Protective role of intratympanic nigella sativa oil against gentamicin induced hearing loss

Accepted Manuscript Protective role of intratympanic nigella sativa oil against gentamicin induced hearing loss Deniz Tuna Edizer, MD, MSc, Ozgur Yigit, MD, Zehra Cinar, MD, Mehmet Gul, PhD, Eyyup Kara, PhD, Birgul Yigitcan, PhD, Duygu Hayır, PhD, Ahmet Atas, PhD PII:

S0165-5876(17)30136-2

DOI:

10.1016/j.ijporl.2017.03.034

Reference:

PEDOT 8478

To appear in:

International Journal of Pediatric Otorhinolaryngology

Received Date: 26 January 2017 Revised Date:

26 March 2017

Accepted Date: 29 March 2017

Please cite this article as: D.T. Edizer, O. Yigit, Z. Cinar, M. Gul, E. Kara, B. Yigitcan, D. Hayır, A. Atas, Protective role of intratympanic nigella sativa oil against gentamicin induced hearing loss, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.03.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT PROTECTIVE ROLE OF INTRATYMPANIC NIGELLA SATIVA OIL AGAINST GENTAMICIN INDUCED HEARING LOSS

Deniz Tuna Edizer, MD, MSc1, Ozgur Yigit, MD, Prof1, Zehra Cinar, MD1, Mehmet Gul,

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PhD, Prof2, Eyyup Kara, PhD3, Birgul Yigitcan, PhD2, Duygu Hayır, PhD3, Ahmet Atas, PhD, Prof3

Istanbul Training and Research Hospital, Department of Otorhinolaryngology, Istanbul,

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1

Turkey

Inonu University Medical Faculty, Department of Histology and Embryology, Malatya,

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2

Turkey 3

Istanbul University, Faculty of Health Sciences, Department of Audiology, Istanbul, Turkey

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Istanbul University, Cerrahpasa Medical School, Department of Otorhinolaryngology,

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Correspondence:

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Istanbul, Turkey

Deniz Tuna Edizer, MD, MSc

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Adress: Izmir Bozyaka Egitim ve Arastirma Hastanesi, KBB Klinigi Saim Cikrikci Cad. No:59

PK: 35170, Bozyaka – Izmir – Turkey Phone: +905335714289 Fax: +902322614444 e-mail: [email protected]

ACCEPTED MANUSCRIPT PROTECTIVE ROLE OF INTRATYMPANIC NIGELLA SATIVA OIL AGAINST

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GENTAMICIN INDUCED HEARING LOSS

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ACCEPTED MANUSCRIPT ABSTRACT Objective: Aminoglycosides, used to combat with life-threatening infections, have a substantial risk of hearing loss. Nigella sativa is an annual herbaceous plant and used for treatment of many diseases for ages. We aimed to investigate the protective role of

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intratympanic nigella sativa oil against gentamicin induced hearing loss in an animal model. Methods and Materials: Twenty eight guinea pigs were randomly divided into four groups: i- control, ii- Intratympanic nigella sativa oil (IT-NSO), iii- Intraperitoneal gentamicin (IP-G)

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and iv- Intraperitoneal gentamicin and intratympanic nigella sativa oil (IP-G + IT-NSO). Preoperative and postoperative hearing thresholds were determined with auditory brainstem

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response with click and 8 kHz tone-burst stimuli. Histological analysis of the cochlea specimens were performed under light microscope. Semiquantitative grading of the histological findings was carried out and compared between the groups. Results: Highest posttreatment hearing thresholds were detected in IP-G group. Posttreatment

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mean hearing threshold of the IP-G group with click stimulus was significantly higher than the IP-G + IT-NSO group (p=0.004). whereas the difference was not significant with 8 kHz tone-burst stimulus (p=0.137). Both IP-G and IP-G + IT-NSO groups had significantly higher

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hearing thresholds compared to control and IT-NSO groups (p>0.05). Histological examination of the control and IT-NSO groups demonstrated normal appearance of cochlear

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nerve, stria vascularis and organ of Corti. IP-G group showed the most severe histological alterations including hydropic and vacuolar degenerations, hair cell damage and deformation of the basilar mambrane. Histological evidence of damage was significantly reduced in IP-G + IT-NSO group compared to IP-G group. Conclusion: Addition of intratympanic NSO to systemic gentamicin was demonstrated to have beneficial effects in hearing thresholds which was supported by histological findings.

