In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6C patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1 mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention.

BACKGROUND: Senior-Loken syndrome is a rare genetic disorder that presents with nephronophthisis and retinal degeneration, leading to end-stage renal disease and progressive blindness. The most frequent cause of juvenile nephronophthisis is a mutation in the nephronophthisis type 1 (NPHP1) gene. NPHP1 encodes the protein nephrocystin-1, which functions at the transition zone (TZ) of primary cilia. METHODS: We report a 9-year-old Senior-Loken syndrome boy with NPHP1 deletion, who presents with bilateral vision decrease and cystic renal disease. Renal function deteriorated to require bilateral nephrectomy and renal transplant. We performed immunohistochemistry, H&E staining, and electron microscopy on the renal sample to determine the subcellular distribution of ciliary proteins in the absence of NPHP1. RESULTS: Immunohistochemistry and electron microscopy of the resected kidney showed disorganized cystic structures with loss of cilia in renal tubules. Phosphoinositides have been recently recognized as critical components of the ciliary membrane and immunostaining of kidney sections for phosphoinositide 5-phosphatase, INPP5E, showed loss of staining compared to healthy control. Ophthalmic examination showed decreased electroretinogram consistent with early retinal degeneration. CONCLUSION: The decreased expression of INPP5E specifically in the primary cilium, coupled with disorganized cilia morphology, suggests a novel role of NPHP1 that it is involved in regulating ciliary phosphoinositide composition in the ciliary membrane of renal tubular cells.

PURPOSE: To investigate genetics, electrophysiology and clinical course of KCNV2-associated retinopathy in a cohort of children and adults. STUDY DESIGN: Multicenter international clinical cohort study. METHODS: Review of clinical notes and molecular genetic testing. Full-field electroretinography (ERG) incorporating the international standards were reviewed and quantified and compared with age and recordings from control subjects. RESULTS: In total 230 disease-associated alleles were identified from 117 patients, corresponding to 75 different KCNV2 variants, with 28 being novel. The mean age of onset was 3.9 years old. All patients were symptomatic before the age of 12 years (age range: 0-11 years). Decreased visual acuity was present in all patients, and four other symptoms were common: reduced color vision (78.6%), photophobia (53.5%), nyctalopia (43.6%), and nystagmus (38.6%). After a mean follow of 8.4 years, the mean best corrected visual acuity (BCVA, ±SD) decreased from 0.81 LogMAR (0.27 LogMAR) to 0.90 LogMAR (0.31 LogMAR). Full-field ERGs showed pathognomonic waveform features. Quantitative assessment revealed a wide range of ERG amplitudes and peak times, with a mean rate of age-associated reduction indistinguishable from the control group. Mean amplitude reductions for the DA 0.01 ERG, DA 10 ERG a-wave, LA30Hz and LA3 ERG b-wave were 55%, 21%, 48% and 74% respectively. Peak times showed stability across 6 decades. CONCLUSION: In KCNV2-retinopathy full-field ERGs are diagnostic, and consistent with largely stable peripheral retinal dysfunction. Report No.1 highlights the severity of the clinical phenotype and established a large cohort of patients, emphasizing the unmet need for trials of novel therapeutics.

Purpose: To evaluate the performance of a deep learning algorithm in the detection of referral-warranted diabetic retinopathy (RDR) on low-resolution fundus images acquired with a smartphone and indirect ophthalmoscope lens adapter. Methods: An automated deep learning algorithm trained on 92,364 traditional fundus camera images was tested on a dataset of smartphone fundus images from 103 eyes acquired from two previously published studies. Images were extracted from live video screenshots from fundus examinations using a commercially available lens adapter and exported as a screenshot from live video clips filmed at 1080p resolution. Each image was graded twice by a board-certified ophthalmologist and compared to the output of the algorithm, which classified each image as having RDR (moderate nonproliferative DR or worse) or no RDR. Results: In spite of the presence of multiple artifacts (lens glare, lens particulates/smudging, user hands over the objective lens) and low-resolution images achieved by users of various levels of medical training, the algorithm achieved a 0.89 (95% confidence interval [CI] 0.83-0.95) area under the curve with an 89% sensitivity (95% CI 81%-100%) and 83% specificity (95% CI 77%-89%) for detecting RDR on mobile phone acquired fundus photos. Conclusions: The fully data-driven artificial intelligence-based grading algorithm herein can be used to screen fundus photos taken from mobile devices and identify with high reliability which cases should be referred to an ophthalmologist for further evaluation and treatment. Translational Relevance: The implementation of this algorithm on a global basis could drastically reduce the rate of vision loss attributed to DR.

