Direct detection of reactive nitrogen species in experimental autoimmune uveitis

Abstract

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)-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.

Fig. 1

ESR spin trapping of nitric oxide in the retina of EAU rats. ESR spectra were recorded from two inflamed retinas from Lewis rats 12 days post-immunization. Ninety minutes before sacrifice, the eyes were injected intravitreally with the spin trap, (MGD)₂-Fe²⁺; after isolation, the retinas were incubated with (MGD)₂-Fe²⁺ for 30 minutes before ESR recording. Each experiment was repeated three times, using a total of six eyes from different animals, and a representative spectrum is shown. normal control: non-immunized retina without spin trap; EAU control: EAU retina without spin trap; normal + (MGD)₂-Fe²⁺: non-immunized retina with spin trap; EAU + (MGD)₂-Fe²⁺: EAU retina with spin trap; (MGD)₂-Fe²⁺-NO: nitric oxide generated from SNAP in vitro reacted with spin trap.

Fig. 2

Effect of AG on the ESR spin trapping of nitric oxide in the EAU retina. Lewis rats with EAU were injected intravitreally with either AG or PBS (as control) two hours prior to the injection of the spin trap, (MGD)₂-Fe²⁺. Following enucleation, the retina was incubated with the spin trap for 30 minutes before ESR measurement. Each experiment was repeated three times using a total of six eyes. Representative ESR spectra are shown. EAU+(MGD)₂-Fe²⁺: EAU retina with spin trap; EAU+(MGD)₂-Fe²⁺+ AG: EAU retina with spin trap plus AG.

Fig. 3

Luminol chemiluminescence of EAU and control retinas. The retina and choroid from Lewis rats were collected at the peak of inflammation, 12 days post-immunization. Luminol (0.2 µ g/ml) was added to the tissues, and the chemiluminescence measurement commenced immediately with a Packard liquid scintillation analyzer set at an out-of coincidence mode for single photon counting. Six sets of retina/choroid were used for each sample, and each measurement was repeated three times, using a total of 18 eyes for each experimental category. Chemiluminescence is expressed as mean±standard error for each category (n=3). An ANOVA single-parameter comparison between maximal chemiluminescent count in EAU vs. that in non-immunized control eyes indicated significance at p<0.0001.

Fig. 4

Effects of bicarbonate, SOD, L-NAME, AG, and AG + SOD on luminol chemiluminescence of EAU retina/choroids. The enhancement by the bicarbonate (50 mM, n=3) addition was first determined by counting six sets of retina/choroid with and without bicarbonate. The countings from the bicarbonate/luminol/retina system were then used as a background level for evaluating the inhibition displayed by SOD (5270 units/sample; n=3), L-NAME (1 mM, n=3), AG (5 mM, n=3), and SOD (5270 units) + AG (5 mM) (n=3). The counts without the inhibitor were established first, and then the inhibitor was added. The counting was continued until it became stabilized (60 minutes). A set of six retinas and choroids were used for one measurement. Measurements were repeated three times. All eyes used were 12 days post-immunization. Single-variable ANOVA was used to compare counts with the inhibitor groups to bicarbonate/luminal/chemiluminescence without inhibitors. ^*p<0.005; ^**p<0.005; ^***p<0.0001. Control: non-immunized retina; EAU: EAU retina; HCO₃^-: EAU retina + bicarbonate; L-NAME: EAU retina + bicarbonate + L-NAME; AG: EAU retina + bicarbonate + AG; SOD: EAU retina + bicarbonate + SOD; AG + SOD: EAU retina + bicarbonate + SOD + AG.