*2.7. CRO Prevented the Peripheral Demyelination of the Sciatic Nerve Induced by EAE*

We have previously demonstrated that EAE decreases sciatic nerve myelin thickness when quantified through the g-ratio estimative (i.e., dividing axon diameter by the fiber diameter) [48]. Here, we checked whether CRO would have a beneficial effect on EAEinduced demyelination.

In order to address this question, the distal portion of the sciatic nerve was collected on the 17th and the 28th days after immunization and its morphology was analyzed (Figure 8A) by transmission electron microscopy. The results demonstrated the presence of intact fibers, with a similar distribution of small and large diameter myelinic fibers, non-myelinic fibers, and Schwann cell nuclei in the control group (CFA). At the peak of the disease (17th day after immunization), EAE animals showed no reduction in myelin sheath thickness (Figure 8C). However, on the 28th day after immunization (Figure 8D), a decrease in the sciatic nerve myelin thickness, defined by an increase in the g-ratio when compared with control animals, was detected. Importantly, animals treated with a single dose of CRO on the fifth day after immunization have a thickness of myelin sheaths similar to the control group. This data shows that CRO prevents EAE-induced demyelination, which may preserve the nerve conduction and contribute to the analgesic effect.

**Figure 8.** Effect of CRO on the peripheral nerve myelin sheath of animals with EAE. Animals were sedated, euthanized, perfused and the sciatic nerve was collected for evaluation by transmission electron microscopy, 7500× magnification. Representative microscopic fields of transverse sections of the peripheral nerve stained with toluidine blue solution ((**A**), panel). The G-ratio (**B**) was measured using ImageJ software for evaluation of axonal myelin content on the 17th (**C**) and 28th days (**D**) after immunization. Data represent the mean ± SEM of 3 animals per group. \* *p* < 0.05 compared to the control group (CFA). # *p* < 0.05 compared to the untreated group (EAE). One-way ANOVA was used, followed by Tukey's post-hoc test.
