Experimental Autoimmune Uveoretinitis In Rats Research Articles

Treatment of experimental autoimmune uveoretinitis with different natural compounds.

Uveitis is an important eye disease that potentially causes loss of sight. Although extensive studies have been conducted on uveitis, the exact pathogenesis remains to be determined. The effects of treatment with natural compounds on an experimental autoimmune uveoretinitis (EAU) rat model were examined in the present study. A total of 25 rats were divided into 5 groups: Alkaloids (n=5), saponins (n=5), flavonoids (n=5), phenols (n=5), and the normal saline group (n=5). The rats in each group were treated with an intraperitoneal injection of proper alkaloids (berberine hydrochloride), saponins (steroidal saponins), flavonoids (baicalein), or phenols (chlorogenic acid) or physiological saline, respectively. The rats' aqueous humour and crystalline lens was then observed under the slit lamp periodically, looking for signs of inflammation. After 2 weeks, the rats were sacrificed and the degree of pathological changes on their eyeballs under different treatment methods were determined using an optical microscope. The expression of the interleukin (IL)-17 gene in the ocular tissues of the rats was assessed via RT-PCR and western blot analysis. Apoptosis on the rats' retinal tissues was detected using flow cytometry. The results showed that rats injected with phenols (chlorogenic acid) had serious ocular vascular dilatation, iris hemorrhage and purulent exudation; those injected with alkaloids (berberine hydrochloride) and flavonoids (baicalein) had a more mild form of inflammation; and those administered saponins (steroidal saponins) had only mild inflammation signs. Following detection of IL-17 mRNA and protein expression levels in the ocular tissues of rats of the five groups, it was found that their expression was lowest in the saponin-treated group and the other differences in expression were all statistically significant (P<0.05). A comparison with other groups revealed that cell apoptosis in the eyes of rats in the saponin group was the most prominent, reflecting a beneficial decrease in the amount of inflammatory cells on the lesion. Based on these findings, natural compounds such as saponins (steroidal saponins), alkaloids (berberine hydrochloride), and flavonoids (baicalein), but not phenols (chlorogenic acid), can inhibit the clinical symptoms of EAU in rats to a certain extent and reduce cell apoptosis. The most promising results in the present study were obtained using steroidal saponins.

MicroRNAs in retina during development of experimental autoimmune uveoretinitis in rats

PurposeTo determine the changes in the expression profiles of microRNAs (miRNAs) in retinas during the development of experimental autoimmune uveoretinitis (EAU) in rats.MethodsThe levels of interleukin-1β (IL-1β) and monocyte chemoattractant...

Correlation of optical coherence tomography with clinical and histopathological findings in experimental autoimmune uveoretinitis

Optical coherence tomography (OCT) is becoming the state-of-the-art method for the non-invasive imaging of a variety of ocular diseases. The aim of this study was to assess the application of OCT for the in vivo monitoring and follow-up of pathological changes during experimental autoimmune uveoretinitis (EAU) in rats. Initially we established OCT imaging in healthy brown Norway rats and correlated it with retinal histology. Subsequently, we induced EAU and imaged animals by OCT throughout the pre-peak, peak, and post-peak phases of the disease. The sensitivity of OCT imaging was determined by comparison with clinical EAU and histopathology scores obtained ex vivo at several time points throughout the disease course. Our data demonstrate that OCT imaging of the healthy rat retina closely correlates with histological observations and allows the clear visualization of all retinal layers. After induction of EAU, the first pathological changes could be detected by OCT at day (d) 8 post-immunization (p.i.) which corresponded to the time point of clinical disease onset. An increase in retinal thickness (RT) was detected from d10 p.i. onwards which peaked at d16 p.i. and decreased again to near control levels by d20 p.i. We introduce a novel semi-quantitative OCT scoring which correlates with histopathological findings and complements the clinical scores. Therefore, we conclude that OCT is an easily accessible, non-invasive tool for detection and follow-up of histopathological changes during EAU in rats. Indeed, significant differences in RT between different stages of EAU suggest that this OCT parameter is a sensitive marker for distinguishing disease phases in vivo.

