Xiaoguang Cao

Nutrient Supplementation with n3 Polyunsaturated Fatty Acids, Lutein, and Zeaxanthin Decrease A2E Accumulation and VEGF Expression in the Retinas of Ccl2/Cx3cr1-Deficient Mice on Crb1rd8 Background

The Age-Related Eye Diseases Study 2 (AREDS2) clinical trial is assessing the effects of higher dietary xanthophyll (lutein and zeaxanthin) and long-chain n3 polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on progression to advanced age-related macular degeneration (AMD). This studys purpose was to examine the retinal effects of the AREDS2 formulation on Chemokine (C-C motif) ligand 2 (Ccl2(-/-))/CX3C chemokine receptor 1 (Cx3cr1(-/-)) mice on Crumbs homolog 1 retinal degeneration phenotype 8 (Crb1(rd8)) background (DKO), which develop focal retinal lesions with certain features similar to AMD. DKO and C57BL/6N rd8 background mice (WT) were bred and randomized into 4 groups. Two groups, WT mice on AREDS2 diet (A-WT) and DKO mice on AREDS2 diet (A-DKO), were supplemented daily with 1.76 μmol of lutein, 35.1 μmol of zeaxanthin, 215 μmol EPA, and 107 μmol of DHA, and 2 control groups, WT mice on control diet (C-WT) and DKO mice on control diet (C-DKO), were fed an isocaloric diet. All mice had monthly fundus photos and were killed after 3 mo for biochemical and histologic analyses. After 3 mo, 81% of A-DKO mice had lesion regression compared with 25% of C-DKO mice (P < 0.05). Toxic retinal 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E) concentrations were significantly lower in A-DKO compared with C-DKO mice. The outer nuclear layer thickness in A-DKO mice was significantly greater than that in C-DKO mice. Retinal expression of inducible nitric oxide synthase (iNos) tumor necrosis factor-α (Tnf-α), Cyclooxygenase-2 (Cox-2), interleukin1beta (IL-1β), and vascular endothelial growth factor (Vegf) was significantly lower in A-DKO compared with C-DKO mice. Xanthophylls and LCPUFAs have antiinflammatory, neuroprotective, and antiangiogenic properties. Our data provide potential mechanisms by which the AREDS2 formula has a protective effect on retinal lesions in DKO mice.

Naloxone Ameliorates Retinal Lesions in Ccl2/Cx3cr1 Double-Deficient Mice via Modulation of Microglia

PURPOSE The role of naloxone, an opioid receptor antagonist, on microglial inhibition and neuroprotective effects has been reported in lipopolysaccharide (LPS)-induced neurodegeneration and light-induced photoreceptor degeneration. The authors evaluated the effects of naloxone on Ccl2(-/-)/Cx3cr1(-/-) (DKO) mice, a murine model of age-related macular degeneration (AMD). METHODS Two-month-old DKO and wild-type controls were given daily intraperitoneal injections of naloxone or PBS for 2 months. Animals were examined monthly by funduscopy. Ocular tissue was analyzed histologically and in retinal flat mount preparations. Ocular A2E was measured using HPLC. Quantitative RT-PCR analyzed TNF-α, IL-1β, IL-10 and TLR4 transcripts in the DKO eyes and LPS activated culture microglial cells. Serum nitrite was measured using Griess colorimetric reaction. RESULTS Naloxone ameliorated the clinical progression and severity of retinal lesions in the DKO mice compared with those of untreated controls. Histopathology also showed less focal retinal degeneration in the treated DKO mice than in controls. The aggregation of microglia in the outer retina in DKO mice was significantly reduced in naloxone-treated animals compared with control untreated DKO. Ocular TNF-α, IL-1β, and TLR4 transcripts and A2E were significantly lower in naloxone-treated DKO animals and cultured microglial cells than in controls, as were serum nitrite levels. CONCLUSIONS Naloxone significantly reduces the progress of retinal lesions in DKO mice. Naloxone modulates microglia accumulation and activation at the site of retinal degeneration, which may be mediated by inhibition of the proinflammatory molecules of NO, TNF-α, and IL-β. The potential therapeutic effects of naloxone on retinal degeneration, including AMD, warrants further investigation.

