Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation

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Antiproliferative, Apoptotic, and Autophagic Activity of Ranibizumab, Bevacizumab, Pegaptanib, and Aflibercept on Fibroblasts: Implication for Choroidal Neovascularization

Purpose. Choroidal neovascularization (CNV) is one of the most common complications of retinal diseases accompanied by elevated secretion of vascular endothelial growth factor (VEGF). Intravitreal anti-VEGFs (ranibizumab, bevacizumab, pegaptanib, and aflibercept) can suppress neovascularization, decrease vascular permeability and CNV size, and, thereby, improve visual function. The antiproliferative, apoptotic, and autophagic effect of anti-VEGF drugs on fibroblasts found in CNVs has not been yet explored. Methods. Concentration-dependent cellular effects of the four anti-VEGFs were examined in L929 fibroblasts over a 5-day period. The cell survival, mitotic and polykaryocytic indices, the level of apoptosis and autophagy, and the cellular growth kinetics were all assessed. Results. The anti-VEGFs could inhibit the survival, mitotic activity, and proliferation as well as increase the cellular heterogeneity, apoptosis, and autophagy of the fibroblasts in a dose-dependent manner. Cellular growth kinetics showed ranibizumab to be less aggressive, but three other anti-VEGFs showed higher antiproliferative and apoptotic activity and expressed negative cellular growth kinetics. Conclusions. The antiproliferative, apoptotic, and autophagic activity of anti-VEGFs upon fibroblasts may explain the cellular response and the etiology of CNV involution in vivo and serve as a good study model for CNV in vitro

Comparative cyto-histological study of needle tip aspirates and entry sites after intravitreal injection using different needle types

A comparison of the cellular content of needle tip aspirates and entry sites after transconjunctival intravitreal injection (IVI) using different needle types was performed. White outbred rats and human cadaver eyes were used for IVI by hypodermic 27 gauge (G) and 30G needles, and spinal anesthesia Pencan 27G needles. Aspiration of vitreous for quantitative morphological and cell cultivation analysis, as well as cyto-histological analysis of aspirates and entry sites were carried out. The most common cells in the aspirates from all needle types were conjunctival epithelial-, ciliary body non-pigmented epithelial- and sclerocyte-like cells and granular proteins. Crystallized vitreous specimens were present in each aspirate. The entry sites of hypodermic needles showed marked trauma in all wall layers of rat and human eyes accompanied by cellular destruction and hemorrhages. Pencan 27G needle caused less tissue trauma with partial reposition of sclerocytes. Transconjunctival IVIs with hypodermic 27G and 30G, and Pencan 27G needles result in trauma of all layers of the eyeball. The possible consequences of cellular content being cut and injected into the eye, as well as the entry site wound shape deserve future consideration and improvements

Resveratrol as Inducer of Autophagy, Pro-Survival, and Anti-Inflammatory Stimuli in Cultured Human RPE Cells.

Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD).Cultured ARPE-19 cells were exposed to 10 and 50 μM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively.Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells.Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD

Intraoperative optical coherence tomography assisted analysis of pars Plana vitrectomy for retinal detachment in morning glory syndrome: a case report

Abstract Background The pathogenesis of non-rhegmatogenous retinal detachment (non-RRD) associated with morning glory syndrome (MGS) is not established, as well as best surgical approach to treat RD. Our purpose was to analyse intraoperative optical coherence tomography data (iOCT) in all steps of pars plana vitrectomy (PPV) for non-RRD in MGS, in order to follow pathophysiological aspects of the disease and to understand the tissues behaviour during surgical workflow. Case presentation Intraoperative spectral domain optical coherent tomography (iSD-OCT) assisted PPV using Rescan 700 (Carl Zeiss Meditech, Jena, Germany) with epiretinal membrane (ERM) and internal retinal membrane (ILM) peeling, and air endotamponade was performed on the only eye of a 21 years old female with non-RRD associated with MGS. BCVA, pre-, intra- and postoperative OCT were performed along with standard ocular examination. iOCT video and snapshots were analysed intra- and postoperatively using post-processing approach using graphic software. The progression of non-RRD resulted in best corrected visual acuity (BCVA) decrease from 0.8 to 0.2. Triamcinolone enhanced iOCT imaging revealed strong vitreous traction and adhesion above the macula and optic disc. Internal limiting membrane was peeled under iOCT control to prevent the peeling of inner layers of the retinal schisis. No retinal break was detected, and only air endotamponade was performed. The retina reattached during first 4 weeks of follow-up with gradual resolution of intraretinal- and subretinal fluid, and remained stable in 12 months. BCVA improved to 0.8. Conclusion Based on iSD-OCT findings we assume that non-RRD in this case of MGS is caused primarily by the vitreous traction with further possible formation of the retinal breaks. Retinal reattachment reached only with air endotamponade strongly advocates the tractional component of non-RRD and retinal schisis assotiated with MGS. Early PPV for central non-RRD and retinal schisis with the use of iOCT can be performed in more safe and controlled manner and has to be considered to reduce the risk of retinal break formation and to prevent the central vision loss

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells—A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell’s biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy—a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells—A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell’s biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy—a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD

Clinical significance of signal shadowing during intraoperative optical coherence tomography-assisted vitreoretinal surgery

Abstract This study aimed to analyze the clinical significance of signal shadowing during intraoperative optical coherence tomography (iOCT)-assisted vitreoretinal surgery caused by vitreoretinal instruments, tissue dyes, and vitreous substitutes, and to objectively quantify its impact on iOCT imaging. This is a retrospective observational study of postoperative image analysis from one hundred seventeen (117) patients who underwent iOCT-assisted vitrectomy. The image data were divided into three groups: vitreoretinal instruments, tissue dyes, and vitreous substitutes. The data was then processed using graphic software to measure the grade of picture quality distortion and compared to paired image controls without clinically perceptive interference, then analyzed statistically. The intraocular portion of all studied vitreoretinal instruments caused a high average gray level interference compared to controls ranging from 32 to 68% reduction, obscuring the area of interest significantly. The tips of the instruments produced low-grade shadowing, allowing the underlying tissue to be distinguished. The analyzed dyes demonstrated a wide interference range: ICG (− 75.12%), and triamcinolone (− 26.13%) showed dose-dependent high shadowing, while VITREODYNE™ (49.3%) and brilliant blue G (14.06%) exhibited no perceived distortions whilst increasing average gray levels. All analyzed vitreous substitutes (air, SF6, C3F8, PFCL, and silicone oil) showed an insignificant shadowing effect on iOCT. Certain dyes and vitreous substitutes produce a negligible shadowing effect compared to controls and other dyes, providing an advantage during real-time iOCT imaging. All analyzed vitreoretinal instruments showed a significant interference that should prompt the development of new imaging techniques or the implementation of materials with low-grade interference to overcome a clinically relevant shadowing effect on iOCT, maximizing the technology’s visual accuracy and surgical diagnostic aid proficiency