*2.5. CRO Mitigate EAE-Induced IL-17 and TNF-α Release in the Spinal Cord at the Peak of the Disease*

IL-17 and TNF-α are proinflammatory cytokines that activate T cells and other immune cells to produce and release a variety of cytokines and chemokines, and to express cell adhesion molecules [46]. Figure 6 shows that the levels of IL-17 and TNF-α are increased in the spinal cord of EAE animals at the peak of disease. Of interest, the treatment with CRO on the 5th day prevents EAE-induced IL-17 and TNF-α release. Moreover, the protocol of five doses of CRO does not prevent IL-17 and TNF-α release (Figure 6).

**Figure 6.** CRO effect on cytokine release in the spinal cord of EAE animals. Evaluation of the expression of IL-17 (**A**) and TNF-α (**B**) cytokines was analyzed by multiplex assay. The animals were immunized, euthanized, and the lumbar portion of the spinal cord was collected on the 17th day after immunization. Data represent the mean ± SEM of 5–7 animals per group. \* *p* < 0.05 compared to the CFA group. # *p* < 0.05 compared to the untreated group (EAE). One-way ANOVA was used, followed by Tukey's post-hoc test.

### *2.6. CRO Not Interfered with the Nerve Growth Factor Expression in the Spinal Cord of Animals with EAE*

Nerve growth factor (NGF) is a neurotrophin associated with the differentiation and survival of numerous neurons localized in the peripheral and CNS [47]. It was next investigated if CRO could interfere with the expression of NGF in the lumbar section of the spinal cord (L3–L6) on the 28th day after immunization. Our data confirm the results of the literature showing that NGF expression is reduced in the spinal cord of EAE mice; however, CRO does not change those levels (Figure 7).

**Figure 7.** Effect of CRO in neurotrophic factor in the spinal cord of animals immunized with MOG35–55. Animals, on the 28th day after immunization, were sedated and euthanized for the spinal cord collection. The expression of neural growth factor (NGF) was evaluated by western blotting. Beta-actin was used as the loading control. Results were normalized by CFA group expression. Results are expressed as the mean ± SEM of 5 animals per gropu. \* *p* < 0.05 in relation to the CFA group. One-way ANOVA followed by Tukey's post-hoc test.
