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 apoptotic and proliferative characteristics of the retinal vascular end othelial cells (RVECs) of the 1～16 weeks diabetic rats and p53 and bcl-2 expressions of the rats,in order to probe the pathogenic mechanism of diabetic retinopathy(DR). Methods Models of diabetic Wistar rats were made by alloxan venous injection.The retinal blood vessels were filled by ink,the wholemounts and paraffin-embedded sections of the retinas were made,TUNEL staining and Immunohistochemical ABC staining were used,and light microscopy was taken,in succession. Results Apoptosis of the RVECs was not found.Compared with control group,the morphologic features of the RVECs and the structure of the retinal blood vessels remained unchanged.In the period from the 10th to the 16th week,the immunohistochemical stain of PCNA,BrdU,p53,and bcl-2 for RVECs revealed positive results,but there was no any sign of the RVECs stacking and proliferating or new blood vessels forming in the retinas.In control group,the reaction of immunological stain of the aforementioned parameters was negative. Conclusions No accelerated apoptosis and proliferation of the RVECs in the 1～16 week diabetic rats happen after alloxan injection.Almost all of the RVECs were stimulated to enter the cell cycle in the 10th week.Expression of p53 and bcl-2 might play an important role in stabilizing the RVECs in early stage of diabetes. (Chin J Ocul Fundus Dis, 1999, 15: 157-159)
ObjectiveTo predict as well as bioinformatically analyze the target genes of has-miR-451. MethodsmiRBase, miRanda, TargetScan and PicTar were used to predict the target genes of hsa-miRNA-451. The functions of the target genes were demonstrated by Gene Ontology and pathway enrichment analysis. P < 0.05 was set as statistically significant. Results18 target spots of hsa-miRNA-451 were predicted by 3 databases or prediction software at least. The functions of the target genes were enriched in proliferation and development of epithelial cells and regulation of kinase activity (P < 0.05). Pathway analysis showed that transforming growth factor-beta signaling pathway, mitogen-activated protein kinase signaling pathway, epidermal growth factor signaling pathway, Wnt signaling pathway and mammalian target of rapamycin signaling pathway were significantly enriched (P < 0.05). Conclusionhsa-miRNA-451 might be involved in various signaling pathways related to proliferation and development of epithelial cells.
The pathogenesis of diabetic retinopathy (DR) is more complex. For the upstream of traditional pathogenesis, to looking for unifying mechanism theory which proposed in foundation of common promoters and the latest view of DR may be the result of chronic inflammation. Both of them provide the basic and clinical theraby of DR with new direction. Therefore, there are many related issues still needs to intensive study. （Chin J Ocul Fundus Dis，2008，24：237-239）
ObjectiveTo observe the protective effect of Zhicao Tea Mixture on Müller cells and the expression of inflammatory factors in mice with diabetic retinopathy.MethodsSeventy-five C57BL/6J mice were randomly divided into the normal control group, diabetes mellitus (DM) group, low concentrations group, medium concentrations group and high concentrations group, with 16 mice in each group. The diabetes model of mice in all groups except the normal control group were established by intraperitoneal injection of STZ (60 mg/kg). Four weeks after the successful modeling, the Zhicao Tea Mixture with low (30 ml/kg), medium (60 ml/kg) and high concentrations (120 ml/kg) were respectively administered by gavage. Weight and blood glucose of mice in each group were measured every two weeks. After 8 weeks, Western blot method was used to detect the mice retina Müller cells activation marker gelatinous fibrous acidic protein (GFAP). Immunofluorescence was performed to detect the expression GFAP and glutamine synthetase (GS). Real-time quantitative PCR (RT-qPCR) and ELISA were used to determine the mRNA and protein expression levels of mouse retinal VEGF, TNF-α, IL-1β and IL-6 respectively.ResultsThe weight of mice in the DM group was lower than that of the normal control group, and the blood glucose was increased. Zhicao Tea Mixture had no effect on the weight of DM mice, but had a significant hypoglycemic effect. The GFAP expression (t=38.318, P＜0.001) in the retina of mice in the DM group was increased and GS expression (t=29.737, P＜0.001) was decreased compared with the control group. The GFAP expression (t=13.677, 19.387, 16.305; P＜0.05) in the retina of mice in the low, medium and high concentrations group were decreased and GS expression (t=5.170, 19.399, 6.705; P＜0.05) were increased compared with the DM group. The expressions of retinal inflammatory factors VEGF, TNF-α, IL-1β and IL-6 in DM group all increased, while the expressions of the above-mentioned inflammatory factors in the retina of mice decreased in the low, medium and high concentrations group.ConclusionZhicao Tea Mixture can decrease the blood glucose of DM mice and reduces the diabetic retinal inflammatory response.
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.
ObjectiveTo observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.
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 effects of high concentr at ion glucose on the calcium-activated potassium channel of rabbits′ retinal Müller cells. Methods The rabbits′retinal Müller cells were cultured in vitro under the condition of high concentration glucose, and identified by immunohistochemical staining and transmission electron microscopy. Patch-clamp technique was used to observe the changes of the calcium-activated potassium channel of retinal Müller cells caused by high concentration glucose at different time.Results High concentration glucose could inhibit the calcium-activated potassium channel of cultured retinal Müller cells in a time-dependent manner. Conclusion High concentration glucose may reduce the biological functions of Müller cells by inhibiting calcium-activated potassium channel. (Chin J Ocul Fundus Dis，2003，19：164-167)