4.2. EAE Induction Evaluation and Tissue Sampling

Experiments were performed in female Lewis rats of 12 weeks of age. EAE was induced by s.c. tail base injection of 30 μg of MBP_72–85 dissolved in 100 μL saline and emulsified in 100 μL complete Freund’s adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 400 μg of H37Ra Mycobacterium tuberculosis (Difco, Detroit, MI, USA). Rats were weighed and assessed daily for clinical signs according to the following criteria: 0, normal; 0.5, weight loss; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; 5, moribund or dead. Moribund animals were sacrificed. Rats were allowed free access to food and water throughout the experiment. Rats were maintained under conventional conditions in the experimental animal unit of the Hellenic Pasteur Institute. All experimental protocols were evaluated and approved by the Institutional Protocol Evaluation Committee and the national Hellenic authorities (National Health Authority (PKA) licenses EL25BIO011, EL25BIO012 and EL25BIO013) and conformed to EU guidelines.