2.5. Counting the instances of scratching behavior and calculating the scratching response

Scratching behavior was evaluated according to the method described by Kuraishi et al, with minor modifications [14]. Animals were housed in a cage in the observation room to acclimatize for 2 h before the challenge with 2.5% OA, after which their behavior was recorded.

Animals were recorded by video and monitored for scratching reactions. Scratching behavior was defined as an interrupted cluster of rapid hind limb movements that were precisely directed to the eye. Scratching behavior was counted within 1 h from OA exposure on days 15 and 19. Scratching responses were calculated based on the number of such behaviors on day 19 divided by the number of these behaviors recorded on day 15 for the same individual. This experiment was independently performed six times.