Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

MACULAR MICROVASCULAR NETWORKS IN HEALTHY PEDIATRIC SUBJECTS.

PURPOSE: To report optical coherence tomography angiography (OCTA) values in healthy pediatric eyes and to identify factors that may modify these values. METHODS: In this prospective observational cross-sectional study, macular OCTA images were acquired from healthy pediatric patients. Main outcome measures were 1) foveal avascular zone (FAZ) area at the level of the superficial retinal capillary plexus (SCP); 2) SCP and deep retinal capillary plexus (DCP) perfusion density (based on the area of vessels); 3) SCP and DCP vessel density (based on a map with vessels of 1-pixel width); and 4) CC perfusion density. Multiple regression analysis was performed to assess the effect of age, sex, ethnicity, refraction, and foveal macular thickness (FMT) on OCTA parameters. RESULTS: Seventy-seven eyes from 52 subjects (23 male and 29 female) were included in analysis. Mean age was 11.1 ± 3.3 years (range = 5.0-17.0 years). Twenty-nine (55.8%) subjects were white, 14 (27.0%) Hispanic, 8 (15.4%) Asian, and 1 (1.8%) African-American. Mean refraction was -0.1 ± 2.4 diopters (D) (range = -5.75 to +9.0 D). Mean FMT was 248.6 ± 18.6 µm. Larger FAZ area was significantly associated with older age (P = 0.014). Furthermore, larger FAZ area was associated with reduced FMT (P < 0.0001). Male sex was associated only with increased SCP perfusion density (P = 0.042). Increased CC perfusion density was associated with younger age (P = 0.022). CONCLUSION: We report data for pediatric OCTA parameters in healthy subjects. Several variables influence the density of macular microvascular networks, and these factors should be considered in the OCTA study of pediatric eye disorders.

Assessment of Corneal Changes Associated with Topical Antiglaucoma Therapy Using in vivo Confocal Microscopy.

PURPOSE: Ocular surface disease (OSD) is highly prevalent in eyes treated with chronic, topical antiglaucoma (A/G) therapy. The purpose of this study was to utilize in vivo confocal microscopy (IVCM) to evaluate the corneal morphology, including characteristics of corneal epithelial cells, presence of epithelial dendritic cells (DCs), and characteristics of subbasal nerve plexus, of eyes under topical A/G therapy versus normal eyes. METHODS: Central corneal images were prospectively captured from 30 eyes of 16 patients under topical A/G therapy (>6 months) and 20 normal control eyes, using IVCM (HRT 3 RCM, Heidelberg, Germany). Demographic data were collected, as well as information on the types and duration of A/G therapy. In addition, OSD index (OSDI) score, tear film breakup time, Schirmer 1 test results, density of epithelial wing cells (WCs) and basal cells (BCs), subbasal nerve features (density, tortuosity, and reflectivity), and presence of DCs were all assessed and recorded by trained Doheny Image Reading Center graders. RESULTS: IVCM findings of 30 glaucomatous eyes and 20 normal control eyes were analyzed. The mean OSDI score was 8.72 in controls and 32.06 in patients under A/G therapy (p = 0.002). Nerve fiber density, nerve fiber reflectivity, and BC density were all decreased in the A/G group (1,789.07 ± 785.70 µm/frame, 2.79 ± 0.83, 6,457.67 ± 692.55 cells/mm2, respectively) as compared to controls (2,815.981 ± 563.77 µm/frame, 3.52 ± 0.50, 7,854.13 ± 1,073.69 cells/mm2, respectively) (p < 0.05), whereas the decrease in WC density was statistically nonsignificant (p = 0.5). Nerve tortuosity and DC density were both significantly greater in the A/G eyes (3.00 ± 0.57, 71.24 ± 61.74 cells/mm2, respectively) compared to controls (2.10 ± 0.42, 34.08 ± 11.70 cells/mm2, respectively) (p < 0.05). Tear film breakup time and Schirmer 1 test results were significantly lower in the A/G group as compared to controls (p < 0.001). CONCLUSIONS: Using IVCM, our study identified significant microstructural alterations in the corneas of eyes treated with topical A/G therapy. In addition, our study also revealed that glaucoma patients treated with topical A/G therapy report significantly higher OSDI scores compared to controls. Thus, IVCM may be a useful tool in providing structural parameters to correlate with the functional OSDI assessments in the evaluation of ocular surface toxicity associated with topical A/G therapy.

