*2.2. CRO Reverts EAE-Induced Motor Impairment*

To evaluate the efficacy of CRO after the onset of motor impairment, CRO was administered for five consecutive days (one dose by day), starting from the 12th day of immunization. The effectiveness of this protocol was compared with the single-dose protocol. The EAE groups showed a progressive increment in clinical score from the 11th day after immunization, peaking at the 16th day. Both groups treated with CRO showed lower clinical signs scores when compared to the untreated group, showing the potential of CRO to interfere with motor impairment even after its onset as well as to prevent it (Figure 2A,B). At this point, the analgesic effect of CRO was not evaluated, since it is not possible to apply the nociceptive test due to the motor impairment. The body weight of the animals was measured during the course of the disease and, as expected, EAE interfered with the weight gain of the animals (Figure 2C). Crotalphine did not alter the EAE-induced weight loss (Figure 2C).

**Figure 2.** Evaluation of CRO effect on clinical signs and body weight of animals immunized with MOG35–55 and treated before or after motor impairment. Animals were immunized with MOG35–55 and treated with CRO in a single dose on the fifth day after immunization (blue arrow), or in five doses (green arrows, one daily dose for five consecutive days starting on the 12th day after immunization). Clinical signs were evaluated daily (**A**), and, for statistical analysis, the area under the curve was evaluated (**B**). Body weight (g) was measured during the course of the disease (**C**). Data represent mean ± SEM 8–10 animals per group. \* *p* < 0.05 compared to the CFA group. # *p* < 0.05 compared to the untreated group (EAE). A one-way ANOVA test was used, followed by Tukey's post-hoc test in the area under the curve.
