The 100 most-cited articles in Investigative Ophthalmology & Visual Science, 2018

AMD is a major cause of legal blindness in older adults approachable through multidisciplinary research involving human tissues and patients. AMD is a vascular-metabolic-inflammatory disease, in which two sets of extracellular deposits, soft drusen/basal linear deposit (BLinD) and subretinal drusenoid deposit (SDD), confer risk for end-stages of atrophy and neovascularization. Understanding how deposits form can lead to insights for new preventions and therapy. The topographic correspondence of BLinD and SDD with cones and rods, respectively, suggest newly realized exchange pathways among outer retinal cells and across Bruch's membrane and the subretinal space, in service of highly evolved, eye-specific physiology. This review focuses on soft drusen/BLinD, summarizing evidence that a major ultrastructural component is large apolipoprotein B,E-containing, cholesterol-rich lipoproteins secreted by the retinal pigment epithelium (RPE) that offload unneeded lipids of dietary and outer segment origin to create an atherosclerosis-like progression in the subRPE-basal lamina space. Clinical…

Purpose: To achieve reproducible imaging of the choriocapillaris and associated flow voids using swept-source OCT angiography (SS-OCTA). Methods: Subjects were enrolled and SS-OCTA was performed using the 3 × 3 mm scan pattern. Blood flow was identified using the complex optical microangiography (OMAG) algorithm. The choriocapillaris was defined as a slab from the outer boundary of Bruch's membrane (BM) to approximately 20 μm below BM. Compensation for the shadowing effect caused by the RPE and BM complex on the choriocapillaris angiogram was achieved by using the structural information from the same slab. A thresholding method to calculate the percentage of flow voids from a region was developed based on a normal database. Results: Twenty normal subjects and 12 subjects with drusen were enrolled. SS-OCTA identified the choriocapillaris in normal subjects as a lobular plexus of capillaries in the central macula and the lobular arrangement became more evident toward the periphery. In all eyes, signal compensation resulted in fewer choriocapillaris flow voids with improved…

Purpose: Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing. Methods: Corneal MSCs (cMSCs) were isolated from human cadaver corneas. The secretome was collected after 72 hours and exosomes were isolated using differential ultracentrifugation. Morphology and size of exosomes were examined by electron microscopy and dynamic light scattering. Expression of CD9, CD63, and CD81 by cMSC exosomes was evaluated by western blotting. Cellular uptake of exosomes was studied using calcein-stained exosomes. The effect of exosome on wound healing was measured in vitro using a scratch assay and in vivo after 2-mm epithelial debridement wounds in mice. Results: cMSC exosomes were morphologically round and main population ranged between 40 and 100 nm in diameter. They expressed CD9, CD63, and CD81, and did not express GM130, Calnexin,…

Purpose: Dry eye is a major ocular pathology worldwide. Although dry eye is a multifactorial disease, recent studies have shown that chronic immunologic processes have a pivotal role in its pathogenesis, characterized by the infiltration of immune cells in the lacrimal glands, elevated levels of tear inflammatory cytokines, and increased density of immune cells in the cornea and conjunctiva. This review describes the recent advances in understanding the relationship between dry eye and inflammation. Methods: This narrative review is based on searches of recent international literature using terms related to the immune response in dry eye, and includes clinical trials, animal experiments, and expert reviews. Results: Although dry eye presents clinically as tear film instability associated with corneal/conjunctival epithelial disorders, Meibomian gland dysfunction, and decreased visual function, recent laboratory and clinical studies have indicated inflammation in the lacrimal glands, Meibomian glands, conjunctiva, cornea, and aqueous tears. Furthermore, inflammation at these…

Reactive oxygen species (ROS) are produced as a by-product during the mitochondrial respiration of the oxygen and potentially able to damage the tissues. Oxidative stress occurs as a result of the disruption of the balance between the anti-oxidant system and the pro-oxidant system found in cells. It has been accepted that overexpression of ROS can be induced in the ocular surface as a result of many acute and chronic diseases and even in normal aging. Recent studies demonstrated that oxidative stress damages the ocular surface and plays an important role in the mechanism of dry eye disease. There is a need to investigate the therapeutic modalities employing topical/systemic use of antioxidants in dry eye disease. This review will summarize the recent studies showing the important relationship between oxidative stress and dry eye disease.

Purpose: While millions of individuals show early age-related macular degeneration (AMD) signs, yet have excellent vision, the risk of progression to advanced AMD with legal blindness is highly variable. We suggest means of artificial intelligence to individually predict AMD progression. Methods: In eyes with intermediate AMD, progression to the neovascular type with choroidal neovascularization (CNV) or the dry type with geographic atrophy (GA) was diagnosed based on standardized monthly optical coherence tomography (OCT) images by independent graders. We obtained automated volumetric segmentation of outer neurosensory layers and retinal pigment epithelium, drusen, and hyperreflective foci by spectral domain-OCT image analysis. Using imaging, demographic, and genetic input features, we developed and validated a machine learning-based predictive model assessing the risk of conversion to advanced AMD. Results: Of a total of 495 eyes, 159 eyes (32%) had converted to advanced AMD within 2 years, 114 eyes progressed to CNV, and 45 to GA. Our predictive model differentiated converting…

The definition and diagnostic criteria of dry eye disease (DED) proposed by the Japan Dry Eye Society and other countries are reviewed. The first definition and criteria of DED in Japan were proposed in 1995. In that report, DED was considered a disease of tears, which subsequently damaged ocular epithelia. The presence of subjective symptoms was not included in the criteria. In 2006, a new definition proposed that interactions between the tear film and ocular surface epithelia play important roles in DED. The presence of subjective symptoms, including visual disturbances, changes in tears, and epithelial damage were proposed to be major components of DED, and eyes positive for all three components were diagnosed as "definitive dry eye." A third version was proposed in 2016, which emphasized unstable tear films as a core finding in DED. Following this guideline, eyes with an abnormally low tear film breakup time and the presence of subjective symptoms are considered to have DED. The current definition and criteria are different from those proposed in other countries. For example,…

Purpose: To develop an automated method of localizing and discerning multiple types of findings in retinal images using a limited set of training data without hard-coded feature extraction as a step toward generalizing these methods to rare disease detection in which a limited number of training data are available. Methods: Two ophthalmologists verified 243 retinal images, labeling important subsections of the image to generate 1324 image patches containing either hemorrhages, microaneurysms, exudates, retinal neovascularization, or normal-appearing structures from the Kaggle dataset. These image patches were used to train one standard convolutional neural network to predict the presence of these five classes. A sliding window method was used to generate probability maps across the entire image. Results: The method was validated on the eOphta dataset of 148 whole retinal images for microaneurysms and 47 for exudates. A pixel-wise classification of the area under the curve of the receiver operating characteristic of 0.94 and 0.95, as well as a lesion-wise area under the precision…

AMD is the leading cause of blindness in developed countries. The dry form of AMD, also known as atrophic AMD, is characterized by the death of RPE and photoreceptors. Currently, there are no treatments for this form of the disease due in part to our incomplete understanding of the mechanism causing AMD. Strong experimental evidence from studies of human donors with AMD supports the emerging hypothesis that defects in RPE mitochondria drive AMD pathology. These studies, using different experimental methods, have shown disrupted RPE mitochondrial architecture and decreased mitochondrial number and mass, altered content of multiple mitochondrial proteins, increased mitochondrial DNA damage that correlates with disease severity, and defects in bioenergetics for primary RPE cultures from AMD donors. Herein, we discuss a model of metabolic uncoupling that alters bioenergetics in the diseased retina and drives AMD pathology. These data provide the rationale for targeting the mitochondria in the RPE as the most efficacious intervention strategy if administered early, before vision loss and…

Purpose: We evaluate how deep learning can be applied to extract novel information such as refractive error from retinal fundus imaging. Methods: Retinal fundus images used in this study were 45- and 30-degree field of view images from the UK Biobank and Age-Related Eye Disease Study (AREDS) clinical trials, respectively. Refractive error was measured by autorefraction in UK Biobank and subjective refraction in AREDS. We trained a deep learning algorithm to predict refractive error from a total of 226,870 images and validated it on 24,007 UK Biobank and 15,750 AREDS images. Our model used the "attention" method to identify features that are correlated with refractive error. Results: The resulting algorithm had a mean absolute error (MAE) of 0.56 diopters (95% confidence interval [CI]: 0.55-0.56) for estimating spherical equivalent on the UK Biobank data set and 0.91 diopters (95% CI: 0.89-0.93) for the AREDS data set. The baseline expected MAE (obtained by simply predicting the mean of this population) was 1.81 diopters (95% CI: 1.79-1.84) for UK Biobank and 1.63 (95% CI: 1.60-1.67)…

Purpose: To investigate whether sheets of retina organoids derived from human embryonic stem cells (hESCs) can differentiate, integrate, and improve visual function in an immunodeficient rat model of severe retinal degeneration (RD). Methods: 3D hESC-derived retina organoids were analyzed by quantitative PCR and immunofluorescence. Sheets dissected from retina organoids (30-65 days of differentiation) were transplanted into the subretinal space of immunodeficient rho S334ter-3 rats. Visual function was tested by optokinetic testing and electrophysiologic recording in the superior colliculus. Transplants were analyzed at 54 to 300 days postsurgery by immunohistochemistry for donor and retinal markers. Results: Retina organoids contained multiple retinal cell types, including progenitor populations capable of developing new cones and rods. After transplantation into an immunodeficient rat model of severe RD, the transplanted sheets differentiated, integrated, and produced functional photoreceptors and other retinal cells, according to the longer human developmental timetable.…

Post-LASIK dry eye is the most common postoperative dry eye after ophthalmic surgeries. The clinical signs of post-LASIK dry eye include positive vital staining of the ocular surface, decreased tear breakup time and Schirmer test values, reduced corneal sensitivity, and decreased functional visual acuity. The symptoms and signs usually last for about 1 month after LASIK. A small number of patients continue to experience symptoms more than 1 year postoperatively. It has been suggested that the loss of corneal innervation caused by flap-making is the major cause, affecting the corneal-lacrimal gland, corneal-blinking, and blinking-meibomian gland reflexes, resulting in decreased aqueous and lipid tear secretion and mucin expression. A new type of corneal refractive surgery, SMILE, which has less impact on corneal nerves, induces less postoperative dry eye, supporting the association between corneal denervation and postoperative dry eye. As LASIK enhancement by flap-lifting induces fewer dry eye symptoms and signs than initial surgery, factors other than neurotrophic effects may be…

