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Ultrastructural Analysis and ABR Alterations in the Cochlear Hair-Cells Following Aminoglycosides Administration in Guinea Pig

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Ultrastructural Analysis and ABR Alterations in the Cochlear Hair-Cells Following Aminoglycosides Administration in Guinea Pig Global Journal of Otolaryngology ISSN 2474-7556 Research Article Glob J Otolaryngol Volume 15 Issue 4 - May 2018 Copyright © All rights are reserved by Mohammed A Akeel DOI: 10.19080/GJO.2018.15.555916 Ultrastructural Analysis and ABR Alterations in the Cochlear Hair-cells Following Aminoglycosides Administration in Guinea Pig Mohammed A Akeel* Department of Anatomy, Faculty of Medicine, Jazan University, Jazan, Kingdom of Saudi Arabia Submission: April 18, 2018; Published: May 08, 2018 *Corresponding author: Mohammed A Akeel, Faculty of Medicine, Jazan University, P.O.Box 114, Jazan, Kingdom of Saudi Arabia. Tel: ; Email: Abstract Aims: since their introduction in the 1940. The aim of the present work was to characterize ultrastructural alterations of the sensory hair-cell following different aminoglycosides Aminoglycosides administration, (AG) antibiotics intratympanic were the first VS ototoxic intraperitoneal agents to and highlight to correlate the problem them with of drug-induced auditory brainstem hearing responses and vestibular (ABR). loss, Methods: streptomycin, gentamycin, or netilmicin; respectively, via the peritoneal route. The next four groups received similar treatment via trans- tympanic route. A Thetotal treatment of 48 adult was guinea administered pigs were dividedfor seven into consecutive 8 groups; sixdays. animals On day in 10,each ABR group. was The utilized first fourfor hearing groups receivedevaluation saline and (control),scanning electron microscopy (SEM) examination of the sensory organs was used for morphological study. Results: Streptomycin produced the most severe morphological changes and a higher elevation of ABR thresholds, followed, in order, by gentamycin and netilmicin. Netilmicin ototoxicity observed in both systemic and transtympanic routes was low because of lesser penetration into the inner ear or/ and lower intrinsic toxicity. Conclusion: Streptomycin cochleotoxicity lesion involves destruction of the sensory hair cells of the cochlear epithelium which was netilmicin. Moreover, we demonstrated the presence of surface scavenger microphages in close association with auditory hair cells in the normal physiologicalquantified functionally conditions. and Such ultrastructurally. cells may form Streptomycin part of the resident was the immune most cochleotoxic surveillance among of organ the of AG Corti. administrated, followed by gentamycin and Keywords: ABR: Auditory Brainstem Responses; Guinea pig; (AGs): Aminoglycosides; SEM: Scanning Electron Microscopy Introduction embryonic formation of the mammalian cochlea. In mammalian The sensory hair cells within the cochlea of the mammalian injury, no hair cell replacement is observed, as opposed to birds, inner ear convert sounds into receptor potentials when their where regenerative mechanisms produce new sensory cells and restore the auditory function. Notably, in neonates, certain cochlea contains two types of hair cells (HC): inner and outer projecting stereocilia are deflected. The organ of Corti of the cell populations of the organ of Corti are multipotent; they can hair cells (IHCs and OHCs), which differ in function. It has been proliferate and generate otic spheres, as well as differentiate appreciated for over two decades that although IHCs act as and express hair cell markers, after being mechanically isolated the primary receptor cells for the auditory system, the OHCs and grown in culture [3]. In spite of the introduction of act as motor cells. OHCs respond to variation in potential, and in vitro new classes of antibiotics, the aminoglycosides: streptomycin, change length at rates unequalled by other motile cells. The gentamycin and netilmicin still remain the primary agents of forces generated by outer hair cells are capable of altering the choice in treating serious gram-negative infections [4]. It is delicate mechanics of the cochlear partition, increasing hearing well known that the use of aminoglycosides antibiotics carries sensitivity and frequency selectivity [1]. a risk of cochleotoxicity. Yet they are still used because of their The mammalian organ of Corti contains at least seven effectiveness and low cost [5,6]. The potential for cochleotoxicity different supporting cell (SC) types: Deiters’ cells, pillar cells, of aminoglycosides has been investigated by many clinical Hensens’ cells, inner phalangeal cells, inner border cells, Claudius’ cells, and Boettcher cells. All of these supporting cell cochleotoxic risk are the total daily dose (in illigrams/per studies [7-9]. Three primary factors identified with increased types are required for normal hearing