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 effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=−26.01, 22.26; P=0.00, 0.00), and group E (t=−10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, −9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=−0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.
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.
Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1alpha;) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1alpha; anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1alpha; sense oligonucleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5% glucose solution caudalis veins injection, group C and group D rats received HIF-1alpha; ASODN and HIF-1alpha; SODN caudalis veins injection, respectively(025 mg/kg).The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD18 positive neutrophil cell was（44.93plusmn;3.60）% in group B，（18.66plusmn;1.52）% in group A，（31.66plusmn;4.72）% in group C，（51.00plusmn;5.66）% in group D. Compared with each other groups，the differences are statistically significant （F=42.46, Plt;0.001）. The number of positive staining cells of retinal leukocyte was （46.16plusmn;10.68）in group A，（133.83plusmn;20.43）in group B,（99.83plusmn;9.28）in group C，（121.33plusmn;10.23） in group C. Compared group B with group C，the number of positive staining cells raised about 2.89 times；compared group B with group C and D，the differences are statistically significant （P=0.12，95% confidence interval -3.69~28.69）. Conclusions In vivo, HIF-1alpha; can decreased the expression of CD18 on neutrophils from diabetic ratsprime; peripheral blood and the collection of retinal leukostasis in the diabetic animals. HIF-1alpha; may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy. （Chin J Ocul Fundus Dis，2008，24：268-271）
Objective To observe the expression of N-cadherin in streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) ratsprime;retinae. Methods Celiac injection with 65 mg/kg STZ was performed on 20 rats to set up the diabetic model, and celiac injection with the same volume citrate buffer was performed on other 20 SD rats as the control. Vascular permeability was detected by Evans blue method. The expression of N-cadherin in both normal and STZ-induced diabetic ratsprime;retinae and trypsinase-digested retinal microvessels were detected by immunohistochemistry method and Western blotting analysis. Results Retinal vascular permeability increased 68%, 91% and 125% 4, 8, and 12 weeks, respectively, after diabetic models was induced (Plt;0.005). In the control group, the expression of N-cadherin was detected in the outer and inner plexiform layer, inner nuclear layer，ganglion cell layer,internal limiting membrane and between retinal endothelial cells and pericytes. However, the expression of N-cadherin significantly decreased in STZ-induced diabetic rats retinae at the 12th week. The results of Western blotting analysis showed that the expression of N-cadherin obviously decreased as the diabetic retinopathy developed. Conclusion The decrease of expression of Ncadherin in the retinae of STZ-induced diabetic rats suggests that N-cadherin may participate in the development of diabetic retinopathy at the early stage. (Chin J Ocul Fundus Dis,2007,23:269-272)
Objective To observe the effect of celecoxib on the expression vascular endothelial growth factors (VEGF) in diabetic rats. Methods Thirty-six wistar rats were used to establish the diabetic models by intraperitoneal injection with streptozotocin. The diabetic rats were divided into 2 groups: diabetic group (n=18) and celecoxib group (n=18). Celecoxib (50 mg/kg) was administered orally to the rats in celecoxib group and the physiological saline with the same volume was given orally to the rats in diabetic group. Eighteen else rats were in normal control group. All of the rats were executed 3 months later. The expression of VEGF protein was detected by immunohistochemistry method. Reverse transcription-polymerase chain reaction（RT-PCR） analysis was used to examine the expression of retinal VEGF mRNA and cyclooxygenase-2 mRNA. Results Lower positive expression of VEGF mRNA and cyclooxygenase-2 mRNA, weakly positive action of immunohistochemistry of VEGF, and lower expression of VEGF protein were detected in normal control group; in the diabetic group, the expression of VEGF mRNA and cyclooxygenase-2 mRNA increased obviously comparing with which in the control group (Plt;0.05), and the bly positive action of immunohistochemistry of VEGF and increased expression of VEGF protein were detected (Plt;0.01); in celecoxib group, the expression of VEGF mRNA was lower than that in the diabetic group (Plt;0.05), the expression of cyclooxygenase-2 mRNA didnprime;t decrease much (Pgt;0.05), the positive action of immunohistochemistry of VEGF decreased, and the expression of VEGF protein decreased (Plt;0.01). Conclusion By inhibiting the activation of cyclooxygenase-2, celecoxib can inhibit the expression of retinal VEGF mRNA and protein in diabetic rats induced by streptozotocin. (Chin J Ocul Fundus Dis,2007,23:265-268)