Search Results (9)

Background: Hyalocytes are the main vitreal cell types with critical functions in health and vitreoretinal diseases. Our aim was to develop cultures of human hyalocytes and verify the retention of their initial cellular features after 3 and 6 days of culturing (3 d and 6 d) by analyzing and comparing a few morphological and functional parameters. Methods: Vitreous samples (n = 22) were collected and vitreous cells and bead-enriched hyalocytes were developed and compared (3 d vs. 6 d cultures). Vitreous and conditioned media were tested for collagen, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), nerve growth factor (NGF), matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and alpha-smooth muscle actin (αSMA) expression (ELISA, array/IP/WB, RT-PCR). Cells were observed at light and fluorescent microscopy (phenotypical properties) and tested for their 3D collagen gel contraction abilities. Results: An increased expression of collagens, vimentin, fibronectin, and the MMP9/TIMP1 ratio were observed in vitreous tissues. In 3 d cultures, collagens and MMP9 were upregulated while the related tissue-enzymes were deregulated. Vitreous samples also showed high levels of TGFβ1, VEGF, and NGF, and this protein signature was retained at 3 d while decreased at 6 d. The original phenotype (low αSMA) was retained at 3 d from seeding while an increased αSMA expression was observed at 6 d; NGF/trkA^NGFR was expressed in cultured hyalocytes and partially drives the collagen retraction. Conclusions: The vitreous print comparison between untouched and cultured hyalocytes allowed us, on one side, to select 3 d cultures and, on the other, to highlight the neuroprotective/contractile NGF in vitro hyalocytes effects. The possibility of scoring reactive hyalocytes would represent an interesting aspect of screening the vitreoretinal interface severity. Full article

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia–vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia–vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia–neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction. Full article

The use of Vascular Endothelial Growth Factor inhibitors (VEGFi) has become prevalent in the field of medicine, given the high incidence of various pathological conditions necessitating VEGF inhibition within the general population. These conditions encompass a range of advanced neoplasms, such as colorectal cancer, non-small cell lung cancer, renal cancer, ovarian cancer, and others, along with ocular diseases. The utilization of VEGFi is not without potential risks and adverse effects, requiring healthcare providers to be well-prepared for identification and management. VEGFi can be broadly categorized into two groups: antibodies or chimeric proteins that specifically target VEGF (bevacizumab, ramucirumab, aflibercept, ranibizumab, and brolucizumab) and non-selective and selective small molecules (sunitinib, sorafenib, cabozantinib, lenvatinib, regorafenib, etc.) designed to impede intracellular signaling of the VEGF receptor (RTKi, receptor tyrosine kinase inhibitors). The presentation and mechanisms of adverse effects resulting from VEGFi depend primarily on this distinction and the route of drug administration (systemic or intra-vitreal). This review provides a thorough examination of the causes, recognition, management, and preventive strategies for VEGFi toxicities with the goal of offering support to oncologists in both clinical practice and the design of clinical trials. Full article

The quail (Coturnix coturnix, Linnaeus 1758), a notable model used in developmental biology, is a precocial bird species in which the processes of retinal cell differentiation and retinal histogenesis have been poorly studied. The purpose of the present research is to examine the retinogenesis in this bird species immunohistochemically and compare the results with those from previous studies in precocial and altricial birds. We found that the first PCNA-negative nuclei are detected at Stage (St) 21 in the vitreal region of the neuroblastic layer, coinciding topographically with the first αTubAc-/Tuj1-/Isl1-immunoreactive differentiating ganglion cells. At St28, the first Prox1-immunoreactive nuclei can be distinguished in the vitreal side of the neuroblastic layer (NbL), but also the first visinin-immunoreactive photoreceptors in the scleral surface. The inner plexiform layer (IPL) emerges at St32, and the outer plexiform layer (OPL) becomes visible at St35—the stage in which the first GS-immunoreactive Müller cells are distinguishable. Newly hatched animals show a well-developed stratified retina in which the PCNA-and pHisH3-immunoreactivies are absent. Therefore, retinal cell differentiation in the quail progresses in the stereotyped order conserved among vertebrates, in which ganglion cells initially appear and are followed by amacrine cells, horizontal cells, and photoreceptors. Müller glia are one of the last cell types to be born. Plexiform layers emerge following a vitreal-to-scleral gradient. Finally, our results suggest that there are no significant differences in the timing of different events involved in retinal maturation between the quail and the chicken, but the same events are delayed in an altricial bird species. Full article

Rhino-orbital cerebral mucor mycosis is a rare disease entity, where retinal involvement is described in the literature mostly as CRAO. However, pathological studies have shown mucor invading the choroid and retina with a neutrophilic reaction. So, it is pertinent that retinal inflammation secondary to invading mucor has some role in microstructural changes seen in the vitreous and retina of these patients. This novel study aims to describe the vitreal and retinal features of patients with vision-threatening rhino-orbital cerebral mucor mycosis and how they evolve on spectral domain optical coherence tomography (SD-OCT). This study shall also provide insight into the pathophysiology of these vitreoretinal manifestations by in vitro analysis of the exenterated orbital content. Fifteen eyes of fifteen patients with vision-threatening ROCM treated with standard care were enrolled in this study and underwent complete ophthalmic examination, serial colour fundus photography, and SD-OCT for both qualitative and quantitative analysis, at baseline and follow-up visits. SD-OCT on serial follow-up revealed thickening and increased inner-retinal reflectivity at presentation followed by thinning of both, other features such as the loss of the inner-retinal organized layer structure, external limiting membrane (ELM) disruption, necrotic spaces in the outer retina, and hyperreflective foci. Vitreous cells with vitreous haze were also seen. There was a significant reduction in CMT, inner and outer retinal thickness, total retinal thickness (all p < 0.05) with time, the quantum of reduction concentrated primarily to the inner retina. In summary, in vivo and in vitro analysis revealed that early microstructural changes were primarily a result of retinal infarctions secondary to thrombotic angioinvasion. With the late microstructural changes, there was possible sequelae of retinal infarction with some contribution from the inflammation, resulting from mucor invading the choroid and retina. Full article

