Comparing the transcriptome of developing native and iPSC-derived mouse retinae by single cell RNA sequencing.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from mouse native retinae and induced pluripotent stem cell (iPSC)-derived retinal organoids at four matched stages of development spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal organoids broadly recapitulate the native developmental trajectories. However, we observe relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in retinal organoids. We demonstrate that genes causing human retinopathies are enriched in cell-type specifying genes and identify a subset of disease-causing genes with expression profiles that are highly conserved between human retinae and murine retinal organoids. This study provides a resource to the community that will be useful to assess and further improve protocols for ex vivo recapitulation and study of retinal development.

Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration.

It has become increasingly important to understand how retinal inflammation is regulated because inflammation plays a role in retinal degenerative diseases. Lipocalin 2 (LCN2), an acute stress response protein with multiple innate immune functions, is increased in ATP-binding cassette subfamily A member 4 (Abca4) -/- retinol dehydrogenase 8 (Rdh8) -/- double-knockout mice, an animal model for Stargardt disease and age-related macular degeneration (AMD). To examine roles of LCN2 in retinal inflammation and degeneration, Lcn2-/-Abca4-/-Rdh8-/- triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn2-/-Abca4-/-Rdh8-/- mice as compared with Abca4-/-Rdh8-/- mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as Ccl8, Ccl2, and Cxcl10 To further probe a possible regulatory role for LCN2 in retinal inflammation, we examined the in vitro effects of LCN2 on NF-κB signaling in human retinal pigmented epithelial (RPE) cells differentiated from induced pluripotent stem cells derived from healthy donors. We found that LCN2 induced expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in these RPE cells and could inhibit the cytotoxic effects of H2O2 and LPS. ELISA revealed increased LCN2 levels in plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration as compared with healthy controls. Finally, overexpression of LCN2 in RPE cells displayed protection from cell death. Overall these results suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration.

Pilot evaluation of short-term changes in macular pigment and retinal sensitivity in different phenotypes of early age-related macular degeneration after carotenoid supplementation.

PURPOSE: To investigate the response of carotenoid supplementation in different phenotypes of early age-related macular degeneration (AMD) by measuring macular pigment optical density (MPOD) and retinal sensitivity. METHODS: Consecutive patients with only medium/large drusen and only reticular pseudodrusen (RPD) and age-matched and sex-matched controls were enrolled. At baseline, participants underwent a complete ophthalmological examination including measurement of best-corrected visual acuity (BCVA), MPOD and retinal sensitivity. Patients were put on vitamin supplementation (lutein 10 mg/day, zeaxanthin 2 mg/day) and 3 months later underwent a repeated ophthalmological examination. RESULTS: Twenty patients with medium/large drusen, 19 with RPD and 15 control subjects were included. At baseline, in controls, mean MPOD and BCVA were significantly higher compared with RPD (p=0.001 and p=0.01) but similar to medium/large drusen (p=0.9 and p=0.4). Mean retinal sensitivity was significantly higher in controls compared with RPD and medium/large drusen (for all p<0.0001). After 3 months of carotenoid supplementation the mean MPOD significantly increased in RPD (p=0.002), thus showing no more difference compared with controls (p=0.3); no significant changes were found in mean retinal sensitivity and BCVA (p=0.3 and p=0.7). Medium/large drusen did not show significant changes on MPOD, retinal sensitivity and BCVA (p=0.5, p=0.7 and p=0.7, respectively). CONCLUSIONS: Patients with early AMD, especially RPD phenotype, show lower macular sensitivity and MPOD than controls. After supplementation, MPOD significantly increased in RPD. These results suggest different pathophysiology for RPD as compared with medium/large drusen and may open new ways to identifying further therapeutic targets in this phenotype of early AMD.

ADAPTIVE OPTICS IMAGING OF FOVEAL SPARING IN GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION.