Key words: Nigella sativa; gentamicin; hearing loss; intratympanic 2

ACCEPTED MANUSCRIPT INTRODUCTION Aminoglycosides are relatively potent antibiotics used against aerobic Gram-negative bacteria including Enterobacteriaceae and Pseudomonas spp., tuberculosis, neonatal sepsis and some other life threatening infections [1-4]. Aminoglycosides display concentration

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dependent bactericidal activity rather than bacteriostatic potential by inhibiting protein synthesis at the ribosomes as well as by pore-forming effect on cell membranes [2,4-6]. Relative selectivitiy for bacterial ribosomes lowers the affinity of aminoglycosides for

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mammalian ribosomes [4]. Ototoxicity and nephrotoxicity are the main adverse effects that should be managed carefully, however neuromuscular block may also be prominent with

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aminoglycosides [2,6]. Ototoxicity appears only after days or weeks of begining of treatment [6]. Risk for ototoxicity becomes more prominent with increasing dose, frequency of administration and duration of treatment [7]. Genetic predisposition, which was described as a mitochondrial DNA A1555G point mutation, is also a well documented risk factor for

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aminoglycoside ototoxicity [4].

The severity of hearing impairment may even increase following cessation of aminoglycoside administration [6]. However, broad spectrum of activity and low cost still

countries [8-10].

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make this class of antibiotics one of the most commonly used agents especially in developing

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The incidence of aminoglycoside induced hearing loss may be seen in up to 25% of

patients [2,9]. Hair cells are the main targets of aminoglycoside ototoxicity, however spiral ganglion and stria vascularis may also be affected [1,4]. Gentamicin induced hearing loss appears secondary to apopotic mechanisms triggered by reactive oxygen species which lead to hair cell death [2,11,12]. Mitochondrial damage is also an important feature of hair cell death [13,14]. Oxygen free radicals are generated by iron-gentamicin complexes [8,10]. Gentamicin-induced hearing loss is generally symmetrical,

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ACCEPTED MANUSCRIPT bilateral and irreversible. Outer hair cells especially at the basal turn are more vulnerable to gentamicin toxicity [15,16]. Hence, higher frequencies are affected initially, however, hearing thresholds at lower frequencies also worsen at later stages [1,17]. Nigella sativa is an annual herbaceous plant and used for treatment of many diseases

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for ages [18]. It is also known as black seed or black cumin and has a bitter taste [19]. Thymoquinone is the predominant active compound of nigella and has considerable antioxidant effects. The effectiveness of nigella sativa against hypertension, hyperlipidemia,

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diabetes mellitus, asthma and infectious, neoplastic and immune-mediated diseases was previously demonstrated in many clinical and experimental studies [19-22].

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This study was designed to evaluate the effectiveness of intratympanic nigella sativa against the ototoxicity of systemic gentamicin administration.

METHODS

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Twenty eight Dunkin-Hartley male adult albino guinea pigs (8 weeks, 600-800 g) were used in this study. Local Committee on Animal Research approved the surgical interventions (2013 / 94). The study was conducted in accordance with the guidelines of

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animal care and use for experimental procedures. Animals had free access to food and water and kept under standard laboratory conditions. Guinea pigs were randomly divided into 4

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groups, with seven animals in each group: i- Intratympanic (IT) saline (control), ii- IT nigella sativa oil (IT-NSO) once a week for three weeks,

iii- IP gentamicin (IP-G) (100 mg/kg) daily for three weeks, iv- IP gentamicin (100 mg/kg) daily for three week + IT NSO once a week for three weeks (IP-G + IT-NSO).

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ACCEPTED MANUSCRIPT Animals were anesthesized by ketamine 40 mg/kg (Ketalar, Eczacibasi, Turkey) and xylazine 10 mg/kg (Rompun, Bayer, Germany), and all of the procedures, except for intraperitoneal injections, were performed under general anesthesia. All intratympanic injections were performed to the right ear.

