Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2528–2533, March 1997 Medical Sciences Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: Implications for the treatment of multiple sclerosis D. CRAIG HOOPER*, OMAR BAGASRA†, JOSEPH C. MARINI*, ANNA ZBOREK‡, S. TSUYOSHI OHNISHI§, RHONDA KEAN*, JEAN M. CHAMPION*, ASHIT B. SARKER†, LISA BOBROSKI†, JOHN L. FARBER¶, TAKAAKI AKAIKEi, HIROSHI MAEDAi, AND HILARY KOPROWSKI* *Center for Neurovirology, Department of Microbiology and Immunology, Dorrance H. Hamilton Laboratories, Section of Molecular Virology, Division of Infectious Diseases, and †Departments of Medicine and ¶Pathology, Thomas Jefferson University, Philadelphia, PA 19107; ‡Instytut Onkologii, Zaklad Biologii Nowotworow, 44–100 Gliwice, Poland; §Philadelphia Biomedical Research Institute, King of Prussia, PA 19406; and iDepartment of Microbiology, Kumamoto University School of Medicine, Kumamoto 860, Japan Contributed by Hilary Koprowski, December 23, 1996 ABSTRACT In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) tobeprevalent in theplaqueareasofpostmortembraintissue frompatientswithMS.Inaddition,wehaveobtainedevidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demon- stration of the presence of activated macrophages expressing high levelsof iNOSandevidenceofperoxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease. Evidence for the activation of the enzyme (iNOS) responsible for the production of the free radical nitric oxide (NO) in an immuneresponseand the releaseofNOin thecentralnervous systems (CNS) of animals with experimental allergic enceph- alomyelitis (EAE) or viral encephalomyelitis (1–6) has led to further study of the role of NO and its products in CNS pathology. Convincing evidence for the involvement of NO overproduction in the pathogenesis of EAE comes from studies that have correlated spinal cord and brain NO levels with clinical symptoms in animals with EAE (1, 5). The relevance of these animal studies to multiple sclerosis (MS) is emphasized by the fact that high levels of mRNA specific for iNOScanbeobserved inbrain lesions fromMSpatients (7, 8). In this case, therehasasyetbeennoconsensusas to thenature of the iNOS-positive cells with either cells of the monocyte lineage or astrocytes being implicated by different studies (7, 8).Todeterminemore specifically thenatureandoriginof the activated cells expressing iNOS in the MS brain we have expanded our analysis of MS brain tissue specimens using a refined combination of immunohistochemistry and reverse transcriptase in situ PCR (RT-IS-PCR). Several possibilities exist regarding the nature of NO in- volvement in the pathogenesis of CNS lesions. A variety of destructive effects have been postulated, both through direct interaction with cell surface and internal molecules (9) and through chemical interactions with oxygen species, such as superoxide (O22), to form other potentially more toxic inter- mediates (10, 11). One of these, peroxynitrite (ONOO2), the reaction product of NO and superoxide, is a potent oxidant (12), which can be formed in an inflammatory response and cancauseavarietyof toxiceffects, including lipidperoxidation (12) and tyrosine nitration (13). It has therefore been sug- gested that peroxynitrite may be responsible for a significant proportion of the damage attributed to NO. Thus, peroxyni- trite may be an important factor in the generation of CNS lesions. Indeed, in a previous study we found evidence of tyrosine nitration in the brain tissue of MS patients (7). To further clarify the direct or indirect role of NO and peroxyni- trite in thepathogenesisofCNS lesions,weanalyzed theeffect of substances thought to inhibit iNOS activation or to selec- tively scavengeNOorperoxynitriteon thedevelopmentof the clinical symptoms of EAE in an acute model. MATERIALS AND METHODS Brain Tissue Specimens. Brain tissue was obtained post mortem from patients who had suffered with MS for at least 10years.Brain tissuewasremovedwithin12hrofdeathduring autopsy at Thomas Jefferson University and areas with and withoutplaqueswere immediately frozenondry iceandstored at 2808C until use. The diagnoses of MS, based on clinical presentation, were confirmed by the post mortem histopatho- logical examination of brain tissues. All MS brains were found to contain pathognomonic MS plaques. In