Purpose: The identification of target pathways to block excessive angiogenesis while simultaneously restoring physiological vasculature is an unmet goal in the therapeutic management of ischemic retinopathies. pNaKtide, a cell-permeable peptide that we have designed by mapping the site of α1 Na/K-ATPase (NKA)/Src binding, blocks the formation of α1 NKA/Src/reactive oxygen species (ROS) amplification loops and restores physiological ROS signaling in a number of oxidative disease models. The aim of this study was to evaluate the importance of the NKA/Src/ROS amplification loop and the effect of pNaKtide in experimental ischemic retinopathy. Methods: Human retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelium (ARPE-19) cells were used to evaluate the effect of pNaKtide on viability, proliferation, and angiogenesis. Retinal toxicity and distribution were assessed in those cells and in the mouse. Subsequently, the role and molecular mechanism of NKA/Src in ROS stress signaling were evaluated biochemically in the retinas of mice exposed to the well-established protocol of oxygen-induced retinopathy (OIR). Finally, pNaKtide efficacy was assessed in this model. Results: The results suggest a key role of α1 NKA in the regulation of ROS stress and the Nrf2 pathway in mouse OIR retinas. Inhibition of α1 NKA/Src by pNaKtide reduced pathologic ROS signaling and restored normal expression of hypoxia-inducible factor 1-α/vascular endothelial growth factor (VEGF). Unlike anti-VEGF agents, pNaKtide did promote retinal revascularization while inhibiting neovascularization and inflammation. Conclusions: Targeting α1 NKA represents a novel strategy to develop therapeutics that not only inhibit neovascularization but also promote physiological revascularization in ischemic eye diseases.

PURPOSE: To investigate the inter-individual variability in duration of anti-vascular endothelial growth factor (VEGF) treatment effect in neovascular age-related macular degeneration (nvAMD). DESIGN: Prospective observational multi-centered study. PARTICIPANTS: Forty-eight patients with nvAMD treated with anti-VEGF injections were included. Both treatment naive (n=25) as well as patients who had previously received treatment with ranibizumab (n=23) more than one month prior to their enrollment were recruited. METHODS: Patients received injection with ranibizumab (0.5 mg/0.05 ml) and were followed weekly for 4 weeks with spectral-domain OCT (SD-OCT) assessing the time to maximal reduction of central retinal thickness (CRT) and the presence of intraretinal and subretinal fluid. Other data collected included age, gender, visual acuity, axial length, lens status, and previous injections. The Shapiro-Wilk test was used to examine normal distributions for all variables. Correlations were examined by calculating Spearman's correlation coeficient. Distributions of quantitative variables are described as means (±SD). Qualitative variables are summarized by counts and percentage. MAIN OUTCOME MEASURES: Time to maximal reduction of CRT and intra- and subretinal fluid after ranibizumab injection. RESULTS: A total of 48 eyes of 48 patients (age 74.8±8.3 years, 62.5% female, 52% treatment naive, 35.4% pseudophakic) were assessed. Two-thirds (64.6%) reached maximal CRT reduction earlier than the standard 4-week interval: 6.3% at 1 week postinjection, 22.9% at 2 weeks postinjection, and 35.4% at 3 weeks postinjection. Only 35.4% of patients had maximal CRT reduction at 4 weeks. Twenty percent of treatment-naive and 34.8% of non-naive patients had a week-4 CRT that was >35 μm thicker than the earlier occuring lowest CRT value (nadir). The time to maximal CRT reduction was not related to axial length, age, lens status, or history of injections. CONCLUSIONS: Optimal dosing interval for maximal CRT reduction may be less than 4 weeks for a significant proportion of patients. Most patients will be classified as complete responders if intervals less than 4 weeks are used to assess anti-VEGF treatment response. Disease load rather than eye size appears to be the driver of anti-VEGF treatment duration and therefore, dosing interval needs to be optimized in the cohort of short-term responders.

