Perivenular Capillary Loss: An Early, Quantifiable Change in Macular Telangiectasia Type 2.

Purpose: To evaluate differences in parafoveal vascular density surrounding arterioles and venules in type 2 macular telangiectasia (MacTel). Methods: Thirty-seven eyes (20 subjects) diagnosed with MacTel and 16 healthy eyes (10 subjects) were imaged with optical coherence tomography angiography between March 2016 and June 2019 in this single-center, observational, cross-sectional study. Arterioles and venules were manually identified, and perivascular density was generated using a custom MATLAB code. The primary outcome measure was the ratio of periarteriolar to perivenular vascular density (arteriovenous [A/V] capillary ratio) in the superficial and deep capillary plexuses across MacTel stages. The main secondary outcome measures were overall parafoveal vascular density (VD), periarteriolar VD, and perivenular VD. Results: In the superficial capillary plexus (SCP), the A/V capillary ratio was significantly higher in MacTel subjects than controls (0.914 vs. 0.892; P = 0.0044). The greatest differences occurred between controls and nonproliferative MacTel subjects without optical coherence tomography evidence of disease (P = 0.0055). A/V capillary ratios progressed in a nonlinear fashion with MacTel severity, increasing from nonproliferative disease (0.912) to intraretinal proliferative disease (0.931), then decreasing in subretinal proliferative disease (0.905). Parafoveal VD in the SCP was lower in MacTel subjects than controls only in subretinal proliferative disease (P = 0.0130). Conclusions: The A/V capillary ratio of the SCP is a quantifiable metric of vascular pathology in MacTel that occurs earlier than decline in parafoveal VD. Elevated A/V capillary ratios in MacTel are consistent with an early, disproportionately perivenular disruption in the SCP. Translational Relevance: Findings inform MacTel pathogenesis through revealing early perivenular capillary loss and offer a new quantitative metric for earliest stage MacTel.

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

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

Visible-light optical coherence tomography oximetry based on circumpapillary scan and graph-search segmentation.

Visible-light optical coherence tomography (vis-OCT) enables retinal oximetry by measuring the oxygen saturation of hemoglobin (sO2) from within individual retinal blood vessels. The sO2 calculation requires reliable estimation of the true spectrum of backscattered light from the posterior vessel wall. Unfortunately, subject motion and image noise make averaging from multiple A-lines at the same depth position challenging, and lead to inaccurate sO2 estimation. In this study, we developed an algorithm to reliably extract the backscattered light's spectrum. We used circumpapillary scanning to sample the vessels repeatedly at the same location. A combination of cross-correlation and graph-search based segmentation extracted the posterior wall locations. Using measurements from 100 B-scans as a gold standard, we demonstrated that our method achieved highly accurate measures of sO2 with minimal bias. In addition, we also investigated how the number of repeated measurements affects the accuracy of sO2 measurement. Our method sets the stage for large-scale studies of retinal oxygenation in animals and humans.

The Correlation of Pars Plana Incision and Transient Hypotony After Silicone Oil Removal.

BACKGROUND AND OBJECTIVE: To evaluate the correlation between pars plana incision and transient hypotony after silicone oil removal in aphakic eyes PATIENTS AND METHODS:Twenty-two patients with aphakia and a high degree of myopia with silicone oil tamponade were recruited for this prospective study and randomly scheduled to two groups for silicone oil removal: 3.5-mm corneal incision with suture corneal or 20-gauge pars plana incision with suture. Intraocular pressure (IOP) was measured and fundus and anterior structure were examined preoperatively on the first, third, and seventh postoperative day and at 1 month after surgery. RESULTS: IOPs in the pars plana group were significantly lower than the corneal group on the first and third day after surgery (P < .001). Nine of the 11 patients in the pars plana group suffered ciliary detachment accompanied by hypotony (IOP < 8 mm Hg), among whom three patients exhibited excessive hypotony (IOP < 5 mm Hg), whereas the ciliary bodies remained tightly attached and IOP was normal in the corneal group. Among these nine patients, six eyes were observed to have multiple silicone oil granules in the supraciliary cavity, three exhibited gaps of pars plana below the scleral incision site, and two had vitreous hemorrhage. Hypotony spontaneously relieved at the seventh postoperative day for all patients. CONCLUSION: Pars plana incision is the crucial cause of ciliary detachment and consequent transient hypotony after silicone oil removal. Using corneal limbus incision in patients with aphakic eyes to avoid the par plana incision is expected to reduce the incidence of hypotony with minimal complications. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e44-e51.].

