Freezing adversely affects measurement of vascular endothelial growth factor levels in human aqueous samples

Sankarathi Balaiya Sandeep Grover Ravi K Murthy Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Aqueous levels of vascular endothelial growth factor (VEGF) can be a surrogate marker of intraocular VEGF activity and a measure of efficacy of anti-VEGF treatment in a variety of vasoproliferative retinal disorders, including diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusion. Measurement of the VEGF level may be adversely affected by premeasurement variables, such as freezing and delay, in sample analysis. We aim to evaluate the effect of storage and delayed measurement of human aqueous VEGF levels in these conditions.Methods: Aqueous samples collected from patients receiving intravitreal injection of bevacizumab for various retinal diseases were divided into two groups. In Group 1, the VEGF levels were analyzed on the same day; in Group 2, the VEGF levels were analyzed after 21 days of freezer storage (-80°C) using immunobead assay. Statistical comparison using a paired t-test was performed between the two groups.Results: Thirty-one aqueous humor samples were collected, and the VEGF concentration for fresh samples was 7.8 ± 5.9 pg/mL (mean ± SD) compared to 6.5 ± 6.0 pg/mL in frozen samples, resulting in a statistically significant difference (P = 0.03).Conclusions: Accurate measurement of the VEGF level is a vital component of clinical decision-making. Delayed analysis of VEGF levels in aqueous samples may result in significant sample degradation and lower levels of measured VEGF.Keywords: VEGF level, aqueous humor, immunobead assay, VEGF storag

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

Sankarathi Balaiya, Ravi K Murthy, Vikram S Brar, Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: 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.Keywords: ultraviolet light, retinal pigment epithelium, retinal ganglion cell, reactive oxygen species, cytochrome

Modified protocol for <it>in vivo</it> imaging of wild-type mouse retina with customized miniature spectral domain optical coherence tomography (SD-OCT) device

<p>Abstract</p> <p>This protocol outlines and evaluates a modified scanning procedure for a customized spectral domain optical coherence tomography (SD-OCT) imaging apparatus within the wild-type C57Bl/6 mouse posterior segment. This modified protocol allows for the capture of a 50 degree field of view spanning 3 mm by 3 mm perimeter with the optic disc as the central point. By utilizing this scanning protocol a more reliable measurement of retinal thickness can be achieved outside the fluctuating region of the optic disc. This protocol, when applied to this high resolution device, enables non-invasive <it>in vivo</it> histological imaging and biometric assessment of the various layers of the rodent posterior segment within a 20 – 30 min procedural time-frame. This protocol could establish a standardized method for evaluating morphological changes, with this commercial SDOCT device, when assessing longitudinal disease pathophysiology and treatment response in mouse models for future vision science research.</p

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

Clinical Study 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 ( = 14) with a decrease in CSF thickness greater than 10% from the baseline (improved group). Group 2 included patients ( = 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 ( = 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

Sirtuins Expression and Their Role in Retinal Diseases

Sirtuins have received considerable attention since the discovery that silent information regulator 2 (Sir2) extends the lifespan of yeast. Sir2, a nicotinamide adenine dinucleotide- (NAD-) dependent histone deacetylase, serves as both a transcriptional effector and energy sensor. Oxidative stress and apoptosis are implicated in the pathogenesis of neurodegenerative eye diseases. Sirtuins confer protection against oxidative stress and retinal degeneration. In mammals, the sirtuin (SIRT) family consists of seven proteins (SIRT1–SIRT7). These vary in tissue specificity, subcellular localization, and enzymatic activity and targets. In this review, we present the current knowledge of the sirtuin family and discuss their structure, cellular location, and biological function with a primary focus on their role in different neuroophthalmic diseases including glaucoma, optic neuritis, and age-related macular degeneration. The potential role of certain therapeutic targets is also described

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

To report normative data for retinal thickness in wild-type C57BL/6 mouse utilizing a miniature SD-OCT system.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.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.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