Multiple sclerosis (MS) is a Central Nervous System inflammatory demyelinating disease that has as primary symptoms losses of sensory and motor functions, including chronic pain. To date, however, few studies have investigated the mechanisms of chronic pain in animal models of MS since locomotor impairments render difficult its evaluation. It was previously demonstrated that in the MOG35-55-induced EAE, an animal model of MS, the hypernociception appears before the onset of motor disability, allowing for the study of these two phenomena separately. Here, we evaluated the effect of crotoxin (CTX), a neurotoxin isolated from the Crotalus durissus terrificus snake venom that displays, at non-toxic dose, antinociceptive, anti-inflammatory and immunomodulatory effects, in the pain and in symptoms progression of EAE. The pain threshold of female C57BL/6 mice decreased at the 4th day after immunization, while the first sign of disease appeared around the 11st-12nd days, coinciding with the onset of motor abnormalities. CTX (40 µg/kg, s.c.) administered in a single dose on the 5th day after immunization, induced a long-lasting analgesic effect (5 days), without interfering with the clinical signs of the disease. On the other hand, when crotoxin was administered for 5 consecutive days, from 5th - 9th day after immunization, it induced analgesia and also reduced EAE progression. The antinociceptive effect of crotoxin was blocked by Boc-2 (0.5 mg/kg, i.p.), a selective antagonist of formyl peptide receptors, by NDGA (30 μg/kg, i.p.), a lipoxygenase inhibitor and by atropine sulfate (10 mg/kg, i.p.), an antagonist of muscarinic receptors, administered 30 min before CTX. CTX was also effective in decreasing EAE clinical signs even when administered after its onset. Regarding the interactions between neurons and immunocompetent cells, CTX, in vitro, was able to reduce T cell proliferation, decreasing Th1 and Th17 and increasing Treg cell differentiation. Furthermore, in EAE model, the treatment with 5 consecutive doses of CTX inhibited IFN-γ-producing T cells, GM-CSF-producing T cells, reduced the frequency of activated microglia/macrophages within the CNS and decreased the number of migrating cell to spinal cord and cerebellum at the peak of the disease. These results suggest that CTX is a potential treatment not only for pain alteration but also for clinical progression induced by the disease as well as an useful tool for the development of new therapeutic approaches for the multiple sclerosis control.

中文翻译：

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Crotoxin 下调促炎细胞并减轻 MOG35-55 诱导的实验性自身免疫性脑脊髓炎的疼痛，这是一种多发性硬化症的动物模型

多发性硬化症 (MS) 是一种中枢神经系统炎性脱髓鞘疾病，其主要症状是感觉和运动功能丧失，包括慢性疼痛。然而，迄今为止，很少有研究调查 MS 动物模型中慢性疼痛的机制，因为运动障碍导致其评估变得困难。先前已经证明，在 MOG35-55 诱导的 EAE（一种 MS 动物模型）中，在运动障碍发作之前出现过度伤害感受，允许分别研究这两种现象。在这里，我们评估了 crotoxin (CTX)，一种从 Crotalus durissus terrificus 蛇毒中分离的神经毒素，在无毒剂量下，在 EAE 的疼痛和症状进展中显示出镇痛、抗炎和免疫调节作用。雌性 C57BL/6 小鼠的痛阈在免疫后第 4 天下降，而疾病的第一个迹象出现在第 11-12 天左右，与运动异常的发作相吻合。CTX (40 µg/kg, sc) 在免疫后第 5 天以单剂量给药，可产生持久的镇痛作用（5 天），而不会干扰疾病的临床症状。另一方面，当从免疫后第 5 天至第 9 天连续 5 天施用 crotoxin 时，它会引起镇痛并减少 EAE 的进展。crotoxin 的镇痛作用被甲酰肽受体的选择性拮抗剂 Boc-2 (0.5 mg/kg, ip)、脂肪氧化酶抑制剂 NDGA (30 μg/kg, ip) 和硫酸阿托品 (10 mg/kg) 阻断。 kg, ip)，毒蕈碱受体拮抗剂，在 CTX 前 30 分钟给药。即使在其发病后给药，CTX 也能有效减少 EAE 临床症状。关于神经元和免疫活性细胞之间的相互作用，体外 CTX 能够减少 T 细胞增殖，减少 Th1 和 Th17 并增加 Treg 细胞分化。此外，在 EAE 模型中，连续 5 个剂量的 CTX 治疗抑制了产生 IFN-γ 的 T 细胞、产生 GM-CSF 的 T 细胞，降低了中枢神经系统内活化的小胶质细胞/巨噬细胞的频率，并减少了迁移细胞的数量。脊髓和小脑处于疾病高峰期。