Hyperglycemia mediates endothelial cell dysfunction through a number of potential mechanisms that could result in the decrease of retinal blood flow early in diabetes. The aim of this study was to explore the role of endothelin receptor A (ETA) in the early decrease of retinal blood flow in diabetic mice. Diabetes was induced by streptozotocin, then ~1 wk later the mice were administered drinking water with or without the ETA receptor antagonist atrasentan (7.5 mg/kg/day) for the following 3 weeks. Non-diabetic age-matched mice with or without atrasentan were included as controls. For each mouse, measurements of retinal vascular diameters and red blood cell (RBC) velocities were obtained via intravital microscopy for the 5–7 feed arterioles (and draining venules) extending out of (and into) the optic disk, and from these values, flow rates and wall shear rates were calculated. Additionally, the number of retinal capillaries was counted by fluorescent immunostaining of platelet-endothelial cell adhesion molecule-1 (PECAM-1). Diabetes induced statistically significant decreases in RBC velocity, flow rate, and wall shear rate, with these alterations partially inhibited by atrasentan. No changes were observed in PECAM-1 expression among groups. The changes induced by diabetes, and the attenuation provided by atrasentan, were greater in the smaller retinal arterioles. In summary, ETA appears to play a role in the early decreases in retinal blood flow in a mouse model of diabetes.

The mechanisms of early diabetes-induced decreases in retinal blood flow have yet to be fully determined. The aim of this study was to explore the hypothesis that 20-hydroxyeicosatetraenoic acid (20-HETE) plays a role in the early decrease of retinal hemodynamics in diabetic mice. 20-HETE has been implicated previously in the diabetes-enhanced vasoconstriction of mesenteric and renal vessels; however, its role in the diabetic retinal microcirculation has not been investigated. Diabetes was induced by multiple low-dose injections of streptozotocin (STZ; 50 mg/kg for 5 consecutive days), then ~2 weeks later the mice were administered daily intraperitoneal injections with or without the 20-HETE inhibitor HET0016 (2.5 mg/kg/day) for the following 2 weeks. Non-diabetic age-matched mice were included as controls. Intravital microscopy was used to obtain measurements of retinal vascular diameters and red blood cell (RBC) velocities for the feed arterioles and draining venules extending out of and into the optic disk. From these values, wall shear rates and blood flow rates were calculated. Diabetes induced approximate 30–40% decreases in RBC velocity, wall shear rate, and blood flow rate. These decreases were attenuated to 5–10% in the mice given HET0016. In summary, the 20-HETE inhibitor HET0016 is able to attenuate the retinal hemodynamic changes induced by diabetes.

We conclude that retinal blood flow rate is reduced significantly in the diabetic rat, with a substantial decrease of flow through the capillaries due to shunting of blood through the superficial layer, allowing rapid transit from arterioles to venules.

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW=155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW=376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.