Foveolar Drusen Decrease Fixation Stability in Pre-Symptomatic AMD.

Purpose: This study aims at linking subtle changes of fixational eye movements (FEM) in controls and in patients with foveal drusen using adaptive optics retinal imaging in order to find anatomo-functional markers for pre-symptomatic age-related macular degeneration (AMD). Methods: We recruited 7 young controls, 4 older controls, and 16 patients with presymptomatic AMD with foveal drusen from the Silversight Cohort. A high-speed research-grade adaptive optics flood illumination ophthalmoscope (AO-FIO) was used for monocular retinal tracking of fixational eye movements. The system allows for sub-arcminute resolution, and high-speed and distortion-free imaging of the foveal area. Foveal drusen position and size were documented using gaze-dependent imaging on a clinical-grade AO-FIO. Results: FEM were measured with high precision (RMS-S2S = 0.0015 degrees on human eyes) and small foveal drusen (median diameter = 60 µm) were detected with high contrast imaging. Microsaccade amplitude, drift diffusion coefficient, and ISOline area (ISOA) were significantly larger for patients with foveal drusen compared with controls. Among the drusen participants, microsaccade amplitude was correlated to drusen eccentricity from the center of the fovea. Conclusions: A novel high-speed high-precision retinal tracking technique allowed for the characterization of FEM at the microscopic level. Foveal drusen altered fixation stability, resulting in compensatory FEM changes. Particularly, drusen at the foveolar level seemed to have a stronger impact on microsaccade amplitudes and ISOA. The unexpected anatomo-functional link between small foveal drusen and fixation stability opens up a new perspective of detecting oculomotor signatures of eye diseases at the presymptomatic stage.

Multimodal phenotyping of foveal hypoplasia in albinism and albino-like conditions: a pediatric case series with adaptive optics insights.

Aim of the present study is to evaluate the relationship between genetic and phenotypic data in a series of patients affected by grade I and II of foveal hypoplasia with stable fixation and good visual acuity using multimodal imaging techniques. All patients underwent complete clinical and instrumental assessment including structural Optical Coherence Tomography (OCT), OCT Angiography and Adaptive Optics (AO) imaging. Central macular thickness (CMT), inner nuclear layer (INL), vessel density in superficial capillary plexus were the main variables evaluated with OCT technology. Cone density, cone spacing, cone regularity, cone dispersion and angular density were the parameters evaluated with AO. Genetic evaluation and trio exome sequencing were performed in all affected individuals. Eight patients (3 males and 5 females) with a mean age of 12.62 years (range 8-18) were enrolled. The mean best corrected visual acuity (BCVA) was 0.18 ± 0.13 logMAR, mean CMT was 291.9 ± 16.6 µm and INL was 26.2 ± 4.6 µm. The absence of a foveal avascular zone (FAZ) was documented by examination of OCT-A in seven patients in the superficial capillary plexus. However, there was a partial FAZ in the deep plexus in patients P5 and P8. Of note, all the patients presented with major retinal vessels clearly crossing the foveal center. All individuals exhibited a grade I or II of foveal hypoplasia. In 5 patients molecular analyses showed an extremely mild form of albinism caused by compound heterozygosity of a TYR pathogenic variant and the hypomorphic p.[Ser192Tyr;Arg402Gln] haplotype. One patient had Waardenburg syndrome type 2A caused by a de novo variant in MITF. Two patients had inconclusive molecular analyses. All the patients displayed abnormalities on OCT-A. Photoreceptor count did not differ from normal subjects according to the current literature, but qualitative analysis of AO imaging showed distinctive features likely related to an abnormal pigment distribution in this subset of individuals. In patients with foveal hypoplasia, genetic and multimodal imaging data, including AO findings, can help understand the physiopathology of the foveal hypoplasia phenotype. This study confirms that cone density and visual function can both be preserved despite the absence of a pit.

Detection of capillary abnormalities in early diabetic retinopathy using scanning laser ophthalmoscopy and optical coherence tomography combined with adaptive optics.