Analysis of ocular inflammation in anterior chamber-involving uveitis using swept-source anterior segment OCT.

PURPOSE: To evaluate the utility of swept-source (SS) optical coherence tomography (OCT) to objectively analyze the degree of anterior chamber (AC) inflammation. METHODS: Thirty-eight eyes of 32 patients with uveitis and 20 control eyes were enrolled. SS OCT B-scans were obtained, and the number of cells in the B-scans was counted using two methods: (1) manual grading by Point Picker plug-in of Image J ( http://bigwww.epfl.ch/thevenaz/pointpicker/ ) and (2) automated grading by the Image J Particle Analysis algorithm ( http://imagej.net/Particle_Analysis ). The automated and manual AC cell counts were correlated with the Standardization of Uveitis Nomenclature score. RESULTS: The average numbers of AC inflammatory cells counted by the automated method were 8 ± 4.0, 18 ± 3.0, 42 ± 14.0, 81 ± 32.0, 117 ± 57.0, and 275 ± 67.0 cells/mm2 for grades 0, 0.5 + , 1 + , 2 + , 3 + , and 4 + , respectively. For the same clinical categories, the average manual cell counts were 6 ± 4.0, 18 ± 3.0, 34 ± 14.0, 72 ± 32.0, 92 ± 43.0, and 168 ± 65.0 cells/mm2, respectively. Zero cells were detected in the AC of healthy eyes. The automated and manual methods were highly correlated (R = 0.98, p < 0.001) and showed good correlation with the clinical grading (R = 0.88, p < 0.001). A mean AC particle size of 117.4 ± 108.8 µm was obtained by the automated method. CONCLUSIONS: Quantification of the AC cells imaged by SS AS-OCT shows good correlation with categorical clinical severity assessments in uveitis eyes. This approach may provide a more objective method for monitoring uveitis and response to uveitis therapy.

Role of in vivo confocal microscopy in the diagnosis of infectious keratitis.

PURPOSE: To investigate the utility of in vivo confocal microscopy (IVCM) in the diagnosis of infectious keratitis (IK). METHODS: Retrospective chart review of 46 patients with a final diagnosis of IK were included in the study. All patients received IVCM corneal imaging using the Heidelberg Retinal Tomography III system. All available scans were randomized and analyzed in a masked fashion. Sensitivity and specificity of IVCM in diagnosing bacterial keratitis (BK), Acanthamoeba keratitis (AK), fungal keratitis (FK), and HSV viral keratitis (VK) were assessed. RESULTS: The pooled sensitivity and specificity of IVCM in identifying atypical IK (AK and FK cases combined) were 85.3% (95% CI 68.2-94.5%) and 100% (95% CI 74.7-100%), respectively. The sensitivity and specificity of IVCM in identifying BK were 66.7% (95% CI 35.4-88.7%) and 89.2% (95% CI 73.4-96.5%), respectively. The sensitivity and specificity of IVCM in identifying VK were 100% (95% CI 46.3-100%) and 93.2% (95% CI 80.3-98.2%). Additionally, IVCM was able to make the correct diagnosis in 8 out of the 11 atypical keratitis cases misdiagnosed clinically. In the AK subgroup, IVCM was more accurate than clinical assessment (16 vs. 11). In the FK subgroup, IVCM were as accurate as clinical assessment, but did correct one misdiagnosed cases by identfying fungal hyphae. CONCLUSION: IVCM is an non-invasive imaging modality that can rapidly and accurately diagnose IK even for experienced corneal specialists. In complex cases of polymicrobial infection, IVCM may guide the correct clinical diagnosis and initiation of the appropriate treatment.

Impact of Multiple En Face Image Averaging on Quantitative Assessment from Optical Coherence Tomography Angiography Images.

