Periorbital mechanical allodynia (PMA)
The measurement of PMA was performed by using the up-and-down paradigm as described previously [18]. Animals were allocated in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds. The apparatus consists of an individual clear three-walled plexiglass box (5x5) with an opening for the tail and one for the head and front paws, located on a platform to allow the operator to access the periorbital area. The box size allowed for head and forepaw movements but prevented the animal from turning around inside it. One day before the first behavioral observations, mice were habituated to the apparatus. On the day of the experiment, after 20 minutes of adaptation inside the chamber, a series of 7 Von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 1.0, and 1.4 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 seconds to elicit a positive response. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. The stimulation was initiated with the 0.16 g filament. The absence of response after 5 s led to the use of a filament with increased weight, whereas a positive response led to the use of a weaker (i.e. lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores.
The mechanical threshold was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum) initially on basal (before stress) and on 1, 3, 7, 10 and 14-days post-stress induction. PMA was also measured before ST or NST induction (Basal), after ST or NST induction (7 days after), and 1 and 4 hours after BIBN4096BS/vehicle treatment.