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)-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

Immunohistochemical localization of inserted iNOS in the transgenic mouse retina.

<p>Rabbit polyclonal anti-iNOS (primary antibody) and Alexa Fluor 568 goat anti-rabbit IgG (secondary antibody) were used for the staining. A: retina from transgenic homozygote; and B: retina from C57BL/6 control. Retinal layers marked are: GCL: ganglion cell layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; and IS: photoreceptor inner segments. Note the intense staining of iNOS specifically in the IS and OPL and low intensity, dispersed staining in INL and IPL. In the control, only some low grade, non-specific staining is seen.</p

Detection of apoptotic cells in retina of iNOS transgenic mice.

<p>Retinal apoptosis was detected using <i>In Situ</i> Cell Death Detection Kit, TMR (trimethyl rhodamine) red, and the free 3′-OH from the DNA strand breaks are detected by modified nucleotides in an enzymatic reaction (TUNEL reaction). A: Apoptotic cells without nuclear staining; B: Apoptotic cells after DAPI staining; and C: C57BL/6 control retinal section with both TUNEL and DAPI staining. A large number of apoptotic cells were found specifically in the outer nuclear layer and none in the other retinal layers. A small number of apoptotic cells (3–4 cells) were routinely seen in the entire segment of control retinal sections. Substantially more apoptotic cells are visible in A without DAPI staining, since following merging with DAPI, some apoptotic cells were buried under the ONL nuclear staining.</p

Immunohistochemical localization of tyrosine-nitrated proteins in transgenic mouse retina.

<p>Antibodies used for the confocal immunolocalization are rabbit polyclonal anti-nitrotyrosine and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Propidium iodide (red) was used for the nuclear staining. The intense localization was seen specifically and uniformly in the photoreceptor inner segments (IS) and outer plexiform layer (OPL). These locations are known mitochondria-rich areas. A: retina section from homozygote; B: retina section from control. Retina layers labeled are: INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: photoreceptor inner segments and OS: photoreceptor outer segments.</p

Detection of tyrosine-nitrated proteins in iNOS-overexpressed transgenic mouse retina.

<p>The total proteins were electrophoresed on 15% polyacrylamide gel and were probed with rabbit polyclonal anti-nitrotyrosine and biotinylated goat anti-rabbit IgG antibodies. Following enhancement with ABC kit, chromogenic visualization was used for the detection. Enhanced chemiluminescence (ECL)-based visualization was also carried out routinely for the comparison. Western blot analyses were carried out in triplicate and representative results are shown. Lane 1: heterozygote line a; lane 2: heterozygote line b; lane 3: C57BL/6 control; and lane 4: homozygote. Although there are several low-intensity tyrosine-nitrated bands in the background, the major nitrated protein pattern appears to be similar in all zygotes, with a major band near 16 kDa (A: nitrated cytochrome c monomer) and a doublet near 30 kDa (B: nitrated cytochrome c dimer). Further confirmation of these bands is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043089#pone-0043089-g006" target="_blank">Figure 6</a>.</p

Map of opsin promoter-driven iNOS transgene used for generation transgenic mice with retinal iNOS-overexpression.

<p>The 4.4 kb Acc65I/Xhol mouse opsin promoter fragment was isolated and cloned into the Acc65I/Sall sites of pCMVSport6-iNOS. The polyadenylation signal was provided by the existing SV40 polyadenylation sequence which was immediately downstream of the iNOS cDNA. To release the transgene from the vector for microinjection, a double restriction enzymatic digestion employing Acc65I and Pvul was performed.</p