PURPOSE: To investigate the impact of multiple en face image averaging on quantitative measurements of the retinal microvasculature using optical coherence tomography angiography (OCTA). DESIGN: Prospective, observational, cross-sectional case series. PARTICIPANTS: Twenty-one healthy individuals with normal eyes. METHODS: Macular OCTA images were acquired from all participants using the Zeiss Cirrus 5000 with Angioplex OCTA software (Carl Zeiss Meditec, Dublin, CA). Nine OCTA cube scans per eye were obtained and 9 superficial retinal layer (SRL) and deep retinal layer (DRL) en face OCTA image slabs were averaged individually after registration. Quantitative parameters from the retinal microvasculature were measured on binarized and skeletonized OCTA images and compared with single OCTA images without averaging. MAIN OUTCOME MEASURES: Vessel density (VD), vessel length density (VLD), vessel diameter index (VDI), and fractal dimension (FD). RESULTS: Participants with artifact or poor image quality were excluded, leaving 18 eyes for the analysis. After averaging, qualitatively there was apparent reduction in background noise, and fragmented vessels in the images before averaging became continuous with smoother walls and showed sharper contrast in both the SRL and DRL. Binarized and skeletonized derivates of these averaged images also showed fewer line fragments and dots in nonvascular areas and more continuous vessel images than those of images without averaging. In both SRL and DRL, VD (P = 0.0010 and P = 0.0003, respectively), VLD (P < 0.0001 for both), and FD (P < 0.0001 for both) significantly decreased and VDI significantly increased after averaging (P < 0.0001 for both). CONCLUSIONS: Averaging of multiple en face OCTA images improves image quality and also significantly impacts quantitative measurements. Reducing noise that could be misinterpreted as flow and annealing discontinuous vessel segments seem to be major mechanisms by which averaging may be of benefit.

Quantification of Anterior Chamber Cells in Children With Uveitis Using Anterior Segment Optical Coherence Tomography.

PURPOSE: To evaluate the feasibility of anterior segment optical coherence tomography (AS-OCT) for measuring anterior chamber (AC) cells in children with uveitis and to compare different AS-OCT acquisition modes. DESIGN: Validity and reliability analysis. METHODS: We enrolled children younger than 18 years who had uveitis involving the anterior segment and children without eye disease as controls. All underwent clinical grading of AC cells. AC images of each eye were obtained using the Optovue Avanti RTVue XR AS-OCT. Two acquisition modes were used: a single cross-sectional line scan and an 8-line radial scan in an asterisk pattern. Two independent, masked graders counted cells manually on AS-OCT images. Rater agreement was assessed using intraclass correlation (ICC). RESULTS: Included were 30 children (59 eyes) with uveitis (median age 13.0 years, range 3-17 years) and 20 control children (40 eyes, median age 10.5 years, range 4-17 years). The number of eyes assigned each clinical grade of cells were as follows: none, 32 (54%); 0.5+, 12 (20.3%); 1+, 5 (8.5%); 2+, 8 (13.6%); 3+, 2 (3.4%). ICC of graders for line and radial scan protocols were 0.87 and 0.90. There was no significant difference between acquisition modes for pooled grader results (95% CI for difference: -0.04 to 0.14). ICC of cell counts between line and radial scan protocols was 0.85 (95% CI: 0.69-0.90). No control eyes had cells on AS-OCT images. CONCLUSIONS: Quantification of AC cell in children with uveitis is feasible with AS-OCT and has excellent reliability between different graders and acquisition modes.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Choriocapillaris flow impairment surrounding geographic atrophy correlates with disease progression.