Enhanced HtrA2/Omi Expression in Oxidative Injury to Retinal Pigment Epithelial Cells and Murine Models of Neurodegeneration

PURPOSE To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H(2)O(2)-induced oxidative stress in human RPE, in the Ccl2(-)(/)(-)Cx3cr1(-)(/)(-) double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice. METHODS Oxidative stress was induced in ARPE-19 cells by 1 mM H(2)O(2) for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice. RESULTS H(2)O(2)-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H(2)O(2)-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi(-)(/)(-) photoreceptors and RPE. CONCLUSIONS These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.

Evidence for enhanced tissue factor expression in age-related macular degeneration.

Tissue factor (TF) is the primary initiator of blood coagulation. In addition to hemostasis, TF can initiate intracellular signaling and promote inflammation and angiogenesis, the key processes underlying the pathogenesis of age-related macular degeneration (AMD). AMD, the leading cause of irreversible blindness among the elderly, involves many genetic and environmental risk factors, including oxidative stress and inflammation. In this study, TF expression was examined in human AMD tissue and in the eyes of a model of AMD, the Ccl2−/−/Cx3cr1−/− (DKO) mouse, as well as in the ARPE-19 cell line after lipopolysaccharide (LPS) and H2O2 stimulation. Total RNA was extracted from tissue samples and further analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate TF protein expression. In the human retina, a 32-fold increase of TF mRNA expression was detected in AMD macular lesions compared with normal maculae. TF protein expression was also enhanced in human AMD maculae. Similarly, TF transcript and protein expression were moderately increased in retinal lesions, neuroretinal tissue, and cultured RPE cells of DKO mice compared with age-matched wild-type mice. TF expression level correlated with age in both wild-type and DKO mice. In order to better understand how AMD might lead to enhanced TF expression, 1, 5, and 10 μg/ml LPS as well as 100 and 200 μM H2O2 were used to stimulate ARPE-19 cells for 24 and 2 h, respectively. LPS treatment consistently increased TF transcript and protein expression. H2O2 alone or in combination with LPS also moderately enhanced TF expression. These results indicate that upregulated TF expression may be associated with AMD, and inflammatory and oxidative stress may contribute to TF expression in AMD eyes.

Morning Glory Syndrome Associated with Posterior Lenticonus

The clinical features of the morning glory syndrome (MSG) are demonstrated in a 12-year-old male patient with the posterior lenticonus in the left eye. This patient had retinal detachment in the left eye. A complete ocular examination was performed and the patient underwent a pars plana vitrectomy of the left eye. Slit-lamp examination revealed the posterior lenticonus with the posterior subcapsular opacities in the left eye. The fundus showed the symptoms of MGS. The discs were pink and deeply excavated, surrounded by a ring of chorioretinal pigmentary disturbance. The retina has remained reattached for six months after surgery. Although most cases of MGS present with retinal and vitrea abnormalities, it may also occur in association with the lens anomalies, including the posterior lenticonus and subcapsular cataract. This association may be helpful to explore the pathogenesis of MGS.

Partial thickness sclerectomy and intravitreal anti-VEGF therapy for intractable uveal effusion syndrome

PurposeTo report a case series of three patients with intractable uveal effusion syndrome (UES), treated with partial thickness sclerectomy and intravitreal anti-VEGF therapy.MethodsThree patients with intractable UES were included. All patients underwent intravitreal anti-VEGF therapy to facilitate resolution of uveal effusion. The concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF and VEGF in aqueous humor were measured.ResultsAfter the last intravitreal injection, all three eyes had total resolution of the chorioretinal detachment or subretinal fluid. One eye experienced improvement in visual acuity. All patients were free from recurrence during the follow-up period. Aqueous IL-6, IL-8 and VEGF concentrations were elevated in all cases.ConclusionsOur current data provided the evidence that VEGF was increased in eyes with intractable UES and anti-VEGF therapy was effective, suggesting that partial thickness sclerectomy and intravitreal anti-VEGF therapy could be a new choice for intractable UES.