AIMS: To compare microaneurysm (MA) counts using ultrawide field colour images (UWF-CI) and ultrawide field fluorescein angiography (UWF-FA). METHODS: Retrospective study including patients with type 1 or 2 diabetes mellitus receiving UWF-FA and UWF-CI within 2 weeks. MAs were manually counted in individual Early Treatment Diabetic Retinopathy Study (ETDRS) and extended UWF zones. Fields with MAs ≥20 determined diabetic retinopathy (DR) severity (0 fields=mild, 1-3=moderate, ≥4=severe). UWF-FA and UWF-CI agreement was determined and UWF-CI DR severity sensitivity analysis adjusting for UWF-FA MA counts performed. RESULTS: In 193 patients (288 eyes), 2.4% had no DR, 29.9% mild non-proliferative DR (NPDR), 32.6% moderate (NPDR), 22.9% severe NPDR and 12.2% proliferative DR. UWF-FA MA counts were 3.5-fold higher (p<0.001) than UWF-CI counts overall, 3.2x-fold higher in ETDRS fields (p<0.001) and 5.3-fold higher in extended ETDRS fields (p<0.001) and higher in type 1 versus type 2 diabetes (p<0.001). In eyes with NPDR on UWF-CI (n=246), UWF-FA images had 1.6x-3.5x more fields with ≥20 MAs (p<0.001). Fair agreement existed between imaging modalities (k=0.221-0.416). In ETDRS fields, DR severity agreement increased from k=0.346 to 0.600 when dividing UWF-FA counts by a factor of 3, followed by rapid decline in agreement thereafter. Total UWF area agreement increased from k=0.317 to 0.565 with an adjustment factor of either 4 or 5. CONCLUSIONS: UWF-FA detects threefold to fivefold more MAs than UWF-CI and identifies 1.6-3.5-fold more fields affecting DR severity. Differences exist at all DR severity levels, thus limiting direct comparison between the modalities. However, correcting UWF-FA MA counts substantially improves DR severity agreement between the modalities.

The tightly structured neural retina has a unique vascular network comprised of three interconnected plexuses in the inner retina (and choroid for outer retina), which provide oxygen and nutrients to neurons to maintain normal function. Clinical and experimental evidence suggests that neuronal metabolic needs control both normal retinal vascular development and pathological aberrant vascular growth. Particularly, photoreceptors, with the highest density of mitochondria in the body, regulate retinal vascular development by modulating angiogenic and inflammatory factors. Photoreceptor metabolic dysfunction, oxidative stress, and inflammation may cause adaptive but ultimately pathological retinal vascular responses, leading to blindness. Here we focus on the factors involved in neurovascular interactions, which are potential therapeutic targets to decrease energy demand and/or to increase energy production for neovascular retinal disorders.

: To examine factors decreasing participation in school-based vision programs from parent and teacher perspectives.: We conducted 41 semi-structured focus groups (20 parent groups, 21 teacher/staff groups), at 10 Baltimore and 11 Chicago public elementary and middle schools offering school-based vision programs. School-based vision programs provided vision screening, eye exams, and eyeglasses if needed. Focus groups ranged in size from 2-9 participants (median = 5). Sessions were recorded, transcribed, and coded through an iterative process to develop themes using inductive analysis.: Ninety parents and 117 teachers/staff participated. Participants identified five major factors decreasing participation in school-based vision programs: (1) challenges with the consent form, including distribution, collection, and literacy and language barriers; (2) having existing eye care; (3) misunderstandings about the program, especially related to cost and insurance; (4) difficulty raising parental awareness of the program; and (5) certain attitudes towards vision, eye care, and school-based programs, including low prioritization of eye care, mistrust of the program, fear of sharing private information, not believing their child needs glasses, and reluctance accepting 'subsidized' services.: Parents and teachers identified important structural barriers to participation (i.e., consent form challenges and low parental awareness) and specific reasons for non-participation (i.e., attitudes, misunderstanding of the program, existing eye care) in school-based vision programs. Effective strategies are needed to facilitate return of consent forms and promote awareness of school-based vision programs among parents. Programs should also target services towards those currently without access to eye care and increase awareness about paediatric vision needs.

