Phenotype of transgenic mice overexpressed with inducible nitric oxide synthase in the retina.

BACKGROUND: Unlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS) along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage. METHODS: For construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process. RESULTS: The insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina. CONCLUSIONS: We have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the oxidative stress-mediated retinal degeneration, a leading cause of blindness.

Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling.

PURPOSE: Recent discovery of microRNAs and their negative gene regulation have provided new understanding in the pathogenesis of inflammatory diseases. This study demonstrated microRNA expression profiling and their likely role in sympathetic ophthalmia, using formalin-fixed, paraffin embedded samples. METHODS: Two groups of four enucleated globes (total eight globes) from patients with clinical and histopathological diagnosis of SO (experimental samples) and one group of four age-matched, noninflamed enucleated globes (control samples) were used. Human genome-wide microRNA PCR array was performed and results were subjected to bioinformatics calculation and P values stringency tests. The targets were searched using the recently published and periodically updated miRWalk software. Quantitative real-time PCR and immunohistochemical staining were performed to confirm the validated targets in the mRNA and in the protein levels, respectively. RESULTS: No microRNA was significantly upregulated in SO, but 27 microRNAs were significantly downregulated. Among these, four microRNAs (hsa-miR-1, hsa-let-7e, hsa-miR-9, and hsa-miR-182) were known to be associated with the inflammatory signaling pathway. Only hsa-miR-9 has the validated targets, tumor necrosis factor-α, and nuclear factor kappa B1, which have been previously shown to be associated with mitochondrial oxidative stress-mediated photoreceptor apoptosis in eyes with SO. CONCLUSIONS: Identification of altered levels of microRNAs by microRNA expression profiling may yield new insights into the pathogenesis of SO by disclosing specific microRNA signatures. In the future these may be targeted by synthetic microRNA mimic-based therapeutic strategies.

Mitochondrial oxidative DNA damage in experimental autoimmune uveitis.

PURPOSE: In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress early in uveitis, before macrophage cellular infiltration. The purpose of this study was to determine whether oxidative mitochondrial DNA damage occurs early in EAU, before leukocyte infiltration. METHODS: Lewis rats were immunized with S-antigen mixed with complete Freund adjuvant (CFA) to induce EAU. Nonimmunized animals and animals injected with CFA served as controls. Animals were killed on days 3, 4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was assessed using a novel long quantitative polymerase chain reaction technique. TUNEL staining to detect apoptosis and immunohistochemical detection of leukocyte infiltration in EAU retinas were also performed at these times. RESULTS: Mitochondrial DNA damage occurred early in EAU, from day 4 to day 12. In the early phase of EAU (days 4-7), there was no inflammatory cell infiltration. On day 12 inflammatory cells infiltrated the retina and uvea. Nuclear DNA damage occurred later in EAU at day 12. Neither mitochondrial nor nuclear DNA damage was detected in the controls. TUNEL-positive staining for apoptosis was detected only at day 12 in EAU retina. CONCLUSIONS: Oxidative mitochondrial DNA damage begins at day 4 in EAU, supporting the view that oxidative stress selectively occurs in the mitochondria in the early phase of EAU, before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal degeneration in EAU.

Elevated retina-specific expression of the small heat shock protein, alphaA-crystallin, is associated with photoreceptor protection in experimental uveitis.

PURPOSE: During the early phase of experimental autoimmune uveitis (EAU), before macrophages infiltrate the retina and uvea, photoreceptor mitochondrial oxidative stress, nitration of photoreceptor mitochondrial proteins, and release of cytochrome c have been observed. However, no apoptosis has been detected during this phase. In this study, alphaA-crystallin upregulation in the retina and its antiapoptotic protective role were evaluated in early EAU. METHODS: Gene microarrays were first used to identify upregulated genes in retinas with early EAU. Among highly upregulated crystallins, alphaA was confirmed by real-time polymerase chain reaction and Western blot, and the site of upregulation was localized by immunohistochemistry. The association of alphaA-crystallin to nitrated cytochrome c and interaction with a procaspase-3 subunit was assayed. Photoreceptor apoptosis in alphaA knockout mice was compared with that in wild-type animals with EAU, by using the terminal transferase dUTP nick-end labeling assay and polymerase chain reaction. RESULTS: In early EAU, alphaA-crystallin was increased 33-fold, and the site of increase was localized to the photoreceptor inner segments. This crystallin suppressed apoptosis by associating with the nitrated cytochrome c and p24. The association with nitrated cytochrome c, in particular, appeared to be restricted to nitrated cytochrome c, and thus, no association of non-nitrated cytochrome c was detected. The knockout mice showed signs of EAU development early and showed apoptosis in the retina; no such changes were seen in the wild-type control animals. CONCLUSIONS: alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.

