Objective Methods Ninety male Wister rats were randomly divided into normal control group, diabetic group and FTY720 group, thirty rats in each group. Diabetes was induced by giving a single intraperitoneal injection of streptozocin. FTY720 group was administered with FTY720 at a dose of 0.3 mg/kg by oral gavage daily for 3 months after establishment of diabetes. All rats were used for experiments following intervention for 3 months in FTY720 group. Immunohistochemical staining was used to observe the expression and distribution of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and the positive cells were counted. Real-time reverse transcription PCR was used to measure mRNA expression of ICAM-1 and VCAM-1. Fluorescein isothiocyanate-Concanavalin A perfusion was used to detect retinal leukocytes adhesion. Evans blue (EB) perfusion was used to analyze retinal vascular permeability. Immunofluorescence staining was used to detect retinal inflammatory cells infiltration. Results In diabetic group, both ICAM-1(t=12.81) and VCAM-1 (t=11.75) positive cells as well as their mRNA expression (t=16.14, 9.59) were increased compared with normal control group, with statistical significance (P < 0.05). In FTY720 group, both ICAM-1(t=-9.93) and VCAM-1 (t=-6.61) positive cells as well as their mRNA expression (t=-15.28, -6.10) were decreased compared with diabetic group, with statistical significance (P < 0.05). Retinal leukocytes adhesion (t=16.32) and EB permeability (t=17.83) were increased in diabetic group compared with normal control group, while they were decreased in FTY720 group compared with diabetic group(t=-9.93, -11.82),with statistical significance (P < 0.05). There were many CD45 positive leukocytes infiltration in retina of diabetic group, including CD11b positive macrophage/activated microglia, while both of them were little in FTY720 group. Conclusions FTY720 can decrease retinal leukocytes adhesion, reduce retinal vascular permeability and inflammatory cells infiltration, which is associated with down-regulation of ICAM-1 and VCAM-1.
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal＋balanced salt solution (BSS) group (group B), normal＋netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 sub-groups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM＋netrin-1 100 μg/ml group (group G), DM＋netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59;P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=−42.72,P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77;P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=−1.13, 0.93, 1.04;P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81;P=0.00, 0.00), but negative correlation to the vascular permeability (r=−0.61,P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1.
ObjectiveTo investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. MethodsA lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. ResultsImmunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P < 0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P < 0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P < 0.05).The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45;P < 0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P < 0.05). ConclusionsThe intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=−4.17, −7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=−30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=−6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
Metabolic memory means if the hyperglycemia can't be controlled at early stage of diabetes, chronic complications such as diabetic retinopathy (DR) will continue to develop even if the blood glucose level maintains normal level at later stage. Oxidative stress plays an important role in the "metabolic memory" of DR, which interacts with the nitrative stress, advanced glycation end products, genetic modification and endoplasmic reticulum stress in the pathogenesis of DR. Further elucidation of the relationship between oxidative stress and "metabolic memory" of DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.