BACKGROUND: To describe adaptive optics (AO) imaging of foveal sparing in geographic atrophy (GA) secondary to age-related macular degeneration. METHODS: Flood-illumination AO infrared (IR) fundus images were obtained in four consecutive patients with GA using an AO retinal camera (rtx1; Imagine Eyes). Adaptive optics IR images were overlaid with confocal scanning laser ophthalmoscope near-IR autofluorescence images to allow direct correlation of en face AO features with areas of foveal sparing. Adaptive optics appearance of GA and foveal sparing, preservation of functional photoreceptors, and cone densities in areas of foveal sparing were investigated. RESULTS: In 5 eyes of 4 patients (all female; mean age 74.2 ± 11.9 years), a total of 5 images, sized 4° × 4°, of foveal sparing visualized on confocal scanning laser ophthalmoscope near-IR autofluorescence were investigated by AO imaging. En face AO images revealed GA as regions of inhomogeneous hyperreflectivity with irregularly dispersed hyporeflective clumps. By direct comparison with adjacent regions of GA, foveal sparing appeared as well-demarcated areas of reduced reflectivity with less hyporeflective clumps (mean 14.2 vs. 3.2; P = 0.03). Of note, in these areas, en face AO IR images revealed cone photoreceptors as hyperreflective dots over the background reflectivity (mean cone density 3,271 ± 1,109 cones per square millimeter). Microperimetry demonstrated residual function in areas of foveal sparing detected by confocal scanning laser ophthalmoscope near-IR autofluorescence. CONCLUSION: Adaptive optics allows the appreciation of differences in reflectivity between regions of GA and foveal sparing. Preservation of functional cone photoreceptors was demonstrated on en face AO IR images in areas of foveal sparing detected by confocal scanning laser ophthalmoscope near-IR autofluorescence.

WEDGE-SHAPED SUBRETINAL HYPOREFLECTIVITY IN GEOGRAPHIC ATROPHY.

PURPOSE: To describe wedge-shaped subretinal hyporeflectivity, a peculiar spectral domain optical coherence tomography finding in geographic atrophy (GA) areas of atrophic age-related macular degeneration. METHODS: We reviewed the charts of consecutive patients with GA who presented between January 2012 and December 2013. A standardized imaging protocol was performed in all patients, which included blue fundus autofluorescence, and spectral domain optical coherence tomography. RESULTS: Wedge-shaped subretinal hyporeflective lesions were found in 12 of 161 included eyes (11 of 94 consecutive patients, 6 males/5 females, mean age 79.6 ± 9.3 years). On spectral domain optical coherence tomography, regions immediately adjacent to the wedge-shaped subretinal hyporeflective lesions were characterized by absence of the hyporeflective outer nuclear layer, the hyperreflective external limiting membrane, the ellipsoid zone, the interdigitation zone, and the retinal pigment epithelium. On "en face" images, they appeared as round-oval hyporeflectivities delimited by hyperreflective borders, which we interpreted as the outer plexiform layer. Mean GA area was significantly larger in eyes with as compared with eyes without wedge-shaped subretinal hyporeflective lesions. Overall, the dimensions of the wedge-shaped subretinal hyporeflective lesions did not change after a mean of ∼ 15 months. CONCLUSION: Wedge-shaped subretinal hyporeflectivity, a previously unreported peculiar finding in GA areas of atrophic age-related macular degeneration eyes, appears delimited internally by the hyperreflective outer plexiform layer and externally by the hyperreflective Bruch membrane. These lesions, which are detected in eyes with large GA (even though stable over time), should be recognized and distinguished from subretinal fluid (and other exudative signs of age-related macular degeneration) because their presence should not require prompt treatment.

MultiColor imaging in the evaluation of geographic atrophy due to age-related macular degeneration.

