Methods of Studying Retinal Vessels in Health and Diseases

The eye contains transparent media that lack blood vessels, including the cornea, the eye lens, and the vitreous. Light must pass through these media before reaching the photoreceptors at the back of the retina. Specifically, the cornea, the eye lens, and the vitreous lack blood vessels in order to avoid the absorption of the light and the distortion of the images¹. Retinal blood vessels are organized into three layers: the superficial layer, which extends from the optic disc to the periphery within the nerve fiber layer; the middle layer, which is located within the inner plexiform layer; and the deep layer, which is distributed through the outer plexiform layer. This vascular network supplies blood to the inner retina, while the photoreceptors are supplied by diffusion from the choroidal blood vessels. The nourishment of the inner and outer retinas is strictly controlled by the inner and outer blood-retinal barriers (BRBs). Retinal endothelial cells, pericytes, and glial cells constitute the inner BRB. On the other hand, retinal pigment epithelial (RPE) cells, choroidal endothelial cells, and the Bruch’s membrane form the outer BRB. Pathological changes in the retinal blood vessels are the hallmarks of several retinal diseases, such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. These pathological changes include but are not limited to vascular inflammation, the breakdown of the BRB, capillary degeneration, changes in retinal blood flow, and pathological retinal neovascularization (RNV)^2,3,4. Therefore, it is important to develop effective methods to study these changes both in vivo using experimental animals and in vitro by culturing various retinal cells under conditions similar to the biological and biochemical environment in human disease. Researchers can use several experimental approaches to dissect the underlying molecular and cellular mechanisms of retinal vascular diseases. The goal of this methods collection is to share with other researchers the expertise of vision scientists and ophthalmologists in terms of methods for studying normal vascular development and vascular abnormalities in various retinal diseases.

The article by Damsgaard et al. presents a flow-enhanced ultrasound method that allows investigators to image the eye vasculatures in three dimensions without the use of contrast agents⁵. This method overcomes the limitations of other methods due to its stability in imaging the eye behind the pigmented retina. Although the method has been demonstrated in goldfish, it can be applied to all other experimental species with nucleated red blood cells. This method is valuable for visualizing the ocular blood vessels under normal and pathological conditions and could be helpful in studying the ocular blood flow. However, this method is limited by the lack of nucleated red blood cells in mammals. Thus, compared to species with nucleated red blood cells, the flow-enhanced ultrasound only produces blood flow contrast in mammals. Moreover, this method is sensitive to motion noise, such as from respiratory movements, which may create blurry images and artifacts. This motion noise can be adjusted by prospective or retrospective gating. For both 2D and 3D acquisitions, it is crucial to limit motion artifacts caused by the animal moving due to inadequate anesthesia or by an unstable transducer setup. The development of this technique paves the way for the non-invasive in vivo examination of the deep vascular networks in the eye in the vast majority of vertebrates that possess nucleated erythrocytes.

The method presented by Macouzet-Romero et al. evaluates the relative contribution of the retinal vessels to the macular perfusion density using optical coherence tomography (OCT)-angiography⁶. This method characterizes the proportion of an area with circulation from dilated vessels. Of note, vascular dilation can be a response to pathologic conditions. This approach can indirectly outline vasodilation and indirectly identify low oxygenation in retinal vascular diseases, such as diabetic retinopathy (DR), central vein occlusion, or age-related macular degeneration (AMD). Using this method, the major findings of Macouzet-Romero et al.⁶ were that the contribution of vessels larger than the retinal capillaries to retinal circulation was 18.2% in healthy subjects. Thus, this percentage could be used as a reference to identify early changes in perfusion density due to retinal vascular diseases. For example, the percentage contribution was 2.6% in patients with arterial hypertension and 16.4% in patients with diabetes, suggesting that this method could potentially be used to evaluate the progression of retinal diseases associated with vascular abnormalities.

