EAE induction rats

Eight-week-old male Lewis rats were obtained from Harlan and maintained at the animal facility of the VU University Medical Center. The animals had ad libitum access to food and water and were housed under a 12-h light/dark cycle.

To induce EAE, rats were injected subcutaneously with 20 μg myelin basic protein (MBP) isolated from guinea pig brain and spinal cord (Harlan Laboratories, Horst, The Netherlands) in PBS mixed with complete Freund’s adjuvant (CFA; 4 mg/ml Mycobacterium tuberculosis H37Ra; Difco Laboratories, Detroit, MI, USA). A control group without EAE induction was included (n = 6). EAE animals were treated by intraperitoneal (i.p.) injection with the vehicle (0.05% Tween 80, 2% DMSO in saline) (n = 11) or with 10 μmol/kg SMI 6877002 (n = 11) from days 6 to 11 after the induction of EAE. This phase of the disease is mainly characterized by the recruitment of inflammatory cells to the CNS. Neurological symptoms were scored daily and graded from 0 to 5: 0 = no neurological abnormalities; 0.5 = partial loss of tail tonus; 1 = complete loss of tail tonus; 2 = hind limb paresis; 3 = hind limb paralysis; 4 = paralysis up to the diaphragm; 5 = death. Body weight was measured daily. Animals were sacrificed 14 and 20 days after induction of EAE. All the experimental procedures were approved by the Ethical Committee for Animal Experiments of the VU University Medical Center (VUMC). Scoring of clinical symptoms was performed by an observer who was blinded to the experimental conditions.