Retinal Cell Damage Research Articles

Effect of leptin on the oxidative damage in human retinal pigment epithelial cells

Objective To investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells.Methods Human RPE cells (ARPE-19) were cultured in vitro,and randomly divided into control group and insulin resistance group.RPE cells were treated with 0,10,100 ng/mL leptin for 24,48,72 hours respectively.Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2',7'-dichlorofluorescin-diacetate (DCFH-DA),and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC),and the levels of human 8-oxoguanine DNA glycosylase 1 (hOGG1) expression in lysate were measured by Western blot.Results After 24,48,72 hours,the level of ROS (Control group:F=37.136,37.178,49.634; P＜0.05.Insulin resistance group:F=9.822,28.881,71.150;P＜0.05),8-OHdG (Control group:F =88.643,390.920,1039.276; P ＜ 0.05.Insulin resistance group:F =273.311,299.155,82.237;P＜0.05) and hOGGl (Control group:F=470.062,1073.113,295.456;P＜0.05.Insulin resistance group:F =240.032,592.389,527.760 ; P＜0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group.Under the same leptin concentration,the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group.After 24 hours,the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392,P＞0.05).After 72 hours,the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394,P＜0.05).The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P＜ 0.05).Conclusion Leptin could induce the oxidative damage of RPE cells in normal and insulin resistance status.With the increase of leptin concentration and time extended,the degree of oxidative damage and its repair were both increased.The degree of oxidative repair increased with the increase of leptin concentration,but decreased with time extended. Key words: Retinal pigment epithelium/injuries; Leptin/poisoning; Reactive oxygen species/poisoning

Rapamycin attenuates visible light-induced injury in retinal photoreceptor cells via inhibiting endoplasmic reticulum stress

An extended exposure of the retina to visible light may lead to photochemical damage in retinal photoreceptor cells. The exact mechanism of retinal light damage remains unknown, and an effective therapy is still unavailable. Here, we demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), markedly protected 661W photoreceptor cells from visible light exposure-induced damage at the nanomolar level. We also observed by transmission electron microscopy that light exposure led to severe endoplasmic reticulum (ER) stress in 661W cells as well as abnormal endomembranes and ER membranes. In addition, obvious upregulated ER stress markers were monitored by western blot at the protein level and by quantitative reverse transcription-polymerase chain reaction (RT-PCR) at the mRNA level. Interestingly, rapamycin pretreatment significantly suppressed light-induced ER stress and all three major branches of the unfolded protein response (UPR), including the RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) pathways both at the protein and mRNA levels. Additionally, the inhibition of ER stress by rapamycin was further confirmed with a dithiothreitol (DTT; a classical ER stress inducer)-damaged 661W cell model. Meanwhile, our results also revealed that rapamycin was able to remarkably inhibit the activation of mTOR and its downstream factors eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), p-4EBP1, p70, p-p70, and phosphorylated ribosomal protein S6 kinase (p-S6K) in the light-injured 661W cells. Thus, these data indicate that visible light induces ER stress in 661W cells; whereas the mTOR inhibitor, rapamycin, effectively protects 661W cells from light injury through suppressing the ER stress pathway.

Hypoxia induces cell damage via oxidative stress in retinal epithelial cells

Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries. RD is characterized by abnormal angiogenesis associated with an increase in cell proliferation and apoptosis. Hypoxia could be one of the triggers of the pathogenic mechanism of this disease. A key regulatory component of the cell's hypoxia response system is hypoxia-inducible factor 1 alpha (HIF-1α). It has been demonstrated that the induction of HIF-1α expression can be also achieved in vitro by exposure with cobalt chloride (CoCl2), leading to an intracellular hypoxia-like state. In this study we have investigated the effects of CoCl2 on human retinal epithelium cells (hRPE), which are an integral part of the blood–retinal barrier, with the aim to determine the possible role of oxidative stress in chemical hypoxia-induced damage in retinal epithelial cells. Our data showed that CoCl2 treatment is able to induce HIF-1α expression, that parallels with the formation of reactive oxygen species (ROS) and the increase of lipid 8-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts levels. In addition we observed the activation of the redox-sensitive transcription factor nuclear factor-kappaB (NFkB) by CoCl2 which can explain the increased levels of vascular endothelial growth factor (VEGF). The increased number of dead cells seems to be related to an apoptotic process. Taken together these evidences suggest that oxidative stress induced by hypoxia might be involved in RD development through the stimulation of two key-events of RD such as neo-angiogenesis and apoptosis.