most cases autopsy sampleswereof frontal lobes,whichprovidedmixedwhiteand gray matter. Immunohistochemistry and RT-IS-PCR. To define cell types positive for iNOS mRNA expression in frozen brain sections, immunohistochemistry was performed in conjunc- tionwithRT-IS-PCRasdescribed(7, 14).Stainingwascarried out with a variety of antibodies and lectins: (i) cells of the macrophageymicroglia cell lineage were identified with rho- damine-conjugatedRicinus communisagglutinin (RCA-1) lec- tin (Sigma), which specifically binds these cells (15); (ii) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y942528-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: MS, multiple sclerosis; NO, nitric oxide; EAE, exper- imental allergic encephalomyelitis; RT-IS-PCR, reverse transcriptase in situ PCR; iNOS, inducible nitric oxide synthase; CNS, central nervous system; c-PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide; D609, tricyclodecan-9-xyl-xanthogenate; GFAP, glial fibrillary acidic protein; vWF, von Willebrand Factor. 2528 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 microvascular endothelial cells were distinguished with poly- clonal antibodies specific for von Willebrand factor (vWF; factor VIII-related antigen), which is expressed in the cyto- plasm of human endothelial cells (Sigma) (16); (iii) astrocytes were identified using polyclonal antibodies against glial fibril- lary acidic protein (GFAP) (17); (iv) oligodendrocytes were stained with monoclonal antibodies to 29,39-cyclic nucleotide- 39-phosphodiesterase (CNPase; Sigma), which is specifically expressed in oligodendrocytes and Schwann cells (18); and (v) neurons were identified with a cocktail of three monoclonal antibodies (NF5139, NF160, and NF389), which bind three different neurofilament epitopes (19). Antibodies to nitroty- rosine were purchased from Upstate Biotechnology (Lake Placid, NY). The GFAP- and neurofilament-specific antibod- ies employed were kind gifts from John Trojanowski (Univer- sity of Pennsylvania). All of the primary antibodies used in these studies were unconjugated and developed using appro- priate rhodamine-labeled secondary antibodies (Sigma). The product of RT-IS-PCR for iNOS mRNA was identified by hybridization with a fluorescein isothiocyanate-conjugated oligonucleotide probe specific for the iNOS-amplified se- quence (25-mer oligonucleotide: 59-AACATTGCTGTGAT- CCATAGTFTrC-39).Using this approach,examinationwitha fluorescent microscope usually reveals cytoplasmic staining FIG. 1. Identification of cells expressing iNOS mRNA in MS brain tissue. As described, sections from MS brain were concurrently stained with RT-IS-PCR for iNOS mRNA (green) and a panel of cell-specific reagents (redyorange). The sections were photographed through a fluorescent microscope at a magnification of 31000 using dual filters for orange and green fluorescence. (A) This section, from the region of a plaque, was stained for iNOS mRNA (green) and RCA-1 (orange). (B) A similar section was stained for iNOS mRNA (green) and vWF (yellowyorange). (C) Asimilar sectionwas stained for iNOSmRNA(green)andneurofilamentantigensusingacocktailof threedistinctmonoclonalantibodies (orange). (D) This section, again from the vicinity of aplaque,was stained for iNOSmRNA(green) andCNPase (orange). (E–H)Different viewsof a single sectionstained for iNOSmRNA(green), aswell asGFAP(orange):E, thecenterof theplaque;F, theedgeof theplaque;G, 2mmfromtheplaque; H, 7 mm from the plaque. Medical Sciences: Hooper et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2529 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 for mRNA versus nuclear staining for DNA (20). At least 10,000 cells per stain combination were examined for the immunohistochemistry and mRNA signals. InductionofAcuteEAE.Six- to8-week-oldSWXJ-14 female mice were purchased from The Jackson Laboratory. For most experiments, 3–7 mice were included per treatment group or time point for analysis. EAE was elicited by immunizing mice subcutaneously at two sites on days 0 and 7 with complete Freund’s adjuvant and 100 mg of the peptide PLP139–151 (PLP; HSLGKWLGHPDKF) derived from the sequence of proteolipid protein, a major component of myelin. PLP was synthesized by the peptide synthesis facility of the Kimmel Cancer Center, Thomas Jefferson University. This