PURPOSE: To evaluate the correlation between the choriocapillaris (CC) flow alterations around geographic atrophy (GA) and the GA yearly growth rate (yGR) in patients with dry age-related macular degeneration (AMD). METHODS: We retrospectively reviewed and analyzed spectral domain optical coherence tomography (SD-OCT) and SD-OCT angiography images of consecutive patients with GA acquired using the Cirrus OCT at the Doheny Eye Centers between 2015 and 2017. All eligible patients had one 6 x 6 mm OCTA scan acquired during the first visit (considered as baseline) and two fovea-centered 512 x 128 macular cubes (6 x 6 mm) acquired at baseline and after a minimum of 12 months. MAIN OUTCOME MEASURES: The fundus images from the OCT volumes were used to manually delineate the GA area and calculate the yGR after square root transformation. The en-face angiogram at the level of the CC was analyzed for the percentage of flow voids (FV) outside the atrophic lesion (FVOUT) and in the para- and peri-atrophy regions (FV500 and FV1000 respectively; two concentric 500 µm wide rings around the atrophy edge). These values, together with the difference between FV500 and FV1000 (ΔFV), were then correlated with the corresponding yGR. RESULTS: Thirty-three eyes of 23 patients were eligible for the analysis. The mean yGR was 0.23 ± 0.17 mm/years. At baseline, the mean FVOUT was 41.86 ± 2.71%, while FV500 and FV1000 were 46.4 ± 4.17% and 42.51 ± 2.65% respectively. The mean ΔFV was 3.89 ± 2.6%. While in the univariable analysis, the yGR was significantly associated with FV500 and with ΔFV (both p < 0.001), in multivariable model the association remained significant only with ΔFV (p < 0.001). CONCLUSIONS: Our study reports a correlation between the CC flow impairment around the atrophic lesions and their yGR in patients with GA. If replicated in future longitudinal studies, the choriocapillaris FV in the para-and peri-atrophy regions may prove to be useful parameters for evaluating the prognosis of these eyes.

Correlation between Guttata Severity and Thickness of Descemet's Membrane and the Central Cornea.

Purpose: To characterize and correlate guttata severity, Descemet's membrane thickness (DMT), central cornea thickness (CCT) in corneas with guttae using specular microscopy and spectral-domain optical coherence tomography (SD-OCT) and test the Doheny Image Reading Center (DIRC) specular microscopy-based corneal guttata severity scale. Methods: Forty-nine eyes of 49 patients with guttata and 36 eyes of age-matched of 36 normal controls were enrolled in the study. Three images of the central cornea and four of the peripheral cornea (inferior, superior, nasal and temporal) of each eye were taken using the Konan NSP-9900 specular microscope. A volume scan of the central cornea cross-section was collected on each eye using the Heidelberg Spectralis SD-OCT. The density of endothelial guttata based on specular images was graded on a 0-4 scale, and the Descemet's membrane thickness (DMT) and central corneal thickness (CCT) were manually measured by two trained graders. Results: The DIRC corneal guttata severity scale showed good reproducibility of all corneal endothelial images (weighted Kappa = 0.87). Mean DMT was 16.1 ± 2.4 µm in controls and 25.5 ± 10.9 µm in corneas with guttata (P < 0.001). Mean CCT was 552 ± 26 µm in controls and 603 ± 55 µm in corneas with guttata (P < 0.001). Guttata severity was significantly correlated with both DMT (r = 0.743, P < 0.001) and CCT (r = 0.569, P < 0.001). Age was moderately correlated with DMT (r = 0.472, P = 0.003) and mildly correlated with guttata severity (r = 0.285, P = 0.031), but was not correlated with CCT (r = 0.058, P = 0.681). Guttatta grade 3 corneas displayed an increase in DMT and guttata grade 4 was associated with a significant increase in CCT. Conclusions: The DMT and CCT are increased in corneas with guttata. The higher density of guttae is correlated with increased thickness. Specular microscopy combined with SD-OCT can be used as a good approach to assess the severity of FECD.

Evaluation of Corneal Epithelial Thickness Imaged by High Definition Optical Coherence Tomography in Healthy Eyes.

