*5.5. Pharmacological Treatment with CRO*

Crotalphine (<E-F-S-P-E-N-C-Q-G-E-S-Q-P-C, where <E is pyroglutamic acid and 7C-14C forms a disulfide bond; MW 1534.6 Da) was synthesized by Proteimax Biotecnologia, Brazil (São Paulo, SP, Brazil) as previously described [35], and was administered by oral route, diluted in sterile saline (NaCl 0.9% in distilled water). For dose setting of CRO, animals with EAE were allocated into different groups which received CRO (10, 50 or 100 μg/kg, p.o.) or saline (p.o.), on the fifth day after immunization (1 day after the onset of nociceptive response, the first symptom of the disease). Administration of CRO was based on previous assays showing long-lasting analgesic effects in experimental models of pain [36,37]. CRO (50 μg/kg) was also administered in five repeated doses (one daily dose for five consecutive days) from the 12th day (one day after motor impairment manifestation). The protocol of five consecutive doses was based on previous results demonstrating the greatest difficulty in controlling the symptoms of the disease after the onset of clinical signs [77]; however, it was performed to determine whether crotalphine would be able to interfere with the development of the disease even after the motor impairment trigger.
