Table 1.

Overall studies depicting in vitro and in vivo findings of oxidative stress on RPE.

S.No	Country/Region	Study	Objective	Method used	Effects	References
1	California, USA	in vitro	To validate the RPE cell culture in AMD Pathology	➢Cell culture ➢Morphological analysis	Cytology of human RPE cells are assessed and found as perfect model to study early stages of AMD.	150
2	Boston, USA	in vitro	To characterize the super oxide mechanisms and toxicity prevention.	➢Cell culture ➢Examined porcine RPE cultures to PMA and to L-dioctanoylglycerol.	in vitro release superoxide and possibly H2O2 of RPE may be subject to regulation. Future studies on in vivo recommended.	151
3	Minnesota, USA	in vitro	To understand the role of zinc in the pathogenesis and prevention of AMD.	➢Flat mounts analysis of tissues - fluorescence and confocal microscopy. ➢Histology.	ZPP1 is a superior probe for the detection of zinc in sub-retinal epithelial deposits in human and murine tissue.	152
4	Washington, USA	in vitro	To determine causative pathways which contributes to AMD	➢RPE and fibroblasts culture ➢Differentiation of iPSCs to RPE ➢Immunostaining ➢Cell viability ➢Microscopy imaging ➢PCR ➢Western blot ➢Karyotyping	The SIRT1/PGC-1α pathways contribute to AMD.	153
5	Florida, USA	in vitro	To study POS phagocytosis by RPE from AMD and the effect of hUTC on RPE phagocytosis, and the mechanisms involved.	➢Cell culture ➢Immunofluorescence ➢hUTC isolation and culture ➢RNA sequencing.	RPE phagocytic dysfunction in AMD and the ability of hUTC to treat the dysfunction were analysed.	154
6	California, USA	in vitro	To investigate the molecular mechanism of wound stimulus in RPE cells.	➢Fetal RPE culture ➢Microarray ➢RNA sequencing ➢Bioinformatics ➢Real-time qPCR ➢Immunocytochemistry	In RPE cells, insistent mesenchymal state with wound stimulus is driven by lasting activation of the TGFβ pathway	155
7	Singapore	in vitro	To develop hPSC-derived RPE production and purification system that yields high-quality RPE monolayers.	➢Maintenance of human ESCs and iPSCs ➢Cell culture ➢Immunocytochemistry ➢Microscopic analysis ➢ELISA	Pure functional RPE monolayers from hPSC using simplified 2D cultures along with RPE PLUS protocol were developed.	156
8	Japan	in vitro	To develop a microfluidic co-culture model of the ocular fundus tissue in a challenge to elucidate AMD pathology.	➢Device fabrication ➢Cell culture ➢Characterisation of cells ➢Image processing	Developed a microfluidic that study the development of diseases compounds that stimulate or inhibit the angiogenesis process.	157
9	New York, USA	in vitro	To study the impact of iron and cigarette smoke, on POS processing and its consequence for autofluorescent material accumulation in human RPE cells.	➢Cell culture ➢Characterisation of cells ➢Bio chemical analysis on cultured cells ➢Cell viability ➢Staining ➢Immunocytochemistry	Both environmental factors together inn under study can impair POS processing and leads to increased autofluorescent material accumulation in hiPSC-RPE.	158
10	USA	in vitro	To investigate the potential use of fucoidan for the treatment of exudative AMD.	➢Cell culture ➢MTT assay ➢Proliferation assay ➢Scratch assay ➢Phagocytosis assay ➢Immunocytochemistry	Fucoidan is safe for RPE cells and making it an interesting molecule for further studies in AMD.	159
11	London, UK	in vitro	To systematically develop and validate a reliable method to isolate RPE cells from adult rats.	➢RPE culture ➢Immunochemistry ➢Quantification of RPE marker expression in RPE cell culture	Developed an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells	160
12	Maryland, USA	in vitro	To study the role of Cryba1 gene in the EMT of RPE cells.	➢Human RPE Cell culture ➢Generation of Cryba1 knock out animal. ➢Culture of OCM3 cell line ➢RNA isolation and RT-PCR ➢Microarray ➢Immunofluorescence ➢Immunohistochemistry ➢Transfection studies ➢Co-immunoprecipitation ➢Western blot ➢Wound healing and cell migration assay	Targeting Cryba 1 mutations is a potential therapeutic method for AMD.	161
13	Washington, USA	in vitro	To provide an evidence for altered autophagic function in the pathophysiology of AMD in an in vitro cellular model	➢SNPs genotyping ➢Immunostaining ➢Real time PCR ➢Microscopic analysis ➢Cell viability assay ➢ROS measurement ➢Biochemical assays	The autophagy was selectively dysregulated in AMD	162
14	California, USA	in vitro	To understand the molecular mechanism behind the AMD by transcriptome analysis.	➢Donor eye tissue and RNA purification ➢Identification of disease modules enriched in protein–protein associations ➢RPE-choroid interactome ➢Retina interactome ➢Compilation of AMD-associated genes	Discovered novel global biomarkers, phenotype-specific gene sets, and functional networks associated with AMD.	163
15	New Jersey, USA	in vitro	To develop a model to evaluate RPE transplantation onto human Bruch's Membrane	➢RPE cell culture ➢Microscopic analysis	The adherence property of RPE to normal and diseased human BrM were studied.	164
16	California, USA	in vitro	To investigate the expression of HN in hRPE cells and its effect on oxidative stress–induced cell death, mitochondrial bioenergetics, and senescence	➢RPE cell culture ➢Localization of HN in RPE cells. ➢Protection of RPE Cells from oxidative stress ➢Uptake of FITC-labeled HN peptide by hRPE cells and co-localization with mitochondria ➢Detection of mitochondrial superoxide ➢Western blotting ➢Quantification of HN levels ➢DNA extraction and mtDNA Copy Number measurement ➢Counting mitochondria by TEM ➢Analysis of oxidative stress–induced cellular senescence ➢Trans-epithelial resistance measurements	Suggested HN as a potential therapeutic method of AMD.	165
17	California, USA	in vitro	To develop a potential therapeutic for both dry and wet AMD by redesign a complement-inhibiting peptide.	➢Peptide synthesis ➢Hemolytic assay ➢Apparent solubility measurements ➢RPE culture ➢Immunofluorescence of sub-RPE deposits ➢Confocal imaging and analysis ➢Structural modelling	A novel peptide analog of compstatin is developed that become a therapeutic for the treatment of AMD.	166
18	Jerusalem, Israel	in vitro and in vivo	To analyse the immunosuppressive property of hESC-RPE	➢Cell culture ➢Immunofluorescence ➢Co-culture of PBMCs with RPE Cells ➢Flow cytometry ➢Cytokine quantification ➢Determination of T-cell proliferation and apotosis ➢PCR ➢Transplantation of hESC-RPE cells into RCS rats and their analysis in vivo	Immune properties of hESC-RPE cells is relevant and valuable for clinical transplantation of hESC-RPE cells in retinal degenerations caused by RPE dysfunction	167
19	Switzerland	in vitro and in vivo	To investigate whether BMCs can be induced to express RPE cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo.	➢Model of RPE degeneration ➢RPE preparation ➢Isolation of GFP+ Sca-1+ BMCs ➢Co-culture of GFP+ Sca-1+ BMCs with RPE ➢BMC mobilization ➢FACS ➢Immunocytochemistry	BMCs once mobilized have the ability to respond to signals from damaged RPE, migrate to the altered sub-retinal space, and form a monolayer of cells that express markers of RPE lineage.	168
20	Kentucky, USA	in vitro and in vivo	To gain the potentiality of RPE cells to be regenerative medicine by reprogramming of differentiated somatic cells into iPSCs	➢Animals and cell transplantation ➢Primary RPE cell isolation and iRPE stem cell preparation ➢Photoreceptor and RPE cell differentiation ➢Tumor formation in athymic nude mice ➢Visual OKR assessment ➢Immunofluorescence ➢Immunohistochemistry ➢Real-time qPCR ➢Affymetrix microarray ➢Lentiviral shRNA ➢Western blotting ➢ChIP assay	By activate Hippo signaling pathway we can prepare regenerative medicines which is important in iRPESC reprogramming.	169
21	Finland	in vivo	To investigate the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global dKO mice.	➢Genotyping of NRF-2 and PGC-1α knockout mice ➢Immunomapping ➢Spectral imaging ➢TEM analysis ➢Vacuole area fraction ➢Detection of lipofuscin-like granules in the RPE cells ➢Apoptosis assay ➢Immunohistochemistry of ER stress markers ➢Flat mount and RPE size analysis ➢ERG recordings	The study suggests that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.	170
22	Chinese Mainland	in vitro	To test the potentiality of paeoniflorin to prevent H2O2-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection.	➢Cell culture and drug preparation ➢Cell viability assay ➢Isolation of total RNA and RT–PCR ➢4,6-diamidino-2-phenolindole staining ➢ROS measurement ➢Caspase-3 activity measurement ➢Western blot	Paeoniflorin could protect human RPE cells against H2O2-induced oxidative stress.	171
23	California, USA	in vitro and in vivo	To understand the molecular mechanism behind the damages caused for RPE cells.	➢in vitro human RPE cell viability assay ➢Fundus photography ➢Retinal morphology	The study suggests a possible role for viral dsRNA transcripts in the development of GA and raise awareness of potential toxicity induced by siRNA therapeutics in the eye.	172
24	Madison, Wisconsin	in vitro and in vivo	To investigate the autonomous impact of PEDF and TSP1 on RPE cell function.	➢Isolation and culture of RPE cells ➢FACS ➢Cell proliferation studies ➢Immunofluorescence ➢Scratch wound assays ➢Transwell migration assays ➢PEDF and TSP1 re-expression studies ➢Cell adhesion assays ➢Western blot ➢Phagocytosis assays ➢Proteasome peptidase assays ➢Capillary morphogenesis assays ➢RNA purification and real-time qPCR analysis ➢NO measurements ➢Whole mount staining studies ➢Assessments of ROS	Demonstrated that PEDF and TSP1 play key roles in RPE cell function and subsequently in pathogenesis of AMD.	33
25	Los Angeles, USA	in vivo	To describe the potential of a peptide derived from αB crystallin protein using a NaIO3 induced mouse model of GA	➢Color fundus photography ➢Quantification of retinal degeneration from fundus imaging ➢Quantitative analysis of fluorescence in color fundus images ➢Intra-vitreal pharmacokinetics ➢Pharmacokinetic analysis ➢Retinal histology ➢TUNEL staining ➢Immunofluorescence	The study shows that crySI hold promise as protective agents to prevent RPE atrophy and progressive retinal degeneration in AMD.	173
26	Durham, UK	in vivo	To generate a therapeutic strategy against AMD, that targets through systemic administration of anti-Aβ antibodies.	➢Animal Experiment ➢Human Tissue Procurement ➢Epitope Mapping ➢hAPP Transfection ➢Immunotherapy ➢Histology ➢Immunohistochemistry ➢RPE Flat-Mount Preparations ➢ELISA	The results support the feasibility of immunotherapeutic strategies targeting Aβ as treatments for both early and advanced stages of AMD, especially for those patients in whom Aβ deposition is a feature of their disease.	174
27	Chicago, USA	in vitro	To better understand the cellular and molecular bases for the association between smoking and AMD	➢APRE-19 cell culture ➢Viability assay ➢Western blot ➢LX-PCR ➢Preparation of POS ➢Phagocytic activity assay ➢Cathepsin D enzyme activity assay ➢N-acetyl- b-glucosoamidase enzyme activity ➢RT-PCR ➢Exposure to cigarette smoke assay ➢Immunohistochemistry ➢Ultrastructural analysis	The cigarette smoking may be main causative agent to genetic mutations which contributes to the pathogenesis of AMD in the elderly.	175
28	Newcastle, UK	in vitro and in vivo	To understand the pathology of the disease and the role of environmental, dietary, and lifestyle factors.	➢Human Donors ➢iPSC Generation ➢iPSC Characterization ➢Karyotyping ➢Generation of iPSC-RPE ➢Western Blotting ➢DNA extraction and sequencing ➢Quantitative RT-PCR ➢RNA Sequencing ➢Pigment bleaching ➢Phagocytosis of rod outer segments ➢Immunofluorescence ➢Trans-Epithelial Resistance ➢TEM	The low- and high-risk AMD-RPE cells respond very differently to UV exposure and moreover this provides evidence for UV mediated functional and cellular improvement of AMD-associated cellular changes in high-risk AMD-RPE cells.	176
29	Germany	in vitro and in vivo	To demonstrate the three-dimensional epithelial cyst culture of human pluripotent stem cells leads to the induction of polarized neuroepithelia	➢Maintenance of hESCs and human iPSCs ➢Differentiation of hESCs and human iPSCs in the neuroepithelial cyst model ➢Differentiation of RPE or neural retina cells on transwell filters ➢Transplantation ➢Immunocytochemistry ➢RT-PCR ➢Electron microscopic analyses ➢Measurement of transepithelial resistance ➢Phagocytosis analyses using retinal explant co-culture model ➢in situ hybridization on cyst cryosections	The work highlights the cell biological environment of pluripotent stem cells while culturing can drastically improve differentiation and the subsequent efficacy of therapeutic outcomes.	177
