Indian Journal of Otology

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 26  |  Issue : 3  |  Page : 159--162

Assessment of vestibulotoxicity of neem leaf (Azadirachta indica) in a rat animal model


Rosdan Salim1, Mohd Khairi Md Daud1, Maithrea Suresh Narayanan2, Aziah Ab Rani1,  
1 Department of Otorhinolaryngology-Head and Neck Surgery, School of Medical Sciences, Universiti Sains Malaysia, Kuban Kerian, Kota Bharu, Kelantan, Malaysia
2 Department of Otorhinolaryngology-Head and Neck Surgery, Hospital Sultanah Nora Ismail, Batu Pahat, Johor, Malaysia

Correspondence Address:
Dr. Aziah Ab Rani
Department of Otorhinolaryngology-Head and Neck Surgery, School of Medical Sciences, Universiti Sains Malaysia Health Campus, 16150 Kota Bharu, Kelantan
Malaysia

Abstract

Introduction: Otomycosis is a challenging and frustrating entity for both patients and otolaryngologists for it frequently requires long-term treatment and follow-up, yet the recurrence rate remains high. The management is directed toward intense aural toileting with administration of topical antifungals. There has been increased resistance to classical antifungal agents, typically involving the azoles and polyenes. This has led into the research of medicinal plants as an alternative treatment for treating fungal infections. Objective: This study aims to investigate the possible vestibulotoxic effects of neem leaf (Azadirachta indica), a commonly used home remedy on the inner ear in a rat animal model. Methodology: Twenty healthy, mature Wistar albino rats were divided into three groups: Groups A (eight animals), B (eight animals), and C (four animals). They underwent baseline vestibular parameter testing, following which an endoscopic-guided transtympanic instillation of aqueous and alcohol neem extract was performed into the right middle ear of the rats in Groups A and B, while normal saline was instilled into the right middle ear of rats in Group C. Vestibular parameter testing was repeated on the rats in both groups post instillation of transtympanic neem at 4-h, 24-h, 48-h, 72-h, 1-week, 2-week, and 3-week intervals. Results: There was no deterioration in all vestibular parameters recorded post instillation of neem extract within the middle ear. Conclusion: Neem extract is a safe alternative in the treatment of otomycosis in the presence of tympanic membrane perforation as it poses no vestibulotoxic side effects.



How to cite this article:
Salim R, Md Daud MK, Narayanan MS, Rani AA. Assessment of vestibulotoxicity of neem leaf (Azadirachta indica) in a rat animal model.Indian J Otol 2020;26:159-162


How to cite this URL:
Salim R, Md Daud MK, Narayanan MS, Rani AA. Assessment of vestibulotoxicity of neem leaf (Azadirachta indica) in a rat animal model. Indian J Otol [serial online] 2020 [cited 2021 Mar 2 ];26:159-162
Available from: https://www.indianjotol.org/text.asp?2020/26/3/159/304288


Full Text



 Introduction



Otomycosis is a fungal infection of the external auditory canal which is frequently encountered in a general otolaryngology clinic setting. It is worldwide in distribution with a prevalence ranging from 9% to 30% among patients presenting with signs and symptoms of otitis externa and discharging ears.[1] The incidence of otomycosis varies in accordance with the different climactic conditions with higher prevalence in the hot and humid areas of the tropics and subtropics. Aspergillus niger and Candida albicans are the most common pathogenic fungi isolated in otomycosis. The gold standard of otomycosis treatment is aural toilet, with topical antifungals as an adjunct. However, to date, there has been no standardized therapeutic regimen for otomycosis, which opens up new treatment options including the use of herbal medicine.[2]