PURPOSE: To evaluate corneal epithelial thickness (CET) and corneal thickness (CT) in healthy eyes using spectral domain optical coherence tomography. METHODS: Thirty-six healthy eyes were imaged using the Cirrus high-definition (HD)-optical coherence tomography device. The average CET and CT were assessed using Cirrus Review Software within predefined concentric corneal ring-shaped zones. Specific regions of CET (superior, inferior, temporal, nasal, superonasal, inferotemporal, superotemporal, and inferonasal) were also assessed. The difference between zones was compared between males and females. RESULTS: The average CET was 48.3, 47.1, 46.1, and 45.8 µm in the 4 concentric zones (0-2, 2-5, 5-7, and 7-9 mm), respectively (P < 0.001). The average CT was 533.5, 550.8, and 579.4 µm in the 3 zones (0-2, 2-5, and 5-7 mm), respectively (P < 0.001). There was no statistically significant correlation between CET and CT in any of the measured zones. Males had thicker corneas than did females in each of the 3 CT zones (P < 0.05), but CET did not differ significantly. The CET superonasal-inferotemporal in 2.0 to 5.0 mm and CET superotemporal-inferonasal in 5.0- to 7.0-mm zones were significantly thinner in males than in females (-1.15 vs. 0.9 µm, -3.5 vs. -1.9 µm), respectively (P < 0.05). CONCLUSIONS: Optical coherence tomography-based analysis of CET reveals that it is thinner in the periphery, whereas the total corneal thickness is greater. Although total CT seems to be influenced by sex, CET is not. Regional and sex-based variations in CT may need to be considered when assessing corneal and epithelial alterations in the setting of disease.

Quantitative Analysis of Retinal and Choroidal Vascular Parameters in Patients With Low Tension Glaucoma.

PRéCIS:: By using OCT-A, we observed a reduction of the superficial macular, peripapillary, and optic nerve, as well as the choriocapillaris in eyes with low tension glaucoma, compared with normal controls. PURPOSE: The purpose of this study was to investigate macular and optic disc vascular parameters in patients with low tension glaucoma (LTG) using optical coherence tomography angiography (OCTA). METHODS: In this cross-sectional study, images were prospectively acquired from both eyes of 26 patients with clinically diagnosed LTG and 22 age-matched volunteers with normal healthy eyes using the Zeiss swept-source (SS) OCTA (Plex Elite 9000, Carl Zeiss Meditec). Perfusion density (PD) and vessel length density (VLD) within a 5 mm diameter circle centered over the macula and optic nerve head were analyzed. RESULTS: The final analysis cohort included 49 eyes with LTG and 40 healthy control eyes. Mean age was 60±10 years in the LTG group and 60±17 years in the control group. The LTG group had a statistically significant reduction in PD of the choriocapillaris (CC) compared with normal controls (71.74±8.37% vs. 80.48±3.84%; P<0.001). There was no statistically significant difference in PD between the LTG and control groups for the superficial vascular plexus (SVP), deep capillary plexus (DCP) or the optic nerve head and peripapillary area (ONH+PP) (P>0.05). The LTG group did show statistically significant reductions in VLD compared with normal controls for the SVP (2083.64±153.76 mm/mm vs. 2154.63±144.18 mm/mm; P=0.03) and ONH + PP (1813.76±271.69 mm/mm vs. 1950.23±169.33 mm/mm; P=0.03), whereas the DCP VD was similar between the 2 groups (P>0.05). CONCLUSIONS: Eyes with low tension glaucoma seems to show a lower CC perfusion density, as well as a lower SVP and ONH+PP vessel length density compared to normal eyes.

Representation of Central Endothelial Cell Density by Analysis of Single Best Specular Microscopy Image Regardless of Cell Size Variance.

PURPOSE: The purpose of this study is to evaluate whether a single best image can represent central endothelial cell density (ECD) in corneas of differing cell size coefficient of variance (CV). METHODS: Four hundred one healthy eyes but with variant CV values were enrolled. For each eye, three nonoverlapping central cornea endothelium images were obtained with Konan NSP-9900 specular microscope. ECD and CV were evaluated by two independent graders using the well-established Center method. Only corneas with high image quality rating (IQR) and ECD >800 cell/mm2 by both graders were included in the study. The study sample was stratified into five CV levels (CV ≤ 35; ≥36; ≥38; ≥40; and ≥45). In each CV level, the ECD agreement, ECD variance, and the correlation between the ECD variation and CV values were analyzed. In addition, the ECD intragrader reproducibility and interframe differences were also analyzed for all levels except CV ≤ 35. RESULTS: The study sample includes a total of 278 eyes. High ECD agreement for the two independent graders (intraclass correlation coefficient [ICC] > 0.99), high ECD intragrader reproducibility (ICC > 0.95), low ECD variance (2.0% ± 1.6%, overall), no correlation between the ECD variation and the CV value (P > 0.05), and no significant ECD difference among frames (P > 0.05) was found in any studied CV levels. CONCLUSIONS: CV does not appear to be associated with ECD variance in the central cornea. TRANSLATIONAL RELEVANCE: This finding highlights that in healthy corneas but with high CV values, ECD can be reliably analyzed using one single image of best quality.

