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Objective: To investigate the therapeutic effect and mechanism of erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, on non-proliferative diabetic retinopathy (NPDR). Methods: An experimental research was conducted. Human retinal Müller cells (RMC) were MIO-M1 cells from Moorfields Ophthalmology Hospital and the Institute of Ophthalmology at London University College. MIO-M1 cells were divided into normal, hypertonic, high glucose, high glucose+dimethyl sulfoxide (DMSO), high glucose+erlotinib 0.5 mmol/L, high glucose+erlotinib 1 mmol/L, and high glucose+erlotinib 2 mmol/L groups using a random number table method. Detection of the effect of erlotinib on the proliferation of MIO-M1 cells under high glucose conditions was performed by 5-ethynyl-2'-deoxyuridine (EdU) method. Western blotting (WB) was used to detect the effect of erlotinib on the activation markers of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) protein levels in MIO-M1 cells under high glucose conditions. WB was used to detect the effect of erlotinib on the protein levels of nerve growth factor receptor (p75NTR), vimentin, and cell retinol binding protein (CRALBP) in RMC under high glucose conditions. MIO-M1 cells were divided into normal group, high glucose group, high glucose+DMSO group, and high glucose+erlotinib (1 mmol/L) group using random number table method. The effect of erlotinib on EGFR nuclear translocation under high glucose conditions was detected by cell immunofluorescence staining. Immunoprecipitation was used to detect the effect of erlotinib on the interaction between EGFR and transcription intermediate factor 2 (TIF2) in MIO-M1 cells under high glucose conditions. MIO-M1 cells were randomly divided into normal group, high glucose group, high glucose+DMSO group, high glucose+Myc-DDK empty body group, high glucose+erlotinib group, high glucose+erlotinib+human doublet protein group, high glucose+erlotinib+TIF2 plasmid group, and high glucose+erlotinib+human doublet protein+TIF2 plasmid group. Cell immunofluorescence staining was used to detect the effect of erlotinib on the binding of EGFR and TIF2 in MIO-M1 cells under high glucose conditions through the EGFR/TIF2 axis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the regulatory effect of EGFR and TIF2 binding on cyclin D1 transcription in MIO-M1 cells under high glucose conditions. The mouse model of diabetes retinopathy (DR) was constructed and divided into normal group, DR group, DR+DMSO group, DR+erlotinib 0.25 mg·kg -1 ·d -1 group, DR+erlotinib 0.5 mg·kg -1 ·d -1 group and DR+erlotinib 1 mg·kg -1 ·d -1 group. 25 mice in total, 5 in each group. Tissue immunofluorescence staining was used to detect the expression of RMC activation marker GFAP. The FITC-dextran injection experiment was used to detect the effect of erlotinib on retinal vascular leakage in a murine DR model. Results: Compared with the normal group (32.4%±3.0%), the proportion of EdU positive cells in RMC in the high glucose group (59.2%±3.8%) increased ( P< 0.001). Compared with the high glucose group (59.2%±3.8%), the proportion of EdU positive cells in the high glucose+1 mmol/L erlotinib group (37.6%±4.4%) decreased ( P< 0.001). Compared with the normal group, the expression of GFAP in RMC in the high glucose group increased (1 in the normal group, 2.27±0.11 in the high glucose group, P< 0.001), while the expression of GS decreased (1 in the normal group, 0.32±0.03 in the high glucose group, P< 0.001). 1 mmol/L erlotinib treatment reduced the expression of GFAP in RMC under high glucose conditions (1.32±0.13 and 2.27±0.11, respectively; P< 0.001), and increased the expression of GS (0.71±0.06 and 0.32±0.03, respectively; P< 0.001). The colocalization of EGFR and DAPI in RMC of the high glucose+1 mmol/L erlotinib group was lower than that of the high glucose group (52.2%±4.1% and 76.4%±5.7%, respectively; P< 0.001). The expression of TIF2 or EGFR both increased while using EGF or TIF2 antibodies to precipitate TIF2 or EGFR under high glucose conditions compared to the normal group (1 in the normal group, 2.27±0.20 in the high glucose group, 2.17±0.21 in the EGFR, all P< 0.05). And the expression of TIF2 (1.38±0.10) or EGFR (1.32±0.13) in the high glucose+erlotinib group was lower than that in the high glucose group (2.27±0.20) and the high glucose group (2.17±0.21) (all P< 0.05). The colocalization of EGFR and TIF2 (17.2%±3.9%) and the mRNA level of Cyclin D1 (1.32±0.16) in the RMC of the high glucose+erlotinib group were lower than those in the high glucose group (54.6%±3.7% of EGFR and TIF2 colocalization ratio, 2.58±0.19 of Cyclin D1 mRNA level,all P< 0.05). The high glucose+erlotinib+AREG (EGFR agonist) group, high glucose+erlotinib+Myc DDK-TIF2 plasmid group and high sugar+erlotinib+AREG+Myc-DDK-TIF2 plasmid group EGFR colocalization with TIF2 (colocalization ratios 24.1%±1.9%, 26.0%±2.3%, 35.3%±2.5%) and TIF2 mRNA levels (1.71±0.16, 1.72±0.18, 2.20±0.18). Compared with the high glucose+erlotinib group, The increases were statistically significant (all P< 0.05). Compared to the normal group, the expression of GFAP in mouse retina tissue was increased in the DR group (1 in the normal group, 3.07±0.19 in the DR group, P< 0.001), and 0.5 mg·kg -1 ·d -1 erlotinib (1.73±0.30) significantly reduced the expression of GFAP in the retina of DR group mice ( P< 0.05). Compared to the normal group (3.97±0.47), the DR group (23.13±2.15) showed an increase in fluorescein leakage, while the DR+erlotinib group (11.66±1.45) showed a significant decrease in leakage compared to the DR group (all P< 0.05). Conclusions: Erlotinib inhibits the proliferation and activation of RMC induced by high glucose, inhibits the entry of EGFR into the nucleus, inhibits the binding of EGFR to TIF2 in RMC, and reduces the transcription of Cyclin D1 in RMC by inhibiting the interaction between EGFR and TIF2. At the same time, erlotinib inhibits the proliferation and activation of RMC in the mouse DR model, ameliorating retinal vascular leakage in mice. These results suggest that erlotinib inhibits the activation and proliferation of RMC by downregulating the EGFR/TIF2/Cyclin D1 pathway under high glucose conditions, thereby alleviating the progression of NPDR.

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.

Objective: To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism., Methods: HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1 α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1 α , ROS, VEGF, TNF- α , IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy., Results: High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability ( P < 0.05), and induced inflammation and oxidative stress in hRMECs ( P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening ( P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues ( P < 0.001)., Conclusion: Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.