Expression of Endoplasmic Reticulum Stress Markers GRP78 and CHOP Induced by Oxidative Stress in Blue Light-Mediated Damage of A2E-Containing Retinal Pigment Epithelium Cells

Aims: Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. Methods: ARPE-19 cells were incubated with A2E (10, 25, 50 μ<smlcap>M</smlcap>) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. Results: After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. Conclusion: RPE cells exhibited ROS accumulation and subsequent elevation of GRP78 and CHOP expression after A2E and blue light-induced damage. The ROS scavenger NAC diminished ER stress protein expression, suggesting a connection between ER and oxidative stress in blue light-mediated damage of A2E-containing RPE cells. Besides, GRP78 may play a protective role in it.

2-ethylpyridine, a cigarette smoke component, causes mitochondrial damage in human retinal pigment epithelial cells in vitro

Purpose:Our goal was to identify the cellular and molecular effects of 2-ethylpyridine (2-EP, a component of cigarette smoke) on human retinal pigment epithelial cells (ARPE-19) in vitro.Materials and Methods:ARPE-19 cells were exposed to varying concentrations of 2-EP. Cell viability (CV) was measured by a trypan blue dye exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was detected with a 2’,7’-dichlorodihydrofluorescein diacetate dye assay. The JC-1 assay was used to measure mitochondrial membrane potential (ΔΨm). Mitochondrial redox potential was measured using a RedoxSensor Red kit and mitochondria were evaluated with Mitotracker dye.Results:After 2-EP exposure, ARPE-19 cells showed significantly decreased CV, increased caspase-3/7 and caspase-9 activities, elevated ROS/RNS levels, decreased ΔΨm value and decreased redox fluorescence when compared with control samples.Conclusions:These results show that 2-EP treatment induced cell death by caspase-dependent apoptosis associated with an oxidative stress and mitochondrial dysfunction. These data represent a possible mechanism by which smoking contributes to age-related macular degeneration and other retinal diseases and identify mitochondria as a target for future therapeutic interventions.

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

Long-Term Neuroprotective Effects of NT-4–Engineered Mesenchymal Stem Cells Injected Intravitreally in a Mouse Model of Acute Retinal Injury

Retinal degenerative diseases targeting the RPE and adjacent photoreceptors affect millions of people worldwide. The field of stem cell- and gene-based therapy holds great potential for the treatment of such diseases. The present study sought to graft genetically engineered mesenchymal stem cells (MSCs) that continuously produce neurotrophin-4 (NT-4) into the murine eye after the onset of acute retinal injury. C57BL/6 mice were subjected to acute retinal damage using a low dose of sodium iodate (20 mg/kg of body weight), followed by intravitreal injection of lentivirally modified MSC-NT-4 into the right eye. At 3 months after the MSC transplantation grafted cell survival, retinal function and gene expression were analyzed. Immunofluorescence analysis confirmed that transplanted MSCs survived for at least 3 months after intravitreal injection and preferentially migrated toward sites of injury within the retina. MSC-NT-4 actively produced NT-4 in the injured retina and significantly protected damaged retinal cells, as evaluated by ERG and optical coherence tomography (OCT). Of importance, the long-term therapy with MSC-NT-4 was also associated with induction of prosurvival signaling, considerable overexpression of some subsets of transcripts, including several members of the crystallin β-γ superfamily (Cryba4, Crybb3, Cryba2, Crybb1, Crybb2, Cryba1, and Crygc) and significant upregulation of biological processes associated with visual perception, sensory perception of light stimulus, eye development, sensory organ development, and system development. Transplantation of genetically modified MSCs that produce neurotrophic growth factors may represent a useful strategy for treatment of different forms of retinopathies in the future.