PURPOSE: To compare different imaging modalities and to investigate the ability of MultiColor to evaluate geographic atrophy (GA) due to age-related macular degeneration (AMD). METHODS: Twenty-two consecutive patients with GA underwent MultiColor, colour fundus photography, blue fundus autofluorescence (FAF) (excitation=488 nm; emission >500 nm), near-infrared FAF (NIR-FAF) (excitation=787 nm; emission >800 nm) and spectral-domain optical coherence tomography (SD-OCT) (Spectralis HRA+OCT; Heidelberg Engineering) imaging. Two readers independently measured the size (area) and the width of GA (on horizontal SD-OCT scan cutting the fovea), and evaluated the foveal sparing in each examination. RESULTS: A total of 32 eyes (22 patients, mean age 79.2±8 years) with GA were included. Intragrader and intergrader agreement considering the evaluation of the size and width of GA was high for all the examinations. MultiColor and FAF showed the greatest intergrader agreement for GA area measurement (intraclass correlation (ICC)=0.990, 95% CI 0.980 to 0.995; ICC=0.998, 95% CI 0.996 to 0.999, respectively). SD-OCT showed the highest intergrader agreement of foveal involvement (k=1), followed by MultiColor and NIR-FAF (k=0.68). CONCLUSIONS: We demonstrated that several different imaging modalities currently available in clinical practice are reliable for evaluating GA due to AMD. MultiColor is an excellent tool for the measurement of GA area and width, and for the detection of foveal sparing.

Appearance of medium-large drusen and reticular pseudodrusen on adaptive optics in age-related macular degeneration.

PURPOSE: To investigate the appearance of medium-large drusen and reticular pseudodrusen on adaptive optics (AO). METHODS: In 14 consecutive patients, AO infrared (IR) images were overlaid with confocal scanning-laser-ophthalmoscope IR reflectance images and IR-referenced spectral-domain optical coherence tomography. RESULTS: In eight eyes of six patients, a total of 19 images of medium-large drusen were investigated by AO imaging. En face AO revealed medium-large drusen as highly hyper-reflective round/oval lesions, always centred and/or surrounded by a continuous/discontinuous hyporeflectivity. Cone photoreceptors were detected overlying drusen, appearing either as continuous 'bright' hyper-reflective dots over a 'dark' hyporeflective background, or as continuous 'dark' hyporeflective dots over a 'bright' hyper-reflective background. In eight eyes from eight patients, a total of 14 images of pseudodrusen were investigated by AO imaging. En face AO revealed reticular pseudodrusen as isoreflective lesions, always surrounded by a continuous/discontinuous hyporeflectivity. Cone photoreceptors were detected overlying pseudodrusen as 'bright' hyper-reflective dots over either a hyporeflective or isoreflective background. No 'dark' hyporeflective dots were detected in eyes with reticular pseudodrusen only. Cone photoreceptors were counted on the border of the drusen and pseudodrusen, respectively, and in a visibly healthy zone in its absolute vicinity. A similar decrease in cone appearance was observed for drusen and pseudodrusen (15.7% vs 16.2%). CONCLUSIONS: AO allows differences in reflectivity between medium-large drusen and reticular pseudodrusen to be appreciated. The cone mosaics may be detected as continuous 'bright' hyper-reflective dots overlying/on the border of drusen and pseudodrusen deposits, and possibly as continuous 'dark' hyporeflective dots overlying drusen only.

Multimodal analysis of the progression of Best vitelliform macular dystrophy.

PURPOSE: To investigate the multimodal morphological features in the different stages of Best vitelliform macular dystrophy (VMD) in subjects harboring mutations in the BEST1 gene, and their changes during the progression of the disease. METHODS: In this retrospective observational study performed between January 2007 and December 2012, 21 patients (42 eyes) with Best VMD from eight families with the BEST1 mutation were included. Best-corrected visual acuity (BCVA), fundus autofluorescence (FAF), and spectral domain optical coherence tomography (SDOCT) were evaluated at study entry and at last visit. RESULTS: The mean age of patients was 26.3±17.4 years. Seven new missense mutations in BEST1 were identified. Mean follow-up was 41.1±18.5 months. Mean BCVA was 0.34±0.34 LogMAR at study entry and 0.32±0.33 LogMAR at last follow-up visit (p = 0.2). The overall lesion area on FAF increased from 6.62±4.9 mm² to 7.34±6.1 mm² (p = 0.05). At study entry, on SD-OCT, photoreceptor inner segment ellipsoid portion (ellipsoid zone, EZ) was normal in 15 eyes, disrupted in 14 eyes, and absent in 13 eyes. In two eyes, EZ changed from normal to disrupted during follow-up. Three eyes of three patients showing pseudohypopyon lesions at study entry progressed to vitelliruptive lesions at the last follow-up visit. Three eyes of three patients showing vitelliruptive lesion at study entry reverted to pseudohypopyon lesion with overall enlargement of the lesion size. CONCLUSIONS: Multimodal analysis allowed documenting a continuous material accumulation and reabsorption in Best VMD progression. Blue FAF and SD-OCT could represent noninvasive imaging techniques to monitor Best VMD.

