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Otolaryngology http://oto.sagepub.com/ -- Head and Neck Surgery Oxymetazoline Ototoxicity in a Chinchilla Animal Model Sam J. Daniel, Olubunmi V. Akinpelu, Sofia Sahmkow, W. Robert J. Funnell and Fadi Akache Otolaryngology -- Head and Neck Surgery 2012 146: 114 originally published online 16 August 2011 DOI: 10.1177/0194599811419082 The online version of this article can be found at: http://oto.sagepub.com/content/146/1/114 Published by: http://www.sagepublications.com On behalf of: American Academy of Otolaryngology- Head and Neck Surgery Additional services and information for Otolaryngology -- Head and Neck Surgery can be found at: Email Alerts: http://oto.sagepub.com/cgi/alerts Subscriptions: http://oto.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav >> Version of Record - Dec 22, 2011 OnlineFirst Version of Record - Aug 16, 2011 What is This? Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012 Original Research—Otology and Neurotology Oxymetazoline Ototoxicity in a Chinchilla Animal Model Sam J. Daniel, MD, MSc, FRCSC1,2, Olubunmi V. Akinpelu, MD, MSc2, Sofia Sahmkow, MD1, W. Robert J. Funnell, PhD1, and Fadi Akache, MSc1 Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. Abstract Objective. To investigate possible ototoxic effects of a onetime application of oxymetazoline drops in a chinchilla animal model with tympanostomy tubes. Study Design. A prospective, controlled animal study. Setting. The Research Institute of the Montreal’s Children Hospital, McGill University Health Centre. Subjects and Methods. Ventilation tubes were inserted in both ears of 12 animals. One ear was randomly assigned to receive oxymetazoline drops (0.5 mL). The contralateral ear did not receive any drops, serving as a control ear. Outcome Measures. Distortion product otoacoustic emissions were measured bilaterally for a wide range of frequencies (between 1 and 16 kHz) before and 1 day after the application of oxymetazoline in the experimental ears. Two months later, the animals were sacrificed and all cochleae were dissected out and processed for scanning electron microscopy. Results. In this established chinchilla animal model, the measured distortion product otoacoustic emission amplitudes and the morphological appearance on scanning electron microscopy were similar for both control and experimental ears. Conclusion. Oxymetazoline did not cause ototoxicity in a chinchilla animal model 2 months after a single application via a tympanostomy tube. Otolaryngology– Head and Neck Surgery 146(1) 114–118 Ó American Academy of Otolaryngology—Head and Neck Surgery Foundation 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0194599811419082 http://otojournal.org million operations performed yearly in North America.1-3 It is common practice in many pediatric centers to use oxymetazoline drops (off-label) to control bleeding after a myringotomy and tube insertion or to unblock clots in ventilation tubes.4 Oxymetazoline has also been used off-label to prevent otorrhea after PE tube insertion in some centers.5,6 To date, only one study has assessed the ototoxicity of oxymetazoline, using a model with an osmotic pump implantation delivering the product slowly over a 2-week period. The aim of this study is to evaluate the safety of a one-time application of oxymetazoline drops during tympanostomy tube insertion in our established chinchilla animal model simulating the clinical procedure. Materials and Methods The study was performed on 14 female chinchillas (Chinchilla laniger) under the supervision of the McGill University Animal Care committee that approved and monitored the protocol. These animals had commercial food and water ad libitum and had a mean body weight of 500 6 75 g. Ventilation tubes were inserted in both ears of all animals. In 12 animals, 1 ear was randomly assigned to receive 0.5 mL of oxymetazoline (Drixoral, Schering Plough Canada) at the time of ventilation tube insertion; the other ear received no drops and served as control for that particular animal. Two animals were used as a positive control group. These received 0.5 mL of gentamicin sulfate (Garasone, Schering Plough Canada), in 1 randomly selected ear, to demonstrate the validity of the