Showing papers in "Investigative Ophthalmology & Visual Science in 2016"

Improved Automated Detection of Diabetic Retinopathy on a Publicly Available Dataset Through Integration of Deep Learning.

Abstract: Purpose To compare performance of a deep-learning enhanced algorithm for automated detection of diabetic retinopathy (DR), to the previously published performance of that algorithm, the Iowa Detection Program (IDP)-without deep learning components-on the same publicly available set of fundus images and previously reported consensus reference standard set, by three US Board certified retinal specialists. Methods We used the previously reported consensus reference standard of referable DR (rDR), defined as International Clinical Classification of Diabetic Retinopathy moderate, severe nonproliferative (NPDR), proliferative DR, and/or macular edema (ME). Neither Messidor-2 images, nor the three retinal specialists setting the Messidor-2 reference standard were used for training IDx-DR version X2.1. Sensitivity, specificity, negative predictive value, area under the curve (AUC), and their confidence intervals (CIs) were calculated. Results Sensitivity was 96.8% (95% CI: 93.3%-98.8%), specificity was 87.0% (95% CI: 84.2%-89.4%), with 6/874 false negatives, resulting in a negative predictive value of 99.0% (95% CI: 97.8%-99.6%). No cases of severe NPDR, PDR, or ME were missed. The AUC was 0.980 (95% CI: 0.968-0.992). Sensitivity was not statistically different from published IDP sensitivity, which had a CI of 94.4% to 99.3%, but specificity was significantly better than the published IDP specificity CI of 55.7% to 63.0%. Conclusions A deep-learning enhanced algorithm for the automated detection of DR, achieves significantly better performance than a previously reported, otherwise essentially identical, algorithm that does not employ deep learning. Deep learning enhanced algorithms have the potential to improve the efficiency of DR screening, and thereby to prevent visual loss and blindness from this devastating disease.

Optical coherence tomography angiography

Abstract: Optical coherence tomography angiography (OCTA) is a noninvasive approach that can visualize blood vessels down to the capillary level. With the advent of high-speed OCT and efficient algorithms, practical OCTA of ocular circulation is now available to ophthalmologists. Clinical investigations that used OCTA have increased exponentially in the past few years. This review will cover the history of OCTA and survey its most important clinical applications. The salient problems in the interpretation and analysis of OCTA are described, and recent advances are highlighted.

Optical Coherence Tomography Angiography Vessel Density in Healthy, Glaucoma Suspect, and Glaucoma Eyes.

Abstract: Author(s): Yarmohammadi, Adeleh; Zangwill, Linda M; Diniz-Filho, Alberto; Suh, Min Hee; Manalastas, Patricia Isabel; Fatehee, Naeem; Yousefi, Siamak; Belghith, Akram; Saunders, Luke J; Medeiros, Felipe A; Huang, David; Weinreb, Robert N | Abstract: PurposeThe purpose of this study was to compare retinal nerve fiber layer (RNFL) thickness and optical coherence tomography angiography (OCT-A) retinal vasculature measurements in healthy, glaucoma suspect, and glaucoma patients.MethodsTwo hundred sixty-one eyes of 164 healthy, glaucoma suspect, and open-angle glaucoma (OAG) participants from the Diagnostic Innovations in Glaucoma Study with good quality OCT-A images were included. Retinal vasculature information was summarized as a vessel density map and as vessel density (%), which is the proportion of flowing vessel area over the total area evaluated. Two vessel density measurements extracted from the RNFL were analyzed: (1) circumpapillary vessel density (cpVD) measured in a 750-μm-wide elliptical annulus around the disc and (2) whole image vessel density (wiVD) measured over the entire image. Areas under the receiver operating characteristic curves (AUROC) were used to evaluate diagnostic accuracy.ResultsAge-adjusted mean vessel density was significantly lower in OAG eyes compared with glaucoma suspects and healthy eyes. (cpVD: 55.1 ± 7%, 60.3 ± 5%, and 64.2 ± 3%, respectively; P l 0.001; and wiVD: 46.2 ± 6%, 51.3 ± 5%, and 56.6 ± 3%, respectively; P l 0.001). For differentiating between glaucoma and healthy eyes, the age-adjusted AUROC was highest for wiVD (0.94), followed by RNFL thickness (0.92) and cpVD (0.83). The AUROCs for differentiating between healthy and glaucoma suspect eyes were highest for wiVD (0.70), followed by cpVD (0.65) and RNFL thickness (0.65).ConclusionsOptical coherence tomography angiography vessel density had similar diagnostic accuracy to RNFL thickness measurements for differentiating between healthy and glaucoma eyes. These results suggest that OCT-A measurements reflect damage to tissues relevant to the pathophysiology of OAG.

