The mouse model of oxygen-induced retinopathy (OIR) has been widely used in studies related to retinopathy of prematurity, proliferative diabetic retinopathy and in studies evaluating the efficacy of antiangiogenic compounds. In this model, 7-d-old (P7) mouse pups with nursing mothers are subjected to hyperoxia (75% oxygen) for 5 d, which inhibits retinal vessel growth and causes significant vessel loss. on P12, mice are returned to room air and the hypoxic avascular retina triggers both normal vessel regrowth and retinal neovascularization (NV), which is maximal at P17. neovascularization spontaneously regresses between P17 and P25. although the OIR model has been the cornerstone of studies investigating proliferative retinopathies, there is currently no harmonized protocol to assess aspects of angiogenesis and treatment outcome. In this protocol we describe standards for mouse size, sample size, retinal preparation, quantification of vascular loss, vascular regrowth, NV and neovascular regression.

The mouse retina has been used extensively over the past decades to study both physiologic and pathologic angiogenesis. Over time, various mouse retina models have evolved into well-characterized and robust tools for in vivo angiogenesis research. This article is a review of the angiogenic development of the mouse retina and a discussion of some of the most widely used vascular disease models. From the multitude of studies performed in the mouse retina, a selection of representative works is discussed in more detail regarding their role in advancing the understanding of both the ocular and general mechanisms of angiogenesis.

Tissues with high metabolic rates often use lipid as well as glucose for energy, conferring a survival advantage during feast and famine.1 Current dogma suggests that high-energy consuming photoreceptors depend on glucose.2,3 Here we show that retina also uses fatty acids (FA) β-oxidation for energy. Moreover, we identify a lipid sensor Ffar1 that curbs glucose uptake when FA are available. Very low-density lipoprotein receptor (VLDLR), expressed in tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived FA.4,5 Vldlr is present in photoreceptors.6 In Vldlr−/− retinas, Ffar1, sensing high circulating lipid levels despite decreased FA uptake5, suppresses glucose transporter Glut1. This impaired glucose entry into photoreceptors results in a dual lipid/glucose fuel shortage and reduction in the Krebs cycle intermediate α-ketoglutarate (KG). Low α-KG levels promote hypoxia-induced factor-1α (Hif1a) stabilization and vascular endothelial growth factor (Vegfa) secretion by starved Vldlr−/− photoreceptors, attracting neovessels to supply fuel. These aberrant vessels invading normally avascular photoreceptors in Vldlr−/− retinas are reminiscent of retinal angiomatous proliferation (RAP), a subset of neovascular age-related macular degeneration (AMD)7, associated with high vitreous VEGF levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in neovascular AMD and other retinal diseases.

Retinopathy of prematurity (ROP) is a major complication of preterm birth. It encompasses a spectrum of pathologies that affect vision, from mild disease that resolves spontaneously to severe disease that causes retinal detachment and subsequent blindness. The pathologies are characterized by an arrest in normal retinal vascular development associated with microvascular degeneration. The resulting ischemia and retinal hypoxia lead to excessive abnormal compensatory blood vessel growth. However, this neovascularization can lead to fibrous scar formation and culminate in retinal detachment. Present therapeutic modalities to limit the adverse consequences of aberrant neovascularization are invasive and/or tissue-destructive. In this Review, we discuss current concepts on retinal microvascular degeneration, neovascularization, and available treatments, as well as present future perspectives toward more profound elucidation of the pathogenesis of ROP.Retinopathy of prematurity (ROP) is the major ocular disorder of the neonate (1, 2) and the dominant cause of severe visual impairment in childhood in North America and Europe. ROP is associated with significant sequelae, the most serious being retinal detachment, which results in blindness. However, even milder forms of ROP increase the incidence of pathologies that negatively impact visual acuity, for example, ametropias, refractive errors that reduce visual acuity; strabismus, a condition in which the eyes are not properly aligned, preventing proper binocular vision and adversely affecting depth perception; and disorders of color discrimination (3-6). ROP proceeds following an initial phase of degeneration of the retinal microvasculature (vasoobliteration) (7,8) (Figure 1) that is associated with cessation of progression of vascular growth toward the retinal periphery. In the subsequent phase of the disease, the ensuing retinal ischemia predisposes to abnormal compensatory neovascularization (9, 10). Of the various factors that have been associated with the development of ROP, low birth weight, low gestational age, supplemental oxygen therapy, and its associated relative hyperoxia dominate.The development of the human retinal vasculature commences at approximately the 16th week of gestation and concludes at term (i.e., the 40th week of gestation) (11). Hence, when an infant is born prematurely, its retinal blood supply is incomplete and highly vulnerable to decay. This immaturity in vascular development predisposes the retina to complications. Major advances have been made over the past 30 years in identifying mechanisms implicated in the genesis of ROP. Comprehension of mechanisms underlying this disorder has, in turn, enhanced understanding of the pathogenesis of ischemic retinal vasculopathies in the adult, for example, diabetic retinopathy (a complication of diabetes mellitus) and neovascular forms of age-related macular degeneration (the major cause of visual impairment in adults over 50 years of age).The oxygen-induced retinopathy (OIR) model of ischemic retinop...

