**2. Results**

#### *2.1. CRO Caused Analgesia in EAE-Induced Pain and Attenuated Clinical Signs*

CRO (10, 50, or 100 μg/Kg) or saline was first administered on the fifth day after immunization, in a single dose. The immunized animals (EAE) showed decreased nociceptive threshold (termed hypernociception) on the fourth day after immunization when compared to the complete Freund's adjuvant (CFA) group (control). The hypernociception remained until the last day of evaluation. i.e., 10th day, when the assessment of the pain threshold was stopped due to the onset of motor dysfunctions. The treatment with CRO with doses of 10, 50, and 100 μg/kg, resulted in a partial reversal of the EAE-induced hyperalgesia (Figure 1A). The experiments were performed in the morning and the pain threshold was assessed 1 h after the treatment with CRO.

**Figure 1.** Evaluation of CRO effect in pain sensitivity and clinical signs of animals immunized with MOG35–55. Animals with EAE were treated with a single dose of CRO p.o. on the fifth day, in different doses. Pain sensitivity was measured by the electronic von Frey model (**A**) and motor impairment was evaluated according to a visual scale of clinical signs from 0 to 5 (**B**). For statistical analysis of clinical signs, the area under the curve was evaluated (**C**). Data represent mean ± SEM 8–10 animals per group. # *p* < 0.05 compared to untreated group (EAE). \* *p* < 0.05 compared to the control group (CFA). The two-way ANOVA test, followed by Bonferroni post-hoc test was used in the electronic von Frey test, and the One-way ANOVA test was used, followed by Tukey's post-hoc test, was used in the area under the curve.

The first motor impairment symptom appeared on the 11th day after immunization and increased progressively, peaking around the 17–18th day. CRO, at the dose of 50 μg/kg, reduced the disease severity, represented by lower clinical scores, when compared to the untreated group (Figure 1B,C). This dose was chosen for subsequent experiments.
