2.2. EAE Induction, Disease Scoring, and Experimental Groups

To induce the formation of encephalitogenic T-cells in peripheral lymphatic tissues, the mice were subcutaneously immunized with an emulsion of myelin oligodendrocyte glycoprotein (MOG_35–55) peptide dissolved in complete Freund’s adjuvant followed by intraperitoneal injections of pertussis toxin (PTx) in PBS on the day of and the day after immunization (Hooke Laboratories, Inc., Lawrence, USA) as previously published [4]. Disease severity was scored as follows: 1, The entire tail drops over the observer’s finger when the mouse is picked up by the base of the tail; 2, the legs are not spread apart but held close together when the mouse is picked up by the base of the tail, and mice exhibit a clearly apparent wobbly gait; 3, the tail is limp and mice show complete paralysis of hind legs (a score of 3.5 is given if the mouse is unable to raise itself when placed on its side); 4, the tail is limp and mice show complete hind leg paralysis and partial front leg paresis, and the mouse is minimally moving around the cage but appears alert and feeding; 5, the mouse is euthanized due to severe paralysis. The parameter “disease onset” was defined as the day post immunization when the first clinical deficit (see above) was observed. The parameter “maximum disease score” was defined as the highest clinical score, reached by a mouse at any time-point during the experiment. The parameter “cumulative disease score” was calculated by adding all clinical scores, registered during the experiment for a single mouse.

The following treatment groups were included: Control^DigiGait mice: Non-immunized mice were subjected to gait analyses; EAE^Only mice: EAE was induced by MOG_35-55 immunization + CFA/PTx, but mice were not subjected to gait analyses; EAE^DigiGait mice: EAE was induced by MOG_35-55 immunization + CFA/PTx, and mice were subjected to gait analyses starting at day 6 post immunization; PTx^DigiGait mice: Mice were injected with CFA and PTx, and mice were subjected to gait analyses starting at day 6 post immunization.