Citation

Here, we present a protocol to induce and monitor EAE in female C57BL/6 mice. Females are preferentially chosen, and there is a incidence of women : men of 3:1 in MS. To assess the severity of EAE, we made use of a 3-point scoring sheet. EAE severity is generally scored with respect to the severity of motor dysfunctions. Mice with advanced stages of EAE, i.e. exhibiting a score above 2 should be sacrificed to avoid unnecessary suffering of the animals. Thus, it is recommended to score the mice at close intervals e.g. twice daily as explained in the protocol.  There are several scoring routines employed, reaching from 0-3 points to 0-6 points16,17,18,19 or even more subpoints20. However, we have previously shown that supposedly refined EAE scoring does not lead to any improvement in determining potential statistically significant differences in disease scores between groups, when considering the overall disease severity15. Based on this observation we made use of the 3-point scale to assess disease progression in EAE in order to reduce mouse handling time and thus, distress for mice implementing the 3R rules. Another critical point using the EAE model to study differences between knockout and wild-type mice or differences between treatment groups is setting up a precise experimental planning. When wild-type mice are compared to knockout mice, it is essential to compare littermates originating from heterozygous matings to ensure identical genetic backgrounds of the wild-type and knockout mice compared in the EAE experiment. It is also important to keep the same conditions for all mice included in an experiment, e.g. cage changes, administration of wet food, and especially mouse housing (health state) conditions. In order to avoid cage-specific phenomena induced by the investigator, cross-immunization should be performed. More precisely, if more than one syringe is needed to immunize a certain number of mice, they should be randomly assigned to different syringes used. The latter likewise should be applied for the performance of treatment studies. Last but not least, the health status of mice housed in different animal facilities might impact on disease incidence and severity and thus more21 or less _Mycobacterium tuberculosis_22 might be required to induce a proper clinical disease. Furthermore, the site of injection may impact on disease development. In this protocol we propose to inject relatively small volumes into five different subcutaneous sites: 30 L into two flanks, 20 L into the left and right tail root, and a little droplet into the neck. In other protocols, 50 L depots are placed subcutaneously into the tail root only23 or into the four flanks21. Injections of smaller depots, however, minimize the risk of causing skin irritation, which mice may try to scratch.