Suppression of Experimental Autoimmune Uveoretinitis by Anti-IL-17 Antibody

Purpuse: To determine whether treatment with anti-IL-17 antibody could suppress the intraocular inflammation of experimental autoimmune uveoretinitis (EAU) in rats. Methods: Rats were immunized with interphotoreceptor retinoid binding protein (IRBP) R16 peptide and were treated with anti-IL-17 antibody. Clinical signs of inflammation and ocular histological sections were observed and graded. Cytokine levels of supernatants of cells from draining lymph nodes were measured by enzyme-linked immunosorbent assay (ELISA). Delayed-type hypersensitivity (DTH) and lymphocyte proliferation assay (LPA) were detected. Results: Treatment of EAU with anti-IL-17 antibody delayed the onset of ocular inflammation and markedly inhibited the development of EAU. Antigen-specific DTH and LPA were suppressed, whereas the level of interferon (IFN)-γ produced by draining lymph node cells was increased after treatment with anti-IL-17 antibody. There was no significant change of IL-5 level as compared with control rats. Conclusions: These findings demonstrate that blockade of endogenous IL-17 activity by treatment with anti-IL-17 antibody attenuates EAU in rats. IL-17 rather than IFN-γ plays a crucial role in the development of EAU, and that antagonism of IL-17 could be useful for the treatment of human intraocular autoimmune diseases.

Immunomodulatory effects of linomide in animals immunized with immunopathogenic retinal antigens: dissociation between different immune functions.

Linomide (LS-2616, quinoline-3-carboxamide) has been reported to exert a diverse range of effects on the immune system. On one hand, this drug was found to stimulate the immune system and to enhance activities such as DTH or allograft rejection. On the other hand, linomide was shown to inhibit the induction of experimental autoimmune encephalomyelitis and myasthenia gravis, as well as the development of diabetes in non-obese diabetic (NOD) mice. Here we report the effects of linomide in animals immunized with uveitogenic retinal antigens. Treatment with linomide completely inhibited the development of experimental autoimmune uveoretinitis (EAU) in mice immunized with interphotoreceptor retinoid-binding protein and markedly suppressed EAU in rats immunized with S-antigen (S-Ag). In addition, linomide-treated rats exhibited reduced antibody production and lymphocyte proliferative response to S-Ag. In contrast to these suppressive activities, linomide treatment did not affect the development of adoptively transferred EAU in rats and moderately enhanced the DTH reactions to S-Ag in immunized rats in which EAU and other immune responses to this antigen were suppressed.

Effects of FTY720, a Novel Immunosuppressant, on Experimental Autoimmune Uveoretinitis in Rats

The immunosuppressive properties of FTY720, a novel immunosuppressant obtained by structural modification of ISP-I isolated from the fermentation broth of Isaria sinclairii, were studied in experimental autoimmune uveoretinitis (EAU) in rats. Lewis rats were immunized with S-antigen and treated with FTY720 (0.03, 0.06, 0.1 mg kg-1day-1) or distilled water for 16 days after the immunization. FTY720 suppressed the incidence and intensity of EAU in a dose-dependent manner as demonstrated by clinical and histological examinations. The drug significantly suppressed the serum levels of antibodies to S-antigen and antigens-specific lymphocyte proliferation. The number of peripheral lymphocytes, but not neutrophils, was markedly reduced by FTY720 treatment. FTY720 also suppressed the intensity of EAU when it was given from the day of EAU onset. These results indicate that FTY720 has intense immunosuppressive effects on EAU in rats and may be a potential candidate for use in the treatment of patients with autoimmune uveitis.

Suppressive effects of a novel compound on interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis in rats

The immunosuppressive effect of ethyl O-(N-(pcarboxyphenyl)-carbamoyl)-mycophenolate(CAM) was examined in interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveoretinitis (EAU) in rats. Lewis rats immunized with bovine IRBP were treated with various oral doses of CAM postimmunization. The degree of inflammation was assessed clinically each day and histologically on day 14 or day 20. Production of various cytokines and IRBP-specific antibody, as well as IRBP-specific proliferation response, was assessed. Complete inhibition of EAU in rats, both by clinical and histologic criteria, was achieved with 50 mg/kg CAM when administered daily for 14 days following IRBP immunization. Partial inhibition was observed at lesser doses of CAM. This CAM-mediated response was accompanied by diminished production of cytokines interleukin-2, interferon-γ and tumor necrosis factor-α, as well as a reduction in IRBP-specific antibody production. Furthermore, administration of CAM either in the induction phase only (days 0–7) or in the effector phase only (days 9 or 11 to day 20) was also capable of suppressing EAU, as assessed histopathologically on day 20. We conclude that CAM is effective in suppressing EAU in rats and its mechanism of action appears to involve modulation of T cell function.

Kinetics of intraocular cytokines in the suppression of experimental autoimmune uveoretinitis by type I IFN.

