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A single pool of multipotent retinal progenitor cells give rise to the diverse cell types within the mammalian retina. Such cellular diversity is due to precise control of various cellular processes like cell specification, proliferation, differentiation, and maturation. Circadian clock genes can control the expression of key regulators of cell cycle progression and therefore can synchronize the cell cycle state of a heterogeneous population of cells. Here we show that the protein encoded by the circadian clock gene brain and muscle arnt-like protein-1 (Bmal1) is expressed in the embryonic retina and is required to regulate the timing of cell cycle exit. Accordingly, loss of Bmal1 during retinal neurogenesis results in increased S-phase entry and delayed cell cycle exit. Disruption in cell cycle kinetics affects the timely generation of the appropriate neuronal population thus leading to an overall decrease in the number of retinal ganglion cells, amacrine cells, and an increase in the number of the late-born type II cone bipolar cells as well as the Müller glia. Additionally, the mislocalized Müller cells are observed in the photoreceptor layer in the Bmal1 conditional mutants. These changes affect the functional integrity of the visual circuitry as we report a significant delay in visual evoked potential implicit time in the retina-specific Bmal1 null animals. Our results demonstrate that Bmal1 is required to maintain the balance between the neural and glial cells in the embryonic retina by coordinating the timing of cell cycle entry and exit. Thus, Bmal1 plays an essential role during retinal neurogenesis affecting both development and function of the mature retina. [ABSTRACT FROM AUTHOR]

Purpose: The retina contains the highest concentration of the omega 3 fatty acid, docosahexaenoic acid (DHA), in the body. Although epidemiologic studies showed an inverse correlation between the consumption of omega 3 fatty acids and the prevalence of diabetic retinopathy, there are no data showing the effect of diabetes on retinal DHA in humans. In this study, we measured the DHA content of the retina in diabetic and non-diabetic humans as well as mice and determined the effect of diabetes on retinal thickness and function in mice., Methods: Fatty acid composition was determined by gas chromatography/mass spectroscopy. Retinal thickness in mice was measured by optical coherence tomography and retinal function was measured by electroretinogram (ERG). Expression of selected genes involved in inflammation and lipid metabolism was determined by quantitative real-time PCR (qRT-PCR)., Results: We found a 40% reduction of DHA in peripheral retina and a 25% reduction in the macula of diabetic humans compared with nondiabetic controls. There was a 24% reduction in retinal DHA of type 2 diabetic mice (db/db) compared with the controls (db/+). The retinal thickness was significantly decreased in db/db mice, especially in the inner retina, and the ERG b-wave amplitudes were significantly attenuated. Increased expression of proinflammatory genes was observed in both human and mouse diabetic retinas., Conclusions: Retinal DHA is reduced in diabetic humans and mice, which is associated with a thinning of retina and functional defects in diabetic mice. Enriching retinal DHA through diet may be beneficial in the prevention and treatment of diabetic retinopathy.

Corneal scarring, a significant cause of global blindness, results from various insults, including trauma, infections, and genetic disorders. The conventional treatment to replace scarred corneal tissues includes partial or full-thickness corneal transplantation using healthy donor corneas. However, only 1 in 70 individuals with treatable corneal scarring can undergo surgery, due to the limited supply of transplantable donor tissue. Our research focuses on cell-based strategies, specifically ex vivo-expanded corneal stromal stem cells (CSSCs), to address corneal scarring. Preclinical studies have demonstrated the efficacy of CSSC treatment in reducing corneal inflammation and fibrosis, inhibiting scar formation, and regenerating native stromal tissue. Mechanisms include CSSC differentiation into stromal keratocytes and the expression of regenerative cytokines. Here, we present a good manufacturing practice (GMP)-compliant protocol to isolate and expand human CSSCs. This method paves the way to produce clinical-grade CSSCs for transplantation and clinical trials. Key features • This protocol utilizes surgical skills to dissect human corneal tissues for CSSC isolation. • The yield and features of CSSCs rely on donor tissue quality (freshness) and have donor-to-donor variability. • Up to 0.5 billion CSSCs can be generated from a single cornea specimen, and cells at passage 3 are suitable for treatment uses., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Purpose: To determine endothelial cell density (ECD) from real-world donor cornea endothelial cell (EC) images using a self-supervised deep learning segmentation model., Methods: Two eye banks (Eversight, VisionGift) provided 15,138 single, unique EC images from 8169 donors along with their demographics, tissue characteristics, and ECD. This dataset was utilized for self-supervised training and deep learning inference. The Cornea Image Analysis Reading Center (CIARC) provided a second dataset of 174 donor EC images based on image and tissue quality. These images were used to train a supervised deep learning cell border segmentation model. Evaluation between manual and automated determination of ECD was restricted to the 1939 test EC images with at least 100 cells counted by both methods., Results: The ECD measurements from both methods were in excellent agreement with rc of 0.77 (95% confidence interval [CI], 0.75-0.79; P < 0.001) and bias of 123 cells/mm2 (95% CI, 114-131; P < 0.001); 81% of the automated ECD values were within 10% of the manual ECD values. When the analysis was further restricted to the cropped image, the rc was 0.88 (95% CI, 0.87-0.89; P < 0.001), bias was 46 cells/mm2 (95% CI, 39-53; P < 0.001), and 93% of the automated ECD values were within 10% of the manual ECD values., Conclusions: Deep learning analysis provides accurate ECDs of donor images, potentially reducing analysis time and training requirements., Translational Relevance: The approach of this study, a robust methodology for automatically evaluating donor cornea EC images, could expand the quantitative determination of endothelial health beyond ECD.

Purpose: To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection., Design: Comparative analysis of human eyes., Subjects: Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed., Methods: Globes were evaluated ex-vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for epon-embedding and immunocytochemistry., Main Outcome Measures: Retinal thickness and histopathology, detection of SARS-CoV-2 Spike protein, changes in vascular density, gliosis, and degree of inflammation., Results: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the control. OCT based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes, however the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples., Conclusions: In situ analysis of the retinal tissue suggested that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Background: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues., Methods: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors., Findings: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection., Interpretations: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.