscope and analyzed using a Leica Q Win Plus V3 Image Analysis System (Leica Micros Imaging Solutions Ltd., Cambridge, UK). Assessment of tissue alterations in the organs of Corti, spiral ganglions, and stria vascularis of sections for each specimen was conducted by an experienced histologist who was unaware of the groups. Changes (hydropic and vacuolar degeneration and loss of outer and inner hair cells) in the organs of Corti and in the stria vascularis (edema, vacuolization, and loss of cells) were noted. The changes in the stria vascularis and organs of Corti were scored as absent (0), mild (1), moderate (2) or severe (3). The percentage of degenerated cells in the spiral ganglion was evaluated (Table I).
Statistical Analysis Data analyses were performed using a statistical software package (SPSS 15.0; SPSS Inc., Chicago IL). The values were expressed as mean 6 standard deviation whenever appropriate. The normality of the distributions was tested using the Kolmogorov–Smirnov test. The DPOAE 2f1-f2 recordings were analyzed for the signal-to-noise ratios, which reflected the hearing levels. Right ear values were evaluated in the same group for each frequency. The pre- and postirradiation measurements were analyzed using the Kruskal-Wallis variance analysis test. When a difference was found, the Mann–Whitney U test with Bonferroni correction was used to find the frequency involved. For all comparisons, the statistical significance was determined as P < 0.05.
RESULTS The DPOAE responses for 1000, 2000, and 3000 Hz frequencies in radiotheraphy and control groups showed Karaer et al.: Melatonin Protects Inner Ear Against Radiation
TABLE I. Comparison of Histologic Evaluation of Bilateral Inner Ear Damage Between Groups. Group C n 5 16
Group M n 5 16
Group RT n 5 14
Group M 1 RT n 5 16
Group RT 1 M n 5 16
Edema in Spiral Ganglion Absent (0) Mild (1)
0 0
0 0
0 3
0 12
0 10
Moderate (2)
0
0
7
4
6
0
4
0
0
Severe (3) 0 Epithelial Degeneration in Stria Vascularis Absent (0)
0
0
0
0
0
Mild (1) Moderate (2)
0 0
0 0
3 7
12 4
8 8
Severe (3)
0
0
4
0
0
Edema in Stria Vascularis Absent (0)
0
0
0
0
0
Mild (1)
0
0
0
11
10
Moderate (2) Severe (3)
0 0
0 0
8 6
5 0
6 0
Degenerated Changes in the Organ of Corti Absent (0) Mild (1)
0 0
0 0
0 7
0 7
0 8
Moderate (2)
0
0
7
7
8
Severe (3)
0
0
0
0
0
C 5 control; M 5 melatonin; M 1 RT 5 melatonin and then radiotherapy; RT 5radiotherapy; RT 1 M 5 radiotherapy and then melatonin.
no statistically significant difference (P > 0.05). However, radiotherapy group exhibited significantly decreased DPOAE responses at frequencies of 4000 and 6000 Hz when compared to group C (P < 0.001, P < 0.001, respectively) (Fig. 1). In contrast, DPOAE responses for 4000 and 6000 Hz frequencies both in the melatonin and then radiotherapy group and the radiotherapy and then melatonin group were significantly higher than in the radiotherapy group (P < 0.001, P < 0.001 for the melatonin and then radiotherapy group; P 5 0.015, P 5 0.01 for the radiotherapy and then melatonin group). However, DPOAE responses for 4000 and 6000 Hz in the radiotherapy and then melatonin group were significantly
Fig. 1. The DPOAE responses in group C, group M, and group RT. DPOAE were decreased at 4000 and 6000 Hz in group RT (P < 0.001 and P < 0.001). C 5 control; DPOAE 5 distortion product otoacoustic emission; M 5 melatonin; RT 5radiotherapy.
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higher than in the melatonin and then radiotherapy group (P 5 0.004 and P 5 0.02, respectively) (Fig. 2). Histological examination of the control and melatonin groups showed normal histological appearance of the organs of Corti, spiral ganglions, and stria vascularis. The sections from the radiotherapy group showed histopathological alterations such as hydropic and vacuolar degeneration and loss of cells (both inner and outer hair cells and pillar cells) in the organs of Corti; deformation of basilar membrane in the organs of Corti; fibrinoid material deposition in the scala tympani and scala
Fig. 2. The DPOAE responses in group RT, group M and then RT, and group RT and then M. There were statistically significant increased at 4000 and 6000 Hz of DPOAE values for melatonin treatment and better DPOAE values in group RT and then M (P 5 0.004 and P 5 0.021). C 5 control; DPOAE 5 distortion product otoacoustic emission; M 5 melatonin; RT 5radiotherapy.
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Fig. 3. (a) Histopathological examination in radiotherapy group as material deposition in the scala tympani and scala media. (b) Cytoplasmic and nuclear condensation, neuron loss, and edema in the spiral ganglions. (c) Edema, epithelial degeneration, vacuolization, and loss of cells in the stria vascularis. (d,e,f) Organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in melatonin administration before and after radiotheraphy. Hematoxylin–eosin, 3 40.
media (Fig. 3a); cytoplasmic and nuclear condensation; neuron loss; edema in the spiral ganglions (Fig. 3b); and edema, epithelial degeneration, vacuolization, and loss of cells in the stria vascularis (Fig. 3c). Histopathological evidence from the organs of Corti, spiral ganglions, and stria vascularis damage was markedly reduced in both the melatonin and then radiotherapy group and the radiotherapy and then melatonin group (Fig. 3d,e,f).