immuniza- tionprotocol results inaprogressive,often fatal, formofacute EAE in female SWXJ-14 mice (21, 22). Clinical symptoms in these mice manifest first as an ascending paralysis approxi- mately 13 days after immunization, with rapid disease pro- gression until death at days 16–20. The clinical signs of EAE werescoredas: (i)piloerection, tailweakness; (ii) tailparalysis; (iii) tail paralysis plus hind limb weaknessyparalysis; (iv) tail, hind and fore limb paralysis; (v) moribund. NO Measurement. Semiquantitative measurement of NO levels in thebrain and spinal cordof control andexperimental animals was performed as described (5). Briefly, NO was spin-trapped invivousingdiethyldithiocarbamate(DETC)and iron administered subcutaneously at two separate sites. Mice were euthanized 30 min later, and spinal cord and brain tissue were snap-frozen for analysis by electron paramagnetic reso- nance(EPR)spectroscopy.NOconcentrationswereestimated from EPR spectra using a calibration curve obtained as described (5). Treatment of Acute EAE. To test their action in the treat- ment of the development of acute EAE, the compounds of interest were administered i.p. in PBS beginning on day 4 or 5afterPLPimmunization.Administrationwascontinueddaily until at least 2 days after all untreated PLP-immunized mice became severely paralyzed. Tricyclodecan-9-yl-xanthogenate (D609) inhibits iNOS induction by blocking the activity of phosphatidylcholine-specificphospholipaseC,akeyenzymein the signal transduction pathway leading to iNOS activation (23). Mice were given 1 mg of D609 (Biomol, Plymouth Meeting, PA), a dose which was selected as one-half the amount suspected to have toxicity based on in vitro experi- ments. A water soluble, carboxylated derivative of 2-phenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) was used as a NO scavenger in a dose range proven to be effective in the treatment of endotoxic shock in mice, apparently through the reductionofNOlevels (24, 25).C-PTIO(Dojindo Chemical, Bethesda) was administered at the doses indicated in the figure legends. Uric acid (2,6,8-trihydroxypurine) was used to target peroxynitrite, based on evidence for its per- oxynitrite scavenging activity (26) and its low level of toxicity as a natural product of purine metabolism in vivo. Uric acid, which is poorly soluble in water, was administered as a suspension in anarbitrarily determineddose range as detailed in the figure legends. RESULTS IdentificationofCellsExpressing iNOSmRNAinMSBrain Tissue. In Figs. 1 and 2, cells in sections of brain tissue from MS patients have been double-labeled with RT-IS-PCR for iNOS mRNA (green fluorescein isothiocyanate probe) and antibodies specific for selective cell surface markers (orange stains). In Fig. 1A, the cells staining positively for iNOS- specific mRNA (green) are also stained with RCA-1 (red), a lectin specific for cells of the monocyte lineage, the double stain appearing as patchy yellow. Fig. 1B shows that cells stainedgreen for iNOS-specificmRNAareclearlydistinguish- able from those that stain with antibodies to vWF (yellowy orange) specific for endothelial cells. Fig. 1C, reveals that neurons and neuronal processes, identified by staining with a cocktail of three monoclonal anti-neurofilament antibodies (NF) (orange), are negative for iNOS mRNA (green), which is evident in other cells in the field. In Fig. 1D, oligodendro- cytes stained with anti-CNPase antibody (orange) are distin- guished from cells identified as iNOS mRNA-positive cells (green). Expression of iNOS mRNA in Plaque Versus Nonplaque Areas of MS Brain. If local production of NO is central to the pathogenesisofMSasappears tobethecase inEAE(5), itmay beexpected thatareascontainingplaques inMSbrain sections should exhibit higher accumulations of iNOS mRNA-positive cells thanareaswithoutpathology.AsshowninFig.1E–H, this appears to be the case. Fig. 1E shows an MS plaque area with large numbers of cells resembling monocytes stained green after RT-IS-PCR for iNOS. Massive destruction of astrocytic processes (stained orange with antibodies for the astrocyte- specific marker GFAP) is apparent. Notably, none of the cells positive for iNOS mRNA reacted with the GFAP-specific reagent.Fig. 1F is aphotographof the sameMS-positivebrain section taken at the edge of the MS plaque, showing partial tissue destruction. The astrocytic processes are positive for GFAP (orange) and clearly distinct from the cells staining for iNOS-specific mRNA (green). In Fig. 1G, the same MS brain section is shown2mmaway fromaplaque.Onlya single iNOS mRNA-positive cell (green) can be seen in this area, among a number of intact astrocytic processes stained with anti-GFAP (orange). InFig. 