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Auditory brainstem evoked potentials were measured (GSI Audera ABR system, Grason-Stadler Inc., USA), in a sound-proof room. Auditory brainstem response (ABR) was recorded with sterile needle electrodes. The active electrode was placed at the vertex, the

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reference electrode at the contralateral mastoid and the ground electrode at the ipsilateral mastoid. Following cleansing of any debris from the external ear canal and inspecting the

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tympanic membrane, insert earphones were used for transmission of acoustic signals. Click and tone-burst at 8 kHz stimuli were used with 1024 sweeps, at a rate of 19.6/sn. The hearing thresholds were determined by the lowest threshold (expressed in dB SPL) exploiting a wave V. ABR measurements were performed both before and 1 week after the end of drug

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administrations. Pretreatment and posttreatment hearing thresholds were recorded. Intraperitoneal gentamicin dosage was 100 mg/kg per day, for a total of three weeks. Nigella sativa oil (Origo, Gaziantep, Turkey) (4 ml/kg) was injected directly to the tympanic

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bulla, once a week for a total of three weeks, through the tympanic membrane following sterilization via bacterial filter passage. The volatile component of NSO contains 18.4-24% of

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thymoquinone (2-isopropyl-5-methylbenzo-1, 4-quinone) and 46% of monoterpenes [23,24]. The dose and frequency of NSO and gentamicin administrations were adjusted in accordance with previous studies on humans and animals [8,18,25,26]. At the end of the procedures, all animals were decapitated following intraperitoneal sodium thiopental (120 mg/kg). Tympanic bullae were quickly removed and fixed in 2.5% glutaraldehyde solution, buffered with 0.2 M NaH2PO4 + NaHPO4 (pH = 7.2) at 4°C for five hours. For decalcification, specimens were stored in 10% formic acid solution (renewed every

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ACCEPTED MANUSCRIPT other day) at 4°C for twelve days and then the cochleae were dissected from the tympanic bullae and divided into two halves at the level of the modiolus. Specimens were fixed in 1% osmium tetraxoide (OsO4) at 4°C for three hours, dehydrated in acetone series and embedded in araldite CY 212. Araldite-embedded cochlea specimens were cut into 1 µm thick sections,

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mounted on slides and stained with Toluidine blue. The sections were examined with a Nikon Optiphot-2 light microscope and analysed in Nikon DS-L3 Image Analysis System (Nikon Corporation, Tokyo, Japan).

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Assessment of tissue alterations in Corti organ, stria vascularis and cochlear nerve fibers for each specimen was conducted by an experienced histologist who was blinded to the

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treatment groups. The average number (100 µm2 at random in five different areas) and the diameter of myelinated axons (randomly, in 100 axons) were calculated along with investigation of the edema of the cochlear nerve. Epithelial damage and edema of the stria vascularis and degeneration of the organ of Corti were also assessed. Changes in organ of

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Corti (hydropic and vacuolar degeneration and loss of hair cells), stria vascularis (edema, vacuolization and loss of cells) and cochlear nerve were scored in a semiquantitative way as no change (0), mild (1), moderate (2) or severe (3). Hence, six histological variables were

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investigated: i- axon number, ii- axon diameter, iii- edema of the cochlear nerve, iv- epithelial damage in the stria vascularis, v- edema in the stria vascularis and vi- degeneration of the

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organ of Corti.

SPSS version 15.0 (IBM Corporation, USA) was used for statistical analysis. One

Way Anova and Kruskal Wallis tests were used for comparison of numerical variables. For subgroup analyses Tukey and Mann Whitney U tests were chosen for parametric and nonparametric evaluations, respectively. Chi-square test was used for categorical variables. A p value less than 0.05 was considered statistically significant.

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RESULTS All animals tolerated the interventions well and completed the study uneventfully.

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Recordings of hearing thresholds both before and after interventions and histologic evaluations were performed promptly without any technical problem. Tympanic bullae of the groups treated with intratympanic nigella sativa oil were noted to include some remnants of

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nigella sativa oil.