White Light–Emitting Diodes (LEDs) at Domestic Lighting Levels and Retinal Injury in a Rat Model

Background: Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000–10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model.Objective: We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements.Methods: We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level.Results: H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury.Conclusion: Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich “white” LEDs for general lighting.Citation: Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light–emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269–276; http://dx.doi.org/10.1289/ehp.1307294

Protective effects of placental growth factor on retinal neuronal cell damage

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family. Although it has been reported that PlGF protects against neuronal damage in the brain, little is known about the effects of PlGF in the retina. Therefore, we investigated the effects of PlGF on retinal neuronal cells. To evaluate the effects of PlGF against L-buthionine-(S,R)-sulfoximine (BSO)/glutamate cell death, oxygen-glucose deprivation (OGD)-induced cell death, and light-induced cell death, RGC-5 and 661W cells were used. We evaluated the mechanism responsible for the protective effects of PlGF against retinal neuronal cell death by performing the examinations with U1026, which is a mitogen-activated protein kinase (MEK) inhibitor, and LY294002, which is a phosphoinositide 3-kinase (PI3K) inhibitor. In addition, we measured caspase-3/7 activity in RGC-5 cells and 661W cells. PlGF protected against RGC-5 cell death induced by BSO/glutamate and OGD and against 661W cell death induced by light irradiation. Moreover, an anti-PlGF antibody negated these protective effects. The protective effects of PlGF against OGD-induced RGC-5 cell death and light-induced 661W cell death were suppressed by using an anti-PlGF antibody, U1026, and LY294002. Treatment with PlGF suppressed caspase-3/7 activity in both cell lines. We demonstrated for the first time that PlGF exerts a protective effect by inhibiting the activation of caspase-3/7 through the MEK and PI3K pathway in retinal neuronal cells. These data suggest that PlGF may be an important protective factor in the retina.

Adeno-Associated Virus Mediated Delivery of a Non-Membrane Targeted Human Soluble CD59 Attenuates Some Aspects of Diabetic Retinopathy in Mice

Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC) and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59) that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59) to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ) induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.

The Protective Effects of Bilberry and Lingonberry Extracts against UV Light-Induced Retinal Photoreceptor Cell Damage in Vitro

Bilberry extract (B-ext) and lingonberry extract (L-ext) are currently used as health supplements. We investigated the protective mechanisms of the B-ext and L-ext against ultraviolet A (UVA)-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661W) cells were exposed to UVA following treatment with B-ext and L-ext and their main constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin). B-ext, L-ext, and constituents improved cell viability and suppressed ROS generation. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) were analyzed by Western blotting. B-ext and cyanidin inhibited phosphorylation of p38 MAPK, and B-ext also inhibited phosphorylation of JNK by UVA. L-ext, trans-resveratrol, and procyanidin alleviated the reduction of phosphorylated Akt levels by UVA. Finally, a cotreatment with B-ext and L-ext showed an additive effect on cell viability. Our findings suggest that both B-ext and L-ext endow protective effects against UVA-induced retinal damage.

The association of blue light-induced human retinal pigment epithelium cell damage with intercellular free Ca2+ change in vitro