Objective: To identify differentially expressed proteins between the patients with proliferative diabetic retinopathy (PDR) and vitreous floaters, and explore treatment target for PDR based on isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS Proteomics. Method: Vitreous samples were collected from 28 eyes of patients with PDR and 4 eyes with vitreous floaters, which served as controls. For quantitative proteomics, vitreous samples were combined and proteins extracted and labeled with iTRAQ peptide-tagging reagents. Samples were fractionated by liquid chromatography (LC), analyzed by tandem mass spectrometry (MS/MS) and Gene Ontology (GO) analyses performed on differentially expressed proteins identified in the PDR samples. Results: In the PDR vitreous, 26 proteins were identified that were differentially expressed when compared to the controls. Of these, 7 showed a significant increase ( P< 0.05) and 19 a significant decrease ( P< 0.05)in expression in PDR patients. These included some high abundance proteins including Retinoic acid receptor reactive protein 2 (PDR 1=85.0, PDR 2=83.0, Control 1=119.6, Control 2=120.2, FC=0.710, P= 0.001), Semaphorin-4B(PDR 1=64.4, PDR 2=68.8, Control 1=135.4, Control 2=146.0, FC=0.473, P= 0.023), Apolipoprotein B (PDR 1=104.4, PDR 2=106.6, Control 1=89.0, Control 2=85.3, FC=1.211, P= 0.024), and Heat shock protein 70 (PDR 1=69.3, PDR 2=75.0, Control 1=137.7, Control 2=138.3, FC=0.523, P= 0.026), which are closely related to the pathological mechanism of PDR. GO analysis clustered the differentially expressed genes into three major functional domains: Biological Processes, Molecular Function and Cellular Component. Differential gene expression was found in the categories of cellular metabolism, organonitrogen compound and carbohydrate derivative metabolic processes, transferase activity and transmembrane signaling receptor activity. KEGG Pathway analysis indicate that Chemerin signaling through Akt, Sema4B signaling via PI3K, and HIF-1α signal pathways were all altered in the PDR samples. Conclusions: In this study we identified variations in expression of genes extensively involved in key biological processes in the retina including neovascularization, cellular metabolism and transmembrane signaling, which provide new insights into the pathophysiology of PDR. Extracellular matrix was degraded and endothelial cell migration was induced by Chemerin, in addition, the destruction of blood-retinal barrier and neuronal apoptosis were induced by ApoB. Chemerin and ApoB accelerated the development of PDR. Sema 4B participated in vascular protection, HSP70 conducted anti-apoptosis. These two cytokines protected the retinal neurovascular in PDR patients. Therefore, Chemerin, Sema 4B, ApoB and HSP70 may be the treatment target for PDR. (Chin J Ophthalmol, 2019, 55:769-776) .

Objective: To evaluate the correlation between the expression of VEGF with t-PA and PAI expression in plasma aqueous humor and vitreous of proliferative diabetic retinopathy (PDR)., Methods: It was an experimental study. The concentrations of VEGF t-PA and PAI in plasma, aqueous humor and vitreous body in PDR and normal group were detected by ELISA., Results: The levels of VEGF (53.37 ng/L) and PAI (8.00 µg/L) in the plasma of PDR patients were higher than control group, and there was significant statistically difference between them (Z = -3.437, -5.816; P < 0.01). The levels of t-PA (24.32 µg/L) in plasma of PDR patients and control group was no statistically difference between them (Z = -1.715, P = 0.086). The levels of VEGF (335.00 ng/L) t-PA (5.70 µg/L) and PAI (0.63 µg/L) in the aqueous humor of PDR patients were higher than control group, and there was statistically difference between them (Z = -4.805, -1.967, -4.018; P < 0.05). The levels of VEGF (691.69 ng/L) t-PA (13.05 µg/L) and PAI (4.01 µg/L) in the vitreous of PDR patients were higher than control group , and there was statistically difference between them (Z = -2.807, -2.642, -2.230;P < 0.05). Compare the plasma aqueous humor and vitreous of PDR patients. The levels of VEGF is: vitreous>aqueous humor>plasma. The levels of t-PA is: plasma>vitreous>aqueous humor. The levels of PAI is: plasma and vitreous>aqueous humor. The expression of VEGF was highly correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR (P < 0.05)., Conclusions: VEGF PAI in plasma, VEGF t-PA PAI in aqueous humor and vitreous were increases. The expression of VEGF was positive correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR.

Objective: To investigate the expression of adiponectin and its receptors (AdipoRs) in the retina of normal mice and mice with type 1 diabetes mellitus (T1DM)., Methods: C57BL/6 mice were randomly divided into control group and streptozotocin-induced T1DM group. Two months after the modeling, the total protein and adiponectin protein expression in the retina and choroid were measured using BCA method and enzyme-linked immunosorbent assay, respectively. Quantitative RT-PCR was performed to detect the mRNA expressions of AdipoRs in the retina and choroid, and Western blotting was employed to examine the protein expressions of AdipoRs in the retina., Results: Adiponectin and AdipoRs proteins were expressed in the retina and choroid in normal mice. The expressions of adiponectin and AdipoR1 were up-regulated in the retina of mice with T1DM while AdipoR2 expression exhibited no significant changes., Conclusion: Adiponectin and AdipoR1 may play an important role in the evolvement of type 1 diabetic retinopathy.

Objective: To investigate the protective effect of Qihuang Mingmu capsule (QHMM) on retina of diabetic mice and its impact on VEGF expression., Method: Forty KK/Upj-Ay mice were randomly divided into the model group and high, middle and low dose QHMM (8.32, 4.16, 2.08 g x kg(-1)) groups. Additional 10 C57BL/6 mice were selected as the control group. Mice were orally administered for three months. Their general appearance, fasting blood-glucose (FBG) and glycosylated hemoglobin (HbA1c) were observed. Pathological changes of retina were observed by light microscope and electron microscope. The expressions of vascular endothelial growth factor (VEGF), growth factor receptors-1 (Flt-1) and growth factor receptors-2 (Flk-1) were examined by Real-time PCR (qPCR) and Western blot., Result: QHMM could ameliorate the symptoms of diabetic mice to varying degrees, decrease FBG and HbA1c, alleviate pathological lesions of retina and decrease the expressions of VEGF, Flt-1, Flk-1 mRNA and protein., Conclusion: QHMM has the protective effect on diabetic retinopathy of mice by inhibiting the expressions of VEGF, Flt-1 and Flk-1 and intervening VEGF-VEGFR signal transduction pathway.

Objective: To observe the effects of Huoxue Jiedu Recipe (HJR) on the electroretinogram (ERG) and the expression of glial fibrillary acid protein (GFAP) in the retina tissue of early diabetic rats., Methods: The diabetic rat model was established using one single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). Then the modeled rats were randomly divided into 5 groups, i.e., the model group, the low dose HJR group (3.85 g/kg), the middle dose HJR group (7.70 g/kg), the high dose HJR group (15.40 g/kg), and the Western medicine treatment group (Calcium Dobesilate Capsule, 0.167 g/kg), 8 in each group. A normal control group consisting of 8 rats was also set up, which was given equal volume of distilled water by gastrogavage. All rats were medicated by gastrogavage for 20 weeks. The electroretinograph (ERG) was determined. The amplitudes of wave a and b (the maximal electric reaction for dark-adapted eyes), and the amplitude sum of the oscillatory potentials (OPs) were detected. The integral optical density (IOD), the protein and mRNA expression of GFAP were detected using immunohistochemical assay, Western blot, and fluorescent quantitative PCR., Results: Compared with the normal control group,the amplitudes of wave a, wave b, and OPs decreased in the model group (P<0.01). The protein and mRNA expressions of IOD and GFAP significantly increased (P<0.01). Compared with the model group, the amplitudes of wave b and OPs increased, and the protein and mRNA expressions of IOD and GFAP significantly decreased in each HJR group. The amplitude of wave a in the middle and high dose HJR groups increased. The amplitude of wave b increased and the IOD expression decreased in the Western medicine treatment group, showing statistical difference (P<0.05, P<0.01). There was no statistical difference in each index between the Western medicine treatment group and each HJR group., Conclusion: HJR could attenuate the visual electrophysiological dysfunction in early diabetic rat, showing certain protection on retinal glial cells.