Quantifying Microvascular Density and Morphology in Diabetic Retinopathy Using Spectral-Domain Optical Coherence Tomography Angiography

Abstract: PURPOSE To quantify changes in retinal microvasculature in diabetic retinopathy (DR) by using spectral-domain optical coherence tomography angiography (SD-OCTA). METHODS Retrospective, cross-sectional, observational study of healthy and diabetic adult subjects with and without DR. Retinal microvascular changes were assessed by using SD-OCTA images and an intensity-based optical microangiography algorithm. A semiautomated program was used to calculate indices of microvascular density and morphology in nonsegmented and segmented SD-OCTA images. Microvascular density was quantified by using skeleton density (SD) and vessel density (VD), while vessel morphology was quantified as fractal dimension (FD) and vessel diameter index (VDI). Statistical analyses were performed by using the Student's t-test or analysis of variance with post hoc Tukey honest significant difference tests for multiple comparisons. RESULTS Eighty-four eyes with DR and 14 healthy eyes were studied. Spearman's rank test demonstrated a negative correlation between DR severity and SD, VD, and FD, and a positive correlation with VDI (ρ = -0.767, -0.7166, -0.768, and +0.5051, respectively; P < 0.0001). All parameters showed high reproducibility between graders (ICC = 0.971, 0.962, 0.937, and 0.994 for SD, VD, FD, and VDI, respectively). Repeatability (κ) was greater than 0.99 for SD, VD, FD, and VDI. CONCLUSIONS Vascular changes in DR can be objectively and reliably characterized with SD, VD, FD, and VDI. In general, decreasing capillary density (SD and VD), branching complexity (FD), and increasing average vascular caliber (VDI) were associated with worsening DR. Changes in capillary density and morphology were significantly correlated with diabetic macular edema.

The Development, Commercialization, and Impact of Optical Coherence Tomography.

Abstract: This review was written for the special issue of IOVS to describe the history of optical coherence tomography (OCT) and its evolution from a nonscientific, historic perspective. Optical coherence tomography has become a standard of care in ophthalmology, providing real-time information on structure and function - diagnosing disease, evaluating progression, and assessing response to therapy, as well as helping to understand disease pathogenesis and create new therapies. Optical coherence tomography also has applications in multiple clinical specialties, fundamental research, and manufacturing. We review the early history of OCT describing how research and development evolves and the important role of multidisciplinary collaboration and expertise. Optical coherence tomography had its origin in femtosecond optics, but used optical communications technologies and required advanced engineering for early OCT prototypes, clinical feasibility studies, entrepreneurship, and corporate development in order to achieve clinical acceptance and clinical impact. Critical advances were made by early career researchers, clinician scientists, engineering experts, and business leaders, which enabled OCT to have a worldwide impact on health care. We introduce the concept of an "ecosystem" consisting of research, government funding, collaboration and competition, clinical studies, innovation, entrepreneurship and industry, and impact - all of which must work synergistically. The process that we recount is long and challenging, but it is our hope that it might inspire early career professionals in science, engineering, and medicine, and that the clinical and research community will find this review of interest.

Normative Data for Vascular Density in Superficial and Deep Capillary Plexuses of Healthy Adults Assessed by Optical Coherence Tomography Angiography.

Abstract: PURPOSE To establish a normative database for vascular density (VD) and foveal avascular zone (FAZ) at the superficial (SCP) and deep capillary plexus (DCP) in healthy subjects with optical coherence tomography (OCT) angiography. METHODS The study was a retrospective chart review of healthy patients who had undergone OCT angiography imaging. A 3- × 3-mm area, centered on the fovea, was scanned for all the study eyes. The automated segmentation allowed separate analysis of the SCP, the DCP, and a comprehensive C-scan including both vascular layers. On the obtained images, VD and FAZ measurements were computed. Interobserver reproducibility and intraobserver repeatability were also assessed. RESULTS A total of 135 eyes of 70 subjects (51% male) were analyzed. The mean age was 48.3 ± 17.5. We divided patients into group 1, from 20 to 39 years of age; group 2, from 40 to 59 years; and group 3, age 60 years or older. At the level of the SCP, mean VD and mean FAZ ± SD were, respectively, 52.58 ± 3.22% and 0.28 ± 0.1 mm2. At the level of the DCP, mean VD and mean FAZ were 57.87 ± 2.82% and 0.37 ± 0.12 mm2, respectively. The mean VD was significantly higher (P < 0.05) in DCP compared with SCP in all Early Treatment Diabetic Retinopathy Study (ETDRS) sectors and in all age groups. Vascular density was higher in women than in men after 60 years (P < 0.01). After adjustment on the signal strength index (SSI), the mean VD remained directly correlated with the age range and sex. The mean FAZ area was lower in group 3 (P < 0.05). Interobserver reproducibility was 0.78 to 0.99 in SCP and 0.67 to 0.92 in DCP, and intraobserver repeatability was 0.64 to 0.93 in SCP and 0.63 to 0.87 in DCP. CONCLUSIONS Our study has provided, for the first time, age-related VD mapping data using OCT angiography in healthy subjects. The prototype software used in this study may help to improve the concept of VD grading with high inter- and intraexaminer repeatability and interexaminer reproducibility.

Retinal Capillary Density and Foveal Avascular Zone Area Are Age-Dependent: Quantitative Analysis Using Optical Coherence Tomography Angiography.

