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

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

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

OBJECTIVETo study the effects of extracts of ginkgo biloba (EGB) on the levels of nitric oxide (NO) and apoptosis in the retina induced by glutamate by intravitreal injection in adult rabbit.

METHODThe model of apoptosis in retina induced by glutamate by intravitreal injection was established in adult rabbit and EGB was retrobulbarly injected. The levels of NO were measured by spectrophotometer. Retina DNA fragmentation was determined by agarose gel electrophoresis.

RESULTThe levels of NO in retina in experiment groups were significantly increased compared with controls, after treatment with high dosage of EGB, and levels of NO was decreased to normal. They were not decreased by injection of small dosage of EGB. DNA fragmentation of retina apoptosis was detected in experiment groups and small dosage EGB groups.

CONCLUSIONThe retina apoptosis was induced by glutamate by intravitreal injection in adult rabbit and was inhibited by EGB that may be through blocking the generation of NO free radicals.

Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Ginkgo biloba ; chemistry ; Glutamic Acid ; Male ; Nitric Oxide ; metabolism ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Rabbits ; Random Allocation ; Retina ; cytology ; metabolism

The authors presented a case of von Hippel-Lindau's disease. The patient was a 33-year-old man who visited to our hospital with visual disturbance on the right eye for a month and blind in the left eye for 10 years on the 4th May, 1973. On the eye examination, the left eye revealed ocular hypertension, deep chamber, and vitreous opacity which interfered with the visuality of the fundus. The right eye was normotensive and 0.1 incorrectable in visual acuity. The fundus revealed two angioma associated with large feeding vessels, scattered hemorrhages, and massive exudates along the engorged and tortuous vessels at the temporal retina. On general physical examination, no neurological signs were found, but multiple hard corn sized masses were palpated on the both sides of epididymal head. The masses were confirmed as epididymal cyst on microscopic examination. The x-ray findings were pulmonary tuberculosis far advanced on chest, an extrinsic pressure indentation in the floor of bladder on I.V.P., and abnormal intracranial vascularity, on right carotid angiogram. The blood chemistry showed an abnormal increase of amylase and lipase titers.
Adult ; Amylases ; Chemistry ; Exudates and Transudates ; Head ; Hemangioma ; Hemorrhage ; Humans ; Lipase ; Ocular Hypertension ; Physical Examination ; Retina ; Spermatocele ; Thorax ; Tuberculosis, Pulmonary ; Urinary Bladder ; Visual Acuity ; Zea mays

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 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

OBJECTIVETo investigate the distribution, changes and a possible role for retinal dopamine transporter (DAT) in experimental myopia in chickens.

METHODSTwo-day-old chickens were divided into four groups. Chicken eyes were fitted with lenses of -10D,-20D and translucent goggles unilaterally. Normal eyes were used as controls. After 3 wk, all chickens were given an intramuscular injection of (125)I-beta-CIT 2beta-carbomethoxy-3beta-(4-iodophenyl)tropane and sacrificed two hours post injection. Retinal pigment epithelium (RPE) and the neural retina were obtained together or RPE was dissected out from the neural retina. Radioactive DAT from each specimen was assayed by gamma-counter.

RESULTSRetinal DAT was detected in RPE specimens rather than in the neural retina in all eyes. Radioactive DAT in myopic eyes was higher, compared with control eyes.

CONCLUSIONSRetinal DAT is mainly located in the RPE and may be involved in the formation of lens induced myopia (LIM) and form deprivation myopia (FDM). These methods may provide a new approach for further studying the role of the dopamine system in experimental myopia.

Animals ; Chickens ; Dopamine Plasma Membrane Transport Proteins ; Eye ; growth & development ; Membrane Glycoproteins ; Membrane Transport Proteins ; analysis ; physiology ; Myopia ; metabolism ; Nerve Tissue Proteins ; Retina ; chemistry

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

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