Background Investigating the association of blue light-induced damage of retinal pigmenepithelial (RPE) cellwith intracellulaCa2+ conteniimportanfounderstanding the mechanism of retinal disorders.Objective Thistudy wato establish blue light-induced damage model of human RPE celland explore the relationship between the damage of RPE cell and intracellulaCa2+ content.MethodHuman RPE cellwere cultured and passaged.Cell vitality waassayed by trypan blue staining.Fourth-generation cellwere used in these experiments.The cellwere exposed to blue lighwith an intensity of (2000±500)lx fo3,6,9 o12 hours,and the rate of apoptosiwaassayed by TUNEL to assesthe optimal irradiation time focellcultured.The cellwere then randomized into the withouirradiation group,irradiation only group,nifedipine group,ligh+ nifedipine group,(-) BayK8644 group and ligh+ (-) BayK8644 group.The laseconfocal microscope waused to determine the fluorescence intensity of intracellulafree Ca2+ aan excitation wavelength of 488 nm and an emission wavelength of 505 nm.The cell imagewere analyzed using computesoftware.The differenceof fluorescence intensity among the differengroupwere compared by one-way analysiof variance.ResultTrypan blue staining showed thathe viability of RPE cellwamore than 90％ afteculturing and passaging.No apoptoticell waseen aftelighexposure fo3 hours.However,differennumberof apoptoticellappeared aftelighexposure fo6,9 and 12 hours.The fluorescence intensity of intracellulafree Ca2+ in the nifedipine group wasignificantly lowethan thaof the withouirradiation group othe ligh+ nifedipine group(both aP=0.000).Lasescanning confocal microscopy showed thathe fluorescence intensitieof intracellulafree Ca2+ in the irradiation only group,(-) BayK8644 group,ligh+ (-)BayK8644 group were highethan thaof the withouirradiation group,with statistical significancebetween them(all P=0.000).No significandifferencewere found in the fluorescence intensity of intracellulafree Ca2+ between the ligh+ nifedipine group and withouirradiation group awell abetween the (-)BayK8644 group and the ligh+(-) BayK8644 group(P =0.339,P =0.410).ConclusionThe optical conditionfoblue light-induced RPE cell damage were exposure of blue-ligha(2000± 500) lx fo6 hourand culturing the cellfo24 hours.Blue lighexposure can induce damage of human RPE cellprobably by triggering the increase of intracellulafree Ca2+ concentration. Key words: Light/damage; Retinal pigmented epithelium; Calcium ion/intercellular, free, antagonist,agonist ; Apoptosis

Intravitreal Fasudil Combined With Bevacizumab for Persistent Diabetic Macular Edema

D espite recent advances in pharmacotherapy for diabeticmacular edema (DME), satisfyinganatomical and visual results are not always achieved. Improved understandingof themechanismsunderlyingdiabetic retinopathy may pave the way for new treatment modalities for DME that is refractory to current therapies. In addition to high vascular endothelial growth factor levels,1 increased activity of the Rho/ROCK pathway has recently been demonstrated in retinal vessels of diabetic rats.2 Experimental studies have demonstrated that fasudil hydrochloride, a potentROCK inhibitor, can suppress leukocyte adhesionandpreventneutrophil-induced retinal endothelial cell damage.2 In this pilot study, we evaluated the anatomical and visual outcomes of a novel treatmentmodality including intravitreal injection of fasudil combinedwith intravitreal bevacizumab in patients with DME resistant to previous injections of intravitreal bevacizumab alone.

Advances in our understanding of diabetic retinopathy.

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Dietary Omega-3 Fatty Acids Deficiency Affects the Glutamatergic Transport System in Rat Retina: Modulatory Effects after High Intraocular Pressure

Glutamate excitotoxicity has been postulated as a putative mechanism involved in the physiopathology of glaucoma, a disease that can cause retinal cell damage. Thus, the modulation of glutamatergic parameters is a putative therapeutic target to prevent excitotoxic retinal injury. Here, we investigated the effect of dietary omega-3 fatty acids (w3) in the retinal glutamate transport system in basal and ischemic conditions. Female Wistar rats were divided into two groups: w3 diet (w3 group) and w3 deficient-diet (D group). Their pups, at 60 days old, were used for the experiments. Retinal ischemia, a mechanism involved in the physiopathology of glaucoma, was induced by high intraocular pressure (HIOP, 140 180 mmHg for 45 min) to impair retinal blood flow. Analyses were performed 7 days after ischemia. The D group showed a decreased glutamate uptake in basal conditions and after HIOP when compared to the w3 group. After HIOP, there was a decrease in glutamate uptake in the D group that was not observed in the w3 group (p w3 group presented higher levels of GLT-1 compared to the D group in basal and ischemic conditions. After HIOP, EAAC1 was increased in both groups, while GLT-1 increased only in the D group, compared to basal levels. GLAST and EAAT5 presented no alterations. The modulation of the glutamatergic system by dietary w3 fatty acids points to a potential mechanism by which w3 PUFAs exert beneficial effects in the retina.