Most cases of Stickler syndrome are due to autosomal-dominant COL2A1 gene mutations leading to abnormal type II collagen. Ocular findings include axial eye lengthening with vitreal degeneration and early-onset glaucoma, which can result in vision loss. Although COL2A1 is a major player in cartilage and bone formation, its specific role in eye development remains elusive. We investigated the role of Col2a1a in neural crest migration and differentiation during early zebrafish eye development. In situ hybridization, immunofluorescence, live imaging, exogenous treatments [10 μM diethylaminobenzaldehyde (DEAB), 100 nM all-trans retinoic acid (RA) and 1–3% ethanol (ETOH)] and morpholino oligonucleotide (MO) injections were used to analyze wildtype Casper (roy−/−;nacre−/−), TgBAC(col2a1a::EGFP), Tg(sox10::EGFP) and Tg(foxd3::EGFP) embryos. Col2a1a colocalized with Foxd3- and Sox10-positive cells in the anterior segment and neural crest-derived jaw. Col2a1a expression was regulated by RA and inhibited by 3% ETOH. Furthermore, MO knockdown of Col2a1a delayed jaw formation and disrupted the ocular anterior segment neural crest migration of Sox10-positive cells. Interestingly, human COL2A1 protein rescued the MO effects. Altogether, these results suggest that Col2a1a is a downstream target of RA in the cranial neural crest and is required for both craniofacial and eye development. Full article

Light-activated liposomes permit site and time-specific drug delivery to ocular and systemic targets. We combined a light activation technology based on indocyanine green with a hyaluronic acid (HA) coating by synthesizing HA–lipid conjugates. HA is an endogenous vitreal polysaccharide and a potential targeting moiety to cluster of differentiation 44 (CD44)-expressing cells. Light-activated drug release from 100 nm HA-coated liposomes was functional in buffer, plasma, and vitreous samples. The HA-coating improved stability in plasma compared to polyethylene glycol (PEG)-coated liposomes. Liposomal protein coronas on HA- and PEG-coated liposomes after dynamic exposure to undiluted human plasma and porcine vitreous samples were hydrophilic and negatively charged, thicker in plasma (~5 nm hard, ~10 nm soft coronas) than in vitreous (~2 nm hard, ~3 nm soft coronas) samples. Their compositions were dependent on liposome formulation and surface charge in plasma but not in vitreous samples. Compared to the PEG coating, the HA-coated liposomes bound more proteins in vitreous samples and enriched proteins related to collagen interactions, possibly explaining their slightly reduced vitreal mobility. The properties of the most abundant proteins did not correlate with liposome size or charge, but included proteins with surfactant and immune system functions in plasma and vitreous samples. The HA-coated light-activated liposomes are a functional and promising alternative for intravenous and ocular drug delivery. Full article

In many vitreal diseases, the surgeon removes the natural vitreous and replaces it with silicone oils, gases, or balanced salt solutions to fill the eyeball and hold the retina in position. However, these materials are often associated with complications and have properties that differ from natural vitreous. Herein, we report an extension of our previous work on the synthesis of a biomimetic hydrogel that is composed of thiolated gellan as an analogue of type II collagen and poly(methacrylamide-co-methacrylate-co-bis(methacryloyl)cystamine), a polyelectrolyte, as an analogue of hyaluronic acid. This thermosensitive hydrogel can be injected into the eye as a viscous solution at 45 °C. It then forms a physical gel in situ when it reaches body temperature, and later forms disulfide covalent crosslinks. In this article, we evaluated two different formulations of the biomimetic hydrogels for their physical, mechanical, and optical properties, and we determined their biocompatibility with several cell lines. Finally, we report on the progress of the four-month preclinical evaluation of our bio-inspired vitreous substitute in comparison to silicone oil or a balanced salt solution. We assessed the eyes with a slit-lamp examination, intraocular pressure measurements, electroretinography, and optical coherence tomography. Preliminary results are very encouraging for the continuing evaluation of our bio-inspired hydrogel in clinical trials. Full article

This study was designed to determine the influence of microvesicles (MVs) derived from multipotent stromal cells isolated from human adipose tissue (hASCs) on retinal functionality in dogs with various types of retinal degeneration. The biological properties of hASC-MVs were first determined using an in vitro model of retinal Muller-like cells (CaMLCs). The in vitro assays included analysis of hASC-MVs influence on cell viability and metabolism. Brain-derived neurotrophic factor (BDNF) expression was also determined. Evaluation of the hASC-MVs was performed under normal and oxidative stress conditions. Preliminary clinical studies were performed on ten dogs with retinal degeneration. The clinical studies included behavioral tests, fundoscopy and electroretinography before and after hASC-MVs intra-vitreal injection. The in vitro study showed that CaMLCs treated with hASC-MVs were characterized by improved viability and mitochondrial potential, both under normal and oxidative stress conditions. Additionally, hASC-MVs under oxidative stress conditions reduced the number of senescence-associated markers, correlating with the increased expression of BDNF. The preliminary clinical study showed that the intra-vitreal administration of hASC-MVs significantly improved the dogs’ general behavior and tracking ability. Furthermore, fundoscopy demonstrated that the retinal blood vessels appeared to be less attenuated, and electroretinography using HMsERG demonstrated an increase in a- and b-wave amplitude after treatment. These results shed promising light on the application of cell-free therapies in veterinary medicine for retinal degenerative disorders treatment. Full article