Projection-Resolved OCT Angiography of Microvascular Changes in Paracentral Acute Middle Maculopathy and Acute Macular Neuroretinopathy.

Purpose: To identify the microvascular changes associated with paracentral acute middle maculopathy (PAMM) and acute macular neuroretinopathy (AMN) and to improve our understanding of the relevant involvement of the three retinal capillary plexuses using projection-resolved optical coherence tomography angiography (PR-OCTA). Methods: This was a retrospective study of 18 eyes with AMN or PAMM imaged with OCTA. We used cross-sectional PR-OCTA to localize reduced flow signal to the superficial (SCP), middle (MCP), or deep capillary plexus (DCP) or choriocapillaris that corresponded to inner retinal PAMM or outer retinal AMN lesions on OCT. Results: Five eyes with AMN showed outer retinal disruption on OCT associated with reduced DCP flow signal. All three eyes with AMN and follow-up had recovery of DCP flow. Thirteen eyes with PAMM showed middle retinal disruption on OCT associated with reduced flow signal in both the MCP and DCP. Of these, five also had reduced flow signal in the SCP. All 10 eyes with PAMM and follow-up showed variable recovery of flow signal in one or more plexuses. PAMM reperfusion was primarily arterial in nature. Three eyes with PAMM and no evidence of MCP reperfusion experienced severe thinning of the inner nuclear layer (INL), while seven eyes with robust MCP flow signal recovery showed relative preservation of INL thickness. Conclusions: Using PR-OCTA, we found that AMN was associated with reduced DCP flow signal, while PAMM was associated with reduced MCP and DCP flow signal and occasionally the SCP. The MCP appears to be important in sustaining INL thickness in these eyes.

Volume-Rendered Projection-Resolved OCT Angiography: 3D Lesion Complexity Is Associated With Therapy Response in Wet Age-Related Macular Degeneration.

Purpose: To explore whether quantitative three-dimensional (3D) analysis of choroidal neovascularization (CNV) using projection-resolved optical coherence tomography angiography (PR-OCTA) is associated with treatment response in neovascular age-related macular degeneration (nAMD). Methods: Retrospective, cross-sectional study of 51 eyes of 49 patients undergoing individualized anti-VEGF therapy for nAMD. Patients were classified as "good" or "poor" responders, requiring injections at less or more frequently than 6-week intervals, respectively. Cross-sectional PR-OCTA images were used to measure the distance between Bruch's membrane and highest CNV flow signal. The number of flow layers within the CNV and the distance between these flow layers (CNV flow thickness) were also analyzed. Two masked, independent graders measured the PR-OCTA parameters. We used 3D volume-rendered PR-OCTA to confirm the number of CNV flow layers and further evaluate CNV complexity. Results: Poor responders had significantly greater distance between Bruch's membrane and highest CNV flow signal (P < 0.01), greater number of CNV flow layers (P = 0.022), and greater CNV flow thickness (P < 0.01). Volume-rendered PR-OCTA images confirmed the number of CNV flow layers. Conclusions: Cross-sectional and 3D volume-rendered PR-OCTA provides a novel approach for quantifying CNV complexity. Our results suggest that CNV acquiring more complex 3D vascular structure are associated with more frequent long-term anti-VEGF therapy, reflecting a particular pattern of normalization or complex CNV remodeling process that characterizes these less responsive eyes.