Neem leaf (Azadirachta indica) has been long valued for its therapeutic properties. The beneficial effects of different parts of neem are attributed to its biologically active principle “Azadirachtin.”[3] Studies have demonstrated that Malaysian neem leaf extracts inhibit the growth of pathogenic fungi in vitro.[4] Neem leaves have long since been in use in the primary health-care setting, primarily in the Indian subcontinent but also in African countries.[5] This demonstrates the successful utilization of the action of its phytoconstituent alkaloids, glycosides, flavonoids, and saponins, which act as the defensive mechanism of plants against different pathogens.[6] Every part of the neem tree has been used as traditional medicine to remedy various human ailments, and it is regarded as the “village dispensary.”[7]

Although its ability to inhibit the growth of pathogenic fungi causing otomycosis has been well demonstrated,[8],[9],[10] the ototoxicity profile of this medicinal plant has yet to be explored. This is a pilot study to determine the ototoxic effect of transtympanic application of neem extract, specifically vestibulotoxicity, in a rat animal model.

 Methodology



Preparation of neem leaf extract

One kilogram of Malaysian neem leaves was collected from a single 10–12-year-old tree in Hospital USM (USM Herbarium voucher no. 11337). The leaves collected were healthy without any fungal or bacterial infection.

The leaves were washed with distilled water and dried in an oven at 45°C for 2 days. Then, the dried leaves were grinded into smaller coarse powder form and stored in a tightly sealed glass container.

Extraction by using a Soxhlet apparatus was chosen for the preparation of the sample. Two types of solvent, the aqueous and ethanol 70%, were used. Three hundred grams of ethanol neem extract and 300 g of aqueous neem extract were obtained after intensive freeze drying. These powder form extracts were used to establish the exact concentration required in this research, which is 5% aqueous extract and 5% ethanol extract of neem.

Preparation of the rat animal model

Twenty healthy, mature Wistar albino rats weighing between 300 g and 400 g were used for this study. They consisted of ten male and ten female rats aged 3 months. They were housed two to four/cage in standard Makrolon cages (280 mm × 520 mm × 145 mm) with wood shavings as bedding. They were kept in a room with 12 h on/off light cycles, simulating the standard day and night rhythm, at constant temperature (20°C ± 2°C) and humidity (55.5%). The rats were divided into three groups: Groups A (eight animals), B (eight animals), and C (four animals). This study was approved by the Animal Ethics Committee of University Sains Malaysia (Number of Animal Ethics Approval: USM/IACUC/2018/(111) (908).

The animals in each group underwent four baseline vestibular parameters' testing. The vestibular function parameters tested were dyskinetic head movements and circling behavior, tail-hanging test, air-righting reflex, and contact inhibition of the air-righting reflex.

The animals were observed for the presence or absence of the following signs while the observer was blinded to the treatments given. The signs of vestibulotoxicity were assessed using previously published procedures as follows.[11]

Dyskinetic head movements and circling

The rats were placed individually in an observation chamber and were observed for dyskinetic head movements (head weaving) and circling for a period of 2 min.

Tail hanging

The rats were lifted by the tail, and the response was carefully observed and rated as follows: 0 – straight body posture with extension of forelimbs toward the earth (normal), 1 – slightly bending the body ventrally (intermediate response), and 2 – persistently bending the body, sometimes crawling up toward its tail (severe response).

Air-righting reflex

The rats were held supine and dropped from a height of 30–40 cm onto a foam cushion. The response was graded as follows: 0 – successful in righting and landing squarely on their feet (normal), 1 – poor righting or landing on the side (intermediate response), and 2 – completely failed in righting and landing on the back (severe response).

Contact inhibition of righting reflex

The rats were placed supine on a horizontal surface, and another horizontal surface was slightly placed in contact with the soles of the supine animal's feet. The rating was performed as follows: 0 – animal rights successfully (normal); 1 – partial righting, animal does some efforts (intermediate response); and 2 – complete loss of righting, animal is facing up the feet and walking with respect to the upper surface (severe response).

