**4. Discussion**

Fibromyalgia is a multisymptomatic and multifactorial disease [20]. The pathophysiological mechanisms by which the disease is characterize include, among others, changes in sensory perception of pain [52], oxidative stress and inflammation [18] with damage to myelinated and nonmyelinated nerve fibers [16]. In our study, animals subjected to fibromyalgia showed increased pain sensitivity in mechanical allodynia and thermal hyperalgesia. Moreover, this enhanced sensibility was coupled with depression symptoms, as indicated by the rat behaviour in the forced swim test. Several evidence, in fact, indicates that the depression-like symptoms in rats increased allodynia and hyperalgesia under the condition of fibromyalgia [3]. A concomitant treatment of melatonin and folic acid was able to reduce the increased pain sensibility and the depression-like behaviour with more efficacy than them single administration. Increasing evidence suggests that enhanced oxidative stress and nitric oxide are involved in the fibromyalgia pathophysiology and increase the severity of the symptoms [53,54]. We are in line with literature [55,56]; our data also underlines that the oxidative and nitrosative stress induces neurogenic inflammation which is responsible for the perpetuation of pain [16]. The oxidants and antioxidants equilibrium is unbalanced in this pathology [32]: increased lipid peroxidation was detected in rats subjected to fibromyalgia, while superoxide dismutase, nonprotein thiols and catalase were significantly decreased [49]. Thanks to its antioxidant properties, a combined treatment of melatonin and folic acid was also able to reduce these parameters better than the single administration. While free oxygen radicals oxidize membrane phospholipids amplifying lipid peroxidation, nitric oxide excessively produced by iNOS reacts with superoxide anions yielding the toxic oxidizing agen<sup>t</sup> peroxynitrite. It nitrates tyrosine residues, causing changes in protein function and structure that induce tissue damage. Peroxynitrite in turn activates PARP, a single-strand break DNA repair enzyme that acts by synthesizing chains of ADP-ribose [50]. To product ADP-ribose monomers, the obligate substrate is NAD+. PARP hyper-activation depletes NAD+ cellular reserves of leading to ATP depletion, cellular dysfunction, and death. Melatonin plus folic acid administration decreased nitrotyrosine and PARP staining induced by fibromyalgia better than the single administration of the two substances. This increased oxidative stress is able to induce mast cells activation [57]. Systemic mastocytosis [58] is commonly experience in patients a ffected by fibromyalgia [59,60]. Several pieces of evidence show the importance of mast cells activation in this disease and [61,62] comorbid disorders [63] such as neuroimmune interactions [64] and painful conditions, [65,66]. Mast cells reside near the nerve fibers, which give them the possibility to migrate for modulating nociception and neural activity [59,67–69]. As result of their migration and degranulation, there is an important release of pro-inflammatory, vasoactive and neuro-sensitizing mediators [70]. In particular, an increased expression of cytokines (IL-1β and TNFα) [71,72] and growth factors (NGF and VEGF) [73–75] that contribute to the maintenance of inflammation and pain have been detected in both nerve and brain [76–79]. An associate administration of melatonin and folic acid was able to decrease mast cells infiltration and the related increased expression of pro-inflammatory cytokines and vasoactive and neuro-sensitizing mediators with more e fficacy than them single administration. Mast cells also communicate with microglia [60,80,81]. In the contests of pain, microglia in the thalamus is responsible for maintaining the pain sensation even after the original stimulus is over [82,83]. The concomitant treatment of melatonin and folic acid was able to reduce the increased microglia activation, assessed by Iba1 and CD11b expression, with more e fficacy than their single administration.
