OBJECTIVETo explore the effects of rat bone mesenchymal stem cell (BMSC) transplantation on retinal neovascularization, and to observe the changes of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factors (VEGF) in rats with oxygen-induced retinopathy (OIR).

METHODSSeventy-two seven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control (CON), model (OIR) and BMSC transplantation. In the BMSC transplantation group, BMSCs were transplanted 5 days after oxygen conditioning. The phosphate buffered saline of the same volume was injected in the CON and OIR groups. The OIR model was prerpared according to the classic hyperoxygen method. At seven days after transplantation, retinal neovascularization was examined by retinal flat-mount staining and hematoxylin eosin (HE) staining. The expression of HIF-1α and VEGF proteins was examined by immunohistochemistry staining and Western blot analysis.

RESULTSThe retinal flat-mount staining results showed that the vessels were well organized in the CON group, but the vessels were irregularly organized, and lots of nonperfusion areas were observed in the OIR group. The large vessels were a bit circuitous, the retinal vessels were relatively organized, and less nonperfusion areas were noted in the BMSC transplantation group. The HE staining results showed that many neovessels and preretinal neovascular (pre-RNC) cells were observed on the internal limiting membrane in the OIR group. There were less pre-RNC cells in the BMSC transplantation group compared with the OIR group (P<0.01). The immunohistochemistry analysis showed that more HIF-1αand VEGFcells were observed in the OIR group compared with the CON group, and less HIF-1αand VEGFcells were observed in the BMSC transplantation group compared with OIR group (P<0.05). The Western blot analysis showed the expression of HIF-1α and VEGF proteins in the OIR group was significantly higher than that in the CON group. The expression of HIF-1α and VEGF proteins in the BMSC transplantation group was lower than that in the OIR group (P<0.01).

CONCLUSIONSBMSC transplantation therapy could alleviate retinal neovascularization in OIR rats, and its mechanisms might be associated with the inhibition of the expression of HIF-1α and VEGF proteins.

Animals ; Animals, Newborn ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Male ; Mesenchymal Stem Cell Transplantation ; Rats ; Rats, Sprague-Dawley ; Retina ; chemistry ; Retinal Neovascularization ; prevention & control ; Retinopathy of Prematurity ; metabolism ; therapy ; Vascular Endothelial Growth Factor A ; analysis

OBJECTIVETo investigate the effect of MiR-200b on human retinal endothelial cells (hRECs) cultured in high glucose and explore the mechanism.

METHODShRECs cultured in high glucose or in normal media were examined for MiR-200b mRNA expression using real-time PCR. The effect of MiR-200b transfection on hREC proliferation in high-glucose culture was evaluated with MTT assay, and real-time PCR and Western blotting were performed to determine vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) expression in the transfected cells.

RESULTSThe cells in high-glucose culture showed significantly decreased MiR-200b expression and active proliferation. Compared with those in normal control cells, VEGF and TGFβ1 mRNA and protein expressions increased markedly in cells cultured in high glucose (P<0.05). MiR-200b transfection of the cells caused significantly increased cellular expression of MiR-200b but decreased expression levels of VEGF and TGFβ1 mRNA and protein, and suppressed hREC proliferation in high glucose culture (P<0.05).

CONCLUSIONMiR-200b can regulate REC growth and proliferation by changing VEGF and TGFβ1 expressions and thus play a role in the pathogenesis and progression of diabetic retinopathy.

Blotting, Western ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Diabetic Retinopathy ; Endothelial Cells ; cytology ; Glucose ; chemistry ; Humans ; MicroRNAs ; metabolism ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Retina ; cytology ; Transfection ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo optimize the hydrolysis process of linarin by response surface methodology, and to use the model of aldose reductase to study the acacetin's activity of aldose reductase inhibitory.

METHODThe model of acacetin enzyme in vitro was established by the determination of fluorescence absorption of NADPH, the inhibition rate of acacetin aldose reductase was calculated, and then the IC50 of hydrolysis was obtained. The hydrolysis process of linarin hydrolysis condition was optimized by using response surface method.

RESULTThe results indicated that the IC50 of acacetin (2.74 mg x L(-1)) was less than the IC50 of linarin (3.53 mg x L(-1)). Hydrolyzation time of 7.4 h, sulphuric acid concentration of 0.54 mol x L(-1) and the ratio of material to liquid of 3 : 1 were the optimum conditions.

CONCLUSIONHydrolyzate acacetin has preferable inhibitory activity of aldose reductase. The optimized hydrolysis condition of linarin is convenient to use with good predictability.

