Open Field Test for Anxiety Evaluation

The evaluation methodology was based on previous studies, with slight modifications (26 (link)–28 (link)). Rodents display characteristic of thigmotaxis to a new environment. Therefore, the central zone represents a threatening situation, while the peripheral area is relatively safe. Experiments were conducted in a location shielded from incidental noise. The space is a circular arena with a diameter of 97 and 42 cm high walls. Lighting with 850 nm far-red LEDs was used and any anxiety induced by the bright illumination was minimized. The room temperature was maintained at 25°C± 2°C during data acquisition. Each mouse was recorded for 10 min and then returned to the group housing. Mice were observed by video recording (FDR-AX700, Sony, Tokyo, Japan), and the time spent in the center, frequency of entering the center, and time spent standing were recorded for 10 min. An increase in the frequency and time spent staying in the center of the space indicated a decrease in anxiety. Video analysis was performed with TruScan software (Version 2.2; Coulbourn Instruments, Allentown, PA, USA). After each test, the space was cleaned with a 70–75% alcohol solution to avoid the effect of the odor of the previous animal on the next one.