Impact of reticular pseudodrusen on macular function.

PURPOSE: To investigate the impact of reticular pseudodrusen on macular function using microperimetry. METHODS: Eighteen consecutive patients (18 eyes) with reticular pseudodrusen (Group 1), and without medium/large drusen, underwent microperimetry. Eighteen age-matched and sex-matched subjects (18 eyes) with typical drusen and without pseudodrusen (Group 2) also underwent microperimetry. Macular sensitivity was assessed by microperimetry and compared between the two Groups. RESULTS: Mean age of patients with reticular pseudodrusen and with typical drusen was 77.3 ± 6.8 years and 75.0 ± 9.9 years, respectively (P = 0.4), and 61.1% and 61.1% were women, respectively. Mean best-corrected visual acuity was 0.14 ± 0.09 logarithm of the minimum angle of resolution and 0.13 ± 0.09 logarithm of the minimum angle of resolution (P = 0.8) in Group 1 and Group 2, respectively. Microperimetry revealed a significant difference in overall mean macular sensitivity ("square 7 × 7"; 49 points) between Group 1 and Group 2 (5.9 ± 1.7 dB vs. 8.8 ± 2.4 dB, P < 0.001). Both mean central macular sensitivity ("square 3 × 3"; 9 points) and mean peripheral macular microperimetric sensitivity (overall "square 7 × 7" - central "square 3 × 3"; 40 points) were significantly reduced in Group 1 compared with Group 2 (central macular sensitivity: 6.9 ± 1.7 dB vs. 8.9 ± 2.6 dB in Group 1 and Group 2, respectively; P = 0.01; peripheral macular sensitivity: 5.7 ± 1.8 dB vs. 8.7 ± 2.3 dB in Group 1 and Group 2, respectively; P < 0.001). In Group 1, mean peripheral sensitivity was reduced when compared with mean central sensitivity (5.7 ± 1.8 dB vs. 6.9 ± 1.7 dB, P = 0.01), whereas in Group 2, mean sensitivity was similar in both peripheral and central macula (8.7 ± 2.3 dB vs. 8.9 ± 2.6 dB, P = 0.4). CONCLUSION: Eyes with reticular pseudodrusen present a greater extent of reduced sensitivity than eyes with typical drusen.

Appearance of regressing drusen on optical coherence tomography in age-related macular degeneration.

OBJECTIVE: To describe and interpret a multilaminar sub-retinal pigment epithelium (RPE) intense hyper-reflectivity observed in vivo in eyes clinically diagnosed with regressing drusen. DESIGN: Observational case series. PARTICIPANTS: Twenty-three consecutive patients clinically diagnosed with regressing calcific drusen due to nonneovascular age-related macular degeneration (AMD). METHODS: Patients were submitted to confocal scanning laser ophthalmoscopy (cSLO) fundus imaging and "eye-tracked" spectral-domain optical coherence tomography (SD-OCT). MAIN OUTCOME MEASURES: Localization and possible origin and composition of the multilaminar sub-RPE hyperreflectivity. RESULTS: Thirty eyes of 23 consecutive patients (8 male and 15 female; mean age, 82.7±10.1 years) showing on SD-OCT an intense multilaminar sub-RPE hyperreflectivity, which matched with regressing calcific drusen as visualized by cSLO infrared (IR) and MultiColor (Heidelberg Engineering, Heidelberg, Germany) images, were included in this study. The multilaminar hyperreflectivity was found to localize to beneath the RPE and above the outer Bruch's membrane (oBM) layer. A mean of 1.2 multilaminar sub-RPE hyperreflectivities per SD-OCT scan were identified by 2 readers. The SD-OCT analysis allowed the 2 readers to describe 3 different types of sub-RPE hyperreflectivity. "Type 1" laminar/multilaminar hyperreflectivity (found in 24 scans of 12 eyes) was characterized by an intense signal originating from what we interpreted as the inner Bruch's membrane (iBM) layer. "Type 2" multilaminar hyperreflectivity (found in 130 scans of 27 eyes) was characterized by an intense signal originating from the oBM layer. "Type 3" multilaminar fragmented hyperreflectivity (found in 22 scans of 11 eyes) was characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. CONCLUSIONS: We describe a novel SD-OCT finding appearing as multilaminar sub-RPE intense hyper-reflectivity observed in vivo in eyes with regressing drusen. This multilaminar sub-RPE hyperreflectivity could be interpreted as layers of lipid mineralization (membranous debris also called "lipoprotein-derived debris" developing calcification), internal and external to the basement membrane, with different degrees of fragmentation.