Topographic distribution of choriocapillaris flow deficits in healthy eyes.

PURPOSE: To evaluate the topographic distribution of the choriocapillaris (CC) flow deficits in a population of healthy subjects. METHODS: Using a swept-source optical-coherence tomography angiography (SS-OCTA) device, two repeated volume 6 x 6 mm and 3 x 3 mm scans were acquired in healthy subjects at the Doheny-UCLA Eye Centers. The en-face CC angiogram was binarized and analyzed for percentage of flow deficits (FD%) using a grid of progressive, concentric rings covering a circular area with a diameter of 2.5 mm (in the 3 x 3 mm scans) and 5 mm (in the 6 x 6 mm scans). The FD% for each ring was plotted against the distance from the fovea. The linear trendline of the resulting curve was analyzed and the slope (m) and intercept (q) were computed. RESULTS: Seventy-five eyes of 75 subjects were enrolled and divided into three subgroups based on age (year ranges: 21-40, 41-60 and 61-80). For the entire cohort and within each subgroup, there was a significant association between distance from the fovea and FD% in both 3X3 mm and 6X6 mm scans, with flow deficits increasing with closer proximity to the foveal center. Age was a significant predictor for both m and q for both scan patterns, with older subjects showing a steeper slope. CONCLUSIONS: In SS-OCTA images, the topographic distribution of CC flow deficits varies with distance from the fovea and age. In particular, the FD% tends to decrease from the fovea towards the periphery, with a steeper decline with advancing age. These normal trends may need to be accounted for in future studies of the CC in disease.

Multiple enface image averaging for enhanced optical coherence tomography angiography imaging.

PURPOSE: To investigate the effect of multiple enface image averaging on image quality of the optical coherence tomography angiography (OCTA). METHODS: Twenty-one normal volunteers were enrolled in this study. For each subject, one eye was imaged with 3 × 3 mm scan protocol, and another eye was imaged with the 6 × 6 mm scan protocol centred on the fovea using the ZEISS Angioplex™ spectral-domain OCTA device. Eyes were repeatedly imaged to obtain nine OCTA cube scan sets, and nine superficial capillary plexus (SCP) and deep capillary plexus (DCP) were individually averaged after registration. RESULTS: Eighteen eyes with a 3 × 3 mm scan field and 14 eyes with a 6 × 6 mm scan field were studied. Averaged images showed more continuous vessels and less background noise in both the SCP and the DCP as the number of frames used for averaging increased, with both 3 × 3 and 6 × 6 mm scan protocols. The intensity histogram of the vessels dramatically changed after averaging. Contrast-to-noise ratio (CNR) and subjectively assessed image quality scores also increased as the number of frames used for averaging increased in all image types. However, the additional benefit in quality diminished when averaging more than five frames. Averaging only three frames achieved significant improvement in CNR and the score assigned by certified grades. CONCLUSION: Use of multiple image averaging in OCTA enface images was found to be both objectively and subjectively effective for enhancing image quality. These findings may of value for developing optimal OCTA imaging protocols for future studies.

Reproducibility of Central Corneal Thickness Measurements in Normal Eyes Using the Zeiss Cirrus 5000 HD-OCT and Pentacam HR.

