*5.4. Evaluation of Pain Sensitivity: Determination of Mechanical Hyperalgesia by Electronic von Frey Test*

The animals were habituated in the experimental room 30 min before the measurements. The mice were individually placed in bottomless acrylic boxes. On the bottom of

the boxes, a wire mesh allows access to the plantar surface of the hind paw of the animal. A polypropylene disposable tip of 0.5 mm2 with increasing perpendicular force was applied on the plantar surface of the hind limb until the animal shows the paw withdrawal reaction, thus indicating its nociceptive threshold [99]. The tip is coupled to a transducer, which indicates the value in grams of the force applied to the animal's paw; the accuracy of the device is 0.1 g and the device records the maximum force applied (Insight Equipamentos Ltda., Ribeirao Preto, SP, Brazil). Pain sensitivity was evaluated before (baseline) and at different periods after immunization with MOG35–55, before they presented motor alterations.
