To simulate the posterior vitreous detachment (PVD) in the rabbit, 1 IU hyaluronidase and/or 0.2 ml of perfluoropropane gas was intravitreally injected. Ophthalmoscopic, light microscopic examination prepared by cryotechnique, electron microscopic examination, and electroretinogram were done on the 3rd and 28th postoperative days. As a result, the eyes undergone simultaneous intravitreal injection of 1 IU hyaluronidase and 0.2 ml perfluoropropane gas showed membranous structure split from the internal limiting membrane of the superior retina in 3 days after injection. The eyes also demonstrated membranous structure separated from the superior retina after 28 days, simulating vitreous detachment. On the contrary, neither agent alone induced vitreous detachment. No toxic retinal changes associated with simultaneous intravitreal injection of 1 IU hyaluronidase and 0.2 ml perfluoropropane gas were observed. Therefore, with a future support by histologic examination other than cryotechnique and by immunohistochemical analysis, the simultaneous intravitreal injection of perfluoropropane gas and hyaluronidase would be a promising method to induce vitreous detachment in non-vitrectomized eye.
Animals ; Drug Combinations ; Electroretinography ; Eye Diseases/chemically induced/pathology ; Fluorocarbons/*toxicity ; Hyaluronoglucosaminidase/*toxicity ; Injections ; Rabbits ; Retina/drug effects/physiology ; Vitreous Body/*drug effects/pathology

We quantitatively analyzed the changes of the (retinal) threshold sensitivities between normal status and dilated emmetropic status, and between dilated emmetropic status and -1.0D induced myopic status. Three consecutive visual field examinations by Humphrey Field Analyzer C-30-2 threshold test and STATPAC program were performed in 36 eyes of 21 normal subjects who ranged in age from 23 to 40 years. The results were as follows: 1) There was a statistically significant decrease (0.87 +/- 0.91dB)in the mean threshold sensitivity within the central 30 degrees at the dilated emmetropic status (27.90dB) as compared with the normal status (28.77dB)(p = 0.0001). 2) There was a statistically significant decrease(0.99 +/- 0.61dB) at -1.0D induced myopic status(26.91dB) as compared with the dilated emmetropic status(27.90dB)(p = 0.0001). 3) After dilation, the mean threshold sensitivity between 20 and 30 degrees of field showed statistically significant decrease as compared with those within the central 20 degrees of visual field(p < 0.05). In case of -1.0D induced myopia, there were uniform decreases in the mean threshold sensitivities within the central 30 degrees of visual field (p > 0.05). Therefore, we emphasize the importance of consistent pupillary size and correct refractive state in performing the serial automated visual field tests.
Adult ; Female ; Humans ; Male ; Myopia/etiology/*physiopathology ; Pupil/drug effects ; Pupil Disorders/chemically induced/*physiopathology ; Retina/physiology ; Sensory Thresholds/*physiology ; Tropicamide/pharmacology ; Visual Field Tests ; Visual Fields/*physiology

BACKGROUNDRetinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition.

METHODSRPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3.

RESULTSCCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05).

CONCLUSIONLow-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.

Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; physiology ; Cell Hypoxia ; drug effects ; physiology ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retina ; cytology ; Stem Cells ; cytology ; drug effects ; Triamcinolone Acetonide ; pharmacology

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4^-/-RDH8^-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4^-/-RDH8^-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
ATP-Binding Cassette Transporters/physiology* ; Alcohol Oxidoreductases/physiology* ; Animals ; Cell Survival/drug effects* ; Cells, Cultured ; Humans ; Inactivation, Metabolic ; Mice ; Retina/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Swine ; Tretinoin/pharmacology*

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

Animals ; Cells, Cultured ; Ginkgo biloba ; Glutamic Acid ; toxicity ; Immunohistochemistry ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Neurons ; drug effects ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; pathology

The aim was to investigate the proteolytic activity of plasmin and its long-term complications. Plasmin was injected into the vitreous cavity of rabbits' eyes. Slit lamp biomicroscopy and electroretinography were performed. Rabbits were serially sacrificed at four months, and globes fixated and prepared for light and transmission electron microscopy. In both the plasmin-injected and control eyes, electroretinography showed a transient decrease in the amplitude, but this recovered to the baseline level in a week. Under the light microscope, the plasmin-treated eyes had a smooth retinal surface, implying separation of the vitreous cortex from the retina. In the control eyes, the collagen fibers remained on the retinal surface. By transmission electron microscopy, the plasmin-treated eyes showed a vitreous-free retinal surface, but no vitreoretinal separation was observed in the control eyes. Plasmin induces a cleavage between the vitreous and the internal limiting membrane, with no long-term complications, so may be a useful pharmacologic adjunct to vitrectomy.
Animals ; Electroretinography ; Fibrinolysis/*drug effects ; Fibrinolytic Agents/*pharmacology ; Injections ; Plasmin/*pharmacology ; Rabbits ; Research Support, Non-U.S. Gov't ; Retina/drug effects/physiology ; Vitreous Body/*drug effects ; Vitreous Detachment/*chemically induced/pathology

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1

BACKGROUNDNeural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion cells in acute diabetes in rats.

METHODSDiabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin (STZ). Expression and localization of caspase-3 and apoptosis-inducing factor (AIF) proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling (TUNEL) assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes.

RESULTSTwo weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes (P < 0.05). Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3.

CONCLUSIONCaspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.

Animals ; Apoptosis ; drug effects ; physiology ; Apoptosis Inducing Factor ; metabolism ; Blood Glucose ; metabolism ; Blotting, Western ; Body Weight ; Caspase 3 ; metabolism ; Caspase Inhibitors ; Caspases ; metabolism ; Diabetes Mellitus, Experimental ; In Situ Nick-End Labeling ; Male ; Oligopeptides ; administration & dosage ; pharmacology ; Rats ; Rats, Wistar ; Retina ; metabolism ; Retinal Ganglion Cells ; cytology ; metabolism