DISCUSSION In this study, we showed that administration of melatonin after or even before ionizing radiation could prevent radiotherapy-dependent hearing loss in higher frequencies in an animal model. The prevention of irradiation-dependent hearing loss was greater in the radiotherapy and then melatonin group than in the melatonin and then radiotherapy group. Furthermore, the histopathological findings confirmed that administration of melatonin was protective of cells in the spiral ganglions, stria vascularis, and organs of Corti. In the present study, we used Cobalt 60 for irradiation. Previous studies showed that treatment with Co-60 results in better performance when compared with other treatment methods.15 The Co-60 provides a strong option for improving the quality of life for patients with cancer. Indeed, its low cost, uncomplicated maintenance, and relative ease of availability may provide a viable treatment option.16 Laryngoscope 125: October 2015
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In this study, 60 Gy radiotherapy was given to the rats. Previous studies found that the cochlear damage occur in various irradiation dosages. The histopathological findings in the ear after irradiation were well summarized by Linskey and Johnstone.3 They noted that two principal sites of damage are the hair cells of the cochlea and the stria vascularis. The researchers found that the outer hair cells are more sensitive than the inner hair cells for gamma irradiation damage.17 Bohne et al.18 found that the damage in the organ of Corti occur in 31% of animals at 30 to 40 Gy compared to 62% of animals at 60 to 90 Gy. Winther19–21 exposed guinea pigs to single doses of photon radiation ranging from 10 to 70 Gy and identified no hair cell changes with doses 20 Gy. Outer hair cell damage began to occur at 40 Gy, and vestibular sensory epithelial cells were damaged with single photon doses of 70 Gy.20,21 Similarly, Nagel and Schafer22 exposed guinea pigs to single-dose photon radiation of 35 to 70 Gy. They noted no hair cell damage in short follow-up for doses 35 Gy. Only outer hair cells were affected at 40 to 45 Gy, whereas inner hair cells began to be affected at 50 Gy. It is known that gamma knife surgery produces tissue damage through vascular injury.23 After gamma knife surgery, the blood vessels exhibit narrowing caused by endothelial proliferation, which tend to occur within days after irradiation and lead to ischemia and necrosis.24 In a clinical study, the researchers showed that ischemia and necrosis caused by irradiation damage can occur with Karaer et al.: Melatonin Protects Inner Ear Against Radiation
fractioned radiation doses 50 Gy or greater.25 Wackym et al.26 proposed that limiting the dose of radiation to < 4 Gy to the cochlea would likely reduce vascular injury to the stria vascularis and improve hearing outcomes. However, a typical gamma knife surgery dose for vestibular schwannoma is between 12 and 13 Gy, which is approximately equivalent to a fractioned dose of 48 to 50 Gy.26 Therefore, it is likely that small cochlear vasculature, such as in the stria vascularis, may be affected by a single fraction of gamma knife surgery. In a clinical study, Massager et al.27 reported in a series of 82 patients with vestibular schwannoma treated with a marginal dose of 12 Gy (median radiation dose delivered to cochlea 4.15 Gy). They found that a statistically significant association between higher cochlear volume radiation dose and deterioration of hearing was reported in these patients. Likewise, Lasak et al.28 reported 33 patients with vestibular schwannoma undergoing gamma knife surgery. Eighteen of their patients received cochlear volume doses greater than 4.75 Gy, and they found that these higher radiation doses were associated with hearing loss. In the present study, we found that the most prominent hearing loss occurred at frequencies of 4000 to 6000 Hz. In agreement with this study, Akmansu et al. found that the most prominent irradiation-dependent hearing loss occurred at 3000 to 6000 Hz.29 The loss of hearing at higher frequencies that was previously reported by several investigators indicates a greater susceptibility of the basal turn of the cochlea to radiation damage because this is responsible for the detecting higher frequencies.6 To the best of our knowledge, this is the first experimental study showing possible beneficial effects of melatonin on radiation-dependent inner ear damage. In this study, we found that melatonin was protective of cells in spiral ganglions, stria vascularis, and organs of Corti in rats administered a dose of irradiation of 60 Gy. When melatonin was administered after irradiation, the prevention ability was greater. In previously articles, melatonin was effective agent against damage produced by irradiation in several organs, including the liver, lung, lymphatic tissue, and lens.30–33 Melatonin was put forward as a potent free radical scavenger and an antiapoptotic agent in utricular hair cells.34 The studies observed that melatonin attenuated hairy cell loss caused by exposure to an ototoxic level of gentamicin.35,36
CONCLUSION In conclusion, these are pioneering results for studies that will be performed with melatonin to protect from radiation toxicity in the inner ear. It would be worthwhile to study the effects of melatonin supplements in radiation-treated cancer patients in the hope of reducing radiation-induced toxicity.
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