2.5. NOR test

The NOR tests were performed according to a previous study (Rokot et al., 2021) and are shown in the schedule diagram in Figure 1B. Each mouse was placed in an empty 60 cm × 60 cm × 70 cm box with black walls and an open top for video recording. The mice were allowed to acclimate to the environment for 10 min (habituation phase) and returned to their home cages. Two objects of the same color, shape, and size were placed on opposite sides of each box. The mice were placed in the boxes, where they could freely explore for 10 min (Phase I), and returned to their home cages. The objects were removed from the boxes. The mice were placed in the cleaned, empty boxes for 10 min (resting phase) and returned to their home cages. The objects used in Phase I were replaced in the boxes. However, one item was placed in the same position as before (familiar), whereas the other was placed in a different position. The mice were placed in the boxes, allowed to explore freely for 10 min (Phase II), and returned to their home cages. The objects were removed from the boxes. The mice were then placed again in the cleaned, empty boxes for 10 min (resting phase). A familiar object and novel object of a different color, shape, and size were placed in the same positions as in Phase I. The mice were placed in the boxes and allowed to explore for 10 min (Phase III). All objects and the box were cleaned with 70% ethanol to remove any residual odors after each phase. Object exploration was defined as touching it with the nose but climbing onto it or chewing it was not considered exploration. In contrast, a mouse that sniffed the object and climbed was considered to have explored it. The NOR index was calculated as follows: (time of exploration of the new object–time of exploration of the familiar object)/(time of exploration of the new object + time of exploration of the familiar object). A schematic diagram of the NOR test procedure is shown in Figure 2A.

Schematic illustration of novel object recognition (NOR) test and NOR indices. All mice in Sham/solid (n = 9), Sham/powder (n = 10), and Malocclusion/powder group (n = 9) were subjected to NOR test on Day 28 (A). NOR indices in Phases I (B), II (C), and III (D) were calculated. Data are represented as means ± SEM. Differences were considered significant at *p < 0.05 and **p < 0.01.