Contact inhibition of righting reflex
The rats were positioned supine on a horizontal surface, and another horizontal surface was placed slightly in touch with the supine animal’s feet soles. The rating was done as follows: 0-the animal successfully righted itself (normal), 1-partial righting, in which the animal exerts some effort (intermediate response), and 2-complete loss of righting, in which the animal faces up its feet and walks on the upper surface (severe response).
Ketamine (75-90 mg/kg, Ketalar, Pfizer, Istanbul, Turkey) and xylazine (5-8 mg/kg, Rompun, Bayer, Leverkusen, Germany) were used to anesthetize the rats intraperitoneally. The pedal reflex was used to determine the depth of anaesthesia, and further anaesthesia was provided in half-dose increments as needed. Each rat’s external ear canal and tympanic membrane were examined following general anaesthesia using a zero-degree rigid Karl Storz surgical scope. Rats with abnormal external ear canals or a perforated tympanic membrane were excluded from this study.
After the baseline vestibular parameters testing, an endoscopic-guided transtympanic instillation of aqueous and ethanol henna extract was conducted into the right middle ear of the rats in Group A and B, respectively (Figure 3). At the same time, normal saline was instilled in the right middle ear of rats in Group C. A total of 0.1ml instillation of aqueous, ethanol extract, and saline for each rat was sufficient to fill the whole middle ear cavity of the rat.
The rats were given time to recover from the anaesthesia. The four vestibular parameters testing were assessed on the rat’s post-instillation of transtympanic Henna at intervals of 4 hours, 24 hours, 48 hours, 72 hours, one week, two weeks, and three weeks.
The rats would then be euthanized intraperitoneally with thiopental sodium (200 mg/kg Pentothal; Abboth, Campoverde di Aprilla, Italy).