| Objectives:To observe the morphological changes in early streptozotocin (STZ) induced diabetes mellitus (DM) rats retina and evaluate the value of STZ-induced DM rats as animal models of early diabetic retinopathy (DR); detect the expression of VEGF mRNA and PEDF mRNA in DM rats retina at different time points and explore the inter-relationship between VEGF and PEDF and its impact on the occurrence and development of DR, and further evaluate the value of STZ-induced DM rats as animal models of early DR from the perspective of cytokines. Using lOw STZ-DM rats as an animal model of early DR, detect altered retinal microRNAs (miRNAs) expression profile and explore the roles of these miRNAs in the pathogenesis of DR.Methods:64 male SD rats (body weight 180±20g) were randomly divided into the CON group and the DM group, DM group with DM induced by STZ intraperitoneal injection and CON group as control. Blood glucose and body weight were determined at 2w,4w,6w,8w and 10w after intraperitoneal injection. They were detected that the expression abundance of VEGF mRNA and PEDF mRNA in random samples of rats retina by real-time quantitative PCR (qRT-PCR) at each time point. At 10w, the morphological changes in random samples of rats retina were observed by HE staining, stretched preparation of FITC-Dextran perfused retinal blood vessels and transmission electron microscopy (TEM); the expression and location of VEGF and PEDF in retina were detected by immunohistochemical staining; and the signitificantly differently expressed miRNAs between DM Group and CON group's retina were screened out by miRNAs microarray. The expression abundance of these miRNAs were detected via qRT-PCR at each time point. Possible target genes of these miRNAs were predicted through bioinformatics analysis.Results:1. The ideal animal model of DM (STZ-induced) was successfully established. Before intraperitoneal injection, the average body weight of DM group has no significant difference comparing with CON group (P>0.05); after injection, DM group body weight had no obvious increase, or even decrease at late stage, CON group body weight increased gradually, the average body weight of DM group has significant difference comparing with CON group at 10w (P<0.001). Before injection, the average blood glucose of DM group has no significant difference comparing with CON group (P>0.05); after injection 72h, the average blood glucose of DM group (26.63±4.54mmol/l) has significant difference comparing with CON group (6.37±0.49mmol/l) (P<0.001), DM group blood glucose>16.7mmol/l and CON group 5.6～7.4mmol/l in～10w. Retina HE staining showed that 10w DM rats retina appear capillary dilatation, interstitial edema; CON group has no obvious abnormalities. Stretched preparation of FITC-Dextran perfused retinal blood vessels showed 10w DM rats retina appear vascular tortuosity, caliber irregularity, but no leakage, microvascular tumor or retinal non-perfusion area; CON group rats retinal vessel diameter is uniform and branching is natural and smooth. The TEM showed DM group retina's ultrastructural changes:thicker capillary basement membrane; digitations of capillary endothelial cell; mitochondrial swelling, cristae disruption and vacuolar degeneration in capillary endothelial cell, pericyte, bipolar cells and ganglion cells; and the number of membranous disc (photoreceptor cell's outer segment) decreased and the gap between membranous disc widened.2. qRT-PCR analysis showed that VEGF mRNA could be detected in the retina of CON group and the expression had no significant changes with time; in DM group, the expression began to increase at 2w, and was significantly higher with 3 times than that in CON group at 6w (P<0.05), reached to 6 times at 10w (P<0.001). PEDF mRNA was also expressed in the retina of CON group, but in DM group, the expression began to decrease at 2w, and was significantly lower with 1/2 than that in CON group at 4w (P<0.001), declined to 1/3 at 10w (P<0.001). Immunohisto-chemical staining showed that, CON group:VEGF mainly distributed in the inner nuclear layer and ganglion cell layer, PEDF mainly distributed in the inner nuclear layer and ganglion cell layer, the rest layer had some positive expression; DM group:the expression of VEGF was significantly increased comparing with CON group (P<0.001), all layers were observed with strong expression; however, the expression intensity of PEDF was significantly decreased comparing with CON group (P<0.001), and mainly in the ganglion cell layer and inner plexiform layer.3. miRNA microarray assay showed that a large number of miRNAs present in retinal tissues. Comparing with CON group, the expression of miR-182, miR-96, miR-183, miR-211, miR-204, miR-124, miR-592, miR-190b, miR-502-3p, miR-363, miR-29c* and miR-210 were significantly increased; and miR-10b, miR-10a, miR-219-2-3p, miR-144, miR-338*, miR-199a-3P, miR-451, miR-34a, miR-542-5p, miR-142-3p, miR-223, and miR-18a significantly decreased. qRT-PCR analysis showed:the expression levels of most of increased expression miRNAs in the retina gradually increased with the course of disease; the expression levels of the decreased expression miRNAs gradually decreased with the course of disease. Bioinformatic analysis showed that these microRNAs with significantly different expression may be closely related to many genes regulating DR, such as miR-29c* and miR-199a targeting to Vegfa, miR-363 targeting to PEDF.Conclusions:1. 10w STZ-induced DM rats retina have morphological changes that correspond with the early stage of DR, and can be used as animal models of the early background diabetic retinopathy (BDR). The method of model establishment is simple, economic, with shorter time, and has good reproducibility and high success rate.2. The dynamic balance between VEGF (a major angiogenic factor) and PEDF (a potential angiogenesis inhibitor) play vital roles in regulating retinal vascular leakage and neovascularization. Under physiological conditions, the expression of VEGF is lower in the retina, while PEDF has comparative advantage, which is necessary for maintaining the integrity of retinal blood vessels and inhibiting neovascularization. The expression of VEGF in 10w DM rats retina was increased, while PEDF decreased. The imbalance of VEGF and PEDF will lead to retinal vascular leakage and neovascularization. These indicate that the imbalance may be responsible for the occurrence and development of DR, but also further evidence that lOw STZ-DM rats can be used as an animal model of early BDR from the perspective of cytokine.3. miRNAs expression in retina of BDR rat model had significant differences comparing with that of normal controls; some miRNAs expression levels increased gradually with the course of development, while some expression levels decreased gradually; these miRNAs with significantly different expression probably target to many genes relating to DR. The above results indicate that miRNAs probably play important roles in regulating the occurrence and development of DR. |
