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Review
, 31 (11), 4665-4681

Animal Models of Ocular Angiogenesis: From Development to Pathologies

Affiliations
Review

Animal Models of Ocular Angiogenesis: From Development to Pathologies

Chi-Hsiu Liu et al. FASEB J.

Abstract

Pathological angiogenesis in the eye is an important feature in the pathophysiology of many vision-threatening diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration, as well as corneal diseases with abnormal angiogenesis. Development of reproducible and reliable animal models of ocular angiogenesis has advanced our understanding of both the normal development and the pathobiology of ocular neovascularization. These models have also proven to be valuable experimental tools with which to easily evaluate potential antiangiogenic therapies beyond eye research. This review summarizes the current available animal models of ocular angiogenesis. Models of retinal and choroidal angiogenesis, including oxygen-induced retinopathy, laser-induced choroidal neovascularization, and transgenic mouse models with deficient or spontaneous retinal/choroidal neovascularization, as well as models with induced corneal angiogenesis, are widely used to investigate the molecular and cellular basis of angiogenic mechanisms. Theoretical concepts and experimental protocols of these models are outlined, as well as their advantages and potential limitations, which may help researchers choose the most suitable models for their investigative work.-Liu, C.-H., Wang, Z., Sun, Y., Chen, J. Animal models of ocular angiogenesis: from development to pathologies.

Keywords: choroidal neovascularization; corneal angiogenesis; macular degeneration; retinal vasculature; retinopathy.

