*2.3. Experimental Groups*

Then, rats were randomly divided into several groups (*n* = 10 for each):

Group 1. Sham + vehicle: Rats were injected subcutaneously with vehicle (distilled water with a final concentration of 0.5% acetic acid) instead of reserpine and treated orally with saline for 21 days starting from 3 days after first vehicle injection.

Group 2. Sham + melatonin: Rats were injected subcutaneously with vehicle (distilled water with a final concentration of 0.5% acetic acid) instead of reserpine and treated orally with melatonin (10 mg/kg) for 21 days starting from 3 days after first vehicle injection.

Group 3. Sham + folic acid: Rats were injected subcutaneously with vehicle (distilled water with a final concentration of 0.5% acetic acid) instead of reserpine and treated orally with folic acid (3 mg/kg) for 21 days starting from 3 days after first vehicle injection.

Group 4. Sham + melatonin + folic acid (Mel + Fol): Rats were injected subcutaneously with vehicle (distilled water with a final concentration of 0.5% acetic acid) instead of reserpine and treated orally with melatonin (10 mg/kg) and folic acid (3 mg/kg) for 21 days starting from 3 days after first vehicle injection.

Group 5. Reserpine + vehicle: Rats were subjected to injection of reserpine as previously described and treated orally with vehicle (saline) for 21 days starting from 3 days after first reserpine injection.

Group 6. Reserpine + melatonin: Rats were subjected to injection of reserpine as previously described and treated orally with melatonin (10 mg/kg) for 21 days starting from 3 days after first reserpine injection.

Group 7. Reserpine + folic acid: Rats were subjected to injection of reserpine as previously described and treated orally with folic acid (3 mg/kg) for 21 days starting from 3 days after first reserpine injection.

Group 8. Reserpine + melatonin + folic acid (Mel + Fol): Rats were subjected to injection of reserpine as previously described and treated orally with folic acid (3 mg/kg) and melatonin (10 mg/kg) for 21 days starting from 3 days after first reserpine injection.

The dose and route of administration of folic acid and melatonin were chosen based on previous studies [39,40]. Twenty-one days after reserpine injection blood was collected, animals were sacrificed and brain and sciatic nerves were harvested for histological, immunohistochemical and western blot analysis.

## *2.4. Von Frey Hair Test*

Mechanical allodynia was evaluated using a dynamic plantar Von Frey hair aesthesiometer on day 0 and 3, 5, 7, 14, and 21 days post-injection (Bio-EVF4; Bioseb, Vitrolles, France) as previously described [41]. The device encloses a force transducer furnished with a plastic tip. When pressure is applied to the tip, the force applied is recorded. The tip was applied to the plantar area of the hind leg, and a rising upward force was exerted until the paw was withdrawn. The withdrawal threshold was defined as the force, expressed in grams, at which the mouse removed the paw.