Purpose: To investigate the benefit of bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) as an intravitreal (ivit) therapy in two rat models of glaucoma and to determine and identify candidate miRNA involved in the mechanism. Methods: sEV were isolated from human BMSC and fibroblasts and ivit injected into adult rats after induction of elevated IOP. IOP was elevated using either intracameral injection of microbeads or laser photocoagulation of circumferential limbal vessels and the trabecular meshwork. Retinal nerve fiber layer (RNFL) thickness was measured using optical coherence tomography, positive scotopic threshold response (pSTR) recorded using ERG, and RNA binding protein with multiple splicing (RBPMS+) retinal ganglion cell (RGC) counted using retinal wholemounts. sEV miRNA were sequenced using RNAseq. Results: sEV isolated from BMSC promoted significant neuroprotection of RGC while preventing RNFL degenerative thinning and loss of pSTR. sEV proved therapeutically efficacious when ivit injected every week or every month, but ineffective with…

Purpose: To determine the prevalence, risk factors, and impact of myopic macular degeneration (MMD) on visual impairment and functioning among adults in Singapore. Methods: A comprehensive eye examination, including subjective refraction, axial length, and visual acuity (VA) measurements, was performed in adults aged ≥40 years in the Singapore Epidemiology of Eye Diseases (SEED) study. From fundus photographs, MMD was graded using the International META-PM classification. Vision-specific functioning (VSF) was assessed with a validated visual-functioning questionnaire (VF-11) using Rasch analysis. Results: A total of 8716 phakic subjects were included in this analysis. The mean age (± SD) was 57.2 ± 9.5 years (33.5% Malays, 33.2% Indians, and 33.3% Chinese). The prevalence of myopia (spherical equivalent [SE] ≤ -0.5 diopters [D]) and high myopia (SE ≤ -5.0 D) was 35.7% and 6.0%, respectively. The age-standardized prevalence of MMD was 3.8% (95% confidence interval [CI], 3.4-4.3%). The prevalence of MMD was 7.7% among low to moderate myopes, and 28.7% among high myopes. The prevalence…

Purpose: The purpose of this review was to provide detailed insights into the pathophysiology of myofibroblast-mediated fibrosis (scarring or late haze) after corneal injury, surgery, or infection. Method: Literature review. Results: The epithelium and epithelial basement membrane (EBM) and/or endothelium and Descemet's basement membrane (BM) are commonly disrupted after corneal injuries, surgeries, and infections. Regeneration of these critical regulatory structures relies on the coordinated production of BM components, including laminins, nidogens, perlecan, and collagen type IV by epithelial, endothelial, and keratocyte cells. Whether a cornea, or an area in the cornea, heals with transparency or fibrosis may be determined by whether there is injury to one or both corneal basement membranes (EBM and/or Descemet's BM) and delayed or defective regeneration or replacement of the BM. These opaque myofibroblasts, and the disordered extracellular matrix these cells produce, persist in the stroma until the EBM and/or Descemet's BM is regenerated or replaced. Conclusions: Corneal stromal…

Purpose: The purpose of this study was to evaluate the correlation between quantity of intraretinal hyperreflective foci (HRF) in the eye with intermediate AMD and progression to late AMD. Methods: Volume optical coherence tomography (OCT) scans from 114 eyes of 114 patients were retrospectively reviewed. HRF were assessed both qualitatively and quantitatively. Five sequential en face slabs from midretina were thresholded to isolate the HRF. These five slabs were recombined, and HRF area was measured in the whole 6 × 6-mm image (HRFTOT) and within the central 3-mm (HRF3mm) and 5-mm (HRF5mm) regions. These measurements were correlated with the development of late AMD (defined as choroidal neovascularization [CNV] and/or complete RPE and photoreceptor atrophy [cRORA]) after 1 year of follow-up. Results: HRF area in all three regions showed significant correlations with progression to late AMD: R = 0.610 for HRF3mm, R = 0.622 for HRF5mm, and R = 0.614 for HRFTOT (all P < 0.001). Correlations remained significant with progression to cRORA alone, though not for progression to CNV alone.…

Purpose: To detect the retinal microvascular impairment using optical coherence tomography angiography (OCT-A) in patients with Parkinson's disease (PD) and find a correlation between the microvascular impairment and the neuronal damage. Methods: This is a prospective, observational study including 49 eyes from 38 PD patients in their early stages and 34 eyes from 28 healthy controls with comparable age range. Macula microvasculature was evaluated with the spectral-domain optical coherence tomography (SD-OCT) angiography and intraretinal layer thickness evaluated with the SD-OCT. A custom algorithm was used for custom segmentation of retinal thickness and quantification of the superficial and deep microvascular density of the macula, respectively. Results: PD patients showed reduced microvascular density in most of the areas of the whole retina. In the superficial retinal capillary plexus, statistical difference (P < 0.01) was seen in the total annular zone (TAZ), superior, temporal, inferior, and nasal zones. In PD patients, there was a strong correlation between the average…

Purpose: To describe an automated algorithm to quantify the foveal avascular zone (FAZ), using optical coherence tomography angiography (OCTA), and to compare its performance for diagnosis of diabetic retinopathy (DR) and association with best-corrected visual acuity (BCVA) to that of extrafoveal avascular area (EAA). Methods: We obtained 3 × 3-mm macular OCTA scans in diabetic patients with various levels of DR and healthy controls. An algorithm based on a generalized gradient vector flow (GGVF) snake model detected the FAZ, and metrics assessing FAZ size and irregularity were calculated. We compared the automated FAZ segmentation to manual delineation and tested the within-visit repeatability of FAZ metrics. The correlations of two conventional FAZ metrics, two novel FAZ metrics, and EAA with DR severity and BCVA, as determined by Early Treatment Diabetic Retinopathy Study (ETDRS) charts, were assessed. Results: Sixty-six eyes from 66 diabetic patients and 19 control eyes from 19 healthy participants were included. The agreement between manual and automated FAZ delineation had a…

In December 2010 and January 2012, 3% diquafosol sodium ophthalmic solution and 2% rebamipide ophthalmic suspension, respectively, appeared first in Japan as prescription drugs for the treatment of dry eye (DE). Since then, not only the diagnosis and treatment but also the understanding of the pathophysiology of DE have greatly advanced, and a new concept of layer-by-layer diagnosis and treatment for DE, respectively termed "tear-film-oriented diagnosis" (TFOD) and "tear-film-oriented therapy" (TFOT) was born. This new concept is currently in the process of expanding from Japan to other Asian countries. TFOD is the method used for the differential diagnosis of DE, which includes aqueous-deficiency DE (ADDE), decreased wettability DE (DWDE), and increased evaporation DE (IEDE), through the dynamics of tear film (TF) and breakup patterns (BUPs) after the eye is opened. BUPs and/or each diagnosed DE subtype are/is able to distinguish the insufficient components of the ocular surface that are responsible for each BUP in a layer-by-layer fashion. Aqueous fluid, membrane-associated mucins…

Purpose: To assess the connection among arterioles, venules, and capillaries in three retinal capillary plexuses using optical coherence tomography angiography (OCTA). Methods: This was a prospective, cross-sectional, observational study including 20 eyes of 10 healthy subjects. En face and cross-sectional OCTA images were segmented to study the superficial (SCP), middle (MCP), and deep capillary plexuses (DCP). Using thin slabs and manual segmentation within the three plexuses, we examined the connections between the large-caliber superficial vessels within a 3 × 3 mm2 OCTA scan (arterioles and venules) and the smaller capillaries in each plexus. Results: Twenty eyes of 10 healthy subjects (5 females; average age of 30.8 ± 6.3 years) were included in the analysis. We identified vascular interconnections linking the superficial arterioles and venules with capillaries in each plexus (SCP, MCP, and DCP). We found capillaries in the DCP crossed the horizontal raphe. Conclusions: Our findings show that each of the three capillary plexuses in the parafovea has its own feeding arteriolar…

AMD pathobiology was irreversibly changed by the recent discovery of extracellular cholesterol-containing deposits in the subretinal space, between the photoreceptors and retinal pigment epithelium (RPE), called subretinal drusenoid deposits (SDDs). SDDs strikingly mirror the topography of rod photoreceptors in human macula, raising the question of whether an equivalent process results in a deposition related to foveal cones. Herein we propose that AMD's pathognomonic lesion-soft drusen and basal linear deposit (BLinD, same material, diffusely distributed)-is the leading candidate. Epidemiologic, clinical, and histologic data suggest that these deposits are most abundant in the central macula, under the fovea. Strong evidence presented in a companion article supports the idea that the dominant ultrastructural component is large apolipoprotein B,E-containing lipoproteins, constitutively secreted by RPE. Lipoprotein fatty acids are dominated by linoleate (implicating diet) rather than docosahexaenoate (implicating photoreceptors); we seek within the retina cellular relationships and…

Purpose: To quantify microvasculature changes in the superficial (SCP), middle (MCP), and deep capillary plexuses (DCP) in diabetic retinopathy (DR). Methods: Retrospective cross-sectional study at a tertiary academic referral center, in which 26 controls (44 eyes), 27 diabetic subjects without retinopathy (44 eyes), 32 subjects with nonproliferative retinopathy (52 eyes), and 27 subjects with proliferative retinopathy (40 eyes) were imaged with optical coherence tomography angiography (OCTA). Outcome measures included parafoveal vessel density (VD), percentage area of nonperfusion (PAN), and adjusted flow index (AFI) at the different plexuses. Results: MCP VD and MCP AFI decreased with worsening DR, while PAN increased, mirroring changes within the DCP. The fitted regression line for MCP and DCP AFI were significantly different than the SCP, while DCP PAN differed from SCP PAN with disease progression. Higher SCP AFI and PAN were different in eyes with diabetes without retinopathy compared with controls. Unexpectedly, sex was found to independently influence MCP VD and AFI with…

Purpose: To evaluate the association between retinal microvasculature (vascular density) on optical coherence tomography-angiography (OCT-A) and the cardiovascular profile of patients hospitalized for acute coronary syndrome (ACS). Methods: EYE-Myocardial Infarction (EYE-MI) study is a prospective cross-sectional study in the Cardiology Intensive Care Unit of Dijon University Hospital. Retinal OCT-A was performed for each patient within 2 days after admission. Superficial retinal capillary plexus (SCP) vascular density was measured. The population was divided into tertiles according to OCT-A data. Results: Overall, 237 cases were retained for analysis. Patients in the tertile with the lowest retinal vascular density (RVD) were older, and more frequently had systemic hypertension and diabetes. Moreover, American Heart Association (AHA) risk and Global Registry of Acute Coronary Events (GRACE) scores were higher and left ventricular ejection fraction (LVEF) was lower in these patients. In multivariate analysis, the AHA risk score (odds ratio [OR], 1.06; 95% confidence interval [CI],…