Abstract: Purpose The purpose of this study was to quantify retinal capillary density and the foveal avascular zone (FAZ) area in normal subjects according to age, using optical coherence tomography angiography (OCTA). Methods All eyes in this cross-sectional study underwent OCTA using RTVue XR Avanti with AngioVue. OCTA scans were analyzed and processed, and vessel density and FAZ dimensions were calculated. Results A total of 113 normal eyes from 70 subjects were included (30 males, 40 females; mean 48 ± 20 years of age). The mean vessel density and FAZ dimensions were significantly smaller in the superficial retinal capillary plexus (SCP) than in the deep retinal capillary plexus (DCP), using quantitative OCTA analysis (all P< 0.0001). With 3 × 3-mm scans, the mean vessel density was 13.431 ± 1.758 mm-1 in the SCP, 18.812 ± 1.796 mm-1 in the DCP, and 5.913 ± 1.308 mm-1 and 10.447 ± 1.262 mm-1 with 6 × 6-mm scans in the SCP and DCP, respectively. Mean FAZ areas were 0.289 ± 0.108 mm2 at the SCP and 0.614 ± 0.200 mm2 at the DCP. Age was a predictor of SCP and DCP vessel density and FAZ area in the SCP. Vessel density decreased 0.0393 mm-1 (0.26%) per year in the SCP and 0.0574 mm-1 (0.27%) per year in the DCP. FAZ areas increased 0.0014 mm2 (0.63%) and 0.0011 mm2 (0.20%) per year in the SCP and DCP, respectively. Conclusions SCP and DCP vessel density decreased with increasing age, while FAZ area increased with age. Normal age-matched measurements provide important standardized values that may facilitate management of retinal vascular disorders.

Projection-Resolved Optical Coherence Tomographic Angiography

Abstract: Shadowgraphic projection artifacts from superficial vasculature interfere with the visualization of deeper vascular networks in optical coherence tomography angiography (OCT-A). We developed a novel algorithm to remove this artifact by resolving the ambiguity between in situ and projected flow signals. The algorithm identifies voxels with in situ flow as those where intensity-normalized decorrelation values are higher than all shallower voxels in the same axial scan line. This \"projection-resolved\" (PR) algorithm effectively suppressed the projection artifact on both en face and cross-sectional angiograms and enhanced depth resolution of vascular networks. In the human macula, the enhanced angiograms show three distinct vascular plexuses in the inner retina and no vessels in the outer retina. We demonstrate that PR OCT-A cleanly removes flow projection from the normally avascular outer retinal slab while preserving the density and continuity of the intermediate and deep retinal capillary plexuses.

Subretinal Transplantation of Embryonic Stem Cell-Derived Retinal Pigment Epithelium for the Treatment of Macular Degeneration: An Assessment at 4 Years.

Abstract: Advanced macular degeneration is an important cause of vision loss in the United States with over 2 million people affected by the disease. Despite substantial progress in the development of new therapies for wet AMD, the severe visual impairment associated with geographic atrophy in dry AMD or Stargardt disease remains untreatable. Recently, two phase I/II studies involving 18 patients with these diseases have demonstrated that it is possible to safely implant human embryonic stem cell-derived RPE (hESC-RPE) in an attempt to rescue photoreceptors and visual function. The anatomical and functional results are encouraging, with more than half of treated patients experiencing sustained improvements in visual acuity and demonstrating evidence of possible cellular engraftment. However, any conclusions remain tempered by the relatively short follow-up time, lack of a formal control group, poor initial visual acuity, and small number of patients. Aside from an instance of postoperative infectious endophthalmitis, no adverse events related to the cell therapy, such as hyperproliferation, tumorigenicity, or rejection-related inflammation were noted in this initial cohort of 18 patients. These first-in-human safety studies have opened the door to future studies enrolling patients with less advanced disease, treating other diseases that result in RPE loss, employing shorter immunosuppressive regimens, and using alternative strategies for RPE transplantation such as sheets of cells with or without scaffolding to mimic Bruch's membrane. The ultimate goal of these initial safety studies is to promote continued translation of complex biological therapies into meaningful treatment strategies that may address unmet medical needs.

Swept-Source OCT Angiography Imaging of the Foveal Avascular Zone and Macular Capillary Network Density in Diabetic Retinopathy.

Abstract: PURPOSE We compared the area of the foveal avascular zone (FAZ) and macular capillary network density at different retinal layers using swept-source optical coherence tomography angiography (OCT-A) in normal individuals and patients with diabetic retinopathy (DR). METHODS Images (a 3 × 3 mm cube centered on the fovea) were acquired in 40 eyes of 22 normal individuals and 28 eyes of 18 patients with varying levels of DR using a swept-source OCT-A device (central wavelength 1050 nm; A-scan-rate of 100,000 scans per second). En face images of the retinal vasculature were generated from the superficial and deep retinal layers (SRL/DRL). Quantitative analysis of the vessel density (VD) and FAZ area was performed. Vessel density was assessed as the ratio of the retinal area occupied by vessels. RESULTS Among the DR subjects (mean age, 72 years; 61% male), 35.7% of the eyes had mild, 35.7% moderate, and 7.1% severe nonproliferative DR (NPDR), and 21.4% and proliferative DR (PDR). The mean FAZ area in patients with DR and in normal individuals was 0.518 and 0.339 mm2, respectively, for the SRL (P = 0.003), and 0.615 and 0.358 mm2, respectively, for the DRL (P < 0.001). The mean VD (ratio) at the SRL and DRL was statistically significantly lower in patients with DR (SRL, P < 0.001; DRL, P = 0.028). CONCLUSIONS Swept-source OCT-A of the microcirculation in eyes of patients with DR can be used to quantitatively demonstrate alterations in the FAZ and VD in the SRL/DRL of the macula compared to normal eyes. Future longitudinal studies may use these metrics to evaluate changes over time or in response to treatment.