Optical coherence tomography angiography of retinal vascular occlusions produced by imaging-guided laser photocoagulation.

Retinal vascular occlusive diseases represent a major form of vision loss worldwide. Rodent models of these diseases have traditionally relied upon a slit-lamp biomicroscope to help visualize the fundus and subsequently aid delivery of high-power laser shots to a target vessel. Here we describe a multimodal imaging system that can produce, image, and monitor retinal vascular occlusions in rodents. The system combines a spectral-domain optical coherence tomography system for cross-sectional structural imaging and three-dimensional angiography, and a fluorescence scanning laser ophthalmoscope for Rose Bengal monitoring and high-power laser delivery to a target vessel. This multimodal system facilitates the precise production of occlusions in the branched retinal veins, central retinal vein, and branched retinal arteries. Additionally, changes in the retinal morphology and retinal vasculature can be longitudinally documented. With our device, retinal vascular occlusions can be easily and consistently created, which paves the way for futures studies on their pathophysiology and therapeutic targets.

OCT angiography and visible-light OCT in diabetic retinopathy.

In recent years, advances in optical coherence tomography (OCT) techniques have increased our understanding of diabetic retinopathy, an important microvascular complication of diabetes. OCT angiography is a non-invasive method that visualizes the retinal vasculature by detecting motion contrast from flowing blood. Visible-light OCT shows promise as a novel technique for quantifying retinal hypoxia by measuring the retinal oxygen delivery and metabolic rates. In this article, we discuss recent insights provided by these techniques into the vascular pathophysiology of diabetic retinopathy. The next milestones for these modalities are large multicenter studies to establish consensus on the most reliable and consistent outcome parameters to study diabetic retinopathy.

Stochastic fluorescence switching of nucleic acids under visible light illumination.

We report detailed characterizations of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Although the fluorescent emission from nucleic acids under the visible light illumination has long been overlooked due to their apparent low absorption cross section, our quantitative characterizations reveal the high quantum yield and high photon count in individual fluorescence emission events of nucleic acids at physiological concentrations. Owing to these characteristics, the stochastic fluorescence switching of nucleic acids could be comparable to that of some of the most potent exogenous fluorescence probes for localization-based super-resolution imaging. Therefore, utilizing the principle of single-molecule photon-localization microscopy, native nucleic acids could be ideal candidates for optical label-free super-resolution imaging.

Choriocapillaris Nonperfusion is Associated With Poor Visual Acuity in Eyes With Reticular Pseudodrusen.

PURPOSE: To study choriocapillaris blood flow in age-related macular degeneration (AMD) using optical coherence tomography angiography (OCTA) and study its correlation to visual acuity (VA) in eyes with reticular pseudodrusen (RPD) vs those with drusen without RPD (drusen). DESIGN: Cross-sectional study. METHODS: Patients with either drusen or RPD in early AMD underwent OCTA imaging of the superior, inferior, and/or nasal macula. We quantified "percent choriocapillaris area of nonperfusion" (PCAN) in eyes with RPD vs those with drusen. We assessed the repeatability of PCAN and its correlations with VA. RESULTS: Twenty-nine eyes of 26 patients with RPD and 21 eyes of 16 age-matched AMD patients with drusen were included. Qualitatively, the choriocapillaris in areas with RPD showed focal dark regions without flow signal on OCTA (nonperfusion). The repeatability coefficient of PCAN was 0.49%. Eyes with RPD had significantly greater PCAN compared with eyes with drusen (7.31% and 3.88%, respectively; P < .001). We found a significant correlation between PCAN and VA for the entire dataset (r = 0.394, P = .005). When considering eyes with RPD separately, this correlation was stronger (r = 0.474, P = .009) but lost significance when considering eyes with drusen separately (r = 0.175, P = .45). CONCLUSIONS: Eyes with RPD have significantly larger areas of choriocapillaris nonperfusion compared with eyes with drusen and no RPD. The correlation between PCAN and VA in this RPD population provides a potential mechanistic explanation for vision compromise in RPD compared with other forms of drusen in AMD.

Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice.

PURPOSE: This study sought to determine the earliest time-point at which evidence of choroidal neovascularization (CNV) could be detected with visible-light optical coherence tomography angiography (vis-OCTA) in a mouse model of laser-induced CNV. METHODS: Visible light-OCTA was used to study laser-induced CNV at different time-points after laser injury to monitor CNV development and measure CNV lesion size. Measurements obtained from vis-OCTA angiograms were compared with histopathologic measurements from isolectin-stained choroidal flatmounts. RESULTS: Choroidal neovascularization area measurements between the vis-OCTA system and isolectin-stained choroidal flatmounts were significantly different in area for days 2 to 4 postlaser injury, and were not significantly different in area for days 5, 7, and 14. Choroidal neovascularization area measurements taken from the stained flatmounts were larger than their vis-OCTA counterparts for all time-points. Both modalities showed a similar trend of CNV size increasing from the day of laser injury until a peak of day 7 postlaser injury and subsequently decreasing by day 14. CONCLUSIONS: The earliest vis-OCTA can detect the presence of aberrant vessels in a mouse laser-induced CNV model is 5 days after laser injury. Visible light-OCTA was able to visualize the maximum of the CNV network 7 days postlaser injury, in accordance with choroidal flatmount immunostaining. Visible light-OCTA is a reliable tool in both detecting the presence of CNV development, as well as accurately determining the size of the lesion in a mouse laser-induced CNV model.

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

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

Inner retinal oxygen metabolism in the 50/10 oxygen-induced retinopathy model.

Retinopathy of prematurity (ROP) represents a major cause of childhood vision loss worldwide. The 50/10 oxygen-induced retinopathy (OIR) model mimics the findings of ROP, including peripheral vascular attenuation and neovascularization. The oxygen metabolism of the inner retina has not been previously explored in this model. Using visible-light optical coherence tomography (vis-OCT), we measured the oxygen saturation of hemoglobin and blood flow within inner retinal vessels, enabling us to compute the inner retinal oxygen delivery (irDO2) and metabolic rate of oxygen (irMRO2). We compared these measurements between age-matched room-air controls and rats with 50/10 OIR on postnatal day 18. To account for a 61% decrease in the irDO2 in the OIR group, we found an overall statistically significant decrease in retinal vascular density affecting the superficial and deep retinal vascular capillary networks in rats with OIR compared to controls. Furthermore, matching the reduced irDO2, we found a 59% decrease in irMRO2, which we correlated with a statistically significant reduction in retinal thickness in the OIR group, suggesting that the decreased irMRO2 was due to decreased neuronal oxygen utilization. By exploring these biological and metabolic changes in great detail, our study provides an improved understanding of the pathophysiology of OIR model.

Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow.

Capitalizing on the optical absorption of hemoglobin, photoacoustic microscopy (PAM) is uniquely capable of anatomical and functional characterization of the intact microcirculation in vivo. However, PAM of the metabolic rate of oxygen (MRO2) at the microscopic level remains an unmet challenge, mainly due to the inability to simultaneously quantify microvascular diameter, oxygen saturation of hemoglobin (sO2), and blood flow at the same spatial scale. To fill this technical gap, we have developed a multi-parametric PAM platform. By analyzing both the sO2-encoded spectral dependence and the flow-induced temporal decorrelation of photoacoustic signals generated by the raster-scanned mouse ear vasculature, we demonstrated-for the first time-simultaneous wide-field PAM of all three parameters down to the capillary level in vivo.

A Mouse Model for Laser-induced Choroidal Neovascularization.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch's membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD. Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Fully motorized optical-resolution photoacoustic microscopy.

We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 µm lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 µm with 20 A-lines and 50 Hz volumetric-scan rate over a FOV of 50 µm × 50 µm with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10 mm × 8 mm FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.