Objective: To study the effects of simvastatin on the expression of connective tissue growth factor (CTGF) that cause fibrosis in the vitreous and retina after intravitreal injection in diabetic rats, and to explore the safety of this procedure., Methods: It was an experimental study. Forty adult male Wistar rats were randomly divided into normal control group (10 rats) and diabetes mellitus group (30 rats). Four months later, according to whether treated with simvastatin or not, the diabetes mellitus group randomly divided into simvastatin intervention group (20 rats) and diabetic positive control group (10 rats). Simvastatin was injected into the vitreous in the simvastatin intervention group, but not in the diabetic positive control group. Seven days later, after the examination of electroretinogram (ERG), all rats were sacrificed, and their eyeballs were enucleated. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry method were performed to determine the expression of CTGF in the vitreous and retina. Terminal DNA transferase-mediated dUTP nick end labeling (TUNEL) method was used to detect apoptosis of retina cells. Concentration of CTGF in the vitreous, retinal expression of CTGF, retinal cellular apoptosis index, ERG-b wave and oscillatory potentials (OPs) of rats in each group were compared using analysis of variance LSD test methods., Results: No systemic toxic reactions, no lens opacity and no endophthalmitis occurred after injection of simvastatin into the vitreous of rats. Concentrations of CTGF in vitreous of simvastatin intervention group, diabetes positive control group and normal control group rats were 359.21 µg/L, 478.47 µg/L and 210.78 µg/L, respectively (F = 252.366, P < 0.05). The levels of CTGF in the vitreous of simvastatin intervention group and diabetic positive control group was significantly higher than that of the normal control group and showed significant difference (t = 12.123, 23.816;P < 0.05). CTGF levels in simvastatin intervention group were significantly lower than those in diabetic positive control group, and the difference was statistically significant. (t = 11.693, P < 0.05). The results of immunohistochemical staining and TUNEL staining were negative in the normal control group. Retinal expression of CTGF in simvastatin intervention group and diabetic positive control group were (26.60 ± 2.95)% and (42.31 ± 2.59)%, respectively. Retinal expression of CTGF in simvastatin intervention group was reduced as compared to the diabetic positive control group, the difference was statistically significant (t = 12.112, P < 0.05). Retinal cellular apoptosis index of simvastatin intervention group and diabetic positive control group was 0.19 ± 0.02 and 0.25 ± 0.03, respectively. Retinal cellular apoptosis index of simvastatin intervention group was significantly lower than that in diabetic positive control group (t = 4.745, P < 0.05). ERG revealed no significant changes. In simvastatin intervention group as compared with diabetic positive control group., Conclusions: Simvastatin could inhibit the expression of CTGF in the vitreous body and retina in diabetic rats. Intravitreal injection of simvastatin is a relatively safe procedure.

Objective: To study the cell apoptosis and the expression of connective tissue growth factor (CTGF) in the retina of diabetic rats and to explore their contributions to the changes of microcirculation., Methods: It was a experiment study. Fifty-five adult male Wistar rats were divided into two groups, normal control group (CON, 10 rats) and diabetes mellitus group (DM, 45 rats). The 30 surviving rats in the DM group were further divided into 3 groups based on the time of observation, 2 month (DM2), 4 month (DM4) and 6 month (DM6) groups, with 10 rats in each group. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). Expression of CTGF was determined by immunohistochemical study. Retinal vessels were observed by retinal digest stretched periodic acid Schiff (PAS) staining., Results: Immunohistochemical staining and TUNEL staining revealed negative results in normal control group. Retinal cell apoptosis index increased gradually from DM2 to DM6, the differences between any two groups were statistically significant (t(2-4, 2-6, 4-6) = 21.432, 50.843, 29.410; P < 0.05). Expression of CTGF in the retina increased from DM2-DM6, the differences between any two groups were statistically significant (t(2-4, 2-6, 4-6) = 15.345, 26.316, 10.971; P < 0.05). PAS staining of retinal blood vessels obtained negative results in the CON and DM2 groups. Part of retinal capillaries were slightly stiff and narrow in DM4 group. Retinal capillaries in DM6 group were trunk stiff and were narrowed obviously. The number of pericytes was reduced in DM4, and progressed following the course of diabetes. The number of pericytes in the DM2 group did not different from that in the CON group (t = 0.875, P = 0.387). The number of pericytes in the DM4 and DM6 group were significantly decreased as compared to the CON group (t = 3.367, 6.667; P < 0.05). Retinal cellular apoptosis index had a significant positive correlation to the expression of CTGF (r = 0.958, P < 0.05). Number of pericytes was significantly correlated (negative correlation) with retinal cellular apoptosis index and the expression of CTGF (r = -0.540, -0.595; P < 0.05)., Conclusions: The appearances of cellular apoptosis and fibrosing factor CTGF in the retina of diabetic rats occurred earlier than the changes of microcirculation and the number of capillary pericytes.

Ocular neovascularization is the primary cause of blindness in a wide range of ocular diseases. The vascular endothelial growth factor A (VEGF-A) is the key factor involved in ocular angiogenesis, which can cause eye diseases through the development of pathological angiogenesis and increase of vascular permeability. There are two families of VEGF-A isoforms formed by alternative splicing, the angiogenic VEGF-A family (VEGF(xxx)), known to contribute to ocular neovascularization, and the anti-angiogenic VEGF-A family (VEGF(xxx)b), which is found in normal ocular tissues but downregulated in human diabetic retinopathy. The first member of the VEGF(xxx)b family to be isolated was VEGF(165)b. It can significantly reduce preretinal neovascularization without inhibition of physiological intraretinal angiogenesis. As the studies on the VEGF(xxx)b family proceed more deeply, controlling the balance of VEGF(xxx) to VEGF(xxx)b isoforms may be therapeutically valuable in the treatment of angiogenic eye diseases such as diabetic retinopathy and age-related macular degeneration.

