Non-invasive quantification of corneal vascularization using anterior segment optical coherence tomography angiography.

The presence of corneal vascularization (CV) interferes with the angiogenic and immune privilege of the cornea, risking rejection in eyes following keratoplasty. Pre-operative (lymph)-angioregression is a promising therapeutic approach, but objective monitoring by non-invasive CV imaging is needed. The purpose of this study was to investigate anterior-segment optical coherence tomography angiography (AS-OCTA) for CV visualization and quantification, and to show its superiority over slit-lamp photography in high-risk eyes scheduled for keratoplasty. This institutional pilot study included 29 eyes of 26 patients (51 ± 16 years, 8 female) with significant CV scheduled for keratoplasty that were imaged by slit-lamp photography (Zeiss SL 800) and AS-OCTA (Zeiss Plex Elite 9000). After manual corneal layer segmentation correction, CV maximum/relative depth was measured with the inbuilt software. Slit-lamp photographs and AS-OCTA images were compared for visualization of vascular details. Angiotool software allowed a semi-automated determination of CV-related parameters in the vascular complex of AS-OCTA images. The predominant causes of CV were the herpes simplex virus keratitis (n = 7) and chemical burn (n = 4). Visualization of vascular morphology in AS-OCTA was superior to slit-lamp photography in all except one eye. Vascular metrics including total vessel length, number of junctions/endpoints, junction density, lacunarity, and vessel area/density were defined using Angiotool, with CV depth localization despite scarring and opacification. AS-OCTA proved effective for angioregressive treatment monitoring. AS-OCTA enables non-invasive and objective three-dimensional visualization of corneal vascularization superior to slit-lamp photography, and could be a precious tool for monitoring angioregressive preconditioning prior to keratoplasty.

Optical coherence tomography angiography in herpetic leucoma.

BACKGROUND: Herpes simplex virus (HSV) keratitis remains a leading infectious cause of blindness worldwide. Although all forms of HSV keratitis are commonly recurrent, the risk is greatest in stromal keratitis, which is the most likely to result in corneal scarring, thinning, and neovascularization. Recent studies showed the ability of Optical Coherence Tomography Angiography (OCTA) to detect and study vascular abnormalities in the anterior segment, including abnormal corneal vessels. This study intends to investigate the potential of OCTA device to image and describe quantitatively the vascularization in eyes diagnosed with herpetic leucoma and to discuss and review the usefulness of this technique in this pathology. METHODS: A Cross-sectional study was made, including 17 eyes of 15 patients with leucoma secondary to herpetic keratitis. All eyes underwent anterior segment Slit-Lamp photography (SLP), and OCTA with en-face, b-scans and c-scans imaging. The vessel density (VD) was analyzed in the inferior, nasal and temporal corneal margin in all patients, and in the central area, in eyes with central corneal neovascularization (CoNV). The measurements were calculated after binarization with ImageJ software, using OCTA scans with 6 × 6 mm in a depth of 800 µm. RESULTS: Patients included had a mean age 53.267 ± 21.542 (years ± SD). The mean total vessel area was 50.907% ± 3.435%. VD was higher in the nasal quadrant (51.156% ± 4.276%) but there were no significant differences between the three analyzed areas (p = 0.940). OCTA was able to identify abnormal vessels when SLP apparently showed no abnormal vessels; OCTA was able to distinguish between larger and smaller vessels even in central cornea; OCTA scans allowed the investigation of several corneal planes and the relation of them with clinical findings. CONCLUSIONS: OCTA can be useful in both qualitative and quantitative follow-up of patients and may become a non-invasive alternative to objectively monitor treatment response in eyes with corneal vascularization due to herpetic infection.

Efficacy of a new automated method for quantification of corneal neovascularisation.

