Direct Detection of Reactive Nitrogen Species in Experimental Autoimmune Uveitis

PURPOSE: 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-Fe2+, 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-Fe2+-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.

Chemiluminescence Study of Pe roxynitrite in Experimental Autoimmune Uveitis

The purpose of this study is to evaluate the extent of peroxynitrite generation in the pool of radicals/oxidants from intraocular inflammation using luminol-dependent chemiluminescence(LDCL)method. S-antigen induced uveitis was produced in Lewis rats. The rats were killed at the peak of inflammation, and the retinas and choroids were collected for the LDCL. Sodium bicarbonate was used to confirm the peroxynitrite signal. Superoxide dismutase(SOD), N-nitro-L-arginine methyl ester(LNAME)and aminoguanidine(AG)were tried to evaluate the inhibitory effect of superoxide and nitric oxide. LDCL counts for 6 inflamed and 6 control retina/choroid preparations were 66, 429+/-413 cpm and 13, 941+/-105 cpm, respectively(p<0.01). In the presence of bicarbonate, emission was increased by 125.3+/-6.6%(n=6)and the signal was sustained for 2 hours. SOD, L-NAME and AG suppressed the LDCL by 39.8+/-6.1%(n=3), 20.4+/-4.4%(n=3) and 35.9+/-4.0 %(n=3), These observations suggest that peroxynitrite contributes considerably to the generation of the total pool of reactive species by the cellular infiltrate. The presence of a high level of peroxynitrite, a potent oxidizing and nitrating agent, in inflamed retina may cause irreversible tissue damage to the photoreceptors.