The systemic administration of IFN-alpha/beta was previously found to suppress inflammation in rats with experimental autoimmune uveoretinitis (EAU); however, an effect on the systemic immune response was not identified. In order to investigate an immunological basis for suppression at the intraocular level, rats immunized with interphotoreceptor retinoid-binding protein (IRBP) were administered daily intramuscular injections of 10(5) IU IFN-alpha/beta and cytokines were measured by ELISA in intraocular extracts prepared by ultrasonification at various timepoints throughout the course of EAU. In control EAU, intraocular concentrations of IFN-gamma were found to be non-detectable on day 8 before the onset of inflammation, significantly elevated on day 12 at peak inflammation (182+/-106 pg/ml), then non-detectable again on day 16 after inflammation had begun to subside. In contrast, intraocular IFN-gamma in IFN-alpha/beta-treated rats remained non-detectable or low at all timepoints. Measurement of intraocular IL-2 revealed no difference between the two groups of rats. Intraocular IL-4 concentrations were elevated in rats treated with IFN-alpha/beta, although this cytokine was also detected in the same range in controls as well as normal rats. Finally, intraocular IL-10 was non-detectable on day 8, significantly elevated at peak inflammation on day 12 (588+/-139 pg/ml), then decreased to low levels on day 16 in control EAU rats, while remaining non-detectable or low in IFN-alpha/beta-treated rats. These results suggest that acute inflammation in IRBP-induced EAU in rats involves both IFN-gamma and IL-10 at the local intraocular level, and that systemic administration of IFN-alpha/beta inhibits EAU via a mechanism that involves suppression of both cytokines.

Intraocular cytokine quantification of experimental autoimmune uveoretinitis in rats

Elevations of inflammatory cytokines have been implicated in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in rats, although such analysis has relied on indirect methods of assessment such as measurement of mRNA content. In this study, we examined the feasibility of directly measuring cytokine concentrations in intraocular extracts prepared by ultrasonic disruption. Cytokines were measured by ELISA in eyes from EAU-induced Lewis rats immunized with interphotoreceptor retinoid-binding protein (IRBP), and compared to eyes from rats immunized with adjuvant only and from normal rats. Interferon-? (IFN-?), tumor necrosis factor-a (TNF-a), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were detectable in EAU eyes at near peak inflammation, with IFN-? achieving the highest mean concentration (331 pg/ml). In eyes from rats immunized with adjuvant only and in normal eyes, IFN-?, TNF-a, and IL-10 were nondetectable. IL-2 and IL-4 were detected at significantly lower mean concentrations (32.3 pg/ml and 69.4 pg/ml, respectively) compared to EAU eyes (217 pg/ml and 230 pg/ml, respectively); IL-4 was also detected in eyes from rats immunized with adjuvant alone (141 pg/ml). Thus, a direct method of measuring intraocular cytokine concentrations was successfully applied to reveal an elevation of IFN-?, TNF-a, IL-2, IL-4, and IL-10 in EAU eyes from rats immunized with IRBP, compared to rats immunized with adjuvant alone and to normal rats. These cytokine elevations reflect the local intraocular environment near peak inflammation, and suggest an important role for these cytokines in the mechanisms of onset and resolution of EAU in rats.

Effect of type I interferon on experimental autoimmune uveoretinitis in rats

In order to establish a scientific basis for the potential use of type I interferons (IFNs) in clinical uveitis, we examined the effect of a preparation of IFN-a/ß on experimental autoimmune uveoretinitis (EAU). Lewis rats were immunized with bovine interphotoreceptor retinoid-binding protein (IRBP) and given daily intramuscular injections of 10 5 IU mouse natural IFN-a/ß. Intraocular inflammation was assessed by slit-lamp biomicroscopy and histopathological examination. Rats treated daily with IFN-a/ß starting on the day of IRBP immunization showed decreased intraocular inflammation as well as a slight delay in onset of inflammation when compared to control rats. This effect was also observed to a lesser extent in rats treated during either the induction phase of EAU only, or starting immediately after the onset of inflammation in the effector phase of EAU. Measurement of IRBP-stimulated splenocyte proliferation and serum anti-IRBP antibody subtypes did not reveal a significant difference between IFN-a/ß-treated rats and control rats. Measurement of cytokine production by IRBP-stimulated splenocytes in vitro showed significantly decreased TNF-a for IFN-a/ß-treated rats compared to control, but no difference for IFN-?, IL-2, IL-4, and IL-10. These results indicate that systemic administration of IFN-a/ß suppresses IRBP-induced EAU in rats, and suggest that such suppression may be mediated in part by a reduction in TNF-a production.