Paucibacterial Microbiome and Resident DNA Virome of the Healthy Conjunctiva

Abstract: Purpose To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques. Methods Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses. Results Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65% of all conjunctiva samples tested. Conclusions Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.).

Quantification of Vessel Density in Retinal Optical Coherence Tomography Angiography Images Using Local Fractal Dimension.

Abstract: PURPOSE To evaluate a fully automated local fractal dimension method to quantify vessel density and foveal avascular zone (FAZ) area in optical coherence tomography angiography (OCTA) images. METHODS Fifty-two healthy Asian Indian eyes underwent imaging prospectively with OCTA system. Superficial and deep retinal vascular plexus was imaged. Local fractal analysis was applied to the OCTA images. A scan area of 3 × 3 mm was selected in the superficial and deep retinal layers. Foveal avascular zone area and vessel density were quantified in circular and sectoral zones around the fovea. A unique contour map of vessel density and dropout zones was developed to perform regional comparisons. RESULTS Foveal avascular zone of superficial (0.35 ± 0.013 mm2) and deep (0.49 ± 0.012 mm2) retinal vascular plexus was segmented. The agreement between the manually segmented and local fractal dimension segmented FAZ area was 0.97 (95% confidence interval [CI]: 0.94-0.98) and did not change significantly with age (P = 0.94 and 0.21, respectively). The vessel density was greater in the deep than the superficial retinal vascular plexus (P < 0.0001). When the image was subdivided into sectors around the FAZ, inferior sector had greater vessel density than the others (temporal, superior, and nasal) in both superficial and deep retinal vascular plexus (P < 0.05). These observations were similar to recent studies on animal retinal vasculature map. CONCLUSIONS A novel implementation of local fractal dimension to calculate vessel density and FAZ area was demonstrated. Age did not impact vessel density but sectoral analyses showed greater vessel density in the inferior zone.

Linking Retinal Microvasculature Features With Severity of Diabetic Retinopathy Using Optical Coherence Tomography Angiography.

Abstract: Purpose To correlate retinal vascular features with severity and systemic indicators of diabetic retinopathy (DR) using optical coherence tomography angiography (OCTA). Methods A total of 209 eyes of 122 type 2 diabetes mellitus patients with DR and 60 eyes of 31 normal Indian subjects underwent OCTA imaging. The diabetic retinopathy patients were graded as having either nonproliferative diabetic retinopathy (NPDR: mild, moderate, and severe NPDR using Early Treatment Diabetic Retinopathy Study classification) or proliferative diabetic retinopathy (PDR). Local fractal analysis was applied to the superficial and deep retinal OCTA images. Foveal avascular zone area (FAZ in mm2); vessel density (%); spacing between large vessels (%); and spacing between small vessels (%) were analyzed. Sensitivity and specificity of vascular parameters were assessed with receiver operating characteristics (ROC) curve. Results Normal eyes had a significantly lower FAZ area, higher vessel density, and lower spacing between large and small vessels compared with DR grades (P 0.05). Conclusions Spacing between the large vessels in the superficial and deep retinal layers had superior diagnostic performance than overall vessel density.

Fractal Dimensional Analysis of Optical Coherence Tomography Angiography in Eyes With Diabetic Retinopathy.

Abstract: Purpose We used fractal dimensional analysis to analyze retinal vascular disease burden in eyes with diabetic retinopathy using spectral-domain optical coherence tomography angiography (OCTA). Methods A retrospective study was performed of 13 eyes with diabetic retinopathy without diabetic macular edema and 56 control eyes. Optical coherence tomography angiography images were acquired using the RTVue XR Avanti. Automated segmentation was obtained through the superficial and deep capillary plexuses for each eye. Grayscale OCTA images were standardized and binarized using ImageJ. Fractal box-counting analyses were performed using Fractalyse. Fractal dimensions (FD) as well as software-generated vascular density analyses of the superficial and deep capillary plexuses were compared between diabetic and control eyes using 2-tailed t-tests and 1-way multivariate ANOVA (MANOVA) analyses. Results The superficial and deep plexuses from diabetic and control eyes were analyzed. The average FD for diabetic eyes was significantly lower than control eyes for the superficial (P = 4.513 × 10-3) and deep (P = 2.653 × 10-3) capillary plexuses. In diabetic eyes, the vascular density also was significantly reduced in the superficial (P = 8.068 × 10-5) and deep (P = 3.120 × 10-6) capillary plexuses. One-way MANOVA showed a significant difference between diabetic and control eyes. Conclusions The OCTA FD is significantly reduced in the superficial and deep capillary plexuses in eyes with diabetic retinopathy. Applying fractal analysis to OCTA imaging holds the potential to establish quantitative parameters for microvascular pathology.

Gut Microbial Alterations Associated With Protection From Autoimmune Uveitis.

