Fig. 2

Evaluation of chemical and mechanical corneal sensitivity and anxiety-like behavior of sham and DED animals on d21. a Chemical corneal sensitivity was evaluated using a drop of capsaicin (100 μM) and recording the palpebral closure time for 5 min. b The corneal mechanical threshold was measured using von Frey filaments. c Elevated plus maze: the mouse was placed in the center area of the maze with its head directed toward a closed arm and time (s) in the open arms was recorded for 5 min. d Black and white test: The mouse was placed in the middle of a brightly illuminated chamber and time in the white zone (%) was recorded for 5 min. e cFos immunoreactivity in the amygdala of sham and DED mice; cFos⁺ cells (black arrows). Scale bar = 100 μm. Regions corresponding to the images are depicted in coronal diagrams taken from the Paxinos atlas [28]. f Quantification of the number of cFos⁺ cells in the amygdala of sham and DED animals. Total number of mice = 48. Number of mice included in the analysis = 48. Capsaicin and von Frey test: n = 6 sham and DED animals for each test. Elevated plus maze and black and white tests: n = 15 sham and DED animals; cFos immunoreactivity in amygdala: n = 3 sham and DED animals. *P < 0.05 relative to the sham group. Results are expressed as the mean ± SEM. For statistical analysis, the Kolmogorov–Smirnov test was performed followed by a parametric t-test (capsaicin test, von Frey test, elevated plus maze test, and black and white test) or nonparametric Mann-Whitney test (quantification of the number of cFos⁺ cells in the amygdala) using GraphPad Prism version 7.00 (GraphPad Software, La Jolla, CA, USA)