The mean hearing thresholds before and after drug administrations are given in table 1

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and figure 1. Pretreatment hearing thresholds were not significantly different between the groups (p>0.05). Highest posttreatment hearing thresholds with both click and 8 kHz toneburst stimuli were detected in IP-G group. Both IP-G and IP-G + IT-NSO groups had significantly higher hearing thresholds with click and 8 kHz tone-burst stimuli than the

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control and IT-NSO groups (p<0.05). IT-NSO group had significantly higher mean hearing thresholds than the control group, with both click and 8 kHz tone-burst stimuli (p<0.001 for each). Posttreatment mean hearing threshold of the IP-G group with click stimulus was

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significantly higher than the IP-G + IT-NSO group (p=0.004). However, posttreatment mean hearing threshold of the IP-G group with 8 kHz tone-burst stimulus was not significantly

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different from the IP-G + IT-NSO group (p=0.137). Histological examination of the control and IT-NSO groups demonstrated normal

appearance of cochlear nerve, stria vascularis and organ of Corti (Figures 2-4). IP-G group showed the most severe histopathological alterations including hydropic and vacuolar degenerations, cellular loss (both inner and outer hair cells and supporting cells) in the organ of Corti and deformation of the basilar mambrane (Figure 2C). Axonal loss, degeneration and edema of the cochlear nerve, and irregularities and condensations of the epithelial cell nuclei

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ACCEPTED MANUSCRIPT along with edematous large vacuoles and separation of the epithelial basement membrane in stria vascularis were other apparent findings (Figures 3C and 4C). Histological evidence of damage of the organ of Corti, cochlear nerve and stria vascularis was markedly reduced in IPG + IT-NSO group compared to IP-G group (Figures 2D, 3D and 4D).

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Results of the six histological variables mentioned previously in the methods section are given in table 2. Axon number and diamater were almost identical in control and IT-NSO groups with no statistical significance (p=0.896 and p=0.749, respectively). Both the control

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and IT-NSO groups had significantly higher axon number and larger axon diameter than the IP-G and IP-G + IT-NSO groups (p<0.05). Edema of the cochlear nerve, epithelial damage

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and edema of the stria vascularis and degeneration of the organ of Corti were not observed in control and IT-NSO groups. IP-G group had significantly lesser axon number and smaller axon diameter than the IP-G + IT-NSO group (p=0.017 and p=0.006, respectively). Score of the edema of the cochlear nerve was significantly higher in the IP-G group than the IP-G +

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IT-NSO group (p=0.01). Scores of the epithelial damage and edema of the stria vascularis and degeneration of the organ of Corti were also higher in the IP-G group than the IP-G + IT-NSO group, however, the differences were not significantly different (p=0.298, p=0.122 and

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p=0.254 respectively).

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DISCUSSION

Generation of oxygen free radicals leading to hair cell damage is intimately related to

aminoglycoside ototoxicity [5]. Hair cell damage was reported to be dose-dependent [27]. Outer hair cells were reported to be more susceptible to the effects of aminoglycosides compared to inner hair cells [28]. Disruption of mitochondrial protein synthesis and activation of glutamartergic receptors are other proposed mechanisms that were reported to be associated with aminoglycoside ototoxicity [14]. However, oxygen free radical damage of the

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ACCEPTED MANUSCRIPT hair cells is the most widely investigated subject especially from the perspective of prevention of aminoglycoside ototoxicity with antioxidants. In this study, we tried to examine the role of nigella sativa oil on gentamicin induced ototoxicity. Nigella sativa consists of various components including thymoquinone,

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unsaturated fatty acids, nigellicine, flavonoids, limonene, carvone and soluble fiber [19]. Anti-oxidative, anti-inflammatory, anti-hypertensive and anti-cancer effects of nigella sativa were described previously [20,29]. No serious side effects were reported in both human and

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animal studies [19]. Its beneficial effects on various clinical and biochemical parameters of metabolic syndrome and cardiovascular risk factors in obese women were also reported [19].