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.

Photoreceptor mitochondrial tyrosine nitration in experimental uveitis.

PURPOSE: In experimental autoimmune uveitis (EAU), phagocytes are thought to be the primary cells in the initiation and maintenance of pathologic tissue damage through the release of cytotoxic agents. Recently, the presence of nitric oxide synthase has been shown in mammalian mitochondria. In this study, the effect of mitochondrial peroxynitrite on the modification of cellular proteins was evaluated in the early phase of uveitis, before the infiltration of leukocytes. METHODS: Tyrosine nitration in proteins was detected by UV/Vis (visible) absorption and Western blot analysis. The identity of the nitrated protein was obtained by liquid chromatography-tandem mass spectrometry. The release of cytochrome c was assessed in whole retinal extract and in isolated mitochondria. The protein nitration in the inflamed retina was also localized by immunohistochemistry. RESULTS: Before the leukocyte infiltration in the early phase of EAU, the mitochondria-originated peroxynitrite initiated the inflammatory insult by specifically nitrating three mitochondrial proteins. In vitro nitration of the control retina by peroxynitrite donor resulted in nonspecific nitration of all major retinal proteins. After nitration, cytochrome c was displaced from its original binding site in the respiratory chain. Further, the nitration appeared to commence in the early phase of inflammation, on postimmunization day 5, long before the peak of inflammation on day 14. Immunohistochemically, tyrosine-nitrated proteins were localized exclusively in the photoreceptor inner segments, which are known to be densely populated with mitochondria. CONCLUSIONS: These data indicate that mitochondrial proteins are the prime targets of inactivation by the mitochondrial peroxynitrite and that photoreceptor mitochondria initiate the subsequent irreversible retinal damage in experimental uveitis.

Peroxynitrite-induced apoptosis in photoreceptor cells.

PURPOSE: To study the mechanisms of peroxynitrite-induced photoreceptor cell damage, using retinal cultures and a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). METHODS: Retinal explants obtained from 20-day-old Lewis rat pups, were exposed to SIN-1 for varying lengths of time at varying concentrations. Apoptosis in the photoreceptor cells was detected using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a DNA fragmentation assay. Selected retinal samples were processed for an ultrastructural analysis to confirm apoptosis. The retinas exposed to SIN-1 were tested for the expression of caspase-3 by immunohistochemistry and a Western blot analysis. The retinas were also evaluated for the prevention of apoptosis in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). RESULTS: The retinal explants exposed to SIN-1 showed a significant increase in the presence of TUNEL-positive photoreceptor cells. Similarly lineal increases in TUNEL-positive cells were seen in the presence of increasing concentrations of SIN-1. DNA ladder formation was seen with the exposure of SIN-1. Ultrastructurally, SIN-1 exposed retinas revealed typical apoptotic changes in the photoreceptor cell nuclei. The retinas preincubated with urate for 6 hours and exposed to SIN-1 for 16 hours showed significantly fewer TUNEL-positive cells compared to the retinas exposed to SIN-1 alone (p < 0.05). Moreover, the retinas exposed to SIN-1 showed the expression of caspase-3. This expression, as well as the number of apoptotic photoreceptors, significantly decreased in the presence of Z-VAD-fmk. CONCLUSIONS: These results show that peroxynitrite induces apoptosis in photoreceptor cells and that such retinal damage appears to be mediated by caspase-3. The apoptotic process can be minimized by peroxynitrite scavenger urate, as well as by the caspase inhibitor Z-VAD-fmk.

Effects of a new dexamethasone-delivery system (Surodex) on experimental intraocular inflammation models.

BACKGROUND: To investigate the effects of a new biodegradable dexamethasone drug delivery system, Surodex, in two experimental intraocular inflammation models; endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU). METHODS: Surodex was inserted into the right anterior chambers (ACs) of rats. In the EIU experiment, protein concentration, cell infiltration, and myeloperoxidase (MPO) activity in the aqueous humor were measured 24 h after injection. Eyes were evaluated histopathologically. In the EAU experiment, firstly, Surodex was administered at various days after immunization. Then, Surodex was administered on day 9 and eyes were evaluated histopathologically. Intraocular cytokine levels (IFN-gamma and IL-4) were investigated. RESULTS: In the EIU experiments, eyes with Surodex exhibited significantly reduced inflammation compared with contralateral controls. Protein concentrations, cell infiltrations, as well as MPO activity were reduced. In the EAU experiments, all rats with Surodex given on days 0 or 7 showed no or significantly reduced inflammation in both eyes. Rats treated on day 12 developed reduced inflammation only in the treated eyes. IFN-gamma levels were significantly lower in the eyes with Surodex, whereas IL-4 was not detectable. CONCLUSIONS: This new, biodegradable corticosteroid drug-delivery system is highly effective in suppressing intraocular inflammation, and should be a useful tool to manage uveitis in humans.