Proliferative fibrotic membranes in Proliferative diabetic retinopathy (PDR) cause severe harm to the patients' visual function. It is critical to inhibit the occurrence and development of fibrosis in order to preserve the visual function of the patients in advanced stage of diabetic retinopathy. In recent years, reports about inhibiting neovascularization in PDR are common. It is rare to find reports about inhibiting fibrosis formation. The application of angiotensin converting enzyme inhibitor, vascular endothelial growth factor receptor antagonist, HMG coenzyme A reductase inhibitors and steroids could inhibit the formation of fibrosis at varying degrees by targeting different pathways. Therefore, it is necessary to summarize the basis of current research conditions for the inhibition of the formation of proliferative fibrotic membranes in PDR, which will provide extensive ideas for the treatment of PDR and fibrosis formation.

Purpose: To explore the role of vascular endothelial growth factor and interleukin-6 in the aqueous humor in the pathogenesis of diabetic retinopathy(DR)., Methods: Aqueous humor samples were collected in type 2 diabetic patients during cataract surgery. The classification of DR was assigned based on the fundus examination and fluorescein angiography after cataract surgery. The experiment group included non-diabetic retinopathy (NDR,21 cases),background diabetic retinopathy (BDR,26 cases) and Proliferative diabetic retinopathy (PDR,19 cases). The control group was the healthy cataract patients (20 cases). The levels of VEGF and IL-6 in the aqueous humor was assessed using the ELISA technique., Results: The level of VEGF in NDR,BDR and PDR cases were(240.30+/-26.15)pg/ml, (292.27+/-58.91)pg/ml and (477.41+/-91.01)pg/m1, respectively. The level of IL-6 in NDR, BDR and PDR cases were (160.83+/-33.41)pg/ml, (238.60+/-62.23)pg/ml and (389.13+/-90.35)pg/ml,respectively. The levels of VEGF and IL-6 in the aqueous humour from the control group were (140.58+/-26.27) pg/ml,(82.72+/-21.53)pg/ml.The level of VEGF and IL-6 were significantly different in experiment and control groups(F=113.67,p<0.01; F=106.53, p<0.01). In the aqueous humor of diabetic patients increased significantly(p<0.O1). The level of VEGF and IL-6 was significantly correlated with each other in experiment group (r=0.995,p<0.O1). The levels of VEGF(r=0.792,0.826,0.841,p<0.01)and IL-6(r=0.829, 0.817.0.896,p<0.01)were also significantly correlated with the duration of DR., Conclusions: VEGF and IL-6 play important roles in the development of DR. The levels of these two factors are correlated with each other in DR patients.

Objective: To investigate whether hyperglycemia affect the expression of glycosyltransferases and Enzyme-catalyzed glycosylation in the retina of diabetic rat., Method: It was an experimental study. RT-PCR was used to analyze the mRNA level of six glycosyltransferases in the retina of streptozocin diabetic rats; Lectin blot assay with RCA-I was performed to investigate the level of Galβ 1→4GlcNAc or N-glycans on total retinal glycoproteins., Results: mRNA level of six glycosyltransferases in the retina of streptomycin diabetic rats and of normal rat is: O-linked N-acetylglucosamine transferase (0.1650 ± 0.1160 versus 0.1160 ± 0.036); UDP-Gal: betaGlcNAc beta1,3-galactosyltransferase (0.0186 ± 0.0122 versus 0.0152 ± 0.0047); alpha 1,4-galactosyltransferase (0.0040 ± 0.0040 versus 0.0054 ± 0.0022); mannoside acetylglucosaminyltransferase 1 (0.0228 ± 0.0166 versus 0.0187 ± 0.0050); UDP-glucose ceramide glucosyltransferase (0.0129 ± 0.0096 versus 0.0116 ± 0.0040); UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase 1 (0.0157 ± 0.0010 versus 0.0081 ± 0.0016). The mRNA level of UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase 1 was up-regulated in the retina of streptomycin diabetic rats (t = 6.847, P = 0.002). Consistent with this, the level of Galβ 1→4GlcNAc of glycoproteins in streptomycin diabetic rat's retina was strengthened compared with that in control rats., Conclusions: The results from this study showed that Hyperglycemia could up-regulate the expression of UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase 1 and strengthen the level of Galβ 1→4GlcNAc of glycoproteins in the retina of streptomycin diabetic rats. Our initial results will contribute to the research for the relation between the Enzyme-catalyzed Glycosylation and the etiopathogenesis of diabetic retinopathy.

Objective: To detect the expression of vascular endotheilal growth factor (VEGF), stromal cell-derived factor-1alpha (SDF-1alpha), and its receptor CXCR-4 in the retinopathy of diabetic rats, and to explore the relationship between those factors and diabetic-retinopathy (DR)., Methods: Diabetes was induced in 40 rats with a single intraperitional injection of streptozotocin(STZ). Experimental rats were randomly divided into M1 (diabetic for 1 month), M3 (diabetic for 3 months), and M5 (diabetic for 5 months) groups, and another 10 rats served as a normal control group (NC). Retinal vascular status was observed by transmission electron microscope. After retinal stretched preparation, VEGF, SDF-1alpha and CXCR-4 immunohistochemistry were done. Retinal VEGF, SDF-1alpha, and CXCR-4 mRNA were detected by semi-quantitative RT-PCR. Protein expression was measured by Western blot., Results: Under transmission electron microscope, change in vascular status was found in M1 to M5 groups, but not in the NC group. The changes became increasingly serious with the prolongation of the disease. By immunohistochemistry, we found the expression of VEGF, SDF-1alpha, and CXCR-4 on the retina increased gradually. It increased after injecting STZ for 1 month and increased significantly after 5 months. VEGF, SDF-1alpha, and CXCR-4 mRNA expression increased obviously after injecting STZ for 1 month and increased significantly after 5 months. Western blot showed that protein of VEGF, SDF-1alpha, and CXCR-4 had no change after injecting STZ for 1 month. It began to increase in the M3 group and increased most in the M5 group., Conclusion: The expression of VEGF, SDF-1alpha, and CXCR-4 on the retina in retinopathy of diabetic rats increases gradually with the prolongation of the disease. It is an important factor for diabetic retinopathy.

Objective: To explore the expression of vascular endothelial growth factor of vitreous in patients with proliferative diabetic retinopathy, and to analyze the relative factors., Methods: It was a case-control study. Data of 50 eyes of 50 PDR patients who underwent vitrectomy were retrospectively analyzed. The vitreous fluid samples were obtained during surgery. The VEGF level in vitreous fluid was determined by enzyme linked immunosorbent assay, and the expression of vascular endothelial growth factor of vitreous in patients with proliferative diabetic retinopathy was analyzed. The mean follow-up was 9 months with a range from 6 to 26 months. The VEGF levels in vitreous fluid were compared between PDR patients and normal control, and silicone oil tamponade group and without silicone oil tamponade group by t-test. The changes of VEGF levels in vitreous fluid in eyes with regression, stabilization, and progression were analyzed by analysis of variance. The effects of VEGF levels in vitreous fluid on PDR were analyzed by analysis of variance., Results: The mean VEGF level of vitreous was (592.4801 +/- 587.4267) ng/L in PDR patients, and it was (131.3022 +/- 26.9192) ng/L in normal control. VEGF level was significantly higher in PDR patients than that in normal control (t = 3.2315, P < 0.05). There were 10 eyes (20%) with progression of PDR, 10 eyes (20%) with stabilization, and 30 eyes (60%) with regression. Vitreous level of VEGF was significantly higher in eyes with progression of PDR than that in eyes with stabilization or regression of PDR (q = -3.3187, -4.0843; P < 0.05). Vitreous level of VEGF was significantly higher in eyes without retinal photocoagulation than that in eyes underwent panretinal photocoagulation or local photocoagulation (q = -4.2187, -3.9672; P < 0.05)., Conclusion: There is a correlation between the expression of VEGF of vitreous and the severity of PDR. The expression of VEGF of vitreous presents a relatively lower level in patients with stabilization and regression of PDR.

