Objective To observe the inhibition effect of the hypoxia inducible factor-1alpha; (HIF-1alpha;) specific siRNA on the expression of vascular endothelial growth factor (VEGF) mRNA in retinal tissues in diabetic rat. Methods This is a randomized controlled study. HIF-1alpha; specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector. Fifty-four healthy Sprague Dawley (SD) rats were divided into control group (15 rats) and experimental group (39 rats). The experimental rats were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into diabetic retinopathy (DR) group (15 rats), vector group (12 rats) and gene therapy group (12 rats). LipofectamineTM2000 mixed with pSilencer2.1-U6neo plasmid or HiF-1alpha; siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively. Nothing was transfected into DR and control group. The expression of VEGF mRNA in retinas was measured by real-time RT-PCR. The inhibition efficiency of VEGFmRNA was calculated at 24, 48, 72 hours and 1 week after injection respectively. Significant differences between groups were evaluated by oneway analysis and LSD-t analysis. Results HIF-1alpha; siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group, increased obviously in the DR and vector group, decreased in the gene therapy group. There was no statistically significant between DR and vector group (t=0.669，0.142,0.151，0.025；P=0.514，0.889，0.882，0.980). The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group (t=8.768, 13.695, 11.285, 8.253;P=0.000). The inhibition efficiency of VEGFmRNA was 32.76%, 43.60%, 47.70%, 50.86% at 24, 48, 72 hours and 1 week after injection. Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1alpha; siRNA recombinant plasmid.
Objective To observe the effect of diabetic retinopathy on endothelial progenitor cells (EPCs) from peripheral blood. Methods Sixty male Wistar rats were divided into control group and diabetes group. The rats in diabetes group were induced with streptozotocin (STZ) injection for diabetic retinopathy model. Flow cytometry was used to identify and count the number of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after injection. All eyeballs were examined by hematoxylin and eosin (HE) staining, periodic acidSchiff's (PAS) staining of trypsin-digested retinal vessels flat preparation and transmission electron microscope. EPCs count, and the relationship between DR morphological changes and EPCs count were compared and analyzed. Results The quantity of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after STZ injection were 25plusmn;7, 28plusmn;8, 39plusmn;7, 43plusmn;7 cells per 200 000 monocytes respectively, which decreased compared with the control group 45plusmn;4 cells per 200 000 monocytes (F=8.933，Plt;0.01). The quantity of EPCs was gradually increased at 1 week, 1, 3 and 6 months after STZ injection, accompanied with responsive pathological changes of retinal structure and vessels. The thickness of retina at 1 week and 1 month after injection were reduced slightly. The number of retinal ganglion cells reduced, with the time passing by. Endothelial cells were edema, mitochondrial was swollen, capillary basement membrane was thicken, lumen was significant stenosis, lumen occlusion and retinal artery aneurysm were observed at 6 months after STZ injection. Conclusion The number of EPCs increases gradually throughout the development of DR.
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 observe the abnormal expression of alpha;A-crystallin protein in neural retina in type 2 diabetic rats via proteomic technique.Methods Twenty-eight male Sprague-Dawley (SD) rats were randomly divided into the normal control and the diabetic experimental groups with 14 rats in each group.A type 2 diabetes rat model (T2DM) was set up in the diabetic experimental group by feeding high fat diet combined with peritoneal injection of low dose streptozotocin (STZ);the successful diabetes model is with the randomlydetected blood glucose of ＞16.7 mmol/L.The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution and were fed with normal diet.All of the animals were sacrificed by decapitation 56 days after the induction of diabetes.The eyes were enucleated and the neural retina layers were carefully peeled off and preserved.The total neural retinal proteins were extracted from the control and diabetic groups, respectively,and then subjected to two dimensional gel electrophoresis (2-DE).Some different proteins spots were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements.Western blot and indirect immunofluorescence (IMF) were used to confirmed that alpha;A-crystallin protein expression was upregulated in diabetic retina.Results An average of （3122plusmn;37） spots in normal retinas and（2702plusmn;21）spots in diabetic group were found by 2-DE image analysis software; about 150 spots in 2-DE gel of diabetic retinae exhibited statistically significant variations (t＞2.77,P＜0.05).Compared with normal rats' retinae, diabetic ones presented 68 protein spots of up regulation expression and 82 of downregulation expression in 2DE gel.Furthermore,20 of the 150 protein spots were identified by mass spectrometry.The points of 2369 and 1048 in 2-DE gel, showing high expression in diabetic retinal tissues, were identified as alpha;A-crystallin via PMF.Western blot validated that the expression level of alpha;A-crystallin in diabetic neural retina was much higher than that in the control group. Significantly increased expression of alpha;A-crystallin in nuclear retina in diabetic group was also observed by IMF. Fluorescence was mainly seen in the retinal nuclear layer;alpha;A-crystallin aggregation was detected in the perinuclear region of neurons.Conclusion The expression of alpha;A-crystallin increases in neural retina of early T2DM rats.
Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
Objective To observe the degradation regulation of ubiquitinproteasome inhibitor nuclear factor kappa;B（NF-kappa;B）and its inhibitory signal protein Ikappa;B kinase in earlier period diabetic retinopathy（DR）,and the effects on retinal ganglion cells (RGC) apoptosis.Methods Forty healthy adult Wistar rats were randomly divided into control (group A）,DR（group B）,DR+lowconcentration MG132 treated （group C）and DR+high concentration MG132 treated（group D）groups,10 rats in each group.After 6 and 8 weeks,the results of body masses and fasting blood glucose (FBG) were detected,the expression of NF-kappa;B and Ikappa;B were observed by immunohistochemistry respectively.RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Results The expression of NF-kappa;B was upregulated in group B compared with group A,its expression decreased in group D compared with group B; but the expression of Ikappa;B was contrary to NF-kappa;B; RGC apoptosis was followed a similar pattern with the expression of NF-kappa;B; the differences among them were statistically significant (P＜0.01).Compared the expression of NF-kappa;B，Ikappa;B and RGC apoptosis in group C and D, there were no statistically significant differences(P＞0.05).Conclusion Ubiquitin-proteasome inhibitor MG132 can block the activation of NF-kappa;B,inhibit ubiquitination of Ikappa;B degradation and RGC apoptosis.
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)