*2.4. CRO Decreased EAE-Induced Glial Cells Immunoreactivity in the Spinal Cord*

Besides leucocyte infiltration, activation of resident CNS cells likewise contributes to the pathogenesis of EAE [44,45]. Next, the effect of CRO on EAE-induced astrocytes and microglia activation in the dorsal horn of the spinal cord was evaluated. Immunoreactivity for GFAP (glial fibrillary acidic protein), an astrocyte marker, and for Iba-1, a microglia marker was evaluated on the17th (clinical signs peak) and 28th (period of stabilization of clinical signs) days. The results show that EAE increases the reactivity for GFAP (Figure 4A,B) and Iba-1 (ionized calcium-binding adaptor-1) (Figure 5A,B) when compared with the control group (CFA), in both periods. On the other hand, the single-dose and five doses CRO protocol revert astrocyte and microglia activation induced by EAE (Figures 4 and 5).

**Figure 4.** CRO effect on immunoreactivity for astrocytes in the spinal cord of EAE animals. Animals immunized with MOG35–55 and treated or untreated with CRO were perfused for the spinal cord collection. Representative 20× microscopic field of transverse sections of the spinal cord (L3-L6) stained with GFAP ((**A**), panel). Immunohistochemistry analysis on 17th (**B**) and 28th days (**C**) after immunization. Immunoreactivity (IR) was quantified using Image J software. Data represent mean ± SEM of 3 animals per group. # *p* < 0.05 compared to EAE group. \* *p* < 0.05 compared to control group (CFA). A one-way ANOVA test was used, followed by Tukey's post-hoc test.

**Figure 5.** Evaluation of immunoreactivity for microglia in the spinal cord of animals with EAE and treated with CRO. Animals immunized with MOG35–55 and treated or untreated with CRO were perfused and had the spinal cords (L3-L6) collected. Representative 20× microscopic field of transverse sections of the spinal cord (L3-L6) stained with Iba-1 ((**A**), panel). Immunohistochemistry analysis on the 17th (**B**) and 28th (**C**) days after immunization. Immunoreactivity was quantified using Image J software. Data represent mean ± SEM of 3 animals per group. # *p* < 0.05 compared to EAE + sal group. \* *p* < 0.05 compared to control group (CFA). A one-way ANOVA test was used, followed by Tukey's post-hoc test.