Contact lens wearers complain of various types of contact lens discomfort (CLD), which may result in the discontinuation of contact lenses. CLD is often associated with dry eye disease. A contact lens divides the tear film into two layers: the pre- and post-lens tear film. This change leads to instability of the pre-lens tear film, thinning of pre- and post-lens tear film thickness, and increased friction between the contact lens and the ocular surface. The Japanese Dry Eye Society recommends the diagnosis of tear film abnormality first (tear film-oriented diagnosis [TFOD]) and the treatment of dry eye disease based on TFOD (tear film-oriented therapy [TFOT]). These concepts can be applied for contact lens-associated dry eye disease (CLADE). Noninvasive tear film breakup time, tear volume evaluation, vital staining, and assessment of Meibomian glands are performed to evaluate the tear film. On vital staining analysis of CLADE, lid wiper epitheliopathy and conjunctival edge staining are major findings. In TFOT of CLADE, secretagogues of water or mucins, such as diquafosol and…

Purpose: To determine if bone marrow-derived stem cell (BMSC) small extracellular vesicles (sEV) promote retinal ganglion cell (RGC) neuroprotection in the genetic DBA/2J mouse model of glaucoma for 12 months. Methods: BMSC sEV and control fibroblast-derived sEV were intravitreally injected into 3-month-old DBA/2J mice once a month for 9 months. IOP and positive scotopic threshold responses were measured from 3 months: IOP was measured monthly and positive scotopic threshold responses were measured every 3 months. RGC neuroprotection was determined in wholemounts stained with RNA binding protein with multiple splicing (RBPMS), whereas axonal damage was assessed using paraphenylenediamine staining. Results: As expected, DBA/2J mice developed chronic ocular hypertension beginning at 6 months. The delivery of BMSC sEV, but not fibroblast sEV, provided significant neuroprotective effects for RBPMS+ RGC while significantly reducing the number of degenerating axons seen in the optic nerve. BMSC sEV significantly preserved RGC function in 6-month-old mice, but provided no benefit at 9 and…

Purpose: To evaluate peripapillary vessel density and morphology in patients with diabetes mellitus (DM) without clinical signs of diabetic retinopathy (DR) and with mild, nonproliferative DR and to correlate with peripapillary nerve fiber layer (NFL) thickness. Methods: One hundred seventeen eyes (34 healthy controls, 54 patients with DM without DR [noDR group] and 24 patients with mild DR [DR group]) were prospectively evaluated. All subjects underwent peripapillary and macular optical coherence tomography angiography (OCT-A). Peripapillary NFL thickness was also recorded. OCT-A slab of radial peripapillary plexus (RPC) and macular superficial capillary plexus (SCP) were analysed in order to calculate perfusion density (PD) and vessel density (VD). Further an image analysis of RPC slab was performed to identify number of branches (NoB) and total branches length (tBL). Results: In peripapillary area there was a significant decrease in VD (P = 0.003), NoB (P < 0.001), and tBL (P < 0.001) in noDR group versus controls; PD values were not different among groups (P = 0.126); there was…

Purpose: Corticosteroids remain the mainstay of treatment for inflammatory diseases almost 80 years after their first clinical use. Topical ophthalmic formulations of corticosteroids have been available to treat disease of the anterior segment of the eye, but the approval of corticosteroids to treat vitreoretinal diseases, including vein occlusion, diabetic macular edema, and uveitis, has occurred only recently. Although most diseases respond to corticosteroid therapy, some patients are resistant to this therapy and side effects, including cataract and elevated intraocular pressure, can limit their use. The purpose of this review is to detail the basic science of corticosteroids focusing on differences in potency, drug delivery, pharmacokinetics, and gene activation, and how these differences affect safety and efficacy in the treatment of diabetic macular edema. Methods: A review was conducted of basic science and pharmacology of the corticosteroids used to treat diabetic macular edema. Results: Clinically available corticosteroids not only have differing potency and…

Purpose: There is ample evidence that retinal dopamine (DA) is involved in the biochemical signaling cascade that controls emmetropization, but it is unknown how its release depends on the spectral composition of ambient light. We have studied DA release, refractive development, and growth in chicken eyes that were exposed to light of different spectral bands, and had either normal vision or were covered by frosted diffusers to induce myopia. Methods: Experiment 1: After spending the night in the dark, chicks were exposed to white room light (spectral range, 430-630 nm) or kept in the dark. Additional chicks were unilaterally exposed to blue (peak at 470 nm), red (620 nm), or UV lighting (375 nm) for 30 minutes and their fellow eyes covered with black occluders to minimize light exposure. Experiment 2: In the second experiment, chicks wore diffusers over one eye to induce deprivation myopia and were raised for 5 days in either white room light or in lighting supplied by UV, blue, or red light-emitting diodes (LEDs). Refractive states were recorded daily with infrared…

Purpose: To apply computational techniques to wide-angle swept-source optical coherence tomography (SS-OCT) images to identify novel, glaucoma-related structural features and improve detection of glaucoma and prediction of future glaucomatous progression. Methods: Wide-angle SS-OCT, OCT circumpapillary retinal nerve fiber layer (cpRNFL) circle scans spectral-domain (SD)-OCT, standard automated perimetry (SAP), and frequency doubling technology (FDT) visual field tests were completed every 3 months for 2 years from a cohort of 28 healthy participants (56 eyes) and 93 glaucoma participants (179 eyes). RNFL thickness maps were extracted from segmented SS-OCT images and an unsupervised machine learning approach based on principal component analysis (PCA) was used to identify novel structural features. Area under the receiver operating characteristic curve (AUC) was used to assess diagnostic accuracy of RNFL PCA for detecting glaucoma and progression compared to SAP, FDT, and cpRNFL measures. Results: The RNFL PCA features were significantly associated with mean deviation (MD) in both…

Purpose: To investigate the associations between soluble CD14 (sCD14), a cytokine released by microglia and macrophages, and hyperreflective foci (HF) and various characteristics of spectral-domain optical coherence tomography (SD-OCT) in diabetic macular edema (DME). Methods: Sixty-nine eyes from 51 patients with DME and 28 eyes from 28 control subjects were studied. sCD14 levels in the aqueous humor (AH) were measured using ELISA before bevacizumab injection (IVB), and the associations between sCD14 and visual acuity, the number of HF, retinal volume, and the DME pattern were assessed. Results: sCD14 levels were higher in DME patients than in the control subjects (29.9 ± 41.6 pg/mL versus 8.1 ± 3.6 pg/mL, P < 0.001). sCD14 levels in diffuse edema were higher than those in focal edema (50.0 ± 65.3 pg/mL versus 19.8 ± 14.7 pg/mL, P = 0.039). The number of HF in the inner retina of patients with diffuse edema was significantly higher than that in patients with focal edema (4.4 ± 2.3 vs. 2.6 ± 2.1, P = 0.001), but no difference was found in the outer retina (5.8 ± 3.4 vs. 5.0 ± 3.9, P…

Mucins, which play important roles on the ocular surface in wettability, lubrication, and barrier function, are classified into two categories: secreted mucins and membrane-associated mucins. The most important secreted mucin on the ocular surface is MUC5AC, which is secreted by the conjunctival goblet cells. In the human conjunctiva, goblet cells are present in higher concentrations in the fornix, inferior nasal bulbar, and the lid wiper on the lid margin. The number of conjunctival goblet cells and MUC5AC expression/secretion are decreased in a patient with dry eye. In Japan, drugs that stimulate mucin secretion or increase the number of conjunctival goblet cells are commercially available. A P2Y2 receptor, diquafosol, stimulates tear fluid secretion from conjunctival epithelial cells and promotes mucin secretion from conjunctival goblet cells. Rebamipide was marketed originally as an oral therapeutic drug to treat gastritis in Japan. Topical rebamipide increases numbers of goblet cells in the bulbar conjunctiva and the lid wiper area of palpebral conjunctiva. Many researchers…

Purpose: To develop a deep learning approach to digitally stain optical coherence tomography (OCT) images of the optic nerve head (ONH). Methods: A horizontal B-scan was acquired through the center of the ONH using OCT (Spectralis) for one eye of each of 100 subjects (40 healthy and 60 glaucoma). All images were enhanced using adaptive compensation. A custom deep learning network was then designed and trained with the compensated images to digitally stain (i.e., highlight) six tissue layers of the ONH. The accuracy of our algorithm was assessed (against manual segmentations) using the dice coefficient, sensitivity, specificity, intersection over union (IU), and accuracy. We studied the effect of compensation, number of training images, and performance comparison between glaucoma and healthy subjects. Results: For images it had not yet assessed, our algorithm was able to digitally stain the retinal nerve fiber layer + prelamina, the RPE, all other retinal layers, the choroid, and the peripapillary sclera and lamina cribrosa. For all tissues, the dice coefficient, sensitivity,…

The Early Treatment Diabetic Retinopathy Study (ETDRS) and other standardized classification schemes have laid a foundation for tremendous advances in the understanding and management of diabetic retinopathy (DR). However, technological advances in optics and image analysis, especially optical coherence tomography (OCT), OCT angiography (OCTa), and ultra-widefield imaging, as well as new discoveries in diabetic retinal neuropathy (DRN), are exposing the limitations of ETDRS and other classification systems to completely characterize retinal changes in diabetes, which we term diabetic retinal disease (DRD). While it may be most straightforward to add axes to existing classification schemes, as diabetic macular edema (DME) was added as an axis to earlier DR classifications, doing so may make these classifications increasingly complicated and thus clinically intractable. Therefore, we propose future research efforts to develop a new, comprehensive, and clinically useful classification system that will identify multimodal biomarkers to reflect the complex pathophysiology of DRD and…

Purpose: Lens epithelial cell (LEC) conversion to myofibroblast is responsible for fibrotic cataract surgery complications including posterior capsular opacification. While transforming growth factor beta (TGFβ) signaling is important, the mechanisms by which the TGFβ pathway is activated post cataract surgery (PCS) are not well understood. Methods: RNA-seq was performed on LECs obtained from a mouse cataract surgery model at the time of surgery and 24 hours later. Bioinformatic analysis was performed with iPathwayGuide. Expression dynamics were determined by immunofluorescence. Results: The LEC transcriptome is massively altered by 24 hours PCS. The differentially expressed genes included those important for lens biology, and fibrotic markers. However, the most dramatic changes were in the expression of genes regulating the innate immune response, with the top three altered genes exhibiting greater than 1000-fold upregulation. Immunolocalization revealed that CXCL1, S100a9, CSF3, COX-2, CCL2, LCN2, and HMOX1 protein levels upregulate in LECs between 1 hour and 6 hours PCS and peak…

