Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut

Henrik Hasseldam, Rune Skovgaard Rasmussen, Flemming Fryd Johansen

Multiple sclerosis is widely accepted as an inflammatory disease. However, studies indicate that degenerative processes in the CNS occur prior to inflammation. In the widely used animal model experimental autoimmune encephalomyelitis (EAE), we investigated the significance of degenerative processes from mitochondrial membrane potentials, reactive oxidative species, cell death markers, chemokines, and inflammatory cell types in brain, spinal cord, and optic nerve tissue during the effector phase of the disease, before clinical disease was evident. Sixty-two rats were placed in eight groups, n = 6 to 10. Four groups were immunized with spinal cord homogenate emulsified in complete Freund's adjuvant (one served as EAE group), three groups were immunized with complete Freund's adjuvant only, and a control group was injected with phosphate buffered saline only. Groups were sacrificed 3, 5, 7, or 12-13 days after the intervention and analyzed for early signs of CNS degeneration. Loss of mitochondrial membrane potential and oxidative changes was observed days before clinical disease debut at day 9.75 ± 0.89. The early mitochondrial changes were not associated with cytochrome C release, cleavage of caspases 9 (38/40 kDa) and 3 (17/19 kDa), and cleavage of PARP (89 kDa) or spectrin (120/150 kDa), and apoptosis was not initiated. Axonal degeneration was only present at disease onset. Increases in a range of cytokines and chemokines were observed systemically as a consequence of immunization with complete Freund's adjuvant, whereas the encephalitogenic emulsion induced an upregulation of the chemokines Ccl2, Ccl20, and Cxcl1, specifically in brain tissue, 7 days after immunization. Five to seven days after immunization, subtle decreases in the mitochondrial membrane potential and an increased reactive oxygen species burden in brain tissue were observed. No cell death was detected at these time-points, but a specific expression pattern of chemokines indicates activity in the CNS, several days before clinical disease debut.