Indocyanine green increases light‐induced oxidative stress, senescence, and matrix metalloproteinases 1 and 3 in human RPE cells

Indocyanine green (ICG) is a commonly used vital dye for macular surgery. Recent reports implicate that its use might be associated with less favourable results regarding postoperative visual outcome and damage of retinal cells, and atrophic degeneration of the retinal pigment epithelium (RPE) has been described. This study investigates the effects of ICG on light-induced senescence of RPE cells. Primary human RPE cells were either pre-incubated with ICG in concentrations of 0.005% and 0.05% or not and then exposed to white light. After 10 min of irradiation viability, induction of intracellular reactive oxygen species (ROS) and senescence-associated β-galactosidase activity (SA β-Gal) were determined. Expression and secretion of matrix metalloproteinases (MMPs) 1 and 3 and their mRNA were determined by RT-PCR and ELISA. Light exposure decreased RPE cell viability by 46%. Treatment with 0.005% and 0.05% ICG alone decreased RPE cell viability by 7% and 21%. In addition, expression of ROS, SA β-Gal, and MMP-1 and 3 was significantly increased. When 0.005% and 0.05% ICG treatments were combined with light exposure, viability decreased by 69% and 82% compared to the untreated control. Effects on the expression of ROS, SA β-Gal, and MMP-1 and 3 were, depending on the ICG dose, significantly increased when cells were pre-incubated with ICG and then illuminated. In this study, pretreatment with ICG significantly increased light-induced oxidative stress and senescence. This might indicate a potential, supplementary mechanism that could explain RPE alterations and reduced functional results after ICG-assisted internal limiting membrane peeling.

Protective effects of astragaloside IV on high-glucose-induced damage of retinal ganglion cells

To explore the protective effects of astragaloside IV on retinal ganglion cells (RGC) incubated under high glucose. Cultured RGC-5 were divided into 3 groups: control, high glucose and high glucose+astragaloside IV. The survival rate of cell was measured by CCK-8 kit and flow cytometry. The changes of mitochondrial membrane potential were monitored by confocal laser scanning microscopy while those of RGC-5 mitochondria observed by electron microscopy. When the cell survival rate was 100% in the control group, the survival rate improved after 1 h 5, 10, 50, 100, 200 µg/ml astragaloside IV pretreatment and high-glucose culture. The cell survival rates for 100 mg/L, 200 mg/L of astragaloside IV were 81.6%, 80.0%, the difference from the high glucose group (66%) was significant (P < 0.05) and 100 mg/L of astragaloside IV was the best protection concentration. The apoptotic rate for 100 mg/L astragaloside IV pretreated group of RGC-5 cell was 6.1%. And it could partially inhibit the RGC-5 cell apoptosis caused by high glucose (8.2%) (P < 0.05). In the high-glucose group, the cells showed marked increases in mitochondrial swelling, especially for cristae. Numerous normal mitochondria were observed in 100 mg/L astragaloside IV pretreated group. Astragaloside IV could elevate mitochondrial membrane potential and alleviate mitochondrial swelling. Astragaloside IV can protect RGC from high-glucose induced damage and may benefit the patients with diabetic retinopathy.