The method presented by Subirada et al. evaluates different parameters of vascular alterations (the extent of capillary degeneration [avascular areas], pathological retinal neovascularization [neovascular areas], dilatation and tortuosity of the retinal arterioles) in experimental models of ischemic retinopathies, such as oxygen-induced retinopathy (OIR) and experimental diabetes⁷. Of note, OIR reproduces retinopathy of prematurity and some aspects of proliferative diabetic retinopathy. The ability to quantify the extent of retinal neovascularization (RNV) or capillary degeneration in experimental models is critical for studying the underlying molecular and cellular mechanisms, as well as for testing the possible therapeutic effects of pharmacological or molecular interventions. In summary, this article presents classical, well-established, and reproducible techniques to quantify some of the most relevant vascular parameters considered in clinical practice.

The article by Vaglienti et al. presents methods to measure the levels of reactive oxygen species (ROS) in Müller glial cells⁸. The role of oxidative stress in retinal microvascular dysfunction in ischemic retinopathies has been established and has encouraged researchers to investigate the extent to which reactive oxygen species (ROS) contribute to the breakdown of the BRB and RNV both in vivo and in vitro^9,10,11,12. Vaglienti et al. use 2',7'-dichlorofluorescein diacetate (DCFD) to evaluate the levels of intracellular generation of reactive oxygen species (ROS). This method is based on the diffusion of DCFD into the cell and then the oxidization of DCFD by ROS into 2’, 7’ –dichlorofluorescein (DCF). DCF is highly fluorescent and can be detected by flow cytometry or confocal microscopy. This method can image and measure the levels of ROS generation in both retinal cells and tissues.

The article by Tomaszewski et al. demonstrates the isolation and growth of mouse retinal pigment epithelial (RPE) cells¹³. RPE cells contribute to the outer BRB and play an essential role in photoreceptor hemostasis and integrity. RPE cell dysfunction is a hallmark of various retinal diseases, including but not limited to AMD and DR. Thus, in vitro studies of RPE cell function in health and disease are critical for understanding the molecular and cellular mechanisms of several vascular retinal diseases. For this purpose, primary RPE cell isolation could be an important tool for studying the underlying mechanisms and pathogenesis of different eye diseases, as well as for proposing new therapeutic applications for those diseases. This technique enables the study of changes in RPE cell structure and function and their impact on the outer BRB in AMD and DR. Moreover, the technique can be applied to study changes in RPE cells isolated from wild-type C57BL6 mice and different genetically modified mice and to test different pharmacological compounds as therapeutic targets for AMD¹⁴.

The article by Elmasry et al. presents an ex vivo experimental model that can be used to study neurovascular retinal diseases by culturing retinal explants for an extended period¹⁵. In this ex vivo system, retinal explants present intact vascular and glial networks and retinal neurons for up to 2 weeks in culture. Organotypic retinal explants have emerged as a reliable tool for studying retinal diseases, such as DR and degenerative retinal diseases^16,17. Compared to other existing techniques, the use of retinal explants supports both in vitro retinal cell cultures and in vivo animal models by adding a unique feature that allows for the examination of the crosstalk between various retinal cells (e.g., vascular, glial, and neuronal cells) under the same biochemical parameters and independent of systemic variables. The explant cultures maintain various retinal cells together in the same environment and retain the intercellular physical and molecular interactions. Moreover, studies have shown that retinal explants can preserve the morphological structure and functionality of the cultured retinal cells. In summary, retinal explants can provide a suitable platform for investigating the crosstalk between various retinal cells in response to biochemical changes, such as hyperglycemia and hypoxia, similar to those observed in diabetic retinopathy (DR) or retinopathy of prematurity (ROP). This approach also allows the testing of therapeutic interventions under a restricted biochemical environment. Retinal explant culture represents a controllable technique and very flexible experimental model that allows numerous pharmacological manipulations and can uncover several molecular mechanisms of retinal diseases.