MicroRNA (miRNA) is a non-coding RNA that can regulate the expression of many target genes, and it is widely involved in various important physiological activities. MiR-124-3p was found to associate with the normal development of retinal vessels in our previous study, but the mechanism of its anti-angiogenic effect on pathological retinal neovascularization still needed to be explored. Therefore, this study aimed to investigate the effect and mechanism of miR-124-3p on retinal neovascularization in mice with oxygen-induced retinopathy (OIR). Here, we found that intravitreal injection of miR-124-3p agomir attenuated pathological retinal neovascularization in OIR mice. Moreover, miR-124-3p preserved the astrocytic template, inhibited reactive gliosis, and reduced the inflammatory response as well as necroptosis. Furthermore, miR-124-3p inhibited the signal transducer and activator of transcription 3 (STAT3) pathway and decreased the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor. Taken together, our results revealed that miR-124-3p inhibited retinal neovascularization and neuroglial dysfunction by targeting STAT3 in OIR mice. Full article

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H₂O₂. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research. Full article

Traumatic brain injury (TBI) is among the main causes of sudden death after head trauma. These injuries can result in severe degeneration and neuronal cell death in the CNS, including the retina, which is a crucial part of the brain responsible for perceiving and transmitting visual information. The long-term effects of mild–repetitive TBI (rmTBI) are far less studied thus far, even though damage induced by repetitive injuries occurring in the brain is more common, especially amongst athletes. rmTBI can also have a detrimental effect on the retina and the pathophysiology of these injuries is likely to differ from severe TBI (sTBI) retinal injury. Here, we show how rmTBI and sTBI can differentially affect the retina. Our results indicate an increase in the number of activated microglial cells and Caspase3-positive cells in the retina in both traumatic models, suggesting a rise in the level of inflammation and cell death after TBI. The pattern of microglial activation appears distributed and widespread but differs amongst the various retinal layers. sTBI induced microglial activation in both the superficial and deep retinal layers. In contrast to sTBI, no significant change occurred following the repetitive mild injury in the superficial layer, only the deep layer (spanning from the inner nuclear layer to the outer plexiform layer) shows microglial activation. This difference suggests that alternate response mechanisms play a role in the case of the different TBI incidents. The Caspase3 activation pattern showed a uniform increase in both the superficial and deep layers of the retina. This suggests a different action in the course of the disease in sTBI and rmTBI models and points to the need for new diagnostic procedures. Our present results suggest that the retina might serve as such a model of head injuries since the retinal tissue reacts to both forms of TBI and is the most accessible part of the human brain. Full article

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports a potential role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes. Full article

Fucoidans are polysaccharides and constituents of cell walls of brown algae such as Laminaria hyperborea (LH). They exhibit promising effects regarding age-related macular degeneration (AMD). However, the safety of this compound needs to be assured. The focus of this study lies on influences of an LH fucoidan on the retinal pigment epithelium (RPE). The high-molecular weight LH fucoidan Fuc1 was applied to primary porcine RPE cells, and a tetrazolium (MTT) cell viability assay was conducted. Further tests included a scratch assay to measure wound healing, Western blotting to measure expression of retinal pigment epithelium-specific 65 kDa protein (RPE65), as well as immunofluorescence to measure uptake of opsonized fluorescence beads into RPE cells. Lipopolysaccharide was used to proinflammatorily activate the RPE, and interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion was measured. RPE/choroid cultures were used to assess vascular endothelial growth factor (VEGF) secretion. Real-time polymerase chain reaction (real-time PCR) was performed to detect the gene expression of 91 different genes in a specific porcine RPE gene array. Fuc1 slightly reduced wound healing, but did not influence cell viability, phagocytosis or RPE65 expression. Fuc1 lowered IL-6, IL-8 and VEGF secretion. Furthermore, Fuc1 did not change tested RPE genes. In conclusion, Fuc1 does not impair RPE cellular functions and shows antiangiogenic and anti-inflammatory activities, which indicates its safety and strengthens its suitability concerning ocular diseases. Full article

Retina can receive incidental γ-ray exposure from various sources. For example, although radiation therapy is a crucial tool for managing head and neck tumors, patients may develop ocular complications as collateral damage from accidental irradiation. Recently, there has been concern that retinal irradiation during space flight may compromise mission goals and long-term quality of life after space travel. Previously, in our in vitro model, we proved that immature retinal cells are more vulnerable to γ-radiation than differentiated neurons. Here, we investigate if a low-dose pre-irradiation (0.025 Gy), known to have a protective effect in various contexts, can affect DNA damage and oxidative stress in cells exposed to a high dose of γ-rays (2 Gy). Our results reveal that pre-irradiation reduces 2 Gy effects in apoptotic cell number, H2AX phosphorylation and oxidative stress. These defensive effects are also evident in glial cells (reduction in GFAP and ED1 levels) and antioxidant enzymes (catalase and CuZnSOD). Overall, our results confirm that rat retinal cultures can be an exciting tool to study γ-irradiation toxic effects on retinal tissue and speculate that low irradiation may enhance the skill of retinal cells to reduce damage induced by higher doses. Full article

