The pathogenesis of diabetic retinopathy is complicated. The vast network of multiple factors including unifying mechanism, inflammatory reaction, neuron degeneration and metabolic memory of glucose, and the four established pathogenic molecular pathways are hotspots of mechanism research for diabetic retinopathy. Nevertheless, these researches may be only one corner of the ldquo;icebergrdquo; of DR mechanism, and we still face enormous challenges in DR mechanism research. Collaboration with multiple disciplines to study the relationship between DR and diabetes and other systemic diseases, search novel therapy targets may increase the result in an unexpected windfall for DR basic research.
Objective To observe the oxidative damage of mtDNA, apoptosis and expression of adhesion molecules in retinal capillary cells of diabetic rat with different disease courses. Methods One hundred Sprague-Dawley rats were randomly divided into the control group and the experimental group. The rats of experimental group were induced with streptozotocin (STZ) injection creating a diabetic model. Then they were divided into DR1m, DR2m DR3m group according to disease courses. The rats of control group were divided into NR1m, NR2m, NR3m group. Rat retinal capillaries were prepared, and then the contents of undamaged mtDNA were examined by Southern blot combined with Fpg. The expression of cyclooxygenase (COX)-1 encoded by mtDNA and transcription factors A (mtTFA) mRNA were detected by real-time quantitative polymerase chain reaction (RT-PCR). Apoptosis and expression of intercellular adhesion molecule-1 (ICAM-1) were detected by terminal dUT nick endlabeling (TUNEL) immuno-fluorescence and immunohistochemistry respectively. Results The contents of undamaged mtDNA in rats of DR1m, DR2m, DR3m were less than those of NR1m、NR2m、NR3m. The contents of undamaged mtDNA in diabetic rats decreased with the increase of disease courses. In addition, the mRNA levels of COX-1 and mtTFA were downregulated in diabetic rats. The positive cells of TUNEL and ICAM-1TUNEL and ICAM-1 in diabetic rats increased with the increase of disease courses. Conclusion With the increase of disease courses, mtDNA damage and apoptotic cells are increased, while the expression of mRNA encoded by mtDNA and ICAM-1 decreased in retinal capillary cells in diabetic rats.
Objective To observe the relationship between endothelial constitutive nitric oxide synthase (ecNOS) genetic polymorphism and diabetic retinopathy（DR）of non insulindependent diabetes mellitus (NIDDM) patients of the Han nationality.Methods A total of 166 patients who clinical diagnosed with NIDDM as case group, 85 cases of patients (cataract or fracture) and healthy subjects without diabetes, hypertension and kidney disease,over 40 years old of age and without consanguinity between each other were selected as normal control group. Case group were divided into non-DR (NDR) group, nonproliferative-DR (BDR) group and proliferativeDR (PDR) group according to the result of fundus fluorescein angiography. Case group and normal control group subjects all were Han nationality. DNA was extracted from peripheral venous blood; the fourth 27 base pairs (bp) repeat polymorphism of ecNOS gene by was measured by polymerase chain reaction (PCR). Results The 27 bp repeat sequences within the ecNOS gene present in the Han nationality,allele b repeat 5 times, alleles a repeat 4 times. PCR results showed that there are 2 alleles and 3 genotypes in normal control, NDR, BDR and PDR group. The frequency of genotype bb、ab、aa were 80%, 16.5%, 3.5% in normal subjects; 77.2％, 13.9%, 8.9％ in NDR group; 80.5％, 17.1%,2.4％ in BDR group;78.3％, 13%, 8.7％ in PDR group,respectively. The allele frequency (chi;2 =1.841) and gene frequency (chi;2=3.847) were not statistically significant (P＞0.5) in normal control,NDR,BDR and PDR group. Logistic regression analysis showed that there is no relation between DR and ecNOS duplicated gene polymorphism. Conclusions There is 27 bp repeated polymorphism in 4th intron of ecNOS gene, which may not be associated with the DR of NIDDM in the Han nationality.