OBJECTIVES: To determine the repeatability and reproducibility of Central Corneal Thickness (CCT) measurements using two different anterior segment imaging modalities, including those obtained with the new anterior segment lens attachments for the Cirrus 5000 HD-OCT. METHODS: A total of 32 eyes from 16 normal volunteers (8 male, 8 female) were enrolled in this prospective study. CCT was measured by the same examiner using the Cirrus 5000 HD-OCT and Pentacam HR. The results of CCT obtained by each method were averaged and compared using t-test analysis. The agreement between the measurement methods was evaluated. Coefficient of Repeatability (CoR) and Intra-Class Correlation Coefficient (ICC) were computed. RESULTS: The mean measurements taken with the Cirrus OCT anterior chamber lens (CCTAC), HD cornea lens (CCTHDC) and pachymetry scans (CCTPach) were 545.35 ± 31.02, 537.87 ± 26.82, and 532.04 ± 29.82 µm, respectively. The mean CCT obtained with the Pentacam (CCTPent) was 545.51 ± 30.71 µm. CCTPent were significantly higher than CCTHDC and CCTPach (p< 0.0001). In contrast, the CCTPent and CCTAC were similar (p=0.87). CCT, as evaluated by the two different instruments, showed excellent correlation (r > 0.98, p< 0.0001) with an ICC > 0.99 (95% CI, 0.97 - 0.99). CoR was the highest for CCTPach (3.7 ± 1.4, 95% CI (3.0- 4.6)). CONCLUSION: CCT measurements from the Cirrus OCT using the new anterior segment lens attachments and the Pentacam HR are highly correlated. This should allow the use of a standardized correction factor if necessary to inter-relate the measurements between the two devices.

Increased choriocapillaris vessel density in amblyopic children: a case-control study.

PURPOSE: To investigate the choriocapillaris in children with amblyopia, using optical coherence tomography angiography (OCT-A). METHODS: Patients with amblyopia and age-matched controls were prospectively imaged using OCT-A. On OCT-A, the choriocapillaris measures 30 µm starting 31 µm posterior to the retinal pigment epithelium. The section of choriocapillaris under superficial retinal vessels was excluded from analysis to avoid shadowing or projection artifacts. The main outcome measure was choriocapillaris vessel density. Secondary outcome measures were foveal macular thickness and parafoveal macular thickness. RESULTS: A total of 20 eyes of 16 patients with amblyopia and 25 eyes of 25 controls were included. Mean age of amblyopic subjects was 7.6 ± 3.6 years; of controls, 9.3 ± 2.2 years (P = 0.10). Mean refractive error of subjects was 4.3 ± 6.2 D; of controls, 0.0 ± 1.6 D (P = 0.004). Mean choriocapillaris vessel density was 74.8 ± 5.8 in the amblyopic group and 71.1 ± 3.6 in the control group, which was significant even after adjusting for age and refractive error (P = 0.012). There was no difference between groups in foveal macular thickness or parafoveal macular thickness; however, outer parafoveal macular thickness (the inner boundary of the inner nuclear layer to the retinal pigment epithelium outer boundary) was significantly greater in amblyopic eyes than in control eyes, even after adjusting for age and refractive error (203 ± 11 µm and 189 ± 12 µm, resp. [P = 0.014]). CONCLUSIONS: In our study cohort, amblyopic eyes were found to have increased choriocapillaris vessel density as well as a greater outer parafoveal macular thickness, which may be due to alterations in outer retinal maturation.

cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-ß production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-ß, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Choriocapillaris Imaging Using Multiple En Face Optical Coherence Tomography Angiography Image Averaging.

Importance: Imaging of the choriocapillaris in vivo is challenging with existing technology. Optical coherence tomography angiography (OCTA), if optimized, could make the imaging less challenging. Objective: To investigate multiple en face image averaging on OCTA images of the choriocapillaris. Design, Setting, and Participants: Observational, cross-sectional case series at a referral institutional practice in Los Angeles, California. From the original cohort of 21 healthy individuals, 17 normal eyes of 17 participants were included in the study. The study dates were August to September 2016. Exposures: All participants underwent OCTA imaging of the macula covering a 3 × 3-mm area using OCTA software (Cirrus 5000 with AngioPlex; Carl Zeiss Meditec). One eye per participant was repeatedly imaged to obtain 9 OCTA cube scan sets. Registration was first performed using superficial capillary plexus images, and this transformation was then applied to the choriocapillaris images. The 9 registered choriocapillaris images were then averaged. Quantitative parameters were measured on binarized OCTA images and compared with the unaveraged OCTA images. Main Outcome and Measure: Vessel caliber measurement. Results: Seventeen eyes of 17 participants (mean [SD] age, 35.1 [6.0] years; 9 [53%] female; and 9 [53%] of white race/ethnicity) with sufficient image quality were included in this analysis. The single unaveraged images demonstrated a granular appearance, and the vascular pattern was difficult to discern. After averaging, en face choriocapillaris images showed a meshwork appearance. The mean (SD) diameter of the vessels was 22.8 (5.8) µm (range, 9.6-40.2 µm). Compared with the single unaveraged images, the averaged images showed more flow voids (1423 flow voids [95% CI, 967-1909] vs 1254 flow voids [95% CI, 825-1683], P < .001), smaller average size of the flow voids (911 [95% CI, 301-1521] µm2 vs 1364 [95% CI, 645-2083] µm2, P < .001), and greater vessel density (70.7% [95% CI, 61.9%-79.5%] vs 61.9% [95% CI, 56.0%-67.8%], P < .001). The distribution of the number vs sizes of the flow voids was skewed in both unaveraged and averaged images. A linear log-log plot of the distribution showed a more homogeneous distribution in the averaged images compared with the unaveraged images. Conclusions and Relevance: Multiple en face averaging can improve visualization of the choriocapillaris on OCTA images, transforming the images from a granular appearance to a level where the intervascular spaces can be resolved in healthy volunteers.

Repeatability and Reproducibility of Superficial Macular Retinal Vessel Density Measurements Using Optical Coherence Tomography Angiography En Face Images.

Importance: The repeatability and reproducibility of quantitative metrics from optical coherence tomographic angiography (OCTA) must be assessed before these data can be confidently interpreted in clinical research and practice. Objective: To evaluate the repeatability and reproducibility of OCTA-derived retinal vascular quantitative metrics. Design, Setting and Participants: In this cross-sectional study, 21 healthy volunteers (42 eyes) and 22 patients with retinal disease (22 eyes), including 14 with age-related macular degeneration, 3 with epiretinal membrane, 2 with diabetic retinopathy, 2 with myopic macular degeneration, and 1 with retinal vein occlusion, were enrolled. Participants were recruited from September 1 through November 31, 2016. Each eye underwent 3 repeated scans with 3 instruments for a total of 9 acquisitions. Eyes were randomly assigned to scanning with a 3 × 3-mm or 6 × 6-mm pattern. Eyes were excluded from subsequent analysis if any acquisition had a signal strength of less than 7. Repeatability (defined as the agreement in measurements within a device) and reproducibility (defined as the agreement between devices of the same type) were assessed by intraclass correlation coefficient (ICC) and coefficient of variation. Exposures: All eyes underwent scanning using 3 separate devices. Main Outcomes and Measures: Vessel length density (VLD) and perfusion density (PD) of the superficial retinal vasculature. Results: A total of 21 healthy volunteers (8 men and 13 women; mean [SD] age, 36 [6] years) and 22 patients with retinal disease (15 men and 7 women; mean [SD] age, 79 [9] years) underwent evaluation. Of these, 40 of 42 normal eyes and 15 of 22 eyes with retinal disease met signal strength criteria and were included in this analysis. The ICC among the 3 consecutive scans ranged from 0.82 to 0.98 for VLD and from 0.83 to 0.95 for PD. The coefficient of variation (CV) ranged from 2.2% to 5.9% for VLD and from 2.4% to 5.9% for PD. For reproducibility, the ICC ranged from 0.62 to 0.95 and the CV was less than 6% in all groups. The agreement was highest for the 3 × 3-mm pattern in the inner ring (ICC range, 0.92 [95% CI, 0.85-0.96] to 0.96 [95% CI, 0.93-0.98]) and 6 × 6-mm pattern in the outer ring (ICC range, 0.93 [95% CI, 0.86-0.97] to 0.96 [95% CI, 0.92-0.98]). Conclusions and Relevance: Vessel length density and PD of the superficial retinal vasculature can be obtained from OCTA images with high levels of repeatability and reproducibility but can vary with scan pattern and location.