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases. Full article

To describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature, and retinal nerve fiber layer thickness, which are critical for inner retinal tissue homeostasis and survival. Astrocyte and capillary distribution, retinal nerve fiber (RNFL), and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: −7.01 ± 1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: −0.74 ± 0.4D). Untreated marmoset eyes grew normally, their RNFL thickened and their astrocyte numbers were associated with RNFL thickness. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased GFAP-immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels. The myopic changes in retinal astrocytes, vasculature, and retinal nerve fiber layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated. Full article

Purpose: Gene therapy actually seems to have promising results in the treatment of Leber Congenital Amaurosis and some different inherited retinal diseases (IRDs); the primary goal of this strategy is to change gene defects with a wild-type gene without defects in a DNA sequence to achieve partial recovery of the photoreceptor function and, consequently, partially restore lost retinal functions. This approach led to the introduction of a new drug (voretigene neparvovec-rzyl) for replacement of the RPE65 gene in patients affected by Leber Congenital Amaurosis (LCA); however, the treatment results are inconstant and with variable long-lasting effects due to a lack of correctly evaluating the anatomical and functional conditions of residual photoreceptors. These variabilities may also be related to host immunoreactive reactions towards the Adenovirus-associated vector. A broad spectrum of retinal dystrophies frequently generates doubt as to whether the disease or the patient is a good candidate for a successful gene treatment, because, very often, different diseases share similar genetic characteristics, causing an inconstant genotype/phenotype correlation between clinical characteristics also within the same family. For example, mutations on the RPE65 gene cause Leber Congenital Amaurosis (LCA) but also some forms of Retinitis Pigmentosa (RP), Bardet Biedl Syndrome (BBS), Congenital Stationary Night Blindness (CSNB) and Usher syndrome (USH), with a very wide spectrum of clinical manifestations. These confusing elements are due to the different pathways in which the product protein (retinoid isomer-hydrolase) is involved and, consequently, the overlapping metabolism in retinal function. Considering this point and the cost of the drug (over USD one hundred thousand), it would be mandatory to follow guidelines or algorithms to assess the best-fitting disease and candidate patients to maximize the output. Unfortunately, at the moment, there are no suggestions regarding who to treat with gene therapy. Moreover, gene therapy might be helpful in other forms of inherited retinal dystrophies, with more frequent incidence of the disease and better functional conditions (actually, gene therapy is proposed only for patients with poor vision, considering possible side effects due to the treatment procedures), in which this approach leads to better function and, hopefully, visual restoration. But, in this view, who might be a disease candidate or patient to undergo gene therapy, in relationship to the onset of clinical trials for several different forms of IRD? Further, what is the gold standard for tests able to correctly select the patient? Our work aims to evaluate clinical considerations on instrumental morphofunctional tests to assess candidate subjects for treatment and correlate them with clinical and genetic defect analysis that, often, is not correspondent. We try to define which parameters are an essential and indispensable part of the clinical rationale to select patients with IRDs for gene therapy. This review will describe a series of models used to characterize retinal morphology and function from tests, such as optical coherence tomography (OCT) and electrophysiological evaluation (ERG), and its evaluation as a primary outcome in clinical trials. A secondary aim is to propose an ancillary clinical classification of IRDs and their accessibility based on gene therapy’s current state of the art. Material and Methods: OCT, ERG, and visual field examinations were performed in different forms of IRDs, classified based on clinical and retinal conditions; compared to the gene defect classification, we utilized a diagnostic algorithm for the clinical classification based on morphofunctional information of the retina of patients, which could significantly improve diagnostic accuracy and, consequently, help the ophthalmologist to make a correct diagnosis to achieve optimal clinical results. These considerations are very helpful in selecting IRD patients who might respond to gene therapy with possible therapeutic success and filter out those in which treatment has a lower chance or no chance of positive results due to bad retinal conditions, avoiding time-consuming patient management with unsatisfactory results. Full article

An imbalance of homeostasis in the retina leads to neuron loss and this eventually results in a deterioration of vision. If the stress threshold is exceeded, different protective/survival mechanisms are activated. Numerous key molecular actors contribute to prevalent metabolically induced retinal diseases—the three major challenges are age-related alterations, diabetic retinopathy and glaucoma. These diseases have complex dysregulation of glucose-, lipid-, amino acid or purine metabolism. In this review, we summarize current knowledge on possible ways of preventing or circumventing retinal degeneration by available methods. We intend to provide a unified background, common prevention and treatment rationale for these disorders and identify the mechanisms through which these actions protect the retina. We suggest a role for herbal medicines, internal neuroprotective substances and synthetic drugs targeting four processes: parainflammation and/or glial cell activation, ischemia and related reactive oxygen species and vascular endothelial growth factor accumulation, apoptosis and/or autophagy of nerve cells and an elevation of ocular perfusion pressure and/or intraocular pressure. We conclude that in order to achieve substantial preventive or therapeutic effects, at least two of the mentioned pathways should be targeted synergistically. A repositioning of some drugs is considered to use them for the cure of the other related conditions. Full article