The rats were anesthetized with ketamine (75–90 mg/kg, Ketalar, Pfizer, Istanbul, Turkey) and xylazine (5–8 mg/kg, Rompun, Bayer, Leverkusen, Germany) through intraperitoneal injection. The depth of anesthesia was determined by pedal reflex, and additional anesthesia was administered in half dose increments as required.

Following the administration of general anesthesia, the external ear canal and tympanic membrane of each rat were examined using a 0° rigid Karl Storz surgical scope.

After the first vestibular parameter testing, an endoscopic-guided transtympanic instillation of aqueous and alcohol neem extract was performed into the middle ear of the rats in Groups A and B, while normal saline was instilled in the right tympanic membrane of rats in Group C.

The rats were given adequate time to recover from the anesthesia. The four vestibular parameters' testing was repeated on the rats in both groups post instillation of transtympanic neem at 4-h, 24-h, 48-h, 72-h, 1-week, 2-week, and 3-week intervals.

The rats were later euthanized with thiopental sodium (200 mg/kg Pentothal; Abboth, Campoverde di Aprilla, Italy) through intraperitoneal injection.

 Results



In Group A (n = 8) where the rats were subjected to transtympanic instillation of 5% aqueous neem extract, there was no vestibular dysfunction observed at 4 h, 24 h, 24 h, 48 h, 72 h, 1 week, 2 weeks, and 3 weeks post-instillation. Similar results were obtained in Group B (n = 8) where the rats were subjected to transtympanic instillation of 5% ethanol neem extract and in Group C (n = 4) where the control group received transtympanic instillation of normal saline.

In view of the score of 0 obtained for both pretesting and posttesting parameters, statistical analysis was unable to be applied to this study. There was no deterioration in all vestibular parameters recorded post instillation of both aqueous and ethanol neem extracts within the middle ear, which shows that neem does not pose any vestibulotoxic effects following its application onto the middle ear mucosa.

In this study, we used twenty Wistar albino rats consisting of four male rats and four female rats each in Groups A and B. For Group C which served as a control, two male rats and two female rats were used. The rats were 3 months old at the time this study was carried out and weighed between 300 and 400 g each.

 Discussion



Otomycosis or fungal otitis externa has typically been described as fungal infection of the external auditory canal with infrequent complications involving the middle ear. Although rarely life-threatening, the disease process presents a challenging and frustrating entity for both patients and otolaryngologists for it frequently requires long-term treatment and follow-up, yet the recurrence rate remains high.[12] Patients with fungal otitis externa should be treated with intense debridement and cleansing, with administration of topical antifungals.[13] The main advantages of these topical drugs include a high concentration at the affected site and decreased systemic side effects.

Clinical resistance is defined as the failure to eradicate a fungal infection despite the administration of an antifungal agent with in vitro activity against the organism. There has been increased resistance to classical antifungal agents, typically involving the azoles and polyenes.[14] This has led to research of medicinal plants as an alternative treatment for treating fungal infections.[7],[8],[10],[15],[16]

The present study explores the potential vestibulotoxic effect of neem extract. Despite already being used as a home remedy for treating ear itchiness and discomfort, the safety profile of using neem leaves and its potential vestibulotoxic effect has yet to be studied.[1] Ototoxicity is defined as the tendency of certain therapeutic agents and other chemical substances to cause functional impairment and cellular degeneration of the tissues of the inner ear, which can be divided into cochleotoxicity and vestibulotoxicity.[17]

We chose to assess the vestibulotoxic side effect of neem upon application into the middle ear mucosa. This is because the disturbance of balance as a drug side effect is often underestimated by clinicians, whereas patients often regard it as part of their disease and impairment of their general status. In patients, most studies on drug-induced ototoxicity are limited to the cochleotoxic effect, whereas reports on the vestibulotoxic effect are relatively rare. Hence, it is important to ensure an early diagnosis to prevent vestibulototoxic effects induced by drugs,[18] especially those used as home remedies or as first-line treatment in primary health-care units.