This study tested if a high-resolution, multi-modal, multi-scale retinal imaging instrument can provide novel information about structural abnormalities in vivo. The study examined 11 patients with very mild to moderate non-proliferative diabetic retinopathy (NPDR) and 10 healthy subjects using fundus photography, optical coherence tomography (OCT), OCT angiography (OCTA), adaptive optics scanning laser ophthalmoscopy (AO-SLO), adaptive optics OCT and OCTA (AO-OCT(A)). Of 21 eyes of 11 patients, 11 had very mild NPDR, 8 had mild NPDR, 2 had moderate NPDR, and 1 had no retinopathy. Using AO-SLO, capillary looping, inflections and dilations were detected in 8 patients with very mild or mild NPDR, and microaneurysms containing hyperreflective granular elements were visible in 9 patients with mild or moderate NPDR. Most of the abnormalities were seen to be perfused in the corresponding OCTA scans while a few capillary loops appeared to be occluded or perfused at a non-detectable flow rate, possibly because of hypoperfusion. In one patient with moderate NPDR, non-perfused capillaries, also called ghost vessels, were identified by alignment of corresponding en face AO-OCT and AO-OCTA images. The combination of multiple non-invasive imaging methods could identify prominent microscopic abnormalities in diabetic retinopathy earlier and more detailed than conventional fundus imaging devices.

Photocoagulation or sham laser in addition to conventional anti-VEGF therapy in macular edema associated with TelCaps due to diabetic macular edema or retinal vein occlusion (TalaDME): a study protocol for a multicentric, French, two-group, non-commercial, active-control, observer-masked, non-inferiority, randomized controlled clinical trial.

BACKGROUND: Macular edema (ME) results from hyperpermeability of retinal vessels, leading to chronic extravasation of plasma components into the retina and hence potentially severe visual acuity loss. Current standard of care consists in using intravitreal injections (IVI), which results in a significant medical and economic burden. During diabetic retinopathy (DR) or retinal vein occlusion (RVO), it has recently been shown that focal vascular anomalies (capillary macro-aneurysms, also termed TelCaps) for telangiectatic capillaries may play a central role in the onset, early recurrence, and/or persistence of ME. Since targeted photocoagulation of TelCaps may improve vision, identification, and photocoagulation of TelCaps, it may represent a way to improve management of ME. OBJECTIVE: The Targeted Laser in (Diabetic) Macular Edema (TalaDME) study aims to evaluate whether ICG-guided targeted laser (IGTL), in association with standard of care by IVI, allows reducing the number of injections during the first year of treatment compared with IVI only, while remaining non-inferior for visual acuity. METHODS: TalaDME is a French, multicentric, two-arms, randomized, sham laser-controlled, double-masked trial evaluating the effect of photocoagulation of TelCaps combined to IVI in patients with ME associated with TelCaps. Patients with vision loss related to center involved ME secondary to RVO or DR and presenting TelCaps are eligible. Two hundred and seventy eyes of 270 patients are randomized in a 1:1 ratio to standard care, i.e., IVI of anti-VEGF solely (control group) or combined with IGTL therapy (experimental group). Stratification is done on the cause of ME (i.e., RVO versus diabetes). Anti-VEGF IVI are administered to both groups monthly for 3 months (loading dose) and then with a pro re nata regimen with a monthly follow-up for 12 months. The primary endpoint will be the number of IVI and the change in visual acuity from baseline to 12 months. Secondary endpoints will be the changes in central macular thickness, impact on quality of life, cost of treatment, and incremental cost-utility ratio in each groups. KEY SAFETY: Rare but severe AE linked to the use of IVI and laser, and previously described, are expected. In the sham group, rescue laser photocoagulation may be administered by the unmasked investigator if deemed necessary at month 3. DISCUSSION: The best management of ME associated with TelCaps is debated, and there have been no randomized study designed to answer this question. Given the fact that TelCaps may affect 30 to 60% of patients with chronic ME due to DR or RVO, a large number of patients could benefit from a specific management of TelCaps. TalaDME aims to establish the clinical and medico-economic benefits of additional targeted laser. The results of TalaDME may raise new recommendations for managing ME and impact healthcare costs. TRIAL REGISTRATION: EudraCT: 2018-A00800-55/ NCT03751501. Registration date: Nov. 23, 2018.

Comparison between Two Adaptive Optics Methods for Imaging of Individual Retinal Pigmented Epithelial Cells.

