**3. Discussion**

MS is a chronic inflammatory disease of autoimmune origin which, demyelinates neurons from the CNS. Components of the CNS are recognized as antigens, more specifically the myelin sheath of the axons. T lymphocyte-mediated CNS aggression is observed

in MS [49], as well as in the activation of glial cells (microglia and astrocytes) in both the spinal cord and brain regions [50,51], innate immune cell activation, and cytokines and chemokines release [52]. This inflammatory response greatly alters the properties of neurons, leading to demyelination and axonal loss [53], as well as to several motor, cognitive and sensory alterations [54,55].

Presently, the effect of CRO on disease development was evaluated. CRO is an analgesic peptide, firstly identified in the venom of *Crotalus durissus terrificus* snake [35], which induces long-lasting antinociceptive effect in animals, observed in acute and chronic pain models, which do not induce some of the side effects observed for analgesic drugs, such as alteration in spontaneous motor and general activity or the development of tolerance to its antinociceptive effect after prolonged treatment [35–37]. It was previously demonstrated by our group that CRO induces analgesia mediated by the activation of CB2 cannabinoid receptors [38]. First, we evaluated the disease development regarding motor impairment through the observation of clinical signs. The results showing that CRO ameliorates the EAE-induced clinical signs corroborate data from the literature, where the administration of a phytocannabinoid, an agonist of CB2 receptors, attenuated the clinical severity of the disease in mice, both preventively and after the onset of clinical signs [56].

In addition to the motor impairment, central neuropathic pain is observed in animal models of EAE, occurring due to prolonged inflammation in the spinal cord, resulting in activation of glial cells and aggression to the myelin sheath, causing painful hypersensitivity [31,57]. Thus, activation of spinal astrocytes and microglia seems to be a key element in the generation and maintenance of peripheral neuropathic pain [58,59], reducing the nociceptive threshold of animals before neurological dysfunction occurs [60]. It was presently shown that CRO partially reverses EAE-induced mechanical hypersensitivity. Importantly, the analgesic effect was already observed 1 h after its administration. Considering the rapid analgesic effect observed, as well as the fact that hypernociception is detected before the onset of clinical signs, it is assumed that the observed pain does not depend on the onset of central inflammation and that the observed analgesic effect is due to mechanisms independent of the anti-inflammatory observed effect.

As previously pointed out, central inflammation is a key factor in the progressing of EAE as well as MS [53–55]. Our data clearly demonstrated that crotalphine is capable of preventing the cell influx to the CNS. However, in addition to migrated cells, resident cells also play an important role in the onset of disease development. Microglial reactivity is manifested by morphological changes, modifications in the expression of surface molecules, and secretion of several substances such as cytokines, trophic factors, and chemokines [61–63]. Specifically, in the acute period of EAE, microglia reactivity was detected along with increased clinical symptomatology. Treatment with a microglial inhibitor or microglia depletion has been shown to induce beneficial effects on EAE symptoms, demonstrating that microglial cells play a role in the pathogenesis of EAE [64,65]. In addition, it has been reported that activated microglia may release a large variety of molecules, which may contribute to immune cell recruitment and the spread of inflammatory response [64–67]. Astrocytes also play important roles in the development of chronic pain, producing neurotoxic mediators, cytokines, and chemokines, with proinflammatory activity [21,68,69]. Here, the literature findings were reproduced, showing that EAE increases microglial and astrocytes labeling, both at the peak of the disease and in the late period [31,61]. Importantly, CRO decreased astrocytes and microglia reactivity in both periods, observed in the dorsal horn of the spinal cord, a region related to the nociceptive pathway [70]. In addition to that, histological analysis of the spinal cord showed that CRO decreases cell infiltrates in EAE mice, suggesting a correlation with reduced glial activation, TNF-α cytokine production, and lower clinical signs. These results point to the reduction of the neuroinflammation induced by CRO as a key factor for the antinociceptive effect and the improvement of clinical signs.