Abstract: PURPOSE To investigate the contribution of the gut microbiota to the pathogenesis of uveitis. METHODS Experimental autoimmune uveitis (EAU) in B10.RIII mice was induced using interphotoreceptor binding protein peptide. Mice were treated with oral or intraperitoneal (IP) antibiotics. Effector (Teff) and regulatory (Treg) T lymphocytes were identified using flow cytometry; 16S rRNA gene sequencing and qPCR were performed on gastrointestinal (GI) contents. RESULTS Broad-spectrum (four antibiotics given simultaneously) oral, but not IP, antibiotics reduced mean uveitis clinical scores significantly compared with water-treated animals (0.5 vs. 3.0, P 0.99 for IP). Both oral metronidazole (P = 0.02) and vancomycin (P < 0.0001) alone decreased inflammation, whereas neomycin (P = 0.7) and ampicillin (P = 0.4) did not change mean uveitis scores. Oral broad-spectrum antibiotics increased Tregs in the GI lamina propria of EAU animals at 1 week, and in extraintestinal lymphoid tissues later, whereas Teff and inflammatory cytokines were reduced. 16S sequencing of GI contents revealed altered microbiota in immunized mice compared with nonimmunized mice, and microbial diversity clustering in EAU mice treated with uveitis-protective antibiotics. Experimental autoimmune uveitis mice also demonstrated gut microbial diversity clustering associated with clinical score severity. CONCLUSIONS Oral antibiotics modulate the severity of inducible EAU by increasing Tregs in the gut and extraintestinal tissues, as well as decreasing effector T cells and cytokines. 16S sequencing suggests that there may be protective and, conversely, potentially uveitogenic, gut microbiota. These findings may lead to a better understanding of how uveitis can be treated or prevented by modulating the gut microbiome.

A Review of Adaptive Optics Optical Coherence Tomography: Technical Advances, Scientific Applications, and the Future.

Abstract: Special Issue A Review of Adaptive Optics Optical Coherence Tomography: Technical Advances, Scientific Applications, and the Future Ravi S. Jonnal, 1 Omer P. Kocaoglu, 2 Robert J. Zawadzki, 1 Zhuolin Liu, 2 Donald T. Miller, 2 and John S. Werner 1 1 Vision Science and Advanced Retinal Imaging Laboratory, University of California-Davis, Sacramento, California, United States School of Optometry, Indiana University, Bloomington, Indiana, United States Correspondence: Ravi S. Jonnal, 4860 Y Street, Suite 2400, Sacra- mento, CA 95817, USA; rsjonnal@ucdavis.edu. Submitted: January 8, 2016 Accepted: May 29, 2016 Citation: Jonnal RS, Kocaoglu OP, Zawadzki RJ, Liu Z, Miller DT, Werner JS. A review of adaptive optics optical coherence tomography: technical ad- vances, scientific applications, and the future. Invest Ophthalmol Vis Sci. 2016;57:OCT51–OCT68. DOI:10.1167/iovs.16-19103 P URPOSE . Optical coherence tomography (OCT) has enabled ‘‘virtual biopsy’’ of the living human retina, revolutionizing both basic retina research and clinical practice over the past 25 years. For most of those years, in parallel, adaptive optics (AO) has been used to improve the transverse resolution of ophthalmoscopes to foster in vivo study of the retina at the microscopic level. Here, we review work done over the last 15 years to combine the microscopic transverse resolution of AO with the microscopic axial resolution of OCT, building AO-OCT systems with the highest three-dimensional resolution of any existing retinal imaging modality. M ETHODS . We surveyed the literature to identify the most influential antecedent work, important milestones in the development of AO-OCT technology, its applications that have yielded new knowledge, research areas into which it may productively expand, and nascent applications that have the potential to grow. R ESULTS . Initial efforts focused on demonstrating three-dimensional resolution. Since then, many improvements have been made in resolution and speed, as well as other enhancements of acquisition and postprocessing techniques. Progress on these fronts has produced numerous discoveries about the anatomy, function, and optical properties of the retina. C ONCLUSIONS . Adaptive optics OCT continues to evolve technically and to contribute to our basic and clinical knowledge of the retina. Due to its capacity to reveal cellular and microscopic detail invisible to clinical OCT systems, it is an ideal companion to those instruments and has the demonstrable potential to produce images that can guide the interpretation of clinical findings. Keywords: adaptive optics, optical coherence tomography, retinal imaging T he 25 years since the advent of optical coherence tomography (OCT) 1 have brought countless improvements in its axial resolution, speed, and sensitivity. The main effort to improve OCT’s transverse (or lateral) resolution has been to combine it with adaptive optics (AO), a union that has been investigated in just a few labs. Nevertheless, AO-OCT now plays important roles in the production of knowledge about the retina and its diseases. Chief among these is its potential to resolve ambiguities present in clinical and research-grade conventional (non-AO) OCT systems. Because AO-OCT reveals the microstructure that makes up clinically observable features, it is indispensable as a scientific companion to conventional OCT. Understanding the differences between the OCT image and the AO-OCT image requires that we describe some of the technical details of AO-OCT imaging and image processing, and how these are motivated by the fundamental biological and physical properties of the living human retina. the retina’s laminar structure. Because retinal diseases often impact this laminar structure, OCT has been used extensively to detect and assess disease and monitor therapeutic efficacy, to such an extent that it has become a standard of ophthalmic care. Each retinal layer contains structural and functional subunits such as cells, organelles, and capillaries, most of which are too small to be resolved by conventional OCT systems such as the commercial systems employed in the clinic (Fig. 1). While cellular imaging is presently of limited interest to clinicians, it is of great interest to basic science researchers, who stand to learn much about these structures and their functions by imaging them in situ. Moreover, an understanding of the gross structural changes observed in clinical OCT images of disease and recovery depends crucially on knowing their microscopic constituents. Cellular imaging of the retina will continue to expand our understanding of retinal disease, and is therefore of great translational significance. The Spatial Scale of Retinal Features Optical Factors Affecting Transverse Resolution The human retina consists of approximately a dozen layers of mostly transparent tissue, together less than half a millimeter thick. The axial resolution afforded by OCT lends itself to imaging The point-spread function (PSF) is a measure of the quality of an imaging system, describing how light originating from a point in tissue manifests in the image of that tissue. The PSF has both iovs.arvojournals.org j ISSN: 1552-5783 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/Journals/IOVS/935468/ on 11/08/2016 OCT51

