**3. Results**

#### *3.1. E*ff*ect of Folic Acid and Melatonin Treatment on Behavioral Defects Induced by Reserpine Injection*

Mechanical hyperalgesia was evaluated by a von Frey test. Reserpine injection produced a significant decrease in paw-withdrawal threshold in response to von-Frey hair stimulation in vehicle treated rats compared to sham groups (Figure S1). Mel + Fol treatment significantly increased the paw-withdrawal threshold in reserpine-treated rats, compared to melatonin and folic acid (Figure 1A). In addition, the e ffect of Mel + Fol treatment on pain sensitivity was tested by subjecting rats to hot plate and tail-flick tests. Reserpine injection produced an increased pain sensitivity in vehicle group compared to control groups (Figure S1). Mel + Fol treatment displayed an antinociceptive effect in hot plate (Figure 1B) and tail-flick tests (Figure 1C) in reserpine-treated rats, compared to melatonin and folic acid. The depressive-like behavior was evaluated by the forced swimming test. Reserpine injection increased the immobility time in reserpine-vehicle treated animals, compared to the sham groups (Figure S1). Mel + Fol treatment significantly decreased the immobility time in reserpine-treated rats, compared to melatonin and folic acid (Figure 1D).

**Figure 1.** <sup>E</sup>fficacy of folic acid and melatonin administration on behavioral changes reserpine-induced. Behavioral tests: (**A**) Von Frey test, (**B**) hot plate test, (**C**) tail-flick test, (**D**) forced swimming test (D). A *p*-value < 0.05 was considered significant. \* *p* < 0.05 vs. sham, ◦ *p* < 0.05 vs. vehicle, ◦◦ *p* < 0.01 vs. vehicle, \*\*\* *p* < 0.001 vs. sham, ◦◦◦ *p* < 0.001 vs. vehicle.

#### *3.2. E*ff*ect of Folic Acid and Melatonin Treatment on Lipid Peroxidation and Anti-Oxidant Profile Induced by Reserpine Injection*

It has been shown that oxidative stress is implicated in the pathogenesis of fibromyalgia [16]. Lipid peroxide levels were increased in reserpine-vehicle treated rats compared to sham groups (Figure S2). Treatment with Mel + Fol caused a significant reduction in lipid peroxide in reserpine-treated rats, compared to melatonin and folic acid (Figure 2A). The enzymatic activity of superoxide dismutase (Figure 2B), non-protein thiols (Figure 2C), and catalase (Figure 2D) significantly decreased in the reserpine-vehicle treated rats compared to the sham groups (Figure S2). This reduction was significantly restored with in animals treated with Mel + Fol, compared to melatonin and folic acid.

**Figure 2.** <sup>E</sup>fficacy of folic acid and melatonin administration on oxidative stress reserpine-induced. (**A**) Estimation of lipid peroxidation, (**B**) estimation of non protein thiols, (**C**) estimation of superoxide dismutase, (**D**) estimation of catalase. A *p*-value < 0.05 was considered significant. ◦ *p* < 0.05 vs. vehicle, ◦◦ *p* < 0.01 vs. vehicle, \*\*\* *p* < 0.001 vs. sham, ◦◦◦ *p* < 0.001 vs. vehicle.

#### *3.3. E*ff*ect of Folic Acid and Melatonin Treatment on Nitrosative Stress and PARP Expression Induced by Reserpine Injection*

Twenty-one days after reserpine injection, we also investigated nitrotyrosine and PARP expression associated with oxidative stress by immunohistochemistry. Increased nitrotyrosine and PARP expression was found in brain tissue sections of reserpine-vehicle treated (Figure 3B,F,H,N) rats compared with the sham groups (Figure 3A,F,G,N and Figure S3). Treatment with Mel + Fol caused a significant reduction in nitrotyrosine (Figure 3E,F) and PARP expression (Figure 3M,N) in reserpine-treated rats, compared to melatonin (Figure 3D,F,L,N) and folic acid (Figure 3C,F,I,N).

**Figure 3.** <sup>E</sup>fficacy of folic acid and melatonin administration on nitrityrosine and PARP expression reserpine-induced. Immunohistochemistry evaluation of nitrityrosine expression in (**A**) sham, (**B**) vehicle, (**C**) folic acid, (**D**) melatonin, (**E**) melatonin plus folic acid and (**F**) graphical quantification. Immunohistochemistry evaluation of PARP expression in (**G**) sham, (**H**) vehicle, (**I**) folic acid, (**L**) melatonin, (**M**) melatonin plus folic acid, and (**N**) graphical quantification. A *p*-value < 0.05 was considered significant, ◦◦ *p* < 0.01 vs. vehicle, \*\*\* *p* < 0.001 vs. sham, ◦◦◦ *p* < 0.001 vs. vehicle.