The Retinal Pigment Epithelium (RPE) plays a prominent role in diseases such as age-related macular degeneration, but imaging individual RPE cells is challenging due to their high absorption and low autofluorescence emission. The RPE lies beneath the highly reflective photoreceptor layer (PR) and contains absorptive pigments, preventing direct backscattered light detection when the PR layer is intact. Here, we used near-infrared autofluorescence adaptive optics scanning laser ophthalmoscopy (NIRAF AOSLO) and transscleral flood imaging (TFI) in the same healthy eyes to cross-validate these approaches. Both methods revealed a consistent RPE mosaic pattern and appeared to reflect a distribution of fluorophores consistent with findings from histological studies. Interestingly, even in apparently healthy RPE, we observed dynamic changes over months, suggesting ongoing cellular activity or alterations in fluorophore distribution. These findings emphasize the value of NIRAF AOSLO and TFI in understanding RPE morphology and dynamics.

Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Early remodeling and loss of light-induced dilation of retinal small arteries in CADASIL.

A major hurdle to therapeutic development in cerebral small vessel diseases is the lack of in-vivo method that can be used repeatedly for evaluating directly cerebral microvessels. We hypothesised that Adaptive Optics (AO), which allows resolution images up to 1-2 µm/pixel at retinal level, could provide a biomarker for monitoring vascular changes in CADASIL, a genetic form of such condition. In 98 patients and 35 healthy individuals, the wall to lumen ratio (WLR), outer and inner diameter, wall thickness and wall cross-sectional area were measured in a parapapillary and/or paramacular retinal artery. The ratio of vessel diameters before and after light flicker stimulations was also calculated to measure vasoreactivity (VR). Multivariate mixed-model analysis showed that WLR was increased and associated with a larger wall thickness and smaller internal diameter of retinal arteries in patients. The difference was maximal at the youngest age and gradually reduced with aging. Average VR in patients was less than half of that of controls since the youngest age. Any robust association was found with clinical or imaging manifestations of the disease. Thus, AO enables the detection of early functional or structural vascular alterations in CADASIL but with no obvious link to the clinical or imaging severity.

Light sheet fluorescence microscopy of cleared human eyes.

We provide here a procedure enabling light sheet fluorescence microscopy (LSFM) of entire human eyes after iDISCO + -based clearing (ClearEye) and immunolabeling. Demonstrated here in four eyes, post-processing of LSFM stacks enables three-dimensional (3D) navigation and customized display, including en face viewing of the fundus similarly to clinical imaging, with resolution of retinal capillaries. This method overcomes several limitations of traditional histology of the eyes. Tracing of spatially complex structures such as anterior ciliary vessels and Schlemm's canal was achieved. We conclude that LSFM of immunolabeled human eyes after iDISCO + -based clearing is a powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.

Multimodal high-resolution retinal imaging using a camera-based DMD-integrated adaptive optics flood-illumination ophthalmoscope.

We demonstrate the feasibility of a multimodal adaptive optics flood-illumination ophthalmoscope, able to provide both bright-field and dark-field images (such as phase contrast). The multimodality was made possible by integrating a digital micromirror device (DMD) at the illumination path to project a sequence of complementary high-resolution patterns into the retina. Through a versatile post-processing method that digitally selects backscattered or multiply scattered photons, we were able: (1) to achieve up to four-fold contrast increase of bright-field images when imaging the photoreceptor mosaic and nerve fibers; and (2) to visualize translucent retinal features such as capillaries, red blood cells, vessel walls, ganglion cells, and photoreceptor inner segments through phase contrast.

Severe macular complications in glaucoma: high-resolution multimodal imaging characteristics and review of the literature.

PURPOSE: To describe imaging characteristics of severe macular complications occurring in glaucoma and discuss available treatments. METHODS: Retrospective case series of glaucomatous patients with macular retinoschisis (MR) and/or serous retinal detachment (SRD). Patients underwent a complete ophthalmological examination and multimodal imaging including retinography, SD-OCT, fluorescein and indocyanine green angiography (FA & ICGA) and adaptive optics (AO). RESULTS: Ten eyes (8 patients) were included. Initial BCVA was 1.04 ± 1.12 logMAR and IOP was 24.0 ± 9.3mmHg. All eyes presented with MR while SRD was present in 5 eyes (5 patients), with a central macular thickness of 573 ± 152 µm. FA and ICGA allowed to exclude leakage in all cases. A focal lamina cribrosa defect (LCD) was found in four eyes (4 patients) using OCT, with AO providing en-face visualization of the defect in one eye. Outer retinal hole was present in 3 eyes (3 patients). No visual improvement or resolution of the macular retinoschisis was observed in eyes with medical or surgical IOP control (N = 9). Vitrectomy with internal membrane limiting peeling and gas tamponade was performed in one eye with good visual results. CONCLUSIONS: Multimodal high-resolution imaging is essential to diagnose severe macular complications associated with advanced glaucoma.