model. Ototoxicity was expected in the ear under test in this group to confirm that the drops indeed reached the cochlea through the method described here. Keywords oxymetazoline, ototoxicity, tympanostomy tubes, scanning electron microscopy Received September 27, 2010; revised July 8, 2011; accepted July 14, 2011. M yringotomy with ventilation tube placement has become one of the most common surgical procedures performed on children, with more than 1 1 2 Department of Otolaryngology, Head and Neck Surgery, McGill University Montreal Children’s Hospital, Montreal, Quebec, Canada Corresponding Author: Sam J. Daniel, MD, MSc, FRCSC, Department of Otolaryngology, Head and Neck Surgery, McGill University, Montreal Children’s Hospital, 2300 Tupper Avenue, Montreal, Quebec, Canada H3H 1P3 Email: [email protected] Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012 Daniel et al 115 Sample Size Estimation Based on an initial pilot study, setting a at 0.05, a minimum absolute difference of 15, and a power of 0.80, a sample size of 8 animals was deemed to be sufficient. We used 12 animals, considering the possibility of unexpected events leading to animal loss during the study. Surgical Procedure The animals were anaesthetized using intraperitoneal injections of ketamine (35 mg/kg) and xylazine (5 mg/kg) and then positioned on their sides to allow for examination of the external auditory canal and tympanic membrane. Any debris or wax seen was cleared off using a curette. With a speculum in the external auditory canal and under an operating microscope (Zeiss, Germany), an incision was made in the anterior, inferior quadrant of the tympanic membrane. Afterward, a Reuter Bobbin tube (1.14 mm wide, Medtronic Xomed Inc, Jacksonville, Florida) was inserted in the eardrum. This procedure was carried out on both ears of each of the animals. Oxymetazoline Application The head of the animal was positioned such that the external auditory canal of the randomly selected experimental ear faced upward. Then 0.5 mL of oxymetazoline drops was instilled directly over the opening of the ventilation tube. The head was maintained in that position for 5 minutes. ethanol solutions, 30% (15 minutes), 50% (15 minutes), 70% (15 minutes), 80% (15 minutes), 95% (15 minutes), and 100% (15 minutes). Finally, they were critical-point dried, mounted on stubs, and coated with gold in a sputter coater. The samples were analyzed under a field-emission scanning electron microscope (Hitachi S4700). The control and experimental micrographs were compared visually with regard to the shape of the stereocilia, outer hair cell loss, and inner hair cell loss using the method described by Korver.7 Statistical Analysis The difference in DPOAE amplitudes between the control ear and the treated ear was calculated for each animal, at each frequency, before and after oxymetazoline application. We also calculated the 95% confidence intervals for each of the differences reported both at baseline and post oxymetazoline. Results Gentamicin Group The 2 ears that were given Garasone (gentamicin) as a positive control group had severe damage to the inner and outer hair cells and loss of architecture of the stereocilia, indicating that in this model drops applied through the tympanostomy tubes reached the inner ear in sufficient quantity to cause damage. Both gentamycin and oxymetazoline are marketed in aqueous forms. Assessment of Ototoxicity Using the DPOAE Test DPOAEs We used the Smart distortion product otoacoustic emission (DPOAE) high-frequency software/hardware package (Intelligent Hearing Systems, Miami, Florida). The tests were performed in a quiet environment at all times. Thermal stability of the animal was maintained at 36°C with an infrared lamp at 2 feet above the operating table. All tests were done using inhalational anesthesia induction with 5% isofluorane and maintenance with 2% isofluorane. The otoacoustic emissions were recorded for both ears between 1 and 16 kHz. Two-tone stimuli at 55 and 65 dB SPL were emitted with a frequency ratio (F1/ F2) of 1.22 and averaged 32 times. The 2F1-F2 DPOAE amplitude was used in assessing hearing function. Otoacoustic emission measurements were used to compare the response of the control and tested ears before (baseline) and 1 day after oxymetazoline application. Baseline DPOAEs were similar in both control and experimental ears, showing that the experimental and control ears were similar at the onset of the experiment. Table 1 shows the amplitudes obtained for control and experimental ears at low, middle, and high frequencies at baseline and post treatment, while Figure 1 shows the apparent similarities in the values obtained for both groups. The 95% confidence intervals for the means of the differences between the control and experimental ears at baseline and post oxymetazoline treatment for each frequency are shown in Table 2. These do not reveal any significant difference between the treated ears and untreated ears in each animal. Assessment of Ototoxicity Using Scanning Electron Microscopy Two months after the instillation of oxymetazoline, all animals were sacrificed by an overdose of anesthetic using 40 mg/kg of pentobarbital. After decapitation, the cochleae were removed quickly and washed in a saline solution. They were then fixed in 1% glutaraldehyde for 1 hour and left in a 3% glutaraldehyde solution for 3 days at 4°C. The bone covering the scala vestibuli and the cochlear duct was removed. The specimens were then dehydrated in a series of Scanning Electron Microscopy Twenty four cochleae were processed (12 control, 12 experimental). Two cochleae (1 experimental and 1 control from 2 different animals) were rejected for poor processing. The scanning electron microscopy analysis of all remaining cochleae did not show any damage in the structure of the inner ear. Both control and experimental ears had no discernible hair cell loss in the basal turns. The outer and inner hair cell layers were intact, there was no loss of hair cells, and the integrity of the stereocilia was preserved, indicating that oxymetazoline did not cause any hair cell damage. The basal turns of the cochleae were studied, as this is where most ototoxicity would manifest itself if present. Figures 1 and 2 show representative electron microscopy sections of Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012 116 Otolaryngology–Head and Neck Surgery 146(1) Table 1. Mean Distortion Product Otoacoustic Emission Changes in the Control and Experimental Ears 1 Control ears F2, kHz 1.1 Baseline, dB 20.2 Post treatment 0.8 SD 2.5 Change 1 Experimental ears F2, kHz 1.1 Baseline, dB 21.2 Post treatment 20.2 SD 3 Change 1 2 3 4 5 6 7 8 9 10 11 12 1.3 21.5 20.5 2 1 1.55 0.7 2.7 1.5 2 1.85 4.8 7.8 2 3 4.42 12.3 14.3 2 2 5.24 20.8 23.8 0.5 3 6.24 24.2 27.2 0.2 3 7.42 26.3 30.3 1 4 8.84 22.1 24.1 1 2 10.5 19.7 22.7 1.8 3 12.5 17.1 19.1 1 2 14.8 18.2 20.2 0.8 2 1.3 20.9 2.3 2 3.2 1.55 4.6 6.6 1 2 1.85 7.2 11.2 0.4 4 4.42 14.1 17.1 2 3 5.24 21.6 23.1 1 1.5 6.24 25.3 25.9 0.1 0.6 7.42 29.5 34.5 0.5 5 8.84 23.6 24.6 3 1 10.5 22.1 24.1 0.5 2 12.5 19.2 22.2 2 3 14.8 20.6 23.6 1 3 Abbreviations: SD, standard deviation. Figure 1. Representative electron microscopy section of outer hair cells from a cochlea exposed to oxymetazoline revealing normal structures and architecture. outer hair cells from oxymetazoline and gentamicin exposed cochleas, respectively. Discussion Oxymetazoline HCl in aqueous solution is an antihistamine decongestant that has been described by some authors as having antibiotic properties with no observed ototoxic effects.5,6 Drixoral contains oxymetazoline HCl 0.05% in aqueous solution with nonmedicinal ingredients including benzalkonium chloride, edetate disodium, propylene glycol, and water. Being an adrenergic a-agonist and a directly acting sympathomimetic, Drixoral is used as a vasoconstrictor to relieve nasal congestion.8 The sympathomimetic action of oxymetazoline constricts the smaller arterioles of the nasal passages, producing a prolonged decongestant effect for up to 12 hours.8,9 This property is most likely responsible for its hemostatic effect at the myringotomy incision site. It is not uncommon to have some bleeding at the time of myringotomy incision. Although this is rarely significant, controlling it can prolong the time of surgery, and its occurrence can later lead to blockage of the PE tube.10 The prophylactic use of oxymetazoline or the related xylometazoline to prevent the occurrence of blocked tubes was the practice of 13% of