The contribution of Th17 cells and its effector cytokine signature, IL-17, is well described as promoters of the induction and progression of MS/EAE [71–73]. The role of

Th1 cells in the process is known, but Th17 cells have a greater proliferative capacity than Th1 cells, in addition to being able to cross the blood-brain barrier (BBB) more easily [71]. Here, it was confirmed that IL-17 levels are up-regulated in the EAE group. Of interest, CRO prevented the IL-17 increase in the spinal cord (17th day). It is evident that IL-17 is a key cytokine in the EAE model [74]. In fact, anti-IL17A treatment showed satisfactory results in attenuating the development of EAE [75], for example, in reducing the clinical signs and improving the histological findings. In addition, IL-17 knockout mice exhibited delayed onset, reduced clinical scores, and early recovery after immunization [76]. Data from our group also demonstrated the importance of the IL-17 to the development of EAE using crotoxin, the main neurotoxin from the venom of the *Crotalus durissus terrificus* snake. In these studies, crotoxin attenuated clinical signs by inhibiting the release of IL-17 and reducing CD4+IL-17+ cell proliferation in lymph nodes [77]. These data point out that the reduction of this pro-inflammatory cytokine is one of the main factors that contribute to CRO attenuation of disease progression. Importantly, although the central release of IL-17 was decreased, the proliferation of Th17 cells in the lymph nodes, at the onset of the immune process, was not altered by CRO (data not shown). In addition to IL-17, the profile of TNF-α was also investigated, since IL-17 and TNF-α are the two major cytokines involved in MS [78–80]. Our results demonstrated that a single dose of CRO reversed EAE-induced TNF-α release in the spinal cord at the peak of the disease. The contribution of TNF-α to the development and progression of MS is well established. However, despite the pro-inflammatory actions of TNF-α, the use of anti-TNF-α monoclonal antibodies in MS patients, in addition to being ineffective, promotes an unexpected worsening of the disease [81], indicating that this cytokine also plays a protective role. Several studies have shown that the pathogenic and homeostatic activities of TNF-α are mediated by distinct cellular and molecular pathways and depend on the type of TNF-α receptor that is activated. Astrocytes, oligodendrocytes, and Treg cells express the TNFR2 receptor; the activation of this receptor mediates neuronal survival, re-myelination, and acts on immunoregulation. On the other hand, activation of TNFR1 receptors induces neuroinflammation and demyelination. Substances that activate CB2 type cannabinoid receptors act on glial cells and neurons, inhibiting TNF-α release and having antioxidant action [82]. Thus, the decrease in TNF-α levels presently observed may be a consequence of CB2 receptor activation, and since this decrease is partial, it is plausible that the remaining TNF-α would continue to exert its neuroprotective effect.

The increased chemokines levels detected in EAE makes the BBB more permeable to inflammatory cells; thus peripheral macrophages infiltrate the CNS [83]. Microglia and macrophages are considered important in the development of EAE and actively contribute to the pathogenesis and progression of the disease [84], causing the release of proinflammatory cytokines and leading to gliosis, inflammation tissue damage, and demyelination, culminating in neuronal death (neurodegeneration) in the CNS [61,85,86]. During the acute stage of EAE, NGF and its tyrosine kinase receptor (TrkA) expression are decreased in the CNS in rats [87]. Furthermore, studies suggest that NGF is responsible for the induction of axonal regeneration, survival, maintenance, and the differentiation of oligodendrocytes, as well as for facilitating the migration and proliferation of oligodendrocyte precursors to the myelin injury sites, a key role in the recovery of demyelinating diseases and stimulating recovery from neuroinflammatory diseases, including EAE [88–90]. On the 28th day after immunization, the expression of neurotrophin NGF was analyzed. In our study, corroborating data from the literature, a reduction of NGF in the spinal cord of the EAE animals was observed; however, it was not prevented by CRO.

It is noteworthy that MS is a disease that affects the CNS and that the literature is scarce regarding the effects of EAE on the demyelination of peripheral nerves. To verify possible peripheral demyelination in this animal model of MS and whether the treatment with CRO can interfere in this process, the morphology of the sciatic nerve was analyzed by transmission electron microscopy. This methodology has been applied for the evaluation of peripheral neurodegeneration, such as demyelination and the effect of neuroprotective therapies on these processes [91]. It was presently observed that this autoimmune disease alters the structure of the sciatic nerve myelin sheath of animals. Our results, together with results previously published [92], indicate that sciatic nerve demyelination may contribute to the neuropathic pain detected in this EAE model. In contrast to previous knowledge about the expression of myelin basic protein (MBP) and MOG exclusively at the CNS, it is currently known that these proteins are expressed in the peripheral nervous system and can therefore be attacked by autoantibodies, contributing to peripheral demyelination [93–96]. Interestingly, treatment with a single dose of CRO induces improvement in myelin fiber thickness. These results are promising and indicate that CRO may prevent demyelination or promote remyelination. Further studies to confirm these hypotheses are necessary.
