Sankarathi Balaiya

A review: role of ultraviolet radiation in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the western world. The retina is highly susceptible to photochemical damage from continuous exposure of light and oxygen. The cornea and the lens block a major portion of the ultraviolet (UV) radiation from reaching the retina (<295 nm). The relationship between UV light exposure and AMD is unclear, although short wavelength radiation and the blue light induce significant oxidative stress to the retinal pigment epithelium. Epidemiologic evidence indicates a trend toward association between severity of light exposure and AMD. In this review, we discuss type 1 and type 2 photochemical damage that occurs in response to UV exposure. We examine the impact of different doses of exposure to UV radiation and the subsequent production of oxidative stress in AMD. Local and systemic protective mechanisms of the retina including antioxidant enzymes and macular pigments are reviewed. This article provides a review of possible cellular and molecular effects of UV radiation exposure in AMD and potential therapies that may prevent blindness resulting from this disease.

Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor-2α

The aim of this work was to study the chemical composition of Allium obliquum L., A. senescens L. subsp. montanum (Fries) Holub, and A. schoenoprasum L. subsp. schoenoprasum. Sulphur-containing compounds analysis was performed by an LC-MS method, the identification and quantification of polyphenolic compounds through a HPLC-UV-MS method, and the presence of five sterols was simultaneously assessed by HPLC-MS-MS. Alliin was identified only in A. obliquum and A. senescens subsp. montanum extracts, whilst allicin was present in all extracts, with higher amounts in A. schoenoprasum and A. obliquum. The pattern of phenol carboxylic acids shows the presence of p-coumaric and ferulic acids in all species. Isoquercitrin was identified in A. obliquum and A. schoenoprasum, and rutin in A. senescens subsp. montanum and A. schoenoprasum. Luteolin and apigenin were identified only in A. obliquum. All three species contain glycosides of kaempferol and quercetol. β-Sitosterol and campesterol were identified in all species. The results obtained showed significant differences in the composition of the three Allium species.

Tumor necrosis factor-alpha (TNF-α) levels in aqueous humor of primary open angle glaucoma.

Purpose: Tumor necrosis factor alpha (TNF-α), a macrophage/monocyte derived pluripotent cytokine is associated with tissue ischemia, neuronal damage and remodeling. The physiological level of TNF-α in aqueous humor of normal and glaucomatous eyes is unknown. In this study, we evaluated the TNF-α levels in aqueous in patients with primary open angle glaucoma (POAG) and compared them to controls. Methods: 50–100 μL of undiluted aqueous humor samples were obtained from eyes of 32 POAG patients who underwent cataract extraction, trabeculectomy or aqueous shunt implantation. Controls were obtained from 32 normal subjects who underwent routine cataract surgery. TNF-α levels were quantified using singleplex bead immunoassay analysis. Results: The average TNF-α level in POAG samples was 2.72 ± 1.5 pg/mL (mean ± SD). The average TNF-α level in normal samples was 1.59 ± 0.46 pg/mL (mean ± SD). Significant increase of TNF-α levels in POAG samples was noted in comparison to normal samples (P < 0.001). Conclusion: TNF-α levels are elevated in aqueous in patients with POAG compared to normal subjects based on highly sensitive Luminex® bead immunoassay and may be a reliable biomarker in the progression of glaucoma.

Evaluation of sirtuin role in neuroprotection of retinal ganglion cells in hypoxia.

PURPOSE. Hypoxia-induced apoptosis is responsible for reduced retinal ganglion cell (RGC) viability in a variety of chronic ocular disorders. Sirtuin 1 (SIRT1) plays an important role in preserving cell viability during hypoxia. We investigated the role of SIRT1 in sustaining RGC viability in an in vitro model of hypoxia. METHODS. Staurosphorine-differentiated RGCs (RGC-5) received varying hypoxic concentrations (100-500 μM) of cobalt chloride (CoCl2) for 24 hours. Hypoxia-induced cell viability was assessed by WST-1 assay. The role of SIRT1 in promoting viability was determined indirectly via sirtinol (SIRT1 inhibitor). Hypoxia-induced apoptosis was evaluated by measuring stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) and caspase 3 activity. Vascular endothelial growth factor (VEGF) was measured to ascertain the influence of SIRT1. RESULTS. CoCl2 concentrations greater than 100 μM resulted in significantly reduced RGC viability (P=0.01). CoCl2 treatment increased SIRT1 levels significantly (P<0.01): 100 (6.5-fold), 200 (6-fold), 300 (3.5-fold), and 400 μM (4.5-fold). Phosphorylated SAPK/JNK increased 36-fold (200 μM CoCl2 concentration), then plateaued at the 300- (25-fold) and 400-μM (27.8-fold) CoCl2 concentrations (P<0.01). CoCl2 and sirtinol treatment increased Caspase 3 activity (P<0.05). VEGF release was significantly higher than control at the 100-μM CoCl2 concentrations (P<0.01). Sirtinol reduced RGC viability, SIRT1 levels, and VEGF release (P<0.01) while having greater effect on SAPK/JNK phosphorylation. CONCLUSIONS. SIRT1 significantly influences RGC viability. Sirtinols effect reflects the interaction SIRT1 has with apoptotic signaling proteins. This investigation demonstrated SIRT1 importance in forestalling the effects of hypoxia-induced apoptosis.