BACKGROUND/AIMS: To evaluate the efficacy of a new automated method for quantification of corneal neovascularisation (NV). METHODS: An in-house software for automated measurement of corneal NV was developed. Anterior segment photographs (ASPs) of 81 consecutive patients with corneal NV were analysed using our newly developed software. Manual measurements were performed by three independent examiners using ImageJ software V.1.48 (National Institute of Health, Bethesda, Maryland, USA). Interobserver reliability of the automated and manual methods, and correlations between the results of both methods were evaluated. RESULTS: The automated method showed a strong interexaminer reliability (intraclass correlation coefficient (ICC)=0.994), which was slightly better than the manual method (ICC=0.958). A significant correlation was found between the results of both methods (p<0.001 for all three examiners). The time spent for analysis of each ASP was significantly reduced in the automated method compared with the manual method (p<0.001 for all three examiners). CONCLUSIONS: Our newly developed automated method for quantification of corneal NV was more reproducible and time-saving compared with the manual method. Our method can be useful for diagnosis and monitoring diseases causing corneal NV.

Anterior Segment Optical Coherence Tomography Angiography in Patients Following Cultivated Oral Mucosal Epithelial Transplantation.

PURPOSE: To analyze corneal neovascularization using anterior segment optical coherence tomography angiography (AS-OCTA) in patients following cultivated oral mucosal epithelial sheet transplantation (COMET). DESIGN: Observational case series. METHODS: Nine eyes in 7 patients were analyzed. Four images of corneal quadrant were obtained by AS-OCTA from each patient during follow-up post-COMET in the Department of Ophthalmology at Osaka University Hospital. The depth of corneal neovascularization was evaluated using en face and B-scan images. Each quadrant image was classified as 1 of the following 5 types: stromal, predominantly stromal, epithelial, predominantly epithelial, or avascular. The image quality of slit-lamp photography and AS-OCTA was graded from 0 to 4. Manually segmented images of the epithelial and stromal vessels were obtained. MAIN OUTCOME MEASURES: Depth and image quality of corneal neovascularization following COMET. RESULTS: Six patients were male and 1 was female. The mean patient age was 61.3 ± 19.1 years. Thirty-six quadrant images were obtained, of which 4 (11.1%) were stromal, 16 (44.4%) were predominantly stromal, 3 (8.3%) were epithelial, 11 (30.6%) were predominantly epithelial, and 2 (5.6%) were avascular. The image quality obtained by AS-OCTA was significantly better than that obtained by slit-lamp photography (2.38 ± 0.94 vs 2.03 ± 0.90; P = .021). Segmentation images clearly demonstrated both epithelial and stromal vasculatures individually. CONCLUSIONS: AS-OCTA is useful for evaluation of depth of corneal neovascularization and has the potential to distinguish between conjunctivalization and stromal neovascularization following COMET. Findings on AS-OCTA could contribute to clinical decision making, given that retreatment is required for conjunctivalization after COMET.

In vivo confocal microscopy of the inflamed anterior segment: A review of clinical and research applications.

In vivo confocal microscopy (IVCM) allows non-invasive imaging of the living human cornea, specifically enabling the detection of immune cells in the healthy and diseased ocular anterior segment. Studies using IVCM have provided insight into the effects of contact lens wear on corneal Langerhans cell density and morphology, and the effects of eye drops on conjunctiva-associated lymphoid tissue. IVCM has also been shown to be a useful adjunctive diagnostic tool in distinguishing infective and non-infective uveitis and in diagnosing atypical infective keratitis. In the research setting, this technology has enhanced our understanding of the role of inflammatory cells in corneal neuropathy and angiogenesis. In vivo-ex vivo correlation using animal models has helped overcome some of the difficulties in identifying cell type on IVCM images. As highlighted in this review, currently there are multiple established, and emerging, clinical and research applications for IVCM in the inflamed anterior segment.

Localization of Corneal Neovascularization Using Optical Coherence Tomography Angiography.