Optical Coherence Tomography Angiography Analysis of Perfused Peripapillary Capillaries in Primary Open-Angle Glaucoma and Normal-Tension Glaucoma.

Abstract: Purpose To compare perfused peripapillary capillary density in primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and normal patients using optical coherence tomography angiography (OCT-A). Methods A retrospective review of POAG, NTG, and normal patients imaged with OCT-A was performed. En face OCT angiograms identifying peripapillary vessels were obtained using a spectral-domain OCT system (Avanti RTVue-XR). A custom image analysis approach identified perfused peripapillary capillaries, quantified perfused capillary density (PCD), and generated color-coded PCD maps for 3.5- and 4.5-mm-diameter scans. We compared PCD values, PCD maps, standard automated perimetry (Humphrey visual field [HVF]) parameters, and OCT retinal nerve fiber layer (RNFL) thickness analyses across all groups. Results Forty POAG, 26 NTG, and 26 normal patients were included. Annular PCD in POAG (34.24 ± 6.76%) and NTG (37.75 ± 3.52%) patients was significantly decreased compared to normal patients (42.99 ± 1.81%) in 4.5-mm scans (P < 0.01 and P < 0.01, respectively). Similar trends and statistical significances were seen in 3.5-mm scans. Linear regression analysis resulted in moderate correlations between annular PCD values and other glaucomatous parameters. Pearson coefficients comparing annular PCD from 4.5-mm scans in POAG and NTG groups to HVF mean deviation, HVF pattern standard deviation, and average RNFL thickness all showed statistical significance (P < 0.05). Color maps showed that POAG and NTG patients had a reduction of perfused capillaries that progressed in size when comparing early, moderate, and severe glaucoma groups. Conclusions Optical coherence tomography angiography can uniquely identify changes in peripapillary PCD in glaucoma patients. Optical coherence tomography angiography may offer insights into the pathophysiology of glaucomatous damage and risk factors for disease progression.

Retinal Capillary Network and Foveal Avascular Zone in Eyes with Vein Occlusion and Fellow Eyes Analyzed With Optical Coherence Tomography Angiography.

Abstract: PURPOSE To evaluate the perifoveolar retinal capillary network at different depths and to quantify the foveal avascular zone (FAZ) in eyes with retinal vein occlusion (RVO) compared with their fellow eyes and healthy controls using spectral-domain optical coherence tomography angiography (SD-OCTA). METHODS We prospectively recruited 23 patients with RVO including 15 eyes with central RVO (CRVO) and 8 eyes with branch RVO (BRVO), their fellow eyes, and 8 age-matched healthy controls (8 eyes) for imaging on prototype OCTA software within RTVue-XR Avanti. The 3 × 3 mm and 6 × 6 mm en face angiograms of superficial and deep retinal capillary plexuses were segmented. Perifoveolar retinal capillary network was analyzed and FAZ was quantified. RESULTS Decrease in vascular perfusion at the deep plexus was observed in all eyes with CRVO (8/8, 100%) and BRVO (6/6, 100%) without cystoid macular edema, and in 8 of 15 (53%) and 2 of 8 (25%) of the fellow eyes, respectively. Vascular tortuosity was observed in 13 of 15 (87%) CRVO and 5 of 8 (63%) BRVO eyes. Collaterals were seen in 10 of 15 (67%) CRVO and 5 of 8 (63%) BRVO eyes. Mean FAZ area was larger in eyes with RVO than their fellow eyes (1.13 ± 0.25 mm2 versus 0.58 ± 0.28 mm2; P = 0.007) and controls (1.13 ± 0.25 mm2 versus 0.30 ± 0.09 mm2; P < 0.0001), and in fellow eyes of RVO patients when compared to controls (0.58 ± 0.28 mm2 versus 0.30 ± 0.09 mm2; P = 0.01). CONCLUSIONS Spectral-domain OCTA reveals abnormalities at different levels of perifoveolar retinal capillary network and is able to quantify the FAZ in RVO. Longitudinal studies may be considered to evaluate the clinical utility of OCTA in RVO and other retinal vascular diseases.

Optical Coherence Tomography Angiography Evaluation of the Parafoveal Vasculature and Its Relationship With Ocular Factors.

