PURPOSE: To investigate the effect of bevacizumab (Avastin; Genentech, San Francisco, CA, USA) on vascular endothelial growth factor (VEGF) expression and inflammation in fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: Fibrovascular membranes from 19 eyes of 18 patients with PDR were studied using immunohistochemistry and analyzed in the following 3 groups; group 1: 4 inactive PDR eyes, group 2: 10 active PDR eyes treated preoperatively with adjunctive intravitreal bevacizumab, group 3: five active PDR eyes not treated preoperatively with bevacizumab. Immunohistochemical staining for VEGF, CD31 and CD68 were done. RESULTS: The immunoreactivity to VEGF and CD 31-positive blood vessels was significantly higher in membranes from group 3 than group 1 (p = 0.007 for VEGF, 0.013 for CD 31-positive vessels). Intravitreal bevacizumab caused a reduction in VEGF expression and vascular densities in 4 out of 10 (40%) excised membranes from eyes with PDR. However, six membranes (60%) in group 2 still demonstrated relatively strong VEGF expression and high vascular density. Infiltration of macrophages was observed in 16 out of the 19 membranes, and the density of macrophages was increased in group 2 compared with group 1 (p = 0.043). CONCLUSION: Intravitreal bevacizumab injections caused some reduction in VEGF expression and vascular densities in a limited number of active PDR patients. A single intravitreal bevacizumab injection may not be enough to induce complete blockage of VEGF and pathologic neovascularization in active PDR patients. Repeated injections, panretinal photocoagulation and/or PPV may be necessary following intravitreal bevacizumab to reinforce the anti-VEGF effect of the drug.
Adult ; Angiogenesis Inhibitors/*therapeutic use ; Antibodies, Monoclonal/*therapeutic use ; Diabetic Retinopathy/*drug therapy/*metabolism ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Vascular Endothelial Growth Factor A/*metabolism

OBJECTIVETo 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.

METHODSThe 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.

RESULTSCompared 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.

CONCLUSIONHJR could attenuate the visual electrophysiological dysfunction in early diabetic rat, showing certain protection on retinal glial cells.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; physiopathology ; Diabetic Retinopathy ; drug therapy ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; Electroretinography ; Male ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; metabolism ; physiopathology

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.
Rats ; Animals ; Diabetic Retinopathy/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mitogen-Activated Protein Kinase 3/metabolism* ; Interleukin-1beta/metabolism* ; Methylene Blue/pharmacology* ; Phosphorylation ; Rats, Sprague-Dawley ; MAP Kinase Signaling System ; Diabetes Mellitus, Experimental/drug therapy* ; Superoxide Dismutase/metabolism*

OBJECTIVETo 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.

METHODSDiabetic 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.

RESULTTSP-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.

CONCLUSIONPioglitazone 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.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetic Retinopathy ; drug therapy ; metabolism ; Hypoglycemic Agents ; therapeutic use ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; therapeutic use ; Thrombospondin 1 ; biosynthesis ; genetics

BACKGROUNDHematopoietic stem cells (HSCs) can be used to deliver functionally active angiostatic molecules to the retinal vasculature by targeting active astrocytes and may be useful in targeting pre-angiogenic retinal lesions. We sought to determine whether HSC mobilization can ameliorate early diabetic retinopathy in mice.

METHODSMice were devided into four groups: normal mice control group, normal mice HSC-mobilized group, diabetic mice control group and diabetic mice HSC mobilized group. Murine stem cell growth factor (murine 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. Immunohistochemical double staining was conducted with anti-mouse rat CD31 monoclonal antibody and anti-BrdU rat antibody.

RESULTSMarked HSCs clearly increased after SCF plus G-csf-mobilization. Non-mobilized diabetic mice showed more HSCs than normal mice (P=0.032), and peripheral blood significantly increased in both diabetic and normal mice (P=0.000). Diabetic mice showed more CD31 positive capillary vessels (P=0.000) and accelerated endothelial cell regeneration. Only diabetic HSC-mobilized mice expressed both BrdU and CD31 antigens in the endothelial cells of new capillaries.

CONCLUSIONAuto-mobilized adult hematopoietic stem cells advance neovasculature in diabetic retinopathy of mice.

Animals ; Diabetic Retinopathy ; drug therapy ; Female ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Hematopoietic Cell Growth Factors ; therapeutic use ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Immunohistochemistry ; Mice ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism

BACKGROUNDDiabetic retinopathy (DR) is a leading cause of visual impairment and blindness among the people of occupational age. To prevent the progress of retina injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE) have been reported to be effective in treating diabetic complications, while little is discussed about the functional protein changes.

METHODSWe used streptozotocin (STZ) to induce diabetes in rats. GSPE (250 mg/kg body weight per day) were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin and advanced glycation end products (AGEs) were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate retina protein profiles among control, STZ-induced diabetic rats, and GSPE treated diabetic rats.

RESULTSGSPE significantly reduced the AGEs of diabetic rats (P < 0.05). Moreover, GSPE significantly suppressed the vascular lesions of central regions, decreased capillary enlargements and neovascularization, similar to those of the control rats under light microscope. Eighteen proteins were found either up-regulated or down-regulated in the retina of STZ-induced diabetic rats. And seven proteins in the retina of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in many important biological processes such as heat shock, ubiquitin-proteasome system, cell proliferation, cell growth and glucose metabolism.

CONCLUSIONSThese findings might promote a better understanding for the mechanism of DR, and provide novel targets for evaluating the effects of GSPE therapy.

Animals ; Blood Glucose ; drug effects ; metabolism ; Body Weight ; drug effects ; Diabetes Mellitus, Experimental ; complications ; metabolism ; pathology ; Diabetic Retinopathy ; drug therapy ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Glycated Hemoglobin A ; metabolism ; Glycation End Products, Advanced ; metabolism ; Grape Seed Extract ; Male ; Plant Extracts ; pharmacology ; Proanthocyanidins ; pharmacology ; Proteomics ; methods ; Rats ; Rats, Wistar

OBJECTIVETo investigate the protective effect of Qihuang Mingmu capsule (QHMM) on retina of diabetic mice and its impact on VEGF expression.

METHODForty 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.

RESULTQHMM 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.

CONCLUSIONQHMM 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.

Animals ; Capsules ; administration & dosage ; Diabetic Retinopathy ; drug therapy ; genetics ; metabolism ; prevention & control ; Drugs, Chinese Herbal ; administration & dosage ; Gene Expression ; drug effects ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Protective Agents ; administration & dosage ; Retinal Diseases ; drug therapy ; genetics ; metabolism ; prevention & control ; Vascular Endothelial Growth Factor A ; genetics ; metabolism