Purpose: To investigate the levels of stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) in the vitreous of patients with proliferative diabetic retinopathy., Methods: The levels of SDF-1 and VEGF in the vitreous of 30 eyes of 30 patients with proliferative diabetic retinopathy (PDR) and 12 eyes of 12 patients with idiopathic macular hole (MH) were measured by enzyme-linked immunosorbent assay. Vitreous fluid samples were obtained by vitrectomy., Results: The vitreous concentration of VEGF was significantly higher in eyes with PDR (2,865.87 +/- 387.85 pg/ml) than in eyes with idiopathic macular hole (142.42 +/- 21.03 pg/ml, P < 0.0001). The vitreous level of SDF-1 was also significantly higher in eyes with PDR (298.40 +/- 24.57 pg/ml) than in eyes with idiopathic macular hole (86.91 +/- 15.89 pg/ml, P < 0.0001). The vitreous concentration of SDF-1 correlated significantly with that of VEGF in eyes with PDR ([correlation coefficient] r = 0.62, P < 0.001)., Conclusion: Vitreous levels of both SDF-1 and VEGF in patients with PDR are significantly higher than those of nondiabetic patients. SDF-1 may be correlated with VEGF in angiogenesis in PDR.

Objective: To study the autoimmune injuries on diabetic retinopathy (DR) and the protective effects of immunosuppressive therapy with cyclosporine A (CsA) on the DR of streptozotocin (STZ)-induced diabetic rats., Methods: STZ-induced diabetic rats were randomly divided into 3 groups: group 1: diabetic group without any treatment; group 2: insulin-treated group; group 3: CsA-treated group which was further divided into 2 subgroups: subgroup A: CsA was given 1 week before hyperglycemia appeared and subgroup B: CsA was given one week after hyperglycemia appeared. Subgroup A and subgroup B were further subdivided into 3 groups respectively, based on the dose of CsA (1, 4 and 8 mg x kg(-1) x d(-1)). As a control group, normal rats were also simultaneously monitored. The pathologic changes in the retina were investigated with HE stain and the deposition of immunoglobulins was detected with immunohistochemistry and immunofluorescent microscopy., Results: After 8 week, the deposition of IgG, IgA and IgM was quite significant in the retina of diabetic rats. The data also suggested that insulin treatment had no effects on the DR. In contrast,with CsA intervention, the deposition of immunoglobulins on the retina of diabetic rats vanished., Conclusions: Autoimmune injuries were shown, in the present study, to play a critical role in the pathogenesis of diabetic retinopathy. Immunosuppressive treatment with CsA showed protective effects by inhibiting the deposition of immunoglobulins in the retina of diabetic rats.

Objective: To observe the role of blood glucose control on morphology changes of retinal vessels and retinal vascular endothelial growth factor (VEGF) expression in early diabetic rats., Methods: Diabetes was induced in 8-week-old male Wistar rats by a single intraperitoneal injection of Streptozotocin (STZ). After 3 weeks, the animals were randomly divided into four groups: (1) normal rats group, (2) diabetic rats group, (3) diabetic rats with glucose controlled group, received intensive insulin therapy for 20 days, and (4) diabetic rats with uncontrolled blood glucose group, received unregular insulin therapy, which caused the abrupt fluctuation of glucose. After the treatment period, the animals were perfused with 30 mg/ml Fluorescein isothiocyanate-dextran (FITC-Dextran). The eyes were enucleated and fixed in 4% paraformaldehyde immediately. Retinal flat mounts and paraffin sections were prepared. Morphology of impaired retinal vessels and retinal VEGF expression were assessed by immunofluorescence stain and image analysis., Results: The flat-mounted retinas showed more FITC-Dextran aggregated locally at vessels and capillary meandered or dilated in STZ-diabetic rats with uncontrolled blood glucose than in STZ-diabetic rats with controlled blood glucose. Retinal VEGF expression decreased in group 3 compared with group 2 or with group 4. The statistical difference was significant (P < 0.05)., Conclusion: Strict control of blood glucose by insulin could decrease VEGF expression in retina and protect retinal vessels from impairing in early STZ-diabetic rats.

Objective: To investigate the expression of stromal cell-derived factor 1 (SDF-1) in retina of experimental diabetic rats., Methods: Thirty four healthy male Wistar rats of six to eight weeks old were chosen and divided randomly into 3 groups: normal control (CON, n = 8), diabetes mellitus 2 months (DM2M, n = 14) and 3 months (DM3M, n = 12). Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). At the end point, immunochemistry and digital photomicrography analysis were employed to localize and quantitate the expression of SDF-1 in the retinas. The ratios of integrated optical density (IOD) to area of interest (AOI) were calculated to evaluate the intensities of positive staining. Values of z and t, as well as P value of each, were determined by Wilcoxon-Mann-Whitney test and Student's t-test, respectively. Immunofluorescent double-staining was used to indicate the co-expression of SDF-1 and hypoxia-inducible factor-1 (HIF-1), a pivotal transcription factor activated by hypoxia. The SDF-1 mRNA of the retina from DM2M (n = 4) and CON (n = 2) was detected by real-time RT-PCR. Copies of SDF-1 mRNA were then adjusted by the copies of beta-actin mRNA in each sample. Differences between groups were compared by a Student's t test., Results: Positive immunohistochemical staining for SDF-1 was found in each group, most of which located in the inner part of the retina, especially at the inner nuclear layer (INL) and ganglion cell layer (GCL). The inter-quartile range of IOD/AOI of SDF-1 was between 0.6 x 10(-2) to 1.5 x 10(-2) in diabetic group, and 1.1 x 10(-3) to 2.4 x 10(-3) in control group (z = -3.359, P < 0.001). There was no significant difference between DM2M and DM3M (t = 0.952, P > 0.05). Co-expression of SDF-1 and HIF-1 was found in some cells in the inner part of the retina of diabetic rats. Copies of SDF-1 mRNA in DM2M were 2.3 x 10(-4) +/- 1.2 x 10(-4), and those in CON were 2.9 x 10(-4) +/- 1.0 x 10(-4). The ratios of them to beta-actin were 3.3 x 10(-2) +/- 8.5 x 10(-3) for DM2M and 1.7 x 10(-2) +/- 5.7 x 10(-3) for CON (t = 2.33, P < 0.05)., Conclusion: Possibly promoted by HIF-1, expression of SDF-1 is increased in the retinas of diabetic rats, which may play an important role in the mechanism of diabetic retinopathy.