Adaptive Optics Flood Illumination Ophthalmoscopy in Nonhuman Primates: Findings in Normal and Short-term Induced Detached Retinae.

Objective: To describe adaptive optics flood illumination ophthalmoscopy (AO-FIO) of the photoreceptor layer in normal nonhuman primates (NHPs) and in the case of a short-term induced retinal detachment (RD). Design: Longitudinal fundamental research study. Subjects: Four NHPs were used to image normal retinae with AO-FIO (in comparison with 4 healthy humans); 2 NHPs were used to assess the effects of RD. Intervention: The photoreceptor layer (cone mosaic metrics, including cone density, cone spacing, and cone regularity) was followed with AO-FIO imaging (rtx1, Imagine Eyes) during a surgically induced RD in 2 NHPs using a vehicle solution containing dimethyl sulfoxide, classically used as a chemical solvent. We also performed functional testing of the retina (full-field and multifocal electroretinogram [ERG]). Main Outcome Measures: Correlation of cone mosaic metrics (cone density, spacing, and regularity) between normal retinae of NHPs and humans, and cone metrics, power spectrum, and ERG wave amplitudes after RD. Results: Imaging features were very similar in terms of cone reflectivity, cell density, regularity, and spacing values, showing strong positive correlations between NHPs and humans. After RD, AO-FIO revealed several alterations of the cone mosaic slowly recovering during the 3 months after the reattachment, which were not detected functionally by ERG. Conclusions: These results demonstrate by in vivo AO-FIO imaging the transient structural changes of photoreceptors after an RD in the primate retina. They also provide an interesting illustration of the AO-FIO potential for investigating photoreceptor toxicity during preclinical studies in NHPs with a high translatability to human studies. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Retinal vascular density in CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy).

Background and objective: Retinal vascular density (VD) measured using optical coherence tomography with angiography (OCTA) has been suggested as a potential marker of intracerebral vascular changes in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). We aimed to determine whether VD is related to the clinical and imaging manifestations of the disease. Methods: OCTA was performed in 104 CADASIL patients (parallel to their clinical and imaging assessment) and in 83 healthy individuals. Results: A significant reduction of VD related to age was detected in patients and controls in the superficial and deep vascular plexus of the whole foveal or parafoveal retinal area (p<0.0001). After adjustment for age, these parameters were found significantly lower in patients than in controls (p<0.03). Multivariable analysis did not show any association between retinal VD and history of stroke, modified Rankin Scale or Mini-Mental Status Examination scores. No significant association was found with MRI lesions either. Conclusion: In CADASIL, retinal VD is decreased early and progresses with ageing but does not appear related to the severity of clinical or imaging manifestations.

Change in Cone Structure Over 24 Months in USH2A-Related Retinal Degeneration.

PURPOSE: To describe cone structure changes using adaptive optics scanning laser ophthalmoscopy (AOSLO) in the Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A) study. DESIGN: Multicenter, longitudinal natural history study. METHODS: AOSLO images were acquired at 4 centers, twice at baseline and annually for 24 months in this natural history study. For each eye, at least 10 regions of interest (ROIs) with ≥50 contiguous cones were analyzed by masked, independent graders. Cone spacing Z-scores, standard deviations from the normal mean at the measured location, were compared between graders and tests at baseline. The association of cone spacing with clinical characteristics was assessed using linear mixed effects regression models weighted by image quality score. Annual rates of change were calculated based on differences between visits. RESULTS: Fourteen eyes of 14 participants were imaged, with 192 ROIs selected at baseline. There was variability among graders, which was greater in images with lower image quality score (P < .001). Cone spacing was significantly correlated with eccentricity, quality score, and disease duration (P < .02). On average, the cone spacing Z-score increased 0.14 annually (about 9%, P < .001). We observed no significant differences in rate of change between disease type (Usher syndrome or retinitis pigmentosa), imaging site, or grader. CONCLUSIONS: Using current methods, the analysis of quantitative measures of cone structure showed some challenges, yet showed promise that AOSLO images can be used to characterize progressive change over 24 months. Additional multicenter studies using AOSLO are needed to advance cone mosaic metrics as sensitive outcome measures for clinical trials. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Revisiting malignant hypertension: rationale and design of the 'HAMA cohort', on behalf of the ESH working group 'hypertension and the kidney'.