Aldehyde Reductase ; antagonists & inhibitors ; metabolism ; Animals ; Diabetic Retinopathy ; enzymology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Enzyme Inhibitors ; chemistry ; pharmacology ; Flavones ; chemistry ; Glycosides ; chemistry ; pharmacology ; Glycosylation ; Humans ; Hydrolysis ; Male ; Rats ; Rats, Wistar ; Retina ; enzymology

To evaluate the effect of chlorogenic acid (CGA), a polyphenol abundant in coffee, on retinal vascular leakage in the rat model of diabetic retinopathy, Sprague-Dawley rats were divided into four groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with 10 and 20 mg/kg chlorogenic acid intraperitoneally daily for 14 days, respectively. Blood-retinal barrier (BRB) breakdown was evaluated using FITC-dextran. Vascular endothelial growth factor (VEGF) distribution and expression level was evaluated with immunohistochemistry and Western blot analysis. Expression of tight junction proteins, occludin and claudin-5, and zonula occludens protein, ZO-1 was also evaluated with immunohistochemistry and Western blot analysis. BRB breakdown and increased vascular leakage was found in diabetic rats, with increased VEGF expression and down-regulation of occludin, claudin-5, and ZO-1. CGA treatment effectively preserved the expression of occludin, and decreased VEGF levels, leading to less BRB breakdown and less vascular leakage. CGA may have a preventive role in BRB breakdown in diabetic retinopathy by preserving tight junction protein levels and low VEGF levels.
Animals ; Blood-Retinal Barrier/*drug effects ; Chlorogenic Acid/metabolism/*pharmacology ; Claudin-5/metabolism ; Dextrans/chemistry ; Diabetes Mellitus, Experimental/complications/metabolism/*pathology ; Diabetic Retinopathy/etiology/prevention & control ; Down-Regulation ; Fluorescein-5-isothiocyanate/chemistry ; Male ; Occludin/metabolism ; Rats ; Rats, Sprague-Dawley ; Retina/*metabolism ; Tight Junction Proteins/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; Zonula Occludens-1 Protein/metabolism

BrdU (5-Bromo-2'-deoxyuridine) is usually used to label the mitotic cells as well as to trace reagent in cell transplation. However, BrdU could also exert some side effect on cellular biological characteristics upon inappropriate use. To explore the appropriate concentration of BrdU for labelling retinal progenitor cells (RPCs), we co-cultured Embryonic day (E) 17. 5 RPCs with different concentrations of BrdU, which were 0.2, 1, 5 and 10 micromol/L, respectively. After 48 hours, the RPCs were proliferation- or differentiation-cultured. Immunofluorescence was used to detect the BrdU-positive ratio and differentiation potential. Cell count was used to evaluate proliferation ability, and lactate dehydrogenase (LDH) release assay was used to monitor cytotoxicity. The results showed that 0.2 micromol/ L BrdU could not label RPCs clearly, while BrdU of 1, 5 or 10 micromol/L could label the RPCs with similar ratios. 1 micromol/L BrdU displayed no obvious cytotoxicity and showed no obvious effect on the proliferation and differentiation ability. However, 5 micromol/L or 10 micromol/L BrdU could evidently inhibit RPCs proliferation, partly due to the cytotoxicity effect. Furthermore, 10 micromol/L BrdU could inhibit the differentiation of RPCs towards MAP2-positive nerve cells, but showed no influence on the differentiation of RPCs towards GFAP- and glutamine synthetase positive glial cells. This study suggested that 1 micromol/L BrdU could be an appropriate concentration for RPCs labelling and could efficiently label RPCs without obvious side effect.
Animals ; Bromodeoxyuridine ; chemistry ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Proliferation ; Embryo, Mammalian ; Immunohistochemistry ; Mice ; Retina ; cytology ; Staining and Labeling ; Stem Cells ; cytology

OBJECTIVETo study the effect of Vaccinium uliginosum L., (VU) on the electroretinogram (ERG) and retinal pathological changes in rabbits after light-induced damage.

METHODSTwenty-eight Chinchilla rabbits were randomly divided into four groups: administration beforehand (A), administration after injury (B), light injury without administration (C), and blank (D) groups. After a 4-week administration of VU homogenate at 4.8 g/(kg·d) once a day in group A, ERG in groups A, B and C were recorded according to the standards set by the International Society for Clinical Electrophysiology of Vision (ISCEV). Except for group D, the groups were then exposed to strong light. Just after that, group A stopped receiving VU treatment and group B started to receive it. Then ERGs in all groups were recorded after 1 day, 1 week, and 2 weeks. Throughout the whole process groups which were not fed with VU were fed with normal saline. Finally, the tissues and structures of all the groups were observed and the thickness of the outer nuclear layers (ONL) was measured.

