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      Optical Coherence Tomography Angiography in Mice: Comparison with Confocal Scanning Laser Microscopy and Fluorescein Angiography

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          Abstract

          Purpose

          Optical coherence tomography angiography (OCT-A) allows noninvasive visualization of retinal vessels in vivo. OCT-A was used to characterize the vascular network of the mouse retina and was compared with fluorescein angiography (FA) and histology.

          Methods

          In the present study, OCT-A based on a Heidelberg Engineering Spectralis system was used to investigate the vascular network in mice. Data was compared with FA and confocal microscopy of flat-mount histology stained with isolectin IB4. For quantitative analysis the National Cancer Institute's AngioTool software was used. Vessel density, the number of vessel junctions, and endpoints were measured and compared between the imaging modalities.

          Results

          The configuration of the superficial capillary network was comparable with OCT-A and flat-mount histology in BALBc mice. However, vessel density and the number of vessel junctions per region of interest ( P = 0.0161 and P = 0.0015, respectively) in the deep vascular network of BALBc mice measured by OCT-A was significantly higher than with flat-mount histology. In C3A.Cg -Pde6b+Prph2Rd2/J mice, where the deep capillary plexus is absent, analysis of the superficial network provided similar results for all three imaging modalities.

          Conclusion

          OCT-A is a helpful imaging tool for noninvasive, in vivo imaging of the vascular plexus in mice. It may offer advantages over FA and confocal microscopy especially for imaging the deep vascular plexus.

          Translational Relevance

          The present study shows that OCT-A can be employed for small animal imaging to assess the vascular network and offers advantages over flat-mount histology and FA.

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          Most cited references22

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          A Computational Tool for Quantitative Analysis of Vascular Networks

          Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time - and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called “branching index” (branch points / unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge.
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            Oxygen-induced retinopathy in the mouse.

            To develop oxygen-induced retinopathy in the mouse with reproducible and quantifiable proliferative retinal neovascularization suitable for examining pathogenesis and therapeutic intervention for retinal neovascularization in retinopathy of prematurity (ROP) and other vasculopathologies. One-week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then to room air. A novel fluorescein-dextran perfusion method has been developed to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in 6 microns sagittal cross-sections. Cross-sections were also stained for glial fibrillary acidic protein (GFAP). Fluorescein-dextran angiography delineated the entire vascular pattern, including neovascular tufts in flat-mounted retinas. Hyperoxia-induced neovascularization occurred at the junction between the vascularized and avascular retina in the mid-periphery. Retinal neovascularization occurred in all the pups between postnatal day 17 and postnatal day 21. There was a mean of 89 neovascular nuclei per cross-section of 9 eyes in hyperoxia compared to less than 1 nucleus per cross-section of 8 eyes in the normoxia control (P < 0.0001). Proliferative vessels were not associated with GFAP-positive astrocyte processes. The authors have described a reproducible and quantifiable mouse model of oxygen-induced retinal neovascularization that should prove useful for the study of pathogenesis of retinal neovascularization as well as for the study of medical intervention for ROP and other retinal angiopathies.
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              Development and pathology of the hyaloid, choroidal and retinal vasculature.

              During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.
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                Author and article information

                Journal
                Transl Vis Sci Technol
                Transl Vis Sci Technol
                tvst
                tvst
                tvst
                Translational Vision Science & Technology
                The Association for Research in Vision and Ophthalmology
                2164-2591
                18 August 2016
                August 2016
                : 5
                : 4
                : 11
                Affiliations
                [1]Department of Ophthalmology and Department of Clinical Research Inselspital, Bern University Hospital, and University of Bern, Switzerland
                Author notes
                Correspondence: Martin S. Zinkernagel, MD, PhD, Department of Ophthalmology, Inselspital, Bern University hospital, Universität Bern, CH-3010 Bern, Switzerland. email: martin.zinkernagel@ 123456insel.ch

                *Helena Giannakaki-Zimmermann and Despina Kokona contributed equally to this work.

                Article
                tvst-05-04-11 TVST-16-0356
                10.1167/tvst.5.4.11
                4997887
                f37413bb-00e4-43e0-af0b-8694206a3e1f

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

                History
                : 21 March 2016
                : 29 June 2016
                Categories
                Articles

                optical coherence angiography,confocal microscopy,fluorescein angiography,confocal scanning microscopy,retinal vasculature

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