Direct detection of reactive nitrogen species in experimental autoimmune uveitis.
OBJECTIVE
Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)(2)-Fe(2+), as a spin trap.


METHODS
Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer.


RESULTS
The ESR three-line spectrum (g=2.04; a(N)=12.5 G) obtained was characteristic of the adduct [(MGD)(2)-Fe(2+)-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite.


CONCLUSIONS
The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.
Bae
Sun Ryang
SR
Department of Ophthalmology & Laboratory of Visual Science, College of Medicine, The Catholic University of Korea, Daejon St. Marys Hospital, Jung-gu, Daejon, Korea. sunrbae@yahoo.com
Wu
Guey Shuang
GS
Sevanian
Alex
A
Schultz
Brian E
BE
Zamir
Ehud
E
Rao
Narsing A
NA
D000818:Animals; D019393:Arrestin / Q000276:immunology; D001327:Autoimmune Diseases / Q000276:immunology / Q000378:metabolism*; D002829:Choroid / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D011917:Rats, Inbred Lew; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D013012:Sorbitol / Q000031:analogs & derivatives; D013113:Spin Labels; D019097:Spin Trapping; D013859:Thiocarbamates; D014605:Uveitis / Q000276:immunology / Q000378:metabolism*
D019393:Arrestin; D010446:Peptide Fragments; D026361:Reactive Nitrogen Species; D013113:Spin Labels; D013859:Thiocarbamates; D013012:Sorbitol; C041262:N-methyl-D-glucamine dithiocarbamate