Marrow-derived activated macrophages are required during the effector phase of experimental autoimmune uveoretinitis in rats

Purpose. Experimental autoimmune uveoretinitis (EAU), an established model for human endogenous (autoimmune) posterior uveitis, is a CD4+ T cell-mediated disease inducible in Lewis rats by intradermal inoculation with retinal antigens. Immunohistochemical studies have previously documented the lymphocyte profiles during various stages of the disease process. The purpose of the present study was to investigate the role of macrophages in EAU.Methods. EAU was induced in Lewis rats, and the effect of macrophage depletion, using the drug dichlorodimethylene diphosphonate (Cl2MDP) encapsulated in liposomes and administered intravenously, was assessed based on the clinical and histological profile of the disease.Results. The results have shown that in control animals macrophages occur early, feature prominently throughout the course of the disease and display considerable heterogeneity: marrowderived ED1+ cells and ED3+ cells are the major infiltrating cells, with many cells also expressing ED7 and ED8. In contrast, few cells expressed the ED2 antigen during EAU, even though ED2+ “resident” macrophages occur in the normal choroid. Macrophage depletion, using intravenously injected dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, caused a delay in the onset and a reduction in the severity of EAU when administered during the “effector” stage of the disease, i.e. 9–11 days after inoculation with retinal antigen. The delay in disease onset was greater when liposomes were mannosylated and was accompanied by a reduction in the overall inflammatory cell infiltrate into the eye and reduced tissue damage. In addition, there was a reduction in the level of expression of MHC Class II antigen and CR3 (ED7) antigen, a marker of macrophage activation, in Cl2MDP-treated animals compared to controls.Conclusion. These results suggest that blood-borne, activated macrophages are major effectors of tissue damage during EAU. Curr. Eye Res. 17: 426–437, 1998.

Choroidal mast cell dynamics during experimental autoimmune uveoretinitis in rat strains of differing susceptibility

Choroidal mast cells have been implicated in the pathogenesis of experimental autoimmune uveoretinitis (EAU). The aim of the present study was to examine the dynamics of choroidal mast cells during the course of EAU in rat strains of varying susceptibility. Histochemical staining showed choroidal mast cell degranulation in Lewis rats, a highly susceptible strain, commenced nine days post-immunisation, and peaked at day 11, at which time the percentage of degranulated choroidal mast cells (32. 5± 4%) was significantly higher than controls (15. 3 ± 3%; p <0.05). At day 14, mean choroidal mast cell density was significantly reduced (from 23. 6 ± 1/mm(2) tot 16. 2 ± 2/mm(2); p<0.05) and early signs of choroidal mast cell regeneration were evident. Immunisation of PVG/C (moderate susceptibility) and Brown Norway (very low susceptibility) rats produced a similar pattern of morphological changes. Onset of clinical signs in Lewis rats, which possess approximately 1100 to 1800 choroidal mast cells per eye, occurred one day following commencement of choroidal mast cell degranulation but prior to the peak of degranulation. However, in PVG/C and Brown Norway rats, which possess only approximately 70 and 110 choroidal mast cells per eye respectively, onset of disease was not temporally linked to commencement of degranulation. Production of antigen-specific IgE during the course of EAU was extremely low in all three strains. These results indicate that choroidal mast cells may be important in the pathogenesis of EAU in Lewis rats but not in PVG/C or Brown Norway rats and that non-IgE mediated degranulation may play a role in disease induction.

Cyclosporine therapy of guinea-pig autoimmune uveoretinitis induced with autologous retina.

Pigmented guinea-pigs were immunized by a single bilateral footpad injection of their own retina from one enucleated eye, mixed with complete Freund's adjuvant. Ocular inflammation started on the third week, and was still detectable in the fourth month. Daily subcutaneous injections of Cyclosporine (CsA) (20 mg/kg) from the day of immunization totally prevented the ocular disease, clinically and histologically, confirming earlier experiments in S-antigen induced experimental autoimmune uveoretinitis in rats. When CsA injections (20 mg or 10 mg/kg) were started at the onset time of EAU, a rapid regression of the inflammation occurred. This curative therapy was given during two months. No relapse was observed after having stopped the treatment. On the fourth month, only mild lesions of photoreceptors were found histologically, compared to extensive retinal destruction in controls. Most control guinea-pigs developed antibodies to antigens contained in guinea-pig retinal extract as tested by ELISA. The serum antibody content was lower in treated than in control animals. The autologous model of EAU in pigmented guinea-pigs seems to be very appropriate for testing immunosuppressive drugs in conditions close to clinical practice.