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.

Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of autoimmune inflammatory disorders that was directly responsible for the discovery of drugs currently used to treat MS [1] [2] [3] [4] . Mice are often used for EAE, with C57BL/6 mice being a popular strain based on the availability of genetic variants. C57BL/6 mice induced with EAE exhibit chronic disease progression with onset around day 10 postinduction. Infiltration of the spinal cord parenchyma and cerebellum are characteristic of the histopathology of these animals, with absence of infiltration in the cortical parenchyma 5 . Additionally, cortical lesions and demyelination in the brain are hallmarks of the disease [6] [7] [8] [9] , which are relatively absent in C57BL/6 mice. Therefore, it may be preferable when possible to use SJL mice, which have relapsing-remitting disease and lesions found in both the brain and spinal cord that appear similar to those in MS 10 . Treatment cannot be classified as neuroprotective if immune cells never reach the CNS. Therefore, this protocol makes use of flow cytometric analysis of brains, spinal cords, and spleens from EAE mice to determine effects of treatment on immune cell infiltration into the CNS and proliferation of immune cells in the periphery, as previously demonstrated 2. Assessment of immune cell infiltration in brain and spinal cord 1. Cut brains and spinal cords into smaller pieces using sterile scissors. Crush with the plunger from a 3 ml syringe over a 70 μm cell strainer into a new 50 ml tube while rinsing the strainer with media. Bring each tube to a 50 ml volume with media. Centrifuge at 453 x g for 5 min to pellet cells. 2. Aspirate supernatant and resuspend pellet in 4 ml of 40% density gradient media. Carefully overlay the 40% density gradient containing cells on top of 2 ml of 70% density gradient in a new 15 ml conical tube-pipette very slowly onto the wall of the conical tube to ensure proper layering of the gradient. Spin at 796 x g for 20 min at RT without a brake. 3. Carefully remove the upper myelin layer from the gradient with a 1 ml transfer pipette, then remove viable cells at the interface with a 1 ml transfer pipette and transfer to a new 15 ml conical tube. Bring the tube to 15 ml with media and centrifuge at 448 x g for 10 min. 4. Resuspend pellet in 200 μl media and place into one well of a 96-well round-bottom plate (each sample from each animal will go into its own well). Centrifuge the plate at 410 x g for 5 min. 5. Flick off the supernatant and resuspend pellet in 200 μl of restimulation media (RPMI supplemented with 10% FCS, 100 IU/ ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1x non-essential amino acids, 1 mM sodium pyruvate, and 55 μM βmercaptoethanol, plus 50 ng/ml phorbol myristate acetate (PMA), 750 ng/ml ionomycin, and the protein transport inhibitor Brefeldin A). Place the plate in an incubator at 37 o C for 4 hr. NOTE: PMA and ionomycin restimulation results in activation of all T lymphocytes-regardless of their antigen specificity in order to assess the total number of each T cell subset in the given tissue. However, antigen-specific effector T cell responses can be assessed in various ways, including restimulating cells with MOG peptide in the presence of Brefeldin A 15