Science and medicine have become increasingly "human-centric" over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human-derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post-transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre-designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue-engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell-cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step-by-step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial- and endothelial-stromal co-cultures and isolation of extracellular vesicles. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolating and culturing human corneal keratocytes and fibroblasts Basic Protocol 2: Isolating and culturing human corneal epithelial cells Basic Protocol 3: Isolating and culturing human corneal endothelial cells Basic Protocol 4: 3D corneal stromal construct assembly Basic Protocol 5: 3D corneal epithelial-stromal construct assembly Basic Protocol 6: 3D corneal endothelial-stromal construct assembly Basic Protocol 7: Isolating extracellular vesicles from corneal cell conditioned medium Support Protocol: Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity. Changes to DNA methylation patterns over time form the basis of ageing clocks, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Purpose: During the COVID-19 pandemic, there is growing concern that patients are forgoing necessary care. Emergency departments (ED) represent an important site of eye care. We analyzed patterns of ED visits at an eye-specific ED since the declaration of the public health crisis. Materials and Methods: In this retrospective, cross-sectional single center study, medical records of 6744 patients who presented to the Massachusetts Eye and Ear ED between March 1st and April 30th in 2018, 2019, and 2020 were studied. The primary outcome measures were total volume of ED visits, proportion of urgent ED visits, and proportion of surgical visits. Results: Overall, the median number of daily visits to the ED decreased by 18 visits per day since the declaration of public health guidelines (interquartile range, 9-24, p < 0.001). This accounted for a 32% decrease in the total volume of ED visits in 2020 compared to prior years during the study period (p < 0.001). There was a 9% increase in the proportion of primary diagnoses considered urgent (p = 0.002). The proportion of visits requiring urgent surgery increased by 39% (p = 0.004). Conclusion: The total number of eye-specific ED visits dropped compared to prior years while the proportion of urgent visits increased. Patients were likely more reluctant to seek eye care, deferring less urgent evaluation.

CD147 is a widely expressed matrix metalloproteinase inducer involved in the regulation of cell migration. The high glycosylation and ability to undergo oligomerization have been linked to CD147 function, yet there is limited understanding on the molecular mechanisms behind these processes. The current study demonstrates that the expression of Golgi α1,2-mannosidase I is key to maintaining the cell surface organization of CD147 during cell migration. Using an in vitro model of stratified human corneal epithelial wound healing, we show that CD147 is clustered within lateral plasma membranes at the leading edge of adjacent migrating cells. This localization correlates with a surge in matrix metalloproteinase activity and an increase in the expression of α1,2-mannosidase subtype IC (MAN1C1). Global inhibition of α1,2-mannosidase I activity with deoxymannojirimycin markedly attenuates the glycosylation of CD147 and disrupts its surface distribution at the leading edge, concomitantly reducing the expression of matrix metalloproteinase-9. Likewise, treatment with deoxymannojirimycin or siRNA-mediated knockdown of MAN1C1 impairs the ability of the carbohydrate-binding protein galectin-3 to stimulate CD147 clustering in unwounded cells. We conclude that the mannose-trimming activity of α1,2-mannosidase I coordinates the clustering and compartmentalization of CD147 that follows an epithelial injury.