Lipid signaling is dysregulated in many diseases with vascular pathology, including cancer, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. We have previously demonstrated that diets enriched in ω-3 polyunsaturated fatty acids (PUFAs) effectively reduce pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy, in part through metabolic products that suppress microglialderived tumor necrosis factor–α. To better understand the protective effects of ω-3 PUFAs, we examined the relative importance of major lipid metabolic pathways and their products in contributing to this effect. ω-3 PUFA diets were fed to four lines of mice deficient in each key lipid-processing enzyme (cyclooxygenase 1 or 2, or lipoxygenase 5 or 12/15), retinopathy was induced by oxygen exposure; only loss of 5-lipoxygenase (5-LOX) abrogated the protection against retinopathy of dietary ω-3 PUFAs. This protective effect was due to 5-LOX oxidation of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxy-docosahexaenoic acid (4-HDHA). 4-HDHA directly inhibited endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator–activated receptor γ (PPARγ), independent of 4-HDHA’s anti-inflammatory effects. Our study suggests that ω-3 PUFAs may be profitably used as an alternative or supplement to current anti–vascular endothelial growth factor (VEGF) treatment for proliferative retinopathy and points to the therapeutic potential of ω-3 PUFAs and metabolites in other diseases of vasoproliferation. It also suggests that cyclooxygenase inhibitors such as aspirin and ibuprofen (but not lipoxygenase inhibitors such as zileuton) might be used without losing the beneficial effect of dietary ω-3 PUFA.

The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) IntroductionProliferative retinopathies (PRs) are traditionally perceived as disorders limited to the microvasculature because of the characteristic profuse and deregulated growth of retinal vessels. 1 The mechanisms by which neovessels grow toward the vitreous and fail to revascularize ischemic zones are thought to result from high concentrations of proangiogenic factors such as VEGF in the vitreous of PR patients. However, if such an explanation were sufficient, retinal glial cells (astrocytes and Müller cells) 2 and neurons 3 that produce vast amounts of growth factors under hypoxic conditions should retain vessels on the retinal surface and ensure revascularization of the retina proper. It is, therefore, compelling to hypothesize the presence of a vasorepulsive force originating from the significantly hypoxic avascular retina that repels neovessels away from the vaso-obliterated retina and grows toward the vitreous.Neurovascular cross-talk shapes vascular development but has received limited attention in the pathology setting. In PRs, evidence points to an early decline in the function of ischemic regions of the neural retina, as shown by multifocal electroretinogram (mfERG). 4,5 Throughout the vaso-obliterative phase of retinopathy, the local retinal environment is hostile to both vasculature and neurons. 6 After blood vessel degeneration, neurons are metabolically starved and undergo several adaptive cellular changes to counter the ischemic state of the tissue. 3,6 If adequate vascular supply is not reinstated in time to salvage deprived neurons, it is conceivable that these severely hypoxic cells may mount a repulsive front in an attempt to shunt metabolic resources away from the perishing ischemic tissue toward less affected regions of the retina. In the process, excessive production of VEGF 7 induces exaggerated neovascularization at the periphery of the ischemic and repulsive zones into the pre-retinal region (normally devoid of vasculature), because reestablishing a vascular network to neurons that are unsalvageable would be wasteful.Given their established role in influencing endothelial cell (EC) behavior, classic neuronal guidance cues may ...

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Age‐related macular degeneration in its neovascular form (NV AMD) is the leading cause of vision loss among adults above the age of 60. Epidemiological data suggest that in men, overall abdominal obesity is the second most important environmental risk factor after smoking for progression to late‐stage NV AMD. To date, the mechanisms that underscore this observation remain ill‐defined. Given the impact of high‐fat diets on gut microbiota, we investigated whether commensal microbes influence the evolution of AMD. Using mouse models of NV AMD, microbiotal transplants, and other paradigms that modify the gut microbiome, we uncoupled weight gain from confounding factors and demonstrate that high‐fat diets exacerbate choroidal neovascularization (CNV) by altering gut microbiota. Gut dysbiosis leads to heightened intestinal permeability and chronic low‐grade inflammation characteristic of inflammaging with elevated production of IL‐6, IL‐1β, TNF‐α, and VEGF‐A that ultimately aggravate pathological angiogenesis.