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In this experimmental study, the effects of intraperitoneal gentamicin were evaluated audiologically and histologically and whether the use of nigella sativa oil had advantages against gentamicin cochleotoxicity was investigated. We demonstrated that intratympanic nigella sativa oil had some deleterious effects on auditory function based on the findings of

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elevated hearing thresholds. In fact, effects of nigella sativa on cochlear function was not reported previously. Systemic administration of gentamicin resulted in significant hearing deterioration demonstrated with both click and 8 kHz tone-burst stimuli. Addition of

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intratympanic nigella sativa oil to systemic gentamicin yielded gains in hearing thresholds compared to systemic gentamicin alone. However, hearing gain with click stimulus was

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significant, whereas it was not significant with 8 kHz tone-burst stimulus. Since gentamicin ototoxicity was previously reported to be more pronounced at the basal turn and hence at higher frequencies, the dose of the nigella sativa oil might be less than necessary to prevent the cochleotoxic effects of gentamicin completely [2,8]. These findings may point to the beneficial effects of nigella sativa against the cochlear ototoxicity of the gentamicin. When we examine the results of histological investigation, axon number and diameter of the control and IT-NSO groups were almost identical with no significant difference. No

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ACCEPTED MANUSCRIPT sign of edema of the cochlear nerve and stria vascularis, epithelial damage of the stria vascularis and degeneration of the organ of Corti were noted in the IT-NSO group. These findings are consistent with the hypothesis that nigella sativa does not lead to auditory dysfunction.

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Since the histological changes following intratympanic nigella sativa oil were almost identical to control group, the above mentioned limited increase in hearing thresholds associated with intratympanic nigella sativa oil can be attributed to accumulation within the

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tympanic bulla and interference with sound transmission.

Histological evidence of edema of the cochlear nerve, epithelial damage and edema of

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the stria vascularis and degeneration of the organ of Corti were significantly lesser in IP-G + IT-NSO group than the IP-G group. Better histological findings in IP-G + IT-NSO group may point to the protective effects of nigella sativa against the cochleotoxic effects of gentamicin. Systemically administered aminoglycosides are postulated to enter the endolymph via

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trans-strial trafficking from strial capillaries or by traversing the blood labyrinth barrier into perilymph and then into endolymph [30,31]. Aminoglycosides in the endolymph contact with the cochlear hair cells and react with iron to generate oxygen free radicals leading to damage

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to hair cells and auditory neurons [2,32]. Intrinsic antioxidant mechanisms located within the inner ear usually become insufficient to counteract the effects of aminoglycosides and free

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radical damage results in apoptotic injury to the hair cells [8]. Ototoxicity of aminoglycosides can be ameliorated by using antioxidants. In fact, this is the focus of many previous experimental desings in which a variety of agents including trimetazidine, garlic derivatives, Korean red ginseng, melatonin, coenzyme-Q and galangin were used to protect against the gentamicin as well as for cisplatin ototoxicity [1,2,8,30,33,34]. Use of iron chelators is another option to decrease the ototoxicity associated with aminoglycosides [35].

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ACCEPTED MANUSCRIPT Nigella sativa belongs to the Ranunculaceae family. Thymoquinone, considered as the main component, has strong anti-oxidative effects as well as anti-inflammatory, anti-cancer, anti-microbial, anti-diabetic and neuroprotective effects [29,36,37]. Tayman et al. demonstrated reduction of the oxidative stress and induction of endogenous anti-oxidant

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enzymes with the use of nigella sativa oil in a hyperoxic lung injury model [25]. Effectiveness of nigella sativa against the nephrotoxic effects of gentamicin was investigated previously and this effect was attributed to its antioxidant properties [18,29,38,39].

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Penetration of NSO into the inner ear was not investigated in this study. However, in review of the literature, no data was available regarding the permeability of the NSO into the

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cochlear fluids.

In this study, use of intratympanic nigella sativa oil in addition to systemic gentamicin was demonstrated to have beneficial effects on hearing thresholds and histological changes compared to systemic gentamicin alone. Gentamicin induced hearing loss may be partially

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prevented with the use of intratympanic nigella sativa oil.

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Acknowledgements

This work was supported by Istanbul Training and Research Hospital, Committee of Education and Planning (821, 2013).

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ACCEPTED MANUSCRIPT [36] N.S. Korany, B.A. Ezzat. Prophylactic effect of green tea and Nigella sativa extracts against fenitrothion-induced toxicity in rat parotid gland. Arch Oral Biol. 56 (2011) 13391346. [37] S. Hosseinian, A. Khajavi Rad, M.A. Hadjzadeh, N. Mohamadian Roshan, S. Havakhah,

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S. Shafiee. The protective effect of Nigella sativa against cisplatin-induced nephrotoxicity in rats. Avicenna J Phytomed. 6 (2016) 44-54.

