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Locacorten Vioform Ototoxicity Upon Guinea Pig Middle Ear Application ORIGINAL ARTICLE J Audiol Otol 2018;22(2):75-79 pISSN 2384-1621 / eISSN 2384-1710 https://doi.org/10.7874/jao.2017.00199 Locacorten Vioform Ototoxicity Upon Guinea Pig Middle Ear Application Owen Woods and Issam Saliba Division of Otolaryngology-Head and Neck Surgery, Sainte-Justine University Hospital Center, University of Montreal, Montreal, Canada Background and Objectives: Locacorten Vioform (Novartis UK) is frequently prescribed for otomycosis. Its component, Clioquinol, also has anti-bacterial properties. Up to this point, its ototoxic potential has not been evaluated. Our objective aims to evaluate Locacorten Vio- form’s potential ototoxicity when applied directly to the middle ear cavity. Materials and Methods: We performed an experimental prospective animal study in our animal research center with 20 Hartley guinea pigs divided into 2 groups. The first group (experimental) was treated with Locacorten Vioform in one ear and with a physiologic saline solution in the other. The second group (positive control) was treated with concentrated gentamycin in one ear and physiologic saline in the other. Auditory brainstem response measurements were ob- Received July 28, 2017 tained before and after three sets of injections. Statistics were analyzed using a variance Revised November 20, 2017 analysis with repeated measures. The histological state of cochlear outer hair cells was com- Accepted December 5, 2017 pared between the two groups using scanning electron microscopy. Results: Average hear- Address for correspondence ing loss in ears treated with Locacorten Vioform was 32.1 dB, compared with a 2.5 dB aver- Issam Saliba, MD, FRCSC age loss in the saline-treated ears. Ears treated with gentamycin lost an average of 33.0 dB. Division of Otolaryngology- There were clinically and statistically significant differences between the two ears of the guin- Head and Neck Surgery, ea pigs in both groups (p<0.001). Scanning electron microscopy revealed severe perico- Sainte-Justine University chlear and cochlear inflammation and ossification in the Locacorten Vioform-treated ears. Hospital Center, Gentamycin caused significant destruction of outer hair cell architecture. Conclusions: Lo- University of Montreal, cacorten Vioform induces a hearing loss similar to that caused by gentamycin when applied 3175, Côte Sainte-Catherine, directly to the middle ear of a guinea pig model. Electron microscopy indicates a pericochlear Montréal (QC) H3T 1C5, Canada and cochlear inflammatory reaction with ossification. Tel +514-345-4857 J Audiol Otol 2018;22(2):75-79 Fax +514-507-9014 KEY WORDS:0 Locacorten Vioform · Gentamycin · Ototoxicity · Inner hair cell · Outer hair cell. E-mail [email protected] Introduction predisposes to otomycosis. Most patients present with unilat- eral ear pain and aural fullness, with possible persistent dis- Otomycosis is a common cause of otalgia and aural full- charge and mild hearing loss. Physical examination varies de- ness, particularly in patients recently treated with a topic anti- pending on the offending agent and extent of disease, but bacterial agent. It is defined as a fungal infection of the external typically reveals a white crust lining the external auditory ca- or middle ear, or of an open mastoid cavity. Patients with nal with a normal tympanic membrane (TM). Culture reveals’ chronic otitis media are at increased risk of developing otomy- Candida albicans, Candida parapsilosis, and Aspergillus fu- cosis. The use of topical quinolones has also been shown to migatus in 95% of cases [2]. When black crusts are identified, increase its prevalence [1]. Aspergillus infection should be suspected. Mainstay of treat- As with all fungal infections, a hot and humid environment ment consists of cleaning the infected ear, keeping it dry and applying topical antifungal cream or ointment. This is an Open Access article distributed under the terms of the Cre- ative Commons Attribution Non-Commercial License (http://creative- Not all antifungal agents are appropriate for otomycosis commons.org/licenses/by-nc/4.0/) which permits unrestricted non-com- treatment. Acetic acid and gentian violet have known ototoxic mercial use, distribution, and reproduction in any medium, provided the original work is properly cited. potential and should therefore be avoided [3,4]. Several alter- Copyright © 2018 The Korean Audiological Society and Korean Otological Society 75 Locacorten Vioform Ototoxicity in Guinea Pig native agents have proven their safety in animal studies, includ- was regulated to approximately 3-5% for induction and then ing ciclopirox, clotrimazole, miconazole, and nystatin [4-7]. approximately 1-3% for maintenance. In order to reduce hy- Locacorten Vioform (Novartis UK, London, UK) is a topi- pothermia related to anesthesia, we used a thermal support cal antifungal agent with three components: clioquinol, flu- until the animal is fully recovered from anesthesia [10]. All methasone and a polyethylene glycol