In our study, we used Wistar albino rats which are a commonly used species in otologic studies. The middle and inner ear structures are almost similar to humans, where the rat cochlear has two and a half turns which makes them a suitable model for vestibulotoxicity and cochleotoxicity studies.[19] All the rats were at 3 months of age. This is because age correspondence between mice and human is now widely recognized, especially regarding neural development. Mice of 3 months old are considered as equivalent at human of age 20–26 years.[20]

The inner ear can be exposed to chemical agents through diffusion from the middle ear through transtympanic injection. This is a route that has been increasingly used for therapeutic drug delivery in humans suffering from auditory and vestibular diseases. In laboratory animals, transtympanic exposure is a well-established model for ototoxicity studies.[21] To date, there has been no studies assessing the vestibulotoxicity of an transtympanically administered natural compound.

Two Malaysian neem extracts known for its fungicidal effect were used, which are aqueous extract and alcohol extract of neem. The antifungal effect of Malaysian neem leaf extract against two common otomycotic pathogenic fungi which are C. albicans and A. niger has been studied in vitro. The minimum inhibitory concentration of Malaysian neem aqueous extract against C. albicans was 11.91 g/ml, neem ethanol extract against C. albicans was 5.16 g/ml, neem aqueous extract against A. niger was 7.73 g/ml, and neem ethanol extract against A. niger was 9.25 g/ml. Statistical analysis showed that the antifungal activity of C. albicans is better in ethanol neem than aqueous extract, but aqueous neem extract is better than ethanol extract for A. niger,[4] which is why both extracts were tested in this study.

In order to facilitate administration of the drug into the ear canal and ensuring absorption of the drug into the middle ear mucosa and inner ear, a transtympanic injection of neem at a concentration of 5% of both aqueous and ethanol solvents was used.[8] This concentration has been proven to produce adequate antifungal effect, while at the same time is in a form of diluted thin solution suitable to be administered into the ear in drop form by clinicians and patients alike.

The four vestibular parameters tested were dyskinetic head movements and circling behavior, tail-hanging test, air-righting reflex, and contact inhibition of air-righting reflex.[21] For each parameter tested, the rats were rated from 0 to 2 where 0 showed no abnormalities seen, 1 showed mild abnormalities, and 2 showed marked abnormalities observed. Circling behavior can be defined as a stereotypical circling behavior, while in dyskinetic head movements, we observed for intermittent extreme backward extension of the neck as well as unilateral head tilting toward the side of the vestibular damage.

In the tail-hanging test, rats with impaired vestibular function bend ventrally, sometimes “crawling” up toward their tails, thus tending to occipital landing. Unilateral lesions cause body rotation around the tail axis rather than ventral bending. Normal rats would extend their forelimbs and exhibit a “landing response” when lifted by the tail.

In our study, a baseline vestibular parameter observation was carried out to evaluate rats with normal vestibular function as an inclusion criterion for the study. Post transtympanic instillation, they were repeated at 4-h, 24-h, 48-h, 72-h, 1-week, 2-week, and 3-week intervals. This is because the presentation of acute vestibular changes in rats can be observed in as early as 4 h and up to 3 weeks posttranstympanic instillation, as rats can restore normal postural-locomotor function following transient unilateral vestibular insult.[20]

All rats were given transtympanic injection of the neem extract into the right ear for practical observational purposes, as a vestibular dysfunction would cause the affected rats to tilt their heads uniformly to the right.

 Conclusion



The aqueous and ethanol extracts of neem have been evaluated in vivo in this study. Both extracts were found to be safe to be administered into the middle ear of rats without causing any vestibular dysfunction up to 3 weeks post administration. Whether neem possesses any cochleotoxic effects remains an open question for future investigations.

Financial support and sponsorship

This study was financially supported by the Malaysian Society of Otorhinolaryngologists – Head and Neck Surgeons (MSO HNS) Research Grant 2018.

Conflicts of interest

There are no conflicts of interest.

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