Abstract: PURPOSE To determine the size and characteristics of the superficial and deep foveal avascular zone (FAZ) in healthy adults by using optical coherence tomography angiography (OCT-A), and to ascertain the effects of demographic and ocular parameters on the FAZ size. METHODS In a prospective cohort study of 117 healthy volunteers, foveal-centered 3 × 3-mm OCT-A scans were manually graded by certified graders to determine the size of the superficial and deep FAZ. Multiple linear regression analyses were performed to evaluate the impact of demographics and ocular factors, including central retinal thickness (CRT), choroidal thickness, axial length (AL), and spherical equivalent (SE) on superficial and deep FAZ areas. RESULTS The mean age of the participants was 22.5 years, with mean AL of 25.4 mm and mean SE of -4.3 diopters. The mean CRT was 262.8 μm (range, 220-316 μm). The mean superficial FAZ area was 0.24 mm2, while the deep FAZ area was 0.38 mm2 (P < 0.001). Females had a larger superficial (P < 0.001) and deep FAZ (P < 0.001). On univariate linear regression, both superficial and deep FAZ areas had significant correlations with CRT, sex, AL, and SE, but not with age. By multiple linear regression analysis, in normal eyes, superficial FAZ area varied significantly with CRT and sex. Among eyes with high myopia, both superficial and deep FAZ varied significantly with CRT, sex, and choroidal thickness. CONCLUSIONS The superficial and deep FAZ areas varied significantly among healthy eyes. Factors such as CRT, sex, SE, AL, and choroidal thickness influence the size of the FAZ.

Peripapillary Retinal Nerve Fiber Layer Vascular Microcirculation in Glaucoma Using Optical Coherence Tomography–Based Microangiography

Abstract: Purpose To investigate the vascular microcirculation changes in the retinal nerve fiber layer (RNFL) in normal, glaucoma suspect, and open-angle glaucoma (OAG) groups using optical coherence tomography-based microangiography (OMAG). Methods One eye from each subject was scanned with a Cirrus HD-OCT 5000-based OMAG prototype system montage scanning protocol centered at the optic nerve head (ONH). Blood flow signals were extracted using OMAG algorithm. Retinal nerve fiber layer vascular microcirculation was measured by calculating the blood flux index and vessel area density within a 1.2-mm width annulus centered at the ONH with exclusion of big retinal vessels. One-way ANOVA were performed to analyze the RNFL microcirculation among groups. Linear-regression models were constructed to analyze the correlation between RNFL microcirculation and clinical parameters. Discrimination capabilities of the flow metrics were assessed with the area under the receiver operating characteristic curve (AROC). Results Twenty normal, 26 glaucoma suspect, and 42 OAG subjects were enrolled. Eyes from OAG subjects and glaucoma suspects showed significantly lower blood flux index compared with normal eyes (P ≤ 0.0015). Retinal nerve fiber layer blood flow metrics showed significant correlations with visual field indices and structural changes in glaucomatous eyes (P ≤ 0.0123). Similar discrimination capability of blood flux index compared with RNFL thickness was found in both disease groups. Conclusions Peripapillary RNFL vascular microcirculation measured as blood flux index by OMAG showed significant differences among OAG, glaucoma suspect, and normal controls and was significantly correlated with functional and structural defects. Retinal nerve fiber layer microcirculation measurement using OMAG may help physicians monitor glaucoma.

The mitochondrial-derived peptide humanin protects RPE cells from oxidative stress, senescence, and mitochondrial dysfunction

Abstract: Purpose To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress-induced cell death, mitochondrial bioenergetics, and senescence. Methods Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5-10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress-induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment. Results A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress-induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death. Conclusions Humanin protected RPE cells against oxidative stress-induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.

New Insights Into Microaneurysms in the Deep Capillary Plexus Detected by Optical Coherence Tomography Angiography in Diabetic Macular Edema.

Abstract: Purpose: To study the association between the distributions of microaneurysms detected by en face optical coherence tomography angiography (OCTA) and diabetic macular edema (DME). Methods: The study design was a retrospective chart review of 27 patients (33 eyes) with DME. The eyes were scanned using OCTA (6 × 6 mm) and spectral-domain (SD) OCT macular cube. Each of the images of the capillary plexus was overlaid onto the image of the topographic map, and the densities of the microaneurysms were measured by ImageJ software. The association between the distribution of microaneurysms and macular edema was evaluated. Results: For microaneurysms in areas with and without edema, 77.3 ± 8.1% of these microaneurysms were located in the deep capillary plexuses (DCP). However, in areas of edema where the retinal thickness was more than 400 μm, 91.3 ± 9.1% of the microaneurysms were found in the DCP. This difference was statistically significant (P < 0.001). In the macular edema area, there was a significantly higher density of microaneurysms in the DCP compared to the superficial capillary plexuses (1.71/mm2 vs. 0.17/mm2, P < 0.001). There was also a significant correlation between the macular volume and the density of microaneurysms in the DCP in edema (r = 0.63, P < 0.001). Conclusions: Our study demonstrated a high proportion of microaneurysms in the DCP, as well as a novel association between the distributions of microaneurysms detected by OCTA and DME. Results also indicated that microaneurysms located in the DCP contribute to the pathogenesis of DME.

Netarsudil Increases Outflow Facility in Human Eyes Through Multiple Mechanisms.

Abstract: Purpose Netarsudil is a Rho kinase/norepinephrine transporter inhibitor currently in phase 3 clinical development for glaucoma treatment. We investigated the effects of its active metabolite, netarsudil-M1, on outflow facility (C), outflow hydrodynamics, and morphology of the conventional outflow pathway in enucleated human eyes.

