Evaluation of grimacing pain behavior
The grimace scale quantifies changes in a few “action units” including orbital tightening, nose-cheek bulge, whisker tightening, and ear position, and orbital tightening, nose bulge, cheek bulge, ear position, and whisker change for mice. Then, animals were placed in a Plexiglas chamber (5x5) of von Frey test for 60 minutes. This time, we capture one image every 2 minutes. Thus, face images were screened, labeled, randomly scrambled, and scored, with the experimenter blinded to the treatment groups and identity of each image. 30 images were blinded selected for each animal—per treatment condition or time-point—and on each image, each action unit was given a score of 0, 1, or 2, as previously described [36]. Mean grimace scores were calculated as the average score across all the action units. This behavior scale was evaluated on days 1-, 7-, and 14-days post-stress induction or in NST animals. Grimace was also measured before ST or NST induction (Basal), after ST or NST induction (7 days after), and 1 and 3 hours after BIBN4096BS/vehicle treatment.