Objective To observe the opticin expression in the eyes of nonobese diabetes (NOD) mice and nondiabetic NOD mice.Methods Twenty NOD mice were divided into diabetic group (experimental group) and nondiabetic group (control group). All the mice were killed by cervical dislocation method.The eyes were harvested, and the vitreous, retina and sclera were separately collected. Western blot and realtime reverse transcriptionpolymerase chain reaction（RT-PCR）were respectively used to determine opticin protein and OPTC mRNA levels.Results The opticin protein level in the vitreous and retina was lower in the experimental group（t=4.42,4.58;P=0.002,0.002）,but is same in thesclera between the 2 groups（t=0.27,P=0.794）.OPTCmRNA level was vitreousgt;retinagt;sclera. OPTCmRNA levels of vitreous and retina in diabetic group were significantly lower（t=3.30,2.48；P=0.01,0.04）; there was no statistical significant on OPTC mRNA of sclera between two groups（t=0.27,P=0.80）.Conclusion Expression of opticin was suppressed in retina and vitreous of diabetic mice.
Objective To investigate the protective effect of blocking the signal path of p38 mitogen activated protein kinase on blood retinal barrier (BRB) and retinal ganglion cells (RGC) in early diabetic rats.Methods A total of 60 Wistar rats were divided into the control and diabetes group, with 30 rats in each group. Diabetes was induced in rats in diabetes group by peritoneal injection of streptozotocin (STZ);the plasma glucose level of ＞16.7 mmol/L indicated that the diabetes model was set up successfully.The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution. IgG leakage method was used to measure the damage of BRB function and vascular leakage. The expression and localization of caspase-3 and vascular endothelial growth factor (VEGF) in retina of diabetic rats were examined by immunohistochemistry analyses.Two weeks after the establishment of the diabtes model, the rats in diabtes group underwent intravitreal injection with SB203580, a p38 inhibitor;six weeks after the injection, the expression of caspase-3 and VEGF was detected, and the number of apoptosis RGC was counted via immunofluorescence technique.Results In the contral group, IgG staining located in the blood vessels with little leakage; while the IgG leakage was much more obvious in the diabetes group eight weeks after the establishment of the model. Six weeks after intravitreal injection with SB203580, the leakage decreased in diabtes rats. The results of semiquantitative analysis and fluorescence immunohistochemistry showed that compared with the results in diabetes rats 8 weeks after intravitreal injection (2.9 times much more than that in the control group), the fluorescence expression of VEGF decreased in diabetes rats six weeks after intravitreal injection (1.8 times much more than that in the control group).The apoptisis RGC number in rats 6 weeks after intravitreal injection of SB203580 was much less than that in rats without intravitreal injection (t＝5.731, Plt;0.01). Conclusions SB203580 can alleviate the disruption of BRB and apoptosis of RGC in early diabetes rats, which suggests that p38 MAPK pathways appear to be directly involved in the pathogenesis of early diabetic retinopathy.
Objective To quantitatively assess the damage of blood-retinal barrier (BRB) in rats with diabetic retinopathy using dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI). Methods Forty 3-week-old Sprague-Dawley (SD) rats were randomly divided into experiment and control group. The rats in experiment group underwent intraperitoneal injection of streptozotocin. The rats with blood glucose over 16.65 mmol/L and ldquo;+++rdquo; of urine glucose were considered as diabetes and were further divided into four subgroups according to the course of diabetes mellitus (2, 4, 6, and 8 months).The rats in control group underwent intraperitoneal injection with the same volume of buffer and were divided into four subgroups (with 5 rats in each subgroup) according to the coordinate age of rats in experimental group.All of the eyeballs were scanned by DCE MRI and enucleation was performed after intraperitoneal injection with pentobarbitone.The data were analyzed by SPSS 12.0 statistical software.Results All the rats in experiment group became diabetic models. There was no obvious BRB permeability in control group and in 2- and 4-months experiment group.The average BRB permeability rate in 6 and 8 month experiment groups were (0.1399plusmn;0.0065) and (0.1816plusmn;0.2756) mm3/min respectively (Z=-2.121, Plt;0.05). Retinal edema and cellular disorganization appeared at 4 months and became more severe when diabetes course extended.Conclusions DCE MRI can measure the BRB permeability rate accurately and assess the extent of BRB damage quantitatively in rats with diabetic retinopathy.
Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 SpragueDawley（SD）rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi.Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope.Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat mounts. The density of microglial cells which phagocytosed DiI particles in the RGC layer significantly increased in the 3month diabetes group（P＜0.01）. The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group（P＜0.05）. However there were more microglial cells in the RGC layer in the 3- month diabetes group than the 1-month diabetes group（P＜0.0001）. Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats.