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Myopia is an increasingly prevalent public health concern globally, with a complex pathogenesis involving the interplay of multiple signaling pathways and genes. The Wnt signaling pathway plays a crucial role in biological processes such as cell proliferation, differentiation, apoptosis, and tissue remodeling, and its role in myopia development has garnered significant attention in recent years. Studies have demonstrated that the Wnt signaling pathway influences the occurrence and progression of myopia by regulating the proliferation, differentiation, and apoptosis of retinal cells(including RPE cells and ipRGCs), as well as the proliferation of scleral fibroblasts and the expression of extracellular matrix components(such as type I collagen), thereby affecting scleral remodeling and axial length elongation. This paper summarizes the roles of the Wnt signaling pathway in myopia development within different ocular tissues(retina and sclera)and explores potential myopia prevention and treatment strategies based on this pathway, providing insights for further research and clinical management of myopia.

Three-dimensional bioprinting (3DP) is transforming the field of regenerative medicine by enabling the precise fabrication of complex tissues, including the retina, a highly specialized and anatomically complex tissue. This review provides an overview of 3DP's principles, its multi-step process, and various bioprinting techniques, such as extrusion-, droplet-, and laser-based methods. Within the scope of biomimicry and biomimetics, emphasis is placed on how 3DP potentially enables the recreation of the retina's natural cellular environment, structural complexity, and biomechanical properties. Focusing on retinal tissue engineering, we discuss the unique challenges posed by the retina's layered structure, vascularization needs, and the complex interplay between its numerous cell types. Emphasis is placed on recent advancements in bioink formulations, designed to emulate retinal characteristics and improve cell viability, printability, and mechanical stability. In-depth analyses of bioinks, scaffold materials, and emerging technologies, such as microfluidics and organ-on-a-chip, highlight the potential of bioprinted models to replicate retinal disease states, facilitating drug development and testing. While challenges remain in achieving clinical translation—particularly in immune compatibility and long-term integration—continued innovations in bioinks and scaffolding are paving the way toward functional retinal constructs. We conclude with insights into future research directions, aiming to refine 3DP for personalized therapies and transformative applications in vision restoration. [ABSTRACT FROM AUTHOR]

Retinal pathologies are major causes of vision impairment and blindness in humans, and inherited retinal diseases (IRDs), such as retinitis pigmentosa, Leber congenital amaurosis, and Stargardt disease, greatly contribute to this problem. In vitro disease modeling can be used for understanding the development of pathology and for screening therapeutic pharmaceutical compounds. In the preclinical research phase, in vitro models complement in vivo models by reducing animal studies, decreasing costs, and shortening research timelines. Additionally, animal models may not always accurately replicate the human disease phenotype. This review examines the types of cells that can be used to create in vitro IRD models, including retina-specific cell lines, primary retinal cells, induced pluripotent stem cells (iPSCs), and more. Special attention is given to mesenchymal stem cells (MSCs), which are characterized by various isolation sources, relative ease of isolation, and straightforward differentiation. MSCs derived from bone marrow (BM), adipose tissue (AT), dental tissue (DT), umbilical cord (UC), and other sources can differentiate into retinal cells, including photoreceptor cells and retinal pigment epithelial (RPE) cells, dysfunction of which is most commonly associated with IRDs. Subsequent differentiation of MSCs into retinal cells can be carried out via various methods: culturing in induction media supplemented with certain growth factors, co-culturing with retinal cells or in their conditioned media, or regulating gene expression with viral vector-delivered transcription factors (TFs) or microRNAs (miRNAs). Compared to the popular iPSCs, for example, MSC-based models are significantly cheaper and faster to obtain, making them more feasible for large-scale drug screening. Nevertheless, the existing differentiation methods need further optimization for this promising platform to receive the success it deserves. [ABSTRACT FROM AUTHOR]

Retinal pathologies are major causes of vision impairment and blindness in humans, and inherited retinal diseases (IRDs), such as retinitis pigmentosa, Leber congenital amaurosis, and Stargardt disease, greatly contribute to this problem. In vitro disease modeling can be used for understanding the development of pathology and for screening therapeutic pharmaceutical compounds. In the preclinical research phase, in vitro models complement in vivo models by reducing animal studies, decreasing costs, and shortening research timelines. Additionally, animal models may not always accurately replicate the human disease phenotype. This review examines the types of cells that can be used to create in vitro IRD models, including retina-specific cell lines, primary retinal cells, induced pluripotent stem cells (iPSCs), and more. Special attention is given to mesenchymal stem cells (MSCs), which are characterized by various isolation sources, relative ease of isolation, and straightforward differentiation. MSCs derived from bone marrow (BM), adipose tissue (AT), dental tissue (DT), umbilical cord (UC), and other sources can differentiate into retinal cells, including photoreceptor cells and retinal pigment epithelial (RPE) cells, dysfunction of which is most commonly associated with IRDs. Subsequent differentiation of MSCs into retinal cells can be carried out via various methods: culturing in induction media supplemented with certain growth factors, co-culturing with retinal cells or in their conditioned media, or regulating gene expression with viral vector-delivered transcription factors (TFs) or microRNAs (miRNAs). Compared to the popular iPSCs, for example, MSC-based models are significantly cheaper and faster to obtain, making them more feasible for large-scale drug screening. Nevertheless, the existing differentiation methods need further optimization for this promising platform to receive the success it deserves.

As part of the central nervous system (CNS), the retina senses light and also conducts and processes visual impulses. The damaged development of the retina not only causes visual damage, but also leads to epilepsy, dementia and other brain diseases. Recently, we have reported that copper (Cu) overload induces retinal developmental defects and down-regulates microtubule (MT) genes during zebrafish embryogenesis, but whether the down-regulation of microtubule genes mediates Cu stress induced retinal developmental defects is still unknown. In this study, we found that microtubule gene stmn4 exhibited obviously reduced expression in the retina of Cu overload embryos. Furthermore, stmn4 deficiency (stmn4−/−) resulted in retinal defects similar to those seen in Cu overload embryos, while overexpression of stmn4 effectively rescued retinal defects and cell apoptosis occurred in the Cu overload embryos and larvae. Meanwhile, stmn4 deficient embryos and larvae exhibited reduced mature retinal cells, the down-regulated expression of microtubules and cell cycle-related genes, and the mitotic cell cycle arrests of the retinal cells, which subsequently tended to apoptosis independent on p53. The results of this study demonstrate that Cu stress might lead to retinal developmental defects via down-regulating expression of microtubule gene stmn4, and stmn4 deficiency leads to impaired cell cycle and the accumulation of retinal progenitor cells (RPCs) and their subsequent apoptosis. The study provides a certain referee for copper overload in regulating the retinal development in fish. [ABSTRACT FROM AUTHOR]

Objective To study the role and mechanism of mir-199-3p in the pathogenesis of diabetic retinopathy (DR). Methods The diabetic model of rats was established by injection of streptozotocin. The expression of miR-199-3p and vascular endothelial growth factor A (VEGFA) in the retina of rats was detected by RT-qPCR. The targeting relationship between miR-199-3p and VegfA in retinal cells was studied by dual luciferase assays. Apoptosis of retinal cells was evaluated by TUNEL staining. Protein of VEGFA and transforming growth factor-β(TGF-β), Hepatocyte growth factor (HGF), pigment epithelium derived factor (PEDF) were detected by Western blot. Results The retinal staining showed that the pathological changes such as retinal capillary dilation and vascular curvature in diabetic rats. The results of reverse transcription showed that the expression of miR-199-3p decreased and VEGFA increased in the retina of diabetic rats significantly. The targeting relationship between miR-199-3p and VegfA was confirmed by dual luciferase assays. The low-expression of miR-199-3p significantly reduced the number of retinal ganglion cells and enhance the apoptosis of retinal cells, while the low expression-miR-199-3p and VEGFA significantly reversed the effects of miR-199-3p on retinal cells(P

Non-adherent culture is critical for the transdifferentiation of stem cells from mesoderm to neuroectoderm. Sphere culture has been reported to directly induce the adipose tissue cells to neural stem cells. Here we aimed to evaluate continuous non-adherent culture on the transdifferentiation potential of human adipose-derived stem cells (ASCs) into retinal lineage. Human ASCs were induced into retinal lineage by the treatment of noggin, dickkopf-related protein 1, and IGF-1 (NDI) under adherent and non-adherent culture. The NDI induction treatment with the adherent culture for 21 days promoted robust expression of retinal markers in the induced ASCs as compared to those without NDI induction on the adherent culture. With continuous non-adherent culture for 21 days, human ASCs could highly express retinal marker genes even without NDI induction treatment as compared to those on the adherent culture. The combination of continuous non-adherent culture with the NDI induction did not show a significant upregulation of the retinal marker expression as compared to either NDI induction with the adherent culture or continuous non-adherent culture without NDI induction treatment. In summary, both non-adherent culture and NDI induction medium could independently promote the transdifferentiation of human ASCs into retinal lineage. Yet, their combination did not produce an enhancement effect.

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There are limited treatments currently available for retinal diseases such as age-related macular degeneration (AMD). Cell-based therapy holds great promise in treating these degenerative diseases. Three-dimensional (3D) polymeric scaffolds have gained attention for tissue restoration by mimicking the native extracellular matrix (ECM). The scaffolds can deliver therapeutic agents to the retina, potentially overcoming current treatment limitations and minimizing secondary complications. In the present study, 3D scaffolds made up of alginate and bovine serum albumin (BSA) containing fenofibrate (FNB) were prepared by freeze-drying technique. The incorporation of BSA enhanced the scaffold porosity due to its foamability, and the Maillard reaction increased crosslinking degree between ALG with BSA resulting in a robust scaffold with thicker pore walls with a compression modulus of 13.08 KPa suitable for retinal regeneration. Compared with ALG and ALG-BSA physical mixture scaffolds, ALG-BSA conjugated scaffolds had higher FNB loading capacity, slower release of FNB in the simulated vitreous humour and less swelling in water and buffers, and better cell viability and distribution when tested with ARPE-19 cells. These results suggest that ALG-BSA MR conjugate scaffolds may be a promising option for implantable scaffolds for drug delivery and retinal disease treatment. [ABSTRACT FROM AUTHOR]

Introduction: Neurosphere-free transdifferentiation of bone marrow stem cells into Retinal Pigment Epithelium (RPE) and Retinal Cells (RCs) in vitro could offer an exceptional opportunity to study cell replacement in degenerative eye diseases. Thus, a simple and efficient protocol for retinal cells production from transdifferentiation of rat BMSCs in the neurosphere-free state is reported. Methods: Extracted BMSCs from hooded pigment rats were exposed to a single-step protocol, including neurosphere-free, containing a cocktail medium that induced transdifferentiation into Retinal Pigment Epithelium (RPE) and retinal cells. Results: The results showed morphological differentiation changes in vitro. Also, the expressed retinal pigment epithelium and retinal cell markers, such as retinal orthodenticle homeobox 2 (23.45%), retinal pigment epithelium protein 65 (91.54%), cellular retinaldehyde-binding protein (91.21%), vascular endothelial growth factor (94.79%), rhodopsin (57.19%), glial fibrillary acidic protein (28.33%), and neurofilament 200 (24.55%). Conclusion: Overall, these findings showed that a protocol without using basic fibroblast growth factor, epidermal growth factor, and B-27 supplements could generate RPE and retinal cells in vitro.

The detection of moving objects is a trivial task performed by vertebrate retinas, yet a complex computer vision task. Object-motion-sensitive ganglion cells (OMS-GC) are specialised cells in the retina that sense moving objects. OMS-GC take as input continuous signals and produce spike patterns as output, that are transmitted to the Visual Cortex via the optic nerve. The Hybrid Sensitive Motion Detector (HSMD) algorithm proposed in this work enhances the GSOC dynamic background subtraction (DBS) algorithm with a customised 3-layer spiking neural network (SNN) that outputs spiking responses akin to the OMS-GC. The algorithm was compared against existing background subtraction (BS) approaches, available on the OpenCV library, specifically on the 2012 change detection (CDnet2012) and the 2014 change detection (CDnet2014) benchmark datasets. The results show that the HSMD was ranked overall first among the competing approaches and has performed better than all the other algorithms on four of the categories across all the eight test metrics. Furthermore, the HSMD proposed in this paper is the first to use an SNN to enhance an existing state of the art DBS (GSOC) algorithm and the results demonstrate that the SNN provides near real-time performance in realistic applications.

Several signaling pathways and transcription factors control the cell fate in its in vitro development and differentiation. The orchestrated use of these factors results in cell specification. In coculture methods, many of these factors secrete from host cells but control the process. Today, transcription factors required for retinal progenitor cells are well known, but the generation of these cells from mesenchymal stem cells is an ideal goal. The purpose of the paper is to review novel methods for retinal progenitor cell production and selecting a set of signaling molecules in the presence of adult retinal pigment epithelium and extraocular mesenchyme acting as inducers of retinal cell differentiation.

AIM: To analyze the function and mechanism of Apelin-13 in preventing the apoptosis of retinal Müller cells induced by hypoxia.METHODS: In the research, the retinal Müller cells are regarded as research subjects, and the control group, hypoxia group and experiment group are set up. The cells of control group are cultivated in normal environment. The cells of hypoxia group are cultivated in hypoxia environment. The cells of experiment group are cultivated in hypoxia environment and are treated with the Apelin-13(1μmol/L). MTT method is used to monitor the changing of the cell viability, and the crystal violet staining method is adopted to observe the cell morphology. In addition, the immunofluorescence staining method is used to test the expression of GFAP and YAP and the TUNEL staining method is used to monitor the cell apoptosis situation and the apoptosis index is calculated. The protein staining method is used to observe the changing of the expression of p-LATS1, p-YAP, LATS1 and YAP protein. RESULTS:The separated and extracted Müller cells grow on the wall and show elongation, polygon and circular shapes. The cytoplasm is plentiful and the cell nucleus show circular shape. The GFAP expression of the cell is positive. The treatment with 0.1, 1, 10μmol/L Apelin-13 can obviously prevent the Müller cell viability decreasing induced by hypoxia(PPPPPPCONCLUSION: Apelin-13 can be used to prevent the retinal Müller cells apoptosis caused by the hypoxia, which may be related to the regulation of YAP into the nucleus.

Introduction: Neurosphere-free transdifferentiation of bone marrow stem cells into Retinal Pigment Epithelium (RPE) and Retinal Cells (RCs) in vitro could offer an exceptional opportunity to study cell replacement in degenerative eye diseases. Thus, a simple and efficient protocol for retinal cells production from transdifferentiation of rat BMSCs in the neurosphere-free state is reported. Methods: Extracted BMSCs from hooded pigment rats were exposed to a single-step protocol, including neurosphere-free, containing a cocktail medium that induced transdifferentiation into Retinal Pigment Epithelium (RPE) and retinal cells. Results: The results showed morphological differentiation changes in vitro. Also, the expressed retinal pigment epithelium and retinal cell markers, such as retinal orthodenticle homeobox 2 (23.45%), retinal pigment epithelium protein 65 (91.54%), cellular retinaldehydebinding protein (91.21%), vascular endothelial growth factor (94.79%), rhodopsin (57.19%), glial fibrillary acidic protein (28.33%), and neurofilament 200 (24.55%). Conclusion: Overall, these findings showed that a protocol without using basic fibroblast growth factor, epidermal growth factor, and B-27 supplements could generate RPE and retinal cells in vitro. [ABSTRACT FROM AUTHOR]

Age‑related macular degeneration (AMD) is an ocular disease that threatens the visual function of older adults worldwide. Key pathological processes involved in AMD include oxidative stress, inflammation and choroidal vascular dysfunction. Retinal pigment epithelial cells and Müller cells are most susceptible to oxidative stress. Traditional herbal medicines are increasingly being investigated in the field of personalized medicine in ophthalmology. Triptonide (Tn) is a diterpene tricyclic oxide, the main active ingredient in the extract from the Chinese herbal medicinal plant Tripterygium wilfordii , and is considered an effective immunosuppressant and anti‑inflammatory drug. The present study investigated the potential beneficial role of Tn in retinal oxidative damage in order to achieve personalized treatment for early AMD. An oxidative stress model of retinal cells induced by H 2 O 2 and a retinal injury model of mice induced by light and N‑Methyl‑D‑aspartic acid were constructed. In vitro , JC‑1 staining, flow cytometry and apoptosis assay confirmed that low concentrations of Tn effectively protected retinal cells from oxidative damage, and reverse transcription‑quantitative PCR and western blotting analyses revealed that Tn reduced the expression of retinal oxidative stress‑related genes and inflammatory factors, which may depend on the PI3K/AKT/mTOR‑induced Nrf2 signaling pathway. In vivo , by retinal immunohistochemistry, hematoxylin and eosin staining and electroretinogram assay, it was found that retinal function and structure improved and choroidal neovascularization was significantly inhibited after Tn pretreatment. These results suggested that Tn is an efficient Nrf2 activator, which can be expected to become a new intervention for diseases such as AMD, to inhibit retinal oxidative stress damage and pathological neovascularization.

Several signaling pathways and transcription factors control the cell fate in its in vitro development and differentiation. The orchestrated use of these factors results in cell specification. In coculture methods, many of these factors secrete from host cells but control the process. Today, transcription factors required for retinal progenitor cells are well known, but the generation of these cells from mesenchymal stem cells is an ideal goal. The purpose of the paper is to review novel methods for retinal progenitor cell production and selecting a set of signaling molecules in the presence of adult retinal pigment epithelium and extraocular mesenchyme acting as inducers of retinal cell differentiation. [ABSTRACT FROM AUTHOR]

Mathematical modelling of non‐linearity due to charge injection phenomenon with variation in desired characteristics of complementary metal oxide semiconductor (CMOS) image sensor (CIS) and correlated double sampling (CDS) circuits is presented. Existing suppression strategies of charge injection effect for CIS and CDS circuits lack in accuracy because of the absence of knowledge of its effect with variation in major device parameters related to switching transistors viz. width (W), length (L), sense node capacitance (Cpd) and photon current (Iph). Therefore, variations in these parameters under the effect of charge injection have been experimentally studied. Based on the outcomes, it can be concluded that four‐parameter logistic regression symmetrical sigmoid function is the best fit for the non‐linearity introduced. Also, the devised mathematical model could be utilised as an activation function to train the biological neurons in CIS centred biomedical applications. A brief illustration of the same has been included for electrical stimulation of retinal cells. Further, the higher values of Iph, Cpd and scaling of switching transistors as Wmin and L > Lmin can prove effective in reducing the non‐linearity. Contrary to previous studies, the higher value of Cpd utilising the normal photodiode found suitable for charge injection suppression in CIS. [ABSTRACT FROM AUTHOR]

AIM: To investigate whether the human olfactory mucosa mesenchymal stem cells (OM-MSCs) can differentiate into photoreceptor cells in vitro. METHODS: Through the olfactory mucosa adherent method, olfactory mucosa was isolated, cultured and identified in vitro among mesenchymal stem cells. The cell surface markers were analyzed by flow cytometry, induced to differentiate into retinal photoreceptor cells in vitro, and the expression of rhodopsin was observed and identified by Immunofluorescence and Western blot methods. RESULTS: OM-MSCs from human were spindle cell-based, and showing radial colony arrangement. OM-MSCs were negative for CD34, CD45 and CD105, but positive for CD73 and CD90. Following induction, a strong positive reaction was produced by photoreceptor specific marker rhodopsin in the cells.

Light transmission efficiency by porcine retinal intermediate filaments in function of the external potential difference recorded at 5 °C for 546 nm light. Completely filled capillary matrix is assumed. • Electric resistance and energy transfer along intermediate filaments (IFs) measured. • Electric field control of energy transfer along IFs. • The recorded I/U plot of IFs was that of a semiconductor with 0.62 eV band gap. We report an experimental method for simultaneous measurement of the electric properties and energy transfer by natural biological fibers. We found that energy transfer efficiency along intermediate filaments (IFs) isolated from porcine retina varied with the potential difference applied to these IFs. The maximum measured effect amounted to 25% of the transfer efficiency increase. The recorded I/U plots characteristic of the IFs demonstrate the behavior of a semiconductor with 0.62 eV band gap. These results show that the image signal might be modulated by the potential difference across the cell membrane, facilitating image contrast enhancement in the brain. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a degenerative disease of the retina where the molecular mechanism involves the production of vascular endothelial growth factor (VEGF), a factor of poor prognosis of the progression of the disease. Previous studies have shown that resveratrol, a polyphenol of grapevines, can prevent VEGF secretion induced by stress from retinal cells. Considering the fundamental role played by VEGF in development and progression of AMD, we investigate the potential effect of red wine extract (RWE) on VEGF secretion and its signaling pathway in human retinal cells ARPE-19. To examine the effect of RWE in ARPE-19, a quantitative and qualitative analysis of the RWE was performed by HPLC MS/MS. We show for the first time that RWE decreased VEGF-A secretion from ARPE-19 cells and its protein expression in concentration-dependent manner. RWE-induced alteration in VEGF-A production is associated with a down of VEGF-receptor 2 (VEGF-R2) protein expression and its phosphorylated intracytoplasmic domain. Subsequently, the activation of kinase pathway is disturbing and RWE prevents the phosphorylation of MEK and ERK 1/2 in human retinal cells ARPE-19. Finally, this study sheds light on the interest that the use of polyphenolic cocktails could represent in a prevention strategy.

Background aims We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line Methods Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line Results Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology Conclusions We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines. [ABSTRACT FROM AUTHOR]

In recent years, mesenchymal stem cells (MSC) have been considered the most effective source for regenerative medicine, especially due to released soluble paracrine bioactive components and extracellular vesicles. These factors, collectively called the secretome, play crucial roles in immunomodulation and in improving survival and regeneration capabilities of injured tissue. Recently, there has been a growing interest in the secretome released by retinal cytotypes, especially retinal pigment epithelium and Müller glia cells. The latter trophic factors represent the key to preserving morphofunctional integrity of the retina, regulating biological pathways involved in survival, function and responding to injury. Furthermore, these factors can play a pivotal role in onset and progression of retinal diseases after damage of cell secretory function. In this review, we delineated the importance of cross-talk between MSCs and retinal cells, focusing on common/induced secreted factors, during experimental therapy for retinal diseases. The cross-link between the MSC and retinal cell secretomes suggests that the MSC secretome can modulate the retinal cell secretome and vice versa. For example, the MSC secretome can protect retinal cells from degeneration by reducing oxidative stress, autophagy and programmed cell death. Conversely, the retinal cell secretome can influence the MSC secretome by inducing changes in MSC gene expression and phenotype., Competing Interests: Conflict-of-interest statement: The authors declare no conflicts of interest for this article., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization, which is seen in age-related macular degeneration. This study investigates whether retinal cells cultured individually or as cocultures with endothelial cells and maintained in the horizontally rotating bioreactor will express more VEGF and VEGF receptors. We measured the expression of VEGF isotypes and VEGF receptors for cells maintained in monolayer and horizontally rotating bioreactor culture at various times by using reverse transcription PCR and Western blot analysis. Retinal cells showed a twofold increase in VEGF-A mRNA expression after five days of culture in the bioreactor, compared with monolayer cultures (77±3 vs 42±2, P<.006). Further, we found that the expression of mRNA for VEGF-A growth factor was increased fivefold for retinal cells cocultured with endothelial cell (52±4 for one day vs 240±15, P<.001, cultured in the bioreactor for five days). Where the expression of VEGF receptors (FLK-1 and FLT-4) was low for monolayer retinal cultures, we found the expression of both VEGF receptors was higher after 5, 10, and 15 days of culture. Increased expression of these receptors was also found for cocultured retinal/endothelial cells. Further, we found that cultured retinal cells showed higher VEGF-C protein expression compared to monolayer cultures. Our protein analysis data showed that the expression of VEGF-A was increased by twofold (780±30 for one day vs 1520±36 for five days, P<.001 ) after five days in bioreactor cocultures. These findings suggest that retinal/endothelial cell coculture in the horizontally rotating bioreactor may be a very good model for investigating the role of growth factors in the neovascularization seen in human ocular disorders. [ABSTRACT FROM AUTHOR]

ObjectiveWe tested the effect of two phosphodiesterase type-5 (PDE5) inhibitors, sildenafil and tadalafil, on ophthalmic artery (OA) blood flow velocity and investigated the presence of the PDE5 enzyme on human retinal tissue in comparison with the PDE6 enzyme localization.MethodsUsing Colour Doppler ultrasonography (CDU) we investigated, in 30 healthy young subjects (27.8 years of age; range, 24.3–33.7 years), the effects of a single oral dose of sildenafil (100 mg), tadalafil (20 mg), and placebo on OA blood flow velocity. Western blot for PDE6 and PDE5 protein expression was performed on frozen samples of human retina, testis, sperm, skin, and corpus cavernosum. Immunohistochemistry was performed on two ocular globes from dead donors.ResultsCDU showed a relationship between the administration of PDE5 inhibitors and OA blood flow velocity modifications in a time-dependent manner. Western blot and immunohistochemical analysis showed PDE6 and PDE5 presence in human retinal tissue and gave a map of its distribution.ConclusionWe demonstrated that (a) tadalafil and sildenafil are able to modify the OA flux in a time-dependent manner; (b) the PDE5 enzyme is expressed on retinal and choroid vasculature (smooth muscle and endothelial cells), on ganglion and bipolar cells; (c) human retinal tissues express the PDE6 enzyme in the rod and cone photoreceptors; (d) visual side effects after PDE5 inhibitors administration may be linked to a specific effect on the PDE5 enzyme; and (e) the PDE5 enzyme may have a physiologic role on ganglion and bipolar cells that need to be further investigated.Eye (2008) 22, 144–149; doi:10.1038/sj.eye.6702908; published online 22 June 2007 [ABSTRACT FROM AUTHOR]

A long-term ‘memory’ of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus α-lipoic acid in the last week. In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-β, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant α-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H2DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with α-lipoic acid added to the last week of normalised glucose. These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation. [ABSTRACT FROM AUTHOR]

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes. [ABSTRACT FROM AUTHOR]

Background: Diabetic retinopathy (DR) is a common microvascular complication of diabetes and one of the most common causes of visual impairment and blindness. However, it is not yet known how abnormal retinal cell subpopulations contribute to disease progression., Methods: In this study, we used the Gene Expression Omnibus database to construct a single-cell atlas of DR and healthy samples to explore changes in the abundance of different cell subpopulations, and identify the molecular pathways potentially involved in DR., Results: Our results showed that DR was associated with significantly reduced numbers of bipolar cells, Müller glia, retinal pigment epithelial cells, and cone photoreceptors, but was also associated with significantly greater numbers of pericytes, rod photoreceptors, anaplastic cells, and microglia. Our results suggest that subpopulations of Müller glia, microglia, endothelial cells, and bipolar cells in DR tissues may be involved in various oxidative stress- and inflammation-related pathways., Conclusions: In summary, we showed that Müller glia, endothelial cells, microglia, and bipolar cells in DR tissues are involved in oxidative stress- and inflammation-related pathways, which may contribute to the progression of the disease and ultimately lead to visual impairment and blindness. This study will provide a theoretical basis for further exploring the specific mechanism of DR., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-1546/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0–75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a degenerative disease of the retina where the molecular mechanism involves the production of vascular endothelial growth factor (VEGF), a factor of poor prognosis of the progression of the disease. Previous studies have shown that resveratrol, a polyphenol of grapevines, can prevent VEGF secretion induced by stress from retinal cells. Considering the fundamental role played by VEGF in development and progression of AMD, we investigate the potential effect of red wine extract (RWE) on VEGF secretion and its signaling pathway in human retinal cells ARPE-19. To examine the effect of RWE in ARPE-19, a quantitative and qualitative analysis of the RWE was performed by HPLC MS/MS. We show for the first time that RWE decreased VEGF-A secretion from ARPE-19 cells and its protein expression in concentration-dependent manner. RWE-induced alteration in VEGF-A production is associated with a down of VEGF-receptor 2 (VEGF-R2) protein expression and its phosphorylated intracytoplasmic domain. Subsequently, the activation of kinase pathway is disturbing and RWE prevents the phosphorylation of MEK and ERK 1/2 in human retinal cells ARPE-19. Finally, this study sheds light on the interest that the use of polyphenolic cocktails could represent in a prevention strategy. [ABSTRACT FROM AUTHOR]

Degeneration of the retina can lead ultimately to devastating irreversible vision loss, such as in inherited retinitis pigmentosa and age-related macular degeneration. Currently there is no cure to prevent retinal degeneration. Quantitative cell-based assays can be used to test potential drugs that prevent the death of retinal cells . Here, we describe in detail three semi-automated cell-based protocols to identify retinoprotective factors with two retinal cell lines, rat R28 cells and mouse 661W cells. In these protocols, cells are induced to undergo death by photo-oxidation stress, growth factor depletion or cytotoxicity with sodium iodate. Pigment epithelium-derived factor, an established neurotrophic factor for retinal cells, was used as a positive control . We discuss how these protocols will prove useful in high-throughput quantitative screening to identify novel therapeutics for retinal disorders., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)