Preparation of the Rat Animal Model
Twenty-four healthy male, mature Wistar Kyoto rats weighing between 300g to 400g were used for this study. They consisted of 24 males aged three months old. They were housed in a typical Macrolon cage (280×520×145mm) with wood shavings as bedding, three to a cage. They were kept in a facility with 12 hours of on/off light cycles, replicating the regular day and night cycle at a constant temperature (20 ± 2 °C) and humidity (55.5%). The rats were classified into three groups: Group A (8 animals), Group B (8 animals), and Group C (8 animals).
The sample size of 24 rats was calculated via the ’resource equation’ method, a commonly used method in animal studies. The inclusion criteria were Wistar male rats with normal vestibular parameters and intact tympanic membrane. The exclusion criteria were Wistar rats with the diseased external ear canal, the abnormal external ear canal, abnormal tympanic membrane, abnormal vestibular profile before perforation of the tympanic membrane and female albino rats. The ARRIVE Guidelines 2.0 was the reporting guideline that has been followed in this study.
The four baseline vestibular parameters were tested in all rats in each group. The parameters tested were dyskinetic head movements and circling behaviour, tail-hanging test, air-righting reflex, and contact inhibition of the air-righting reflex. The animals were observed and rated for the presence or absence of the following signs, while the observers were blinded to the treatments given. The scorings were rated 0, 1, and 2 for every parameter tested. Zero indicates normal vestibular behaviour, while one indicates mild abnormalities, and two indicate severe abnormalities observed. The signs of vestibulotoxicity were assessed using published procedures as per the following [11]: