Imaging the Retinal Vascular Mural Cells In Vivo: Elucidating the Timeline of Their Loss in Diabetic Retinopathy.

BACKGROUND: Vascular mural cells (VMCs) are integral components of the retinal vasculature with critical homeostatic functions such as maintaining the inner blood-retinal barrier and vascular tone, as well as supporting the endothelial cells. Histopathologic donor eye studies have shown widespread loss of pericytes and smooth muscle cells, the 2 main VMC types, suggesting these cells are critical to the pathogenesis of diabetic retinopathy (DR). There remain, however, critical gaps in our knowledge regarding the timeline of VMC demise in human DR. METHODS: In this study, we address this gap using adaptive optics scanning laser ophthalmoscopy to quantify retinal VMC density in eyes with no retinal disease (healthy), subjects with diabetes without diabetic retinopathy, and those with clinical DR and diabetic macular edema. We also used optical coherence tomography angiography to quantify capillary density of the superficial and deep capillary plexuses in these eyes. RESULTS: Our results indicate significant VMC loss in retinal arterioles before the appearance of classic clinical signs of DR (diabetes without diabetic retinopathy versus healthy, 5.0±2.0 versus 6.5±2.0 smooth muscle cells per 100 µm; P<0.05), while a significant reduction in capillary VMC density (5.1±2.3 in diabetic macular edema versus 14.9±6.0 pericytes per 100 µm in diabetes without diabetic retinopathy; P=0.01) and capillary density (superficial capillary plexus vessel density, 37.6±3.8 in diabetic macular edema versus 45.5±2.4 in diabetes without diabetic retinopathy; P<0.0001) is associated with more advanced stages of clinical DR, particularly diabetic macular edema. CONCLUSIONS: Our results offer a new framework for understanding the pathophysiologic course of VMC compromise in DR, which may facilitate the development and monitoring of therapeutic strategies aimed at VMC preservation and potentially the prevention of clinical DR and its associated morbidity. Imaging retinal VMCs provides an unparalleled opportunity to visualize these cells in vivo and may have wider implications in a range of diseases where these cells are disrupted.

Association between macrophage-like cell density and ischemia metrics in diabetic eyes.

We previously showed that macrophage-like cells (MLCs) are increased in eyes with advanced diabetic retinopathy (DR). Here, we hypothesized that MLC density was correlated with ischemia using optical coherence tomography angiography (OCTA) and ultra-widefield fluorescein angiography (UWF-FA). Treatment-naïve diabetic eyes were prospectively imaged with repeated OCTA (average 5.3 scans per eye) and UWF-FA imaging. OCTA images were registered and averaged to generate a superficial capillary plexus (SCP), deep capillary plexus (DCP), and MLC slab. We calculated geometric perfusion deficit (GPD), vessel length density, and vessel density for the SCP and DCP. MLC density was quantified by two masked graders and averaged. Ischemia on UWF-FA was measured to generate a non-perfusion area (NPA) and index (NPI). Since MLC density was non-parametrically distributed, MLC density was correlated with ischemia metrics using Spearman correlations. Forty-five treatment-naïve eyes of 45 patients (59 ± 12 years of age; 56% female) were imaged. We included 6 eyes with no DR, 7 eyes with mild non-proliferative DR (NPDR), 22 moderate NPDR, 4 severe NPDR, and 6 PDR eyes. MLC density between graders was highly correlated (r = 0.9592, p < 0.0001). MLC density was correlated with DCP GPD (r = 0.296, p = 0.049), but no other OCTA ischemia metrics. MLC density was also correlated with UWF-FA NPA (r = 0.330, p = 0.035) and NPI (r = 0.332, p = 0.034). MLC density was correlated with total ischemia on UWF-FA and local DCP GPD. Since both UWF-FA and DCP non-perfusion are associated with higher risk for DR progression, MLC density could be another potential biomarker for DR progression.

NEW METHOD FOR REDUCING ARTIFACTUAL FLOW DEFICITS CAUSED BY COMPENSATION TECHNIQUES IN THE CHORIOCAPILLARIS WITH OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To mitigate artifactual choriocapillaris flow deficits in optical coherence tomography angiography, which are a side effect of inverse structural optical coherence tomography compensation. METHODS: In a modified algorithm, we set pixels in the original structural optical coherence tomography that were greater than one SD above the mean intensity (hyperreflective regions) to the mean pixel intensity of the image to remove hyporeflective regions in the inverse slab. We compared this algorithm to the original using flow deficit density and multiscale structural similarity index obtained from three distinct thresholding methods (local Phansalkar, global MinError (I), and global Li). RESULTS: We included 16 eyes of 16 healthy subjects (31.1 ± 6.9 years, 10 females). Using the modified optical coherence tomography correction, flow deficit density was lower compared with the original algorithm using Phansalkar (P < 0.001) but higher using Li thresholding (P = 0.049). Multiscale structural similarity index was increased after applying the modified algorithm with all three thresholding methods (P < 0.001), indicating a closer relationship to the original optical coherence tomography angiography scan. CONCLUSION: We demonstrate a new method that significantly reduced the introduction of artifactual flow deficits in the choriocapillaris during postprocessing. Given the improved multiscale structural similarity index, we believe our algorithm more accurately represents the choriocapillaris.

Imaging and Biomarkers in Diabetic Macular Edema and Diabetic Retinopathy.

PURPOSE OF REVIEW: Diabetic retinopathy (DR) is the leading cause of acquired vision loss in adults across the globe. Early identification and treatment of patients with DR is paramount for vision preservation. The aim of this review paper is to outline current and new imaging techniques and biomarkers that are valuable for clinical diagnosis and management of DR. RECENT FINDINGS: Ultrawide field imaging and automated deep learning algorithms are recent advancements on traditional fundus photography and fluorescein angiography. Optical coherence tomography (OCT) and OCT angiography are techniques that image retinal anatomy and vasculature and OCT is routinely used to monitor response to treatment. Many circulating, vitreous, and genetic biomarkers have been studied to facilitate disease detection and development of new treatments. Recent advancements in retinal imaging and identification of promising new biomarkers for DR have the potential to increase detection, risk stratification, and treatment for patients with DR.

MULTILEVEL ISCHEMIA IN DISORGANIZATION OF THE RETINAL INNER LAYERS ON PROJECTION-RESOLVED OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To examine the relationship between ischemia and disorganization of the retinal inner layers (DRIL). METHODS: Cross-sectional retrospective study of 20 patients (22 eyes) with diabetic retinopathy presenting to a tertiary academic referral center, who had DRIL on structural optical coherence tomography (OCT) using Spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany) and OCT angiography with XR Avanti (Optovue Inc, Fremont, CA) on the same day. Optical coherence tomography angiography images were further processed to remove flow signal projection artifacts using a software algorithm adapted from recent studies. Retinal capillary perfusion in the superficial capillary plexuses, middle capillary plexuses, and deep capillary plexuses, as well as integrity of the photoreceptor lines on OCT was compared in areas with DRIL to control areas without DRIL in the same eye. RESULTS: Qualitative assessment of projection-resolved OCT angiography of eyes with DRIL on structural OCT demonstrated significant perfusion deficits compared with adjacent control areas (P < 0.001). Most lesions (85.7%) showed superimposed superficial capillary plexus and/or middle capillary plexus nonperfusion in addition to deep capillary plexus nonflow. Areas of DRIL were significantly associated with photoreceptor disruption (P = 0.035) compared with adjacent DRIL-free areas. CONCLUSION: We found that DRIL is associated with multilevel retinal capillary nonperfusion, suggesting an important role for ischemia in this OCT phenotype.

CHARACTERIZATION AND CORRELATION OF "JAMPOL DOTS" ON ADAPTIVE OPTICS WITH FOVEAL GRANULARITY ON CONVENTIONAL FUNDUS IMAGING.

PURPOSE: To describe features characteristic of multiple evanescent white dot syndrome (MEWDS) using adaptive optics scanning laser ophthalmoscopy (AOSLO). METHODS: Six women (seven eyes) who presented with MEWDS between June 2014 and April 2017 underwent ophthalmologic examinations and multimodal imaging including infrared, AOSLO, and spectral domain optical coherence tomography. RESULTS: Bright hyperreflective lesions on AOSLO throughout the course of MEWDS could be correlated to the hyperreflective dots of foveal granularity on infrared imaging without apparent corresponding changes on spectral domain optical coherence tomography. During the acute phase of MEWDS, extrafoveal hyperreflective dots were also visible on AOSLO and infrared and were associated with accumulations of hyperreflective material above the retinal pigment epithelium on spectral domain optical coherence tomography. CONCLUSION: Foveal granularity on conventional fundus imaging could be correlated with hyperreflective lesions visible on AOSLO. We hypothesize that these hyperreflective lesions, "Jampol dots," are the foveal corollaries of the same process associated with the classic "dot" lesions in MEWDS. Based on the intact photoreceptor mosaic on AOSLO, we surmise that this material is accumulating at the level of the retinal pigment epithelium.

RESIDUAL CHOROIDAL VESSELS IN ATROPHY CAN MASQUERADE AS CHOROIDAL NEOVASCULARIZATION ON OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY: Introducing a Clinical and Software Approach.

PURPOSE: To present a postprocessing approach in optical coherence tomography angiography (OCTA) to facilitate the visualization and interpretation of lesions in age-related macular degeneration with coexisting atrophy and choroidal neovascularization (CNV). METHODS: This retrospective study included 32 eyes of 26 patients with atrophy and treated CNV and 8 eyes with treatment-naive geographic atrophy. En face optical coherence tomography slabs highlighting atrophy were pseudocolored and merged with the corresponding OCTA. Cross-sectional optical coherence tomography and postprocessed OCTA were analyzed to identify CNV and normal choroidal vessels in relationship to the atrophy. We correlate the OCTA findings with those in a donor eye with treatment-naive geographic atrophy studied with transmission electronic microscopy. RESULTS: Medium-sized choroidal vessels were displaced anteriorly in areas of atrophy in all 40 eyes (100%), visualized in the choriocapillaris slab in all eyes, and in the outer retinal slab in 30 of 40 eyes (75.0%). Cross-sectional OCTA was used to confirm the presence of CNV. Postprocessing successfully highlighted the CNV and distinguished it from choroidal vessels in atrophy. Donor eye transmission electronic microscopy confirmed the anterior displacement of medium-sized choroidal vessels in geographic atrophy. CONCLUSION: The anterior displacement of larger choroidal vessels in atrophy requires clinician vigilance to avoid misinterpreting these vessels as CNV on en face OCTA. Our proposed postprocessing approach offers a potential solution to facilitate the interpretation of en face OCTA in these cases. In the absence of other tools, clinicians are encouraged to rely on the location of flow relative to Bruch membrane on cross-sectional OCTA flow images.

RETINAL CAPILLARY DENSITY IN PATIENTS WITH BIRDSHOT CHORIORETINOPATHY.

PURPOSE: To quantify retinal capillary density and determine its correlation with visual acuity in patients with birdshot chorioretinopathy (BCR). METHODS: Patients with BCR and age-matched controls were imaged using a commercially available spectral domain optical coherence tomography angiography system (RTVue- XR Avanti; Optovue, Inc). We used the integrated software of the optical coherence tomography angiography device to analyze the foveal avascular zone area and the capillary density in the full retina as well as in the superficial capillary plexus and deep capillary plexus. We assessed the correlation between these parameters and visual acuity. RESULTS: Seventy-four eyes of 42 study participants (37 eyes of 21 BCR and 37 eyes of 21 healthy subjects) were included in this observational cross-sectional study. Capillary density of the full retina, superficial capillary plexus, and deep capillary plexus were significantly decreased in BCR compared with the healthy control group (P < 0.01). Visual acuity in patients with BCR was significantly associated with the capillary density of the superficial capillary plexus, deep capillary plexus, and full retina (P < 0.01) but not with the area of the foveal avascular zone. CONCLUSION: The decrease in visual acuity in patients with BCR is associated with retinal vascular impairment. Vessel density of the retinal capillary plexuses may be a promising imaging biomarker for BCR disease severity.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN ADULT-ONSET FOVEOMACULAR VITELLIFORM DYSTROPHY.

PURPOSE: To determine the ability of optical coherence tomography angiography (OCTA) to detect choroidal neovascularization (CNV) in the pseudohypopyon stage of adult-onset foveomacular vitelliform dystrophy. METHODS: Prospective case series of eight consecutive patients with adult-onset foveomacular vitelliform dystrophy with at least one eye in the pseudohypopyon stage (a total of 14 eyes). Patients were assessed with spectral domain OCT, flourescein angiography, and OCTA. Main outcome measures were the presence or absence of CNV and any unifying patterns that could be identified on OCTA for adult-onset foveomacular vitelliform dystrophy. RESULTS: One (12.5%) of eight eyes in the pseudohypopyon stage had CNV on OCTA, without definitive evidence of CNV on flourescein angiography. Twelve of 14 eyes (86%) had OCTA segmentation errors, giving the false appearance of deep capillary plexus drop out. All 14 eyes (100%) had blockage of flow signal under the vitelliform lesion on OCTA that presented as artifactual loss of flow in the choriocapillaris. CONCLUSION: Optical coherence tomography angiography may be superior to flourescein angiography in detecting CNV in adult-onset foveomacular vitelliform dystrophy, especially in the pseudohypopyon stage. There are common artifacts that must be considered when analyzing vitelliform lesions with OCTA, including segmentation errors and inability to visualize flow under the vitelliform lesion in the choriocapillaris.

CHARACTERIZING PHOTORECEPTOR CHANGES IN ACUTE POSTERIOR MULTIFOCAL PLACOID PIGMENT EPITHELIOPATHY USING ADAPTIVE OPTICS.

PURPOSE: To characterize lesions of acute posterior multifocal placoid pigment epitheliopathy (APMPPE) by multimodal imaging including adaptive optics scanning laser ophthalmoscopy (AOSLO). METHODS: We included patients with APMPPE at different stages of evolution of the placoid lesions. Color fundus photography, spectral domain optical coherence tomography, infrared reflectance, fundus autofluorescence, and AOSLO images were obtained and registered to correlate microstructural changes. RESULTS: Eight eyes of four patients (two women) were included and analyzed by multimodal imaging. Photoreceptor reflectivity within APMPPE lesions was more heterogeneous than in adjacent healthy areas. Hyperpigmentation on color fundus photography appeared hyperreflective on infrared reflectance and on AOSLO. Irregularity of the interdigitation zone and the photoreceptor inner and outer segment junctions (IS/OS) on spectral domain optical coherence tomography was associated with photoreceptor hyporeflectivity on AOSLO. Interruption of the interdigitation zone or IS/OS was associated with loss of photoreceptor reflectivity on AOSLO. CONCLUSION: Irregularities in the reflectivity of the photoreceptor mosaic are visible on AOSLO even in inactive APMPPE lesions, where the photoreceptor bands on spectral domain optical coherence tomography have recovered. Adaptive optics scanning laser ophthalmoscopy combined with multimodal imaging has the potential to enhance our understanding of photoreceptor involvement in APMPPE.

SEMIAUTOMATED QUANTITATIVE APPROACH TO CHARACTERIZE TREATMENT RESPONSE IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION: A Real-World Study.

PURPOSE: To perform a quantitative study of the vascular microstructure in actively treated choroidal neovascularization by optical coherence tomographic angiography. METHODS: Patients undergoing individualized anti-vascular endothelial growth factor therapy of minimum 12 months duration were included in this cross-sectional observational study and imaged using optical coherence tomographic angiography. En face optical coherence tomographic angiography images were analyzed for quantitative features, such as junction density, vessel length, and lacunarity using validated software (Angiotool). Patients were divided into 2 groups depending on their individualized treatment interval: "good responders, treated less frequently than 6 weeks" versus "poor responders, treated every 6 weeks or more frequently." Nonparametric testing was used to assess differences between these groups. RESULTS: Twenty-five eyes of 23 consecutive patients with a median 58-month history of choroidal neovascularization, treated by median of 34 anti-vascular endothelial growth factor injections, were included in the analysis. There was no significant difference between any of the microvascular choroidal neovascularization features between the 2 groups (P > 0.05). CONCLUSION: The semiautomated vessel segmentation software provides an objective and quantitative approach for choroidal neovascularization characterization. The consistently nonsignificant outcomes between the groups may provide evidence to support the "normalization hypothesis." This would suggest that regardless of treatment interval, individualized therapy in these eyes established vessel stability.

LONGITUDINAL QUANTITATIVE EVALUATION OF OUTER RETINAL LESIONS IN ACUTE POSTERIOR MULTIFOCAL PLACOID PIGMENT EPITHELIOPATHY USING OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To quantify the external limiting membrane (ELM) disruption and photoreceptor volume changes in eyes with acute posterior multifocal placoid pigment epitheliopathy (APMPPE) using Spectral Domain Optical Coherence Tomography (SD-OCT) at the acute and resolution phases. METHODS: Retrospective study of 10 eyes of 5 patients with APMPPE. Intact ELM and the Bruch's membrane were manually traced using ImageJ software and their lengths from each scan of the Spectral Domain Optical Coherence Tomography macular volume were summed. The ratio of intact ELM length/Bruch's membrane length was calculated. Also, two-dimensional areas of specific regions of interest were demarcated between the intact ELM and Bruch's Membrane in every cross-sectional B-scan. Total volume of photoreceptors was calculated by multiplying the total area by the distance between B-scans. RESULTS: There was a statistically significant increase in ELM/Bruch's membrane ratio (P value = 0.022), mean photoreceptors volume (P value = 0.028), and a significant linear positive correlation between change of intact ELM/Bruch's membrane ratio and percent change of photoreceptor volume (r = 0.927, P value = 0.001) when comparing baseline and final follow-up visit, independent of total follow-up length. CONCLUSION: Using Spectral Domain-Optical Coherence Tomography, we showed that quantitative evaluation of ELM disruption and the volume of photoreceptor recovery can help us to follow the clinical course of APMPPE.

ACUTE POSTERIOR MULTIFOCAL PLACOID PIGMENT EPITHELIOPATHY ON OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To investigate choroidal involvement in acute posterior multifocal placoid pigment epitheliopathy (APMPPE). METHODS: A retrospective observational case series using multimodal imaging including optical coherence tomography (OCT) angiography. RESULTS: Five patients with APMPPE were included. In most acute lesions, OCT angiography revealed outer retinal and retinal pigment epithelium (RPE) hyperreflective lesions with attenuated OCT signal in the underlying choroid, but careful examination allowed us to identify a single lesion with decreased choriocapillaris flow outside the signal attenuation. Optical coherence tomography angiography obtained after healing of lesions revealed areas of hypointense circular flow voids clustered in groups surrounded by either isointense or hyperintense signal background. Point-by-point evaluation revealed these flow voids did not correspond to areas of RPE thickening or focal pigmentary changes. Larger hypointense lesions were observed and did correlate with pigmentary changes. CONCLUSION: Our case series demonstrates choriocapillaris flow abnormalities in acute APMPPE extending beyond the OCT lesions, and distinct residual vascular abnormalities in healed APMPPE lesions on OCT angiography. Our findings support a primary ischemic insult to the photoreceptors and RPE, but choriocapillaris flow abnormalities could be secondary to (OCT invisible) retinal and RPE involvement. The lack of understanding of the etiology along with the inability to visualize most of the choroid in acute lesions precludes definite conclusions about the true pathogenesis of APMPPE.

ASSESSMENT OF RETINAL BLOOD FLOW IN DIABETIC RETINOPATHY USING DOPPLER FOURIER-DOMAIN OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To evaluate retinal blood flow measurements in normal eyes and eyes with varying levels of diabetic retinopathy (DR) using Doppler Fourier-domain optical coherence tomography (FD-OCT). METHODS: Twenty-two eyes of 19 subjects, 10 with severe nonproliferative DR (NPDR) and 12 with proliferative DR (PDR), were compared with 44 eyes of 40 healthy control subjects. All eyes were scanned by RTvue FD-OCT. Color disk photographs and cube/volume scans of the optic nerve head were obtained. Doppler OCT scans and accessory imaging data were imported into Doppler OCT of Retinal Circulation grading software to calculate TRBF and vascular parameters (e.g., venous and arterial cross-sectional area). Measurements were compared between cases and controls using independent t-tests. RESULTS: Mean TRBF was 44.98 ± 9.80 (range: 30.18-64.58) µL/minute for normal eyes, 35.80 ± 10.48 (range: 20.69-49.56) µL/minute for eyes with severe NPDR, and 34.79 ± 10.61 (range: 16.77-48.9) µL/minute for eyes with PDR. Mean TRBF was significantly lower in eyes with severe NPDR (P = 0.01) and PDR (P = 0.003) than in normal eyes. CONCLUSION: Total retinal blood flow was significantly lower in eyes with severe NPDR and PDR compared with normal eyes. Retinal blood flow determined by Doppler OCT may be a useful parameter for evaluating patients with DR.

DISCORDANCE BETWEEN BLUE-LIGHT AUTOFLUORESCENCE AND NEAR-INFRARED AUTOFLUORESCENCE IN AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To identify the origin and significance of discordance between blue-light autofluorescence (BL-AF; 488 nm) and near-infrared autofluorescence (NI-AF; 787 nm) in patients with age-related macular degeneration (AMD). METHODS: A total of 86 eyes of 59 patients with a diagnosis of AMD were included in this cross-sectional study conducted between March 9, 2015 and May 1, 2015. A masked observer examined the BL-AF, NI-AF, and spectral-domain optical coherence tomography images. Areas with discordance of autofluorescence patterns between NI-AF and BL-AF images were correlated with structural findings at the corresponding location in optical coherence tomography scans. RESULTS: Seventy-nine eyes had discordance between BL-AF and NI-AF. The most common optical coherence tomography finding accounting for these discrepancies was pigment migration accounting for 35 lesions in 21 eyes. The most clinically relevant finding was geographic atrophy missed on BL-AF in 7 eyes. CONCLUSION: Our findings indicate that variations in the distribution of lipofuscin, melanin and melanolipofuscin account for the majority of discordance between BL-AF and NI-AF. Given our finding of missed geographic atrophy lesions on BL-AF in 24% of eyes with geographic atrophy (7/29 eyes), clinicians should consider multimodal imaging, including NI-AF and optical coherence tomography, especially in clinical trials of geographic atrophy.

CHARACTERIZATION OF THE MIDDLE CAPILLARY PLEXUS USING OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN HEALTHY AND DIABETIC EYES.

PURPOSE: To assess the ability of optical coherence tomography angiography to image the retinal middle capillary plexus (MCP), and to characterize the MCP as a unique vascular network separate from the superficial and deep capillary plexus (DCP). METHODS: Healthy and diabetic eyes were imaged using the Avanti XR optical coherence tomography angiography instrument (Optovue Inc, Fremont, CA). Using manual segmentation of the retinal layers, the authors generated en face angiograms to distinguish the three capillary plexuses (superficial capillary plexus, MCP, DCP). RESULTS: In healthy eyes, arterioles gave rise to distinct branches in the MCP, and venules gave rise to prominent vortex like branches in the DCP. The foveal avascular zone was most well-defined at the level of the MCP, and had a larger area in the DCP. In diabetic eyes, the three capillary plexuses showed varying degrees of nonperfusion, including variable shapes and extent of the foveal avascular zone, with loss of border integrity at the MCP. Microaneurysms appeared in all the three capillary plexuses. CONCLUSION: Using customized segmentation analysis in optical coherence tomography angiography, the authors demonstrate that the MCP is qualitatively and functionally distinct from the superficial capillary plexus and DCP, which may help clarify the pathogenesis of different middle retinal ischemic entities and provide new insights into retinal ischemia in diabetic retinopathy.