PURPOSE: This article explores the application of optical coherence tomography angiography (OCTA) in assessing corneal neovascularization (CoNV) and investigates the features of CoNV in eyes with corneal transplantation. METHODS: A pilot, case series, observational study was conducted to enroll patients who underwent corneal transplantation including penetrating keratoplasty (PKP) and deep lamellar keratoplasty (DLKP) with or without additional keratolimbal allograft transplantation. All patients were followed with a series of ophthalmologic examinations including slit-lamp photography and were then imaged with the anterior segment OCTA. RESULTS: The study included 15 eyes of 14 patients (12 men; mean age of 37.4 ± 13.3 years), of which 9 eyes had undergone PKP and 6 eyes DLKP. OCTA was able to clearly identify the features of CoNV in eyes with significant CoNV and to confirm the presence of CoNV in eyes suspected of having CoNV. Four types of CoNV (superficial, stromal, fringe, and recipient-bed CoNV) were discovered by OCTA based on their location and depth. Superficial CoNV was mainly discovered in eyes that had undergone PKP (88.9% ± 11.1%), whereas the recipient-bed CoNV, once thought to be located in the host-graft interface, was discovered to grow in the host cornea only in eyes that had undergone DLKP (83.3% ± 16.7%). Comparing the assessment by 2 masked observers revealed a kappa value of 0.94, indicating excellent agreement. CONCLUSIONS: OCTA can be useful to visualize CoNV, which may be valuable in assessing corneal graft rejection.

Imaging of Corneal Neovascularization: Optical Coherence Tomography Angiography and Fluorescence Angiography.

Purpose: The purpose of this study was to compare optical coherence tomography angiography (OCTA) and indocyanine green angiography (ICGA) for the assessment of corneal neovascularization (CoNV). Methods: Patients with CoNV extending at least 3 mm into the cornea were included. All patients underwent corneal imaging at the same visit. Images were recorded using the AngioVue OCTA system (Optovue, Inc.) with the long corneal adaptor module (CAM-L). ICGA images were recorded with fluorescent filters using the Heidelberg system (HRA2 Scanning Laser Ophthalmoscope; Heidelberg Engineering). Images were graded for quality by two independent observers. Vessel parameters: area, number, diameter, branch and end points, and tortuosity, were compared between devices. Bland-Altman plots were used to assess differences between parameters. Results: Fifteen patients with CoNV predominantly associated with microbial keratitis were included. Mean subjective image quality score was better for ICGA (3.3 ± 0.9) than for OCTA (2.1 ± 1.2, P = 0.002), with almost perfect interobserver agreement for ICGA images (κ = 0.83) and substantial agreement for OCTA images (κ = 0.69). Agreement of grading of all investigated vessel parameters between ICGA and OCT images was slight to moderate, with significant differences found for vessel diameter (-8.98 µm, P = 0.01, 95% limits of agreement [LOA]: -15.89 to -2.07), number of branch (25.93, P = 0.09, 95% LOA: -4.31 to 56.17), and terminal points (49, P = 0.05, 95% LOA: 0.78 to 97.22). Conclusion: Compared with ICGA, current OCTA systems are less precise in capturing small vessels in CoNV complexes, and validation studies are needed for OCTA segmentation software. OCTA, however, complements ICGA by providing evidence of red blood cell flow, which together with depth information, may be helpful when planning treatment of CoNV.

Novel Method to Detect Corneal Lymphatic Vessels In Vivo by Intrastromal Injection of Fluorescein.

