**1. Introduction**

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease which affects more than 2.8 million people worldwide [1]. The autoimmune inflammation that affects the central nervous system (CNS) progressively results in oligodendrocyte injury and demyelination. In the early stages, the axons are preserved, however, with the advance of the disease, the damages are irreversible [2]. The origin of MS is still unclear; however,

**Citation:** Giardini, A.C.; Evangelista, B.G.; Sant'Anna, M.B.; Martins, B.B.; Lancellotti, C.L.P.; Ciena, A.P.; Chacur, M.; Pagano, R.L.; Ribeiro, O.G.; Zambelli, V.O.; et al. Crotalphine Attenuates Pain and Neuroinflammation Induced by Experimental Autoimmune Encephalomyelitis in Mice. *Toxins* **2021**, *13*, 827. https://doi.org/ 10.3390/toxins13110827

Received: 28 October 2021 Accepted: 17 November 2021 Published: 22 November 2021

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the combination of environmental, such as lifestyle and viral exposure, and genetic factors may contribute to the development of the disease [3–6].

The literature shows multifocal inflamed regions in gray and white matter, with oligodendrocyte death and myelin sheath disruption [7,8]. The clinical presentation of MS is highly variable, making the diagnosis of the disease difficult [9]. The inflammatory response is mainly mediated by T helper (Th) cells, which play an essential role in the course of MS [10]. Pro-inflammatory cytokines such as interleukin (IL)-6, IL-17, IL-21, IL-22, IL-23, and tumor necrosis factor (TNF)-α are produced by Th17 cells [10–13]. IL-17, which is one of the main cytokines produced by these cells, acts as an inducer of neutrophil infiltration [13–15]. Th1 cells mediate CNS inflammation by stimulating macrophage infiltration and producing important cytokines including interferon (IFN)-γ, which plays a relevant role in spinal neuroinflammation [10]. In addition, B-lymphocytes play a critical role in tissue damage, propagating inflammation, and spinal demyelination in MS [16,17].

In addition to peripheral immune cells, activation of CNS resident cells also adds to the pathogenesis of MS, particularly microglia and astrocytes. Microglia cells are activated when tissue integrity is disturbed, secreting pro-inflammatory or anti-inflammatory cytokines, controlling the expression of anti-inflammatory molecules, regulating phagocytosis of debris and tissue repair, responses that depend on the changes that occurred in their specific microenvironment. Astrocytes strengthen and contribute to the maintenance of the integrity of the blood-brain barrier, restricting the entry of peripheral immune cells into the CNS [18] and modulating synaptic activity and plasticity [19,20]. These activated glial cells produce chemokines and cytokines, leading to the recruitment of additional immune cells to the CNS parenchyma besides producing exacerbated sensitization stages, including neural hypersensitivity [21]. Pain is one of the sensory alterations presented by MS patients, and can be of inflammatory, neuropathic or skeletal muscle origin [22]. It also affects the quality of life, aggravating symptoms such as depression, sleep disturbance, and mood [23–27]. Importantly, like many other progressive diseases, there is no specific treatment for MS, and therapies focus on delaying disease progression and promoting symptom relief, thus improving the patient's quality of life.

Animal models have provided valuable answers regarding the mechanisms involved in the development and progression of MS and the efficacy of new drugs with therapeutic potential [11,28]. These experimental models reproduce CNS inflammation, demyelination of neurons, and some motor changes observed in MS [28–31], in addition to sensory alterations. Among the sensory alterations, the literature has demonstrated alterations in the pain threshold in both humans and animals that, in the MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) model, arise before the onset of motor alterations [32–34], allowing for the study of pain previous to the first evidence of motor alterations, which would compromise pain sensitivity assessment.

Considering that MS is a chronic neurodegenerative disease whose treatment only delays progression and alleviates symptoms without interfering with the pathologic process, new therapeutic strategies are needed. Crotalphine (CRO) is a 14-amino acid peptide (EFSPENCQGESQPC) containing a disulfide bridge and a pyroglutamic acid [35], capable of inducing a potent and durable antinociceptive effect in acute and chronic pain models (including cancer pain and neuropathic pain) [35–37]. CRO induces antinociception by activating CB2 receptors, which, in turn, induces the release of endogenous peptides, particularly dynorphin A, the endogenous agonist of kappa opioid receptors [38]. It was also observed that CRO desensitizes transient receptor potential ankyrin ion channels (TRPA1) [39], a receptor that has a relevant role in the maintenance of inflammatory hyperalgesia [40]. Based on clinical studies showing the effectiveness of cannabinoids on clinical symptoms in patients with MS, particularly muscle stiffness and spasms, sleep disorders, and neuropathic pain [41,42], the CRO effect on clinical symptoms, neuroinflammation, and axonal demyelination of mice submitted to the MOG35–55-induced experimental autoimmune encephalomyelitis, an animal model of MS, is investigated here.