Purpose: The aim of this study was to determine the association between nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) family, pyrin domain-containing 3 (NLRP3) inflammasome-induced inflammation and disease severity in diabetic retinopathy (DR). Methods: Blood samples were collected from 64 patients with diabetes (DR, 43; without DR, 21) and 25 healthy controls. The protein and mRNA expression levels of NLRP3 inflammasomes in peripheral blood mononuclear cells were determined using western blotting and quantitative real-time reverse transcription-PCR. A total of 82 vitreous samples were obtained from patients with DR (n = 60) and nondiabetic controls (n = 22). All patients were candidates for vitrectomy. Interleukin (IL)-1β and IL-18 in the peripheral blood mononuclear cell culture medium and vitreous fluid were detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence staining for apoptosis-associated speck-like protein with a caspase recruitment domain (ASC) and NLRP3 was performed in fibrovascular membranes from 21 proliferative DR…

Purpose: To investigate gut microbiota composition and fecal metabolic phenotype in patients with acute anterior uveitis. Methods: Fecal DNA was extracted from 78 fecal samples (38 acute anterior uveitis (AAU) patients and 40 family members of patients or sex- and age-matched healthy controls) and then sequenced by high-throughput 16S rDNA analysis. Gas chromatographic mass spectrometry (GC-MS) based metabolomics was performed on 60 fecal samples (30 AAU patients and 30 healthy controls). Results: A significant difference was observed in beta diversity between AAU patients and healthy controls. Eight genera including Roseburia were reduced in AAU patients, and Veillonella was increased in AAU patients as compared with healthy controls. Significance was however lost after false discovery rate (FDR) correction. The expression of seven fecal metabolites including 6-deoxy-D-glucose 1, linoleic acid, N-Acetyl-beta-D-mannosamine 3, shikimic acid, azelaic acid, isomaltose 1 and palmitoleic acid was increased in AAU patients. Linoleic acid showed a significant correlation with Roseburia and…

Purpose: Current evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy. Our main goal was to examine whether, in the diabetic human retina, common proteins and pathways are shared with brain neurodegenerative diseases. Methods: A proteomic analysis was performed on three groups of postmortem retinas matched by age: nondiabetic control retinas (n = 5), diabetic retinas without glial activation (n = 5), and diabetic retinas with glial activation (n = 5). Retinal lysates from each group were pooled and run on an SDS-PAGE gel. Bands were analyzed sequentially by liquid chromatography-mass spectrometry (LC/MS) using an Orbitrap Mass Spectrometer. Results: A total of 2190 proteins were identified across all groups. To evaluate the association of the identified proteins with neurological signaling, significant signaling pathways belonging to the category "Neurotransmitters and Other Nervous System Signaling" were selected for analysis. Pathway analysis revealed that "Neuroprotective Role of THOP1 in Alzheimer's Disease" and "Unfolded…

Measurement of tear film breakup time (TBUT) has become a standard diagnostic procedure in the dry eye clinic. Short TBUT-type dry eye is characterized by TBUT of less than 5 seconds and dry eye symptoms such as ocular fatigue or dryness; the importance of this type has recently been gaining prominence. Patients with this condition show no reduction in tear production and no staining of the ocular surface. The severity of symptoms is almost the same as in dry eye in which the ocular surface is stained with rose bengal or fluorescein. In addition to discomfort, patients suffer from decreased vision due to the unstable tear film, which can be attributed to the relative abnormality of the lipid layer, aqueous layer, and/or mucin layer. The diagnosis should be performed carefully on the basis of the patient's symptoms and the TBUT test, because the ocular surface appears normal and remains unstained.

Purpose: To elucidate the relationship between disorganization of retinal inner layers (DRILs) and retinal function in diabetic patients without diabetic retinopathy (DR) and with nonproliferative DR, but without diabetic macular edema (DME). Methods: Fifty-seven participants with diabetes mellitus (DM) and 18 healthy controls underwent comprehensive ophthalmic examination, fundus photography, and spectral-domain optical coherence tomography. Scans of the fovea were evaluated for the presence of DRIL. Retinal function was evaluated using Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, the quick contrast sensitivity function (qCSF) on the AST Sentio Platform, short-wavelength automated perimetry (SWAP), standard automated perimetry (SAP), and frequency doubling perimetry (FDP). ANOVA and Kruskal-Wallis were used to compare retinal function in subjects with and without DRIL. Tukey-Kramer test and Wilcoxon were used for post hoc analysis. Results: DRIL was identified in 9 of 57 diabetic subjects. DRIL subjects had higher body mass index and longer diabetes duration…

Purpose: To identify the microvascular changes associated with paracentral acute middle maculopathy (PAMM) and acute macular neuroretinopathy (AMN) and to improve our understanding of the relevant involvement of the three retinal capillary plexuses using projection-resolved optical coherence tomography angiography (PR-OCTA). Methods: This was a retrospective study of 18 eyes with AMN or PAMM imaged with OCTA. We used cross-sectional PR-OCTA to localize reduced flow signal to the superficial (SCP), middle (MCP), or deep capillary plexus (DCP) or choriocapillaris that corresponded to inner retinal PAMM or outer retinal AMN lesions on OCT. Results: Five eyes with AMN showed outer retinal disruption on OCT associated with reduced DCP flow signal. All three eyes with AMN and follow-up had recovery of DCP flow. Thirteen eyes with PAMM showed middle retinal disruption on OCT associated with reduced flow signal in both the MCP and DCP. Of these, five also had reduced flow signal in the SCP. All 10 eyes with PAMM and follow-up showed variable recovery of flow signal in one or more plexuses.…

Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells. Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin. Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and…

Purpose: Childhood amblyopia can be treated with binocular games or movies that rebalance contrast between the eyes, which is thought to reduce depth of interocular suppression so the child can experience binocular vision. While visual acuity gains have been reported following binocular treatment, studies rarely report gains in binocular outcomes (i.e., stereoacuity, suppression) in amblyopic children. Here, we evaluated binocular outcomes in children who had received binocular treatment for childhood amblyopia. Methods: Data for amblyopic children enrolled in two ongoing studies were pooled. The sample included 41 amblyopic children (6 strabismic, 21 anisometropic, 14 combined; age 4-10 years; ≤4 prism diopters [PD]) who received binocular treatment (20 game, 21 movies; prescribed 9-10 hours treatment). Amblyopic eye visual acuity and binocular outcomes (Randot Preschool Stereoacuity, extent of suppression, and depth of suppression) were assessed at baseline and at 2 weeks. Results: Mean amblyopic eye visual acuity (P < 0.001) and mean stereoacuity improved (P = 0.045), and mean…

Purpose: Experimental myopia in animal models suggests that bright light can influence refractive error and prevent myopia. Additionally, animal research indicates activation of rod pathways and circadian rhythms may influence eye growth. In children, objective measures of personal light exposure, recorded by wearable light sensors, have been used to examine the effects of bright light exposure on myopia. The effect of time spent in a broad range of light intensities on childhood refractive development is not known. This study aims to evaluate dim light exposure in myopia. Methods: We reanalyzed previously published data to investigate differences in dim light exposure across myopic and nonmyopic children from the Role of Outdoor Activity in Myopia (ROAM) study in Queensland, Australia. The amount of time children spent in scotopic (<1-1 lux), mesopic (1-30 lux), indoor photopic (>30-1000 lux), and outdoor photopic (>1000 lux) light over both weekdays and weekends was measured with wearable light sensors. Results: We found significant differences in average daily light exposure…

Purpose: The extracellular matrix (ECM) of the trabecular meshwork (TM) modulates resistance to aqueous humor outflow, thereby regulating IOP. Glaucoma, a leading cause of irreversible blindness worldwide, is associated with changes in the ECM of the TM. The elastic modulus of glaucomatous TM is larger than age-matched normal TM; however, the biomechanical properties of segmental low (LF) and high flow (HF) TM regions and their response to elevated pressure, are unknown. Methods: We perfused human anterior segments at two pressures using an ex vivo organ culture system. After extraction, we measured the elastic modulus of HF and LF TM regions by atomic force microscopy and quantitated protein differences by proteomics analyses. Results: The elastic modulus of LF regions was 2.3-fold larger than HF regions at physiological (1×) pressure, and 7.4-fold or 3.5-fold larger than HF regions at elevated (2×) pressure after 24 or 72 hours, respectively. Using quantitative proteomics, comparisons were made between HF and LF regions at 1× or 2× pressure. Significant ECM protein differences…

Purpose: The objective of this study was to investigate the pharmacologic characteristics of omidenepag isopropyl (OMDI), a compound developed as a novel intraocular pressure (IOP)-lowering agent, with better IOP control and fewer side effects than other prostanoid receptor agonists such as prostaglandin F receptor (FP) agonists. Methods: Binding activities of OMDI and its hydrolyzed form, omidenepag (OMD), to human recombinant prostanoid receptors (DP1-2, EP1-4, FP, and IP) were evaluated. Based on these binding assays, the agonistic activities of OMDI and OMD were further evaluated using cultured cells expressing selected prostanoid receptors. The pharmacokinetics of OMDI after topical administration was assessed in rabbits by measurement of the concentrations of both OMDI and OMD in aqueous humor. The ocular hypotensive effect of OMDI was evaluated in ocular normotensive rabbits, dogs, and both ocular normotensive and hypertensive monkeys. Results: OMD was determined to be a selective EP2 receptor agonist. OMDI weakly bound to EP1; however, the agonistic activity of OMDI to this…

Purpose: To determine whether combining quantitative optical coherence tomography angiography (OCTA) parameters can achieve high sensitivity and specificity to distinguish eyes with nonproliferative diabetic retinopathy (NPDR) from those with proliferative diabetic retinopathy (PDR) as well as eyes with diabetes and no DR (NoDR) from those with clinical DR (any DR). Methods: This cross-sectional study included 28 eyes (17 patients) with NoDR, 54 eyes (34 patients) with NPDR, and 56 eyes (36 patients) with PDR. OCTA images were processed to quantify the foveal avascular zone (FAZ) area, acircularity, vessel density, skeletonized vessel density, fractal dimension, and intersections and average vessel diameter for the superficial (SCP) and the deep capillary plexus (DCP). Binary logistic regression models were used to identify the OCTA parameters that best distinguished DR severity groups. The area (AUC) under the receiver operating characteristic (ROC) curves, and sensitivity and specificity were calculated for each model. Results: The regression model identified the SCP FAZ area, DCP…

Vision is a highly rhythmic function adapted to the extensive changes in light intensity occurring over the 24-hour day. This adaptation relies on rhythms in cellular and molecular processes, which are orchestrated by a network of circadian clocks located within the retina and in the eye, synchronized to the day/night cycle and which, together, fine-tune detection and processing of light information over the 24-hour period and ensure retinal homeostasis. Systematic or high throughput studies revealed a series of genes rhythmically expressed in the retina, pointing at specific functions or pathways under circadian control. Conversely, knockout studies demonstrated that the circadian clock regulates retinal processing of light information. In addition, recent data revealed that it also plays a role in development as well as in aging of the retina. Regarding synchronization by the light/dark cycle, the retina displays the unique property of bringing together light sensitivity, clock machinery, and a wide range of rhythmic outputs. Melatonin and dopamine play a particular role in this…