Upregulation of chemokine expression in the retinal vasculature in ischemia-reperfusion injury.

PURPOSE: To evaluate chemokine expression at various retinal sites after ischemia-reperfusion injury, using reverse transcription-polymerase chain reaction (RT-PCR) analysis of selected tissue obtained by laser capture microdissection. METHODS: Retinal ischemia was produced in Lewis rats by increasing intraocular pressure for 75 minutes. At 3, 6, 12, and 24 hours after reperfusion, RT-PCR was used to measure the levels of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin (IL)-8, and interferon-gamma-inducible 10-kDa protein (IP-10) mRNA expression in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), and retinal vessels, after laser capture microdissection of these retinal layers. These chemokines were further localized by immunohistochemical methods, using antibodies specific to MCP-1 and MIP-1alpha. Leukocyte infiltration into the retina was detected with immunostaining for leukocyte common antigen. RESULTS: Ischemia-reperfusion induced expression of MCP-1, MIP-1alpha, and MIP-1beta mRNA in the retinal vessels 3 hours after reperfusion. Six hours after reperfusion, expression of these chemokines and IL-8 mRNA was seen in the GCL and INL. Twelve hours after reperfusion, IP-10 mRNA expression was seen in the GCL and INL. Immunoreactive MCP-1 and MIP-1alpha were detected in the GCL, INL, and the retinal vessels 24 hours after reperfusion. No chemokine mRNA expression or immunoreactivity was detected in the ONL at any time. Leukocyte infiltration was noted at 12 hours, increasing markedly 24 hours after reperfusion. CONCLUSIONS: Ischemia-reperfusion retinal injury results in generation of highly chemotactic agents, initially in the retinal vasculature, then in the other inner retinal layers. Such differential chemokine expression may play a role in leukocyte recruitment and selective leukocyte infiltration in the inner retina, leading to retinal damage primarily localized to the ganglion cells and other inner neuronal structures.

Pathogenic role of retinal microglia in experimental uveoretinitis.

PURPOSE: To devise methods for unequivocal identification of activated retinal microglia in experimental autoimmune uveoretinitis (EAU) and to investigate their role in the development of EAU. METHODS: A group of Lewis rats underwent optic nerve axotomy with the application of N-4-(4-didecylaminostyryl)-N methylpyridinium iodide (4Di-10ASP) at the axotomy site. On days 3, 14, and 38 after axotomy, the rats were killed, the eyes were enucleated, and the retinas were stained for OX42. Another group of such axotomized rats were immunized with S-antigen peptide and were killed on days 7 through 12 after the injection with peptide. The enucleated eyes were stained for OX42 and examined by confocal microscope. After axotomy, bone marrow (Y-->X) chimeric rats were injected with S-antigen peptide and were killed on days 10 and 12 after injection. The retinas were evaluated by PCR with Y-specific primers. Finally, a group of axotomized rats was injected with the S-antigen peptide and killed on days 6, 8, 9, and 10 after injection. Their enucleated eyes were examined for microglial expression of TNFalpha and for generation of peroxynitrite. RESULTS: In the axotomized, non-EAU eyes, 4Di-10ASP-labeled ganglion cells were detectable on days 3 and 14, and 4Di-10ASP-containing OX42-positive cells (microglia) were found in the nerve fiber and other inner retinal layers on days 14 and 38. The S-antigen peptide-injected rats showed migration of the microglia (4Di-10ASP-positive and OX42-positive) to the photoreceptor cell layer on day 9, and these cells increased in number at this site on day 10. No macrophages (OX42-positive and 4Di-10ASP-negative) were present at this early stage of EAU, but such cells appeared in the retina on days 11 and 12. PCR of the chimeric EAU retinas showed an absence of the Y chromosome-amplified product on day 10, but the presence of this product was detected on day 12. The expression of TNFalpha and generation of peroxynitrite were noted in the migrated microglia at the photoreceptor cell layer on days 9 and 10 of EAU. CONCLUSIONS: In the early phase of EAU, the microglia migrate to the photoreceptor cell layer where they generate TNFalpha and peroxynitrite. Such microglial migration and activation take place before infiltration of the macrophages. These findings indicate a novel pathogenic mechanism of EAU, in which retinal microglia may initiate retinitis with subsequent recruitment of circulation-derived phagocytes, leading to the amplification of uveoretinitis.