To observe the changes of TSP-1 expression in the retina of STZ-induced rat diabetic mellitus model with pioglitazone and to elucidate the possible mechanism involved and the effects of thiazolidinediones compound pioglitazone on the early stages of diabetic retinopathy. 30 Male SD rats were randomly divided into 3 groups: control group, DM group and pioglitazone treated DM group (DP group). DM group and pioglitazone treated DM group were injected with STZ intraperitoneally. 8 weeks after the onset of diabetes, the expression of TSPI mRNA was quantified in retinal tissue by quantitative reverse transcription-polymerase chain reaction (RT-PCR) respectively. The locations of TSP-1 in three groups were also established by immunohistochemistry. 8 weeks after the onset of diabetes, every layer of the retina tissue had significant TSP-1 positive staining and when compared with the control group and DM group, the pioglitazone treated DM group had higher TSP-1 positive cell number per unit area; There were significant differences among the DM group, pioglitazone treated DM group and control group in TSP-1 mRNA level, DM group and pioglitazone treated DM group had significantly higher TSP-1 mRNA level than the control group. Our results showed that pioglitazone treatment can alleviate the pathological changes of DR and the expression level of TSP-1 was increased in pioglitazone treated DM rats compared with other DM rats. Our study suggests pioglitazone may have a role on early stage of DR by increased TSP-1 expression.

Objective: One of the earliest changes observed in the development of diabetic retinopathy (DR) is the selective loss of pericytes and acellular capillaries. We tested the hypothesis that advanced glycation end products (AGE) might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the activity and effect of caspase-3 at the same time., Methods: Cultured bovine retinal microvascular pericytes (BRPs) were exposed to various concentrations of advanced glycation end products-bovine serum albumin (AGE, 0.47, 1.88, 7.50 micromol/L) for 4 days. We assayed the degree of pericytes apoptosis by fluorescence activated cell sorting, and further measured the caspase-3 activity and the effect of selective caspase-3 inhibitor Z-DEVD-fmk on apoptosis and the of ratio Bcl-2/Bax expression., Results: The results showed that AGE could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls (r = 0.867, P < 0.01), associated with an increase in intracellular caspase-3 activity. Selective caspase-3 inhibitor Z-DEVD-fmk inhibited pericyte apoptosis induced by AGE., Conclusion: These data suggest that the pericyte loss in DR involves an apoptotic process, and that activation of caspase-3 are associated with apoptotic process, which can provide new therapeutic perspectives in diabetic retinopathy.

Objective: To investigate the changes of vascular endothelial cell tight junction protein (occludin) and glial cell morphology as well as their relationship with blood-retinal barrier (BRB) in the retina of diabetic rats., Methods: The distribution of occludin and GFAP were explored by immunofluorescence histochemical studies in the retina of streptozotocin (STZ)-diabetic rats and age-matched control rats. Evans blue was used to evaluate the impairment of BRB., Results: GFAP immunoreactivity was limited to ganglion cell layer and nerve fiber layer in the control retina. GFAP immunoreactivity was significantly increased in ganglion cell layer and nerve fiber layer in one month diabetic rats. GFAP positive Müller cells were increased in three months and six months diabetic rats. Occludin immunoreactivity progressively decreased in three months and six months diabetic rats but not in the one month diabetic rats. Evans blue injection showed a progressive impairment of BRB., Conclusions: Astrocytes activation in the early stage of diabetes plays an important role in the maintaining of the BRB function. But the activation of Müller cells in the later stage destroyed the BRB eventually. These changes are consistent with the concept that BRB changes caused by altered glial-endothelial cell interactions contributes to the occurrence of diabetic retinopathy.

Objective: To investigate the expression of nitric oxide synthase (NOS) in the retina of 8-week-old diabetic rats, and explore the potential molecular mechanisms for the role of NO in diabetic retinopathy (DR)., Methods: Retinal gene expression profile of normal and 8-week-old diabetic rats was constructed with restriction fragment differential display polymerase chain reaction (RFDD-PCR). Bioinformatic analysis of the differentially expressed gene identified the genes coding for 3 subtypes of NOS, namely eNOS, nNOS and iNOS as the candidate genes related to DR, which was verified using semi-quantitative RT-PCR and immunohistochemistry., Results: The results of RFDD-PCR revealed down-regulated expression of eNOS and nNOS and up-regulated iNOS expression in diabetic rat retina. RT-PCR showed that the expression levels of eNOS and nNOS in diabetic rat retina were obviously lower than that in normal retina (0.23-/+0.03 vs 0.32-/+0.03 for eNOS, P<0.05; 0.25-/+0.02 vs 0.36-/+0.02 for nNOS, P<0.05), but the expression level of iNOS obviously higher (0.27-/+0.02 vs 0.20-/+0.03, P<0.05). Immunohistochemistry of healthy retina visualized eNOS-, nNOS- and iNOS-positive cells, all located in the inner nuclear layer (INL) and ganglion cell layer (GCL), and eNOS-positive cells were also found in vascular endothelium. In diabetic retina, the number of eNOS- and nNOS-positive cells was significantly lowered in comparison with normal rat retina (14.33-/+3.19 vs 22.13-/+3.60 for eNOS, P<0.05; 21.87-/+3.62 vs 34.40-/+7.09 for nNOS, P<0.05), but the number of iNOS-positive cells significantly increased (17.60-/+2.58 vs 11.73-/+2.70, P<0.05)., Conclusion: The alterations in eNOS, nNOS and iNOS expression are associated with the deuelopmant and progression of DR.

Thrombospondin1 (TSP1) is a known antiangiogenic factor, but its possible alteration during the early stages of diabetic retinopathy has not been explored yet. The present study sought to investigate the expression of TSP1 using a model of streptozotocin (STZ) induced diabetes in the rat. Diabetes was induced in male SD rats by an intraperitoneal injection of STZ. Age matched animals served as control. 2 months after the onset of diabetes, the expression of TSP1 mRNA was quantified in retinal tissue by quantitative real time reverse transcription-polymerase chain reaction (RT-PCR) respectively. The locations of TSP1 normal and diabetic retinas were also established by immunohistochemistry. 2 months after the onset of diabetes, retinal TSP1 concentrations were significantly increased in the diabetic rats compared with control rats. Diabetes caused the upregulation of TSP1 expression in the layers of the retina 2 months after the induction of diabetes. The upregulation of TSP1 in retinas of diabetic rats suggests a role in experimental diabetic retinopathy. Further studies should address the possible involvement of the TSP1 in the pathophysiology of diabetic retinopathy.