Prevalence of Glaucoma in the United States: The 2005–2008 National Health and Nutrition Examination Survey

Abstract: Purpose To estimate the prevalence of glaucoma in the US population based on optic nerve head photography, to estimate the prevalence of glaucoma awareness, and to identify demographic and ocular risk factors for being unaware of having glaucoma.

OCT Angiography of the Peripapillary Retina in Primary Open-Angle Glaucoma.

Abstract: Purpose The purpose of this study was to investigate the topographic relationship between the decreased parapapillary retinal microvasculature as assessed by optical coherence tomography angiography (OCTA) and retinal nerve fiber layer (RNFL) defect in eyes with primary open-angle glaucoma (POAG) and a localized RNFL defect. Methods The peripapillary retinal circulation was evaluated using the OCTA centered on the optic nerve head in 98 POAG eyes having a localized RNFL defect and 45 healthy control eyes. A vascular impairment (VI) was identified in OCTA by the presence of a sign indicating decreased microvasculature. The frequencies of VI were compared between the POAG and control groups, and the topographic correlation between the VI and the RNFL defect identified in red-free fundus photographs was determined in the POAG group. Results The VI was observed as an area of decreased density of the microvascular network of the retina in 100% of the POAG eyes. The VI exactly coincided with the RNFL defect evident in red-free fundus photographs in terms of both the location and extent (Pearson's correlation coefficient = 0.997 and 0.988, respectively, all P < 0.001). None of the control eyes exhibited VI in OCTA. Conclusions Decreased parapapillary microvasculature of the retina determined by OCTA was found at the location of RNFL defect in POAG patients. This finding suggests that the decreased retinal microvasculature is likely secondary loss or closure of capillaries at the area of glaucomatous RNFL atrophy.

Retinal Microvasculature Alteration in High Myopia.

Abstract: Purpose To investigate the retinal vascular network alterations in highly myopic eyes. Methods Thirty-three highly myopic eyes from 21 subjects and 47 mildly myopic or emmetropic eyes from 24 healthy control subjects were enrolled. Optical coherence tomography angiography (OCTA) was used to image the superficial, deep, and whole retinal vascular plexuses at the macular region. Highly myopic eye images were analyzed after adjusting the ocular magnification using Bennett's formula. Fractal analysis (box counting method, Dbox) representing vessel density was performed in different annular and quadrantile zones of both large vessels and microvessels. Correlations between the vascular density, axial length, and spherical equivalent refractive error were analyzed. Results The average density (Dbox) of the superficial retinal annular zone (0.6-2.5 mm) microvessels was 1.741 ± 0.018 in highly myopic eyes and was shown to be significantly lower than that of the controls (1.773 ± 0.010, P < 0.001). Individual annular zone (bandwidth of 0.16 mm) analysis of highly myopic eyes revealed a significant level of microvessel alteration in all zones compared with the same zones in control eyes (P < 0.001). Furthermore, in the highly myopic group, the microvessel density was significantly correlated with axial length elongation in all three layers (r = -0.38 to -0.48; P < 0.05). Conclusions This study reveals retinal microvascular network alterations in highly myopic eyes, which correlates with axial length elongation. Fractal analysis of the microvasculature by OCTA images may help to characterize the underlying pathophysiological mechanisms involved in high myopia.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.

Abstract: PURPOSE. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPRassociated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS. Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS. Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS. Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Automated Quantification of Nonperfusion in Three Retinal Plexuses Using Projection-Resolved Optical Coherence Tomography Angiography in Diabetic Retinopathy

Abstract: Purpose The purpose of this study was to evaluate an automated algorithm for detecting avascular area (AA) in optical coherence tomography angiograms (OCTAs) separated into three individual plexuses using a projection-resolved technique.

Dendritic remodeling in early-stage glaucoma: Evidence from genetically identified retinal ganglion cell types

Abstract: Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1–2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or “Off” sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

Finite Element Analysis Predicts Large Optic Nerve Head Strains During Horizontal Eye Movements.

Abstract: Purpose We combined finite element (FE) analysis and dynamic magnetic resonance imaging (MRI) to estimate optic nerve head (ONH) strains during horizontal eye movements, and identified factors influencing such strains. We also compared ONH strains (prelamina, lamina cribrosa, and retrolamina strains) induced by eye movements to those induced by IOP. Methods The ocular globes and orbits of a healthy subject were visualized during horizontal eye movements (up to 13°), using dynamic MRI. A baseline FE model of one eye was reconstructed in the primary gaze position, including details from the orbital and ONH tissues. Finite element-derived ONH strains induced by eye movements were compared to those resulting from an IOP of 50 mm Hg. Finally, a FE sensitivity study was performed, in which we varied the stiffness of all ONH connective tissues, to understand their influence on ONH strains. Results Our models predicted that, during horizontal eye movements, the optic nerve pulled the ONH posteriorly. Optic nerve head strains following a lateral eye movement of 13° were large and higher than those resulting from an IOP of 50 mm Hg. These results held true even with variations in connective tissue stiffness. We also found that stiff sclerae reduced lamina cribrosa and prelamina strains during eye movements, but stiff optic nerve sheaths significantly increased those strains. Conclusions Our models predicted high ONH strains during eye movements, which were aggravated with stiffer optic nerve sheaths. Further studies are needed to explore links between ONH strains induced by eye movements and axonal loss in glaucoma.