Purpose: Oxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 regulation in diabetes. Methods: Using human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnmt and Tets were quantified at the Rac1 promoter. The effect of inhibition of Dnmts/Tets (pharmacological inhibitors or short interfering RNA [siRNA]) on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from…

Purpose: To determine the plasma metabolomic signature of primary open-angle glaucoma (POAG). Methods: We compared the metabolomic profiles of plasma from individuals with POAG (n = 36) with age- and sex-matched controls with cataract (n = 27). A targeted metabolomics study was performed using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer. Multivariate analyses were performed using principal component analysis (PCA) and the least absolute shrinkage and selection operator (LASSO) method. Results: Among the 151 metabolites accurately measured, combined univariate and multivariate analyses revealed 18 discriminant metabolites belonging to the carbohydrate, acyl-carnitine, phosphatidylcholine, amino acids, and polyamine families. The metabolomic signature of POAG points to three closely interdependent pathophysiologic conditions; that is, defective mitochondrial oxidation of energetic substrates, altered metabolism resembling that observed in senescence, and a deficiency in spermidine and spermine, both polyamines being involved in the…

Purpose: To systematically compare epithelial-off standard (SCXL) to accelerated corneal collagen cross-linking (ACXL) for the treatment of keratoconus. Methods: PubMed, Embase, the Cochrane Library, and the US trial registry were searched for trials comparing SCXL and ACXL for keratoconus up to October 2017. Standardized mean differences (SMDs) with 95% confidence intervals (CIs) were calculated. Primary outcomes were changes in uncorrected distance visual acuity, maximum keratometry (Kmax), and mean keratometry (mean K). Secondary outcomes were changes in corrected distance visual acuity, mean refractive spherical equivalent, central corneal thickness (CCT), and endothelial cell density (ECD). Results: Eleven trials were included. For primary outcomes, SCXL showed a greater reduction in Kmax (SMD 0.32; 95% CI 0.16, 0.48) than ACXL. For secondary outcomes, the decrease in CCT (SMD 0.32; 95% CI 0.03, 0.61) and ECD (SMD 0.26; 95% CI 0.06, 0.46) was less with ACXL than with SCXL. For the other outcomes, there were no statistically significant differences. Conclusions: SCXL has a…

Purpose: Resveratrol has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (I/R) injury for glaucoma. However, the precise mechanisms for resveratrol's protective effects are still unclear. The aim of this study is to determine whether resveratrol can inhibit RGC apoptosis, retinal gliosis, and inflammation, all of which are critical events in retinal degeneration following I/R injury. Methods: Right retinal ischemia was induced in adult male Sprague Dawley rats by increasing intraocular pressure to 110 mm Hg for 60 minutes, and the left eyes maintained at normal pressure serve as the control. Intraperitoneal injection of resveratrol or control buffer was performed continuously for 3 days from pre- to post-I/R injury and the protective effects were evaluated and compared. RGCs were retrogradely labeled with Fluoro-Gold by injection into superior colliculi. Apoptosis was detected by TUNEL staining. Western blotting and immunostaining for Bax, Bcl-2, and Caspase-3 were used to explore the Bax-associated apoptotic pathway. Gliosis was…

Purpose: To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants. Methods: The coding and noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospectively. Two asymptomatic siblings carrying the same ABCA4 variants as their probands were clinically examined. The penetrance of p.Asn1868Ile was calculated using allele frequency data of ABCA4 variants in non-Finnish European individuals. Results: The p.Asn1868Ile variant was found in cis with known variants in 14/67 probands. In 27/67 probands, we identified p.Asn1868Ile without additional variants in cis, in combination with known, mainly severe ABCA4 variants. In 23/27 probands, the trans configuration was established. Among 27 probands…

Purpose: To investigate how corneal hydration affects the Brillouin frequency of corneal stroma. Methods: From a simple analytical model considering the volume fraction of water in corneal stroma, we derived the dependence of Brillouin frequency on hydration and hydration-induced corneal thickness variation. The Brillouin frequencies of fresh ex vivo porcine corneas were measured as their hydration was varied in dextran solution and water. Healthy volunteers (8 eyes) were scanned in vivo repeatedly over the course of 9 hours, and the diurnal variations of Brillouin frequency and central corneal thickness (CCT) were measured. Results: The measured dependence of Brillouin frequency on hydration, both ex vivo and in vivo, agreed well with the theoretical prediction. The Brillouin frequencies of human corneas scanned immediately after waking were on average ∼25 MHz lower than their daytime average values. For stabilized corneas, the typical variation of Brillouin frequency was ± 7.2 MHz. With respect to CCT increase or swelling, the Brillouin frequency decreased with a slope of -1.06…

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients. Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients. Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no…

Purpose: To investigate diurnal variations in anterior and posterior segment biometry and assess differences between emmetropic and myopic adults. Methods: Healthy subjects (n = 42, 23-41 years old) underwent biometry and spectral-domain optical coherence tomography imaging (SD-OCT) every 4 hours for 24 hours. Subjects were in darkness from 11:00 PM to 7:00 AM. Central corneal thickness, corneal power, anterior chamber depth, lens thickness, vitreous chamber depth, and axial length were measured. Thicknesses of the total retina, photoreceptor outer segments + RPE, photoreceptor inner segments, and choroid over a 6-mm annulus were determined. Results: All parameters except anterior chamber depth demonstrated significant diurnal variations, with no refractive error differences. Amplitude of choroid diurnal variation correlated with axial length (P = 0.05). Amplitude of axial length variation (35.71 ± 19.40 μm) was in antiphase to choroid variation (25.65 ± 2.01 μm, P < 0.001). The central 1-mm retina underwent variation of 5.03 ± 0.23 μm with a peak at 12 hours (P < 0.001), whereas…

Objective: Corneal confocal microscopy (CCM), an in vivo ophthalmic imaging modality, is a noninvasive and objective imaging biomarker for identifying small nerve fiber damage. We have evaluated the diagnostic performance of previously established CCM parameters to a novel automated measure of corneal nerve complexity called the corneal nerve fiber fractal dimension (ACNFrD). Methods: A total of 176 subjects (84 controls and 92 patients with type 1 diabetes) with and without diabetic sensorimotor polyneuropathy (DSPN) underwent CCM. Fractal dimension analysis was performed on CCM images using purpose-built corneal nerve analysis software, and compared with previously established manual and automated corneal nerve fiber measurements. Results: Manual and automated subbasal corneal nerve fiber density (CNFD) (P < 0.0001), length (CNFL) (P < 0.0001), branch density (CNBD) (P < 0.05), and ACNFrD (P < 0.0001) were significantly reduced in patients with DSPN compared to patients without DSPN. The areas under the receiver operating characteristic curves for identifying DSPN were comparable:…

Purpose: It has been known for nearly 50 years that cannabis and the psychoactive constituent Δ9-tetrahydrocannabinol (THC) reduce intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a major cause of blindness. THC likely acts via one of the known cannabinoid-related receptors (CB1, CB2, GPR18, GPR119, GPR55) but this has never been determined explicitly. Cannabidiol (CBD) is a second major constituent of cannabis that has been found to be without effect on IOP in most studies. Methods: Effects of topically applied THC and CBD were tested in living mice by using tonometry and measurements of mRNA levels. In addition the lipidomic consequences of CBD treatment were tested by using lipid analysis. Results: We now report that a single topical application of THC lowered IOP substantially (∼28%) for 8 hours in male mice. This effect is due to combined activation of CB1 and GPR18 receptors each of which has been shown to lower ocular pressure when activated. We also found that the effect was sex-dependent, being stronger in male mice,…

Purpose: To determine the mechanism causing degeneration of the retinal pigment epithelium (RPE) and photoreceptors in mice after an intravenous injection of sodium iodate (NaIO3). Methods: The time-dependent changes in NaIO3-induced retinal degeneration were determined by analyzing the retinal morphology by optical coherence tomographic (OCT) images, histological sections of the retina, physiology of the retina by electroretinography (ERG), and retinal blood flow by laser speckle flowgraphy. In addition, the expression of the genes associated with age-related macular degeneration in humans was assessed in the NaIO3-treated mice by RT-PCR. We also investigated whether macrophages were involved in the NaIO3-induced retinal degeneration. Results: The intravenous injection of 20 mg/kg NaIO3 altered the morphology of the RPE cells and the ERGs transiently. With 40 mg/kg of NaIO3, the degeneration of the RPE cells was still present at 28 days. Aggregated melanin granules were surrounded by zonula occludens protein 1 (ZO-1)-positive cells. In addition, 40 mg/kg of NaIO3 led to a reduction…

Purpose: To investigate the roles of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), the major effector molecules of the Hippo pathway, in TGF-β2-mediated conjunctival fibrosis. Methods: Primary human conjunctival fibroblasts were treated with TGF-β2. The expression of YAP/TAZ was examined by Western blot analyses and immunocytochemistry. The expression of fibrotic proteins and genes were evaluated by Western blot analyses and quantitative real-time PCR, respectively. The effects of YAP/TAZ on fibrotic changes were examined by knockdown experiments and the YAP/TAZ inhibitor, verteporfin. Results: TGF-β2 stabilized YAP/TAZ and subsequently activated Smad2/3, which led to the transcription of fibrotic genes in human primary conjunctival fibroblasts. These fibrotic genes were differently regulated by YAP/TAZ. Notably, α-smooth muscle actin, fibronectin, collagen I, and collagen IV were primarily regulated by YAP. In contrast, CCN family proteins (CTGF and CYR61) depended on both YAP and TAZ. Mechanistically, YAP/TAZ were located in close…

Purpose: Sleep is an essential life habit and closely associated with lifespan, diabetes, hypertension, and mental health. Sleep disorders are a prominent, but overlooked problem in patients with dry eye disease (DED), characterized by a poor sleep index, short sleep duration, long sleep latency, and poor subjective sleep. DED and sleep disorders are more prevalent in women than men. Moreover, sleep quality in DED is worse than in other eye diseases, including glaucoma, retinal diseases, cataract, and allergic and chronic conjunctivitis. Methods: We reviewed the literature for studies investigating sleep and health, eyes and sleep, and DED and sleep, in terms of systemic and eye health, especially in women. Results: Two studies reported that approximately half of patients with DED suffer from poor sleep. The severity of mood disorders, including anxiety and depression, is correlated strongly with sleep disorders in DED, and the symptoms and signs of DED, especially pain, also are correlated with sleep quality. Sleep disorders are documented in primary Sjögren's syndrome and an…

Purpose: Persistent ocular surface pain occurs in moderate to severe dry eye disease (DE); however, the mechanisms that underlie this symptom remain uncertain. The aim of this study was to determine if the transient receptor potential vanilloid ion channels play a role in hypertonic saline (HS)-evoked corneal reflexes in a model for aqueous tear deficient DE. Methods: Eye wipe behavior and orbicularis oculi muscle activity (OOemg) were measured after ocular instillation of HS, capsaicin, or menthol 14 days after exorbital gland removal. Total RNA and protein were measured from anterior eye segment and trigeminal ganglia of sham and DE rats. Results: Eye wipe behavior was enhanced in DE rats after HS and capsaicin instillation, but not after menthol when compared to sham rats. DE rats displayed greater OOemg activity after HS and capsaicin, but not after menthol, compared to sham rats. HS-evoked OOemg activity was reduced by selective TRPV1 antagonists and by coapplication of capsaicin plus QX-314, a charged lidocaine derivative. Menthol did not affect OOemg activity; however,…

Nonneovascular (dry) AMD is a retinal disease with potential for significant central visual impairment. The hallmarks of this disease are macular drusen, RPE alterations, and geographic atrophy (GA). Classification schemes for nonneovascular AMD have evolved over the years as major advances in retinal imaging have enabled a greater understanding of disease pathophysiology. The original classifications of nonneovascular AMD were based on color fundus photography (CFP), while more modern schemes rely on a multimodal imaging approach. Effective diagnosis and management of nonneovascular AMD requires a thorough understanding of its multimodal imaging features as detailed in this review. Future imaging modalities and imaging biomarkers that may aid in diagnosis and management are also discussed.