A genome scan conducted in a multigenerational pedigree with convergent strabismus supports a complex genetic determinism.

A genome-wide linkage scan was conducted in a Northern-European multigenerational pedigree with nine of 40 related members affected with concomitant strabismus. Twenty-seven members of the pedigree including all affected individuals were genotyped using a SNP array interrogating > 300,000 common SNPs. We conducted parametric and non-parametric linkage analyses assuming segregation of an autosomal dominant mutation, yet allowing for incomplete penetrance and phenocopies. We detected two chromosome regions with near-suggestive evidence for linkage, respectively on chromosomes 8 and 18. The chromosome 8 linkage implied a penetrance of 0.80 and a rate of phenocopy of 0.11, while the chromosome 18 linkage implied a penetrance of 0.64 and a rate of phenocopy of 0. Our analysis excludes a simple genetic determinism of strabismus in this pedigree.

Functional characterization and multimodal imaging of treatment-naive "quiescent" choroidal neovascularization.

PURPOSE: To investigate the multimodal morphological and functional characteristics of treatment-naïve "quiescent" choroidal neovascularization (CNV) secondary to AMD. METHODS: Eleven patients with treatment-naïve "quiescent" CNV that consecutively presented over a 6-month period, underwent multimodal morphological and functional assessment (including indocyanine green angiography [ICGA], spectral-domain optical coherence tomography [SD-OCT], microperimetry, and preferential hyperacuity perimeter [PHP]). For the purpose of this study, asymptomatic previously untreated CNVs showing absence of intraretinal/subretinal exudation in two consecutive visits (at least 6 months apart) were defined as treatment-naïve "quiescent" CNV. RESULTS: Eleven eyes of 11 patients (9 females; mean age 76.5 ± 8.5 years) were included. On fluorescein angiography (FA), "quiescent" CNVs appeared as late speckled hyperfluorescent lesions lacking well-demarcated borders. Mid-late phase ICGA allowed visualizing the hyperfluorescent "quiescent" CNV network and delineating the plaque. Mean lesion area (mid-late phase ICGA) appeared larger compared with earliest previous examination performed 23.8 ± 16.0 months before (3.24 ± 2.51 mm(2) vs. 3.52 ± 2.46 mm(2), respectively; P = 0.01). SD-OCT revealed, at the site of "quiescent" CNV, an irregularly slightly elevated RPE, without hyporeflective intraretinal/subretinal fluid, showing a major axis in the horizontal plane, which was characterized by collections of moderately reflective material in the sub-RPE space and clear visualization of the hyperreflective Bruch's membrane. Hypergeometric distribution revealed a significant correlation between microperimetry and PHP with respect to locations of "affected areas" (P = 0.001). CONCLUSIONS: "Quiescent" CNVs are sub-RPE CNVs secondary to AMD, showing absence of intraretinal/subretinal exudation on repeated OCT. "Quiescent" CNVs enlarge over time and may contribute to local reduced retinal sensitivity and metamorphopsia.