Experimental allergic encephalomyelitis (EAE) is a cell-mediated autoimmune disease of the central nervous system (CNS) which has been well characterised ...
166s Biochemical Society Transactions (1997) 25
Reactive oxygen encephalomyelitis
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Gwen S Scott, K Ivor Williams, and Christopher Bolton School of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, UK,BA2 7AY. Experimental allergic encephalomyelitis (EAE) is a cell-mediated autoimmune disease of the central nervous system (CNS)which has been well characterised as an animal model of the human demyelinating disorder, multiple sclerosis. Although the precise pathological mechanisms involved in the aetiology of both diseases remains undefined a potential role for a number of inflammatory mediators has been proposed [11. In particular several investigators have indicated a role for reactive oxygen species (ROS) in disease development [2-61. Consequently, we have examined CNS levels of the ROS, superoxide and hydrogen peroxide in the Lewis rat model of EAE. Male Lewis rats (200-25Og) were inoculated for EAE as described previously [7]. Briefly, each rat received, per hind foot pad, 0.1 ml of an encephalitogenic emulsion composed of equal parts of guinea pig spinal cord, phosphate buffered saline and Freund's incomplete adjuvant supplemented with 10 mg/ml Mycobacteriuin tuberculosis. Complete Freund's adjuvant (CFA) control animals recieved inoculum without spinal tissues. C N S cytosol was prepared from the cerebellum, medulla-pons and cervical spinal cord regions of each animal [8]. Superoxide levels were determined in samples, by measuring spectrophotometrically at 550 nm the reduction of femcytochrome C [9]. C N S cytosol superoxide levels were calculated from the extinction coefficient of femcytochrome C. Hydrogen peroxide levels were quantitated by measuring the oxidation of scopoletin using a fluorescent platereader with excitation at 350 nm and emission at 460 nm [lo]. CNS' cytosol levels of hydrogen peroxide were determined with reference to a series of standards. Following sample protein content determination results were expressed as either n moles superoxiddmg protein or p moles hydrogen peroxiddmg protein. Levels of superoxide in all CNS areas were similar in normal animal (Fig. 1). Superoxide content from CFA-inoculated animals were comparable to values recorded in normal rats (Fig. 1). In contrast, established neurological EAE was characterised by a significant increase in the superoxide content of all isolated CNS preparations (Fig. 1).
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Figure 1. CNS cytosol superoxide levels. Superoxide levels were determined by measuring ferricytcchrome C reduction and expressed as n moles supemxiddmg protein f S.E.M.0Normal Lewis rats, CFA control inoculated Lewis rats, n=6; EAEinoculated Lcwis rats, n =lo. *~