30	New York, USA	in vitro	To compare the ability of intraocular lenses IOLs as to protect RPE cells from light damage mediated by the lipofuscin fluorophore A2E	➢A2E accumulation in the culture ➢Illumination and placement of IOL ➢Detection of nonviable cells	A yellow-tinted IOL that simulates the adult natural lens and protects lipofuscin-containing RPE from blue light damage may reduce the risk for or progression of AMD	178
31	New York, USA	in vitro	To prepare a culture model for AMD studies	➢Primary fibroblast culture ➢Feeder free and non-integration reprogramming method ➢Immunofluorescence ➢Differentiation of human iPSCs into RPE cells ➢TER ➢Phagocytosis assay ➢Flow cytometry ➢Preparation of RPE cell-derived ECM and nitrite-modified ECM ➢Cell attachment assay ➢Measurement of mitochondrial function ➢Microarray analysis	Culture prepared by RPE derived from patients with AMD act as a perfect model for the future studies.	128
32	Florida, USA	in vitro and in vivo	To study the cellular mechanisms linking oxidative stress and inflammation in AMD,	➢Western blot ➢RNA extraction and RT-PCR ➢ELISA	The injured RPE cells may trigger progression toward CNV in smoker patients with dry AMD.	179
33	Maryland, USA	in vivo	To investigate the role of chemokine receptor CXCR5 in the pathogenesis of AMD.	➢PCR genotyping ➢Fundus examination with the retinal-imaging microscope ➢Immunofluorescence staining of sections and whole-mounts ➢Toluidine blue, hematoxylin and eosin staining ➢Oil red O staining ➢TUNEL assay ➢TEM ➢qPCR ➢Western blotting ➢ERG	CXCR5 itself may be involved in the protection of RPE and retinal cells during aging and its loss may lead to AMD-like pathological changes in aged mice.	180
34	Germany	in vitro	To investigate the glycomic changes associated with EMT of RPE cells in vitro.	➢Human Galectin-3: expression, purification, labelling and quality controls ➢Lectin blot analysis ➢Flow cytometry ➢Cell adhesion assay ➢Cell spreading assay ➢Silencing of Mgat5 expression by siRNA and Lenti-CRISPR/Cas9 ➢Western blot ➢RNA isolation and RT-PCR ➢Immunocytochemical Gal-3 localization ➢Lectin histochemistry of intact eyecups	Provide the first evidence that EMT of RPE cells in vitro confers glycomic changes and that these changes are associated with an increased responsiveness to Gal-3.	181
35	Durham, UK	in vivo	To test the hypothesis that the CFH H402 polymorphism contributes to the development of AMD	➢ERG ➢RPE flat mounts ➢Electron microscopy ➢RNA Isolation and RT-PCR of CFH Tissues ➢Western blots of CFH tissues ➢Cholesterol measurement ➢Lipoprotein fractionation	Demonstrated a functional consequence of the Y402H polymorphism in vivo, which promotes AMD-like pathology development and affects lipoprotein levels in aged mice.	182
36	New York, USA	in vitro	To determine the specific role of RPE-autonomous dysfunction in drusen biogenesis and ECM alterations in maculopathies affecting the RPE–ECM complex.	➢Fibroblast culture ➢Generation of hiPSCs ➢Characterization of hiPSCs. ➢CRISPR correction of DHRD patient hiPSCs ➢Screening and characterization of CRISPR-corrected hiPSCs ➢hiPSC culture and differentiation to RPE ➢Electron microscopy ➢Extracellular matrix isolation ➢Transepithelial resistance measurement ➢RPE culture treatments with serum ➢Western blotting ➢Immunocytochemistry ➢RT-PCR	Distinct complement pathway genes were up-regulated in SFD, DHRD, and ADRD hiPSC-RPE cultures, potentially highlighting similar molecular change as earlier reportings in distinct maculopathies affecting the RPE–ECM complex.	183
37	California	in vitro	To develop an RPE cell culture model that mimics drusen formation and triggers complement activation associated with AMD	➢RPE cell culture ➢Immunohistochemistry ➢Electron microscopy	Developed an RPE cell culture model that mimics various aspects of AMD pathology observed in humans.	184