Purpose: We tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models. Methods: Eighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests. Results: PEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes…

Purpose: To explore differences in driving performance of older adults with age-related macular degeneration (AMD) and age-matched controls, and to identify the visual determinants of driving performance in this population. Methods: Participants included 33 older drivers with AMD (mean age [M] = 76.6 ± 6.1 years; better eye Age-Related Eye Disease Study grades: early [61%] and intermediate [39%]) and 50 age-matched controls (M = 74.6 ± 5.0 years). Visual tests included visual acuity, contrast sensitivity, visual fields, and motion sensitivity. On-road driving performance was assessed in a dual-brake vehicle by an occupational therapist (masked to drivers' visual status). Outcome measures included driving safety ratings (scale of 1-10, where higher values represented safer driving), types of driving behavior errors, locations at which errors were made, and number of critical errors (CE) requiring an instructor intervention. Results: Drivers with AMD were rated as less safe than controls (4.8 vs. 6.2; P = 0.012); safety ratings were associated with AMD severity (early: 5.5 versus…

Purpose: To investigate the amount of lenticule decentration following small-incision lenticule extraction (SMILE) by using the Keratron Scout tangential topography difference map, and the relationship between the magnitudes of total decentration and induced corneal higher-order aberrations (HOAs). Methods: This retrospective observational case series study analyzed decentration values obtained from the Keratron Scout tangential topography difference map of 360 eyes (360 patients) that underwent SMILE. Root mean square total HOAs, third order coma aberration, fourth order spherical aberration, as well as individual coefficients for vertical and horizontal coma were measured preoperatively and 3-months postoperatively. Simple linear regression analysis and piecewise regression models were used to determine the relationship between the magnitudes of total decentration and induced corneal HOAs. Results: The mean total decentration distance from the corneal vertex was 0.36 ± 0.22 mm (range, 0.02-1.27 mm). There were significant differences in total HOAs, coma, vertical and horizontal…

Purpose: DARPin molecules are a novel class of small proteins that contain engineered ankyrin repeat domain(s) and bind to target proteins with high specificity and affinity. Abicipar-pegol (abicipar), a DARPin molecule targeting vascular endothelial growth factor-A (VEGF-A), is currently under evaluation in patients with age-related macular degeneration. The pharmacodynamic properties of abicipar were characterized using in vivo and in vitro assays. Methods: The binding affinity of abicipar was assessed using a kinetic exclusion assay (KinExA). In vitro assays evaluated abicipar effects on VEGF-A165-induced calcium mobilization and tube formation in human umbilical vein endothelial cells. Abicipar was tested in vivo in a mouse model of corneal neovascularization and a rabbit model of chronic retinal neovascularization. The efficacies of abicipar and ranibizumab were compared in a rabbit model of VEGF-A165-induced retinal vasculopathy. Results: Abicipar has a high affinity for the soluble isoforms of VEGF-A; binding affinities for human VEGF-A165 are approximately 100-fold greater…

Visual disturbances were included in the definition of dry eye disease in the 2007 Dry Eye Workshop report. As a result, quality of vision (QoV) in dry eye patients has received increased attention. Corneal topography and wavefront sensors have been used to objectively and quantitatively evaluate optical quality, with data showing increases in irregular astigmatism and higher-order aberrations (HOAs) in dry eye patients. Furthermore, ocular optical characteristics are influenced by the tear film, which constantly fluctuates over time. Therefore, dynamic quantitative assessments of optical quality with continuous measurements are essential to understanding QoV in dry eye patients. This review summarizes what is known and what advances have been made in evaluating and understanding QoV in dry eye patients. In particular, corneal topographic and wavefront analyses, conducted both overseas and in Japan, are described.

Purpose: Keratoconus (KC) is the most common corneal ectasia. We aimed to determine the differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC. Methods: From the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape. Results: Using |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis…

Purpose: Myopia is a refractive disorder that degrades vision. It can be treated with atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but the mechanism is unknown. Atropine may block α-adrenoceptors at concentrations ≥0.1 mM, and another potent myopia-inhibiting ligand, mamba toxin-3 (MT3), binds equally well to human mAChR M4 and α1A- and α2A-adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via α2A-adrenoceptors, rather than mAChR M4. Methods: Human mAChR M4 (M4), chicken mAChR M4 (cM4), or human α2A-adrenergic receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; MT3; HIM: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3-quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) was measured using the Dual-Glo Luciferase Assay System. Results: Relative inhibitory potencies of mAChR antagonists at mAChR…

Many studies have revealed that transmembrane mucins, large glycoproteins with heavily glycosylated glycans, are essential for maintaining ocular surface epithelium lubrication and wettability. Recent reports indicate that transmembrane mucins and galectin-3, a chimera type of galectin that binds β-galactoside in the glycan, play a crucial role in maintaining the epithelial glycocalyx barrier. This review summarizes current evidence regarding the role of galectin-3, the role of the three major transmembrane mucins (i.e., MUC1, MUC4, and MUC16), in the maintenance of ocular surface wettability and transcellular barrier. Pathological mechanisms of glycocalyx barrier disruption and epithelial surface wettability decreases in dry eye disease are also summarized. Lastly, new ophthalmic drugs that target transmembrane mucin are described.

Purpose: To analyze retinal and choroidal microvasculature in patients with nonarteritic anterior ischemic optic neuropathy (NAION) by using optical coherence tomography angiography (OCT-A). Methods: In this case-control retrospective observational study, patients with atrophic NAION (at least 3 months after onset of symptoms) and normal subjects underwent a complete ophthalmic examination including spectral-domain OCT, visual field (VF), and OCT-A. Whole en face image vessel density (wiVD) was used to assess retinal blood flow of the radial peripapillary capillaries (RPCs), circumpapillary RPC vessel density (cpVD), superficial capillary plexus (SCP), deep capillary plexus (DCP), and choriocapillaris (CC). Statistical correlations between wiVD measurements and visual acuity, VF parameters, retinal nerve fiber layer (RNFL), and combined thickness of retinal ganglion cell and inner plexiform layers were analyzed. Results: Twenty-four patients (26 eyes) with NAION and 24 age-matched normal controls (NCs) (24 eyes) were included. OCT-A showed significant reduction of the RPC wiVD (P <…

Purpose: Oxidative stress is a causal factor in the development of diabetic retinopathy; however, clinically relevant strategies to treat the disease by augmenting antioxidant defense mechanisms have not been fully explored. We hypothesized that boosting nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant capacity with the novel Nrf2 activator dh404, would protect the retina in diabetes including vision-threatening breakdown of the blood-retinal barrier (BRB) and associated damage to macroglial Müller cells. Methods: Sprague-Dawley rats were randomized to become diabetic or nondiabetic and administered dh404 by gavage for 10 weeks. Complementary in vitro studies were performed in cultured Müller cells exposed to hyperglycemia. Results: In diabetes, dh404 prevented vascular leakage into the retina and vitreous cavity as well as upregulation of the vascular permeability and angiogenic factors, VEGF, and angiopoietin-2, and inflammatory mediators, including TNF-α and IL-6. Müller cells, which maintain BRB integrity and become gliotic in diabetes with increased immunolabeling…

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis. Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay. Results: Lactic acid was significantly increased in the RPE-choroid…

Purpose: To investigate fundus autofluorescence (FAF) lifetimes in patients with nonexudative AMD. Methods: A total of 150 eyes of 110 patients (mean age: 73.2 ± 10.7 years) with nonexudative AMD, as well as a healthy group of 57 eyes in 38 subjects (mean age: 66.5 ± 8.7 years), were included. Investigations were conducted at the University Eye Clinic in Jena, Germany, as well as the Moran Eye Center in Salt Lake City, Utah, USA, using the Heidelberg Engineering Spectralis-based fluorescence lifetime imaging ophthalmoscope (FLIO). A 30° retinal field centered at the fovea was investigated. FAF decays were detected in short (498-560 nm) and long (560-720 nm, LSC) spectral channels. The mean fluorescence lifetimes (τm) were calculated. Optical coherence tomography scans and fundus photographs were also recorded. Results: In patients with nonexudative AMD, FLIO shows a ring-shaped pattern of prolonged τm in the LSC. This pattern occurs in all patients with AMD (including very early stages) and in one-third of the healthy controls. FAF lifetimes were longer with more advanced stages.…

Purpose: To identify potentially pathogenic variants (PPVs) in Chinese familial exudative vitreoretinopathy (FEVR) patients in FZD4, LRP5, NDP, TSPAN12, ZNF408, and KIF11 genes. Methods: Blood samples were collected from probands and their parent(s). Genomic DNA was analyzed by next-generation sequencing, and the sequence of selected variants were validated by Sanger sequencing. The potential pathogenicity of a variant was evaluated by in silico analysis and by cosegregation of the variant with disease. Each proband was subjected to comprehensive retinal examinations, and the severity of FEVR was individually graded for each eye. Whenever possible, fundus fluorescein angiography was obtained and analyzed for parent(s) of each proband. Variation in mutation expressivity was analyzed. Results: Three hundred eighty-nine consecutive FEVR patients from 389 families participated in this study. About 74% of the probands were children younger than 7 years old. One hundred one PPVs, 49 variants with unknown significance (VUS), were identified, including 73 novel PPVs and 38 novel VUS. One…