RESULTS(1) After 4-week feeding with VU, the latency time of ERG in group A became shorter than those in the other groups and the amplitude increased. After being exposed to strong light, the latency time lengthened and amplitude decreased in all the injury groups, but comparing at each time point, the measured values in group A were better than those in group C. With the accumulation of VU, the ERG in group B improved, and finally, all of the detected values became better than those in group C. (2) Retinae in group D were normal in histology and the layers were in order but those in group C became disarranged. The injuries in groups A and B were minor compared with those in group C. The thickness of the ONL in group C was significantly thinner than in the other groups (P=0.000), and that in groups A and B was thicker than that in group C, although thinner than in group D. That in group A was thicker than in group B.

CONCLUSIONSVU can relieve the injury to rabbit retinae exposed to normal day and night rhythm, alleviate the harm caused by light when used beforehand, and repair the light damage to the retina.

Animals ; Electroretinography ; Light ; Plant Extracts ; pharmacology ; Rabbits ; Retina ; drug effects ; pathology ; physiopathology ; radiation effects ; Retinal Cone Photoreceptor Cells ; drug effects ; pathology ; radiation effects ; Retinal Rod Photoreceptor Cells ; drug effects ; pathology ; radiation effects ; Time Factors ; Vaccinium ; chemistry

BACKGROUNDEndothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina.

METHODSEPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 µmol/L CFSE at 37°C was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.

RESULTSEPCs labeled with 5 µmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.

CONCLUSIONEPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.

Animals ; Cells, Cultured ; Endothelial Cells ; chemistry ; cytology ; Fluoresceins ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Retina ; cytology ; Stem Cells ; chemistry ; cytology ; Succinimides ; chemistry

OBJECTIVETo evaluate the hazardous effects of fried potato chips upon the retina of two developmental stages of the albino rats aged 7 and 14 days from parturition.

METHODSPREGNANT RATS WERE ARRANGED INTO TWO GROUPS: control pregnant rats and consequently their delivered newborns until reaching 7 and 14 days old from parturition and fried potato chips group in which pregnant rats at the 6th day of gestation maintained on diet formed of fried potato chips supplied from the market mixed with standard diet at a concentration of 50% per each till 7 and 14 post-partum. Three fold integrated approaches were adopted, namely, histological, ultrastructural and proteomic analysis.

RESULTSHistological examination of the retina of the experimental offsprings revealed many histopathological changes, including massive degeneration, vacuolization and cell loss in the ganglion cell layer, as well as general reduction in retinal size. At the ultrastructural level, the retina of experimental offsprings exhibited number of deformities, including ill differentiated and degenerated nuclear layer, malformed and vacuolated pigment epithelium with vesiculated and fragmented rough endoplasmic reticulum, degenerated outer segment of photoreceptors, as well as swollen choriocapillaris and loss of neuronal cells. Proteomic analysis of retina of the two experimental developmental stages showed variations in the expressed proteins as a result of intoxication which illustrated the adverse toxic effects of fried potato chips upon the retina.

CONCLUSIONSIt can be concluded that the effect of fried potato chips on the development of retina in rats may be due to the presence of acrylamide or its metabolite.

Acrylamide ; toxicity ; Animals ; Animals, Newborn ; Cooking ; methods ; Diet ; methods ; Female ; Histocytochemistry ; Male ; Pigments, Biological ; Pregnancy ; Proteome ; analysis ; Rats ; Retina ; pathology ; Solanum tuberosum ; chemistry ; Ultrasonography

The expression of caveolin-1 and -2 in the retina was examined; Western blot analysis showed that both were present. Immunohistochemistry indicated that caveolin-1 was expressed in the majority of retinal layers, including the ganglion cell layer, inner plexiform layer, outer plexiform layer, and in the vascular endothelial cells of the retina. Caveolin-2 was primarily immunostained in the vessels, but in a few other elements as well. This is the first demonstration of caveolin differential expression in the retina of rats, and suggests that caveolin plays an important role in signal transduction in glial cells and neuronal cells.
Animals ; Caveolin 1/*analysis/immunology ; Caveolin 2/*analysis/immunology ; Gene Expression Regulation ; Immunohistochemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Retina/*chemistry

BACKGROUNDPrevious studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats.

METHODSA single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits.

RESULTSThe pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end.

CONCLUSIONSMNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Female ; I-kappa B Proteins ; analysis ; physiology ; Methylnitrosourea ; toxicity ; NF-KappaB Inhibitor alpha ; NF-kappa B ; analysis ; physiology ; Photoreceptor Cells ; chemistry ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; pathology