otolaryngologists in a recent survey looking at trends in methods used to prevent and treat blocked PE tubes.11 Oxymetazoline has also been used in human subjects to reduce the occurrence of posttympanostomy otorrhea, which is a common complication of PE tube insertion.5 In a recent animal study, oxymetazoline was shown to possess antimicrobial properties, inhibiting middle-ear pathogens; no associated ototoxicity was reported.6 Off-label use of oxymetazoline raises some concerns regarding its safety within the inner ear.12 With a molecular weight of 296.84 g/mol, oxymetazoline will easily cross the round-window membrane (RWM), as it is known that substances whose molecular weights are less than 500 g/mol will rapidly pass through the RWM.13 Miller et al14 demonstrated a reduction of up to 60% in cochlear blood flow when the cochlea was exposed to epinephrine either topically or systemically. Similarly, a 25% reduction in cochlear blood flow was found when phenylephrine was applied to the RWM of gerbils.15 However, the use of phenylephrine in human subjects as a middle-ear decongestant to prevent PE tube obstruction did not produce hearing loss clinically.16 Our chinchilla animal model has been used previously to demonstrate the severe ototoxic and vestibulotoxic effects of gentamicin and the safety of dexamethasone eardrops.17 Our current results clearly indicate that a one-time application of oxymetazoline at the time of ventilation tube insertion does not lead to demonstrable ototoxicity either functionally or morphologically. This corroborates the earlier claim made by Isaacson et al.6 Although extrapolations from animal studies to humans should be done carefully, the chinchilla animal model is a very sensitive model for toxicity due to ototopical drugs. The thickness of its round window is only 10 to 14 mm, compared with 40 to 70 mm in humans, thereby allowing a much easier penetration of the toxic agents into the cochlea.18 It can be deduced, therefore, that oxymetazoline, being safe through the thinner Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012 Daniel et al 117 Table 2. Differences between DPOAE Amplitude Recorded in Control versus Experimental Ears at Baseline and Post Oxymetazoline Treatment for Each Animal across All Tested Frequencies F2, kHz 1.1 1.3 1.55 1.85 4.42 5.24 6.24 7.42 8.84 1.5 12.5 14.8 Mean of Difference at Baseline SD 95% CI Mean of Difference Post Oxymetazoline SD 95% CI 1.06 0.7 1.2 20.7 21.4 20.8 20.6 2.2 1.8 1.5 2.6 1.9 5.6 4.8 6.3 6.4 5.1 6.5 7.1 6.0 5.7 5.5 6.2 4.9 63.17 62.72 63.57 63.63 62.89 63.68 64.02 63.40 63.23 63.12 62.27 62.76 2.33 1.89 2.3 21.8 20.9 21.7 21.2 3.1 2.2 1.9 1.8 1.7 6.9 7.6 8.1 7.9 9.5 8.2 7.7 6.8 8.5 9.1 6.5 7.3 63.91 64.31 64.59 64.48 65.38 64.65 64.65 63.85 64.82 64.53 63.68 64.14 Abbreviations: CI, confidence interval; SD, standard deviation. Conclusion Oxymetazoline (Drixoral) ear drops are sometimes used off label to stop bleeding after tympanostomy tube insertion or to unblock a clotted tube. When tested in a chinchilla animal model, the oxymetazoline drops did not produce any damage to the outer hair cells demonstrable by either DPOAE measurements or SEM findings. Further study of the effects of oxymetazoline on cochlear blood flow and on the electrophysiological activities of the cochlear hair cells would help in making conclusive statements on safety. Author Contributions Figure 2. Representative electron microscopy section of outer hair cells from a cochlea exposed to Garasone, revealing altered architecture and damaged stereocilia. RWM of the chinchilla, may be also safe in the much thicker human RWM. In our study, oxymetazoline was applied once after placement of the ventilation tube to mimic the clinical procedure whereby patients would receive drops of oxymetazoline as a hemostatic agent or to unblock the tympanostomy tube. It is expected that the drops will be present for several minutes only, but their proximity to the RWM allows them to diffuse into the inner ear. This was shown in our positive-control experiment in which severe damage was noted in the outer hair cells, demonstrating that the route of administration was adequate for the drug to get to the RWM. The oxymetazoline used in the animals described in this study was procured from sterile bottles sold over the counter (Drixoral). Since oxymetazoline is not commercially available as eardrops, the risk of contamination remains an issue. Although the pH of oxymetazoline is slightly acidic, we did not observe any symptoms in the animals to suggest pain or discomfort following oxymetazoline application. Sam J. Daniel, conception and design of the study, analysis and interpretation of data, helping write and revise the article critically for important intellectual content, final approval of the version to be published; Olubunmi V. Vakinpelu, acquisition and interpretation of the data, drafting the article, final approval of the version to be published; Sofia Sahmkow, acquisition and interpretation of the data, drafting the article, final approval of the version to be published; W. Robert J. Funnell, interpretation and analysis of the data, participated in the drafting of the article, final approval of the version to be published; Fadi Akache, acquisition of data, participated in the drafting the article, final approval of the version to be published. Disclosures Competing interests: None. Sponsorships: None. Funding source: Canadian Institute of Health Research (CIHR) and the Canadian Foundation for Innovation (CFI) (only role is funding research grant). References 1. Ah-Tye C, Paradise JL, Colborn DK. Otorrhea in young children after tympanostomy-tube placement for persistent middleear effusion: prevalence, incidence, and duration. Pediatrics. 2001;107:1251-1258. 2. Isaacson G, Rosenfeld RM. Care of the child with tympanostomy tubes. Pediatr Clin North Am. 1996;43:1183-1193. Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012 118 Otolaryngology–Head and Neck Surgery 146(1) 3. Schraff SA. Contemporary indications for ventilation tube placement. Curr Opin Otolaryngol Head Neck Surg. 2008;16: 406-411. 4. Friedman RA, Kesser BW. Surgery of ventilation and mucosal disease. In: Brackmann DE, Sleighton C, eds. Otologic Surgery. 2nd ed. Philadelphia, PA: W. B. Saunders; 2001. 5. Kumar VV, Gaughan J, Isaacson G, Szeremeta W. Oxymetazoline is equivalent to ciprofloxacin in preventing postoperative otorrhea or tympanostomy tube obstruction. Laryngoscope. 2005;115:363-365. 6. Isaacson G, Buttaro BA, Mazeffa V, et al. Oxymetazoline solutions inhibit middle ear pathogens and are not ototoxic. Ann Otol Rhinol Laryngol. 2005;114:645-651. 7. Korver KD, Rybak LP, Whitworth C, et al. Round window application of D-methionine provides complete cisplatin otoprotection. Otolaryngol Head Neck Surg. 2002;126:683-689. 8. Canada JR, ed. USP Dictionary of USAN and International Drug Names, 1998. Rockville, MD: The United States Pharmacopeial Convention Inc; 1997:542. 9. Kobinger W. Central a-adrenergic systems as targets for hypotensive drugs. Rev Physiol Biochem Pharmacol. 1978;81:39100. 10. Jeon E, Park Y, Lee S, et al. Lee D, Factors of the blockage of ventilation tubes in the immediate postoperative period; Eur Arch Otorhinolaryngol. 2007;264:1393-1397. 11. Elden LM, Marsh RR. Survey of pediatric otolaryngologists: clinical practice trends used to prevent and treat blocked ventilation ear tubes in children. Int J Pediatr Otorhinolaryngol. 2006;70:1533-1538. 12. Seidman MD. Is oxymetazoline really safe for middle ear use? Laryngoscope. 2005;115:1321-1323. 13. Hamaguchi Y, Morizono T, Juhn SK. Round window membrane permeability to serum albumin in antigen-induced otitis media. Am J Otolaryngol. 1988;9:34-40. 14. Miller JM, Laurikainen EA, Grenman R, et al. Epinephrine induced changes in human cochlear blood flow. Am J Otol. 1994;15:299-306. 15. Ohlsén KA, Baldwin DL, Nuttall AL, et al. Influence of topically applied adrenergic agents on cochlear blood flow. Circ Res. 1991;69:509-518. 16. Altman JS, Haupert MS, Hamaker RA, et al. Phenylephrine and the prevention of post-operative tympanostomy tube obstruction. Arch Otolaryngol Head Neck Surg. 1998;124: 1233-1236. 17. Daniel SJ, Duval M, Sahmkow S, et al. Ototoxicity of topical moxifloxacin in a chinchilla animal model. Laryngoscope. 2007;117:2201-2205. 18. Goycoolea MV, Lundman L. Round window membrane: structure function and permeability: a review. Microsc Res Tech. 1997;36:201-211. Downloaded from oto.sagepub.com at SOCIEDADE BRASILEIRA DE CIRUR on May 14, 2012