38	Durham, UK	in vivo	To investigate the role of Complement factor H CFH in the development of AMD pathology	➢Western blot ➢Hemolytic Assay for the functional measurement of complement activity in mouse plasma ➢ERG ➢Quantification of sub-RPE deposits by electron microscopy ➢Analysis of RPE damage based on flat-mount quantification of multinucleate cells ➢Quantification of ONL thickness and assessment of RPE atrophy on toluidine blue plastic sections ➢Immunohistochemistry ➢ELISA ➢Peripheral blood and extravascular RPE/choroid monocyte analysis with flow cytometry ➢CFH and Lipoprotein binding to heparin-sepharose beads ➢Porcine RPE/BrM lipoprotein/CFH binding assays ➢Aged human RPE/BrM endogenous lipoprotein/CFH competition assays	(i)CFH and lipoproteins compete for binding to heparan sulfate in BrM, leading to lipoprotein accumulation and sub-RPE deposit formation (ii)Detrimental complement activation within sub RPE deposits leads to recruitment of MNPs, RPE damage, and visual function decline.	185
39	USA	in vivo	To cause mitochondrial damage in RPE cells and test for AMD characteristics	➢Knockout Sod2 in mice ➢SD-OCT ➢ERG ➢Immunohistochemistry ➢RNA isolation and qPCR ➢mtDNA copy number analysis ➢ATP measurement	Sod2 knockout decreased RPE function with an increase in oxidative stress.	20
40	Taiwan Region, China	in vitro	In ARPE-19 cell line, NaIO3 can cause ROS production and its effect in cell death.	➢ARPE-19 culture ➢Annexin V-FITC/PI assay ➢ROS identification ➢Western blotting ➢Mitochondrial oxygen consumption assessment ➢Mitochondrial imaging	NaIO3 induced cytosolic ROS production and oxidative stress that resulted with activating signalling pathways that respond cell death mechanisms.	21

RPE: retinal pigment epithelium; PMA: phorbol 12-myristate 13-acetate; ZPP1: Zinpyr-1; AMD: age related macular degeneration; iPSCs: induced pluripotent stem cells; PCR: polymerase chain reaction; SIRT1: sirtuin 1; PGC1α: Peroxisome proliferator-activated receptor gamma co-activator 1-alpha; POS: photoreceptor outer segments; hUTC: human umbilical tissue cells; RNA: ribonucleic acid; TGFβ: transforming growth factor beta; hPSC: human pluripotent stem cell; ELISA: enzyme linked immunosorbent assay; 2D: two dimensional; RPE PLUS: RPE purification by lipoprotein uptake-based sorting; MTT: 4,5-dimethylthiazol-2-yl; VEGF: vascular endothelial growth factor; EMT: epithelial-to-mesenchymal transition; OCM3: uveal melanoma cell line; SNPs: single nucleotide polymorphisms; ROS: reactive oxygen species; HN: humanin; hRPE: human retinal pigment epithelial; FITC: fluorescein isothiocyanate; TEM: transmission electron microscopy; PBMCs: peripheral blood mononuclear cells; hESCs: human embryonic stem cells; RCS: royal college of surgeons; BMCs: bone marrow–derived cells; GFP⁺: green fluorescent protein; iRPE: iPSC-derived retinal pigment epithelium; OKR: optokinetic response; qPCR: quantitative PCR; shRNA: short hairpin RNA; ChIP: chromatin immunoprecipitation; NRF2: Nuclear factor erythroid 2-related factor 2; ER: endoplasmic reticulum; dKO: double knock-out; TER: trans epithelial resistance; ARPE-19: aris-ing retinal pigment epithelium-19; ERG: electroretinography; TLR3: toll-like receptor-3; dsRNA: double stranded ribonucleic acid; GA: geographic atrophy; siRNA: short interfering RNA; PEDF: pigment epithelium derived factor; TSP1: thrombospondin 1; NO: nitric oxide; FACS: fluorescence acting cell sorting; NaIO₃: sodium iodate; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling; ONL: outer nuclear layer; hAPP: human amyloid precursor protein; LX-PCR: long extension polymerase chain reaction; UV: ultra violet; IOLs: intraocular lenses; ECM: extracellular matrix; CNV: choroidal neovascularization; RT-PCR: real time – polymerase chain reaction; CXCR5: C-X-C chemokine receptor type 5; EMT: epithelial to mesenchymal transition; siRNA: small interfering RNA; CRISPR: clustered regularly interspaced short palindromic repeats; CFH: complement factor H; DHRD: Doyne Honeycomb Retinal Dystrophy; SFD: Sorsby's fundus dystrophy; ADRD: autosomal dominant radial drusen; BrM: Bruch's membrane; mtDNA: mitochondrial DNA; UPS: ubiquitin proteasome system; H₂O₂: hydrogen peroxide; SD-OCT: spectral-domain optical coherence tomography; ATP: adenosine triphosphate.