[38] U. Saleem, B. Ahmad, K. Rehman, S. Mahmood, M. Alam, A. Erum. Nephro-protective

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effect of vitamin C and Nigella sativa oil on gentamicin associated nephrotoxicity in rabbits. Pak J Pharm Sci. 25 (2012) 727-730.

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[39] M.M. Sayed-Ahmed, M.N. Nagi. Thymoquinone supplementation prevents the development of gentamicin-induced acute renal toxicity in rats. Clin Exp Pharmacol Physiol.

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34 (2007) 399-405.

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FIGURE LEGENDS:

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Figure 1. Hearing tresholds of the treatment groups with both click and tone-burst stimuli. ABR: Auditory brainstem response; SPL: Sound pressure level; IT-NSO: Intratympanic Nigella Sativa Oil group; IP-G: Intraperitoneal gentamicin group; IP-G + IP-NSO:

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Intraperitoneal gentamicin and inratympanic nigella sativa oil group; preop: preoperative; postop: postoperative.

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Figure 2. Organ of Corti of the treatment groups. A. Control group, B. Intratympanic Nigella Sativa Oil group, C. Intraperitoneal gentamicin group, D. Intraperitoneal gentamicin and inratympanic nigella sativa oil group. Organ of Corti (thick arrow), basilar membrane (thin arrow), tectorial membrane (arrowhead), Toluidine blue stain, 40x.

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Figure 3. Cochlear nerve myelinated axon sections of the treatment groups. A. Control group, B. Intratympanic Nigella Sativa Oil group, C. Intraperitoneal gentamicin group, D. Intraperitoneal gentamicin and inratympanic nigella sativa oil group. Myelinated axon

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sections (arrow), Toluidine blue stain, 100x. Asterix denotes edema and axonal loss. Figure 4. Stria vascularis of the treatment groups. A. Control group, B. Intratympanic Nigella

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Sativa Oil group, C. Intraperitoneal gentamicin group, D. Intraperitoneal gentamicin and inratympanic nigella sativa oil group. Stria vascularis (arrow), Toluidine blue stain, 100x. Prominent edematous vacuoles and epithelial basement membrane seperation in figure 4C.

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ACCEPTED MANUSCRIPT TABLES Table 1. Preoperative and postoperative mean hearing thresholds of treatment groups HEARING THRESHOLD (dB SPL) Postop-click

p

Preop-8 TB Postop-8 TB

p

CONTROL

11.4 ± 7.7

11.4 ± 5.3

1.0

7.9 ± 4.3

8.6 ± 3.6

0.655

IT-NSO

12.9 ± 4.7

23.6 ± 6.3

0.003

2.9 ± 4.7

19.3 ± 6.2

0.001

IP-G

10.0 ± 5.5

61.4 ± 11.0

0.001

5.0 ± 6.5

58.6 ± 11.0

0.001

IP-G + IT-NSO

10.0 ± 6.8

47.9 ± 8.9

0.001

8.6 ± 6.6

50.7 ± 12.7

0.001

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Preop-click

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IT-NSO: Intratympanic nigella sativa oil, IP-G: Intraperitoneal gentamicin, IP-G + IT-NSO: Intraperitoneal gentamicin and intratympanic nigella sativa oil, preop: preoperative, postop:

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postoperative, TB: tone-burst.

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EDE-COC

EP-DAM

EDE-ST

DEG-COR

CONTROL

13.7 ± 1.5

3.1 ± 0.2

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

IT NSO

13.6 ± 1.7

3.1 ±0.1

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

IP G

8.7 ± 0.8

2.5 ± 0.2

1.4 ± 0.5

0.6 ± 0.5

0.7 ± 0.5

1.4 ± 0.5

IP G + IT NSO

10.1 ± 1.1

2.9 ± 0.1

0.4 ± 0.5

0.3 ± 0.5

0.3 ± 0.5

1.1 ± 0.4

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AX-NUM

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AX-DIA: Axon diameter; EDE-COC: Edema in cochlear nerve; EP-DAM: Epithelial damage in stria vascularis; EDE-ST: Edema in stria vascularis; DEG-COR: Degeneration in the organ

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