function [2]. Hair cell kilogram), the course length, and repeated courses of therapy and supporting cell developments are completed during early [9,10]. The mechanisms underlying their selective toxicity to Glob J Otolaryngol 15(4): GJO.MS.ID.555916 (2018) 0064 Global Journal of Otolaryngology the inner ear continue to be unraveled despite more than 70 prior to trans-tympanic (TT) injection or intra-peritoneal (IP) injections. by severe bacterial infections also increase cochlear uptake of years of investigation [11]. Moreover, inflammation caused The sound level was raised in 20- and/or 5-dB steps. At each aminoglycosides and subsequent ototoxicity [12]. Preclinical level, 1000 responses were averaged. Both ears were measured. Animals with abnormal baseline ABR were excluded from the bacterial lipopolysaccharides displayed increased cochlear models with systemic inflammation, induced by low doses of experiment. Then, ABR evaluations were performed on day 10, uptake of aminoglycosides, and enhanced levels of cochleo- i.e. 72 hours after the last aminoglycosides doses were given. All toxicity without altered serum drug levels [13]. The effects of procedures and animal handling described in this protocol were aminoglycosides on neurosensory hair cells of the cochlea similar to those adopted by Murillo-Cuesta et al [18]. ABR are well documented [14-17]. Yet, few comparative analysis results were compared using a three-way ANOVA. The effects of of systematic vs. transtympanic administration of various the frequency (0.5, 1, 2, 4, 8, 16 and 32kHz), on the side treated aminoglycosides and their induced cochleo-toxicity was reported. (right vs. left) and the different aminoglycosides intraperitoneal This was mainly achieved through immunohistochemistry, light (IP) vs. transtympanic (TT) were measured and compared for microscopy examinations and other similar modalities, except the ultrastructural level. The following study, therefore, aims at comprehensive morphological characterization of the cochlear significanceTissue Preparations at p> 0.05. For Scanning Electron Microscope hair cells following aminoglycosides using scanning electron Seventy two hours after giving the last dose of each microscopic (SEM) examination and combining it with auditory aminoglycoside, the animal were anesthetized with brain stem response (ABR) investigation. intraperitoneal injection of diazepam 3mg/kg, perfused with Materials and Methods sterile phosphate buffer solution PBS (with Ca2+ and Mg2+) Animals and Study Groups for 10 minutes in 1% glutraldehyde, 2% paraformaldehyde in and hereafter PBS++. Then the tissues were fixed by perfusion A total of 48 guinea pigs, each weighing 250-300 grams, phosphate-buffer at room temperature (pH 7.4). The temporal were obtained from animal house of the research centre. All bone was excised and the cochlea carefully removed through the animals were kept under optimum conditions. Animal care was in compliance with research regulation and resolution. paraformaldehyde in phosphate-buffer at room temperature (pH The experimental protocol was approved by Ethic Committee micro-dissection and fixed again in 1% glutraldehyde, 2% of Animal Research. Guinea pigs were divided into 8 groups; The cochlea was then washed twice in buffered sucrose for 7.4) for 60 minutes. Decalcification in EDAT was not performed. 5 min each (0.1 M phosphate buffer, 5% sucrose solution). was performed via the peritoneal route (systemic) for seven six in each group. In the first four groups drug administration consecutive days as follow: 2ml of saline solution vehicle only buffered 2% osmium. The tissues were dehydrated in a graded Post fixation was performed at 4°C for 60 min in phosphate- in group I (control), 125mg/kg streptomycin in group II, 50mg/ series of ethanol (40, 50, 70, 80, 90, and twice in 100%), then kg gentamycin in group III and 37.5mg/kg netilmicin, (1-N-ethyl ethanol- acetone (1:1) absolute solution for further 30 minutes, derivative of sisomicin) in group IV. The doses were 10-20 times absolute acetone 100% for additional 30 minutes 3 times 10 higher than the recommended human dosage. For the next minutes each. The tissues were critically point dried in CO four animal groups the aminoglycosides were administrated at drying apparatus CPD 030 and mounted on stubs, coated with 0.25ml/kg in a 4% saline 40mg /ml via trans-tympanic route gold sputter coater SCD005 and examined with Philips Scanning for seven consecutive days through the right external meatus, as Electron Microscopy XL3 at 30kv. follow: saline for group V, streptomycin for group VI, gentamycin Semi-Quantitative Evaluation for group VII and netilmicin for group VIII. The guinea pigs were anesthetized with intraperitoneal injection of diazepam 3 mg/kg Damage of the stereocilia of outer hair cells (OHCs) observed and the animals were kept in prone position to insure the drugs normal, grade 1 (10-50% loss
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