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Metabolomics is the qualitative and quantitative assessment of the metabolites (small molecules 1.5 kDa) in body fluids. The metabolites are the downstream of the genetic transcription and translation processes and also downstream of the interactions with environmental exposures; thus, they are thought to closely relate to the phenotype, especially for multifactorial diseases. In the last decade, metabolomics has been increasingly used to identify biomarkers in disease, and it is currently recognized as a very powerful tool with great potential for clinical translation. The metabolome and the associated pathways also help improve our understanding of the pathophysiology and mechanisms of disease. While there has been increasing interest and research in metabolomics of the eye, the application of metabolomics to retinal diseases has been limited, even though these are leading causes of blindness. In this manuscript, we perform a comprehensive summary of the tools and knowledge required to perform a metabolomics study, and we highlight essential statistical methods for rigorous study design and data analysis. We review available protocols, summarize the best approaches, and address the current unmet need for information on collection and processing of tissues and biofluids that can be used for metabolomics of retinal diseases. Additionally, we critically analyze recent work in this field, both in animal models and in human clinical disease, including diabetic retinopathy and age-related macular degeneration. Finally, we identify opportunities for future research applying metabolomics to improve our current assessment and understanding of mechanisms of vitreoretinal diseases, and to hence improve patient assessment and care.

The retina consists of organized layers of photoreceptors, interneurons, glia, epithelial cells, and endothelial cells. The economic model of supply and demand used to appropriately determine cost is highly applicable to the retina, in which the extreme metabolic demands of phototransduction are met by precisely localized and designed vascular networks. Proper development and maintenance of these networks is critical to normal visual function; dysregulation is characteristic of several devastating human diseases, including but not limited to age-related macular degeneration and diabetic retinopathy. In this article, we focus on the lessons learned from the study of retinal vascular development and how these lessons can be used to better maintain adult vascular networks and prevent retinal diseases. We then outline the vasculotrophic contributions from neurons, retinal pigment epithelium (RPE) cells, and glia (specifically microglia) before we shift our focus to pathology to provide molecular contexts for neovascular retinal diseases. Finally, we conclude with a discussion that applies what we have learned about how retinal cells interact with the vasculature to identify and validate therapeutic approaches for neurovascular disease of the retina.

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor–binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-β plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.