Purpose: To evaluate spontaneous eye blink patterns and their correlations with clinical tests in dry eye disease (DED). Methods: Twenty-five DED patients and 25 healthy subjects were included in this prospective case-controlled study. Clinical evaluations included the Ocular Surface Disease Index (OSDI), lipid layer thickness (LLT), spontaneous eye blink pattern analysis, fluorescein tear film break-up time (FBUT), and so on. Eye blinks were recorded for 20 seconds with a high-speed camera. Eye blink patterns were divided into the following five phases: the eyelid closing phase (ECP), eyelid closed phase (CDP), early opening phase (EOP), late opening phase (LOP), and interblink intervals (IBI). The correlations between blink parameters and clinical tests were analyzed. Results: Compared with the control group, mean ECP, CDP, and EOP were significantly longer in DED patients (P < 0.001, P = 0.029, and P < 0.001, respectively). DED patients also had significantly shorter LOP and blink intervals (both P < 0.001) and more partial blinks as compared with control subjects (P = 0.001).…

Purpose: To characterize the intraocular immune response following transplantation of iPS-derived allogeneic RPE cells into the subretinal space of non-immune-suppressed rhesus macaques. Methods: GFP-labeled allogeneic iPS-derived RPE cells were transplanted into the subretinal space of one eye (n = 6), and into the contralateral eye 1 day to 4 weeks later, using a two-stage transretinal and transscleral approach. Retinas were examined pre- and post-surgery by color fundus photography, fundus autofluorescence, and optical coherence tomography (OCT) imaging. Animals were euthanized between 2 hours and 7 weeks following transplantation. T-cell (CD3), B-cell (CD20), and microglial (Iba1) responses were assessed immunohistochemically. Results: Cells were delivered into the subretinal space in all eyes without leakage into the vitreous. Transplanted RPE cells were clearly visible at 4 days after surgery but were no longer detectable by 3 weeks. In localized areas within the bleb containing transplanted cells, T- and B-cell infiltrates and microglia were observed in the subretinal space…

Purpose: Previous studies suggest that the adenosine receptor antagonist, 7-methylxanthine (7-MX), retards myopia progression. Our aim was to determine whether 7-MX alters the compensating refractive changes produced by defocus in rhesus monkeys. Methods: Starting at age 3 weeks, monkeys were reared with -3 diopter (D; n = 10; 7-MX -3D/pl) or +3D (n = 6; 7-MX +3D/pl) spectacles over their treated eyes and zero-powered lenses over their fellow eyes. In addition, they were given 100 mg/kg of 7-MX orally twice daily throughout the lens-rearing period (age 147 ± 4 days). Comparison data were obtained from lens-reared controls (-3D/pl, n = 17; +3D/pl, n = 9) and normal monkeys (n = 37) maintained on a standard diet. Refractive status, corneal power, and axial dimensions were assessed biweekly. Results: The -3D/pl and +3D/pl lens-reared controls developed compensating myopic (-2.10 ± 1.07 D) and hyperopic anisometropias (+1.86 ± 0.54 D), respectively. While the 7-MX +3D/pl monkeys developed hyperopic anisometropias (+1.79 ± 1.11 D) that were similar to those observed in +3D/pl controls,…

Purpose: To characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula. Methods: RPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23-65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22-65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively. Results: RPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean ± SD: 6026 ± 1590 cells/mm2 at fovea; 4552 ± 1370 cells/mm2 and 3757 ± 1290 cells/mm2 at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.6 at the foveal center…

Meibomian gland dysfunction (MGD) is one of the primary causes of evaporative dry eye. Stagnation of meibum induces an unstable tear film, thus resulting in shortened tear film breakup time and superficial punctate keratopathy (SPK) in the lower cornea and punctate staining of lower bulbar conjunctiva. MGD is sometimes accompanied with inflammation (termed "meibomitis") via the proliferation of bacteria in the meibomian gland and eyelash area. Meibomitis is strongly related to ocular surface inflammation such as corneal cellular infiltrates and neovascularization, SPK, and conjunctivitis. It is difficult to differentiate SPK caused by dry eye from that caused by meibomitis. When clinicians are unaware of the existence of meibomitis, and only aware of SPK on the cornea, they often try to treat SPK as it is caused by dry eye using dry eye-specific eyedrops or even using punctual plugs when conservative therapy is ineffective. However, even when intensive dry eye therapy is applied, it may be unsuccessful until SPK caused by meibomitis is recognized and treated with systemic…

Purpose: To image, describe, and characterize different features visible in the crystalline lens of older adults with and without cataract when imaged three-dimensionally with a swept-source optical coherence tomography (SS-OCT) system. Methods: We used a new SS-OCT laboratory prototype designed to enhance the visualization of the crystalline lens and imaged the entire anterior segment of both eyes in two groups of participants: patients scheduled to undergo cataract surgery, n = 17, age range 36 to 91 years old, and volunteers without visual complains, n = 14, age range 20 to 81 years old. Pre-cataract surgery patients were also clinically graded according to the Lens Opacification Classification System III. The three-dimensional location and shape of the visible opacities were compared with the clinical grading. Results: Hypo- and hyperreflective features were visible in the lens of all pre-cataract surgery patients and in some of the older adults in the volunteer group. When the clinical examination revealed cortical or subcapsular cataracts, hyperreflective features were visible…

Purpose: To map the corneal epithelium using a map measuring 9 mm in diameter and view the effects of age, sex, and axial length. Additionally, we wanted to demonstrate the reproducibility of this technique. Methods: We calculated the epithelial thickness in 220 individuals using an SD-OCT machine with the newly released commercially available algorithm. We included normal eyes with refractive errors between +5 and -6 diopters (D). We excluded patients with an intraocular pressure of >22 mm Hg, history of cataract, previous ocular surgery, or disease and those with corneal pathology. Additionally, we excluded patients with evidence of systemic disease or pregnancy. Lastly, reproducibility was measured in 50 individuals. Results: We found the center of the corneal epithelium to be thicker than the peripheral in all zones except the nasal (P = 0.124). The superior quadrant was found to be the thinnest while the inferior was the thickest. Males had a thicker epithelium in all locations except the superior outer section (P = 0.123). Three zones had a weak correlation with age: outer…

Purpose: We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. Methods: This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Results: Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba…

Purpose: To model the sensitivity of the optic nerve head (ONH) biomechanical environment to acute variations in IOP, cerebrospinal fluid pressure (CSFP), and central retinal artery blood pressure (BP). Methods: We extended a previously published numerical model of the ONH to include 24 factors representing tissue anatomy and mechanical properties, all three pressures, and constraints on the optic nerve (CON). A total of 8340 models were studied to predict factor influences on 98 responses in a two-step process: a fractional factorial screening analysis to identify the 16 most influential factors, followed by a response surface methodology to predict factor effects in detail. Results: The six most influential factors were, in order: IOP, CON, moduli of the sclera, lamina cribrosa (LC) and dura, and CSFP. IOP and CSFP affected different aspects of ONH biomechanics. The strongest influence of CSFP, more than twice that of IOP, was on the rotation of the peripapillary sclera. CSFP had similar influence on LC stretch and compression to moduli of sclera and LC. On some ONHs, CSFP caused…

Purpose: The purpose of this study was to study the effect of minocycline and several neurotrophic factors, alone or in combination, on photoreceptor survival and macro/microglial reactivity in two rat models of retinal degeneration. Methods: P23H-1 (rhodopsin mutation), Royal College of Surgeon (RCS, pigment epithelium malfunction), and age-matched control rats (Sprague-Dawley and Pievald Viro Glaxo, respectively) were divided into three groups that received at P10 for P23H-1 rats or P33 for RCS rats: (1) one intravitreal injection (IVI) of one of the following neurotrophic factors: ciliary neurotrophic factor (CNTF), pigment epithelium-derived factor (PEDF), or basic fibroblast growth factor (FGF2); (2) daily intraperitoneal administration of minocycline; or (3) a combination of IVI of FGF2 and intraperitoneal minocycline. All animals were processed 12 days after treatment initiation. Retinal microglial cells and cone photoreceptors were immunodetected and analyzed qualitatively in cross sections. The numbers of microglial cells in the different retinal layers and number of nuclei…

Purpose: To investigate the macular quantitative parameters interchangeability of three different optical coherence tomography angiography (OCT-A) angiocubes (i.e., 3 × 3, 6 × 6, and 12 × 12 mm) on healthy subjects and patients affected by diabetic retinopathy (DR) and to assess the interrater reliability of such indices across the different scan protocols. Methods: Retrospective study involving 20 eyes of healthy subjects and 20 eyes with DR. All eyes underwent swept-source OCT-A with 3 × 3-, 6 × 6-, and 12 × 12-mm angiocubes centered on the fovea. Foveal avascular zone (FAZ) area and vessel density on 3 × 3-, 6 × 6-, and 12 × 12-mm macular scans were calculated by three independent operators at all retina, superficial, deep, and choriocapillary vascular layers. Interchangeability and interrater reliabilities were estimated using intraclass correlation coefficient (ICC). Results: Interscan reproducibility of FAZ area was very strong (ICC > 0.85) at every plexus. On the contrary, vessel density values significantly varied across different scan sizes (ICC < 0.51). Intrascan…

Purpose: To evaluate foveal function in patients with inherited retinal degenerations (IRD) by measuring visual acuity (VA) after correction of higher-order aberrations. Methods: Adaptive optics scanning laser ophthalmoscopy (AOSLO) was used to image cones in 4 healthy subjects and 15 patients with IRD. The 840-nm scanning laser delivered an "E" optotype to measure AOSLO-mediated VA (AOSLO-VA). Cone spacing was measured at the preferred retinal locus by two independent graders and the percentage of cones below the average density of 47 age-similar healthy subjects was computed. Cone spacing was correlated with best-corrected VA measured with the Early Treatment of Diabetic Retinopathy Study protocol (ETDRS-VA), AOSLO-VA, and foveal sensitivity. Results: ETDRS-VA significantly correlated with AOSLO-VA (ρ = 0.79, 95% confidence interval [CI] 0.5-0.9). Cone spacing correlated with AOSLO-VA (ρ = 0.54, 95% CI 0.02-0.7), and negatively correlated with ETDRS letters read (ρ = -0.64, 95% CI -0.8 to -0.2). AOSLO-VA remained ≥20/20 until cones decreased to 40.2% (CI 31.1-45.5) below normal.…

Purpose: Stargardt disease (STGD1), the most common early-onset recessive macular degeneration, is caused by mutations in the gene encoding the ATP-binding cassette transporter ABCA4. Although extensive genetic studies have identified more than 1000 mutations that cause STGD1 and related ABCA4-associated diseases, few studies have investigated the extent to which mutations affect the biochemical properties of ABCA4. The purpose of this study was to correlate the expression and functional activities of missense mutations in ABCA4 identified in a cohort of Canadian patients with their clinical phenotype. Methods: Eleven patients from British Columbia were diagnosed with STGD1. The exons and exon-intron boundaries were sequenced to identify potential pathologic mutations in ABCA4. Missense mutations were expressed in HEK293T cells and their level of expression, retinoid substrate binding properties, and ATPase activities were measured and correlated with the phenotype of the STGD1 patients. Results: Of the 11 STGD1 patients analyzed, 7 patients had two mutations in ABCA4, 3 patients…

