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.