EAE Induction

Female C57Bl/6 mice {Taconic, Denmark (DK) aged 17 weeks [22.8 ± 0.4 g; early adulthood (13)]} were housed under standard conditions. EAE was induced in C57Bl/6 mice by active immunization with myelin oligodendrocyte glycoprotein (MOG) 35–55 using the kit EK-2110 from Hooke labs (MA, USA), following the manufacturer’s protocol. Briefly, mice were injected subcutaneously (s.c.) at two flanks with 200 µg of MOG 35–55 emulsified in Complete Freund’s adjuvant (CFA; N = 9), or 100 µl of PBS in case of the control mice (N = 6). At 2 and 24 h post-immunization the mice were injected i.p. with 100 µl of 4 µg/ml pertussis toxin (PTX) or 100 µl PBS for the control mice. Mice were monitored daily for clinical signs of disease and assigned a disease score according to the EAE clinical scoring system devised by the Danish Animal Experiments Inspectorate (see below).

Female Lewis rats (Charles River, Germany) aged 14 weeks (219 ± 1 g) were administered an emulsion consisting of: 100 µl complete Freund’s adjuvant (CFA; BD 263810, DK), 200 µg Mycobacterium tuberculosis H37Ra (BD, 231141, DK), 100 µg guinea pig myelin basic protein (MBP; Sigma-Aldrich, DK, M2295), and 100 µl 0.9% saline (14).

Directly after preparation, a total of 200 μl emulsion was administered intradermally to animals for EAE under isoflurane anesthesia at three sites at the base of the tail, totaling 200 µl in volume (N = 10). MBP-EAE and control rats (N = 8) were treated with a small volume of saline twice-daily (100 μl), in accordance with the design of another study in order to limit the use of experimental animals. Since this set of animals was used as controls for a separate therapeutic intervention study, we could not sample blood longitudinally. Therefore, we obtained only terminal plasma samples at peak disease severity.

Studies were conducted to minimize suffering and were approved by the Danish Animal Inspectorate (2015-15-0201-00647 and 2012-DY-2934-00001). Weight was monitored daily throughout the experiment.

Clinical scoring was performed twice daily using the following scale relating to progressive degrees of paralysis: 0, no clinical signs of EAE; 1, abolished tail tone; 2, mild paresis of one or both hind legs; 3, moderate paresis of one or both hind legs; 4, severe paresis of one or both hind legs; 5, paresis of one or both hind legs and incipient paresis of one or both forelegs are deemed moribund. Animals were deemed terminally ill according to predefined humane endpoints designed in consultation with the Danish Animal Inspectorate: animals registering a clinical score of ≥4, or a ≥20% loss of initial body weight.