1H, a viewof the same section fromthe same MS brain, taken 7 mm away from a plaque, is shown. At this FIG. 2. Identification of iNOS mRNA-positive mononuclear cells in the lumen of a blood vessel in the vicinity of an MS plaque (A) and the association of iNOS mRNA-positive cells with accumulations of nitrotyrosine in an MS plaque (B). (A) A brain section containing an MS plaque was stainedwith antibody to vWF(yellowyorange) andwithRT-IS-PCRusinga fluorescein isothiocyanate-labeledprobe specific for iNOSmRNA (green). (B) Asectionof anMSplaquewas stained for iNOSwithRT-IS-PCR(green)andantibodies tonitrotyrosine.The sectionswereprepared, stained, andvisualizedasdescribed inMaterials andMethodsand the legend toFig. 1.The image inAwasphotographically enlargedapproximately 310. 2530 Medical Sciences: Hooper et al. Proc. Natl. Acad. Sci. USA 94 (1997) D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 distance from the plaque, astrocytes stained with anti-GFAP antibodies (orange) show no signs of damage and no iNOS mRNA-positive cells are present. Fig. 2A shows a blood vessel from a MS plaque containing iNOS mRNA-positive mononuclear cells (green), evidently adherent to the luminal surface of the vessel (stained yellowy orange with antibody to vWF). Careful microscopic examina- tion of the slide showed evidence of possible infiltration by diapedesis of the iNOS-positive cell through the vascular endotheliumintothebraintissue(arrows). InFig.2B, a section of an MS plaque area exhibiting monocytes positive for iNOS mRNA (green) and accumulations of cell debris positive for nitrotyrosine residues (yellow) is shown. Effect of an Inhibitor of iNOS Induction, D609, on EAE. Clinical symptoms of EAE in SWXJ-14 mice immunized with PLP failed todevelop inparallelwithuntreated controlswhen themicewere treateddailywith1mgofD609beginning5days after PLP immunization and terminating on day 14 after immunization (Fig. 3). Twenty-four hours after concluding treatment, the first clinical signs of EAE developed, progress- ing into significant disease over the next few days (Fig. 3). Effect of the NO Scavenger PTIO on EAE. Daily treatment witha singledoseof c-PTIO(100mgykg) fromdays5–16after PLP immunization caused a delay in the onset and inhibited theprogressionof the clinical signs ofEAE(Fig. 4).However, at this level of treatment, disease eventually developed (Fig. 4A).Administrationof thesameamountofc-PTIOtwicedaily had a more profound therapeutic effect on the PLP- immunizedmice, delaying theonsetof clinicalEAEbyat least 3 days (Fig. 4B). When c-PTIO treatment was terminated 16 days after immunization the control mice were extensively paralyzed, whereas the treated animals exhibited only tail weakness and piloerection (Fig. 4B). Assessment of NO levels in the brains and spinal cords of the mice 1 day afterwards, revealed that spinal cord NO was only marginally lower in c-PTIO-treatedversusuntreatedmice (Fig. 5). Incontrast, the level of NO in brain tissue of c-PTIO-treated mice was considerably lower than those of the untreated mice (Fig. 5). Effect of the Peroxynitrite Scavenger Uric Acid on EAE. Analysis of SWXJ-14 mice treated with different doses of uric acid beginning 5 days after PLP immunization and continuing through day 15 showed that as little as one dose of 2 mg per day of uric acid can delay the onset of clinical signs of EAE (Fig. 6). Moreover, a daily dose of 20 mg of uric acid completely protected the mice, whereas all the controls be- came severely paralyzed. As detailed in Table 1, on the 16th dayafterPLPimmunization,NOlevels in thebrainsandspinal cords of mice treated with uric acid were significantly higher, like their untreated PLP-immunized counterparts, than those of nonimmunized controls. Nevertheless, three out of four of the uric acid-treated mice showed no signs of disease. FIG. 3. Effect of D609 on the development of EAE in SWXJ-14 mice. SWXJ-14 mice were immunized with PLP as described. Mice