BACKGROUND: Malignant hypertension has not disappeared and is associated with a poor prognosis. Yet, so far, it has received limited attention from the medical community. Guidelines are mainly based on expert consensus and low quality evidences. METHOD: We set up a prospective, multicenter, observational cohort of patients with malignant hypertension. We collect at admission medical history, demographic data, ongoing treatment, clinical parameters, symptoms, care pathways, target organ status and at discharge and during follow up treatment administrated, adverse events, blood pressure level, target organ status. We aim to recruit 500 patients with malignant hypertension in 5 years, with a 5-year follow-up. Our primary objective is to assess the 5 years prognosis of these patients. DISCUSSION: The HAMA (Hypertension Arterielle MAligne, meaning malignant hypertension) registry aims to describe the epidemiology and to assess the prognosis of malignant hypertension in a contemporary multidisciplinary cohort, with emphasis on the diversity of current management and care pathway among the different medical specialties. It may help improving our pathophysiological knowledge, and pave the way to update the definition of this particular form of hypertension. The multidisciplinary network developed in the wake of this project is expected to facilitate the set up therapeutic trials, laying the ground for evidence-based recommendations.

Confocal microscopy of telangiectatic capillaries (TelCaps) and other features of microvascular remodeling following branch retinal vein occlusion.

Branch retinal vein occlusion (BRVO) is a frequent retinal vascular disease that may cause extensive microvascular remodeling leading to severe visual impairment. Little is known regarding the histology of non-neovascular microvascular remodeling. Here, we examined by confocal microscopy the structure of retinal microvessels of a donor eye with longstanding BRVO. The post-mortem retina of a 91-year-old woman that had superotemporal BRVO for 2 years was examined by confocal microscopy after anti-collagen IV (collIV), alpha-smooth muscle cell (αSMA), and anti-von Willebrand factor (vWf) immunolabeling. In the retinal quadrant affected by BRVO, extensive vascular remodeling affected all vessels, from arterioles to venules, including the foveal avascular zone. Most affected vessels were either irregularly dilated or, on the opposite, reduced to micrometric-size CollIV positive, vWf negative, nuclear-staining negative strings. Telangiectatic capillaries of various sizes and shapes were seen, the largest one (233 µm) being located in the parafoveal area. Some telangiectatic capillaries had a thick, multilayered vWf- and CollIV-positive wall, that often occluded the lumen. Other features included double-channeled arterioles. The majority of microvascular abnormalities were devoid of nuclear staining, suggesting extensive loss of endothelial cells. We describe the spectrum of microvascular abnormalities upstream of a longstanding BRVO. This spectrum comprises a large parafoveal telangiectatic capillary corresponding to what has been previously clinically defined as TelCap. The absence of intraluminal nuclear staining in the majority of abnormal vessels raises the hypothesis that the loss of endothelial cells plays a crucial role in the development of the different manifestations of capillary remodeling. The presence of vWF in de-endothelialized vessels suggests deposition of plasma, hence that they may remain perfused. Our work may help to understand the clinical imaging features of TelCaps.

Structural and functional analysis of retinal vasculature in HANAC syndrome with a novel intronic COL4A1 mutation.