Evaluation of ultraviolet light toxicity on cultured retinal pigment epithelial and retinal ganglion cells

Purpose: Our study is aimed at evaluating the role of UVB light in inducing cytotoxicity in an in vitro model. Methods: RGC-5 and ARPE-19 cells were exposed to different time periods of UVB light: 0, 15, 30, and 45 min. They were subsequently examined for changes in cell morphology, cell viability (neutral red uptake assay), generation of reactive oxygen species (ROS), expression of bax, bcl-2 and cytochome C by reverse transcriptase polymerase chain reaction and western blot, respectively. Results: Dose-dependent reduction in cell viability to UVB light was demonstrated with parallel increase in ROS. Increased duration of exposure (>15 minutes), was associated with increased expression of bax and cytochrome C, and absence of bcl-2 expression. Conclusion: UVB light exposure results in cell cytotoxicity. The concomitant generation of ROS and expression of apoptotic markers suggests the role of oxidative stress in UVB-mediated apoptosis in an in vitro model of retinal ganglion and pigment epithelial cells.

Retinal Thickness Normative Data in Wild-Type Mice Using Customized Miniature SD-OCT

Objective To report normative data for retinal thickness in wild-type C57BL/6 mouse utilizing a miniature SD-OCT system. Methods Thirty adult mice (range: 3–5 months) were anesthetized and secured into the Bioptigen Spectral Domain Ophthalmic Imaging System. Right eye SD-OCT images were standardized by centralizing the optic nerve head (ONH) prior to image acquisition. Global and quadrant total retinal thickness (TRT) values were measured from retinal nerve fiber layer to retinal pigment epithelial layer. Posterior segment analyses also included the outer retinal layer (ORL) and inner retinal layer (IRL). Further sublayer analyses of four layers from the ORL and three layers comprising the IRL were also performed. Results The overall mean±SD global TRT in a C57BL/6 mouse model was 204.41±5.19 µm. Quadrant mean TRT values were 204.85±5.81 µm inferiorly, 204.97±6.71 µm nasally, 205.08±5.44 µm temporally, and 202.74±4.85 µm superiorly. Mean±SD thickness for ORL, and IRL were 126.37±10.01 µm, and 107.03±10.98 µm respectively. The mean±SD estimates for the four layers of the ORL were 18.23±2.73 µm, 26.04±4.21 µm, 63.8±6.23 µm, and 19.22±4.34 µm. Mean±SD values for the three IRL sublayers were 27.82±4.04 µm, 59.62±6.66 µm and 19.12±3.71 µm. Conclusion This study established normative values for the total retinal thickness and sublayer thickness for the wild-type C57BL/6 mice. Moreover, it provides a standard of retinal morphology, in a commonly used animal model, for evaluating therapeutic interventions and retinal disease pathophysiology.