Manganese‐enhanced MRI optic nerve tracking: effect of intravitreal manganese dose on retinal toxicity

The aim of this study was to provide data on the dose dependence of manganese-enhanced MRI (MEMRI) in the visual pathway of experimental rats and to study the toxicity of MnCl₂ to the retina. Sprague-Dawley rats were intravitreally injected with 2 μL of 0, 10, 25, 50, 75, 100, 150 and 300 mM MnCl₂, respectively. The contrast-to-noise ratio (CNR) of MEMRI for optic nerve enhancement was measured at different concentrations of MnCl₂. Simultaneously, the toxicity of manganese was evaluated by counting retinal ganglion cells and by retinal histological examination using light microscopy and transmission electron microscopy. The CNR increased with increasing concentration of MnCl₂ up to 75 mM. Retinal ganglion cell densities were reduced significantly when the concentration of MnCl₂ in the intravitreal injection was equal to or greater than 75 mM. Increasing numbers of ribosomes in retinal ganglion cells were first detected at 25 mM of MnCl₂. The retinal toxicity of MnCl₂ at higher concentration also included mitochondrial pathology and cell disruption of retinal ganglion cells, as well as abnormalities of photoreceptor and retinal pigment epithelium cells. It can be concluded that intravitreal injection of MnCl₂ induces retinal cell damage that appears to start from 25 mM. The concentration of MnCl₂ should not exceed 25 mm through intravitreal injection for visual pathway MEMRI in the rat.

Schnurri regulates hemocyte function to promote tissue recovery after DNA damage

Tissue recovery after injury requires coordinated regulation of cell repair and apoptosis, removal of dead cells and regeneration. A critical step in this process is the recruitment of blood cells that mediate local inflammatory and immune responses, promoting tissue recovery. Here we identify a new role for the transcriptional regulator Schnurri (Shn) in the recovery of UV-damaged Drosophila retina. Using an experimental paradigm that allows precise quantification of tissue recovery after a defined dose of UV, we find that Shn activity in the retina is required to limit tissue damage. This function of Shn relies on its transcriptional induction of the PDGF-related growth factor Pvf1, which signals to tissue-associated hemocytes. We show that the Pvf1 receptor PVR acts in hemocytes to induce a macrophage-like morphology and that this is required to limit tissue loss after irradiation. Our results identify a new Shn-regulated paracrine signaling interaction between damaged retinal cells and hemocytes that ensures recovery and homeostasis of the challenged tissue.

Blueberry anthocyanins: protection against ageing and light-induced damage in retinal pigment epithelial cells

Retinal pigment epithelium (RPE) cells are vital for retinal health. However, they are susceptible to injury with ageing and exposure to excessive light, including UV (100-380 nm) and visible (380-760 nm) radiation. To evaluate the protective effect of blueberry anthocyanins on RPE cells, in vitro cell models of replicative senescent and light-induced damage were established in the present study. After purification and fractionation, blueberry anthocyanin extracts (BAE) were yielded with total anthocyanin contents of 31·0 (SD 0·5) % and were used in this study. Replicative senescence of RPE cells was induced by repeatedly passaging cells from the fourth passage to the tenth. From the fifth passage, cultured RPE cells began to enter a replicative senescence, exhibiting reduced cell proliferation along with an increase in the number of β-galactosidase-positive cells. RPE cells maintained high cell viability (P < 0·01) and a low (P < 0·01) percentage of β-galactosidase-positive cells when treated with 0·1 μg/ml BAE. In contrast, after exposure to 2500 (SD 500) lx light (420-800 nm) for 12 h, RPE cells in the positive control (light exposure, no BAE treatment) exhibited premature senescence, low (P < 0·01) cell viability and increased (P < 0·01) vascular endothelial growth factor (VEGF) release compared with negative control cells, which were not subjected to light irradiation and BAE exposure. Correspondingly, BAE is beneficial to RPE cells by protecting these cells against light-induced damage through the suppression of ageing and apoptosis as well as the down-regulation of the over-expressed VEGF to normal level. These results demonstrate that BAE is efficacious against senescence and light-induced damage of RPE cells.