PURPOSE: Mutations of the COL4A1 gene, a major structural protein of vessels, may cause hereditary angiopathy with nephropathy, aneurysms and muscle cramps (HANAC) syndrome. The vascular structure and function of patients with HANAC is poorly known. Here, we report a family with HANAC syndrome associated to a previously unreported mutation in COL4A1. The structure and function of retinal vessels were detailed by adaptive optics ophthalmoscopy (AOO) and optical coherence tomography (OCT) angiography. METHODS: Clinical data from six affected individuals (43 to 72 years old) from a single family comprising two generations were collected. Imaging charts including conventional fundus imaging, OCT-angiography and AOO in static and dynamic (flicker) mode were reviewed. DNA sequencing was done in the proband. RESULTS: DNA sequencing of the proband revealed a heterozygous deletion of COL4A1 (NM_001845) at position 1120 in the intron 20 resulting in the loss of splicing donor site for exon 20 (c.1120 + 2_1120 + 8del heterozygote). Four patients had arterial hypertension, and three had kidney dysfunction, one of which under dialysis. By fundus examination, five had typical retinal arteriolar tortuosity with arteriolar loops. Wall-to-lumen ratio of arteries was within normal limits, that is, lower than expected for hypertensive patients. Several foci of arteriolar irregularities were noted in the two oldest patients. In three affected subjects, evaluation of the neurovascular coupling showed a higher flicker-induced vasodilation than a control population (6 % to 11 %; n < 5 %). CONCLUSIONS: Structural and dynamic analysis of retinal vessels in a HANAC family bearing a previously unreported intronic COL4 mutation was done. In addition to arteriolar tortuosity, we found reduced wall-to-lumen ratio, arteriolar irregularity and increased vasodilatory response to flicker light. These abnormalities were more marked in the oldest subjects. This abnormal flicker response affected also non-tortuous arteries, suggesting that microvascular dysfunction extends beyond tortuosity. Such explorations may help to better vascular dysfunction related to HANAC and hence better understand the mechanisms of end-organ damage.

Three-dimensional characterization of developing and adult ocular vasculature in mice using in toto clearing.

The ocular vasculature is critically involved in many blinding diseases and is also a popular research model for the exploration of developmental and pathological angiogenesis. The development of ocular vessels is a complex, finely orchestrated sequence of events, involving spatial and temporal coordination of hyaloid, choroidal and retinal networks. Comprehensive studies of the tridimensional dynamics of microvascular remodeling are limited by the fact that preserving the spatial disposition of ocular vascular networks is cumbersome using classical histological procedures. Here, we demonstrate that light-sheet fluorescence microscopy (LFSM) of cleared mouse eyes followed by extensive virtual dissection offers a solution to this problem. To the best of our knowledge, this is the first 3D quantification of the evolution of the hyaloid vasculature and of post-occlusive venous remodeling together with the characterization of spatial distribution of various cell populations in ocular compartments, including the vitreous. These techniques will prove interesting to obtain other insights in scientific questions addressing organ-wide cell interactions.

Long Term Time-Lapse Imaging of Geographic Atrophy: A Pilot Study.

Geographic atrophy (GA), the late stage of age-related macular degeneration, is a major cause of visual disability whose pathophysiology remains largely unknown. Modern fundus imaging and histology revealed the complexity of the cellular changes that accompanies atrophy. Documenting the activity of the disease in the margins of atrophy, where the transition from health to disease occurs, would contribute to a better understanding of the progression of GA. Time-lapse imaging facilitates the identification of structural continuities in changing environments. In this retrospective pilot study, we documented the long-term changes in atrophy margins by time-lapse imaging of infrared scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) images in 6 cases of GA covering a mean period of 32.8 months (range, 18-72). The mean interval between imaging sessions was 2.4 months (range, 1.4-3.8). By viewing time-lapse sequences we observed extensive changes in the pattern of marginal hyperreflective spots, which associated fragmentation, increase and/or disappearance. Over the entire span of the follow-up, the most striking changes were those affecting hyperreflective spots closest to margins of atrophy, on the non-atrophic side of the retina; a continuum between the successive positions of some of the hyperreflective spots was detected, both by SLO and OCT. This continuum in their successive positions resulted in a subjective impression of a centrifugal motion of hyperreflective spots ahead of atrophy progression. Such mobilization of hyperreflective spots was detected up to several hundred microns away from atrophic borders. Such process is likely to reflect the inflammatory and degenerative process underlying GA progression and hence deserves further investigations. These results highlight the interest of multimodal time-lapse imaging to document cell-scale dynamics during progression of GA. Clinical Trial Registration: clinicaltrials.gov, identifier: NCT04128150 and NCT04129021.