#### *3.4. E*ff*ect of Folic Acid and Melatonin Treatment on Mast Cells Infiltration induced by Reserpine Injection*

Twenty-one days after reserpine injection, mast cells infiltration and degranulation were assessed by toluidine blue staining. There was a significant up-regulation in mast cell number, which performs a key role in the development of hyperalgesia and in the inflammatory process, both in brain (Figure 4B) and sciatic nerve (Figure 4G) in reserpine-vehicle treated rats, compared to the sham groups (Figure 4A,F and Figure S4). Mel + Fol treatment reduced the number of mast cells both in brain (Figure 4E) and sciatic nerve (Figure 4L) in reserpine-treated rats, more than melatonin (Figure 4D,I) and folic acid (Figure 4C,H).

**Figure 4.** <sup>E</sup>fficacy of folic acid and melatonin administration on mast cells activation reserpine-induced. Evaluation of mast cell degranulation by toluidine blue in brain: (**A**) sham, (**B**) vehicle, (**C**) folic acid, (**D**) melatonin, (**E**) melatonin plus folic acid. Evaluation of mast cell degranulation by toluidine blue in sciatic nerve: (**F**) sham, (**G**) vehicle, (**H**) folic acid, (**I**) melatonin, (**L**) melatonin plus folic acid. 40× magnification is shown.

#### *3.5. E*ff*ect of Folic acid and Melatonin Treatment on Changes in Pro-Inflammatory, Vasoactive and Neuro-Sensitizing Mediators Induced by Reserpine Injection*

Twenty-one days after reserpine injection, IL-1β and TNF-α levels were increased in reserpine-vehicle treated rats, compared to the sham groups (Figure S5). Treatment with Mel + Fol produced a significant reduction in IL-1β (Figure 5A) and TNF-α (Figure 5B) levels in reserpine-treated rats, compared to melatonin and folic acid. Western blot analysis showed NGF and VEGF increased expression in both brain and nerve tissues harvested from reserpine-vehicle treated animals, compared to the sham groups (Figure 5C,D and Figure S5). Mel + Fol administration significantly reduced NGF and VEGF expression in reserpine-treated rats with more efficacy than melatonin and folic acid (Figure 5C,D).

**Figure 5.** <sup>E</sup>fficacy of folic acid and melatonin administration on IL-1β, TNF-<sup>α</sup>, VEGF, and NGF expression reserpine-induced. Elisa kit of (**A**) IL-1β and (**B**) TNF-α levels. Western blots and respectively quantification of VEGF and NGF in (**C**) brain and (**D**) sciatic nerve. A *p*-value < 0.05 was considered significant. \* *p* < 0.05 vs. sham, ◦ *p* < 0.05 vs. vehicle, \*\* *p* < 0.01 vs. sham, ◦◦ *p* < 0.01 vs. vehicle, \*\*\* *p* < 0.001 vs. sham, ◦◦◦ *p* < 0.001 vs. vehicle.

#### *3.6. E*ff*ect of Folic Acid and Melatonin Treatment on Microglia Activation Induced by Reserpine Injection*

Twenty-one days after reserpine injection, we also investigated microglial activation by immunofluorescence. Increased Iba1 and CD11b positive cells were found in brain tissue sections of reserpine-vehicle treated (Figure 6B,G) rats compared with the sham groups (Figure 6A,F and Figure S6). Treatment with Mel + Fol caused a significant reduction in Iba1 (Figure 6E) and Cd11b positive cells (Figure 6L) in reserpine-treated rats, compared to melatonin (Figure 6D,I) and folic acid (Figure 6C,H).

**Figure 6.** <sup>E</sup>fficacy of folic acid and melatonin administration on Iba1 and CD11b expression reserpine-induced. Immunofluorescence of brain Iba1 in (**A**) sham, (**B**) vehicle, (**C**) folic acid, (**D**) melatonin, (**E**) melatonin plus folic acid. Immunofluorescence of brain CD11b in (**F**) sham, (**G**) vehicle, (**H**) folic acid, (**I**) melatonin, (**L**) melatonin plus folic acid. 40× magnification is shown.
