Effects of etanercept on the apoptosis of ganglion cells and expression of Fas, TNF-α, caspase-8 in the retina of diabetic rats.

AIM: To evaluate the effects of etanercept on the expression of Fas, tumor necrosis factor-alpha (TNF-α) and caspase-8 in the early stage of the apoptotic pathway in diabetic rats, and to explore the therapeutic effect of etanercept on diabetic retinopathy. METHODS: A total of 60 Sprague-Dawley (SD) rats were randomly and evenly divided into 3 groups with 20 rats each, including control group, and diabetic groups with or without treatment. Streptozotocin (STZ)-induced diabetic rats were established for diabetic groups. Blood glucose and body weight were measured weekly. All the rats were sacrificed at the 12wk after treatment. The expressions of Fas, TNF-α and caspase-8 in rat retina were quantitatively detected by PCR and Western blot. The leakage of Evan blue was adopted to measure the retinal vascular leakage quantitatively, and to compare it among different groups. TUNEL method was used to compare the amount of apoptotic bodies quantitatively in rat retina ganglion cells under electron microscope. RESULTS: The expressions of Fas, TNF-α and caspase-8 in each group were compared via PCR and Western blot, in which the diabetic group with treatment was lower than those without treatment (P<0.01), but all the diabetic groups were higher than the control group (P<0.01). Evans blue leakage in the diabetic treatment group was lower than those without treatment (P<0.01), but those in the control group was the lowest compared with the other two groups (P<0.01). TUNEL method showed that the apoptotic bodies of retina in the diabetic treatment group was lower than those without treatment (P<0.01), while those in the control group was the lowest compared with the other two groups (P<0.01). CONCLUSION: Etanercept can effectively reduce the expression of Fas, TNF-α and caspase-8, as well as the retinal leakage and retinal cell apoptosis in diabetic rats.

Experimental circumferential canaloplasty with a new Schlemm canal microcatheter.

AIM: To present a new, simple, inexpensive Schlemm canal microcatheter for circumferential canaloplasty in a rabbit model. METHODS: A rabbit glaucoma animal model was established by intravitreal injection of triamcinolone acetonide. Circumferential canaloplasty with a new Schlemm canal microcatheter (patent license number: 201220029850.0) was performed. The Schlemm canal microcatheter was composed of microcatheter wall and lumen. The wall was made of high refractive index plastic optical fiber that could be attached to an illuminant so that the whole lighted microcatheter was visible during circumferential canaloplasty. The lumen could be attached to an injector for injection of viscoelastic during catheterization. Rabbits were divided randomly into the control, model and treatment groups. Intraocular pressure (IOP) was measured with a Tono-pen tonometer pre-operation and 3, 7, 14, 21 and 28d post-operation. Ultrasound biomicroscopy was performed to visualize the Schlemm canal microcatheter in the Schlemm canal and the sclera pool. RESULTS: The Schlemm canal microcatheter could be used to perform circumferential canaloplasty in the rabbit glaucoma animal model. IOP was lower in the treatment group than that in the model group 3, 7, 14 and 28d after operation. There were no significant differences in IOP between the control group and treatment group. The differences among the three groups were statistically significant (3d: F=41.985, P<0.001; 7d: F=65.696, P<0.001; 14d: F=114.599, P<0.001; 28d: F=55.006, P<0.001). CONCLUSION: Circumferential canaloplasty is safe and effective in control of experimental glaucoma model in rabbits.

Infliximab relieves blood retinal barrier breakdown through the p38 MAPK pathway in a diabetic rat model.

