Objective To observe the effect of amniotic homogenate on closing holes in experimental rhegmatogenous retinal detachment and investigate its mechanism. Methods Forty rabbits were randomly divided into group A, B, C and D with 10 rabbits in each group. Group A and C were the treatment groups, and group B and D were the control groups. All eyes of rabbits underwent pars plana vitrectomy, retinectomy, and fluidair exchange. The surface of the breaks was treated with 01 ml amniotic homogenate in experimental groups and 0.1 ml PBS in control groups. At the end of operation, 20% SF6 was tamponaded and the retina reattaced. The animals were executed 14 (group A and B) and 28 days (group C and D) after the surgery. The tissue sections were observed by light microscope, electron microscope and immunocytochemistry method. Results Fourteen days after the surgery, the retina reattached in 6 eyes in group A (60%) and 2 eyes in group B (20%) （P=0.021）. Twenty-eight days after the surgery, the retina reattached in 8 eyes in group C (80%) and 3 eyes in group D (30%) (P=0.046). The difference of the rate of retinal reattachment among the 4 groups were statistical significant (Plt;0.05). Light postoperative inflammation of ocular anterior segment was observed, which was controlled 3-5 days after treated with topical steroids. The result of light microscopy showed that the eyes in treatment groups had multilayer of fibroblastlike cells around the retinal breaks, adhering to the choroid and retinal pigment epithelial cells. The proliferative cells around the retinal breaks obvious less in control groups than that in the treatment groups, and the retina could not adhere to the choroid. The results of electron microscopy were the same as that of light microscopy. Immunohistochemistry staining of the fibroblastlike cells revealed positve glial fibrillary acidic protein, which suggested that the proliferative cells around the retinal breaks were retinal glial cells. Conclusions Amniotic homogenate helps to seal retinal breaks and promote retinal reattachment by stimulating the proliferation of retinal glial cells around the breaks.
Objective To observe the retinal toxicity of intravitreal injection of Bevacizumab (Avastin) in albino rabbit eyes at different doses. Methods Sixteen New Zealand albino rabbits，thirty-two eyes were divided into four groups at random. Three groups were prepared for Avastin experiment, named A, B, C. Each group received intravitreal injection of Avastin at dose 1.25 mg/0.05ml，2.5 mg/0.1ml and 6.25 mg/0.25 ml respectively. The other group named D served as a control, and accepted intravitreal injection of 0.9% normal saline 0.1 ml. Then test it by electroretinagram (ERG) after 1, 2 and 4 weeks. In addition, each group was removing two rabbitprime;s eyes to observe the retinal morphology and ultra structure by light microscope and transmission electron microscopy after intravitreal injection avastin 1, 2 and 4 weeks. Results The ERG pattern and amplitude of each group were normal after intravitreal injection Avastin 1, 2 and 4 weeks. （P＞0.05）Between study and control groups, there was no significant difference in retinal morphology which was observed by light microscope at any stage of the study. By electron microscopic observation, retinal ultramicrostructure was no evident retinal toxicity being tested both at group A and B (1.25 mg/0.05 ml and 2.5 mg/0.1 ml). But at group C (6.25 mg/0.25 ml), significant mitochondrial swelling and hydropic changes were seen in the inner segments of photoreceptors. And there was no improvement of the pathological changes in four weeks. Conclusion It is safe that intravitreal injection of Avastin in rabbitprime;s eyes at dose 1.25 mg or 2.5 mg at single time. （Chin J Ocul Fundus Dis，2008，24：193-196）
Objective To observe the oxidative damage of mtDNA, apoptosis and expression of adhesion molecules in retinal capillary cells of diabetic rat with different disease courses. Methods One hundred Sprague-Dawley rats were randomly divided into the control group and the experimental group. The rats of experimental group were induced with streptozotocin (STZ) injection creating a diabetic model. Then they were divided into DR1m, DR2m DR3m group according to disease courses. The rats of control group were divided into NR1m, NR2m, NR3m group. Rat retinal capillaries were prepared, and then the contents of undamaged mtDNA were examined by Southern blot combined with Fpg. The expression of cyclooxygenase (COX)-1 encoded by mtDNA and transcription factors A (mtTFA) mRNA were detected by real-time quantitative polymerase chain reaction (RT-PCR). Apoptosis and expression of intercellular adhesion molecule-1 (ICAM-1) were detected by terminal dUT nick endlabeling (TUNEL) immuno-fluorescence and immunohistochemistry respectively. Results The contents of undamaged mtDNA in rats of DR1m, DR2m, DR3m were less than those of NR1m、NR2m、NR3m. The contents of undamaged mtDNA in diabetic rats decreased with the increase of disease courses. In addition, the mRNA levels of COX-1 and mtTFA were downregulated in diabetic rats. The positive cells of TUNEL and ICAM-1TUNEL and ICAM-1 in diabetic rats increased with the increase of disease courses. Conclusion With the increase of disease courses, mtDNA damage and apoptotic cells are increased, while the expression of mRNA encoded by mtDNA and ICAM-1 decreased in retinal capillary cells in diabetic rats.
Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFlt-1 on retinal neovascularization (RNV).Methods Recombinant lentivirus sFlt-1（2-3）and sFlt-1（2-4）expressing the sFlt-1 (2-3) and (2-4) immunoglobulinlike regions of sFlt-1 were constructed. 96 seven-day-old C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1: normal control; group 2: experimental control; group 3: sFlt-1（2-3）; group 4: sFlt-1（2-4）.The mice in group 2-4 were exposed to hyperoxia with （75plusmn;2）% O2 for 5 days and then returned to normoxia with 21% O2;the mice received an intravitreal injection with 1 mu;l virus of empty vector, sFlt-1（2-3）,or sFlt-1（2-4）,respectively. Five days later, all mice underwent perfusion fluorecein angiography and retinal wholemont was made to observe the changes of retinal vessels; retinal sections were stained by hematoxylin and eosin and RNV endothelium cell nucleus were counted; vascular endothelial growth factor(VEGF) and VEGF receptor-2 (KDR/Flk-1) protein were measured by Western blot.Results Seventeen days after birth, the retinal area of fluorescein leakage and RNV, RNV nucleus which breaking through inner limiting membrane in group 3 and 4 were smaller or less than that in group 2（P＜0.01）; while VEGF protein didnprime;t changed much （P＞0.05）the expression of KDR/Flk-1 decreased significantly (P＜0.01). Conclusion sFlt-1（2-3）and sFlt-1（2-4）can inhibit the formation of oxygen-induced RNV,the former virus has a better effect.
Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage. Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted. Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05). Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
Objective To investigate the role of ephrin A genes in the development of oxygen induced retinalneovascularization (OIR) in mice.Methods The OIR model was established by oxygen induction in new born C57BL/6J mice.Reversed transcript polymerase chain reaction (RT-PCR) was used to measure the expression levels of ephrin A1-A5 in retinas of mice in experimental and normal control group.Results All of the ephrin A family genes expressed in normal retinas. Ephrin A1 mRNA was significantly higher in OIR group(t=3.19,P=0.019); ephrin A2 mRNA was higher in the 15-day-old OIR retinas(t=3.71,P=0.033）; ephrin A3-A5 mRNA decreased or disappeared in 12 and 13-day-old RNV mice, and increased in 15-day-old OIR mice. Conclusion Ephrin A genes are involved in the development of retina and OIR.
ObjectiveTo observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR). MethodsEPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining. EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group). EPC cells without lentivirus infection was the EPC group. Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups. 168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats). DM was introduced in the latter 3 groups by streptozotocin intravenous injection. Three months later, the rats in the DM-blank control group and DM-intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively. Immunohistochemistry was used to observe the GFP expression in rat retinas. The blood-retinal barrier breakdown was detected by Evans blue (EB) dye. The retinal histopathologic changes were observed by transmission electron microscope. The mRNA level of VEGF, matrix metallproteinases-9 (MMP-9), angiopoietin-1 (Ang-1), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsELISA showed that the levels of TNF-αand IL8 in the supernatant significantly decreased, while the levels of IL10 and VEGF increased (P < 0.05) in EPC-LV-IL10-GFP group. GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P < 0.05), and blank control group (P < 0.05). RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P < 0.05). The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang-1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P < 0.05). ConclusionsIL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR. Furthermore, EPC transplantation can increase the number of EPC and exerted their effect.
Objective To observe the expression of triggering receptor expressed on myeloid cells (TREM), Caspase-3 and interleukin (IL)-6 in optic nerve tissue of ischemic optic neuropathy (ION). Methods Twenty Sprague-Dawley rats were randomly divided into control group and model group, 10 rats in each group. The permanent ligation of bilateral internal carotid arteries (BICA) was performed for 14 days to establish sub-acute ION model as model group. The control group were separated BICA without ligation. The expressions of TREM-1, TREM-2, Caspase-3 and IL-6 in rat retina were detected by reverse transcription PCR and Western blot, respectively. ResultsCompared with the control group, the expressions of TREM-1, Caspase-3, IL-6 mRNA (t=6.058, 7.86, 6.055) and protein (t=9.671, 9.524, 14.501) in the optic nerve tissue of the model group were increased, while the expression of TREM-2 mRNA and protein (t=9.283) was decreased, and the difference was statistically significant (P＜0.05). Conclusion In ischemic optic nerve tissue, TREM-1 mRNA and protein were significantly expressed, the expressions of TREM-2 mRNA and protein decreased significantly.
Objective To observe the effects of simvastatin on endothelial progenitor cells (EPCs) from peripheral blood and retinopathy in rats with diabetic retinopathy (DR). Methods Eighty male Wistar rats were divided into normal control group (group A), DR model group (group B), DR with placebo group (group C), DR with simvastatin group (group D), with twenty rats in each group. The rats in group C and D received streptozotocin (STZ) injection to induce diabetic retinopathy. The rats in the group A and B were not intervened. The rats were gavaged with 20 mg/kg simvastatin (group D) or same volume of distilled water (group C) once a day. Flow cytometry was used to identify and count the number of EPCs from peripheral blood at the 1st，4th and 12th week after injection. Evans blue perfusion was used to detect the bloodretinal barrier (BRB) permeability. Immunohistochemical staining was used to observe the expression of CD31 in the retina. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) was used to measure the mRNA expression of eNOS, iNOS and Ang-1. The correlation between changes of EPCs and DR morphological changes was analyzed. Results At the 1st，4th and 12th week after injection, compared with the group A, the number of EPCs was decreased in group C; Compared with the group B and C, the number of EPCs was increased in group D (t=4.967, 5.648, 6.688, 6.042, 7.392, 7.454;P＜0.05); there was no statistical difference of EPCs number between group B and C (t=0.525, -0.249, -0.619; P＞0.05), group A and D (t=6.733, 2.794, -5.535; P＜0.05) respectively. The structure of retina was continuous and its capillary structure was normal in control group. The retinal layers were edema and the retina developed telangiectatic vessels and many ganglion cells developed vacuolar degeneration in group B and C. The layers tissue of retina was less edema and gradually arranged in group D. The BRB permeability was significantly increased in group B and D compared with group A. The BRB permeability was significantly decreased in group D compared with group B (F=65.808，P＜0.05). The CD31 expression in group D was obviously higher than that in group A, B and C (F=24.799，P＜0.05). The eNOS expression in group B was obviously lower than that in group A, while in group D was obviously higher than that in group B (t=-2.750，2.230；P＜0.05). The iNOS and Ang-1 expression in group B were obviously higher than those in group A, while in group D was obviously lower than that in group B (t=3.881，-1.144，4.244，-1.458；P＜0.05). There was no difference in BRB permeability, eNOS, iNOS and Ang-1 expression between group B and C (t=0.480,-0.877, 0.062, 0.220; P＞0.05). Conclusions Simvastatin could mobilize DR rats peripheral blood EPCs, induce migration and differentiation of retinal endothelial cell, slow down the DR progress by regulating the expression of endothelium factors such as eNOS and iNOS.
Objective To observe the therapeutic effect of mensenchymal stem cells (MSCs) for experimental autoimmune uveitis (EAU). Methods MSCs were obtained from Wistar rats and selected by plastic adherence. Lewis rats were divided into treatment group and control group, six rats in each group. EAU models were induced by immunization with an emulsion (0.2 ml) containing 30 mu;g interphotoreceptor retinoid-binding protein derived peptide R16 and complete Freundprime;s adjuvant. The clinical manifestations of two groups were observed. Nine to 11 day after modeling, 1 ml MSCs suspension, which contained 5times;106 MSCs, were injected into the rats in treatment group via tail vein, and the rats in control group were given equal volume of phosphate buffer solution. Fifteen day after modeling, the eyes were collected to test the proportion of interferon gamma;, interleukin-17 and Foxp3 positive cells by flow cytometry. The clinical scores were analyzed by mixed linear model and statistical analysis of variance of repeated measurement data. The results of flow cytometry were analyzed using independent-sample t test. Results Six days after immunization, mild dilatation and congestion of iris vascular was observed. Nine days after immunization, mild muddy anterior chamber, myosis and absent pupillary reaction to light were observed. Twelve days after immunization, muddy anterior chamber, occlusion of pupil and dimmed or disappeared red reflex were observed, and then inflammation was slowly reduced. From 11 to 15 days after immunization, the clinical score of treatment group was lower than that in control group, the difference was statistically significant (t=2.42, 2.21, 4.16, 5.24, 4.03; P＜0.05). The results of flow cytometry showed that MSCs treatment could decrease the proportion of CD4＋T cells, Th1 cells and Th17 cells, increase the proportion of Treg cells. Conclusion MSCs treatment can ameliorate EAU, up-regulate the expression of Treg cells and down-regulate the expression of CD4＋T cells, Th1 cells and Th17 cells.