| PurposeDiabetic macular edema (DME) is the most common cause of moderate vision loss in working-age Americans. The Early Treatment Diabetic Retinopathy Study (ETDRS) demonstrated that focal laser photocoagulation in the macular provides benefit to patients with DME, but the benefit is modest. In general, patients with DME are counselsed that with multiple focal laser photocoagulation treatments over the course of many months (often years) there is a good chance of preserving their vision, but they should not expect visual improvement.Resent clinical research reported that intravitreous application of glucocorticoids could ameliorate the degree of macular edema and improve visual acuity. Since glucocorticoids has affirmative anti-inflammation effect and also has satisfactory curative effect for inflammatory macular edema, such as uveitis-associated and post-operative macular, so, we speculated that weather inflammatory mechanism and inflammation-associated cytokines can contribute to the development of DME. In our research, we exammed if some inflammation-associated cytokines could express in retinas of STZ induced WISTAR rats and evaluated its vascular leakage induction effect.Methods1. Retinal Capillary Endothelial Cells (BRCEC) culture:Fresh bovine eyes were bisected posterior to the limbus and vitreous was removed by gentle traction with forceps. Retinas were detached from the optic nerve and cut into small segments. Digestion was allowed to continue for 2 hours in 0.25% collagenase. The suspension then was passed through a 10um mesh. Trapped microvessel fragments werecollected in a glass flask by washing three times, then transferred to a 50ml centrifuge tube. The microvessel suspension was centrifuged and resuspended in DMEM supplemented with 15% FBS, 2ng/ml bFGF, 100ng/ml heparin, 100U/mL penicillin and 100ug/mL streptomycin. BRCEC were selected with the character of easier to attachment and to be passaged with a lower concentration of trypsin than pericytes. Immunocytochemistry was performed using a monoclonal antibody against the von Willebrand's factor (VIII factor) for BRCEC identification.2. Reverse Transcription-Polymerase Chain Reaction:RT-PCR analysis was performed to evaluate the transcription of five tight junction proteincontent: zo-1, zo-2, zo-3, claudin-2 and occludin. The primer was designed as:Zo-1 sense: 5'gag ggc gac cag ate etc 3'Zo-1 anti-sense: 5'aaa atg ggt tet gat ata gaa ag 3'Zo-2 sense: 5'acc aga ttc tga agg tga aca 3' Zo-2 anti-sense: 5'ctt etc aca ttc aaa gtg get 3'Zo-3 sense: 5'aac gac gtg ggc ate ttc g 3' Zo-3 anti-sense: 5'atg egg atg tag aag gag tc 3'occludin sense: 5'ctg gat cag gga ata tec ac 3' occludin anti-sense: 5'ctc ttc act ttc ttc tet ata g 3'claudin-2 sense: 5'gct ect cgt ggc get eg 3' claudin-2 anti-sense: 5'cag gag cag cag age age 3'BRCECs incubated in 20mM glucose for 1 or 2 weeks, 100ug/L IL-6 and IL-1, 50mg/L and 100mg/L PGE2 for one day. The total RNA of was extracted with Trizol(GIBCO) and the cDNA of the five tight junction proteins was amplified with RT-PCR.3. Diabetes Mellitus Model Induction:Wistar rats weighing 150 to 200g were used to induce DM by intraperitoneal injection of streptozotocin (Sigma) in 10mM sodium citrate buffer, pH 4.5. Diabetes was confirmed by a blood glucose level higher than 12mmol/L. The control rat was age and sex-matched and was intraperitoneally injected with the same volume of sodium citrate buffer as that used in diabetic rat.4. Examination of Retinal Vascular Leakage:2% Evans blue was intravenously injected and the eye ball was enucleated 10 minutes later. After fixation with 4% paraformaldehyde in 0.1M phosphate buffer, retina was flatmounted on glass slides and observed under microscopy with green fluorescence.5. ImmunohistochemistryAfter retina cryostat sections were made, sections were fixed in acetone at room temperature for 30 minutes, incubated in 0.5% H2O2 for 20 minutes, followed by incubation with 2% normal serum of the same animal as tha...
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