Objective: To explore the effects of pioglitazone on MKP-1 and TSP-1 expression in the early stages of diabetic retinopathy induced by streptozotocin (STZ) and the relevant mechanism in it., Methods: Diabetic rats were induced by an intraperitoneal injection of STZ in SD rats. Thirty male SD rats were randomly divided into 3 groups: diabetes adding pioglitazone group (intragastric administration pioglitazone 20 mg x kg(-1) x d(-1)), diabetes adding BBS group and normal control group. The body weight and blood glucose were measured every two weeks. Eight weeks later, all rats were killed and the expression of TSP-1 and MKP-1 mRNA was quantified in retinal tissue by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) respectively., Result: TSP-1 and MKP-1 concentrations were significantly increased in the diabetic rats' retinal tissue compared to the control rats. Diabetes groups adding pioglitazone caused the upregulation of TSP-1 and MKP-1 expression in the retina among the three groups., Conclusion: Pioglitazone treatment can significantly attenuate the evolutionary in the early stages of experimental diabetic retinopathy. Further studies should address the possible involvement of TSP-1 and MKP-1 in the correlational pathophysiology between pioglitazone and diabetic retinopathy.

Objective: The purpose of the study was to determine whether hematopoietic stem cell (HSCs) mobilization can regulate early diabetic retinopathy in mice., Methods: Mice were divided into four groups: control group, normal mice HSC-mobilized group, diabetic mice control group and diabetic mice HSC-mobilized group. Murine stem cell growth factor (SCF) and recombined human granulocyte colony stimulating factor (rhG-csf) were administered to the mice with diabetes and without diabetes for continuous 5 days to induce autologous HSCs mobilization, and subcutaneous injection of physiological saline was used as control. The changes associated with autologous HSCs mobilization were characterized using flow cytometry, Immunohistochemistry and semiquantitative RT-PCR. Evans blue quantitative test was used to measure the breakdown level of blood-retina barrier., Results: HSCs were marked by CD34-/low and Sca1+ in this study. The acceleration of endothelial cell regeneration was observed. A decrease of VEGF expression due to autologous stem cell mobilization was found. HSCs could increase the content of VEGFR-2 in mouse retina and significantly downregulated the expression of VEGF and ang-2 in diabetic mice., Conclusions: The experiment suggest that autologous HSCs mobilization can be approach of therapeutic vascular reconstruction and functional restoration of blood-retina barrier in mice.

Purpose: To study the retinal expression of HIF-1 alpha in the diabetic NOD mice., Methods: NOD mice were divided into normal control group and diabetes group randomly, sacrificed at 2, 4, 6, 8 or 12 weeks. Eyes were enucleated and retinas were isolated and used for western blot, or eyes were sectioned for immunohistochemical staining for HIF-1 alphal., Results: Compared to the normal control group, blood sugar of diabetes mice increased significantly but the weights decreased significantly (P < 0.01). Western blot of retinal lysates showed low levels of HIF-1 alpha at the 6th weeks that were markedly increased at the 12th weeks in the diabetes group. Immunohistochemistry showed increased staining for HIF-1 in the inner retina, but not in the outer retina., Conclusion: The retinal HIF-1 alpha levels increasing showed that retinal ischemia happening in the early diabetes. HIF-1 alpha may participate in the secondary damage of retinal ischemia induced by early diabetes.

Objective: To further investigate the effect of NO and its synthase on the retinal oxidative damages in diabetes., Methods: Diabetic rat model was induced by injection with streptozotoc. Saline was injected in the controls. The retina was excised and studied 2 w and 20 w after the establishment of the experimental model and the controls. The distribution and content of 3-nitrotyrosine (3-NT) in the retina were analyzed by employing immunohistochemical method and icon manipulation technique. The proteins and mRNA expression of inducible nitric oxide synthetase (iNOS) and neuronal nitric oxide synthetase (nNOS) were determined by employing immunohistochemical method and RT-PCR technique., Results: The 3-NT expression in the retina of diabetic rats increased in the second week of diabetes. The positive cells only distributed in the ganglion cell layer, indicating that the retinal damage of diabetic rats first appeared in the ganglion cell layer. The NT expression increased significantly in the 20 week of diabetes and the positive cells distributed in the whole layers of retina, indicating that the retinal oxidative damages was in progress step by step and the production of NO increased with the progress of diabetes. Compared with the control, the iNOS expression increased and nNOS decreased in the 2 w of diabetes. The former increased further while the latter nearly disappeared in the 20 w, indicating that the increasing production of NO in the retina of diabetic rats was related with the decrease of nNOS expression and the increase of iNOS expression., Conclusion: The diabetic retinal damages first appear in the ganglion cell layer. Later on, the function of the external retinal layers is impaired. The retinal damages at the early stage of diabetes mainly focus on the ganglion cell layer. In the diabetic rat models, the retinal damages are closely related with the increase of NO which results from the decrease of nNOS expression and increase of iNOS expression.

Objective: To observe and estimate the distribution the changes of histology and biochemical parameters in retina of the patient with diabetic retinopathy., Methods: Retinas were divided into quadrants and the lesions of diabetic retinopathy were compared among quadrants. the vasculature was isolated by trypsin-digest, homogenates taken from temporal and nasal quadrant were used to explore regional differences in the expression of Glut-1, PKCbeta and activity of caspase1., Results: Microaneurysms, acellular capillaries and pericytes ghosts were significantly increased in the temporal retina than in the nasal retina. Activity of the pro-inflammatory protease, caspase1, was the only biochemical abnormality which was found significantly difference between quadrants. Glut-1 expression was decreased in diabetes, but there was no significant difference between quadrants. Expression of PKCbeta in both temporal and nasal retina was tended to be greater in diabetic than in non diabetic patients., Conclusion: Retinal microvascular disease does not develop uniformly across the retina, even the different regions are exposed to the same level of hyperglycemia. Regional differences play an important role in the response of the retinal microvasculature to hyperglycemia.

Objective: To study the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in early stage diabetic retinopathy and their mechanisms, so as to guide the clinical work theoretically., Methods: Fifty-five Wistar rats were randomly divided into four groups. Ten rats were used as controls (M group). Streptozocin was injected intraperitoneally to induce diabetes for I month (M(1) group, n = 15), 3 months (M(3) group, n = 15), and 5 months (M(5) group, n = 15). At the experimental ends of each group, the rats were over-anesthetized and their eyeballs were extracted to make digest preparation. In situ hybridization and immunohistochemistry were used to investigate the expression of bFGF and VEGF on retinal vascular. Transmission electron microscopy was used to observe the histology of the retinal vessels., Results: There was no difference in the number of pericytes between M(1) group and M group (P > 0.05), but the number of pericytes was significantly lower in M(3) and M(5) groups than in the M and M(1) groups (P < 0.001). VEGF in situ hybridization showed an expression rate of 34% in the M(5) group. VEGF immunohistochemistry showed an expression rate of 56% in M(5) group. bFGF in situ hybridization showed an expression rate of 78% in M(3) group and an expression rate of 89% in M(5) group. Transmission electron microscopy showed no change in M group. However, it showed swelling of endothelial cells, finger-like process into the capillary cavity, and uneven distribution of heterochromatin in pericytes in M(1) group. Obvious fragmental thickening and splitting of basement membrane, swelling and deformation, finger like process to the capillary cavity, and concentration and margination of heterochromatin in endothelial cells and swelling and deformation of rough surfaced endoplasmic reticulum and mitochondrion in pericytes were seen in the M(3) and M(5) groups, especially the latter., Conclusion: During the course of diabetic retinopathy, morphologic changes of vessels occurs prior to the expression of growth factors, in which the expression of VEGF follows the expression of bFGF.