vehicle. Its antibiotic manipulations, including middle ear injections and animal component, clioquinol, inhibits bacterial and fungal DNA sacrifice were performed under general anesthesia. The guin- structure and function [8]. Though studies report the use of Lo- ea pigs had spontaneous breathing but did not react to stimu- cacorten Vioform going as far back as 1966, no English-lan- lus. Auditory brainstem response (ABR) recordings required guage study has reported its efficacy or safety [9]. us to maintain anesthesia with a mask outside of the container. The goal of this study is to evaluate Locacorten Vioform’s Each animal’s adequate recovery from anesthesia was assured ototoxic potential when exposed to the middle ear of a guinea before leaving it unsupervised. pig model. Middle ear injections Materials and Methods We began our experiment with a microscopic evaluation of each guinea pig’s TM. Debris was cleared as needed. Each an- Animals imal then passed an ABR test within the first week. ABR pro- Twenty Hartley guinea pigs were included in our study. We tocol details are described below. prefer this species because of their accessible cochlea, their We then injected approximately 0.1 cc of the experimental functional anatomy which resembles that of humans, their solution into each ear, or enough to fill the middle ear cavity, widespread use in similar studies and finally because of our using a Whitacre spinal needle (BD, Middletown, NY, USA). experience with this species in our laboratory. Each injection as described above was repeated every second Animals were cared for in accordance with the guidelines of day for a total of three sets of injections. the Canadian Council of Animal Care. The study was ap- At this point we waited a total of two weeks before proceed- proved by our institutional review board and follows the stan- ing with further tests. This waiting time was considered nec- dards of the institutional animal care committee (c-461). essary in order to minimize the amount of residue present at the round window niche, which if considerable could inter- General procedures and group assignment fere with ABR readings as well as SEM analysis. The experiment was performed over eight weeks, begin- Our second ABR recordings were performed during week ning with group assignation and ending with scanning elec- 5. A thorough cleaning and drying of each ear was performed tron microscope (SEM) interpretation. the day before the recordings. It is important to note that all Each guinea pig was randomly assigned to one of two groups: TM were normal and no perforation was identified. an experimental group (group I) and a positive control group (group II). Animals from group I received Locacorten Vio- Auditory brainstem response form (flumethasone pivalate 0.02% and clioquinol 1%) injec- All ABR tests were performed with the same Nicolet Bra- tions in one randomly selected ear and physiological saline voTM device (Nicolet Bravo System; Nicolet Biomedical, (0.9% NaCl) injections in the other. Animals from group II Madison, WI, USA). The same room of the Experimental received gentamycin (40 mg/mL) injections in one randomly Surgery Center was used for all tests. Electrodes were insert- selected ear and physiological saline in the other. Each con- ed subcutaneously. The non-inverting electrodes were placed tralateral ear was thus used as a negative control, allowing us directly above the left and right mastoid processes, the in- to reduce the number of animals required and minimize the verting electrode was placed at the apex and the ground elec- impact of variability between animals. trode was placed in the middle of the animal’s back. Imped- ance was verified and accepted at values inferior to 3.0 kΩ. Anesthesia protocol We used tone burst stimuli emitted through TIP-300 (Bio- Anesthesia was induced by placing the animals in a plastic logic Corp., Madison, WI, USA) earphones to record differ- container with two entrance ports and one exit port. Oxygen ences across a range of frequencies. Tone bursts at 2000, and isoflurane 1% were administered in parallel systems and 4,000, 6,000, and 8,000 Hz were generated with condensation were ventilated out through the exit port. When animal was polarity. Amplitude thresholds were found to the nearest 5 dB. placed in the induction chamber and oxygen flowmeter ad- A total of 1,500 sweeps were averaged out with each recording. justed to approximately 0.5-1.0 L/min, isoflurane vaporizer To be considered a positive reading, a visible wave V needed to 76 J Audiol Otol 2018;22(2):75-79 Woods O, et al. be reproducible beyond a doubt on a minimum of two stimu- Auditory brainstem response lations with the same amplitude. ABR values are provided as absolute dB levels. Descriptive results Initial ABR testing yielded similar results between the two Animal sacrifice and preparation groups. Average threshold values across all tested frequen- All animals were sacrificed by guillotine under general an- cies ranged between 25.6±2.7 dB and 27.4±10.5 dB. After esthesia.
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