Purpose: To test the hypothesis that genes known to cause clinical syndromes featuring myopia also harbor polymorphisms contributing to nonsyndromic refractive errors. Methods: Clinical phenotypes and syndromes that have refractive errors as a recognized feature were identified using the Online Mendelian Inheritance in Man (OMIM) database. One hundred fifty-four unique causative genes were identified, of which 119 were specifically linked with myopia and 114 represented syndromic myopia (i.e., myopia and at least one other clinical feature). Myopia was the only refractive error listed for 98 genes and hyperopia and the only refractive error noted for 28 genes, with the remaining 28 genes linked to phenotypes with multiple forms of refractive error. Pathway analysis was carried out to find biological processes overrepresented within these sets of genes. Genetic variants located within 50 kb of the 119 myopia-related genes were evaluated for involvement in refractive error by analysis of summary statistics from genome-wide association studies (GWAS) conducted by the CREAM Consortium…

Meibography allows observation of meibomian glands in an objective and repeatable manner. Original meibography systems were invasive and not readily adopted by ophthalmology clinics. The development of noncontact infrared meibography allowed the rapid and noninvasive observation of meibomian glands, and such systems have now been widely adopted for standard examinations in dry eye clinics. Noncontact meibography has also spurred research into meibomian glands and has been applied to evaluation of their structure and status in various ocular surface diseases. Although the images obtained by meibography are objective and repeatable, the interpretation of these images is subjective, with the relationship between image features and actual gland structure and composition remaining unclear. Additional clinical and basic research with regard to the interpretation of meibography images is thus necessary. Future improvements to meibography will likely provide new insights into the pathophysiology of meibomian gland diseases as well as enhance its contribution to the diagnosis and evaluation…

Purpose: To investigate variations in chorioretinal vasculatures in fellow eyes of patients with unilateral neovascular age-related macular degeneration (nAMD). Methods: We included fellow eyes of consecutive patients with unilateral nAMD from swept source optical coherence tomography (SS-OCT) angiography database. Vascular and nonvascular indices were determined based on SS-OCT and SS-OCT angiography images. Variation of the vascular or nonvascular index was compared between fellow eyes with and without early AMD. Results: In 146 fellow eyes, 88 (60.3%) had early AMD and 58 (39.7%) had a normal fundus. Vascular density (VD) values of the superficial and deep retinal capillary plexus and choriocapillaris were smaller in eyes with early AMD than in those without (P < 0.001, P = 0.001, P = 0.006, respectively). Flow void area in choriocapillaris was greater in eyes with early AMD than those without (P = 0.015). In 88 fellow eyes with early AMD, vascular indices of the retina were correlated with those of choroid while nonvascular indices were not. Fellow eyes of patients with classic…

Purpose: We used acoustic radiation force optical coherence elastography (ARF-OCE) to map out the elasticity of retinal layers in healthy and diseased in vivo rabbit models for the first time. Methods: A healthy rabbit eye was proptosed and imaged using ARF-OCE, by measuring the tissue deformation after an acoustic force is applied. A diseased retinal inflammation model was used to observe the contrast before and after disease formation. Retinal histologic analysis was performed to identify layers of the retina corresponding with the optical images. Results: The general trend of the retinal layer elasticity is increasing stiffness from the ganglion side to the photoreceptor side, with the stiffest layer being the sclera. In a healthy rabbit model, the mechanical properties varied from 3 to 16 kPa for the five layers that were identified via optical imaging and histology (3.09 ± 0.46, 3.82 ± 0.88, 4.53 ± 0.74, 6.59 ± 2.27, 16.11 ± 5.13 kPa). In the diseased model, we have induced optical damage in a live rabbit and observed a change in the stiffness trend in its retina. Conclusions:…

Purpose: To determine microvasculature changes in the deep optic nerve head (ONH) and peripapillary tissues after trabeculectomy, and to correlate these with changes in the lamina cribrosa (LC) curvature. Methods: Fifty-six eyes with primary open-angle glaucoma that underwent trabeculectomy were included. The optic nerve and peripapillary microvasculature were evaluated in en face images obtained using optical coherence tomography (OCT) angiography (OCTA) before and 3 months after trabeculectomy. The OCTA-derived vessel density (VD) was calculated in each layer segmented into the prelaminar tissue (PLT), LC, peripapillary retina (PR), and peripapillary choroid (PPC). Swept-source OCT volume scanning of ONH was performed on the same day as OCTA to examine the change in LC curvature quantified as the LC curve index (LCCI). Results: At 3 months postoperative, the IOP and LCCI had significantly decreased (both P < 0.001). OCTA images revealed a significant increase in VD in the LC (P = 0.006), but not in the PLT, PR, or PPC. Twenty-six eyes showed both significant LCCI decrease and VD…

Despite strong genetic associations, compelling human histological data and numerous hypotheses generated with supportive animal data, the mechanisms of inflammation or inflammatory control of cell health during progression of age-related macular degeneration arguably remain elusive. This perspective delivers a view that maintaining tissue health requires active immune cellular and tissue pathways, but when responses are perturbed or exaggerated, chronic inflammation is destructive. There are potential pathways and processes to enable understanding and determine how potential causative factors including altered cellular metabolism, senescence, oxidative stress disrupt tissue homeostasis are engaged. Establishing differences in the immune phenotype between normal aging and AMD, and how the inter-relatedness of these triggers contribute to pathobiology is integral for future therapeutic success.

Purpose: Knowledge of whether microorganisms reside in protected niches of the conjunctiva is potentially significant in terms of minimizing risks of contact lens inflammation/infection and endophthalmitis. We define if and how microbial communities from limbal and forniceal conjunctival tissue differ from those on the conjunctival surface. Methods: Human limbal and forniceal conjunctival tissue was obtained from 23 patients undergoing pterygium surgery and analyzed with data from a recent study of conjunctival surface swabs (n = 45). Microbial communities were analyzed by extracting total DNA from tissue samples and surface swabs and sequencing the 16S rRNA gene using the Illumina MiSeq platform. Sequences were quality filtered, clustered into operational taxonomic units (OTUs) at 97% similarity. OTUs associated with blank extraction and sampling negative controls were removed before analysis. Fluorescent in situ hybridization (FISH) was performed on cyrosections of limbal and forniceal conjunctival tissue. Results: There was a significant difference in bacterial community…

Purpose: We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. Methods: Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. Results: MiR-124 expression was correlated with progressive retinal damage, inflammation,…

Purpose: Biomechanical properties changes and α-smooth muscle actin (α-SMA) overexpression are involved in myopia scleral remodeling. However, interactions between altered tissue biomechanics and cellular signaling that sustain scleral remodeling have not been well defined. We determine the mechanisms of mechanotransduction in the regulation of α-SMA expression during myopia scleral remodeling. Methods: Guinea pigs were used to establish a form-deprivation myopia (FDM) model. Protein profiles in myopic sclera were examined using tandem mass spectrometry. Ras homolog gene family member A (RhoA) and α-SMA expressions were confirmed using quantitative (q) RT-PCR and Western blotting. Scleral fibroblasts were cultured and subjected to 4% cyclic strain. Levels of RhoA, rho-associated protein kinase-2 (ROCK2), myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), and α-SMA were determined by qRT-PCR and Western blotting in groups with or without the RhoA siRNA or ROCK inhibitor Y27632. MRTF-A and α-SMA were evaluated by confocal immunofluorescent microscopy and…

Purpose: To describe different patterns of macular pigment (MP) seen in fluorescence lifetime imaging ophthalmoscopy (FLIO) and to analyze ex vivo fluorescence characteristics of carotenoids. Methods: A total of 31 eyes of young healthy subjects, 4 eyes from patients with albinism, 36 eyes with macular telangiectasia type 2 (MacTel), 24 eyes with retinitis pigmentosa, and 1 eye with a macular hole were included in this clinic-based, cross-sectional study. All subjects underwent Heidelberg Engineering FLIO and MP measurements (dual-wavelength autofluorescence). Fundus autofluorescence (FAF) lifetimes of a 30° retinal field were detected in two spectral channels (SSC: 498-560 nm; LSC: 560-720 nm), and amplitude-weighted mean fluorescence lifetimes (τm) were calculated. Additionally, autofluorescence lifetimes of known dilutions of lutein and zeaxanthin were measured in a cuvette in free- and protein-associated states. Results: MP shows a significant inverse correlation to foveal FAF lifetimes measured with FLIO (SSC: r = -0.608; P < 0.001). Different distribution patterns can be…

Purpose: The purpose of this study was to measure the rupture pressure and the biomechanical properties of porcine Bruch's membrane (BM)-choroid complex (BMCC) and the influences of BM on optic nerve head (ONH) tissues. Methods: The biomechanical properties of BMCC were extracted through uniaxial tensile tests of 10 BMCC specimens from 10 porcine eyes; the rupture pressures of BMCC were measured through burst tests of 20 porcine eyes; and the influence of BM on IOP-induced ONH deformations were investigated using finite element (FE) analysis. Results: Uniaxial experimental results showed that the average elastic (tangent) moduli of BMCC samples at 0% and 5% strain were 1.60 ± 0.81 and 2.44 ± 1.02 MPa, respectively. Burst tests showed that, on average, BMCC could sustain an IOP of 82 mm Hg before rupture. FE simulation results predicted that, under elevated IOP, prelamina tissue strains increased with increasing BM stiffness. On the contrary, lamina cribrosa strains showed an opposite trend but the effects were small. Conclusions: BMCC stiffness is comparable or higher than those of…

Purpose: The purpose of this study was to document the distribution of the severity of myopic maculopathy in a cohort of highly myopic patients and to explore the associated risk factors. Methods: A total of 890 Chinese highly myopes aged between 7 and 70 years (median age 19 years) and with spherical refraction -6.00 diopter (D) or worse in both eyes were investigated. All participants underwent detailed ophthalmic examination. Myopic maculopathy was graded into 5 categories according to the International Photographic Classification and Grading System using color fundus photographs: category 0, no myopic retinal lesions, category 1, tessellated fundus only; category 2, diffuse chorioretinal atrophy; category 3, patchy chorioretinal atrophy; category 4, macular atrophy. Category 2 or greater were further classified as clinically significant myopic maculopathy (CSMM). Results: Data from 884 of 890 right eyes were available for analysis. The proportions of category 1, category 2, category 3, and category 4 were 20.0% (177 eyes), 20.2% (178 eyes), 2.6% (23 eyes), and 0.2% (2 eyes),…