PURPOSE: Von Hippel-Lindau (VHL) disease is a hereditary disorder that can lead to ophthalmic manifestations, including retinal capillary hemangioma (RCH). The diagnosis of RCH is often guided by wide-field fluorescein angiography. In some cases, optical coherence tomography angiography (OCT-A) serves as a non-invasive alternative to FA. Herein, we used OCT-A to examine the macular microvasculature in patients with VHL disease. SUBJECTS: Subjects were selected from patients with a diagnosis of VHL. The control group included eyes without retinal diagnosis from patients with an episode of unilateral retinal detachment or trauma and age ≤ 50 years old. METHODS: Subjects were scanned on the Optovue RTVue-XR device to acquire 3mm x 3mm OCT-A images of the superficial (SCP) and deep capillary plexus (DCP). SCP and DCP vessel density (VD) were calculated after the images were binarized. Furthermore, for subjects with RCH, each OCT-A image was divided equally into four quadrants. SCP and DCP VD of quadrants with RCH were compared to those without RCH. T-tests were performed for statistical analysis. RESULTS: 67 eyes with a history of VHL disease were included as study subjects, while 16 eyes were included as controls. Significant increases in VD were found in patients with VHL disease for both the SCP (p = 0.0441) and DCP (p = 0.0344). When comparing quadrants with associated RCH development to those without, we found no significant difference in SCP VD (p = 0.160) or DCP VD (p = 0.484). CONCLUSIONS: OCT-A can detect changes in the retinal microvasculature in the macula of patients with VHL disease. OCT-A imaging may be an additional tool for screening and early detection of patients at risk of developing ocular complications of VHL disease. Future studies should explore subtle progression on OCT-A associated with the pathogenesis and development of RCH, particularly with larger scan patterns.

Importance: Among eyes with center-involved diabetic macular edema (CI-DME) and good visual acuity (VA), randomized clinical trial results showed no difference in VA loss between initial observation plus aflibercept only if VA decreased, initial focal/grid laser plus aflibercept only if VA decreased, or prompt aflibercept. Understanding the initial observation approach is relevant to patient management. Objective: To assess the DRCR Retina Network protocol-defined approach and outcomes of initial observation with aflibercept only if VA worsened. Design, Setting, and Participants: This was a post hoc secondary analyses of a randomized clinical trial of the DRCR Retina Network Protocol V that included 91 US and Canadian sites from November 2013 to September 2018. Participants were adults (n = 236) with type 1 or 2 diabetes, 1 study eye with CI-DME, and VA letter score at least 79 (Snellen equivalent, 20/25 or better) assigned to initial observation. Data were analyzed from March 2019 to November 2019. Interventions: Initial observation and follow-up with aflibercept only for VA loss of at least 10 letters from baseline at 1 visit or 5 to 9 letters at 2 consecutive visits. Follow-up occurred at 8 weeks and then every 16 weeks unless VA or optical coherence tomography central subfield thickness worsened. Main Outcomes and Measures: Whether individuals received aflibercept. Results: Among 236 eyes in 236 individuals (149 [63%] male; median age, 60 years [interquartile range, 53-67 years]) randomly assigned to initial observation, 80 (34%) were treated with aflibercept during 2 years of follow-up. At 2 years, the median VA letter score was 86.0 (interquartile range, 89.0-81.0; median Snellen equivalent, 20/20 [20/16-20/25]). Receipt of aflibercept was more likely in eyes with baseline central subfield thickness at least 300 μm (Zeiss-Stratus equivalent) vs less than 300 μm (45% vs 26%; hazard ratio [HR], 1.98 [95% CI, 1.26-3.13], continuous P = .005), moderately severe nonproliferative diabetic retinopathy (Early Treatment Diabetic Retinopathy Study retinopathy severity level 47) and above vs moderate nonproliferative diabetic retinopathy (retinopathy severity level 43) and below (51% vs 27%; HR, 2.22 [95% CI, 1.42-3.47], ordinal P < .001), and among participants whose nonstudy eye received DME treatment within 4 months of randomization vs not (52% vs 25%; HR, 2.55 [95% CI, 1.64-3.99], P < .001). Conclusions and Relevance: Most eyes managed with initial observation plus aflibercept only if VA worsened maintained good vision at 2 years and did not require aflibercept for VA loss. However, the eyes in the trial were approximately twice as likely to receive aflibercept for VA loss if they had greater baseline central subfield thickness, worse diabetic retinopathy severity level, or a nonstudy eye receiving treatment for DME. Trial Registration: ClinicalTrials.gov Identifier: NCT01909791.