AIM: To clarify the mechanism of infliximab treatment in diabetic macular edema (DME) and to provide a new alternative therapy for DME. METHODS: Rats were randomly divided into the control group, the model group and the infliximab treatment group. A diabetic rat model was created. The concentration of TNF-α in the vitreous body was detected by ELISA. The expressions of B-Raf, p38, claudin-1 and occludin in the retina were detected by Western blot. The integrity of the blood retinal barrier (BRB) was measured using Evan's blue as a tracer. RESULTS: After three months and six months of the diabetes model, the vitreous TNF-α level in the model group was higher than that of the control group. It was also higher in treated group than that of the control group but was lower than that of the model group. The differences among the three groups were statistically significant (at 3mo, F=857.098, P<0.001; 6mo, F=1261.897, P<0.001). The retina B-Raf and p38 levels in the model group were higher than that of the control group. They were also higher in treated group than that of the control group but were lower than that of the model group. The differences among the three groups were statistically significant (B-Raf at 3mo, F=106.596, P<0.001 and at 6mo, F=200.681, P<0.001; p38 at 3mo, F=41.662, P<0.001 and at 6mo, F=67.979, P<0.001). The retina claudin-1 and occludin levels in the model group were lower than that of the control group. They were also lower in treated group than that of the control group but were higher than that of the model group. The differences among three groups were statistically significant (claudin-1 at 3mo, F=139.088, P<0.001 and at 6mo, F=128.415, P<0.001; occludin at 3mo, F=92.733, P<0.001 and at 6mo, F=104.478, P<0.001). The retinal Evans blue leakage in the model group was higher than that of the control group. It was also higher in treated group than that of the control group but was lower than that of the model group. The differences among the three groups were statistically significant (at 3mo, F=447.946, P<0.001; at 6mo, F=1610.732, P<0.001). CONCLUSION: In a diabetic rat model, infliximab may relieve TNF-α induced BRB breakdown via the B-Raf and p38 signaling pathway.

[Protective effects of melatonin on cultural human retinal pigment epithelial cells against oxidative damage in vitro].

OBJECTIVE: To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. METHODS: The RPE cells were seeded and divided into normal control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1 x 10(-7) mol/L, 1 x 10(-6) mol/L, 1 x 10(-5) mol/L and 1 x 10(-4) mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 micromol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic method, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. RESULTS: Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate (t = 2.25, 39.50, 68.42; P < 0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low MDA contents, and low apoptosis rate (P < 0.05). CONCLUSIONS: Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.

Rat model for anterior segment intraocular surgery induced blood-retinal barrier breakdown.

PURPOSE: To develop a practical rat model of blood-retinal barrier (BRB) breakdown induced by anterior segment intraocular surgery. METHODS: A 27-gauge needle attached to infusion tubing running to a bottle of balanced salt solution was inserted through the limbus into the rat anterior chamber. The pressure of the balanced salt solution was oscillated from 0 to 12 mm Hg above the atmospheric pressure for 30 times. The needle was removed and the anterior chamber was formed. Then the eye was exposed vertically to the light of the operating microscope. The contralateral eye was left untreated. The eyes were applied with ofloxacin ophthalmic solution after surgery. At several time points after surgery, the integrity of the BRB was assessed by fluorescence fundus angiography, optical coherence tomography, immunohistochemical staining for serum albumin and quantitative measurement using Evans blue as a tracer. RESULTS: The extravasation of fluorescein, the increased central retinal thickness, strong staining for albumin in the retina and substantially elevated retinal Evans blue leakage demonstrated BRB breakdown after anterior segment intraocular surgery. On the 1st day after surgery, the model group showed a statistically significant elevation in the retinal Evans blue leakage as compared to the contralateral control group and the normal control group. These increased and reached the peak on the 2nd day after surgery and decreased to the point that there was no significant difference as compared to the contralateral control group and the normal control group on the 7th day after surgery. CONCLUSIONS: This study establishes a practical rat model of BRB breakdown induced by anterior segment intraocular surgery.

Culture in vitro modulation of human leukocyte antigen molecules and costimulatory molecules on human retinal pigment epithelium.

PURPOSE: To determine the potential of culture in vitro to alter the human leukocyte antigen (HLA) molecules and costimulatory molecules on human retinal pigment epithelium (RPE). METHODS: Pure RPE were isolated and cultured. Two sets of RPE (normal and activated) were used. Activated RPE were obtained by incubating primary cultures of RPE with recombinant human interferon-gamma. The expression of HLA molecules and costimulatory molecules on human RPE at different passages after culture in vitro were analyzed quantitatively by flow cytometry. RESULTS: In the process of routine culture on culture flask, the duration of culture in vitro was significantly correlated with the expression of HLA-ABC molecules on the normal RPE and the activated RPE (r = -0.893, p < 0.001 and r = -0.964, p < 0.001 respectively), HLA-DR molecule on the activated RPE (r = -0.901, p < 0.001) and intercellular adhesion molecule-1 on the normal RPE (r = 0.961, p < 0.001). There were no correlations between the duration of culture in vitro and the expression of HLA-DR molecule on the normal RPE, intercellular adhesion molecule-1 on the activated RPE, B7-1 and B7-2 molecules on the normal RPE and the activated RPE. CONCLUSIONS: The chronic rejection is the major immunological rejection following RPE allogeneic graft. Culture in vitro modulation of HLA molecules and costimulatory molecules on RPE may increase lymphocyte infiltration and the activated RPE.