were either left untreated (l) or treated with 1 mg of D609 (m) daily from days 5 through 14 after immunization (arrow). Severity scores were graded and are presented as a mean. FIG. 4. Effect of administration of c-PTIO on EAE in SWXJ-14 mice. Four days after immunization with PLP, SWXJ-14 mice were either left untreated (l) or treated (m) either once (A) or twice (B) daily with 2 mg c-PTIO. In both cases, treatment was continued until day 16 after immunization (arrows). Severity scores were graded and are expressed as a mean. FIG.5. Effectofdailyadministrationofc-PTIOonNOlevels inbrain andspinalcordofSWXJ-14miceimmunizedwithPLP.NOlevelsinbrain and spinal cords of the SWXJ-14 mice described in Fig. 4A were semiquantitated using spin-trapping with DETC and EPR spectroscopy at day 17 after immunization with PLP and are presented as a mean. Medical Sciences: Hooper et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2531 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 DISCUSSION Our concurrent analysis of tissue sections from the brains of MS patients with RT-IS-PCR for iNOS mRNA and a battery of stains for subsets of CNS resident or infiltrating cells demonstrated that cells expressing iNOS message also stained with RCA-1 lectin, which selectively binds to cells of the monocyte lineage. Stains for astrocytes, neurons, oligoden- drocytes, Schwann cells, and endothelial cells were not found to react with the iNOS mRNA-positive cells. Cells positive for iNOS mRNA were concentrated in regions immediately sur- rounding the MS plaque areas of extensive damage to white matter andadiminishing gradient of positives andevidenceof damage to astrocytes could be seen at increasing distances from the plaques. Regions further removed (7 mm) from plaques contained few if any iNOS mRNA-positive cells and little evidence of the destruction of astrocytes. Furthermore, nitration of tyrosine was evident in the vicinity of plaques implicating peroxynitrite in the pathogenesis of the lesions. While our reagents were unable to distinguish between monocyteymacrophages and glial cells, it is important to note that we observed cells positive for iNOS mRNA either in the lumen of blood vessels or apparently adherent to the vascular endothelium in sections from MS brain. The apparent emer- gence of iNOS-positive cells from the blood vessel in the MS-plaque area indicates the likelihood that these are mono- cytes that have been activated in peripheral lymphoid tissues prior to infiltrating into the brain. In EAE animal models, inhibition of the induction of iNOS hasbeen successfullyused for treatmentof clinical disease (3). D609 indirectly suppresses severalbiologicalprocesses, includ- ing iNOS activation and tumor necrosis factor-a-induced apoptosis, by inhibiting the activation of phosphatidylcholine- specificphospholipaseC,anenzyme involved in theregulation of the promoter NF-kB (27). In our hands, PLP-immunized SWXJ-14 did not develop EAE while under treatment with 1 mgofD609(Fig.1).Clinical signsofEAEappeared24hrafter discontinuing D609. Because the dosage of D609 was chosen more or less arbitrarily, it is possible that a higher dose or prolonged treatment may have a more lasting effect. Alterna- tively, D609-insensitive pathways leading to the formation of active iNOS may exist (e.g., ref. 28). In the latter case, a scavenger of NO, like c-PTIO, would be expected to be more effective. Indeed, c-PTIO had significant effects on the development and severity of the clinical signs of EAE in our experiments. The therapeutic action of c-PTIO was improved by more frequent administration (Fig. 4B) as expected based on the short biological half-life of c-PTIO in the blood of experimen- tal animals. In fact, it has been reported that 82% of the c-PTIO administered to mice is excreted within 4.5 hr (25), raising the possibility that the therapeutic effects of c-PTIO seen in this study might not entirely depend on its continued presence in vivo. The finding that NO levels in the brains of PLP-immunizedmice treatedwithc-PTIOremainmuch lower than those in spinal cord evidently suggest that the clinical signsofEAE, in thismodel,maybeassociatedwithhigh levels of NO in brain rather than spinal cord (Fig. 5). Even though c-PTIOmayhaveaneffect on theotherbiological rolesofNO in the nervous and circulatory