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated 'knotted' morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neuro-vascular, degenerative processes.

Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of β-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.

The aim of the current study was to investigate the impact of long-acting fibroblast growth factor 21 (FGF21) on retinal vascular leakage utilizing machine learning and to clarify the mechanism underlying the protection. To assess the effect on retinal vascular leakage, C57BL/6J mice were pre-treated with long-acting FGF21 analog or vehicle (Phosphate Buffered Saline; PBS) intraperitoneally (i.p.) before induction of retinal vascular leakage with intravitreal injection of mouse (m) vascular endothelial growth factor 164 (VEGF164) or PBS control. Five hours after mVEGF164 injection, we retro-orbitally injected Fluorescein isothiocyanate (FITC) -dextran and quantified fluorescence intensity as a readout of vascular leakage, using the Image Analysis Module with a machine learning algorithm. In FGF21- or vehicle-treated primary human retinal microvascular endothelial cells (HRMECs), cell permeability was induced with human (h) VEGF165 and evaluated using FITC-dextran and trans-endothelial electrical resistance (TEER). Western blots for tight junction markers were performed. Retinal vascular leakage in vivo was reduced in the FGF21 versus vehicle- treated mice. In HRMECs in vitro, FGF21 versus vehicle prevented hVEGF-induced increase in cell permeability, identified with FITC-dextran. FGF21 significantly preserved TEER compared to hVEGF. Taken together, FGF21 regulates permeability through tight junctions; in particular, FGF21 increases Claudin-1 protein levels in hVEGF-induced HRMECs. Long-acting FGF21 may help reduce retinal vascular leakage in retinal disorders and machine learning assessment can help to standardize vascular leakage quantification.

Plasmacytoid dendritic cells (pDCs) constitute a unique population of bone marrow-derived cells that play a pivotal role in linking innate and adaptive immune responses. While peripheral tissues are typically devoid of pDCs during steady state, few tissues do host resident pDCs. In the current study, we aim to assess presence and distribution of pDCs in naïve murine limbus and bulbar conjunctiva. Immunofluorescence staining followed by confocal microscopy revealed that the naïve bulbar conjunctiva of wild-type mice hosts CD45 CD11c PDCA-1 pDCs. Flow cytometry confirmed the presence of resident pDCs in the bulbar conjunctiva through multiple additional markers, and showed that they express maturation markers, the T cell co-inhibitory molecules PD-L1 and B7-H3, and minor to negligible levels of T cell co-stimulatory molecules CD40, CD86, and ICAM-1. Epi-fluorescent microscopy of DPE-GFP×RAG1 transgenic mice with GFP-tagged pDCs indicated lower density of pDCs in the bulbar conjunctiva compared to the limbus. Further, intravital multiphoton microscopy revealed that resident pDCs accompany the limbal vessels and patrol the intravascular space. In vitro multiphoton microscopy showed that pDCs are attracted to human umbilical vein endothelial cells and interact with them during tube formation. In conclusion, our study shows that the limbus and bulbar conjunctiva are endowed with resident pDCs during steady state, which express maturation and classic T cell co-inhibitory molecules, engulf limbal vessels, and patrol intravascular spaces.