Lutein protects retinal pigment epithelium from cytotoxic oxidative stress

Abstract Context: Lutein (LUT) and zeaxanthin (ZEA) are currently under investigation in clinical trials as prophylactic nutritional agents for age-related macular degeneration (AMD). However, dose used in these trials is empirical and not been investigated in in vitro studies. Objective: In this study, we investigated the dose–response effect of LUT and ZEA in protecting retinal pigment epithelium (RPE) from oxidative stress, a common underlying pathology in AMD. Methods: Three thousand cultured human retinal pigment epithelial cells (ARPE-19) were plated in 72-well plate and after 24 h were exposed to increasing concentrations of hydrogen peroxide (H2O2). ARPE-19 cells were exposed to four different concentrations of LUT (0.5, 1, 2 and 4 µg/mL) and ZEA (0.1, 0.2, 0.4 and 0.8 µg/mL). After 24 h incubation, cells were subjected to oxidative stress induced with H2O2. Cultures containing saline solution and dichloromethane served as controls. Cell viability was assessed using the WST-1 assay. Pathophysiological pathways were evaluated by measuring caspase-3 levels as an indicator of apoptosis induction. Reactive oxygen species (ROS) levels were measured using dihydrorhodamine-123. Results: Cell viability as a percentage of control was 81.3%, 81.1%, and 88.8% at 0.5, 1, and 2 µg/ml, respectively of LUT (p < 0.001). The maximum cytoprotective effect was seen with LUT at 2 μg/mL. ZEA did not show any cytoprotective effect at all concentrations used in the study. Caspase-3 showed a corresponding decrease in levels with LUT (1 and 2 µg/ml). Significant decrease in ROS levels were measured only with LUT at 4 µg/ml (p = 0.02). Discussion and conclusions: Results from our study provide in vitro data to support the epidemiologic studies, which are currently underway to provide evidence that lutein may act as cofactor that modulates processes implicated in AMD pathogenesis.

Aqueous Interleukin-6 Levels Are Superior to Vascular Endothelial Growth Factor in Predicting Therapeutic Response to Bevacizumab in Age-Related Macular Degeneration

Objective. To prospectively evaluate the effect of intravitreal bevacizumab on aqueous levels of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in patients with exudative age-related macular degeneration (AMD) and correlate clinical outcomes with cytokine levels. Methods. 30 eyes of 30 patients with exudative AMD underwent intravitreal injection of bevacizumab three times at monthly intervals. The aqueous samples prior to the 1st injection (baseline) and 3rd injection were analyzed for VEGF and IL-6 levels. Subjects were subgrouped based upon change in the central subfield (CSF) macular thickness on SD-OCT at 8 weeks. Group 1 included patients (n = 14) with a decrease in CSF thickness greater than 10% from the baseline (improved group). Group 2 included patients (n = 16) who had a decrease in CSF thickness 10% or less (treatment-resistant). Results. In subgroup analysis, in both groups 1 and 2 patients, compared to aqueous VEGF, aqueous IL-6 levels showed a better correlation with CSF thickness on SD-OCT (r = 0.72 and 0.71, resp.). Conclusions. Aqueous IL-6 may be an important marker of treatment response or resistance in wet macular degeneration. Future therapeutic strategies may include targeted treatment against both VEGF and IL-6, in patients who do not respond to anti-VEGF treatment alone.

Retinal Thickness Measurement Obtained with Spectral Domain Optical Coherence Tomography Assisted Optical Biopsy Accurately Correlates with Ex Vivo Histology

Background This study determines ‘correlation constants’ between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice. Methods Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as ‘correlation constants’. Results Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (±1.09) and 219.58 µm (±2.67), respectively. The mean ‘correlation constant’ for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the ‘correlation constant’ values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations. Conclusion This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

Effects of Indocyanine green on cultured retinal ganglion cells in-vitro

BackgroundIndocyanine green (ICG) dye is commonly used to stain the inner limiting membrane during macular surgery. There are reports documenting the toxicity of ICG on retinal pigment epithelial cells, with conflicting results in retinal ganglion cells. In the present study, we evaluated the effect of ICG on retinal ganglion cells in vitro.Cultured rat retinal ganglion cells (RGC-5) were exposed to different concentrations of ICG (0.25, 0.5, 1.0, 1.25, & 5 mg/ml) and at various time intervals (1, 5, 15, 30, & 60 minutes). Changes in structural morphology were identified using phase contrast bright field microscopy. Cell viability was quantified using the neutral red assay and cell death was characterized using Annexin-V staining.FindingsSignificant morphologic changes were observed at the 15 and 60 min intervals for all concentrations, where a reduction in cell size and loss of normal spindle shape was noted. A dose dependent decrease in cell viability was observed with increasing concentration of ICG as well as increasing exposure intervals. Compared to control, 48-74% reduction in neutral red uptake at all concentrations for exposures 5 min or greater (p < 0.001). Even at 1 min exposure, a dose dependent decline was observed in cell viability, with a 28-48% decline for doses above 1.25 mg/ml (p = 0.007). Staining with Annexin-V, demonstrated a similar dose and time dependent increase in number of cells exhibiting early apoptosis. A greater than two-fold increase in Annexin-V expression for all doses at exposures greater than 1 min was noted.ConclusionICG dye exhibits toxicity to retinal ganglion cells at clinically relevant doses following 1 min exposure.