Figures

Figure 1.
Figure 1.
Schematic diagram of the ocular vasculature. A) Drawing showing a cross-section of an eyeball. B) An enlarged schematic illustration showing a cross-sectional view of the retinal and choroidal vasculature. Three interconnected layers of retinal vessels are embedded among retinal neurons: the superficial retinal vasculature lies in the nerve fiber layer (NFL) and the intermediate and deep retinal vascular networks along each side of the inner nuclear layer (INL). The choroidal vessels are located beneath RPE and Bruch’s membrane and supply oxygen and nutrients to the outer portion of the retina. GCL, ganglion cell layer; IS/OS, inner segment/outer segment of photoreceptor; ONL, outer nuclear layer.
Figure 2.
Figure 2.
Mouse model of OIR. A) A schematic diagram depicting the mouse OIR procedure. Neonatal mice and their nursing mother are exposed to 75% oxygen from P7 to 12. Excessive oxygen suppresses the development of the retinal vasculature and leads to regression of the existing immature retinal vessels, which results in a central zone of vaso-obliteration. At P12, mice are returned to room air, and the relative hypoxia triggers both normal vessel regrowth toward the vaso-obliteration zone and pathologic retinal neovascularization at the border between the vascular and avascular zones, which also protrude toward the vitreous space. The levels of neovascularization reach maximum severity at P17. B) Representative images of retinal flat mounts with isolectin staining in the normoxic and OIR retinas show the normal retinal vasculature at P7 (left), vaso-obliteration at P12 (middle; the vessel loss area is highlighted with white outline), and pathologic neovascularization at P17 (right; the vessel loss area is highlighted with white dashed line). Scale bar, 1 mm. C) Magnified images of isolectin-stained retinal flat mounts show the normal retinal vasculature from normoxic retinas (left) and abnormal neovessels from OIR retinas (right; image enlarged from the rectangular white box region in panel B at P17). D) Cross-sections of mouse retinas with normoxia (left) and OIR (right) at P17. Preretinal neovascular tufts (arrowheads) arise from the superficial retinal vascular layer of the OIR retina and protrude into the vitreous space. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.
Figure 3.
Figure 3.
Laser-induced CNV model. A) Scheme of laser-induced CNV in mice. Adult mice are anesthetized and pupils are dilated. Laser burns are produced in the 3, 6, 9, and 12 o’clock positions around the optic disc (OD), with the laser focused on the RPE. The presence of a subretinal bubble confirms that the laser impact caused the disruption of Bruch’s membrane and RPE, which is necessary for the induction of successful CNV lesions. At 1 and 2 wk after laser treatment, CNVs growing into the subretinal space are observed and analyzed by using fundus fluorescein angiography (FFA) and dissected choroidal flat mounts with isolectin staining. B) A representative image of FFA from a mouse on d 6 after laser burns shows the formation of CNV (green lesions, arrowheads) beneath the retinal vasculature (green). C) A fluorescence microscopy image of an isolectin-stained flat mount of the mouse choroid/RPE at 1 wk after laser treatment shows the extent of CNV lesions (arrowheads). Scale bars, 500 µm. D) Immunohistologic examination of a cross-sectioned mouse eye at 1 wk after laser photocoagulation shows extension of the CNV lesion (red, stained with isolectin; arrow) into the subretinal space between photoreceptors and RPE. Scale bars, 100 µm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 4.
Figure 4.
Vldlr−/− mouse model of retinal angiomatous proliferation. A) Schematic diagrams (top) show the retinal neuronal layers (purple) and 3 layers of retinal blood vasculature (superficial, intermediate, and deep; red) on cross-section of wild-type (WT) and Vldlr−/− mice, with blood vessels in Vldlr−/− retinas extending from the deep layer toward RPE. Three-dimensional reconstructed images of vascular lesions in the photoreceptor layer of Vldlr−/− retinas compared with the avascular photoreceptor layer in WT retinas (bottom). Blood vessels were stained with isolectin (red, vessel marker). B) Immunohistochemical staining with isolectin (red) and DAPI (blue, nuclear marker) on a Vldlr−/− mouse retinal cross-section shows abnormal vessel growth (arrows) that originates from the deep vessel layers through the normally avascular photoreceptor layer [outer nuclear layer (ONL)] and toward RPE [reprinted from Hua et al. (112), copyright owned by the Association for Research in Vision and Ophthalmology]. Deep, deep retinal vessel; GCL, ganglion cell layer; INL, inner nuclear layer; Int, intermediate retinal vessels; IPL, inner plexiform layer; OPL, outer plexiform layer; Sup, superficial retinal vessel.
Figure 5.
Figure 5.
Lrp5−/− mouse model of deficient intraretinal angiogenesis. A) Isolectin-stained (red) mouse retinal flat mounts show delayed retinal vascular development in Lrp5−/− mice (right) compared with wild-type (WT; left) at P7. White lines indicate the edge of the retinal periphery. B) Schematic representation of the retinal layers and blood vasculature on cross-section of WT and Lrp5−/− mice. C) Immunofluorescence images showing the vasculature in WT and Lrp5−/− mouse retinas. Endothelial cells (red) and nucleus (blue) were visualized by staining with isolectin and DAPI, respectively. D) DAPI-labeled (blue) vitreous bodies that were isolated from WT (left) and Lrp5−/− (right) mice at P8. Lrp5−/− mice retain more persistent hyaloid vasculature compared with WT mice. Deep, deep retinal vessel; GCL, ganglion cell layer; INL, inner nuclear layer; Int, intermediate retinal vessels; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; Sup, superficial retinal vessel.
Figure 6.
Figure 6.
Murine models of corneal angiogenesis. A) Chemical injury model by alkali burn. A filter paper disk that is saturated with 1 N NaOH or control saline solution is applied to the center of the cornea for 30 s, followed by rinsing with saline. At 7–14 d after the alkali burn, corneal angiogenesis can be observed as neovessels growing from the limbus toward the injured region. B) Suture injury model. The cornea is artificially perforated with 10.0 nylon sutures in the experimental animal. Corneal neovascularization can be triggered and observed within 2 wk after the procedure. C) Corneal micropocket assay. A micropocket with a pellet (yellow) that contains proangiogenic factors or vehicle control is created in the stroma of the cornea. At 5–7 d after pellet implantation, corneal neovascularization can be observed and evaluated. D) Transgenic spontaneous models of corneal neovascularization. Genetically engineered mice, such as knockout of Destrin, CD36, or soluble VEGFRs, as well as overexpression of PAX6 in corneas, develop spontaneous corneal neovascularization. E) Representative images of rat corneas that were treated with control solution (Ctrl) or alkali solution-soaked filter papers. The cornea with alkali burn shows neovessels (arrowheads) growing from limbal vessels toward the central corneal burn. F) Mouse corneas with pellet implantation into micropockets. No abnormal corneal angiogenesis is observed with the implant of control buffer–containing pellet, whereas the pellet with angiogenic factor (arrow) induces pathologic vessel growth (arrowheads) from the limbus. Red arrows in panels AD represent the direction of blood vessel growth.

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