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 observe and investigate the effect of HIF-2α in the process of neovascularization of proliferative diabetic retinopathy (PDR).MethodsRetrospective clinical study. From July 2014 to July 2015, 60 eyes of 57 PDR patients diagnosed in Ophthalmology Department of Affiliated Hospital of Qingdao University were included in the study. Twenty-eight eyes of 27 patients received intravitreal injections of 0.5 mg ranibizumab (0.05 ml) at 2-7 days before surgery (ranibizumab group) and other 32 eyes of 30 patients did not (group without ranibizumab). Eighteen eyes of 18 patients with epiretinal membranes were included as controls. Pathological specimens of PDR fibrovascular membrane and premacular membrane were obtained during vitrectomy. The immunohistochemical staining and real-time PCR (RT-PCR) were used to detecting the expression of HIF-2α, Dll4 and VEGF. Kruskal-wallis test was used to compare the expression differences of correlation factors between groups. Spearman correlation analysis was used to analyze the relationship between the two variables.ResultsThe immunohistochemical staining revealed that there were positive expression of HIF-2α, Dll4 and VEGF in all PDR membranes, regardless of the injection of the ranibizumab. The levels of HIF-2α, Dll4 and VEGF protein in the group without ranibizumab were higher than those of the ranibizumab group (t=4.36, 6.01, 4.82; P=0.000, 0.008, 0.016). RT-PCR showed that the differences of the mRNA expression of HIF-2α, Dll4 and VEGF were all statistically significant among the PDR patients and controls (H=18.81,19.60, 20.50; P=0.000, 0.000, 0.000). The expression of HIF-2α, Dll4 and VEGF in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR patients,the expression of HIF-2α, Dll4 and VEGF of the group without ranibizumab was higher than that of the ranibizumab group. The spearman correlation analysis showed that the expression of mRNA between HIF-2α and Dll4, HIF-2α and VEGF were both significantly correlated (r=0.95, 0.87; P＜0.05).ConclusionsThe expression of HIF-2α in the PDR membranes was higher than that of the controls. It is positively correlated with the expression of the DLL4 and VEGF.
Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer (IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant (F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
ObjectiveTo observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).MethodsA total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group, with 10 and 60 rats in each group, respectively. The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model. The rats with blood glucose recovery and death were excluded, and the final 60 rats were included in the statistics. Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer. Rats in the DM group were divided into DM 1 month (DM 1m) group, DM 2 months (DM 2m) group, DM 3 months (DM 3m) group and DM 3m + Anti group, DM 3m + phosphate buffer solution (PBS) group by random number table method, and 10 rats in each group. In the DM 3m+Anti group, 4 μl of anti-Dll-4 polyclonal antibody was injected into the vitreous cavity, and the antibody concentration was 0.25 mg/ml. The DM 3m+PBS group was intravitreally injected with an equal volume of PBS. Five days after the injection, the rats were sacrificed. Rats in the DM 3m group and the normal group were not treated, and were sacrificed 3 months after the model was established. The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining, and the total thickness of the retina was measured. The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR). One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group. The least significant difference t test was used to compare the two groups.ResultsLight microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous, the inner and outer nuclear layers were thinner, the number of cells was reduced, the arrangement was disordered, the edema of outer plexiform layer was obvious, and the microvessels were abnormally dilated. In the DM 3m+Anti group, the edema of outer plexiform layer was lessened than that of the DM 3m group, and the other layers were not significantly different from the DM 3m group. Compared with the normal group, the total retinal thickness of the DM 3m group, the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596, 3.290, 4.286; P=0.000, 0.008, 0.002). Immunohistochemical staining showed that a small amount of Dll4 was positively expressed in the retinal ganglion cell layer of the normal group; a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers. The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly. The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells of DM 3m+Anti group, while the expression of VEGFR-2 was significantly increased. There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group. The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705, 20.871; P＜0.05). Compared with DM 3m group, the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased, and the relative expression of VEGFR-2 mRNA increased (t=2.681, 3.639;P＜0.05). The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513, 0.657; P＜0.05).ConclusionsThe expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats. The expression of Dll-4 is inhibited, the expression of VEGFR-2 is up-regulated, and the plexus edema is alleviated.