systems (29, 30), its strong therapeutic effect in EAE was without notable side effects. We have found that treatment with uric acid, a known scavenger of peroxynitrite, can virtually block clinical disease in SWXJ-14 mice. The fact that mice remain healthy when treatedwithuricaciddespitehavingelevatedNOlevels intheir brains and spinal cords provides evidence for the assumption that NO derivatives may be more toxic in EAE than NO itself. Becauseuric acid is highly insoluble in aqueous solutions (31), a significant proportion of the uric acid administered i.p. as a suspension in PBS accumulated in the peritoneum. It was therefore difficult for us to accurately determine the effective therapeutic dose. In the future, analysis of serum uric acid levels will be used to shed more light on this problem. Since, in contrast to man, mice metabolize uric acid in an additional step to allantoin (32), it is conceivable that this compound, although not known to be a free radical scavenger, may be the protective molecule rather than uric acid. However, based on its knownperoxynitrite binding activity,we speculate that uric acid is the active protective molecule in the mouse EAE system. In this investigation we have shown that treatment with an inhibitor of iNOS activation and scavengers of NO and per- FIG. 6. Effectof theadministrationof variousdosesofuric acidon the development of EAE in SWXJ-14 mice. Beginning on day 5 after immunizationwithPLP,SWXJ-14micewereeither leftuntreated(l) or treated once daily with either 2 mg (D), 4 mg (å), 8 mg (F), or 20 mg(m)ofuricacidgiven i.p.Treatmentwas terminatedonday15after immunization. Severity scores were graded and are expressed as a mean. Table 1. Effect of administration of uric acid on brain and spinal cord NO levels in EAE No. PLP* Uric acid treatment Clinical severity NO brain, mM NO spinal cord, mM 1 No None 0 ;0 ;0 2 No None 0 1.3 0.6 3 Yes None 4 0.9 2.0 4 Yes None 5 3.5 25.0 5 Yes 8 mgyday 4 1.8 1.8 6 Yes 8 mgyday 0 1.9 1.5 7 Yes 20 mgyday 0 3.0 1.4 8 Yes 20 mgyday 0 5.2 4.4 *SWXJ-14 mice, immunized with PLP as detailed in Materials and Methods, were treated once daily with 8 or 20 mg of uric acid i.p. At day 16 after immunization, clinical severity was scored and NO levels in brain and spinal cord were assessed by EPR as described. 2532 Medical Sciences: Hooper et al. Proc. Natl. Acad. Sci. USA 94 (1997) D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 oxynitrite are effective inpreventing thedevelopmentofEAE in an acute model. If these substances all function mechanis- tically as believed, they should also be effective in the treat- ment of chronic recurrent EAE. We are now investigating the mode of action of D609, c-PTIO, and uric acid in mice with chronic recurrent EAE. If treatment is successful under these circumstances,onemayconsider theuseof these substances in the therapy of chronic degenerative CNS diseases of humans such as MS. Thefactors involvedintheformationofplaques inthebrains ofMSpatientshavebeenhithertounknown.Ourdata strongly suggest that activated macrophages are causing these lesions through the production of NO and, subsequently, peroxyni- trite. Inhibitors of these substancesprovide a strong therapeu- tic effect in EAE and, because the inhibitors act at the postinductioneffector stageof thedisease, shouldbeuseful in blocking the development of CNS lesions in MS regardless of its etiology. The properties of c-PTIO are not as well known as those of the natural compound uric acid, initially isolated in the late 18th century (33). In the case of c-PTIO, toxicity studies must beperformedbefore further experimentation,whichmay lead to applications in humans. In the case of uric acid, further studies are necessary to clarify our hypothesis that there is a negative association between uric acid on the one hand and peroxynitriteand itspathogenic role inMSontheother.These studies may include a determination of whether abnormalities in the metabolism of uric acid may have some effect on the incidence or course of MS. For instance, a statistical survey of patients with gout for incidence of MS may indicate that hyperuricemia may preclude the development of MS and provide a basis for the extension of our experimental studies to the treatment of MS with uric acid. 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