PURPOSE: Corneal lymphatic vessels are clinically invisible because of their thin walls and clear lymph fluid. There is no easy and established method for in vivo imaging of corneal lymphatic vessels so far. In this study, we present a novel approach to visualize corneal lymphatic vessels in vivo by injecting intrastromal fluorescein sodium. METHODS: Six- to eight-week-old female BALB/c mice were used in the mouse model of suture-induced corneal neovascularization. Two weeks after the suture placement, fluorescein sodium was injected intrastromally. The fluorescein, taken up by the presumed lymphatic vessels, was then tracked using a clinically used Spectralis HRA + OCT device. Immunohistochemistry staining with specific lymphatic marker LYVE-1 and pan-endothelial marker CD31 was used to confirm the indirect lymphangiography findings. RESULTS: By injecting fluorescein intrastromally, both corneal blood and lymphatic vessels were detected. While the lymphatic vessels were visible as bright vessel-like structures using HRA, the blood vessels appeared as dark networks. Fluorescein-labeled lymphatic vessels were colocalized with LYVE-1 in immunohistochemically stained sections of the same specimen. CONCLUSIONS: Corneal lymphatic vessels can be easily imaged in vivo in the murine model using intrastromal fluorescein injection.

Label-Free In Vivo Imaging of Corneal Lymphatic Vessels Using Microscopic Optical Coherence Tomography.

Purpose: Corneal neovascularization, in particular lymphangiogenesis, is a limiting factor in corneal transplant survival. Novel treatment approaches focus on (selective) inhibition and regression of lymphatic vessels. Imaging clinically invisible corneal lymphatic vessels is a prerequisite for these strategies. Using a murine model, this study investigates whether corneal lymphatic vessels can be imaged using microscopic optical coherence tomography (mOCT). Methods: Corneal neovascularization was induced by intrastromal placement of 11.0 nylon sutures in one eye of BALB/c mice. After 2 weeks, cross-sectional images and volumes of the corneas with a 0.5 mm lateral and axial field of view were acquired using a custom-built mOCT system enabling a resolution of 1 µm at a B-scan rate of 165/s. Three of the six animals received an additional intrastromal injection of India ink 24 hours before the measurement to stain the corneal lymphatic system in vivo. Immunohistochemistry using CD31 and LYVE-1 was used to validate the mOCT findings. Results: Using mOCT, lymphatic vessels were visible as dark vessel-like structures with the lumen lacking a hyperreflective wall and mostly lacking cells. However, individual, slowly moving particles, which most likely are immune cells, occasionally could be observed inside the lumen. In lymphatic vessels of ink-stained corneas, hyperreflection and shadowing underneath was observed. Ink-filled lymphatic vessels were colocalized in consecutive corneal flat mounts of the same specimen. Conclusions: Corneal lymphatic vessels can be imaged using mOCT. This novel approach opens new options for noninvasive clinical imaging of corneal lymphatic vessels for diagnostic and therapeutic indications.

In Vivo Photoacoustic Imaging of Anterior Ocular Vasculature: A Random Sample Consensus Approach.

Visualizing ocular vasculature is important in clinical ophthalmology because ocular circulation abnormalities are early signs of ocular diseases. Photoacoustic microscopy (PAM) images the ocular vasculature without using exogenous contrast agents, avoiding associated side effects. Moreover, 3D PAM images can be useful in understanding vessel-related eye disease. However, the complex structure of the multi-layered vessels still present challenges in evaluating ocular vasculature. In this study, we demonstrate a new method to evaluate blood circulation in the eye by combining in vivo PAM imaging and an ocular surface estimation method based on a machine learning algorithm: a random sample consensus algorithm. By using the developed estimation method, we were able to visualize the PA ocular vascular image intuitively and demonstrate layer-by-layer analysis of injured ocular vasculature. We believe that our method can provide more accurate evaluations of the eye circulation in ophthalmic applications.

[Experimental and morphological study of the effect of antiangiogenic therapy on corneal neovessels].

The article presents the results of an experimental and morphological study of combined antiangiogenic effect of photodynamic therapy with Photosens photosensitizer and Avastin (bevacizumab) on neovascularization process in corneal stroma. The combined approach has demonstrated an advantage over separate application of these methods, what, however, has to be proved clinically.

[Effectiveness of topical bevacizumab in bilateral primary lipid keratopathy]. / Eficacia de bevacizumab tópico en queratopatía lipoidea bilateral primaria.

