Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

Evonuk, Kirsten S.; Moseley, Carson E.; Doyle, Ryan E.; Weaver, Casey T.; DeSilva, Tara M.

To determine if immune cell infiltration is reduced, the spinal cords are removed and processed for flow cytometry analysis at the peak of disease ( Figure 1A, approximately day 18 ). This ensures that the largest number of immune cells have entered into the spinal cord. Entrance of T cells into the CNS is considered the initiating inflammatory event and both Th1 and Th17 cells are found in animal models of EAE as well as MS patients. Taken together, flow cytometric analysis should include assessment of both types of pathogenic T cells. Furthermore, Tregs are wellcharacterized suppressor T cells that dampen disease. Therefore, the percentage of Tregs from a total CD4 + population must be evaluated Figure 5A) . Furthermore, no change in Ki67 + CD4 + cells should be found if proliferation is unaffected ( Figure 5B) . Drug treatments are introduced on day 7 or later to avoid altering initial antigen presentation and T cell activation in the periphery. However, in genetic models proteins are often deleted constitutively during embryogenesis or induced before induction of EAE making splenocyte assessment of high importance.