Objective: To study dynamically the pathological changes of diabetic retinopathy (DR)., Methods: The techniques of light microscopy, immunohistochemical staining, electron microscopy and histochemical electron microscopy were used to quantitatively analyze the Type IV collagen, laminin and their anionic sites in the rat retinal capillary basement membrane (RBM)., Results: RBM became gradually thickened and was accompanied with an increased staining reaction for type IV collagen and laminin and decrease of anionic sites in number., Conclusion: The increase in thickness, in type IV collagen and laminin and the decrease of the anionic sites in the RBM may be the pathological changes of the exudative lesion in DR.

Objectives: To detect and compare the levels of vascular endothelial growth factor (VEGF) in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to investigate the role of VEGF in the pathogenesis of PDR., Methods: Undiluted vitreous samples of 7 normal subjects and 19 patients with PDR were obtained in different ways, and VEGF levels in the vitreous were detected by using enzyme linked immunosorbent assay (ELISA) for quantitative analysis., Results: In the normal group (n = 7), the mean value of VEGF in the vitreous was 0.35 ng/ml (range 0.18-0.60 ng/ ml) in comparison with that of 5.66 ng/ml in the PDR group (n = 19; range 0.84-15.64 ng/ml), there was a significant difference between the two groups statistically (P < 0.05, Primer t test). In the comparison between male (n = 12; range 0.84-15.64 ng/ml) and female PDR group (n = 7; range 1.34-12.34 ng/ml), as their mean values of VEGF in the vitreous were 4.83 ng/ml and 7.08 ng/ml respectively, there was no significant difference between the two groups statistically (P > 0.05)., Conclusions: The VEGF levels in the vitreous of patients with PDR were elevated, and as a neovascular growth factor, VEGF may play a role in the pathogenesis of PDR.

Objective: To evaluate the characteristics of cellular proliferation in different retinal diseases., Methods: Three specific antibodies [to cytokeratin (CK), glial fibrillary acidic protein (GFAP) and actin, respectively] were used. Vitrectomy specimens from 28 cases with different proliferative retinal diseases were studied immunohistochemically., Results: In the vitreous with proliferative vetreoretinopathy (PVR), most of the proliferative cells were derived from glial and retinal pigment epithelial (RPE) cells. In the vitreous of proliferative diabetic retinopathy (PDR), RPE cells might enhance the contraction of proliferative membranes. Increase in actin in firovascular membranes with PDR may play a positive role on the pericytes dropping out from microvessels., Conclusion: The cell proliferation in the vitreous associated with the increase of levels of growth factors in pathological vitreous, possibly, is one of the mechanisms for the deterioration of proliferative retinal diseases.

The insulin-like growth factor I (IGF-I) levels in 67 vitreous samples taken from 57 patients with various proliferative retinal diseases and 10 normal subjects were detected by radioimmunoassay (RIA). The acid-ethanol-extracted (AEE) method was used to remove the IFG-binding protein before the detection. Our results revealed that IGF-I levels were increased to varying extents in these patients' vitreous. The IGF-I levels in the vitreous with proliferative diabetic retinopathy (PDR) and central retinal vein occlusion (CRVO) were significantly higher than those of the controls. In the PDR group, the increased IGF-I level in the vitreous was positively correlated with the level in serum. The increased IGF-I level in vitreous may play an important role in the development and pathogenesis of proliferative retinal diseases. Great differences were found between the native and the AEE treated vitreous, suggesting that the binding protein posed an obvious interference on the detection of IGF-I by RIA.

Risk factors of tissue hypoxia was investigated in 26 patients with diabetic retinopathy (DMR), who compared with 62 patients without DMR. significant higher incidence of DMR was found in patients with poor diabetic control (HbA 1 greater than or equal to 10%), with orthostatic hypotension (OH), with peripheral neuropathy and with atherosclerosis respectively. Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) was lower (P less than 0.05) and venous oxygen tension in pedal dorsum (PvO2) higher (P less than 0.05) in DMR patients than those in uncomplicated DMR patients and normal subjects. There was a rank correlation between DMR severity and PvO2 (r = 0.429, P less than 0.05), which imply that decreased blood oxygen utilization may be related to diabetic microangiopathy. These data suggested that abnormal changes of HbA 1 and 2,3-DPG, decrease of peripheral blood oxygen utilization, atherosclerosis, OH and neuropathy might be risk factors of tissue hypoxia in DMR patients.

Macrophages, monocytes and the expression of HLA-DR antigen in 52 cases of various vitreal and epiretinal membranes in human eyes were studied with the immunohistochemical technique. The results showed that more than 80% of the specimens were macrophage positive and expressed HLA-DR antigen, and monocytes were mainly seen in the specimens of proliferative diabetic retinopathy (41.7%). Macrophages were considered to play an important role in the development of epiretinal membranes, and immuno-response was also one of the related factors.

The biochemical basis of pericyte loss in early diabetic retinopathy is still an open question. In studies on the mechanism by which retinal pericytes degenerate, we first established a bovine retinal capillary pericyte (BRCP) cell line. Subcultured BRCP grown in high (10-40 mmol/L) glucose media were used as an experimental model. We found that high concentrations of glucose suppress the mitotic rate and cell birth rate of cultured BRCP. High concentrations of glucose inhibit myo-inositol transport and result in decreased intracellular myo-inositol content. This inhibition can be partially reversed by sorbinil, an aldose reductase inhibitor (ARI). Myo-inositol is a precursor of inositol phospholipids (IPLs), whose metabolism is responsible for a number of signal transduction processes. Phosphoinositidase (PIase) cleaves the phosphodiester bond of phosphotidylinositol 4,5 diphosphate (PIP2) to produce two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG). Further experiments showed that IP3 and DG synergistically activate BRCP proliferation in vitro. High concentrations of glucose altered the formation of both IPLs and inositol phosphate esters (IPEs) in an organ culture of retinal microvessels. This alteration can be reversed by adding either high concentrations of myo-inositol or ARI to the medium. PIase activity was attenuated to 82% or 55% when glucose in the growth medium was increased from 5 to 15 or 30 mmol/L, respectively. When IPLs from BRCP were analyzed by HPLC and TLC, we observed the reduction of three IPLs, including the substrate of PIase, PIP2.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental pathologic model of pericyte drop-out was established with capillary pericytes in culture medium of high glucose concentration. The high glucose activated the sorbitol pathway, reduced the intracellular myo-inositol content and disturbed the inositol phospholipid metabolism which resulted in a decrease in the inositol trisphosphate (IP3) level. The correlation of suppressed IP3 level with reduced DNA synthesis was evident, suggesting the mechanism that retinal pericytes degenerated in high glucose concentration. In vitro test of adding myo-inositol and/or aldose reductase inhibitor to the high-glucose medium reversed the inositol phospholipid metabolism and the decreased DNA synthesis, which could hold promise for the clinical prevention and treatment of early diabetic retinopathy.