CASE REPORT: A 75-year-old man with bilateral idiopathic lipid keratopathy underwent a penetrating keratoplasty in the left eye. One month later, there was deep corneal neovascularisation extending across the bed and the graft-host interface, with a whitish opacity surrounding the vessels. Topical bevacizumab (25mg/mL) was administered 4 times daily for 2 months with partial regression of corneal neovascularization. DISCUSSION: Topical bevacizumab may be useful in preventing a recurrence of lipid deposition after penetrating keratoplasty in patients with bilateral primary lipid keratopathy, although its long-term efficacy needs to be assessed.

Experimental inhibition of corneal neovascularization by photodynamic therapy with verteporfin.

PURPOSE: To investigate the anti-angiogenic effects of photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization. METHODS: One week after suturing, the localization of verteporfin in the neovascularized cornea was examined through fluorescent microscopy 1 hr after administration. Rabbits were treated with one or two times of photodynamic therapy with verteporfin at 1-week intervals. Analysis of corneal neovascularization was performed by biomicroscopic and histological examinations. RESULTS: Fluorescent microscopy showed green fluorescence in the vascular walls and interstitial tissue of the corneal stroma. The mean percentages of neovascularized corneal area at 3 days, 1 week, and 2 weeks after one time of photodynamic therapy were 90.3% +/- 3.5%, 71.6% +/- 6.2%, and 43.6% +/- 15.1% in treated eyes and 96.4% +/- 1.9% (p = 0.10), 88.6% +/- 4.6% (p = 0.01), and 76.8% +/- 4.4% (p < 0.01) in control eyes, respectively. The mean percentages 3 days, 1 week, and 2 weeks after two times of photodynamic therapy were also significantly lower in treated eyes compared with control eyes. In quantitative histological examination at 1 and 2 weeks after therapy, treated eyes showed significantly less neovascular area and number of vessels than control eyes. CONCLUSIONS: Photodynamic therapy with verteporfin is a safe and useful procedure to reduce experimental corneal neovascularization and can be used to inhibit angiogenesis in the cornea.

Treatment parameters for selective occlusion of experimental corneal neovascularization by photodynamic therapy using a water soluble photosensitizer, ATX-S10(Na).

Time dependent change of an accumulation of an amphiphilic photosensitizer, ATX-S10(Na) on rabbit corneal neovascularization (CoNV) was evaluated by angiography using ATX-S10(Na) as a fluorescent dye on three rabbits. The angiography showed that the dye accumulated on CoNV 3-5 hr after dye injection when the dye in the iris was minimum. The results suggested 3-5 hr after might be the optimal time to start photodynamic therapy (PDT) to occlude CoNV selectively without damage to the surrounding normal tissue such as the iris. Then the optimal treatment parameters in PDT using ATX-S10(Na) for selective occlusion of the CoNV were investigated on rabbit eyes. PDT was performed with two different time intervals between dye injection and laser irradiation of a diode laser (670 nm), different laser doses and three different dye doses on 21 animals. PDT performed immediately after dye injection selectively occluded CoNV with laser irradiations from 30.6 to 38.2 J cm(-2)and a 2 mg kg(-1)dose of ATX-S10(Na), as well as with 15.3 J cm(-2)and a 6 mg kg(-1)dose. PDT performed 4 hr after dye injection with 107.0-152.8 J cm(-2)and a 6 mg kg(-1)dose, as well as with 38.2-53.5 J cm(-2)and a 12 mg kg(-1)dose was also effective. Although PDT performed either immediately or 4 hr after ATX-S10(Na) injection selectively occluded CoNV, the width of the optimal range of radiant exposures seemed wider in PDT performed 4 hr after dye injection. It is supposed that this result is associated with the difference of dye accumulation between in CoNV and in normal tissue as shown by the present angiographical findings.